Sample records for lactate dehydrogenase increased

  1. 21 CFR 862.1440 - Lactate dehydrogenase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... Systems § 862.1440 Lactate dehydrogenase test system. (a) Identification. A lactate dehydrogenase test system is a device intended to measure the activity of the enzyme lactate dehydrogenase in serum. Lactate... hepatitis, cirrhosis, and metastatic carcinoma of the liver, cardiac diseases such as myocardial infarction...

  2. Biochemical and structural characterization of Cryptosporidium parvum Lactate dehydrogenase.

    PubMed

    Cook, William J; Senkovich, Olga; Hernandez, Agustin; Speed, Haley; Chattopadhyay, Debasish

    2015-03-01

    The protozoan parasite Cryptosporidium parvum causes waterborne diseases worldwide. There is no effective therapy for C. parvum infection. The parasite depends mainly on glycolysis for energy production. Lactate dehydrogenase is a major regulator of glycolysis. This paper describes the biochemical characterization of C. parvum lactate dehydrogenase and high resolution crystal structures of the apo-enzyme and four ternary complexes. The ternary complexes capture the enzyme bound to NAD/NADH or its 3-acetylpyridine analog in the cofactor binding pocket, while the substrate binding site is occupied by one of the following ligands: lactate, pyruvate or oxamate. The results reveal distinctive features of the parasitic enzyme. For example, C. parvum lactate dehydrogenase prefers the acetylpyridine analog of NADH as a cofactor. Moreover, it is slightly less sensitive to gossypol inhibition compared with mammalian lactate dehydrogenases and not inhibited by excess pyruvate. The active site loop and the antigenic loop in C. parvum lactate dehydrogenase are considerably different from those in the human counterpart. Structural features and enzymatic properties of C. parvum lactate dehydrogenase are similar to enzymes from related parasites. Structural comparison with malate dehydrogenase supports a common ancestry for the two genes. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Diammonium phosphate stimulates transcription of L-lactate dehydrogenase leading to increased L-lactate production in the thermotolerant Bacillus coagulans strain.

    PubMed

    Sun, Lifan; Li, Yanfeng; Wang, Limin; Wang, Yanping; Yu, Bo

    2016-08-01

    Exploration of cost-effective fermentation substrates for efficient lactate production is an important economic objective. Although some organic nitrogen sources are also cheaper, inorganic nitrogen salts for lactate fermentation have additional advantages in facilitating downstream procedures and significantly improving the commercial competitiveness of lactate production. In this study, we first established an application of diammonium phosphate to replace yeast extract with a reduced 90 % nitrogen cost for a thermotolerant Bacillus coagulans strain. In vivo enzymatic and transcriptional analyses demonstrated that diammonium phosphate stimulates the gene expression of L-lactate dehydrogenase, thus providing higher specific enzyme activity in vivo and increasing L-lactic acid production. This new information provides a foundation for establishing a cost-effective process for polymer-grade L-lactic acid production in an industrial setting.

  4. Genetics Home Reference: lactate dehydrogenase deficiency

    MedlinePlus

    ... dehydrogenase-B pieces (subunits) of the lactate dehydrogenase enzyme. This enzyme is found throughout the body and is important ... cells. There are five different forms of this enzyme, each made up of four protein subunits. Various ...

  5. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... dehydrogenase isoenzymes test system is a device intended to measure the activity of lactate dehydrogenase isoenzymes (a group of enzymes with similar biological activity) in serum. Measurements of lactate...

  6. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... dehydrogenase isoenzymes test system is a device intended to measure the activity of lactate dehydrogenase isoenzymes (a group of enzymes with similar biological activity) in serum. Measurements of lactate...

  7. Increasing the Heme-Dependent Respiratory Efficiency of Lactococcus lactis by Inhibition of Lactate Dehydrogenase

    PubMed Central

    Arioli, Stefania; Zambelli, Daniele; Guglielmetti, Simone; De Noni, Ivano; Pedersen, Martin B.; Pedersen, Per Dedenroth; Dal Bello, Fabio

    2013-01-01

    The discovery of heme-induced respiration in Lactococcus lactis has radically improved the industrial processes used for the biomass production of this species. Here, we show that inhibition of the lactate dehydrogenase activity of L. lactis during growth under respiration-permissive conditions can stimulate aerobic respiration, thereby increasing not only growth efficiency but also the robustness of this organism. PMID:23064338

  8. Evolution of D-lactate dehydrogenase activity from glycerol dehydrogenase and its utility for D-lactate production from lignocellulose.

    PubMed

    Wang, Qingzhao; Ingram, Lonnie O; Shanmugam, K T

    2011-11-22

    Lactic acid, an attractive, renewable chemical for production of biobased plastics (polylactic acid, PLA), is currently commercially produced from food-based sources of sugar. Pure optical isomers of lactate needed for PLA are typically produced by microbial fermentation of sugars at temperatures below 40 °C. Bacillus coagulans produces L(+)-lactate as a primary fermentation product and grows optimally at 50 °C and pH 5, conditions that are optimal for activity of commercial fungal cellulases. This strain was engineered to produce D(-)-lactate by deleting the native ldh (L-lactate dehydrogenase) and alsS (acetolactate synthase) genes to impede anaerobic growth, followed by growth-based selection to isolate suppressor mutants that restored growth. One of these, strain QZ19, produced about 90 g L(-1) of optically pure D(-)-lactic acid from glucose in < 48 h. The new source of D-lactate dehydrogenase (D-LDH) activity was identified as a mutated form of glycerol dehydrogenase (GlyDH; D121N and F245S) that was produced at high levels as a result of a third mutation (insertion sequence). Although the native GlyDH had no detectable activity with pyruvate, the mutated GlyDH had a D-LDH specific activity of 0.8 μmoles min(-1) (mg protein)(-1). By using QZ19 for simultaneous saccharification and fermentation of cellulose to D-lactate (50 °C and pH 5.0), the cellulase usage could be reduced to 1/3 that required for equivalent fermentations by mesophilic lactic acid bacteria. Together, the native B. coagulans and the QZ19 derivative can be used to produce either L(+) or D(-) optical isomers of lactic acid (respectively) at high titers and yields from nonfood carbohydrates.

  9. Evolution of D-lactate dehydrogenase activity from glycerol dehydrogenase and its utility for D-lactate production from lignocellulose

    PubMed Central

    Wang, Qingzhao; Ingram, Lonnie O.; Shanmugam, K. T.

    2011-01-01

    Lactic acid, an attractive, renewable chemical for production of biobased plastics (polylactic acid, PLA), is currently commercially produced from food-based sources of sugar. Pure optical isomers of lactate needed for PLA are typically produced by microbial fermentation of sugars at temperatures below 40 °C. Bacillus coagulans produces L(+)-lactate as a primary fermentation product and grows optimally at 50 °C and pH 5, conditions that are optimal for activity of commercial fungal cellulases. This strain was engineered to produce D(−)-lactate by deleting the native ldh (L-lactate dehydrogenase) and alsS (acetolactate synthase) genes to impede anaerobic growth, followed by growth-based selection to isolate suppressor mutants that restored growth. One of these, strain QZ19, produced about 90 g L-1 of optically pure D(−)-lactic acid from glucose in < 48 h. The new source of D-lactate dehydrogenase (D-LDH) activity was identified as a mutated form of glycerol dehydrogenase (GlyDH; D121N and F245S) that was produced at high levels as a result of a third mutation (insertion sequence). Although the native GlyDH had no detectable activity with pyruvate, the mutated GlyDH had a D-LDH specific activity of 0.8 μmoles min-1 (mg protein)-1. By using QZ19 for simultaneous saccharification and fermentation of cellulose to D-lactate (50 °C and pH 5.0), the cellulase usage could be reduced to 1/3 that required for equivalent fermentations by mesophilic lactic acid bacteria. Together, the native B. coagulans and the QZ19 derivative can be used to produce either L(+) or D(−) optical isomers of lactic acid (respectively) at high titers and yields from nonfood carbohydrates. PMID:22065761

  10. High brain lactate is a hallmark of aging and caused by a shift in the lactate dehydrogenase A/B ratio

    PubMed Central

    Ross, Jaime M.; Öberg, Johanna; Brené, Stefan; Coppotelli, Giuseppe; Terzioglu, Mügen; Pernold, Karin; Goiny, Michel; Sitnikov, Rouslan; Kehr, Jan; Trifunovic, Aleksandra; Larsson, Nils-Göran; Hoffer, Barry J.; Olson, Lars

    2010-01-01

    At present, there are few means to track symptomatic stages of CNS aging. Thus, although metabolic changes are implicated in mtDNA mutation-driven aging, the manifestations remain unclear. Here, we used normally aging and prematurely aging mtDNA mutator mice to establish a molecular link between mitochondrial dysfunction and abnormal metabolism in the aging process. Using proton magnetic resonance spectroscopy and HPLC, we found that brain lactate levels were increased twofold in both normally and prematurely aging mice during aging. To correlate the striking increase in lactate with tissue pathology, we investigated the respiratory chain enzymes and detected mitochondrial failure in key brain areas from both normally and prematurely aging mice. We used in situ hybridization to show that increased brain lactate levels were caused by a shift in transcriptional activities of the lactate dehydrogenases to promote pyruvate to lactate conversion. Separation of the five tetrameric lactate dehydrogenase (LDH) isoenzymes revealed an increase of those dominated by the Ldh-A product and a decrease of those rich in the Ldh-B product, which, in turn, increases pyruvate to lactate conversion. Spectrophotometric assays measuring LDH activity from the pyruvate and lactate sides of the reaction showed a higher pyruvate → lactate activity in the brain. We argue for the use of lactate proton magnetic resonance spectroscopy as a noninvasive strategy for monitoring this hallmark of the aging process. The mtDNA mutator mouse allows us to conclude that the increased LDH-A/LDH-B ratio causes high brain lactate levels, which, in turn, are predictive of aging phenotypes. PMID:21041631

  11. High brain lactate is a hallmark of aging and caused by a shift in the lactate dehydrogenase A/B ratio.

    PubMed

    Ross, Jaime M; Öberg, Johanna; Brené, Stefan; Coppotelli, Giuseppe; Terzioglu, Mügen; Pernold, Karin; Goiny, Michel; Sitnikov, Rouslan; Kehr, Jan; Trifunovic, Aleksandra; Larsson, Nils-Göran; Hoffer, Barry J; Olson, Lars

    2010-11-16

    At present, there are few means to track symptomatic stages of CNS aging. Thus, although metabolic changes are implicated in mtDNA mutation-driven aging, the manifestations remain unclear. Here, we used normally aging and prematurely aging mtDNA mutator mice to establish a molecular link between mitochondrial dysfunction and abnormal metabolism in the aging process. Using proton magnetic resonance spectroscopy and HPLC, we found that brain lactate levels were increased twofold in both normally and prematurely aging mice during aging. To correlate the striking increase in lactate with tissue pathology, we investigated the respiratory chain enzymes and detected mitochondrial failure in key brain areas from both normally and prematurely aging mice. We used in situ hybridization to show that increased brain lactate levels were caused by a shift in transcriptional activities of the lactate dehydrogenases to promote pyruvate to lactate conversion. Separation of the five tetrameric lactate dehydrogenase (LDH) isoenzymes revealed an increase of those dominated by the Ldh-A product and a decrease of those rich in the Ldh-B product, which, in turn, increases pyruvate to lactate conversion. Spectrophotometric assays measuring LDH activity from the pyruvate and lactate sides of the reaction showed a higher pyruvate → lactate activity in the brain. We argue for the use of lactate proton magnetic resonance spectroscopy as a noninvasive strategy for monitoring this hallmark of the aging process. The mtDNA mutator mouse allows us to conclude that the increased LDH-A/LDH-B ratio causes high brain lactate levels, which, in turn, are predictive of aging phenotypes.

  12. Catalytic properties of thermophilic lactate dehydrogenase and halophilic malate dehydrogenase at high temperature and low water activity.

    PubMed

    Hecht, K; Wrba, A; Jaenicke, R

    1989-07-15

    Thermophilic lactate dehydrogenases from Thermotoga maritima and Bacillus stearothermophilus are stable up to temperature limits close to the optimum growth temperature of their parent organisms. Their catalytic properties are anomalous in that Km shows a drastic increase with increasing temperature. At low temperatures, the effect levels off. Extreme halophilic malate dehydrogenase from Halobacterium marismortui exhibits a similar anomaly. Increasing salt concentration (NaCl) leads to an optimum curve for Km, oxaloacctate while Km, NADH remains constant. Previous claims that the activity of halophilic malate dehydrogenase shows a maximum at 1.25 M NaCl are caused by limiting substrate concentration; at substrate saturation, specific activity of halophilic malate dehydrogenase reaches a constant value at ionic strengths I greater than or equal to 1 M. Non-halophilic (mitochondrial) malate dehydrogenase shows Km characteristics similar to those observed for the halophilic enzyme. The drastic decrease in specific activity of the mitochondrial enzyme at elevated salt concentrations is caused by the salt-induced increase in rigidity of the enzyme, rather than gross structural changes.

  13. Lactate dehydrogenase activity drives hair follicle stem cell activation

    PubMed Central

    Aimee, Flores; John, Schell; Abby, Krall; David, Jelinek; Matilde, Miranda; Melina, Grigorian; Daniel, Braas; White Andrew, C; Jessica, Zhou; Nick, Graham; Thomas, Graeber; Pankaj, Seth; Denis, Evseenko; Hilary, Coller; Jared, Rutter; Heather, Christofk; Lowry William, E

    2017-01-01

    Summary While normally dormant, Hair Follicle Stem Cells (HFSCs) quickly become activated to divide during a new hair cycle. The quiescence of HFSCs is known to be regulated by a number of intrinsic and extrinsic mechanisms. Here we provide several lines of evidence to demonstrate that HFSCs utilize glycolytic metabolism and produce significantly more lactate than other cells in the epidermis. Furthermore, lactate generation appears to be critical for the activation of HFSCs as deletion of lactate dehydrogenase (Ldha) prevented their activation. Conversely, genetically promoting lactate production in HFSCs through mitochondrial pyruvate carrier (Mpc1) deletion accelerated their activation and the hair cycle. Finally, we identify small molecules that increase lactate production by stimulating Myc levels or inhibiting Mpc1 carrier activity and can topically induce the hair cycle. These data suggest that HFSCs maintain a metabolic state that allow them to remain dormant and yet quickly respond to appropriate proliferative stimuli. PMID:28812580

  14. Pyruvate dehydrogenase complex and lactate dehydrogenase are targets for therapy of acute liver failure.

    PubMed

    Ferriero, Rosa; Nusco, Edoardo; De Cegli, Rossella; Carissimo, Annamaria; Manco, Giuseppe; Brunetti-Pierri, Nicola

    2018-03-24

    Acute liver failure is a rapidly progressive deterioration of hepatic function resulting in high mortality and morbidity. Metabolic enzymes can translocate to the nucleus to regulate histone acetylation and gene expression. Levels and activities of pyruvate dehydrogenase complex (PDHC) and lactate dehydrogenase (LDH) were evaluated in nuclear fractions of livers of mice exposed to various hepatotoxins including CD95-antibody, α-amanitin, and acetaminophen. Whole-genome gene expression profiling by RNA-seq was performed in livers of mice with acute liver failure and analyzed by gene ontology enrichment analysis. Cell viability was evaluated in cell lines knocked-down for PDHA1 or LDH-A and in cells incubated with the LDH inhibitor galloflavin after treatment with CD95-antibody. We evaluated whether the histone acetyltransferase inhibitor garcinol or galloflavin could reduce liver damage in mice with acute liver failure. Levels and activities of PDHC and LDH were increased in nuclear fractions of livers of mice with acute liver failure. The increase of nuclear PDHC and LDH was associated with increased concentrations of acetyl-CoA and lactate in nuclear fractions, and histone H3 hyper-acetylation. Gene expression in livers of mice with acute liver failure suggested that increased histone H3 acetylation induces the expression of genes related to damage response. Reduced histone acetylation by the histone acetyltransferase inhibitor garcinol decreased liver damage and improved survival in mice with acute liver failure. Knock-down of PDHC or LDH improved viability in cells exposed to a pro-apoptotic stimulus. Treatment with the LDH inhibitor galloflavin that was also found to inhibit PDHC, reduced hepatic necrosis, apoptosis, and expression of pro-inflammatory cytokines in mice with acute liver failure. Mice treated with galloflavin also showed a dose-response increase in survival. PDHC and LDH translocate to the nucleus, leading to increased nuclear concentrations of

  15. Expression of Lactate Dehydrogenase in Aspergillus niger for L-Lactic Acid Production

    PubMed Central

    Dave, Khyati K.; Punekar, Narayan S.

    2015-01-01

    Different engineered organisms have been used to produce L-lactate. Poor yields of lactate at low pH and expensive downstream processing remain as bottlenecks. Aspergillus niger is a prolific citrate producer and a remarkably acid tolerant fungus. Neither a functional lactate dehydrogenase (LDH) from nor lactate production by A. niger is reported. Its genome was also investigated for the presence of a functional ldh. The endogenous A. niger citrate synthase promoter relevant to A. niger acidogenic metabolism was employed to drive constitutive expression of mouse lactate dehydrogenase (mldhA). An appraisal of different branches of the A. niger pyruvate node guided the choice of mldhA for heterologous expression. A high copy number transformant C12 strain, displaying highest LDH specific activity, was analyzed under different growth conditions. The C12 strain produced 7.7 g/l of extracellular L-lactate from 60 g/l of glucose, in non-neutralizing minimal media. Significantly, lactate and citrate accumulated under two different growth conditions. Already an established acidogenic platform, A. niger now promises to be a valuable host for lactate production. PMID:26683313

  16. 21 CFR 862.1440 - Lactate dehydrogenase test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Lactate dehydrogenase test system. 862.1440 Section 862.1440 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test...

  17. A Bacterial Multidomain NAD-Independent d-Lactate Dehydrogenase Utilizes Flavin Adenine Dinucleotide and Fe-S Clusters as Cofactors and Quinone as an Electron Acceptor for d-Lactate Oxidization

    PubMed Central

    Jiang, Tianyi; Guo, Xiaoting; Yan, Jinxin; Zhang, Yingxin; Wang, Yujiao; Zhang, Manman; Sheng, Binbin; Ma, Cuiqing; Xu, Ping

    2017-01-01

    ABSTRACT Bacterial membrane-associated NAD-independent d-lactate dehydrogenase (Fe-S d-iLDH) oxidizes d-lactate into pyruvate. A sequence analysis of the enzyme reveals that it contains an Fe-S oxidoreductase domain in addition to a flavin adenine dinucleotide (FAD)-containing dehydrogenase domain, which differs from other typical d-iLDHs. Fe-S d-iLDH from Pseudomonas putida KT2440 was purified as a His-tagged protein and characterized in detail. This monomeric enzyme exhibited activities with l-lactate and several d-2-hydroxyacids. Quinone was shown to be the preferred electron acceptor of the enzyme. The two domains of the enzyme were then heterologously expressed and purified separately. The Fe-S cluster-binding motifs predicted by sequence alignment were preliminarily verified by site-directed mutagenesis of the Fe-S oxidoreductase domain. The FAD-containing dehydrogenase domain retained 2-hydroxyacid-oxidizing activity, although it decreased compared to the full Fe-S d-iLDH. Compared to the intact enzyme, the FAD-containing dehydrogenase domain showed increased catalytic efficiency with cytochrome c as the electron acceptor, but it completely lost the ability to use coenzyme Q10. Additionally, the FAD-containing dehydrogenase domain was no longer associated with the cell membrane, and it could not support the utilization of d-lactate as a carbon source. Based on the results obtained, we conclude that the Fe-S oxidoreductase domain functions as an electron transfer component to facilitate the utilization of quinone as an electron acceptor by Fe-S d-iLDH, and it helps the enzyme associate with the cell membrane. These functions make the Fe-S oxidoreductase domain crucial for the in vivo d-lactate utilization function of Fe-S d-iLDH. IMPORTANCE Lactate metabolism plays versatile roles in most domains of life. Lactate utilization processes depend on certain enzymes to oxidize lactate to pyruvate. In recent years, novel bacterial lactate-oxidizing enzymes have been

  18. Enzymatic Kinetic Properties of the Lactate Dehydrogenase Isoenzyme C4 of the Plateau Pika (Ochotona curzoniae)

    PubMed Central

    Wang, Yang; Wei, Lian; Wei, Dengbang; Li, Xiao; Xu, Lina; Wei, Linna

    2016-01-01

    Testis-specific lactate dehydrogenase (LDH-C4) is one of the lactate dehydrogenase (LDH) isozymes that catalyze the terminal reaction of pyruvate to lactate in the glycolytic pathway. LDH-C4 in mammals was previously thought to be expressed only in spermatozoa and testis and not in other tissues. Plateau pika (Ochotona curzoniae) belongs to the genus Ochotona of the Ochotonidea family. It is a hypoxia-tolerant species living in remote mountain areas at altitudes of 3000–5000 m above sea level on the Qinghai-Tibet Plateau. Surprisingly, Ldh-c is expressed not only in its testis and sperm, but also in somatic tissues of plateau pika. To shed light on the function of LDH-C4 in somatic cells, Ldh-a, Ldh-b, and Ldh-c of plateau pika were subcloned into bacterial expression vectors. The pure enzymes of Lactate Dehydrogenase A4 (LDH-A4), Lactate Dehydrogenase B4 (LDH-B4), and LDH-C4 were prepared by a series of expression and purification processes, and the three enzymes were identified by the method of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and native polyacrylamide gel electrophoresis (PAGE). The enzymatic kinetics properties of these enzymes were studied by Lineweaver-Burk double-reciprocal plots. The results showed the Michaelis constant (Km) of LDH-C4 for pyruvate and lactate was 0.052 and 4.934 mmol/L, respectively, with an approximate 90 times higher affinity of LDH-C4 for pyruvate than for lactate. At relatively high concentrations of lactate, the inhibition constant (Ki) of the LDH isoenzymes varied: LDH-A4 (Ki = 26.900 mmol/L), LDH-B4 (Ki = 23.800 mmol/L), and LDH-C4 (Ki = 65.500 mmol/L). These data suggest that inhibition of lactate by LDH-A4 and LDH-B4 were stronger than LDH-C4. In light of the enzymatic kinetics properties, we suggest that the plateau pika can reduce reliance on oxygen supply and enhance its adaptation to the hypoxic environments due to increased anaerobic glycolysis by LDH-C4. PMID:26751442

  19. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Lactate dehydrogenase isoenzymes test system. 862.1445 Section 862.1445 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

  20. Purification, properties and immunological relationship of L (+)-lactate dehydrogenase from Lactobacillus casei.

    PubMed

    Gordon, G L; Doelle, H W

    1976-08-16

    The fructose-1,6-bisphosphate-activated L-lactate dehydrogenase (EC 1.1.1.27) from Lactobacillus casei ATCC 393 has been purified to homogenity by including affinity chromatography (cibacronblue-Sephadex-G-200) and preparative polyacrylamide gel electrophoresis into the purification procedures. The enzyme has an Mr of 132000-135000 with a subunit Mr of 34000. The pH optimum was found to be 5.4 insodium acetate buffer. Tris/maleate and citrate/phosphate buffers inhibited enzyme activity at this pH. The enzyme was completely inactivated by a temperature increase from 60 degrees C to 70 degrees C. Pyruvate saturation curves were sigmoidal in the absence of fructose 1,6-bisphosphate. In the presence of 20 muM fructose 1,6-bisphosphate a Km of 1.0 mM for pyruvate was obtained, whereas fructose 1,6-bisphosphate had no effect on the Km of 0.01 mM for NADH. The use of pyruvate analogues revealed two types of pyruvate binding sites, a catalytic and an effector site. The enzyme from L. casei appears to be subject to strict metabolic control, since ADP, ATP, dihydroxyacetone phosphate and 6-phosphogluconate are strong inhibitors. Immunodiffusion experiments with a rabbit antiserum to L. casei lactate dehydrogenase revealed that L. casei ATCC 393 L (+)-lactate dehydrogenase is probably not immunologically related to group D and group N streptococci. Of 24 lactic acid bacterial strains tested only 5 strains did cross-react: L. casei ATCC 393 = L. casei var. rhamnosus ATCC 7469 - L. casei var. alactosus NCDO 680 greater than L. casei UQM 95 greater than L. plantarum ATCC 14917.

  1. Peroxisomal lactate dehydrogenase is generated by translational readthrough in mammals

    PubMed Central

    Schueren, Fabian; Lingner, Thomas; George, Rosemol; Hofhuis, Julia; Dickel, Corinna; Gärtner, Jutta; Thoms, Sven

    2014-01-01

    Translational readthrough gives rise to low abundance proteins with C-terminal extensions beyond the stop codon. To identify functional translational readthrough, we estimated the readthrough propensity (RTP) of all stop codon contexts of the human genome by a new regression model in silico, identified a nucleotide consensus motif for high RTP by using this model, and analyzed all readthrough extensions in silico with a new predictor for peroxisomal targeting signal type 1 (PTS1). Lactate dehydrogenase B (LDHB) showed the highest combined RTP and PTS1 probability. Experimentally we show that at least 1.6% of the total cellular LDHB is targeted to the peroxisome by a conserved hidden PTS1. The readthrough-extended lactate dehydrogenase subunit LDHBx can also co-import LDHA, the other LDH subunit, into peroxisomes. Peroxisomal LDH is conserved in mammals and likely contributes to redox equivalent regeneration in peroxisomes. DOI: http://dx.doi.org/10.7554/eLife.03640.001 PMID:25247702

  2. LDHk, an unusual oxygen-sensitive lactate dehydrogenase expressed in human cancer.

    PubMed Central

    Anderson, G R; Kovacik, W P

    1981-01-01

    An unusual isozyme of lactate dehydrogenase (LDH; L-lactate:NAD+ oxidoreductase, EC 1.1.1.27), LDHk, has been described in cells transformed by the Kirsten murine sarcoma virus (KiMSV). This isozyme appears to contain one or more subunits encoded by the transforming gene of KiMSV and is readily distinguished from other isozymes of LDH. Specifically, it is more basic than other LDH isozymes, has an apparent subunit structure of (35,000)4(22,000)1, is essentially inactive if assayed under a normal atmosphere, and is strongly inhibited by GTP and various related compounds. We have examined human cancer and normal tissue controls for expression of an activity like LDHk. In 11 out of 16 human carcinomas, LDHk activity was increased 10- to 500-fold over the level seen in adjoining nontumor tissue. In contrast, other LDH isozymes were increased by only 2- to 5-fold. Images PMID:6942426

  3. Lactate dehydrogenase activity is inhibited by methylmalonate in vitro.

    PubMed

    Saad, Laura O; Mirandola, Sandra R; Maciel, Evelise N; Castilho, Roger F

    2006-04-01

    Methylmalonic acidemia (MMAemia) is an inherited metabolic disorder of branched amino acid and odd-chain fatty acid metabolism, involving a defect in the conversion of methylmalonyl-coenzyme A to succinyl-coenzyme A. Systemic and neurological manifestations in this disease are thought to be associated with the accumulation of methylmalonate (MMA) in tissues and biological fluids with consequent impairment of energy metabolism and oxidative stress. In the present work we studied the effect of MMA and two other inhibitors of mitochondrial respiratory chain complex II (malonate and 3-nitropropionate) on the activity of lactate dehydrogenase (LDH) in tissue homogenates from adult rats. MMA potently inhibited LDH-catalyzed conversion of lactate to pyruvate in liver and brain homogenates as well as in a purified bovine heart LDH preparation. LDH was about one order of magnitude less sensitive to inhibition by MMA when catalyzing the conversion of pyruvate to lactate. Kinetic studies on the inhibition of brain LDH indicated that MMA inhibits this enzyme competitively with lactate as a substrate (K (i)=3.02+/-0.59 mM). Malonate and 3-nitropropionate also strongly inhibited LDH-catalyzed conversion of lactate to pyruvate in brain homogenates, while no inhibition was observed by succinate or propionate, when present in concentrations of up to 25 mM. We propose that inhibition of the lactate/pyruvate conversion by MMA contributes to lactate accumulation in blood, metabolic acidemia and inhibition of gluconeogenesis observed in patients with MMAemia. Moreover, the inhibition of LDH in the central nervous system may also impair the lactate shuttle between astrocytes and neurons, compromising neuronal energy metabolism.

  4. Properties of lactate dehydrogenase from the isopod, Saduria entomon.

    PubMed

    Mulkiewicz, E; Zietara, M S; Stachowiak, K; Skorkowski, E F

    2000-07-01

    Saduria entomon lactate dehydrogenase (LDH-A4*) from thorax muscle was purified about 89 fold to specific activity 510 micromol NADH/min/mg using Cibacron Blue 3GA Agarose and Oxamate-Agarose chromatographies. The enzyme is a tetramer, with molecular weight of 140 kDa for the native enzyme and 36 kDa for the subunit. The isoelectric point was at pH 5.7. The enzyme possesses high heat stability (T50 = 71.5 degrees C). The optimum pH for pyruvate reduction reaction was 6.5, while for lactate oxidation one, the maximum activity was at pH 9.1. The Km for pyruvate was minimal at 5 degrees C, the average environmental temperature of the isopod. The Km values determined at 30 degrees C and optimal pH for pyruvate reduction and lactate oxidation were 0.18 and 90.04 mM, respectively. Amino acid compositional analyses showed the strongest resemblance of the isopod isoenzyme to cod (Gadus morhua) LDH-C4.

  5. [Evaluation of the increasing serum lactate dehydrogenase caused by recombinant human granulocyte-colony stimulating factor].

    PubMed

    Sawa, Toshiyuki; Yoshida, Tsutomu; Ikoma, Tetsuroh; Toyoda, Miki; Ohno, Yasushi; Fujiwara, Hisayoshi

    2003-01-01

    Increasing serum lactate dehydrogenase (LDH) is often caused by granulocyte-colony stimulating factor (G-CSF) for leukopenia following chemotherapy in patients with lung cancer. To evaluate the increase in LDH, we investigated the significance of its elevation and LDH isozyme during chemotherapy supported by recombinant human G-CSF (rhG-CSF). To exclude effects of liver diseases and chemotherapy-induced liver dysfunction, only patients in whom laboratory findings concerning liver function were within normal range were entered in this study. If leukocyte or neutrophil counts were less than grade 3, subcutaneous injection of 50 micrograms/m2 of filgrastim was given daily until leukocyte counts increased to more than 10,000/mm3. Sixty patients with unresectable lung cancer were enrolled in this study and the LDH isozyme was evaluable in 54 patients. Increasing LDH was observed in 38 patients(70.4%), and LDH isozyme was measured in these 38 patients. Increases in granulocytes and LDH isozymes were found to have a positive correlation. LDH2, LDH3, LDH4 and LDH5 increased significantly after rhG-CSF administration, although LDH 1 did not increase. It was found that a rapid increase in leukocytes by rhG-CSF induced an increase in LDH, especially LDH 3.4. Considering the results of principal component analysis and the distribution ratio of LDH isozymes in neutrophils, it is thought that elevation of LDH is reflected in the rapid production and consumption of neutrophils.

  6. The Core of Allosteric Motion in Thermus caldophilus l-Lactate Dehydrogenase*

    PubMed Central

    Ikehara, Yoko; Arai, Kazuhito; Furukawa, Nayuta; Ohno, Tadashi; Miyake, Tatsuya; Fushinobu, Shinya; Nakajima, Masahiro; Miyanaga, Akimasa; Taguchi, Hayao

    2014-01-01

    For Thermus caldophilus l-lactate dehydrogenase (TcLDH), fructose 1,6-bisphosphate (FBP) reduced the pyruvate S0.5 value 103-fold and increased the Vmax value 4-fold at 30 °C and pH 7.0, indicating that TcLDH has a much more T state-sided allosteric equilibrium than Thermus thermophilus l-lactate dehydrogenase, which has only two amino acid replacements, A154G and H179Y. The inactive (T) and active (R) state structures of TcLDH were determined at 1.8 and 2.0 Å resolution, respectively. The structures indicated that two mobile regions, MR1 (positions 172–185) and MR2 (positions 211–221), form a compact core for allosteric motion, and His179 of MR1 forms constitutive hydrogen bonds with MR2. The Q4(R) mutation, which comprises the L67E, H68D, E178K, and A235R replacements, increased Vmax 4-fold but reduced pyruvate S0.5 only 5-fold in the reaction without FBP. In contrast, the P2 mutation, comprising the R173Q and R216L replacements, did not markedly increase Vmax, but 102-reduced pyruvate S0.5, and additively increased the FBP-independent activity of the Q4(R) enzyme. The two types of mutation consistently increased the thermal stability of the enzyme. The MR1-MR2 area is a positively charged cluster, and its center approaches another positively charged cluster (N domain cluster) across the Q-axis subunit interface by 5 Å, when the enzyme undergoes the T to R transition. Structural and kinetic analyses thus revealed the simple and unique allosteric machinery of TcLDH, where the MR1-MR2 area pivotally moves during the allosteric motion and mediates the allosteric equilibrium through electrostatic repulsion within the protein molecule. PMID:25258319

  7. Deletion of lactate dehydrogenase in Enterobacter aerogenes to enhance 2,3-butanediol production.

    PubMed

    Jung, Moo-Young; Ng, Chiam Yu; Song, Hyohak; Lee, Jinwon; Oh, Min-Kyu

    2012-07-01

    2,3-Butanediol is an important bio-based chemical product, because it can be converted into several C4 industrial chemicals. In this study, a lactate dehydrogenase-deleted mutant was constructed to improve 2,3-butanediol productivity in Enterobacter aerogenes. To delete the gene encoding lactate dehydrogenase, λ Red recombination method was successfully adapted for E. aerogenes. The resulting strain produced a very small amount of lactate and 16.7% more 2,3-butanediol than that of the wild-type strain in batch fermentation. The mutant and its parental strain were then cultured with six different carbon sources, and the mutant showed higher carbon source consumption and microbial growth rates in all media. The 2,3-butanediol titer reached 69.5 g/l in 54 h during fed-batch fermentation with the mutant,which was 27.4% higher than that with the parental strain.With further optimization of the medium and aeration conditions,118.05 g/l 2,3-butanediol was produced in 54 h during fed-batch fermentation with the mutant. This is by far the highest titer of 2,3-butanediol with E. aerogenes achieved by metabolic pathway engineering.

  8. [Characterization of D-lactate dehydrogenase isozymes from a D-lactic acid producing bacterium Sporolactobacillus inulinus].

    PubMed

    Zhang, Danru; Zheng, Lu; Wu, Bin; He, Bingfang

    2016-11-04

    Sporolactobacillus inulinus, a typical homofermentative lactic acid bacterium, is an efficient D-lactic acid producer. Various environment factors affect the productivity of S. inulinus. Glucokinase, phosphofructokinase, pyruvate kinase and lactic dehydrogenase are the key enzymes of D-lactic acid production from glucose by S. inulinus. The characteristics of these enzymes are important in controlling and regulating the fermentation process. According to the genome bioinformatics analysis of S. inulinus CASD, three putative D-lactate dehydrogenases were identified, among which the bifunctional protein had been reported. In this study, we provided insights into the characteristics of the other two D-lactate dehydrogenase isozymes. S. inulinus Y2-8 genome was used as the template to amplify D-lactate dehydrogenase gene (dldh) and D-isomer specific 2-hydroxyacid dehydrogenase gene (dhdh). The two recombinant strains E-pET-28a/dldh and E-pET-28a/dhdh were constructed for enzyme expression. Both recombinants DLDH and DHDH could convert pyruvic acid into D-lactic acid. Enzymes expressed by recombinant strains were purified by Ni-NTA chromatography. The apparent molecular mass of DLDH was approximately 37 kDa by SDS-PAGE analysis, and DLDH showed a high affinity to pyruvate with the Km value of (0.58±0.04) mmol/L. The optimal reaction temperature and pH for DLDH was 35℃ and 6.5, respectively. The apparent molecular mass of DHDH was approximately 39 kDa, and the Km of DHDH toward pyruvate was (1.70±0.08) mmol/L. The optimum catalysis temperature and pH of DHDH were 30℃ and 7.5, respectively. According to the Km and optimal reaction pH, DLDH was suggested as the main catalyst in formation D-lactic acid from pyruvate during the fermentation. The enzymatic properties would contribute to the regulation of the fermentation of S. inulinus.

  9. Lactate dehydrogenase inhibition: exploring possible applications beyond cancer treatment.

    PubMed

    Di Stefano, Giuseppina; Manerba, Marcella; Di Ianni, Lorenza; Fiume, Luigi

    2016-04-01

    Lactate dehydrogenase (LDH) inhibition is considered a worthwhile attempt in the development of innovative anticancer strategies. Unfortunately, in spite of the involvement of several research institutions and pharma-companies, the discovery of LDH inhibitors with drug-like properties seems a hardly resolvable challenge. While awaiting new advancements, in the present review we will examine other pathologic conditions characterized by increased glycolysis and LDH activity, which could potentially benefit from LDH inhibition. The rationale for targeting LDH activity in these contexts is the same justifying the LDH-based approach in anticancer therapy: because of the enzyme position at the end of glycolytic pathway, LDH inhibitors are not expected to hinder glucose metabolism of normal cells. Moreover, we will summarize the latest contributions in the discovery of enzyme inhibitors and try to glance over the reasons underlying the complexity of this research.

  10. Fragment growing and linking lead to novel nanomolar lactate dehydrogenase inhibitors.

    PubMed

    Kohlmann, Anna; Zech, Stephan G; Li, Feng; Zhou, Tianjun; Squillace, Rachel M; Commodore, Lois; Greenfield, Matthew T; Lu, Xiaohui; Miller, David P; Huang, Wei-Sheng; Qi, Jiwei; Thomas, R Mathew; Wang, Yihan; Zhang, Sen; Dodd, Rory; Liu, Shuangying; Xu, Rongsong; Xu, Yongjin; Miret, Juan J; Rivera, Victor; Clackson, Tim; Shakespeare, William C; Zhu, Xiaotian; Dalgarno, David C

    2013-02-14

    Lactate dehydrogenase A (LDH-A) catalyzes the interconversion of lactate and pyruvate in the glycolysis pathway. Cancer cells rely heavily on glycolysis instead of oxidative phosphorylation to generate ATP, a phenomenon known as the Warburg effect. The inhibition of LDH-A by small molecules is therefore of interest for potential cancer treatments. We describe the identification and optimization of LDH-A inhibitors by fragment-based drug discovery. We applied ligand based NMR screening to identify low affinity fragments binding to LDH-A. The dissociation constants (K(d)) and enzyme inhibition (IC(50)) of fragment hits were measured by surface plasmon resonance (SPR) and enzyme assays, respectively. The binding modes of selected fragments were investigated by X-ray crystallography. Fragment growing and linking, followed by chemical optimization, resulted in nanomolar LDH-A inhibitors that demonstrated stoichiometric binding to LDH-A. Selected molecules inhibited lactate production in cells, suggesting target-specific inhibition in cancer cell lines.

  11. Ultrafiltration-LC-MS combined with semi-preparative HPLC for the simultaneous screening and isolation of lactate dehydrogenase inhibitors from Belamcanda chinensis.

    PubMed

    Li, Senlin; Li, Sainan; Tang, Ying; Liu, Chunming; Chen, Lina; Zhang, Yuchi

    2016-12-01

    Stroke represents the fourth leading cause of death in the USA and the second leading cause of death worldwide. Lactate dehydrogenase inhibitors are widely used in the treatment of ischemic stroke and natural products are considered a promising source of novel lactate dehydrogenase inhibitors. In this study, we used PC12 cells to determine the protective effect of extracts from the herb Belamcanda chinensis following toxic challenge. Using ultrafiltration high-performance liquid chromatography coupled with photo-diode array detection and electrospray ionization mass spectrometry, we screened and identified isoflavonoids from Belamcanda chinensis extracts. Semi-preparative high-performance liquid chromatography was then applied to separate and isolate the active constituents. Using these methods, we identified six major compounds in Belamcanda chinensis as lactate dehydrogenase inhibitors: tectoridin, iristectorin A, iridin, tectorigenin, irigenin, and irisflorentin, which were then isolated to >92% purity. This is the first report that Belamcanda chinensis extracts contain potent lactate dehydrogenase inhibitors. Our results demonstrate that the systematic isolation of bioactive components from Belamcanda chinensis guided by ultrafiltration high-performance liquid chromatography coupled with photo-diode array detection and electrospray ionization mass spectrometry represents a feasible and efficient technique that could be extended for the identification and isolation of other enzyme inhibitors. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Reduction of ammonia and lactate through the coupling of glutamine synthetase selection and downregulation of lactate dehydrogenase-A in CHO cells.

    PubMed

    Noh, Soo Min; Park, Jin Hyoung; Lim, Myung Sin; Kim, Jong Won; Lee, Gyun Min

    2017-02-01

    Chinese hamster ovary (CHO) cell cultivation for production of therapeutic proteins is accompanied by production of metabolic wastes, mostly ammonia and lactate. To reduce ammonia production, the glutamine synthetase (GS) system was used to develop therapeutic monoclonal antibody (mAb)-producing CHO cells (SM-0.025). Additionally, the lactate dehydrogenase-A (LDH-A) was downregulated with shRNA to reduce lactate production in SM-0.025. The resulting mAb-producing cell lines (#2, #46, and #52) produced less ammonia than the host cell line during the exponential phase due to GS protein overexpression. LDH-A downregulation in SM-0.025 not only reduced lactate production but also further reduced ammonia production. Among the three LDH-A-downregulated clones, clone #2 had the highest mAb production along with significantly reduced specific lactate and ammonia production rates compared to those in SM-0.025. Waste reduction increased the galactosylation level of N-glycosylation, which improved mAb quality. LDH-A downregulation was also successfully applied to the host cell lines (CHO K1 and GS knockout CHO-K1). However, LDH-A downregulated host cells could not survive the pool-selection process wherein glutamine was excluded and methionine sulfoximine was added to the media. Taken together, LDH-A downregulation in the mAb-producing cell line generated with the GS system successfully reduced both ammonia and lactate levels, improving mAb galactosylation. However, LDH-A downregulation could not be applied to host cell lines because it hampered the selection process of the GS system.

  13. Age related rise in lactate and its correlation with lactate dehydrogenase (LDH) status in post-mitochondrial fractions isolated from different regions of brain in mice.

    PubMed

    Datta, Siddhartha; Chakrabarti, Nilkanta

    2018-04-18

    Rise in brain lactate is the hallmark of ageing. Separate studies report that ageing is associated with elevation of lactate level and alterations of lactate dehydrogenase (LDH)-A/B mRNA-expression-ratio in cerebral cortex and hippocampus. However, age related lactate rise in brain and its association with LDH status and their brain regional variations are still elusive. In the present study, level of lactate, LDH (A and B) activity and LDH-A expression were evaluated in post-mitochondrial fraction of tissues isolated from four different brain regions (cerebral cortex, hippocampus, substantia nigra and cerebellum) of young and aged mice. Lactate levels elevated in four brain regions with maximum rise in substantia nigra of aged mice. LDH-A protein expression and its activity decreased in cerebral cortex, hippocampus and substantia nigra without any changes of these parameters in cerebellum of aged mice. LDH-B activity decreased in hippocampus, substantia nigra and cerebellum whereas its activity remains unaltered in cerebral cortex of aged mice. Accordingly, the ratio of LDH-A/LDH-B-activity remains unaltered in hippocampus and substantia nigra, decreased in cerebral cortex and increased in cerebellum. Therefore, rise of lactate in three brain regions (cerebral cortex, hippocampus, substantia nigra) appeared to be not correlated with the alterations of its regulatory enzymes activities in these three brain regions, rather it supports the fact of involvement of other mechanisms, like lactate transport and/or aerobic/anaerobic metabolism as the possible cause(s) of lactate rise in these three brain regions. The increase in LDH-A/LDH-B-activity-ratio appeared to be positively correlated with elevated lactate level in cerebellum of aged mice. Overall, the present study indicates that the mechanism of rise in lactate in brain varies with brain regions where LDH status plays an important role during ageing. Copyright © 2018 Elsevier Ltd. All rights reserved.

  14. Some Lactobacillus l-Lactate Dehydrogenases Exhibit Comparable Catalytic Activities for Pyruvate and Oxaloacetate

    PubMed Central

    Arai, Kazuhito; Kamata, Takeo; Uchikoba, Hiroyuki; Fushinobu, Shinya; Matsuzawa, Hiroshi; Taguchi, Hayao

    2001-01-01

    The nonallosteric and allosteric l-lactate dehydrogenases of Lactobacillus pentosus and L. casei, respectively, exhibited broad substrate specificities, giving virtually the same maximal reaction velocity and substrate Km values for pyruvate and oxaloacetate. Replacement of Pro101 with Asn reduced the activity of the L. pentosus enzyme toward these alternative substrates to a greater extent than the activity toward pyruvate. PMID:11114942

  15. Role of malate dehydrogenase in facilitating lactate dehydrogenase to support the glycolysis pathway in tumors.

    PubMed

    Mansouri, Siavash; Shahriari, Ali; Kalantar, Hadi; Moini Zanjani, Taraneh; Haghi Karamallah, Mojtaba

    2017-04-01

    High aerobic glycolysis, as one of the hallmarks of cancer cells, requires nicotinamide adenine dinucleotide (NAD + ) as a vital co-factor, to guarantee the flow of glycolysis. Malate dehydrogenase (MDH), as an important enzyme in cancer metabolism, is a source of NAD + additional to lactate dehydrogenase (LDH). The current study aimed to elucidate the kinetic parameters of MDH in human breast cancer and evaluate its supportive role in the glycolysis pathway. The Michaelis-Menten constant (K m ) and maximum velocity (V max ) of MDH were determined in the crude extracts of human breast tumors and healthy tissue samples, which were obtained directly from the operating theatre. To assess the potential role of MDH in supporting glycolysis, the MDH activity was measured when the LDH activity was inhibited by different concentrations of oxamate, an inhibitor of LDH in breast cancer cell lines. The K m of cancerous MDH (C-MDH) was the same as the healthy MDH, although the V max of C-MDH was higher relative to the healthy MDH. Notably, the MDH activity was increased in the MDA-MB-231 cell line, which was treated with the LDH inhibitor (oxamate), but not in the MCF-7 cell line (P<0.05). The higher tendency of C-MDH for NAD + and malate generation in cancer cells is an effective approach for supporting glycolysis. Increasing MDH activity in the absence of LDH demonstrates the supportive role of MDH in glycolysis. Therefore, decreasing MDH activity and expression in a forward reaction may present as a valid molecular target to abolish its potential effect on tumor metabolism.

  16. Lactate dehydrogenase-A is indispensable for vascular smooth muscle cell proliferation and migration.

    PubMed

    Kim, Ji-Hyun; Bae, Kwi-Hyun; Byun, Jun-Kyu; Lee, Sungwoo; Kim, Jung-Guk; Lee, In Kyu; Jung, Gwon-Soo; Lee, You Mie; Park, Keun-Gyu

    2017-10-07

    The proliferation and migration of vascular smooth muscle cells (VSMCs) have been implicated in the pathogenesis of atherosclerosis. Increased aerobic glycolysis is a key feature of cellular phenotypes including cancer and immune cells. However, the role of aerobic glycolysis in the atherogenic phenotype of VSMCs remains largely unknown. Here, we investigated the role of lactate dehydrogenase-A (LDHA), which is a key enzyme for glycolysis, in the proliferation and migration of VSMCs. Activation of primary rat VSMCs with fetal bovine serum (FBS) or platelet-derived growth factor (PDGF) increased their proliferation and migration, glycolytic activity, and expression of LDHA. Wound healing and transwell migration assays demonstrated that small interfering RNA-mediated knockdown of LDHA and pharmacological inhibition of LDHA by oxamate both effectively inhibited VSMC proliferation and migration. Inhibition of LDHA activity by oxamate reduced PDGF-stimulated glucose uptake, lactate production, and ATP production. Taken together, this study shows that enhanced glycolysis in PDGF- or FBS-stimulated VSMCs plays an important role in their proliferation and migration and suggests that LDHA is a potential therapeutic target to prevent vessel lumen constriction during the course of atherosclerosis and restenosis. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Empirical evaluation of a virtual laboratory approach to teach lactate dehydrogenase enzyme kinetics.

    PubMed

    Booth, Christine; Cheluvappa, Rajkumar; Bellinson, Zack; Maguire, Danni; Zimitat, Craig; Abraham, Joyce; Eri, Rajaraman

    2016-06-01

    Personalised instruction is increasingly recognised as crucial for efficacious learning today. Our seminal work delineates and elaborates on the principles, development and implementation of a specially-designed adaptive, virtual laboratory. We strived to teach laboratory skills associated with lactate dehydrogenase (LDH) enzyme kinetics to 2nd-year biochemistry students using our adaptive learning platform. Pertinent specific aims were to:(1)design/implement a web-based lesson to teach lactate dehydrogenase(LDH) enzyme kinetics to 2nd-year biochemistry students(2)determine its efficacious in improving students' comprehension of enzyme kinetics(3)assess their perception of its usefulness/manageability(vLab versus Conventional Tutorial). Our tools were designed using HTML5 technology. We hosted the program on an adaptive e-learning platform (AeLP). Provisions were made to interactively impart informed laboratory skills associated with measuring LDH enzyme kinetics. A series of e-learning methods were created. Tutorials were generated for interactive teaching and assessment. The learning outcomes herein were on par with that from a conventional classroom tutorial. Student feedback showed that the majority of students found the vLab learning experience "valuable"; and the vLab format/interface "well-designed". However, there were a few technical issues with the 1st roll-out of the platform. Our pioneering effort resulted in productive learning with the vLab, with parity with that from a conventional tutorial. Our contingent discussion emphasises not only the cornerstone advantages, but also the shortcomings of the AeLP method utilised. We conclude with an astute analysis of possible extensions and applications of our methodology.

  18. Nuclear lactate dehydrogenase modulates histone modification in human hepatocytes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Castonguay, Zachary; Auger, Christopher; Thomas, Sean C.

    Highlights: • Nuclear LDH is up-regulated under oxidative stress. • SIRT1 is co-immunoprecipitated bound to nuclear LDH. • Nuclear LDH is involved in histone deacetylation and epigenetics. - Abstract: It is becoming increasingly apparent that the nucleus harbors metabolic enzymes that affect genetic transforming events. Here, we describe a nuclear isoform of lactate dehydrogenase (nLDH) and its ability to orchestrate histone deacetylation by controlling the availability of nicotinamide adenine dinucleotide (NAD{sup +}), a key ingredient of the sirtuin-1 (SIRT1) deacetylase system. There was an increase in the expression of nLDH concomitant with the presence of hydrogen peroxide (H{sub 2}O{sub 2})more » in the culture medium. Under oxidative stress, the NAD{sup +} generated by nLDH resulted in the enhanced deacetylation of histones compared to the control hepatocytes despite no discernable change in the levels of SIRT1. There appeared to be an intimate association between nLDH and SIRT1 as these two enzymes co-immunoprecipitated. The ability of nLDH to regulate epigenetic modifications by manipulating NAD{sup +} reveals an intricate link between metabolism and the processing of genetic information.« less

  19. Supplementation of medium with diammonium hydrogen phosphate enhanced the D-lactate dehydrogenase levels leading to increased D-lactic acid productivity.

    PubMed

    Singhvi, Mamata; Jadhav, Akanksha; Gokhale, Digambar

    2013-10-01

    The production of D-lactic acid by Lactobacillus lactis RM2-24 was investigated using modified media to increase the efficiency of the fermentation process. The results indicated that the addition of 5 g/l peptone and 1 g/l (NH4)2HPO4 enhanced D-lactic acid production by 32%, as compared to that obtained from non supplemented media, with a productivity of 3.0 g/l/h. Lactate dehydrogenase (LDH) expression profile in these different media was studied which resulted in appearance of additional LDH isoform produced by cells when they were grown in HSYE supplemented with (NH4)2HPO4. The additional LDH appears to be L-LDH contributing to production of L-lactic acid in the fermented broth. This is totally new information in the lactic acid fermentation and could be very useful to industries engaged in D-lactic acid production. Copyright © 2013 Elsevier Ltd. All rights reserved.

  20. Purification and Properties of White Muscle Lactate Dehydrogenase from the Anoxia-Tolerant Turtle, the Red-Eared Slider, Trachemys scripta elegans

    PubMed Central

    Dawson, Neal J.; Bell, Ryan A. V.; Storey, Kenneth B.

    2013-01-01

    Lactate dehydrogenase (LDH; E.C. 1.1.1.27) is a crucial enzyme involved in energy metabolism in muscle, facilitating the production of ATP via glycolysis during oxygen deprivation by recycling NAD+. The present study investigated purified LDH from the muscle of 20 h anoxic and normoxic T. s. elegans, and LDH from anoxic muscle showed a significantly lower (47%) K m for L-lactate and a higher V max value than the normoxic form. Several lines of evidence indicated that LDH was converted to a low phosphate form under anoxia: (a) stimulation of endogenously present protein phosphatases decreased the K m of L-lactate of control LDH to anoxic levels, whereas (b) stimulation of kinases increased the K m of L-lactate of anoxic LDH to normoxic levels, and (c) dot blot analysis shows significantly less serine (78%) and threonine (58%) phosphorylation in anoxic muscle LDH as compared to normoxic LDH. The physiological consequence of anoxia-induced LDH dephosphorylation appears to be an increase in LDH activity to promote the reduction of pyruvate in muscle tissue, converting the glycolytic end product to lactate to maintain a prolonged glycolytic flux under energy-stressed anoxic conditions. PMID:23533717

  1. Purification and Properties of White Muscle Lactate Dehydrogenase from the Anoxia-Tolerant Turtle, the Red-Eared Slider, Trachemys scripta elegans.

    PubMed

    Dawson, Neal J; Bell, Ryan A V; Storey, Kenneth B

    2013-01-01

    Lactate dehydrogenase (LDH; E.C. 1.1.1.27) is a crucial enzyme involved in energy metabolism in muscle, facilitating the production of ATP via glycolysis during oxygen deprivation by recycling NAD(+). The present study investigated purified LDH from the muscle of 20 h anoxic and normoxic T. s. elegans, and LDH from anoxic muscle showed a significantly lower (47%) K m for L-lactate and a higher V max value than the normoxic form. Several lines of evidence indicated that LDH was converted to a low phosphate form under anoxia: (a) stimulation of endogenously present protein phosphatases decreased the K m of L-lactate of control LDH to anoxic levels, whereas (b) stimulation of kinases increased the K m of L-lactate of anoxic LDH to normoxic levels, and (c) dot blot analysis shows significantly less serine (78%) and threonine (58%) phosphorylation in anoxic muscle LDH as compared to normoxic LDH. The physiological consequence of anoxia-induced LDH dephosphorylation appears to be an increase in LDH activity to promote the reduction of pyruvate in muscle tissue, converting the glycolytic end product to lactate to maintain a prolonged glycolytic flux under energy-stressed anoxic conditions.

  2. Quinoline 3-sulfonamides inhibit lactate dehydrogenase A and reverse aerobic glycolysis in cancer cells

    PubMed Central

    2013-01-01

    Background Most normal cells in the presence of oxygen utilize glucose for mitochondrial oxidative phosphorylation. In contrast, many cancer cells rapidly convert glucose to lactate in the cytosol, a process termed aerobic glycolysis. This glycolytic phenotype is enabled by lactate dehydrogenase (LDH), which catalyzes the inter-conversion of pyruvate and lactate. The purpose of this study was to identify and characterize potent and selective inhibitors of LDHA. Methods High throughput screening and lead optimization were used to generate inhibitors of LDHA enzymatic activity. Effects of these inhibitors on metabolism were evaluated using cell-based lactate production, oxygen consumption, and 13C NMR spectroscopy assays. Changes in comprehensive metabolic profile, cell proliferation, and apoptosis were assessed upon compound treatment. Results 3-((3-carbamoyl-7-(3,5-dimethylisoxazol-4-yl)-6-methoxyquinolin-4-yl) amino) benzoic acid was identified as an NADH-competitive LDHA inhibitor. Lead optimization yielded molecules with LDHA inhibitory potencies as low as 2 nM and 10 to 80-fold selectivity over LDHB. Molecules in this family rapidly and profoundly inhibited lactate production rates in multiple cancer cell lines including hepatocellular and breast carcinomas. Consistent with selective inhibition of LDHA, the most sensitive breast cancer cell lines to lactate inhibition in hypoxic conditions were cells with low expression of LDHB. Our inhibitors increased rates of oxygen consumption in hepatocellular carcinoma cells at doses up to 3 microM, while higher concentrations directly inhibited mitochondrial function. Analysis of more than 500 metabolites upon LDHA inhibition in Snu398 cells revealed that intracellular concentrations of glycolysis and citric acid cycle intermediates were increased, consistent with enhanced Krebs cycle activity and blockage of cytosolic glycolysis. Treatment with these compounds also potentiated PKM2 activity and promoted apoptosis in Snu

  3. Effect of chlorocamphene on the isoenzyme spectrum of lactate dehydrogenase in rat serum and liver.

    PubMed Central

    Kuz'minskaya, U A; Alekhina, S M

    1976-01-01

    Rats were used to study the general activity and the isoenzyme spectrum of lactate dehydrogenase (LDH) during single-instance and long-term introduction of polychlorocamphene. Total lactate dehydrogenase activity decreases in the liver during the single-instance introduction of half the LD50 (120 mg/kg). The isoenzyme spectrum of LDH is characterized by an increase in the quantity of LDH1, LDH2, and LDH3 and by a decrease in the amount of LDH4. The overall LDH activity does not change in blood serum. The isoform ratio changes insignificantly and LDH1 falls, but normalized 15 days after the introduction of the compound. Long-term introduction of polychlorocamphene at levels 1/100 the LD50 dose over 1.3 and 6 months causes a reduction in the overall LDH activity, both in the liver and in the serum. A decrease in the activity of the basic LDH isoenzyme of the liver (LDH5) and a sharp increase in LDH3 are characteristic for the isoenzyme spectrum of the liver. LDH1 and LDH4 decrease and LDH2 and LDH3 increase in blood serum. Beginning with the third month of polychlorocamphene introduction, LDH1 tends to return to normal levels. LDH2, LDH3, and LDH4 do return to normal levels, while LDH5 increases regularly. This results in a reduction of the number of H subunits and an increase of M subunits. This is characteristic of hypoxic states. On comparing the changes in the LDH enzymes of the liver and blood serum, it can be considered that the introduction of polychlorocamphene does not result in an increase in the permeability of the cellular membranes of the liver for LDH isoenzymes, while the observed isoenzyme spectrum shifts in blood serum are either the result of the biosynthesis of the isoforms of this enzyme changed by the compound or the result of the permeability for them of cells of other tissues. PMID:1269500

  4. Purification and Electrophoretic Characterization of Lactate Dehydrogenase from Mammalian Blood: A Different Twist on a Classic Experiment

    ERIC Educational Resources Information Center

    Brunauer, Linda S.

    2016-01-01

    A multiweek protein purification suite, suitable for upper-division biochemistry or biotechnology undergraduate students, is described. Students work in small teams to isolate the enzyme lactate dehydrogenase (LDH) from a nontraditional tissue source, mammalian blood, using a sequence of three column chromatographic procedures: ion-exchange, size…

  5. Altered Kinetics Properties of Erythrocyte Lactate Dehydrogenase in Type II Diabetic Patients and Its Implications for Lactic Acidosis.

    PubMed

    Mali, Aniket V; Bhise, Sunita S; Katyare, Surendra S; Hegde, Mahabaleshwar V

    2018-01-01

    Recent studies have been noted that the erythrocytes from Type II diabetic patients show significantly altered structural and functional characteristics along with the changed intracellular concentrations of glycolytic intermediates. More recent studies from our laboratory have shown that the activities of enzymes of glycolytic pathway changed significantly in RBCs from Type II diabetic patients. In particular the levels of lactate dehydrogenase (LDH) increased significantly. Lactic acidosis is an established feature of diabetes and LDH plays a crucial role in conversion of pyruvate to lactate and reportedly, the levels of lactate are significantly high which is consistent with our observation on increased levels of LDH. Owing to this background, we examined the role of erythrocyte LDH in lactic acidosis by studying its kinetics properties in Type II diabetic patients. Km, Vmax and apparent catalytic efficiency were determined using pyruvate and NADH as the substrates. With pyruvate as the substrate the Km values were comparable but Vmax increased significantly in the diabetic group. With NADH as the substrate the enzyme activity of the diabetic group resolved in two components as against a single component in the controls. The Apparent Kcat and Kcat/Km values for pyruvate increased in the diabetic group. The Ki for pyruvate increased by two fold for the enzyme from diabetic group with a marginal decrease in Ki for NADH. The observed changes in catalytic attributes are conducive to enable the enzyme to carry the reaction in forward direction towards conversion of pyruvate to lactate leading to lactic acidosis.

  6. l-Lactate metabolism in HEP G2 cell mitochondria due to the l-lactate dehydrogenase determines the occurrence of the lactate/pyruvate shuttle and the appearance of oxaloacetate, malate and citrate outside mitochondria.

    PubMed

    Pizzuto, Roberto; Paventi, Gianluca; Porcile, Carola; Sarnataro, Daniela; Daniele, Aurora; Passarella, Salvatore

    2012-09-01

    As part of an ongoing study of l-lactate metabolism both in normal and in cancer cells, we investigated whether and how l-lactate metabolism occurs in mitochondria of human hepatocellular carcinoma (Hep G2) cells. We found that Hep G2 cell mitochondria (Hep G2-M) possess an l-lactate dehydrogenase (ml-LDH) restricted to the inner mitochondrial compartments as shown by immunological analysis, confocal microscopy and by assaying ml-LDH activity in solubilized mitochondria. Cytosolic and mitochondrial l-LDHs were found to differ from one another in their saturation kinetics. Having shown that l-lactate itself can enter Hep G2 cells, we found that Hep G2-M swell in ammonium l-lactate, but not in ammonium pyruvate solutions, in a manner inhibited by mersalyl, this showing the occurrence of a carrier-mediated l-lactate transport in these mitochondria. Occurrence of the l-lactate/pyruvate shuttle and the appearance outside mitochondria of oxaloacetate, malate and citrate arising from l-lactate uptake and metabolism together with the low oxygen consumption and membrane potential generation are in favor of an anaplerotic role for l-LAC in Hep G2-M. Copyright © 2012 Elsevier B.V. All rights reserved.

  7. Lactate Dehydrogenase in Hepatocellular Carcinoma: Something Old, Something New.

    PubMed

    Faloppi, Luca; Bianconi, Maristella; Memeo, Riccardo; Casadei Gardini, Andrea; Giampieri, Riccardo; Bittoni, Alessandro; Andrikou, Kalliopi; Del Prete, Michela; Cascinu, Stefano; Scartozzi, Mario

    2016-01-01

    Hepatocellular carcinoma (HCC) is the most common primary liver tumour (80-90%) and represents more than 5.7% of all cancers. Although in recent years the therapeutic options for these patients have increased, clinical results are yet unsatisfactory and the prognosis remains dismal. Clinical or molecular criteria allowing a more accurate selection of patients are in fact largely lacking. Lactic dehydrogenase (LDH) is a glycolytic key enzyme in the conversion of pyruvate to lactate under anaerobic conditions. In preclinical models, upregulation of LDH has been suggested to ensure both an efficient anaerobic/glycolytic metabolism and a reduced dependence on oxygen under hypoxic conditions in tumour cells. Data from several analyses on different tumour types seem to suggest that LDH levels may be a significant prognostic factor. The role of LDH in HCC has been investigated by different authors in heterogeneous populations of patients. It has been tested as a potential biomarker in retrospective, small, and nonfocused studies in patients undergoing surgery, transarterial chemoembolization (TACE), and systemic therapy. In the major part of these studies, high LDH serum levels seem to predict a poorer outcome. We have reviewed literature in this setting trying to resume basis for future studies validating the role of LDH in this disease.

  8. The diagnostic significance of lactate dehydrogenase isoenzymes in urinary cytology.

    PubMed Central

    Nishikawa, A.; Tanaka, T.; Takeuchi, T.; Fujihiro, S.; Mori, H.

    1991-01-01

    Lactate dehydrogenase (LDH) isoenzyme distribution was examined in 106 urine samples being tested cytologically for evidence of bladder cancer; the samples were selected to have less than 20 leucocytes and erythrocytes per high power field and the LDH pattern determined by electrophoresis. The Papanicolaou stained-smears showed 68 negative, 17 suspicious and 21 positive. The LDH M-fraction of the urinary supernatant in cytologically positive cases was significantly greater than in negative cases, although the latter included a few false negative samples. Some of the false negatives gave positive results for the LDH M-fraction; these results suggest that the determination of LDH isoenzymes in the urine is useful in diagnosing urinary tract cancers, including early stage, and for follow-up of patients with bladder cancers after surgical resection. PMID:2039708

  9. Trehalose Mediated Inhibition of Lactate Dehydrogenase from Rabbit Muscle. The Application of Kramers' Theory in Enzyme Catalysis.

    PubMed

    Hernández-Meza, Juan M; Sampedro, José G

    2018-04-19

    Lactate dehydrogenase (LDH) catalyzes the reduction of pyruvate to lactate by using NADH. LDH kinetics has been proposed to be dependent on the dynamics of a loop over the active site. Kramers' theory has been useful in the study of enzyme catalysis dependent on large structural dynamics. In this work, LDH kinetics was studied in the presence of trehalose and at different temperatures. In the absence of trehalose, temperature increase raised exponentially the LDH V max and revealed a sigmoid transition of K m toward a low-affinity state similar to protein unfolding. Notably, LDH V max diminished when in the presence of trehalose, while pyruvate affinity increased and the temperature-mediated binding site transition was hindered. The effect of trehalose on k cat was viscosity dependent as described by Kramers' theory since V max correlated inversely with the viscosity of the medium. As a result, activation energy ( E a ) for pyruvate reduction was dramatically increased by trehalose presence. This work provides experimental evidence that the dynamics of a structural component in LDH is essential for catalysis, i.e., the closing of the loop on the active site. While the trehalose mediated-increased of pyruvate affinity is proposed to be due to the compaction and/or increase of structural order at the binding site.

  10. Evidence of lactate dehydrogenase-B allozyme effects in the teleost, Fundulus heteroclitus.

    PubMed

    DiMichele, L; Paynter, K T; Powers, D A

    1991-08-23

    The evolutionary significance of protein polymorphisms has long been debated. Exponents of the balanced theory advocate that selection operates to maintain polymorphisms, whereas the neoclassical school argues that most genetic variation is neutral. Some studies have suggested that protein polymorphisms are not neutral, but their significance has been questioned because one cannot eliminate the possibility that linked loci were responsible for the observed differences. Evidence is presented that an enzymatic phenotype can affect carbon flow through a metabolic pathway. Glucose flux differences between lactate dehydrogenase-B phenotypes of Fundulus heteroclitus were reversed by substituting the Ldh-B gene product of one homozygous genotype with that of another.

  11. Resting oxygen consumption varies among lactate dehydrogenase genotypes in the sow bug, Porcellio scaber

    PubMed Central

    Mitton, J. B.; Carter, P. A.; DiGiacomo, A.

    1997-01-01

    Laboratory studies of respiration in the sow bug, Porcellio scaber, reveal that respiration rates are related to genetic variation at the lactate dehydrogenase (Ldh) locus. In population samples taken from Burlington, North Carolina and Pacific Grove, California, respiration rates differed among Ldh genotypes, but not among genotypes at the other enzyme polymorphisms. In both population samples, the respiration rate of the common Ldh homozygote exceeded the respiration rate of the heterozygote by more than 50 per cent. The differences in respiration rates are consistent with previously reported viability differentials at the Ldh polymorphism.

  12. Positive selection on D-lactate dehydrogenases of Lactobacillus delbrueckii subspecies bulgaricus.

    PubMed

    Zhang, Jifeng; Gong, Guangyu; Wang, Xiao; Zhang, Hao; Tian, Weidong

    2015-08-01

    Lactobacillus delbrueckii has been widely used for yogurt fermentation. It has genes encoding both D- and L-type lactate dehydrogenases (LDHs) that catalyse the production of L(+) or D(-) stereoisomer of lactic acid. D-lactic acid is the primary lactate product by L. delbrueckii, yet it cannot be metabolised by human intestine. Since it has been domesticated for long time, an interesting question arises regarding to whether the selection pressure has affected the evolution of both L-LDH and D-LDH genes in the genome. To answer this question, in this study the authors first investigated the evolution of these two genes by constructing phylogenetic trees. They found that D-LDH-based phylogenetic tree could better represent the phylogenetic relationship in the acidophilus complex than L-LDH-based tree. They next investigated the evolutions of LDH genes of L. delbrueckii at amino acid level, and found that D-LDH gene in L. delbrueckii is positively selected, possibly a consequence of long-term domestication. They further identified four amino acids that are under positive selection. One of them, V261, is located at the centre of three catalytic active sites, indicating likely functional effects on the enzyme activity. The selection from the domestication process thus provides direction for future engineering of D-LDH.

  13. Overproduction and nucleotide sequence of the respiratory D-lactate dehydrogenase of Escherichia coli.

    PubMed Central

    Rule, G S; Pratt, E A; Chin, C C; Wold, F; Ho, C

    1985-01-01

    Recombinant DNA plasmids containing the gene for the membrane-bound D-lactate dehydrogenase (D-LDH) of Escherichia coli linked to the promoter PL from lambda were constructed. After induction, the levels of D-LDH were elevated 300-fold over that of the wild type and amounted to 35% of the total cellular protein. The nucleotide sequence of the D-LDH gene was determined and shown to agree with the amino acid composition and the amino-terminal sequence of the purified enzyme. Removal of the amino-terminal formyl-Met from D-LDH was not inhibited in cells which contained these high levels of D-LDH. Images PMID:3882663

  14. Analysis of the Mycoplasma bovis lactate dehydrogenase reveals typical enzymatic activity despite the presence of an atypical catalytic site motif.

    PubMed

    Masukagami, Yumiko; Tivendale, Kelly Anne; Browning, Glenn Francis; Sansom, Fiona Margaret

    2018-02-01

    The lactate dehydrogenase (LDH) of Mycoplasma genitalium has been predicted to also act as a malate dehydrogenase (MDH), but there has been no experimental validation of this hypothesized dual function for any mollicute. Our analysis of the metabolite profile of Mycoplasma bovis using gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) detected malate, suggesting that there may be MDH activity in M. bovis. To investigate whether the putative l-LDH enzyme of M. bovis has a dual function (MDH and LDH), we performed bioinformatic and functional biochemical analyses. Although the amino acid sequence and predicted structural analysis of M. bovisl-LDH revealed unusual residues within the catalytic site, suggesting that it may have the flexibility to possess a dual function, our biochemical studies using recombinant M. bovis -LDH did not detect any MDH activity. However, we did show that the enzyme has typical LDH activity that could be inhibited by both MDH substrates oxaloacetate (OAA) and malate, suggesting that these substrates may be able to bind to M. bovis LDH. Inhibition of the conversion of pyruvate to lactate by OAA may be one method the mycoplasma cell uses to reduce the potential for accumulation of intracellular lactate.

  15. Interaction of cytoplasmic dehydrogenases: quantitation of pathways of ethanol metabolism.

    PubMed

    Vind, C; Grunnet, N

    1983-01-01

    The interaction between xylitol, alcohol and lactate dehydrogenase has been studied in hepatocytes from rats by applying specifically tritiated substrates. A simple model, describing the metabolic fate of tritium from [2-3H] xylitol and (1R) [1-3H]ethanol is presented. The model allows calculation of the specific radioactivity of free, cytosolic NADH, based on transfer of tritium to lactate, glucose and water. From the initial labelling rate of lactate and the specific radioactivity of cytosolic NADH, we have determined the reversible flow through the lactate dehydrogenase catalyzed reaction to 1-5 mumol/min . g wet wt. The results suggest that xylitol, alcohol and lactate dehydrogenase share the same pool of NAD(H) in the cytoplasma. This finding allows estimation of the ethanol oxidation rate by the non-alcohol dehydrogenase pathways from the relative yield of tritium in water and glucose. The calculations are based on a comparison of the fate of the 1-pro-R hydrogen of ethanol and the hydrogen bound to carbon 2 of xylitol or carbon 2 of lactate under identical conditions.

  16. White shrimp Litopenaeus vannamei recombinant lactate dehydrogenase: Biochemical and kinetic characterization.

    PubMed

    Fregoso-Peñuñuri, Ambar A; Valenzuela-Soto, Elisa M; Figueroa-Soto, Ciria G; Peregrino-Uriarte, Alma B; Ochoa-Valdez, Manuel; Leyva-Carrillo, Lilia; Yepiz-Plascencia, Gloria

    2017-09-01

    Shrimp lactate dehydrogenase (LDH) is induced in response to environmental hypoxia. Two protein subunits deduced from different transcripts of the LDH gene from the shrimp Litopenaeus vannamei (LDHvan-1 and LDHvan-2) were identified. These subunits are expressed by alternative splicing. Since both subunits are expressed in most tissues, the purification of the enzyme from the shrimp will likely produce hetero LDH containing both subunits. Therefore, the aim of this study was to overexpress, purify and characterize only one subunit as a recombinant protein, the LDHvan-2. For this, the cDNA from muscle was cloned and overexpressed in E. coli as a fusion protein containing an intein and a chitin binding protein domain (CBD). The recombinant protein was purified by chitin affinity chromatography column that retained the CBD and released solely the full and active LDH. The active protein appears to be a tetramer with molecular mass of approximately 140 kDa and can use pyruvate or lactate as substrates, but has higher specific activity with pyruvate. The enzyme is stable between pH 7.0 to 8.5, and between 20 and 50 °C with an optimal temperature of 50 °C. Two pK a of 9.3 and 6.6, and activation energy of 44.8 kJ/mol°K were found. The kinetic constants K m for NADH was 23.4 ± 1.8 μM, and for pyruvate was 203 ± 25 μM, while V max was 7.45 μmol/min/mg protein. The shrimp LDH that is mainly expressed in shrimp muscle preferentially converts pyruvate to lactate and is an important enzyme for the response to hypoxia. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Epitopes of human testis-specific lactate dehydrogenase deduced from a cDNA sequence

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Millan, J.L.; Driscoll, C.E.; LeVan, K.M.

    The sequence and structure of human testis-specific L-lactate dehydrogenase (LDHC/sub 4/, LDHX; (L)-lactate:NAD/sup +/ oxidoreductase, EC 1.1.1.27) has been derived from analysis of a complementary DNA (cDNA) clone comprising the complete protein coding region of the enzyme. From the deduced amino acid sequence, human LDHC/sub 4/ is as different from rodent LDHC/sub 4/ (73% homology) as it is from human LDHA/sub 4/ (76% homology) and porcine LDHB/sub 4/ (68% homology). Subunit homologies are consistent with the conclusion that the LDHC gene arose by at least two independent duplication events. Furthermore, the lower degree of homology between mouse and human LDHC/submore » 4/ and the appearance of this isozyme late in evolution suggests a higher rate of mutation in the mammalian LDHC genes than in the LDHA and -B genes. Comparison of exposed amino acid residues of discrete anti-genic determinants of mouse and human LDHC/sub 4/ reveals significant differences. Knowledge of the human LDHC/sub 4/ sequence will help design human-specific peptides useful in the development of a contraceptive vaccine.« less

  18. Cloning of D-lactate dehydrogenase genes of Lactobacillus delbrueckii subsp. bulgaricus and their roles in D-lactic acid production.

    PubMed

    Huang, Yanna; You, Chunping; Liu, Zhenmin

    2017-07-01

    Lactobacillus delbrueckii subsp. bulgaricus is a heterogenous lactic acid bacterium that converts pyruvate mainly to D-lactic acid using D-lactate dehydrogenases (D-LDHs), whose functional properties remain poorly characterized. Here, the D-LDHs genes (ldb0101, ldb0813, ldb1010, ldb1147 and ldb2021) were cloned and overexpressed in Escherichia coli JM109 from an inducible pUC18 vector, respectively, and the resulting strains were compared in terms of D-lactic acid production. The strain expressing ldb0101 and ldb1010 gene individually produced more D-lactate than other three strains. Further study revealed that Ldb0101 activity was down-regulated by the oxygen and, therefore, achieved a highest titer of D-lactate (1.94 g/L) under anaerobic condition, and introduction of ldb1010 gene enhanced D-lactate formation (0.94 and 0.85 g/L, respectively) both in aerobic and anaerobic conditions due to a relatively stable q d-lactate . Our results suggested that the enzyme Ldb0101 and Ldb1010 played a role of more importance in D-lactate formation. To the best of our knowledge, we demonstrate for the first time the roles of different D-LDH homologs from L. bulgaricus in D-lactic acid production.

  19. Cloning and polymorphisms of yak lactate dehydrogenase B gene.

    PubMed

    Wang, Guosheng; Zhao, Xingbo; Zhong, Juming; Cao, Meng; He, Qinghua; Liu, Zhengxin; Lin, Yaqiu; Xu, Yaou; Zheng, Yucai

    2013-06-05

    The main objective of this work was to study the unique polymorphisms of the lactate dehydrogenase-1 (LDH1) gene in yak (Bos grunniens). Native polyacrylamide gel electrophoresis revealed three phenotypes of LDH1 (a tetramer of H subunit) in yak heart and longissimus muscle extracts. The corresponding gene, ldhb, encoding H subunits of three LDH1 phenotypes was obtained by RT-PCR. A total of six nucleotide differences were detected in yak ldhb compared with that of cattle, of which five mutations cause amino acid substitutions. Sequence analysis shows that the G896A and C689A, mutations of ldhb gene, result in alterations of differently charged amino acids, and create the three phenotypes (F, M, and S) of yak LDH1. Molecular modeling of the H subunit of LDH indicates that the substituted amino acids are not located within NAD+ or substrate binding sites. PCR-RFLP examination of G896A mutation demonstrated that most LDH1-F samples are actually heterozygote at this site. These results help to elucidate the molecular basis and genetic characteristic of the three unique LDH1 phenotypes in yak.

  20. Cloning and Polymorphisms of Yak Lactate Dehydrogenase b Gene

    PubMed Central

    Wang, Guosheng; Zhao, Xingbo; Zhong, Juming; Cao, Meng; He, Qinghua; Liu, Zhengxin; Lin, Yaqiu; Xu, Yaou; Zheng, Yucai

    2013-01-01

    The main objective of this work was to study the unique polymorphisms of the lactate dehydrogenase-1 (LDH1) gene in yak (Bos grunniens). Native polyacrylamide gel electrophoresis revealed three phenotypes of LDH1 (a tetramer of H subunit) in yak heart and longissimus muscle extracts. The corresponding gene, ldhb, encoding H subunits of three LDH1 phenotypes was obtained by RT-PCR. A total of six nucleotide differences were detected in yak ldhb compared with that of cattle, of which five mutations cause amino acid substitutions. Sequence analysis shows that the G896A and C689A, mutations of ldhb gene, result in alterations of differently charged amino acids, and create the three phenotypes (F, M, and S) of yak LDH1. Molecular modeling of the H subunit of LDH indicates that the substituted amino acids are not located within NAD+ or substrate binding sites. PCR-RFLP examination of G896A mutation demonstrated that most LDH1-F samples are actually heterozygote at this site. These results help to elucidate the molecular basis and genetic characteristic of the three unique LDH1 phenotypes in yak. PMID:23739677

  1. Towards development of aptamers that specifically bind to lactate dehydrogenase of Plasmodium falciparum through epitopic targeting.

    PubMed

    Frith, Kelly-Anne; Fogel, Ronen; Goldring, J P Dean; Krause, Robert G E; Khati, Makobetsa; Hoppe, Heinrich; Cromhout, Mary E; Jiwaji, Meesbah; Limson, Janice L

    2018-05-03

    Early detection is crucial for the effective treatment of malaria, particularly in those cases infected with Plasmodium falciparum. There is a need for diagnostic devices with the capacity to distinguish P. falciparum from other strains of malaria. Here, aptamers generated against targeted species-specific epitopes of P. falciparum lactate dehydrogenase (rPfLDH) are described. Two classes of aptamers bearing high binding affinity and specificity for recombinant P. falciparum lactate dehydrogenase (rPfLDH) and P. falciparum-specific lactate dehydrogenase epitopic oligopeptide (LDHp) were separately generated. Structurally-relevant moieties with particular consensus sequences (GGTAG and GGCG) were found in aptamers reported here and previously published, confirming their importance in recognition of the target, while novel moieties particular to this work (ATTAT and poly-A stretches) were identified. Aptamers with diagnostically-supportive functions were synthesized, prime examples of which are the aptamers designated as LDHp 1, LDHp 11 and rLDH 4 and rLDH 15 in work presented herein. Of the sampled aptamers raised against the recombinant protein, rLDH 4 showed the highest binding to the target rPfLDH in the ELONA assay, with both rLDH 4 and rLDH 15 indicating an ability to discriminate between rPfLDH and rPvLDH. LDHp 11 was generated against a peptide selected as a unique P. falciparum LDH peptide. The aptamer, LDHp 11, like antibodies against the same peptide, only detected rPfLDH and discriminated between rPfLDH and rPvLDH. This was supported by affinity binding experiments where only aptamers generated against a unique species-specific epitope showed an ability to preferentially bind to rPfLDH relative to rPvLDH rather than those generated against the whole recombinant protein. In addition, rLDH 4 and LDHp 11 demonstrated in situ binding to P. falciparum cells during confocal microscopy. The utilization and application of LDHp 11, an aptamer generated against a

  2. Skeletal Muscle Pyruvate Dehydrogenase Phosphorylation and Lactate Accumulation During Sprint Exercise in Normoxia and Severe Acute Hypoxia: Effects of Antioxidants.

    PubMed

    Morales-Alamo, David; Guerra, Borja; Santana, Alfredo; Martin-Rincon, Marcos; Gelabert-Rebato, Miriam; Dorado, Cecilia; Calbet, José A L

    2018-01-01

    Compared to normoxia, during sprint exercise in severe acute hypoxia the glycolytic rate is increased leading to greater lactate accumulation, acidification, and oxidative stress. To determine the role played by pyruvate dehydrogenase (PDH) activation and reactive nitrogen and oxygen species (RNOS) in muscle lactate accumulation, nine volunteers performed a single 30-s sprint (Wingate test) on four occasions: two after the ingestion of placebo and another two following the intake of antioxidants, while breathing either hypoxic gas (P I O 2 = 75 mmHg) or room air (P I O 2 = 143 mmHg). Vastus lateralis muscle biopsies were obtained before, immediately after, 30 and 120 min post-sprint. Antioxidants reduced the glycolytic rate without altering performance or VO 2 . Immediately after the sprints, Ser 293 - and Ser 300 -PDH-E1α phosphorylations were reduced to similar levels in all conditions (~66 and 91%, respectively). However, 30 min into recovery Ser 293 -PDH-E1α phosphorylation reached pre-exercise values while Ser 300 -PDH-E1α was still reduced by 44%. Thirty minutes after the sprint Ser 293 -PDH-E1α phosphorylation was greater with antioxidants, resulting in 74% higher muscle lactate concentration. Changes in Ser 293 and Ser 300 -PDH-E1α phosphorylation from pre to immediately after the sprints were linearly related after placebo ( r = 0.74, P < 0.001; n = 18), but not after antioxidants ingestion ( r = 0.35, P = 0.15). In summary, lactate accumulation during sprint exercise in severe acute hypoxia is not caused by a reduced activation of the PDH. The ingestion of antioxidants is associated with increased PDH re-phosphorylation and slower elimination of muscle lactate during the recovery period. Ser 293 re-phosphorylates at a faster rate than Ser 300 -PDH-E1α during the recovery period, suggesting slightly different regulatory mechanisms.

  3. Skeletal Muscle Pyruvate Dehydrogenase Phosphorylation and Lactate Accumulation During Sprint Exercise in Normoxia and Severe Acute Hypoxia: Effects of Antioxidants

    PubMed Central

    Morales-Alamo, David; Guerra, Borja; Santana, Alfredo; Martin-Rincon, Marcos; Gelabert-Rebato, Miriam; Dorado, Cecilia; Calbet, José A. L.

    2018-01-01

    Compared to normoxia, during sprint exercise in severe acute hypoxia the glycolytic rate is increased leading to greater lactate accumulation, acidification, and oxidative stress. To determine the role played by pyruvate dehydrogenase (PDH) activation and reactive nitrogen and oxygen species (RNOS) in muscle lactate accumulation, nine volunteers performed a single 30-s sprint (Wingate test) on four occasions: two after the ingestion of placebo and another two following the intake of antioxidants, while breathing either hypoxic gas (PIO2 = 75 mmHg) or room air (PIO2 = 143 mmHg). Vastus lateralis muscle biopsies were obtained before, immediately after, 30 and 120 min post-sprint. Antioxidants reduced the glycolytic rate without altering performance or VO2. Immediately after the sprints, Ser293- and Ser300-PDH-E1α phosphorylations were reduced to similar levels in all conditions (~66 and 91%, respectively). However, 30 min into recovery Ser293-PDH-E1α phosphorylation reached pre-exercise values while Ser300-PDH-E1α was still reduced by 44%. Thirty minutes after the sprint Ser293-PDH-E1α phosphorylation was greater with antioxidants, resulting in 74% higher muscle lactate concentration. Changes in Ser293 and Ser300-PDH-E1α phosphorylation from pre to immediately after the sprints were linearly related after placebo (r = 0.74, P < 0.001; n = 18), but not after antioxidants ingestion (r = 0.35, P = 0.15). In summary, lactate accumulation during sprint exercise in severe acute hypoxia is not caused by a reduced activation of the PDH. The ingestion of antioxidants is associated with increased PDH re-phosphorylation and slower elimination of muscle lactate during the recovery period. Ser293 re-phosphorylates at a faster rate than Ser300-PDH-E1α during the recovery period, suggesting slightly different regulatory mechanisms. PMID:29615918

  4. Lactate Dehydrogenase Activity in Gingival Crevicular Fluid as a Marker in Orthodontic Tooth Movement

    PubMed Central

    Alfaqeeh, Sarah A; Anil, Sukumaran

    2011-01-01

    Objectives: This study aims at analyzing the changes in gingival crevicular fluid (GCF) lactate dehydrogenase (LDH) activity during orthodontic movement. Methods: Twenty patients all requiring first premolar extractions were selected and treated with conventional straight wire mechanotherapy. Canine retraction was done using 125 g Nitinol closed coil springs. The maxillary canine on one side served as the experimental site while the contralateral canine served as the control. GCF was collected from the canines before initiation of retraction, then 1 hour after initiating canine retraction, followed by 1 day, 7 days, 14 days and 21 days. GCF LDH levels were estimated and compared with the control site. Results The results revealed significantly higher LDH levels on the 7th, 14th and 21st day at the sites where orthodontic force had been applied. The levels also showed a significant increase from 0 hour to the 21st day. Peak levels were seen on 14th and 21st day following initiation of retraction. Conclusions: The study showed that LDH could be successfully estimated in the GCF and its increased levels could indicate active tooth movement, which could aid the clinician in monitoring active orthodontic tooth movement. PMID:21760863

  5. Misconceptions regarding basic thermodynamics and enzyme kinetics have led to erroneous conclusions regarding the metabolic importance of lactate dehydrogenase isoenzyme expression.

    PubMed

    Bak, Lasse K; Schousboe, Arne

    2017-11-01

    Lactate dehydrogenase (LDH) catalyzes the interconversion of pyruvate and lactate involving the coenzyme NAD + . Part of the foundation for the proposed shuttling of lactate from astrocytes to neurons during brain activation is the differential distribution of LDH isoenzymes between the two cell types. In this short review, we outline the basic kinetic properties of the LDH isoenzymes expressed in neurons and astrocytes, and argue that the distribution of LDH isoenzymes does not in any way govern directional flow of lactate between the two cellular compartments. The two main points are as follows. First, in line with the general concept of chemical catalysis, enzymes do not influence the thermodynamic equilibrium of a chemical reaction but merely the speed at which equilibrium is obtained. Thus, differential distribution of LDH isoenzymes with different kinetic parameters does not predict which cells are producing and which are consuming lactate. Second, the thermodynamic equilibrium of the reaction is toward the reduced substrate (i.e., lactate), which is reflected in the concentrations measured in brain tissue, suggesting that the reaction is at near-equilibrium at steady state. To conclude, the cellular distribution of LDH isoenzymes is of little if any consequence in determining any directional flow of lactate between neurons and astrocytes. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  6. Molecular Characterization of Two Lactate Dehydrogenase Genes with a Novel Structural Organization on the Genome of Lactobacillus sp. Strain MONT4

    PubMed Central

    Weekes, Jennifer; Yüksel, Gülhan Ü.

    2004-01-01

    Two lactate dehydrogenase (ldh) genes from Lactobacillus sp. strain MONT4 were cloned by complementation in Escherichia coli DC1368 (ldh pfl) and were sequenced. The sequence analysis revealed a novel genomic organization of the ldh genes. Subcloning of the individual ldh genes and their Northern blot analyses indicated that the genes are monocistronic. PMID:15466577

  7. [Temperature-switched high-efficiency D-lactate production from glycerol].

    PubMed

    Tian, Kangming; Zhou, Li; Chen, Xianzhong; Shen, Wei; Shi, Guiyang; Singh, Suren; Lu, Fuping; Wang, Zhengxiang

    2013-01-01

    Glycerol from oil hydrolysis industry is being considered as one of the abundent raw materials for fermentation industry. In present study, the aerobic and anaerobic metabolism and growth properties on glycerol by Esherichia coli CICIM B0013-070, a D-lactate over-producing strain constructed previously, at different temperatures were investigated, followed by a novel fermentation process, named temperature-switched process, was established for D-lactate production from glycerol. Under the optimal condition, lactate yield was increased from 64.0% to 82.6%. Subsequently, the yield of D-lactate from glycerol was reached up to 88.9% while a thermo-inducible promoter was used to regulate D-lactate dehydrogenase transcription.

  8. From Gene to Structure: "Lactobacillus Bulgaricus" D-Lactate Dehydrogenase from Yogurt as an Integrated Curriculum Model for Undergraduate Molecular Biology and Biochemistry Laboratory Courses

    ERIC Educational Resources Information Center

    Lawton, Jeffrey A.; Prescott, Noelle A.; Lawton, Ping X.

    2018-01-01

    We have developed an integrated, project-oriented curriculum for undergraduate molecular biology and biochemistry laboratory courses spanning two semesters that is organized around the "ldhA" gene from the yogurt-fermenting bacterium "Lactobacillus bulgaricus," which encodes the enzyme d-lactate dehydrogenase. The molecular…

  9. Divergent lactate dehydrogenase isoenzyme profile in cellular compartments of primate forebrain structures.

    PubMed

    Duka, Tetyana; Collins, Zachary; Anderson, Sarah M; Raghanti, Mary Ann; Ely, John J; Hof, Patrick R; Wildman, Derek E; Goodman, Morris; Grossman, Lawrence I; Sherwood, Chet C

    2017-07-01

    The compartmentalization and association of lactate dehydrogenase (LDH) with specific cellular structures (e.g., synaptosomal, sarcoplasmic or mitochondrial) may play an important role in brain energy metabolism. Our previous research revealed that LDH in the synaptosomal fraction shifts toward the aerobic isoforms (LDH-B) among the large-brained haplorhine primates compared to strepsirrhines. Here, we further analyzed the subcellular localization of LDH in primate forebrain structures using quantitative Western blotting and ELISA. We show that, in cytosolic and mitochondrial subfractions, LDH-B expression level was relatively elevated and LDH-A declined in haplorhines compared to strepsirrhines. LDH-B expression in mitochondrial fractions of the neocortex was preferentially increased, showing a particularly significant rise in the ratio of LDH-B to LDH-A in chimpanzees and humans. We also found a significant correlation between the protein levels of LDH-B in mitochondrial fractions from haplorhine neocortex and the synaptosomal LDH-B that suggests LDH isoforms shift from a predominance of A-subunits toward B-subunits as part of a system that spatially buffers dynamic energy requirements of brain cells. Our results indicate that there is differential subcellular compartmentalization of LDH isoenzymes that evolved among different primate lineages to meet the energy requirements in neocortical and striatal cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Lactic acid-producing yeast cells having nonfunctional L- or D-lactate:ferricytochrome C oxidoreductase cells

    DOEpatents

    Miller, Matthew [Boston, MA; Suominen, Pirkko [Maple Grove, MN; Aristidou, Aristos [Highland Ranch, CO; Hause, Benjamin Matthew [Currie, MN; Van Hoek, Pim [Camarillo, CA; Dundon, Catherine Asleson [Minneapolis, MN

    2012-03-20

    Yeast cells having an exogenous lactate dehydrogenase gene ae modified by reducing L- or D-lactate:ferricytochrome c oxidoreductase activity in the cell. This leads to reduced consumption of lactate by the cell and can increase overall lactate yields in a fermentation process. Cells having the reduced L- or D-lactate:ferricytochrome c oxidoreductase activity can be screened for by resistance to organic acids such as lactic or glycolic acid.

  11. Myasthenia gravis: long-term prognostic value of thymus lactate dehydrogenase isoenzyme pattern of hyperplastic thymus and thymoma.

    PubMed Central

    Szathmáry, I; Selmeci, L; Pósch, E; Szobor, A; Molnár, J

    1985-01-01

    Lactate dehydrogenase (LDH) isoenzyme pattern and the percent of H-subunit content were determined in the thymus of 62 patients (55 with hyperplasia, 7 with tumours) after thymectomy. An increase in LDH1 relative activity indicates that in the thymus of patients with myasthenia gravis the ratio of mature differentiated thymocytes was higher than in the thymus of control subjects. LDH isoenzyme profiles of thymus tumours were similar to those described in other neoplasms, except that thymomas with apparent predominance of epithelial cells and with minimal lymphocytic reaction exhibited a marked elevation only in LDH2 relative activity, presumably associated with the specific (secretory) function of epithelial cells. The elevation of H-subunit content, a parameter characteristic of both thymic components (lymphoid and epithelial), correlated closely with a poor clinical condition in patients several years after surgery. PMID:4031927

  12. Stringency of substrate specificity of Escherichia coli malate dehydrogenase.

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Boernke, W. E.; Millard, C. S.; Stevens, P. W.

    1995-09-10

    Malate dehydrogenase and lactate dehydrogenase are members of the structurally and functionally homologous family of 2-ketoacid dehydrogenases. Both enzymes display high specificity for their respective keto substrates, oxaloacetate and pyruvate. Closer analysis of their specificity, however, reveals that the specificity of malate dehydrogenase is much stricter and less malleable than that of lactate dehydrogenase. Site-specific mutagenesis of the two enzymes in an attempt to reverse their specificity has met with contrary results. Conversion of a specific active-site glutamine to arginine in lactate dehydrogenase from Bacillus stearothermophilus generated an enzyme that displayed activity toward oxaloacetate equal to that of the nativemore » enzyme toward pyruvate (H. M. Wilks et al. (1988) Science 242, 1541-1544). We have constructed a series of mutants in the mobile, active site loop of the Escherichia coli malate dehydrogenase that incorporate the complementary change, conversion of arginine 81 to glutamine, to evaluate the role of charge distribution and conformational flexibility within this loop in defining the substrate specificity of these enzymes. Mutants incorporating the change R81Q all had reversed specificity, displaying much higher activity toward pyruvate than to the natural substrate, oxaloacetate. In contrast to the mutated lactate dehydrogenase, these reversed-specificity mutants were much less active than the native enzyme. Secondary mutations within the loop of the E. coli enzyme (A80N, A80P, A80P/M85E/D86T) had either no or only moderately beneficial effects on the activity of the mutant enzyme toward pyruvate. The mutation A80P, which can be expected to reduce the overall flexibility of the loop, modestly improved activity toward pyruvate. The possible physiological relevance of the stringent specificity of malate dehydrogenase was investigated. In normal strains of E. coli, fermentative metabolism was not affected by expression of the

  13. Distribution of lactate dehydrogenase in healthy and degenerative canine stifle joint cartilage.

    PubMed

    Walter, Eveline L C; Spreng, David; Schmöckel, Hugo; Schawalder, Peter; Tschudi, Peter; Friess, Armin E; Stoffel, Michael H

    2007-07-01

    In dogs, degenerative joint diseases (DJD) have been shown to be associated with increased lactate dehydrogenase (LDH) activity in the synovial fluid. The goal of this study was to examine healthy and degenerative stifle joints in order to clarify the origin of LDH in synovial fluid. In order to assess the distribution of LDH, cartilage samples from healthy and degenerative knee joints were investigated by means of light and transmission electron microscopy in conjunction with immunolabeling and enzyme cytochemistry. Morphological analysis confirmed DJD. All techniques used corroborated the presence of LDH in chondrocytes and in the interterritorial matrix of healthy and degenerative stifle joints. Although enzymatic activity of LDH was clearly demonstrated in the territorial matrix by means of the tetrazolium-formazan reaction, immunolabeling for LDH was missing in this region. With respect to the distribution of LDH in the interterritorial matrix, a striking decrease from superficial to deeper layers was present in healthy dogs but was missing in affected joints. These results support the contention that LDH in synovial fluid of degenerative joints originates from cartilage. Therefore, we suggest that (1) LDH is transferred from chondrocytes to ECM in both healthy dogs and dogs with degenerative joint disease and that (2) in degenerative joints, LDH is released from chondrocytes and the ECM into synovial fluid through abrasion of cartilage as well as through enhanced diffusion as a result of increased water content and degradation of collagen.

  14. Increased titer and reduced lactate accumulation in recombinant retrovirus production through the down-regulation of HIF1 and PDK.

    PubMed

    Rodrigues, A F; Guerreiro, M R; Formas-Oliveira, A S; Fernandes, P; Blechert, A-K; Genzel, Y; Alves, P M; Hu, W S; Coroadinha, A S

    2016-01-01

    Many mammalian cell lines used in the manufacturing of biopharmaceuticals exhibit high glycolytic flux predominantly channeled to the production of lactate. The accumulation of lactate in culture reduces cell viability and may also decrease product quality. In this work, we engineered a HEK 293 derived cell line producing a recombinant gene therapy retroviral vector, by down-regulating hypoxia inducible factor 1 (HIF1) and pyruvate dehydrogenase kinase (PDK). Specific productivity of infectious viral titers could be increased more than 20-fold for single gene knock-down (HIF1 or PDK) and more than 30-fold under combined down-regulation. Lactate production was reduced up to 4-fold. However, the reduction in lactate production, alone, was not sufficient to enhance the titer: high-titer clones also showed significant enrollment of metabolic routes not related to lactate production. Transcriptome analysis indicated activation of biological amines metabolism, detoxification routes, including glutathione metabolism, pentose phosphate pathway, glycogen biosynthesis and amino acid catabolism. The latter were validated by enzyme activity assays and metabolite profiling, respectively. High-titer clones also presented substantially increased transcript levels of the viral genes expression cassettes. The results herein presented demonstrate the impact of HIF1 and PDK down-regulation on the production performance of a mammalian cell line, reporting one of the highest fold-increase in specific productivity of infectious virus titers achieved by metabolic engineering. They additionally highlight the contribution of secondary pathways, beyond those related to lactate production, that can be also explored to pursue improved metabolic status favoring a high-producing phenotype. © 2015 Wiley Periodicals, Inc.

  15. A membrane-associated adenylate cyclase modulates lactate dehydrogenase and creatine kinase activities required for bull sperm capacitation induced by hyaluronic acid.

    PubMed

    Fernández, Silvina; Córdoba, Mariana

    2017-04-01

    Hyaluronic acid, as well as heparin, is a glycosaminoglycan present in the female genital tract of cattle. The aim of this study was to evaluate oxidative metabolism and intracellular signals mediated by a membrane-associated adenylate cyclase (mAC), in sperm capacitation with hyaluronic acid and heparin, in cryopreserved bull sperm. The mAC inhibitor, 2',5'-dideoxyadenosine, was used in the present study. Lactate dehydrogenase (LDH) and creatine kinase (CK) activities and lactate concentration were determined spectrophotometrically in the incubation medium. Capacitation and acrosome reaction were evaluated by chlortetracycline technique, while plasma membrane and acrosome integrity were determined by trypan blue stain/differential interference contrast microscopy. Heparin capacitated samples had a significant decrease in LDH and CK activities, while in hyaluronic acid capacitated samples LDH and CK activities both increased compared to control samples, in heparin and hyaluronic acid capacitation conditions, respectively. A significant increase in lactate concentration in the incubation medium occurred in hyaluronic acid-treated sperm samples compared to heparin treatment, indicating this energetic metabolite is produced during capacitation. The LDH and CK enzyme activities and lactate concentrations in the incubation medium were decreased with 2',5'-dideoxyadenosine treatment in hyaluronic acid samples. The mAC inhibitor significantly inhibited heparin-induced capacitation of sperm cells, but did not completely inhibit hyaluronic acid capacitation. Therefore, hyaluronic acid and heparin are physiological glycosaminoglycans capable of inducing in vitro capacitation in cryopreserved bull sperm, stimulating different enzymatic pathways and intracellular signals modulated by a mAC. Hyaluronic acid induces sperm capacitation involving LDH and CK activities, thereby reducing oxidative metabolism, and this process is mediated by mAC. Copyright © 2017 Elsevier B.V. All

  16. Insufficient filling of vacuum tubes as a cause of microhemolysis and elevated serum lactate dehydrogenase levels. Use of a data-mining technique in evaluation of questionable laboratory test results.

    PubMed

    Tamechika, Yoshie; Iwatani, Yoshinori; Tohyama, Kaoru; Ichihara, Kiyoshi

    2006-01-01

    Experienced physicians noted unexpectedly elevated concentrations of lactate dehydrogenase in some patient samples, but quality control specimens showed no bias. To evaluate this problem, we used a "latent reference individual extraction method", designed to obtain reference intervals from a laboratory database by excluding individuals who have abnormal results for basic analytes other than the analyte in question, in this case lactate dehydrogenase. The reference interval derived for the suspected year was 264-530 U/L, while that of the previous year was 248-495 U/L. The only change we found was the introduction of an order entry system, which requests precise sampling volumes rather than complete filling of vacuum tubes. The effect of vacuum persistence was tested using ten freshly drawn blood samples. Compared with complete filling, 1/5 filling resulted in average elevations of lactate dehydrogenase, aspartic aminotransferase, and potassium levels of 8.0%, 3.8%, and 3.4%, respectively (all p<0.01). Microhemolysis was confirmed using a urine stick method. The length of time before centrifugation determined the degree of hemolysis, while vacuum during centrifugation did not affect it. Microhemolysis is the probable cause of the suspected pseudo-elevation noted by the physicians. Data-mining methodology represents a valuable tool for monitoring long-term bias in laboratory results.

  17. Microcomputer Assisted Interpretative Reporting of Sequential Creatine Kinase (CK) and Lactate Dehydrogenase (LDH) Isoenzyme Determination

    PubMed Central

    Talamo, Thomas S.; Losos, Frank J.; Mercer, Donald W.

    1984-01-01

    We have developed a microcomputer based system for interpretative reporting of creatine kinase (CK) and lactate dehydrogenase (LDH) isoenzyme studies. Patient demographic data and test results (total CK, CK-MB, LD-1, and LD-2) are entered manually through the keyboard. The test results are compared with normal range values and an interpretative report is generated. This report consists of all pertinent demographic information with a graphic display of up to 12 previous CK and LDH isoenzyme determinations. Diagnostic interpretative statements are printed beneath the graphic display following analysis of previously entered test results. The combination of graphic data display and interpretations based on analysis of up to 12 previous specimens provides useful and accurate information to the cardiologist.

  18. Reference values of blood parameters in beef cattle of different ages and stages of lactation.

    PubMed Central

    Doornenbal, H; Tong, A K; Murray, N L

    1988-01-01

    Reference (normal) values for 12 blood serum components were determined for 48 Shorthorn cows (2-10 years old) and their 48 calves, 357 crossbred cows (12-14 years old), 36 feedlot bulls and 36 feedlot steers. In addition, hemoglobin, hematocrit, triiodothyronine, thyroxine and cortisol levels were determined for the crossbred cows, and feedlot bulls and steers. Reference values were tabulated according to sex, age and stage of lactation. Serum concentrations of urea, total protein and bilirubin, and serum activity of aspartate aminotransferase and lactate dehydrogenase increased with age (P less than 0.05), while calcium, phosphorus and alkaline phosphatase decreased with age (P less than 0.05) from birth to the age of ten years. The Shorthorn cows had the highest levels of glucose at parturition (P less than 0.05) with decreasing levels during lactation. Creatinine concentration decreased during lactation and increased during postweaning. Both lactate dehydrogenase and aspartate aminotransferase levels increased (P less than 0.05) during lactation. Urea and uric acid were present at higher concentrations in lactating than nonlactating cows (P less than 0.05). The values reported, based on a wide age range and large number of cattle, could serve as clinical guides and a basis for further research. PMID:3349406

  19. Free energy landscape of the Michaelis complex of lactate dehydrogenase: A network analysis of atomistic simulations

    NASA Astrophysics Data System (ADS)

    Pan, Xiaoliang; Schwartz, Steven

    2015-03-01

    It has long been recognized that the structure of a protein is a hierarchy of conformations interconverting on multiple time scales. However, the conformational heterogeneity is rarely considered in the context of enzymatic catalysis in which the reactant is usually represented by a single conformation of the enzyme/substrate complex. Lactate dehydrogenase (LDH) catalyzes the interconversion of pyruvate and lactate with concomitant interconversion of two forms of the cofactor nicotinamide adenine dinucleotide (NADH and NAD+). Recent experimental results suggest that multiple substates exist within the Michaelis complex of LDH, and they are catalytic competent at different reaction rates. In this study, millisecond-scale all-atom molecular dynamics simulations were performed on LDH to explore the free energy landscape of the Michaelis complex, and network analysis was used to characterize the distribution of the conformations. Our results provide a detailed view of the kinetic network the Michaelis complex and the structures of the substates at atomistic scale. It also shed some light on understanding the complete picture of the catalytic mechanism of LDH.

  20. Free energy surface of the Michaelis complex of lactate dehydrogenase: a network analysis of microsecond simulations.

    PubMed

    Pan, Xiaoliang; Schwartz, Steven D

    2015-04-30

    It has long been recognized that the structure of a protein creates a hierarchy of conformations interconverting on multiple time scales. The conformational heterogeneity of the Michaelis complex is of particular interest in the context of enzymatic catalysis in which the reactant is usually represented by a single conformation of the enzyme/substrate complex. Lactate dehydrogenase (LDH) catalyzes the interconversion of pyruvate and lactate with concomitant interconversion of two forms of the cofactor nicotinamide adenine dinucleotide (NADH and NAD(+)). Recent experimental results suggest that multiple substates exist within the Michaelis complex of LDH, and they show a strong variance in their propensity toward the on-enzyme chemical step. In this study, microsecond-scale all-atom molecular dynamics simulations were performed on LDH to explore the free energy landscape of the Michaelis complex, and network analysis was used to characterize the distribution of the conformations. Our results provide a detailed view of the kinetic network of the Michaelis complex and the structures of the substates at atomistic scales. They also shed light on the complete picture of the catalytic mechanism of LDH.

  1. Melanoma inhibiting activity protein (MIA), beta-2 microglobulin and lactate dehydrogenase (LDH) in metastatic melanoma.

    PubMed

    Cao, M González; Auge, J M; Molina, R; Martí, R; Carrera, C; Castel, T; Vilella, R; Conill, C; Sánchez, M; Malvehy, J; Puig, S

    2007-01-01

    Serum levels of melanoma markers may have a role in monitoring disease evolution in metastatic melanoma. Serial measurements of melanoma inhibiting activity protein (MIA), lactate dehydrogenase (LDH), S-100 and beta2-microglubulin were obtained from 42 metastatic melanoma patients during their biochemotherapy treatment. High pre-treatment serum levels of S-100, LDH, MIA and P2-microglobulin were detected in 50%, 57%, 50% and 24% of the patients, respectively. Only S-100 had prognostic significance for both disease-free (p=0.011) and overall survival (p=0.021). In patients who responded to treatment, S-100 levels decreased significantly from pre-treatment to the time of response (p = 0.050). When patients progressed, levels of MIA and P2-microglobulin increased significantly (p =0.028 and p =0.030, respectively). Correlation with disease evolution was found for S-100, MIA and P2-microglobulin levels. Despite the small sample size of the study, S-100 was a significant prognostic marker for overall survival and disease-free survival.

  2. Metabolic Engineering of Escherichia coli K12 for Homofermentative Production of L-Lactate from Xylose.

    PubMed

    Jiang, Ting; Zhang, Chen; He, Qin; Zheng, Zhaojuan; Ouyang, Jia

    2018-02-01

    The efficient utilization of xylose is regarded as a technical barrier to the commercial production of bulk chemicals from biomass. Due to the desirable mechanical properties of polylactic acid (PLA) depending on the isomeric composition of lactate, biotechnological production of lactate with high optical pure has been increasingly focused in recent years. The main objective of this work was to construct an engineered Escherichia coli for the optically pure L-lactate production from xylose. Six chromosomal deletions (pflB, ldhA, ackA, pta, frdA, adhE) and a chromosomal integration of L-lactate dehydrogenase-encoding gene (ldhL) from Bacillus coagulans was involved in construction of E. coli KSJ316. The recombinant strain could produce L-lactate from xylose resulting in a yield of 0.91 g/g xylose. The chemical purity of L-lactate was 95.52%, and the optical purity was greater than 99%. Moreover, three strategies, including overexpression of L-lactate dehydrogenase, intensification of xylose catabolism, and addition of additives to medium, were designed to enhance the production. The results showed that they could increase the concentration of L-lactate by 32.90, 20.13, and 233.88% relative to the control, respectively. This was the first report that adding formate not only could increase the xylose utilization but also led to the fewer by-product levels.

  3. Kinetic characterization of recombinant Bacillus coagulans FDP-activated l-lactate dehydrogenase expressed in Escherichia coli and its substrate specificity.

    PubMed

    Jiang, Ting; Xu, Yanbing; Sun, Xiucheng; Zheng, Zhaojuan; Ouyang, Jia

    2014-03-01

    Bacillus coagulans is a homofermentative, acid-tolerant and thermophilic sporogenic lactic acid bacterium, which is capable of producing high yields of optically pure lactic acid. The l-(+)-lactate dehydrogenase (l-LDH) from B. coagulans is considered as an ideal biocatalyst for industrial production. In this study, the gene ldhL encoding a thermostable l-LDH was amplified from B. coagulans NL01 genomic DNA and successfully expressed in Escherichia coli BL21 (DE3). The recombinant enzyme was partially purified and its enzymatic properties were characterized. Sequence analysis demonstrated that the l-LDH was a fructose 1,6-diphosphate-activated NAD-dependent lactate dehydrogenase (l-nLDH). Its molecular weight was approximately 34-36kDa. The Km and Vmax values of the purified l-nLDH for pyruvate were 1.91±0.28mM and 2613.57±6.43μmol(minmg)(-1), respectively. The biochemical properties of l-nLDH showed that the specific activity were up to 2323.29U/mg with optimum temperature of 55°C and pH of 6.5 in the pyruvate reduction and 351.01U/mg with temperature of 55°C and pH of 11.5 in the lactate oxidation. The enzyme also showed some activity in the absence of FDP, with a pH optimum of 4.0. Compared to other lactic acid bacterial l-nLDHs, the enzyme was found to be relatively stable at 50°C. Ca(2+), Ba(2+), Mg(2+) and Mn(2+) ions had activated effects on the enzyme activity, and the enzyme was greatly inhibited by Ni(2+) ion. Besides these, l-nLDH showed the higher specificity towards pyruvate esters, such as methyl pyruvate and ethyl pyruvate. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. Lactate dehydrogenase regulation in aged skeletal muscle: Regulation by anabolic steroids and functional overload.

    PubMed

    Washington, Tyrone A; Healey, Julie M; Thompson, Raymond W; Lowe, Larry L; Carson, James A

    2014-09-01

    Aging alters the skeletal muscle response to overload-induced growth. The onset of functional overload is characterized by increased myoblast proliferation and an altered muscle metabolic profile. The onset of functional overload is associated with increased energy demands that are met through the interconversion of lactate and pyruvate via the activity of lactate dehydrogenase (LDH). Testosterone targets many of the processes activated at the onset of functional overload. However, the effect of aging on this metabolic plasticity at the onset of functional overload and how anabolic steroid administration modulates this response is not well understood. The purpose of this study was to determine if aging would alter overload-induced LDH activity and expression at the onset of functional overload and whether anabolic steroid administration would modulate this response. Five-month and 25-month male Fischer 344xF1 BRN were given nandrolone decanoate (ND) or sham injections for 14days and then the plantaris was functionally overloaded (OV) for 3days by synergist ablation. Aging reduced muscle LDH-A & LDH-B activity 70% (p<0.05). Aging also reduced LDH-A mRNA abundance, however there was no age effect on LDH-B mRNA abundance. In 5-month muscle, both ND and OV decreased LDH-A and LDH-B activity. However, there was no synergistic or additive effect. In 5-month muscle, ND and OV decreased LDH-A mRNA expression with no change in LDH-B expression. In 25-month muscle, ND and OV increased LDH-A and LDH-B activity. LDH-A mRNA expression was not altered by ND or OV in aged muscle. However, there was a main effect of OV to decrease LDH-B mRNA expression. There was also an age-induced LDH isoform shift. ND and OV treatment increased the "fast" LDH isoforms in aged muscle, whereas ND and OV increased the "slow" isoforms in young muscle. Our study provides evidence that aging alters aspects of skeletal muscle metabolic plasticity normally induced by overload and anabolic steroid

  5. Changes in creatine kinase, lactate dehydrogenase and aspartate aminotransferase in saliva samples after an intense exercise: a pilot study.

    PubMed

    Barranco, Tomas; Tvarijonaviciute, Asta; Tecles, Fernando; Carrillo, Jose M; Sánchez-Resalt, Cristina; Jimenez-Reyes, Pedro; Rubio, Monica; García-Balletbó, Monserrat; Cerón, Jose J; Cugat, Ramon

    2018-06-01

    The aim of this study was to evaluate changes in the enzymes creatine kinase (CK), lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) in saliva before and after an intense exercise consisting of a futsal match. CK, LDH and AST were analyzed in saliva and serum samples of eleven, injury-free, amateur young men before and 30 minutes, 12 hours and 36 hours after a futsal match. A significant increase in CK, LDH and AST was observed after the game in serum samples. In saliva, although a high interindividual variability was found with some individuals no showing increases, significant increases in CK and LDH were observed after the game. No significant changes were observed in saliva AST after the game. Our study showed for first time that CK and LDH can increase in saliva after an intensive exercise consisting on a futsal match. Results suggest that measurements of CK and LDH in saliva could be potentially used to evaluate possible muscle stress or damage in cases of intensive exercise.

  6. Molecular cloing and bioinformatics analysis of lactate dehydrogenase from Taenia multiceps.

    PubMed

    Guo, Cheng; Wang, Yu; Huang, Xing; Wang, Ning; Yan, Ming; He, Ran; Gu, Xiaobin; Xie, Yue; Lai, Weimin; Jing, Bo; Peng, Xuerong; Yang, Guangyou

    2017-10-01

    Coenurus cerebralis, the larval stage (metacestode or coenurus) of Taenia multiceps, parasitizes sheep, goats, and other ruminants and causes coenurosis. In this study, we isolated and characterized complementary DNAs that encode lactate dehydrogenase A (Tm-LDHA) and B (Tm-LDHB) from the transcriptome of T. multiceps and expressed recombinant Tm-LDHB (rTm-LDHB) in Escherichia coli. Bioinformatic analysis showed that both Tm-LDH genes (LDHA and LDHB) contain a 996-bp open reading frame and encode a protein of 331 amino acids. After determination of the immunogenicity of the recombinant Tm-LDHB, an indirect enzyme-linked immunosorbent assay (ELISA) was developed for preliminary evaluation of the serodiagnostic potential of rTm-LDHB in goats. However, the rTm-LDHB-based indirect ELISA developed here exhibited specificity of only 71.42% (10/14) and sensitivity of 1:3200 in detection of goats infected with T. multiceps in the field. This study is the first to describe LDHA and LDHB of T. multiceps; meanwhile, our results indicate that rTm-LDHB is not a specific antigen candidate for immunodiagnosis of T. multiceps infection in goats.

  7. Lactate dehydrogenase isoenzyme patterns upon chronic exposure to cigarette smoke: Protective effect of bacoside A.

    PubMed

    Anbarasi, Kothandapani; Sabitha, Kuruvimalai Ekambaram; Devi, Chennam Srinivasulu Shyamala

    2005-09-01

    Despite a strong association between cigarette smoking and alarming increase in mortality rate from smoking-related diseases, around 35-40% of the world's population continues to smoke and many more are being exposed to environmental tobacco smoke. Since the role of free radicals and oxidative damage in the pathogenesis of smoking-related diseases has been suggested, bacoside A, a potent antioxidant was tested for its ability to protect against cigarette smoking-induced toxicity in terms of lactate dehydrogenase (LDH) and its isoenzymes. Rats were exposed to cigarette smoke and simultaneously administered with bacoside A, for a period of 12 weeks. Total LDH activity was assayed in serum, lung, heart, brain, liver and kidney, and serum LDH isoforms were separated electrophoretically. Cigarette smoke exposure resulted in significant increase in serum LDH and its isoenzymes with a concomitant decrease in these organs. These alterations were prevented by administration of bacoside A. Excessive oxidants from cigarette smoke is known to cause peroxidation of membrane lipids leading to cellular damage, thereby resulting in the leakage of LDH into the circulation. Bacoside A could have rendered protection to the organs by stabilizing their cell membranes and prevented the release of LDH, probably through its free radical scavenging and anti-lipid peroxidative effect.

  8. Computational analyses of mammalian lactate dehydrogenases: human, mouse, opossum and platypus LDHs.

    PubMed

    Holmes, Roger S; Goldberg, Erwin

    2009-10-01

    Computational methods were used to predict the amino acid sequences and gene locations for mammalian lactate dehydrogenase (LDH) genes and proteins using genome sequence databanks. Human LDHA, LDHC and LDH6A genes were located in tandem on chromosome 11, while LDH6B and LDH6C genes were on chromosomes 15 and 12, respectively. Opossum LDHC and LDH6B genes were located in tandem with the opossum LDHA gene on chromosome 5 and contained 7 (LDHA and LDHC) or 8 (LDH6B) exons. An amino acid sequence prediction for the opossum LDH6B subunit gave an extended N-terminal sequence, similar to the human and mouse LDH6B sequences, which may support the export of this enzyme into mitochondria. The platypus genome contained at least 3 LDH genes encoding LDHA, LDHB and LDH6B subunits. Phylogenetic studies and sequence analyses indicated that LDHA, LDHB and LDH6B genes are present in all mammalian genomes examined, including a monotreme species (platypus), whereas the LDHC gene may have arisen more recently in marsupial mammals.

  9. Computational analyses of mammalian lactate dehydrogenases: human, mouse, opossum and platypus LDHs

    PubMed Central

    Holmes, Roger S; Goldberg, Erwin

    2009-01-01

    Computational methods were used to predict the amino acid sequences and gene locations for mammalian lactate dehydrogenase (LDH) genes and proteins using genome sequence databanks. Human LDHA, LDHC and LDH6A genes were located in tandem on chromosome 11, while LDH6B and LDH6C genes were on chromosomes 15 and 12, respectively. Opossum LDHC and LDH6B genes were located in tandem with the opossum LDHA gene on chromosome 5 and contained 7 (LDHA and LDHC) or 8 (LDH6B) exons. An amino acid sequence prediction for the opossum LDH6B subunit gave an extended N-terminal sequence, similar to the human and mouse LDH6B sequences, which may support the export of this enzyme into mitochondria. The platypus genome contained at least 3 LDH genes encoding LDHA, LDHB and LDH6B subunits. Phylogenetic studies and sequence analyses indicated that LDHA, LDHB and LDH6B genes are present in all mammalian genomes examined, including a monotreme species (platypus), whereas the LDHC gene may have arisen more recently in marsupial mammals. PMID:19679512

  10. SYNAPTOSOMAL LACTATE DEHYDROGENASE ISOENZYME COMPOSITION IS SHIFTED TOWARD AEROBIC FORMS IN PRIMATE BRAIN EVOLUTION

    PubMed Central

    Duka, Tetyana; Anderson, Sarah M.; Collins, Zachary; Raghanti, Mary Ann; Ely, John J.; Hof, Patrick R.; Wildman, Derek E.; Goodman, Morris; Grossman, Lawrence I.; Sherwood, Chet C.

    2014-01-01

    With the evolution of a relatively large brain size in haplorhine primates (i.e., tarsiers, monkeys, apes and humans), there have been associated changes in the molecular machinery that delivers energy to the neocortex. Here we investigated variation in lactate dehydrogenase (LDH) expression and isoenzyme composition of the neocortex and striatum in primates using quantitative Western blotting and isoenzyme analysis of total homogenates and synaptosomal fractions. Analysis of isoform expression revealed that LDH in the synaptosomal fraction from both forebrain regions shifted towards a predominance of the heart-type, aerobic isoforms, LDHB, among haplorhines as compared to strepsirrhines (i.e., lorises and lemurs), while in total homogenate of neocortex and striatum there was no significant difference in the LDH isoenzyme composition between the primate suborders. The largest increase occurred in synapse-associated LDH-B expression in the neocortex, displaying an especially remarkable elevation in the ratio of LDH-B to LDH-A in humans. The phylogenetic variation in LDH-B to LDH-A ratio was correlated with species typical brain mass, but not encephalization quotient. A significant LDHB increase in the sub-neuronal fraction from haplorhine neocortex and striatum suggests a relatively higher rate of aerobic glycolysis that is linked to synaptosomal mitochondrial metabolism. Our results indicate that there is differential composition of LDH isoenzymes and metabolism in synaptic terminals that evolved in primates to meet increased energy requirements in association with brain enlargement. PMID:24686273

  11. Adaptations in lactate dehydrogenase and its isozymes in aging mammalian myocardium: interaction of exercise and temperature.

    PubMed

    Prathima, S; Devi, S A

    1999-04-01

    The responses of the left and right ventricles (LV and RV) to physical conditioning in cold (25 degrees C) and thermoneutral temperatures (35 degrees C), with special reference to lactate dehydrogenase (LDH) and its isoenzyme profile, were studied in the 2-month (young)- and 12-month (middle-aged)-old rats. Moderate hypertrophy was a common observation irrespective of age, region and swim temperature. LV, however, hypertrophied to a significantly lesser extent in the middle-aged, than the RV. Blood Lactate (La) content showed a decline in the trained rather than their untrained counterparts. LDH activity decreased with age. Swim training induced elevations in the enzyme activity. The isoenzyme profile was suitably and efficiently altered in the LV and RV of trained animals to meet the arising O2 demands. The above adaptations were best seen in the young and in the animals trained at thermoneutral temperatures. Thus it is suggested that young age is very apt for initiation of training programs although middle-age is not so late. Swimming in water near body temperature is emphasised as a more preferred environment to cold water, in order to derive maximal exercise-associated beneficial effects.

  12. Malate dehydrogenase of the cytosol. A kinetic investigation of the reaction mechanism and a comparison with lactate dehydrogenase.

    PubMed Central

    Lodola, A; Shore, J D; Parker, D M; Holbrook, J

    1978-01-01

    1. The mechanisms of the reduction of oxaloacetate and of 3-fluoro-oxaloacetate by NADH catalysed by cytoplasmic pig heart malate dehydrogenase (MDH) were investigated. 2. One mol of dimeric enzyme produces 1.7+/-0.4 mol of enzyme-bound NADH when mixed with saturating NAD+ and L-malate at a rate much higher than the subsequent turnover at pH 7.5. 3. Transient measurements of protein and nucleotide fluorescence show that the steady-state complex in the forward direction is MDH-NADH and in the reverse direction MDH-NADH-oxaloacetate. 4. The rate of dissociation of MDH-NADH was measured and is the same as Vmax. in the forward direction at pH 7.5. Both NADH-binding sites are kinetically equivalent. The rate of dissociation varies with pH, as does the equilibrium binding constant for NADH. 5. 3-Fluoro-oxaloacetate is composed of three forms (F1, F2 and S) of which F1 and F2 are immediately substrates for the enzyme. The third form, S, is not a substrate, but when the F forms are used up form S slowly and non-enzymically equilibrates to yield the active substrate forms. S is 2,2-dihydroxy-3-fluorosuccinate. 6. The steady-state compound during the reduction of form F1 is an enzyme form that does not contain NADH, probably MDH-NAD+-fluoromalate. The steady-state compound for form F2 is an enzyme form containing NADH, probably MDH-NADH-fluoro-oxaloacetate. 7. The rate-limiting reaction in the reduction of form F2 shows a deuterium isotope rate ratio of 4 when NADH is replaced by its deuterium analogue, and the rate-limiting reaction is concluded to be hydride transfer. 8. A novel titration was used to show that dimeric cytoplasmic malate dehydrogenase contains two sites that can rapidly reduce the F1 form of 3-fluoro-oxaloacetate. The enzyme shows 'all-of-the-sites' behaviour. 9. Partial mechanisms are proposed to explain the enzyme-catalysed transformations of the natural and the fluoro substrates. These mechanisms are similar to the mechanism of pig heart lactate

  13. Disruption of lactate dehydrogenase and alcohol dehydrogenase for increased hydrogen production and its effect on metabolic flux in Enterobacter aerogenes.

    PubMed

    Zhao, Hongxin; Lu, Yuan; Wang, Liyan; Zhang, Chong; Yang, Cheng; Xing, Xinhui

    2015-10-01

    Hydrogen production by Enterobacter aerogenes from glucose was enhanced by deleting the targeted ldhA and adh genes responsible for two NADH-consuming pathways which consume most NADH generated from glycolysis. Compared with the wild-type, the hydrogen yield of IAM1183-ΔldhA increased 1.5 fold. Metabolic flux analysis showed both IAM1183-ΔldhA and IAM1183-Δadh exhibited significant changes in flux, including enhanced flux towards the hydrogen generation. The lactate production of IAM1183-ΔldhA significantly decreased by 91.42%, while the alcohol yield of IAM1183-Δadh decreased to 30%. The mutant IAM1183-ΔldhA with better hydrogen-producing performance was selected for further investigation in a 5-L fermentor. The hydrogen production of IAM1183-ΔldhA was 2.3 times higher than the wild-type. Further results from the fermentation process showed that the pH decreased to 5.39 levels, then gradually increased to 5.96, indicating that some acidic metabolites might be degraded or uptaken by cells. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Effect of Controlled Ice Nucleation on Stability of Lactate Dehydrogenase During Freeze-Drying.

    PubMed

    Fang, Rui; Tanaka, Kazunari; Mudhivarthi, Vamsi; Bogner, Robin H; Pikal, Michael J

    2018-03-01

    Several controlled ice nucleation techniques have been developed to increase the efficiency of the freeze-drying process as well as to improve the quality of pharmaceutical products. Owing to the reduction in ice surface area, these techniques have the potential to reduce the degradation of proteins labile during freezing. The objective of this study was to evaluate the effect of ice nucleation temperature on the in-process stability of lactate dehydrogenase (LDH). LDH in potassium phosphate buffer was nucleated at -4°C, -8°C, and -12°C using ControLyo™ or allowed to nucleate spontaneously. Both the enzymatic activity and tetramer recovery after freeze-thawing linearly correlated with product ice nucleation temperature (n = 24). Controlled nucleation also significantly improved batch homogeneity as reflected by reduced inter-vial variation in activity and tetramer recovery. With the correlation established in the laboratory, the degradation of protein in manufacturing arising from ice nucleation temperature differences can be quantitatively predicted. The results show that controlled nucleation reduced the degradation of LDH during the freezing process, but this does not necessarily translate to vastly superior stability during the entire freeze-drying process. The capability of improving batch homogeneity provides potential advantages in scaling-up from lab to manufacturing scale. Copyright © 2018 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  15. Salivary lactate dehydrogenase and aminotransferases in diabetic patients

    PubMed Central

    Malicka, Barbara; Skoskiewicz-Malinowska, Katarzyna; Kaczmarek, Urszula

    2016-01-01

    Abstract Diabetes mellitus (DM) is a group of metabolic diseases resulting from impaired insulin secretion and/or action. DM is characterized by hyperglycemia that can lead to the dysfunction or damage of organs, including the salivary glands. The aim of this study was to compare the levels of salivary lactate dehydrogenase (LDH), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) in diabetic patients. The study was approved by the Bioethics Committee of Wroclaw Medical University (Poland). The study comprised 90 adults of both sexes, aged 21 to 57 years. The patients were divided into 3 groups: type 1 diabetics (D1), type 2 diabetics (D2), and a healthy control group (C). Each group consisted of 30 age- and sex-matched subjects. Total protein (P, by Lowry method), LDH, AST, ALT (with Alpha Diagnostics kits), and salivary flow rate were measured in unstimulated mixed saliva. The level of glycosylated hemoglobin (HbA1c) was measured with DCA 2000 Reagent Kit. The obtained data were analyzed using the Mann–Whitney U test and the Spearman rank at a significance level of P < 0.05 with the use of STATISTICA 9.0 software. In comparison with C, D1 presented a significantly higher activity of LDH (P < 0.001), AST (P < 0.001), and ALT (P < 0.01), whereas D2 indicated higher levels of LDH (P < 0.001) and ALT (P < 0.05) compared with C. Comparing D1 to D2, approximately 3-fold higher activity of AST (P < 0.01) and approximately 4.5-fold higher activity of ALT (P < 0.01) was observed. Higher levels of salivary LDH, AST, and ALT in D1 compared with D2 and C confirm that salivary glands of D1 might be attributed to autoimmunological damage associated with the pathomechanism of DM. PMID:27893660

  16. Salivary lactate dehydrogenase and aminotransferases in diabetic patients.

    PubMed

    Malicka, Barbara; Skoskiewicz-Malinowska, Katarzyna; Kaczmarek, Urszula

    2016-11-01

    Diabetes mellitus (DM) is a group of metabolic diseases resulting from impaired insulin secretion and/or action. DM is characterized by hyperglycemia that can lead to the dysfunction or damage of organs, including the salivary glands.The aim of this study was to compare the levels of salivary lactate dehydrogenase (LDH), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) in diabetic patients.The study was approved by the Bioethics Committee of Wroclaw Medical University (Poland). The study comprised 90 adults of both sexes, aged 21 to 57 years. The patients were divided into 3 groups: type 1 diabetics (D1), type 2 diabetics (D2), and a healthy control group (C). Each group consisted of 30 age- and sex-matched subjects. Total protein (P, by Lowry method), LDH, AST, ALT (with Alpha Diagnostics kits), and salivary flow rate were measured in unstimulated mixed saliva. The level of glycosylated hemoglobin (HbA1c) was measured with DCA 2000 Reagent Kit. The obtained data were analyzed using the Mann-Whitney U test and the Spearman rank at a significance level of P < 0.05 with the use of STATISTICA 9.0 software.In comparison with C, D1 presented a significantly higher activity of LDH (P < 0.001), AST (P < 0.001), and ALT (P < 0.01), whereas D2 indicated higher levels of LDH (P < 0.001) and ALT (P < 0.05) compared with C. Comparing D1 to D2, approximately 3-fold higher activity of AST (P < 0.01) and approximately 4.5-fold higher activity of ALT (P < 0.01) was observed.Higher levels of salivary LDH, AST, and ALT in D1 compared with D2 and C confirm that salivary glands of D1 might be attributed to autoimmunological damage associated with the pathomechanism of DM.

  17. Development of L-lactate dehydrogenase biosensor based on porous silicon resonant microcavities as fluorescence enhancers.

    PubMed

    Jenie, S N Aisyiyah; Prieto-Simon, Beatriz; Voelcker, Nicolas H

    2015-12-15

    The up-regulation of L-lactate dehydrogenase (LDH), an intracellular enzyme present in most of all body tissues, is indicative of several pathological conditions and cellular death. Herein, we demonstrate LDH detection using porous silicon (pSi) microcavities as a luminescence-enhancing optical biosensing platform. Non-fluorescent resazurin was covalently attached onto the pSi surface via thermal hydrocarbonisation, thermal hydrosylilation and acylation. Each surface modification step was confirmed by means of FTIR and the optical shifts of the resonance wavelength of the microcavity. Thermal hydrocarbonisation also afforded excellent surface stability, ensuring that the resazurin was not reduced on the pSi surface. Using a pSi microcavity biosensor, the fluorescence signal upon detection of LDH was amplified by 10 and 5-fold compared to that of a single layer and a detuned microcavity, respectively, giving a limit of detection of 0.08 U/ml. The biosensor showed a linear response between 0.16 and 6.5 U/ml, covering the concentration range of LDH in normal as well as damaged tissues. The biosensor was selective for LDH and did not produce a signal upon incubation with another NAD-dependant enzyme L-glutamic dehydrogenase. The use of the pSi microcavity as a sensing platform reduced reagent usage by 30% and analysis time threefold compared to the standard LDH assay in solution. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Physiological and fermentation properties of Bacillus coagulans and a mutant lacking fermentative lactate dehydrogenase activity.

    PubMed

    Su, Yue; Rhee, Mun Su; Ingram, Lonnie O; Shanmugam, K T

    2011-03-01

    Bacillus coagulans, a sporogenic lactic acid bacterium, grows optimally at 50-55 °C and produces lactic acid as the primary fermentation product from both hexoses and pentoses. The amount of fungal cellulases required for simultaneous saccharification and fermentation (SSF) at 55 °C was previously reported to be three to four times lower than for SSF at the optimum growth temperature for Saccharomyces cerevisiae of 35 °C. An ethanologenic B. coagulans is expected to lower the cellulase loading and production cost of cellulosic ethanol due to SSF at 55 °C. As a first step towards developing B. coagulans as an ethanologenic microbial biocatalyst, activity of the primary fermentation enzyme L-lactate dehydrogenase was removed by mutation (strain Suy27). Strain Suy27 produced ethanol as the main fermentation product from glucose during growth at pH 7.0 (0.33 g ethanol per g glucose fermented). Pyruvate dehydrogenase (PDH) and alcohol dehydrogenase (ADH) acting in series contributed to about 55% of the ethanol produced by this mutant while pyruvate formate lyase and ADH were responsible for the remainder. Due to the absence of PDH activity in B. coagulans during fermentative growth at pH 5.0, the l-ldh mutant failed to grow anaerobically at pH 5.0. Strain Suy27-13, a derivative of the l-ldh mutant strain Suy27, that produced PDH activity during anaerobic growth at pH 5.0 grew at this pH and also produced ethanol as the fermentation product (0.39 g per g glucose). These results show that construction of an ethanologenic B. coagulans requires optimal expression of PDH activity in addition to the removal of the LDH activity to support growth and ethanol production.

  19. The determination and arrangement of a combination of enzyme lactate dehydrogenase of bacteria Acinetobacter sp. as a device the identity important bacteria agent composts

    NASA Astrophysics Data System (ADS)

    Sukmawati, D.; Puspitaningrum, R.; Muzajjanah

    2017-07-01

    The number of garbage generated by the industry or society is a usual problem encountered by almost all urban centers, especially large cities such as Jakarta. Waste prevention strategy required quickly and accurately. One strategy for tackling the Junk was getting lactic acid-producing bacteria. It has been shown that lactic acid can increase the acceleration of organic matter such as an overhaul of lignin and cellulose as well as out causing toxic compounds arising from decay. This research will be conducted on the determination and characterization of the enzyme-producing compost bacteria LDH lactate dehydrogenase LDH - which in isolation from the garbage Landfill Rawasari. Methodology: Research carried out consists: isolation of lactic acid-producing bacteria; identification of microscopic, macroscopic and staining Gram; cellulose assay, and optimization of PCR conditions LDH enzymes producing bacteria. Isolation is performed by dilution method and the direct method. As many as 5-point sampling. Each stage is conducted from 10 grams of soil from the top surface of the compost. Isolation results obtained 100 isolate the bacteria. Base on the characteristic of macroscopic and microscopic observations retrieved 14 isolates of bacteria have shaped rods and brought forth a negative kind of Gram positive staining. Bacterial isolates with codes (BK1; BK3; BK4; BK5; BK6; BK7; BK8; BK9; BK10; BK11: BK12; BK 13). The potential bacteria with ability produce lactate dehydrogenase was BK1 and BK3. Base for analysis phylogenetic there was identification bacteria bak1 and bak3 where Acinetobacter sp.

  20. Lactate Dehydrogenase Undergoes a Substantial Structural Change to Bind its Substrate

    PubMed Central

    Qiu, Linlin; Gulotta, Miriam; Callender, Robert

    2007-01-01

    Employing temperature-jump relaxation spectroscopy, we investigate the kinetics and thermodynamics of the formation of a very early ternary binding intermediate formed when lactate dehydrogenase (LDH) binds a substrate mimic on its way to forming the productive LDH/NADH·substrate Michaelis complex. Temperature-jump scans show two distinct submillisecond processes are involved in the formation of this ternary binding intermediate, called the encounter complex here. The on-rate of the formation of the encounter complex from LDH/NADH with oxamate (a substrate mimic) is determined as a function of temperature and in the presence of small concentrations of a protein destabilizer (urea) and protein stabilizer (TMAO). It shows a strong temperature dependence with inverse Arrhenius behavior and a temperature-dependent enthalpy (heat capacity of 610 ± 84 cal/Mol K), is slowed in the presence of TMAO and speeded up in the presence of urea. These results suggest that LDH/NADH occupies a range of conformations, some competent to bind substrate (open structure; a minority population) and others noncompetent (closed), in fast equilibrium with each other in accord with a select fit model of binding. From the thermodynamic results, the two species differ in the rearrangement of low energy hydrogen bonds as would arise from changes in internal hydrogen bonding and/or increases in the solvation of the protein structure. The binding-competent species can bind ligand at or very near diffusion-limited speeds, suggesting that the binding pocket is substantially exposed to solvent in these species. This would be in contrast to the putative closed structure where the binding pocket resides deep within the protein interior. PMID:17483169

  1. Effects and Mechanism of Atmospheric-Pressure Dielectric Barrier Discharge Cold Plasma on Lactate Dehydrogenase (LDH) Enzyme

    NASA Astrophysics Data System (ADS)

    Zhang, Hao; Xu, Zimu; Shen, Jie; Li, Xu; Ding, Lili; Ma, Jie; Lan, Yan; Xia, Weidong; Cheng, Cheng; Sun, Qiang; Zhang, Zelong; Chu, Paul K.

    2015-05-01

    Proteins are carriers of biological functions and the effects of atmospheric-pressure non-thermal plasmas on proteins are important to applications such as sterilization and plasma-induced apoptosis of cancer cells. Herein, we report our detailed investigation of the effects of helium-oxygen non-thermal dielectric barrier discharge (DBD) plasmas on the inactivation of lactate dehydrogenase (LDH) enzyme solutions. Circular dichroism (CD) and dynamic light scattering (DLS) indicate that the loss of activity stems from plasma-induced modification of the secondary molecular structure as well as polymerization of the peptide chains. Raising the treatment intensity leads to a reduced alpha-helix content, increase in the percentage of the beta-sheet regions and random sequence, as well as gradually decreasing LDH activity. However, the structure of the LDH plasma-treated for 300 seconds exhibits a recovery trend after storage for 24 h and its activity also increases slightly. By comparing direct and indirect plasma treatments, plasma-induced LDH inactivation can be attributed to reactive species (RS) in the plasma, especially ones with a long lifetime including hydrogen peroxide, ozone, and nitrate ion which play the major role in the alteration of the macromolecular structure and molecular diameter in lieu of heat, UV radiation, and charged particles.

  2. Inhibiting Sperm Pyruvate Dehydrogenase Complex and Its E3 Subunit, Dihydrolipoamide Dehydrogenase Affects Fertilization in Syrian Hamsters

    PubMed Central

    Sailasree, Purnima; Singh, Durgesh K.; Kameshwari, Duvurri B.; Shivaji, Sisinthy

    2014-01-01

    Background/Aims The importance of sperm capacitation for mammalian fertilization has been confirmed in the present study via sperm metabolism. Involvement of the metabolic enzymes pyruvate dehydrogenase complex (PDHc) and its E3 subunit, dihydrolipoamide dehydrogenase (DLD) in hamster in vitro fertilization (IVF) via in vitro sperm capacitation is being proposed through regulation of sperm intracellular lactate, pH and calcium. Methodology and Principal Findings Capacitated hamster spermatozoa were allowed to fertilize hamster oocytes in vitro which were then assessed for fertilization, microscopically. PDHc/DLD was inhibited by the use of the specific DLD-inhibitor, MICA (5-methoxyindole-2-carboxylic acid). Oocytes fertilized with MICA-treated (MT) [and thus PDHc/DLD-inhibited] spermatozoa showed defective fertilization where 2nd polar body release and pronuclei formation were not observed. Defective fertilization was attributable to capacitation failure owing to high lactate and low intracellular pH and calcium in MT-spermatozoa during capacitation. Moreover, this defect could be overcome by alkalinizing spermatozoa, before fertilization. Increasing intracellular calcium in spermatozoa pre-IVF and in defectively-fertilized oocytes, post-fertilization rescued the arrest seen, suggesting the role of intracellular calcium from either of the gametes in fertilization. Parallel experiments carried out with control spermatozoa capacitated in medium with low extracellular pH or high lactate substantiated the necessity of optimal sperm intracellular lactate levels, intracellular pH and calcium during sperm capacitation, for proper fertilization. Conclusions This study confirms the importance of pyruvate/lactate metabolism in capacitating spermatozoa for successful fertilization, besides revealing for the first time the importance of sperm PDHc/ DLD in fertilization, via the modulation of sperm intracellular lactate, pH and calcium during capacitation. In addition, the

  3. Specific Inhibition of Hepatic Lactate Dehydrogenase Reduces Oxalate Production in Mouse Models of Primary Hyperoxaluria.

    PubMed

    Lai, Chengjung; Pursell, Natalie; Gierut, Jessica; Saxena, Utsav; Zhou, Wei; Dills, Michael; Diwanji, Rohan; Dutta, Chaitali; Koser, Martin; Nazef, Naim; Storr, Rachel; Kim, Boyoung; Martin-Higueras, Cristina; Salido, Eduardo; Wang, Weimin; Abrams, Marc; Dudek, Henryk; Brown, Bob D

    2018-06-15

    Primary hyperoxalurias (PHs) are autosomal recessive disorders caused by the overproduction of oxalate leading to calcium oxalate precipitation in the kidney and eventually to end-stage renal disease. One promising strategy to treat PHs is to reduce the hepatic production of oxalate through substrate reduction therapy by inhibiting liver-specific glycolate oxidase (GO), which controls the conversion of glycolate to glyoxylate, the proposed main precursor to oxalate. Alternatively, diminishing the amount of hepatic lactate dehydrogenase (LDH) expression, the proposed key enzyme responsible for converting glyoxylate to oxalate, should directly prevent the accumulation of oxalate in PH patients. Using RNAi, we provide the first in vivo evidence in mammals to support LDH as the key enzyme responsible for converting glyoxylate to oxalate. In addition, we demonstrate that reduction of hepatic LDH achieves efficient oxalate reduction and prevents calcium oxalate crystal deposition in genetically engineered mouse models of PH types 1 (PH1) and 2 (PH2), as well as in chemically induced PH mouse models. Repression of hepatic LDH in mice did not cause any acute elevation of circulating liver enzymes, lactate acidosis, or exertional myopathy, suggesting further evaluation of liver-specific inhibition of LDH as a potential approach for treating PH1 and PH2 is warranted. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

  4. Homo-D-lactic acid fermentation from arabinose by redirection of the phosphoketolase pathway to the pentose phosphate pathway in L-lactate dehydrogenase gene-deficient Lactobacillus plantarum.

    PubMed

    Okano, Kenji; Yoshida, Shogo; Tanaka, Tsutomu; Ogino, Chiaki; Fukuda, Hideki; Kondo, Akihiko

    2009-08-01

    Optically pure d-lactic acid fermentation from arabinose was achieved by using the Lactobacillus plantarum NCIMB 8826 strain whose l-lactate dehydrogenase gene was deficient and whose phosphoketolase gene was substituted with a heterologous transketolase gene. After 27 h of fermentation, 38.6 g/liter of d-lactic acid was produced from 50 g/liter of arabinose.

  5. Meta-analysis of serum lactate dehydrogenase and prognosis for osteosarcoma.

    PubMed

    Fu, Yucheng; Lan, Tao; Cai, Hongliu; Lu, Anwei; Yu, Wei

    2018-05-01

    A large number of studies have reported the relationships between serum lactate dehydrogenase (LDH) and prognosis of osteosarcoma. However, the result is still controversial and no consensus has been reached. Therefore, we performed a meta-analysis to evaluate the prognostic role of serum LDH in osteosarcoma patients. We performed the systematic computerized search for eligible articles from PubMed, Embase, and Cochrane databases until December 21, 2017. The pooled hazard ratio (HR) and 95% confidence intervals (CIs) of overall survival (OS) and event-free survival (EFS) were obtained to assess the prognostic value of serum LDH. A total of 18 studies with 2543 osteosarcoma patients were included. Overall, 15 studies assessed the elevated serum LDH level on OS and the pooled HR was 1.87 (95% CI = 1.58-2.20). Meanwhile, the pooled HR to evaluate the relationship between serum LDH and EFS in 9 studies was 1.78 (95% CI = 1.51-2.10). The same results were acquired when these studies were stratified by Enneking stage, geographic region, and sample size. No heterogeneity existed between these subgroups (P > .05). Begg's funnel plot and Egger's test (OS: P = .04; EFS: P = .34) showed that possible publication bias might exist in OS studies. Sensitivity analysis suggested the pooled HR was robust. This meta-analysis demonstrates that elevated serum LDH level is apparently associated with lower EFS rate and serum LDH could be a prognostic biomarker for osteosarcoma patients.

  6. INACTIVATION OF LACTATE DEHYDROGENASE BY SEVERAL CHEMICALS: IMPLICATIONS FOR IN VITRO TOXICOLOGY STUDIES

    PubMed Central

    Kendig, Derek M.; Tarloff, Joan B.

    2007-01-01

    Lactate dehydrogenase (LDH) release is frequently used as an end-point for cytotoxicity studies. We have been unable to measure LDH release during studies using para-aminophenol (PAP) in LLC-PK1 cells. When LLC-PK1 cells were incubated with either PAP (0–10 mM) or menadione (0–1000 μM), viability was markedly reduced when assessed by alamar Blue or total LDH activity but not by release of LDH into the incubation medium. In addition, we incubated cells with PAP or menadione and compared LDH activity using two different assays. Both assays confirmed our observation of decreased LDH activity in cell lysates without corresponding increases in LDH activity in incubation media. Using purified LDH and 10 mM PAP, we that PAP produced loss of LDH activity that was inversely proportional to the amount of LDH initially added. In additional experiments, we incubated 0.5 units of LDH for 1 h with varying concentrations of PAP, menadione, hydrogen peroxide (H2O2) or cisplatin. All four chemicals produced concentration-dependent decreases in LDH activity. In previous experiments, inclusion of antioxidants such as reduced glutathione (GSH) and ascorbate protected cells from PAP toxicity. GSH (1 mM) preserved LDH activity in the presence of toxicants while ascorbate (1 mM) only prevented LDH loss induced by PAP. These studies suggest that LDH that is released into the incubation medium is susceptible to degradation when reactive chemicals are present. PMID:17079110

  7. Increased salivary aldehyde dehydrogenase 1 in non-reticular oral lichen planus.

    PubMed

    Mansourian, Arash; Shanbehzadeh, Najmeh; Kia, Seyed Javad; Moosavi, Mahdieh-Sadat

    2017-01-01

    Oral lichen planus is a potentially malignant disorder. One of the malignant transformation markers is cancer stem cells. One of the proposed marker for the detection of cancer stem cells's in head and neck cancer is aldehyde dehydrogenase. Recently it is shown that aldehyde dehydrogenase 1 expression in tissue samples is associated with oral lichen planus malignant transformation. This study evaluates salivary aldehyde dehydrogenase 1 in oral lichen planus. Thirty patients and 30 age and sex-matched healthy volunteers were recruited. Oral lichen planus was diagnosed based on the modified World Health Organization criteria. Subjects in the case group were divided into reticular and non-reticular forms. Unstimulated salivary samples were collected at 10-12 AM. Saliva concentrations of aldehyde dehydrogenase 1 were measured by ELISA. The differences between aldehyde dehydrogenase levels in the oral lichen planus group compared with the control group were not significant but aldehyde dehydrogenase in non-reticular oral lichen planus was significantly higher than that of the reticular form. This is a cross-sectional study, thus longitudinal studies in oral lichen planus may present similar or different results. The mechanism of malignant transformation in oral lichen planus is not defined. Previous analyses revealed that the aldehyde dehydrogenase 1 expression is significantly correlated with increased risk of transformation. This finding is consistent with our results because in the erosive and ulcerative forms of oral lichen planus, which have an increased risk of transformation, salivary aldehyde dehydrogenase 1 was overexpressed. A higher salivary aldehyde dehydrogenase level in non-reticular oral lichen planus can be a defensive mechanism against higher oxidative stress in these groups. Aldehyde dehydrogenase may be one of the malignant transformation markers in oral lichen planus. Further studies are needed for introducing aldehyde dehydrogenase as a prognostic

  8. Increased salivary aldehyde dehydrogenase 1 in non-reticular oral lichen planus*

    PubMed Central

    Mansourian, Arash; Shanbehzadeh, Najmeh; Kia, Seyed Javad; Moosavi, Mahdieh-Sadat

    2017-01-01

    Background Oral lichen planus is a potentially malignant disorder. One of the malignant transformation markers is cancer stem cells. One of the proposed marker for the detection of cancer stem cells's in head and neck cancer is aldehyde dehydrogenase. Recently it is shown that aldehyde dehydrogenase 1 expression in tissue samples is associated with oral lichen planus malignant transformation. Objective This study evaluates salivary aldehyde dehydrogenase 1 in oral lichen planus. Method Thirty patients and 30 age and sex-matched healthy volunteers were recruited. Oral lichen planus was diagnosed based on the modified World Health Organization criteria. Subjects in the case group were divided into reticular and non-reticular forms. Unstimulated salivary samples were collected at 10-12 AM. Saliva concentrations of aldehyde dehydrogenase 1 were measured by ELISA. Results The differences between aldehyde dehydrogenase levels in the oral lichen planus group compared with the control group were not significant but aldehyde dehydrogenase in non-reticular oral lichen planus was significantly higher than that of the reticular form. Limitations of the study This is a cross-sectional study, thus longitudinal studies in oral lichen planus may present similar or different results. Conclusions The mechanism of malignant transformation in oral lichen planus is not defined. Previous analyses revealed that the aldehyde dehydrogenase 1 expression is significantly correlated with increased risk of transformation. This finding is consistent with our results because in the erosive and ulcerative forms of oral lichen planus, which have an increased risk of transformation, salivary aldehyde dehydrogenase 1 was overexpressed. A higher salivary aldehyde dehydrogenase level in non-reticular oral lichen planus can be a defensive mechanism against higher oxidative stress in these groups. Aldehyde dehydrogenase may be one of the malignant transformation markers in oral lichen planus. Further

  9. D-Lactate transport and metabolism in rat liver mitochondria.

    PubMed

    de Bari, Lidia; Atlante, Anna; Guaragnella, Nicoletta; Principato, Giovanni; Passarella, Salvatore

    2002-07-15

    In the present study we investigated whether isolated rat liver mitochondria can take up and metabolize D-lactate. We found the following: (1) externally added D-lactate causes oxygen uptake by mitochondria [P/O ratio (the ratio of mol of ATP synthesized to mol of oxygen atoms reduced to water during oxidative phosphorylation)=2] and membrane potential (Delta(psi)) generation in processes that are rotenone-insensitive, but inhibited by antimycin A and cyanide, and proton release from coupled mitochondria inhibited by alpha-cyanocinnamate, but not by phenylsuccinate; (2) the activity of the putative flavoprotein (D-lactate dehydrogenase) was detected in inside-out submitochondrial particles, but not in mitochondria and mitoplasts, as it is localized in the matrix phase of the mitochondrial inner membrane; (3) three novel separate translocators exist to mediate D-lactate traffic across the mitochondrial inner membrane: the D-lactate/H(+) symporter, which was investigated by measuring fluorimetrically the rate of endogenous flavin reduction, the D-lactate/oxoacid antiporter (which mediates both the D-lactate/pyruvate and D-lactate/oxaloacetate exchanges) and D-lactate/malate antiporter studied by monitoring photometrically the appearance of the D-lactate counteranions outside mitochondria. The D-lactate translocators, in the light of their different inhibition profiles separate from the monocarboxylate carrier, were found to differ from each other in the V(max) values and in the inhibition and pH profiles and were shown to regulate mitochondrial D-lactate metabolism in vitro. The D-lactate translocators and the D-lactate dehydrogenase could account for the removal of the toxic methylglyoxal from cytosol, as well as for D-lactate-dependent gluconeogenesis.

  10. 21 CFR 862.1440 - Lactate dehydrogenase test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... dehydrogenase measurements are used in the diagnosis and treatment of liver diseases such as acute viral hepatitis, cirrhosis, and metastatic carcinoma of the liver, cardiac diseases such as myocardial infarction...

  11. Estrogen-Related Receptor Alpha Modulates Lactate Dehydrogenase Activity in Thyroid Tumors

    PubMed Central

    Mirebeau-Prunier, Delphine; Le Pennec, Soazig; Jacques, Caroline; Fontaine, Jean-Fred; Gueguen, Naig; Boutet-Bouzamondo, Nathalie; Donnart, Audrey; Malthièry, Yves; Savagner, Frédérique

    2013-01-01

    Metabolic modifications of tumor cells are hallmarks of cancer. They exhibit an altered metabolism that allows them to sustain higher proliferation rates in hostile environment outside the cell. In thyroid tumors, the expression of the estrogen-related receptor α (ERRα), a major factor of metabolic adaptation, is closely related to the oxidative metabolism and the proliferative status of the cells. To elucidate the role played by ERRα in the glycolytic adaptation of tumor cells, we focused on the regulation of lactate dehydrogenases A and B (LDHA, LDHB) and the LDHA/LDHB ratio. Our study included tissue samples from 10 classical and 10 oncocytic variants of follicular thyroid tumors and 10 normal thyroid tissues, as well as samples from three human thyroid tumor cell lines: FTC-133, XTC.UC1 and RO82W-1. We identified multiple cis-acting promoter elements for ERRα, in both the LDHA and LDHB genes. The interaction between ERRα and LDH promoters was confirmed by chromatin immunoprecipitation assays and in vitro analysis for LDHB. Using knock-in and knock-out cellular models, we found an inverse correlation between ERRα expression and LDH activity. This suggests that thyroid tumor cells may reprogram their metabolic pathways through the up-regulation of ERRα by a process distinct from that proposed by the recently revisited Warburg hypothesis. PMID:23516535

  12. Secondary metabolites of Mirabilis jalapa structurally inhibit Lactate Dehydrogenase A in silico: a potential cancer treatment

    NASA Astrophysics Data System (ADS)

    Kusumawati, R.; Nasrullah, A. H.; Pesik, R. N.; Muthmainah; Indarto, D.

    2018-03-01

    Altered energy metabolism from phosphorylated oxidation to aerobic glycolysis is one of the cancer hallmarks. Lactate dehydrogenase A (LDHA) is a major enzyme that catalyses pyruvate to lactate in such condition. The aim of this study was to explore LDHA inhibitors derived from Indonesian herbal plants. In this study, LDHA and oxamate molecular structures were obtained from protein data bank. As a standard ligand inhibitor, oxamate was molecularly re-validated using Autodock Vina 1.1.2 software and showed binding energy -4.26 ± 0.006 kcal/mol and interacted with LDHA at Gln99, Arg105, Asn137, Arg168, His192, and Thr247 residues. Molecular docking was used to visualize interaction between Indonesian phytochemicals and LDHA. Indonesian phytochemicals with the lowest binding energy and similar residues with standard ligand was Miraxanthin-III (-8.53 ± 0.006 kcal/mol), Vulgaxanthin-I (-8.46 ± 0.006 kcal/mol), Miraxanthin-II (-7.9 ± 0.2 kcal/mol) and Miraxanthin-V (-7.96 ± kcal/mol). Lower energy binding to LDHA and binding site at these residues was predicted to inhibit LDHA activity better than standard ligand. All phytochemicals were found in Mirabilis jalapa plant. Secondary metabolites in Mirabilis jalapa have LDHA inhibitor property in silico. Further in vitro study should be performed to confirm this result.

  13. Regulation of the Activity of Lactate Dehydrogenases from Four Lactic Acid Bacteria*

    PubMed Central

    Feldman-Salit, Anna; Hering, Silvio; Messiha, Hanan L.; Veith, Nadine; Cojocaru, Vlad; Sieg, Antje; Westerhoff, Hans V.; Kreikemeyer, Bernd; Wade, Rebecca C.; Fiedler, Tomas

    2013-01-01

    Despite high similarity in sequence and catalytic properties, the l-lactate dehydrogenases (LDHs) in lactic acid bacteria (LAB) display differences in their regulation that may arise from their adaptation to different habitats. We combined experimental and computational approaches to investigate the effects of fructose 1,6-bisphosphate (FBP), phosphate (Pi), and ionic strength (NaCl concentration) on six LDHs from four LABs studied at pH 6 and pH 7. We found that 1) the extent of activation by FBP (Kact) differs. Lactobacillus plantarum LDH is not regulated by FBP, but the other LDHs are activated with increasing sensitivity in the following order: Enterococcus faecalis LDH2 ≤ Lactococcus lactis LDH2 < E. faecalis LDH1 < L. lactis LDH1 ≤ Streptococcus pyogenes LDH. This trend reflects the electrostatic properties in the allosteric binding site of the LDH enzymes. 2) For L. plantarum, S. pyogenes, and E. faecalis, the effects of Pi are distinguishable from the effect of changing ionic strength by adding NaCl. 3) Addition of Pi inhibits E. faecalis LDH2, whereas in the absence of FBP, Pi is an activator of S. pyogenes LDH, E. faecalis LDH1, and L. lactis LDH1 and LDH2 at pH 6. These effects can be interpreted by considering the computed binding affinities of Pi to the catalytic and allosteric binding sites of the enzymes modeled in protonation states corresponding to pH 6 and pH 7. Overall, the results show a subtle interplay among the effects of Pi, FBP, and pH that results in different regulatory effects on the LDHs of different LABs. PMID:23720742

  14. Lactate is oxidized outside of the mitochondrial matrix in rodent brain.

    PubMed

    Herbst, Eric A F; George, Mitchell A J; Brebner, Karen; Holloway, Graham P; Kane, Daniel A

    2018-05-01

    The nature and existence of mitochondrial lactate oxidation is debated in the literature. Obscuring the issue are disparate findings in isolated mitochondria, as well as relatively low rates of lactate oxidation observed in permeabilized muscle fibres. However, respiration with lactate has yet to be directly assessed in brain tissue with the mitochondrial reticulum intact. To determine if lactate is oxidized in the matrix of brain mitochondria, oxygen consumption was measured in saponin-permeabilized mouse brain cortex samples, and rat prefrontal cortex and hippocampus (dorsal) subregions. While respiration in the presence of ADP and malate increased with the addition of lactate, respiration was maximized following the addition of exogenous NAD + , suggesting maximal lactate metabolism involves extra-matrix lactate dehydrogenase. This was further supported when NAD + -dependent lactate oxidation was significantly decreased with the addition of either low-concentration α-cyano-4-hydroxycinnamate or UK-5099, inhibitors of mitochondrial pyruvate transport. Mitochondrial respiration was comparable between glutamate, pyruvate, and NAD + -dependent lactate oxidation. Results from the current study demonstrate that permeabilized brain is a feasible model for assessing lactate oxidation, and support the interpretation that lactate oxidation occurs outside the mitochondrial matrix in rodent brain.

  15. Radiation-induced enzyme efflux from rat heart: sedentary animals. [Gamma radiation, lactate dehydrogenase, creative kinase, glutamate oxaloacetate transaminase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    MacWilliam, L.D.; Bhakthan, N.M.G.

    1976-01-01

    Serum levels of lactate dehydrogenase, creatine kinase, and glutamate oxaloacetate transaminase show initial elevations within 12 hr of exposure to 2,000 rads of ..gamma..-radiation to the thoracic region of rats. Significant decreases in heart muscle homogenate levels of these enzymes parallel initial elevations in the serum and may suggest that enhanced leakage of enzymes is a consequence of radiation injury to heart muscle. Insignificant alterations in mitochondrial glutamate oxaloacetate transaminase levels after exposure indicate that in vivo injury to the mitochondria from therapeutic levels of ..gamma..-radiation is questionable. The results support the contention that ionizing radiation instigates alterations in themore » dynamic permeability of membranes, allowing leakage of biologically active material out of the injured cell.« less

  16. Two separate pathways for d-lactate oxidation by Saccharomyces cerevisiae mitochondria which differ in energy production and carrier involvement.

    PubMed

    Pallotta, Maria Luigia; Valenti, Daniela; Iacovino, Michelina; Passarella, Salvatore

    2004-02-15

    In this work we looked at whether and how mitochondria isolated from Saccharomyces cerevisiae (SCM) oxidize d-lactate. We found that: (1). externally added d-lactate causes oxygen uptake by SCM with P/O ratio equal to 1.5; in the presence of antimycin A (AA), P/O ratio was 1.8, differently in the presence of the non-penetrant alpha-cyanocinnamate (alpha-CCN-) no P/O ratio could be measured. Consistently, mitochondrial electrical membrane potential (deltapsi) generation was found, due to externally added d-lactate in the presence of antimycin A, but not of alpha-CCN-. (2). SCM oxidize d-lactate in two different manners: (i). via inner membrane d-lactate dehydrogenase which leads to d-lactate oxidation without driving deltapsi generation and ATP synthesis and (ii). via the matrix d-lactate dehydrogenase, which drives deltapsi generation and ATP synthesis by using taken up d-lactate. (3). Pyruvate newly synthesised in the mitochondrial matrix is exported via the novel d-lactate/pyruvate antiporter. d-Lactate/pyruvate antiport proved to regulate the rate of pyruvate efflux in vitro. (4). The existence of the d-lactate/H+ symporter is also proposed as shown by mitochondrial swelling. The d-lactate carriers and d-lactate dehydrogenases could account for the removal of the toxic methylglyoxal from cytosol, as well as for the d-lactate-dependent gluconeogenesis.

  17. CONVERSION OF LACTATE-C14 TO PROPIONATE BY THE RUMEN MICROFLORA12

    PubMed Central

    Baldwin, R. L.; Wood, W. A.; Emery, R. S.

    1962-01-01

    Baldwin, R. L. (Michigan State University, East Lansing), W. A. Wood, and R. S. Emery. Conversion of lactate-C14 to propionate by the rumen microflora. J. Bacteriol. 83:907–913. 1962.—Rumen microflora enriched on five different diets calculated to present increasing carbohydrate or lactate availability were used to determine the contribution of the randomizing (succinate) and nonrandomizing (acrylate) routes to propionate with lactate-2-C14 and -3-C14 as substrates. Propionate was labeled as though 70 to 90% was formed via the nonrandomizing route. This percentage was highest on diets containing high levels of carbohydrate or lactate or both. Evidence for the presence of succinic dehydrogenase, acetokinase, phosphotransacetylase, and coenzyme A transphorase was obtained with cell-free extracts. Propionate-2-C14 and lactate-2-C14 were converted by extracts to the activated derivatives of acrylate, lactate, propionate, and acetate. PMID:13864343

  18. Stilbene Glucoside, a Putative Sleep Promoting Constituent from Polygonum multiflorum Affects Sleep Homeostasis by Affecting the Activities of Lactate Dehydrogenase and Salivary Alpha Amylase.

    PubMed

    Wei, Qian; Ta, Guang; He, Wenjing; Wang, Wei; Wu, Qiucheng

    2017-01-01

    Chinese herbal medicine (CHM) has been used for treating insomnia for centuries. The most used CHM for insomnia was Polygonum multiflorum. However, the molecular mechanism for CHM preventing insomnia is unknown. Stilbene glucoside (THSG), an important active component of P. multiflorum, may play an important role for treating insomnia. To test the hypothesis, Kunming mice were treated with different dosages of THSG. To examine the sleep duration, a computer-controlled sleep-wake detection system was implemented. Electroencephalogram (EEG) and electromyogram (EMG) electrodes were implanted to determine sleep-wake state. RT-PCR and Western blot was used to measure the levels of lactate dehydrogenase (LDH) and saliva alpha amylase. Spearman's rank correlation coefficient was used to identify the strength of correlation between the variables. The results showed that THSG significantly prolonged the sleep time of the mice (p<0.01). THSG changed sleep profile by reducing wake and rapid eye movement (REM) period, and increasing non-REM period. RT-PCR and Western blot analysis showed that THSG could down-regulate the levels of LDH and saliva alpha amylase (p<0.05). The level of lactate and glucose was positively related with the activity of LDH and saliva alpha amylase (p<0.05), respectively. On the other hand, the activities of LDH and amylase were negatively associated with sleep duration (p<0.05). The levels of lactate and glucose affect sleep homeostasis. Thus, THSG may prevent insomnia by regulating sleep duration via LDH and salivary alpha amylase.

  19. Construction of Mutant Glucose Oxidases with Increased Dye-Mediated Dehydrogenase Activity

    PubMed Central

    Horaguchi, Yohei; Saito, Shoko; Kojima, Katsuhiro; Tsugawa, Wakako; Ferri, Stefano; Sode, Koji

    2012-01-01

    Mutagenesis studies on glucose oxidases (GOxs) were conducted to construct GOxs with reduced oxidase activity and increased dehydrogenase activity. We focused on two representative GOxs, of which crystal structures have already been reported—Penicillium amagasakiense GOx (PDB ID; 1gpe) and Aspergillus niger GOx (PDB ID; 1cf3). We constructed oxygen-interacting structural models for GOxs, and predicted the residues responsible for oxidative half reaction with oxygen on the basis of the crystal structure of cholesterol oxidase as well as on the fact that both enzymes are members of the glucose/methanol/choline (GMC) oxidoreductase family. Rational amino acid substitution resulted in the construction of an engineered GOx with drastically decreased oxidase activity and increased dehydrogenase activity, which was higher than that of the wild-type enzyme. As a result, the dehydrogenase/oxidase ratio of the engineered enzyme was more than 11-fold greater than that of the wild-type enzyme. These results indicate that alteration of the dehydrogenase/oxidase activity ratio of GOxs is possible by introducing a mutation into the putative functional residues responsible for oxidative half reaction with oxygen of these enzymes, resulting in a further increased dehydrogenase activity. This is the first study reporting the alteration of GOx electron acceptor preference from oxygen to an artificial electron acceptor. PMID:23203056

  20. Lactation induces increases in the RANK/RANKL/OPG system in maxillary bone.

    PubMed

    Macari, Soraia; Sharma, Lavanya A; Wyatt, Amanda; da Silva, Janine Maíra; Dias, George J; Silva, Tarcília A; Szawka, Raphael E; Grattan, David R

    2018-05-01

    The underlying causes of maxillary bone loss during lactation remain poorly understood. We evaluated the impact of lactation on physiological and mechanically-induced alveolar bone remodeling. Nulliparous non-lactating (N-LAC) and 21-day lactating (LAC) mice underwent mechanically-induced bone remodeling by orthodontic tooth movement (OTM). Micro-computed tomography (microCT) was performed in the maxilla, femur and vertebra. Tartrate-resistant-acid phosphatase (TRAP) and Masson's trichrome labelling was performed in the maxillary bone and gene expression was determined in the periodontal ligament. The effect of prolactin on osteoclast (OCL) and osteoblast (OBL) differentiation was also investigated in N-LAC and LAC mice. Lactation increased alveolar bone loss in the maxilla, femur and vertebra, while OTM was enhanced. The number of OCL and OBL was higher in the maxilla of LAC mice. OTM increased OCL in both groups; while OBL was increased only in N-LAC but not in LAC mice, in which cell numbers were already elevated. The alveolar bone loss during lactation was associated with increased expression of receptor activator of nuclear factor-KappaB (RANK), RANK ligand (RANKL), and osteoprotegerin (OPG) in the maxilla. OTM induced the same responses in N-LAC mice, whereas it had no further effect in LAC mice. Lactation enhanced differentiation of OCL and OBL from bone marrow cells, and prolactin recapitulated OCL differentiation in N-LAC mice. Thus, lactation increases physiological maxillary bone remodeling and OTM, and both require activation of RANK/RANKL/OPG system. These findings expand our knowledge of lactation-induced osteopenia and have possible impact on clinical practice regarding orthodontic treatments and dental implants in lactating women. Copyright © 2018 Elsevier Inc. All rights reserved.

  1. Regulator LdhR and d-Lactate Dehydrogenase LdhA of Burkholderia multivorans Play Roles in Carbon Overflow and in Planktonic Cellular Aggregate Formation.

    PubMed

    Silva, Inês N; Ramires, Marcelo J; Azevedo, Lisa A; Guerreiro, Ana R; Tavares, Andreia C; Becker, Jörg D; Moreira, Leonilde M

    2017-10-01

    LysR-type transcriptional regulators (LTTRs) are the most commonly found regulators in Burkholderia cepacia complex, comprising opportunistic pathogens causing chronic respiratory infections in cystic fibrosis (CF) patients. Despite LTTRs being global regulators of pathogenicity in several types of bacteria, few have been characterized in Burkholderia Here, we show that gene ldhR of B. multivorans encoding an LTTR is cotranscribed with ldhA encoding a d-lactate dehydrogenase and evaluate their implication in virulence traits such as exopolysaccharide (EPS) synthesis and biofilm formation. A comparison of the wild type (WT) and its isogenic Δ ldhR mutant grown in medium with 2% d-glucose revealed a negative impact on EPS biosynthesis and on cell viability in the presence of LdhR. The loss of viability in WT cells was caused by intracellular acidification as a consequence of the cumulative secretion of organic acids, including d-lactate, which was absent from the Δ ldhR mutant supernatant. Furthermore, LdhR is implicated in the formation of planktonic cellular aggregates. WT cell aggregates reached 1,000 μm in size after 24 h in liquid cultures, in contrast to Δ ldhR mutant aggregates that never grew more than 60 μm. The overexpression of d-lactate dehydrogenase LdhA in the Δ ldhR mutant partially restored the formed aggregate size, suggesting a role for fermentation inside aggregates. Similar results were obtained for surface-attached biofilms, with WT cells producing more biofilm. A systematic evaluation of planktonic aggregates in Burkholderia CF clinical isolates showed aggregates in 40 of 74. As CF patients' lung environments are microaerophilic and bacteria are found as free aggregates/biofilms, LdhR and LdhA might have central roles in adapting to this environment. IMPORTANCE Cystic fibrosis patients often suffer from chronic respiratory infections caused by several types of microorganisms. Among them are the Burkholderia cepacia complex bacteria, which

  2. Increased IMP dehydrogenase gene expression in solid tumor tissues and tumor cell lines

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Collart, F.R.; Chubb, C.B.; Mirkin, B.L.

    1992-07-10

    IMP dehydrogenase, a regulatory enzyme of guanine nucleotide biosynthesis, may play a role in cell proliferation and malignancy. To assess this possibility, we examined IMP dehydrogenase expression in a series of human solid tumor tissues and tumor cell lines in comparison with their normal counterparts. Increased IMP dehydrogenase gene expression was observed in brain tumors relative to normal brain tissue and in sarcoma cells relative to normal fibroblasts. Similarly, in several B- and T-lymphoid leukemia cell lines, elevated levels of IMP dehydrogenase mRNA and cellular enzyme were observed in comparison with the levels in peripheral blood lymphocytes. These results aremore » consistent with an association between increased IMP dehydrogenase expression and either enhanced cell proliferation or malignant transformation.« less

  3. Variants of glycerol dehydrogenase having D-lactate dehydrogenase activity and uses thereof

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wang, Qingzhao; Shanmugam, Keelnatham T.; Ingram, Lonnie O'Neal

    The present invention provides methods of designing and generating glycerol dehydrogenase (GlyDH) variants that have altered function as compared to a parent polypeptide. The present invention further provides nucleic acids encoding GlyDH polypeptide variants having altered function as compared to the parent polypeptide. Host cells comprising polynucleotides encoding GlyDH variants and methods of producing lactic acids are also provided in various aspects of the invention.

  4. Falsely increased plasma lactate concentration due to ethylene glycol poisoning in 2 dogs.

    PubMed

    Hopper, Kate; Epstein, Steven E

    2013-01-01

    To describe false increases in plasma lactate concentration measured on point-of-care analyzers in 2 dogs with ethylene glycol (EG) intoxication. Two dogs presenting with EG intoxication had extreme increases of plasma lactate concentrations recorded on a point-of-care machine. Laboratory analysis by spectrophotometry of lactate concentration determined these lactate measurements to be erroneous. False increases in plasma lactate concentration were demonstrated in 2 out of 3 point-of-care machines tested. Glycolate, a toxic metabolite of EG, can interfere with the measurement of plasma lactate by some analyzers and this may delay the correct diagnosis of EG toxicity if not recognized. © Veterinary Emergency and Critical Care Society 2012.

  5. Cytochemical Localization of Glycolate Dehydrogenase in Mitochondria of Chlamydomonas1

    PubMed Central

    Beezley, Belinda B.; Gruber, Peter J.; Frederick, Sue Ellen

    1976-01-01

    Mildly disrupted cells of Chlamydomonas reinhardi Dangeard were incubated in a reaction medium containing glycolate, ferricyanide, and cupric ions, and then processed for electron microscopy. As a result of the cytochemical treatment, an electron opaque product was deposited specifically in the outer compartment of mitochondria; other cellular components, including microbodies, did not accumulate stain. Incubation with d-lactate yielded similar results, while treatment with l-lactate produced only a weak reaction. Oxamate, which inhibits glycolate dehydrogenase activity in cell-free extracts, also inhibited the cytochemical reaction. These findings demonstrate in situ that glycolate dehydrogenase is localized in mitochondria, and thus corroborate similar conclusions reached on the basis of enzymic studies of isolated algal organelles. Images PMID:16659670

  6. Prognostic significance of serum lactate dehydrogenase levels in Ewing's sarcoma: A meta-analysis.

    PubMed

    Li, Suoyuan; Yang, Qing; Wang, Hongsheng; Wang, Zhuoying; Zuo, Dongqing; Cai, Zhengdong; Hua, Yingqi

    2016-12-01

    A number of studies have investigated the role of serum lactate dehydrogenase (LDH) levels in patients with Ewing's sarcoma, although these have yielded inconsistent and inconclusive results. Therefore, the present study aimed to systematically review the published studies and conduct a meta-analysis to assess its prognostic value more precisely. Cohort studies assessing the prognostic role of LDH levels in patients with Ewing's sarcoma were included. A pooled hazard ratio (HR) with 95% confidence intervals (CIs) of overall survival (OS) or 5-year disease-free survival (DFS) was used to assess the prognostic role of the levels of serum LDH. Nine studies published between 1980 and 2014, with a total of 1,412 patients with Ewing's sarcoma, were included. Six studies, with a total of 644 patients, used OS as the primary endpoint and four studies, with 795 patients, used 5-year DFS. Overall, the pooled HR evaluating high LDH levels was 2.90 (95% CI: 2.09-4.04) for OS and 2.40 (95% CI: 1.93-2.98) for 5-year DFS. This meta-analysis demonstrates that high levels of serum LDH are associated with lower OS and 5-year DFS rates in patients with Ewing's sarcoma. Therefore, serum LDH levels are an effective biomarker of Ewing's sarcoma prognosis.

  7. Lactate shuttles in nature.

    PubMed

    Brooks, G A

    2002-04-01

    Once thought to be the consequence of oxygen lack in contracting skeletal muscle, the glycolytic product lactate is formed and utilized continuously under fully aerobic conditions. "Cell-cell" and "intracellular lactate shuttle" concepts describe the roles of lactate in the delivery of oxidative and gluconeogenic substrates, as well as in cell signalling. Examples of cell-cell shuttles include lactate exchanges between white-glycolytic and red-oxidative fibres within a working muscle bed, between working skeletal muscle and heart, and between tissues of net lactate release and gluconeogenesis. Lactate exchange between astrocytes and neurons that is linked to glutamatergic signalling in the brain is an example of a lactate shuttle supporting cell-cell signalling. Lactate uptake by mitochondria and pyruvate-lactate exchange in peroxisomes are examples of intracellular lactate shuttles. Lactate exchange between sites of production and removal is facilitated by monocarboxylate transport proteins, of which there are several isoforms, and, probably, also by scaffolding proteins. The mitochondrial lactate-pyruvate transporter appears to work in conjunction with mitochondrial lactate dehydrogenase, which permits lactate to be oxidized within actively respiring cells. Hence mitochondria function to establish the concentration and proton gradients necessary for cells with high mitochondrial densities (e.g. cardiocytes) to take up and oxidize lactate. Arteriovenous difference measurements on working cardiac and skeletal muscle beds as well as NMR spectral analyses of these tissues show that lactate is formed and oxidized within the cells of formation in vivo. Glycolysis and lactate oxidation within cells permits high flux rates and the maintenance of redox balance in the cytosol and mitochondria. Other examples of intracellular lactate shuttles include lactate uptake and oxidation in sperm mitochondria and the facilitation of beta-oxidation in peroxisomes by pyruvate-lactate

  8. Stability and activity of lactate dehydrogenase on biofunctional layers deposited by activated vapor silanization (AVS) and immersion silanization (IS)

    NASA Astrophysics Data System (ADS)

    Calvo, Jorge Nieto-Márquez; Elices, Manuel; Guinea, Gustavo V.; Pérez-Rigueiro, José; Arroyo-Hernández, María

    2017-09-01

    The interaction between surfaces and biological elements, in particular, proteins is critical for the performance of biomaterials and biosensors. This interaction can be controlled by modifying the surface in a process known as biofunctionalization. In this work, the enzyme lactate dehydrogenase (LDH) is used to study the stability of the interaction between a functional protein and amine-functionalized surfaces. Two different functionalization procedures were compared: Activated Vapor Silanization (AVS) and Immersion Silanization (IS). Adsorption kinetics is shown to follow the Langmuir model for AVS-functionalized samples, while IS-functionalized samples show a certain instability if immersed in an aqueous medium for several hours. In turn, the enzymatic activity of LDH is preserved for longer times by using glutaraldehyde as crosslinker between the AVS biofunctional surface and the enzyme.

  9. Enhancing the light-driven production of D-lactate by engineering cyanobacterium using a combinational strategy

    NASA Astrophysics Data System (ADS)

    Li, Chao; Tao, Fei; Ni, Jun; Wang, Yu; Yao, Feng; Xu, Ping

    2015-05-01

    It is increasingly attractive to engineer cyanobacteria for bulk production of chemicals from CO2. However, cofactor bias of cyanobacteria is different from bacteria that prefer NADH, which hampers cyanobacterial strain engineering. In this study, the key enzyme D-lactate dehydrogenase (LdhD) from Lactobacillus bulgaricus ATCC11842 was engineered to reverse its favored cofactor from NADH to NADPH. Then, the engineered enzyme was introduced into Synechococcus elongatus PCC7942 to construct an efficient light-driven system that produces D-lactic acid from CO2. Mutation of LdhD drove a fundamental shift in cofactor preference towards NADPH, and increased D-lactate productivity by over 3.6-fold. We further demonstrated that introduction of a lactic acid transporter and bubbling CO2-enriched air also enhanced D-lactate productivity. Using this combinational strategy, increased D-lactate concentration and productivity were achieved. The present strategy may also be used to engineer cyanobacteria for producing other useful chemicals.

  10. Trimethylamine-N-oxide counteracts urea effects on rabbit muscle lactate dehydrogenase function: a test of the counteraction hypothesis.

    PubMed Central

    Baskakov, I; Wang, A; Bolen, D W

    1998-01-01

    Trimethylamine-N-oxide (TMAO) in the cells of sharks and rays is believed to counteract the deleterious effects of the high intracellular concentrations of urea in these animals. It has been hypothesized that TMAO has the generic ability to counteract the effects of urea on protein structure and function, regardless of whether that protein actually evolved in the presence of these two solutes. Rabbit muscle lactate dehydrogenase (LDH) did not evolve in the presence of either solute, and it is used here to test the validity of the counteraction hypothesis. With pyruvate as substrate, results show that its Km and the combined Km of pyruvate and NADH are increased by urea, decreased by TMAO, and in 1:1 and 2:1 mixtures of urea:TMAO the Km values are essentially equivalent to the Km values obtained in the absence of the two solutes. In contrast, values of k(cat) and the Km for NADH as a substrate are unperturbed by urea, TMAO, or urea:TMAO mixtures. All of these effects are consistent with TMAO counteraction of the effects of urea on LDH kinetic parameters, supporting the premise that counteraction is a property of the solvent system and is independent of the evolutionary history of the protein. PMID:9591690

  11. Contributory roles of two l-lactate dehydrogenases for l-lactic acid production in thermotolerant Bacillus coagulans.

    PubMed

    Sun, Lifan; Zhang, Caili; Lyu, Pengcheng; Wang, Yanping; Wang, Limin; Yu, Bo

    2016-11-25

    Thermotolerant Bacillus coagulans is considered to be a more promising producer for bio-chemicals, due to its capacity to withstand harsh conditions. Two L-lactate dehydrogenase (LDH) encoding genes (ldhL1 and ldhL2) and one D-LDH encoding gene (ldhD) were annotated from the B. coagulans DSM1 genome. Transcriptional analysis revealed that the expression of ldhL2 was undetectable while the ldhL1 transcription level was much higher than that of ldhD at all growth phases. Deletion of the ldhL2 gene revealed no difference in fermentation profile compared to the wild-type strain, while ldhL1 single deletion or ldhL1ldhL2 double deletion completely blocked L-lactic acid production. Complementation of ldhL1 in the above knockout strains restored fermentation profiles to those observed in the wild-type strain. This study demonstrates ldhL1 is crucial for L-lactic acid production and NADH balance in B. coagulans DSM1 and lays the fundamental for engineering the thermotolerant B. coagulans strain as a platform chemicals producer.

  12. Plasma Lactate Dehydrogenase Levels Predict Mortality in Acute Aortic Syndromes

    PubMed Central

    Morello, Fulvio; Ravetti, Anna; Nazerian, Peiman; Liedl, Giovanni; Veglio, Maria Grazia; Battista, Stefania; Vanni, Simone; Pivetta, Emanuele; Montrucchio, Giuseppe; Mengozzi, Giulio; Rinaldi, Mauro; Moiraghi, Corrado; Lupia, Enrico

    2016-01-01

    Abstract In acute aortic syndromes (AAS), organ malperfusion represents a key event impacting both on diagnosis and outcome. Increased levels of plasma lactate dehydrogenase (LDH), a biomarker of malperfusion, have been reported in AAS, but the performance of LDH for the diagnosis of AAS and the relation of LDH with outcome in AAS have not been evaluated so far. This was a bi-centric prospective diagnostic accuracy study and a cohort outcome study. From 2008 to 2014, patients from 2 Emergency Departments suspected of having AAS underwent LDH assay at presentation. A final diagnosis was obtained by aortic imaging. Patients diagnosed with AAS were followed-up for in-hospital mortality. One thousand five hundred seventy-eight consecutive patients were clinically eligible, and 999 patients were included in the study. The final diagnosis was AAS in 201 (20.1%) patients. Median LDH was 424 U/L (interquartile range [IQR] 367–557) in patients with AAS and 383 U/L (IQR 331–460) in patients with alternative diagnoses (P < 0.001). Using a cutoff of 450 U/L, the sensitivity of LDH for AAS was 44% (95% confidence interval [CI] 37–51) and the specificity was 73% (95% CI 69–76). Overall in-hospital mortality for AAS was 23.8%. Mortality was 32.6% in patients with LDH ≥ 450 U/L and 16.8% in patients with LDH < 450 U/L (P = 0.006). Following stratification according to LDH quartiles, in-hospital mortality was 12% in the first (lowest) quartile, 18.4% in the second quartile, 23.5% in the third quartile, and 38% in the fourth (highest) quartile (P = 0.01). LDH ≥ 450 U/L was further identified as an independent predictor of death in AAS both in univariate and in stepwise logistic regression analyses (odds ratio 2.28, 95% CI 1.11–4.66; P = 0.025), in addition to well-established risk markers such as advanced age and hypotension. Subgroup analysis showed excess mortality in association with LDH ≥ 450 U/L in elderly, hemodynamically stable

  13. Increased Ventricular Cerebrospinal Fluid Lactate in Depressed Adolescents

    PubMed Central

    Bradley, Kailyn A. L.; Mao, Xiangling; Case, Julia A. C.; Kang, Guoxin; Shungu, Dikoma C.; Gabbay, Vilma

    2016-01-01

    Background Mitochondrial dysfunction has been increasingly examined as a potential pathogenic event in psychiatric disorders, although its role early in the course of major depressive disorder (MDD) is unclear. Therefore, the purpose of this study was to investigate mitochondrial dysfunction in medication-free adolescents with MDD through in vivo measurements of neurometabolites using high-spatial resolution multislice/multivoxel proton magnetic resonance spectroscopy. Methods Twenty-three adolescents with MDD and 29 healthy controls, ages 12–20, were scanned at 3T and concentrations of ventricular cerebrospinal fluid lactate, as well as N-acetyl-aspartate (NAA), total creatine (tCr), and total choline (tCho) in the bilateral caudate, putamen, and thalamus were reported. Results Adolescents with MDD exhibited increased ventricular lactate compared to healthy controls [F(1, 41) = 6.98, p = .01]. However, there were no group differences in the other neurometabolites. Dimensional analyses in the depressed group showed no relation between any of the neurometabolites and symptomatology, including anhedonia and fatigue. Conclusions Increased ventricular lactate in depressed adolescents suggests mitochondrial dysfunction may be present early in the course of MDD; however it is still not known whether the presence of mitochondrial dysfunction is a trait vulnerability of individuals predisposed to psychopathology or a state feature of the disorder. Therefore, there is a need for larger multimodal studies to clarify these chemical findings in the context of network function. PMID:26802978

  14. Minimizing the effects of oxygen interference on l-lactate sensors by a single amino acid mutation in Aerococcus viridansl-lactate oxidase.

    PubMed

    Hiraka, Kentaro; Kojima, Katsuhiro; Lin, Chi-En; Tsugawa, Wakako; Asano, Ryutaro; La Belle, Jeffrey T; Sode, Koji

    2018-04-30

    l-lactate biosensors employing l-lactate oxidase (LOx) have been developed mainly to measure l-lactate concentration for clinical diagnostics, sports medicine, and the food industry. Some l-lactate biosensors employ artificial electron mediators, but these can negatively impact the detection of l-lactate by competing with the primary electron acceptor: molecular oxygen. In this paper, a strategic approach to engineering an AvLOx that minimizes the effects of oxygen interference on sensor strips was reported. First, we predicted an oxygen access pathway in Aerococcus viridans LOx (AvLOx) based on its crystal structure. This was subsequently blocked by a bulky amino acid substitution. The resulting Ala96Leu mutant showed a drastic reduction in oxidase activity using molecular oxygen as the electron acceptor and a small increase in dehydrogenase activity employing an artificial electron acceptor. Secondly, the Ala96Leu mutant was immobilized on a screen-printed carbon electrode using glutaraldehyde cross-linking method. Amperometric analysis was performed with potassium ferricyanide as an electron mediator under argon or atmospheric conditions. Under argon condition, the response current increased linearly from 0.05 to 0.5mM l-lactate for both wild-type and Ala96Leu. However, under atmospheric conditions, the response of wild-type AvLOx electrode was suppressed by 9-12% due to oxygen interference. The Ala96Leu mutant maintained 56-69% of the response current at the same l-lactate level and minimized the relative bias error to -19% from -49% of wild-type. This study provided significant insight into the enzymatic reaction mechanism of AvLOx and presented a novel approach to minimize oxygen interference in sensor applications, which will enable accurate detection of l-lactate concentrations. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Increased plasma leptin attenuates adaptive metabolism in early lactating dairy cows.

    PubMed

    Ehrhardt, Richard A; Foskolos, Andreas; Giesy, Sarah L; Wesolowski, Stephanie R; Krumm, Christopher S; Butler, W Ronald; Quirk, Susan M; Waldron, Matthew R; Boisclair, Yves R

    2016-05-01

    Mammals meet the increased nutritional demands of lactation through a combination of increased feed intake and a collection of adaptations known as adaptive metabolism (e.g., glucose sparing via insulin resistance, mobilization of endogenous reserves, and increased metabolic efficiency via reduced thyroid hormones). In the modern dairy cow, adaptive metabolism predominates over increased feed intake at the onset of lactation and develops concurrently with a reduction in plasma leptin. To address the role of leptin in the adaptive metabolism of early lactation, we asked which adaptations could be countered by a constant 96-h intravenous infusion of human leptin (hLeptin) starting on day 8 of lactation. Compared to saline infusion (Control), hLeptin did not alter energy intake or milk energy output but caused a modest increase in body weight loss. hLeptin reduced plasma glucose by 9% and hepatic glycogen content by 73%, and these effects were associated with a 17% increase in glucose disposal during an insulin tolerance test. hLeptin attenuated the accumulation of triglyceride in the liver by 28% in the absence of effects on plasma levels of the anti-lipolytic hormone insulin or plasma levels of free fatty acids, a marker of lipid mobilization from adipose tissue. Finally, hLeptin increased the plasma concentrations of T4 and T3 by nearly 50% without affecting other neurally regulated hormones (i.e., cortisol and luteinizing hormone (LH)). Overall these data implicate the periparturient reduction in plasma leptin as one of the signals promoting conservation of glucose and energy at the onset of lactation in the energy-deficient dairy cow. © 2016 Society for Endocrinology.

  16. The bifunctional alcohol and aldehyde dehydrogenase gene, adhE, is necessary for ethanol production in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum.

    PubMed

    Lo, Jonathan; Zheng, Tianyong; Hon, Shuen; Olson, Daniel G; Lynd, Lee R

    2015-04-01

    Thermoanaerobacterium saccharolyticum and Clostridium thermocellum are anaerobic thermophilic bacteria being investigated for their ability to produce biofuels from plant biomass. The bifunctional alcohol and aldehyde dehydrogenase gene, adhE, is present in these bacteria and has been known to be important for ethanol formation in other anaerobic alcohol producers. This study explores the inactivation of the adhE gene in C. thermocellum and T. saccharolyticum. Deletion of adhE reduced ethanol production by >95% in both T. saccharolyticum and C. thermocellum, confirming that adhE is necessary for ethanol formation in both organisms. In both adhE deletion strains, fermentation products shifted from ethanol to lactate production and resulted in lower cell density and longer time to reach maximal cell density. In T. saccharolyticum, the adhE deletion strain lost >85% of alcohol dehydrogenase (ADH) activity. Aldehyde dehydrogenase (ALDH) activity did not appear to be affected, although ALDH activity was low in cell extracts. Adding ubiquinone-0 to the ALDH assay increased activity in the T. saccharolyticum parent strain but did not increase activity in the adhE deletion strain, suggesting that ALDH activity was inhibited. In C. thermocellum, the adhE deletion strain lost >90% of ALDH and ADH activity in cell extracts. The C. thermocellum adhE deletion strain contained a point mutation in the lactate dehydrogenase gene, which appears to deregulate its activation by fructose 1,6-bisphosphate, leading to constitutive activation of lactate dehydrogenase. Thermoanaerobacterium saccharolyticum and Clostridium thermocellum are bacteria that have been investigated for their ability to produce biofuels from plant biomass. They have been engineered to produce higher yields of ethanol, yet questions remain about the enzymes responsible for ethanol formation in these bacteria. The genomes of these bacteria encode multiple predicted aldehyde and alcohol dehydrogenases which could be

  17. Engineering of Pyranose Dehydrogenase for Increased Oxygen Reactivity

    PubMed Central

    Krondorfer, Iris; Lipp, Katharina; Brugger, Dagmar; Staudigl, Petra; Sygmund, Christoph; Haltrich, Dietmar; Peterbauer, Clemens K.

    2014-01-01

    Pyranose dehydrogenase (PDH), a member of the GMC family of flavoproteins, shows a very broad sugar substrate specificity but is limited to a narrow range of electron acceptors and reacts extremely slowly with dioxygen as acceptor. The use of substituted quinones or (organo)metals as electron acceptors is undesirable for many production processes, especially of food ingredients. To improve the oxygen reactivity, site-saturation mutagenesis libraries of twelve amino acids around the active site of Agaricus meleagris PDH were expressed in Saccharomyces cerevisiae. We established high-throughput screening assays for oxygen reactivity and standard dehydrogenase activity using an indirect Amplex Red/horseradish peroxidase and a DCIP/D-glucose based approach. The low number of active clones confirmed the catalytic role of H512 and H556. Only one position was found to display increased oxygen reactivity. Histidine 103, carrying the covalently linked FAD cofactor in the wild-type, was substituted by tyrosine, phenylalanine, tryptophan and methionine. Variant H103Y was produced in Pichia pastoris and characterized and revealed a five-fold increase of the oxygen reactivity. PMID:24614932

  18. Metabolic engineering of Methanosarcina acetivorans for lactate production from methane.

    PubMed

    McAnulty, Michael J; Poosarla, Venkata Giridhar; Li, Jine; Soo, Valerie W C; Zhu, Fayin; Wood, Thomas K

    2017-04-01

    We previously demonstrated anaerobic conversion of the greenhouse gas methane into acetate using an engineered archaeon that produces methyl-coenzyme M reductase (Mcr) from unculturable microorganisms from a microbial mat in the Black Sea to create the first culturable prokaryote that reverses methanogenesis and grows anaerobically on methane. In this work, we further engineered the same host with the goal of converting methane into butanol. Instead, we discovered a process for converting methane to a secreted valuable product, L-lactate, with sufficient optical purity for synthesizing the biodegradable plastic poly-lactic acid. We determined that the 3-hydroxybutyryl-CoA dehydrogenase (Hbd) from Clostridium acetobutylicum is responsible for lactate production. This work demonstrates the first metabolic engineering of a methanogen with a synthetic pathway; in effect, we produce a novel product (lactate) from a novel substrate (methane) by cloning the three genes for Mcr and one for Hbd. We further demonstrate the utility of anaerobic methane conversion with an increased lactate yield compared to aerobic methane conversion to lactate. Biotechnol. Bioeng. 2017;114: 852-861. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  19. Induction of triglyceride accumulation and mitochondrial maintenance in muscle cells by lactate

    PubMed Central

    Sun, Jingquan; Ye, Xin; Xie, Minhao; Ye, Jianping

    2016-01-01

    Muscle exercise induces intramuscular triglyceride (TG) accumulation and promotes mitochondrial maintenance in myotubes. However, the mechanism underlying exercise effects remains unknown. In this study, lactic acid was tested as a signaling molecule in C2C12 myotubes to understand the mechanism. Intracellular TG storage was induced in the cells by sodium lactate. The lactate activity was observed with an inhibition of the cAMP-PKA pathway as indicated by a reduction in the phosphorylation status of CREB (pCREB). Induction of pCREB signal by forskolin was blocked by pretreatment of cells with lactate. The impact of lactate on mitochondrial function was examined with a focus on the activities of two enzymes, MCAT (malonylCoA:ACP transferase) and PDH (pyruvate dehydrogenase). The enzyme activities were induced in the cells by lactate. Expression of the lactate receptor (GPR81) and lactate transporters (MCT1/4) were induced as well by lactate. The lactate activities were observed at concentrations between 4–64 mM, and were not dependent on the increase in intracellular pyruvate. Pyruvate treatment did not generate the same effects in the cells. Those results suggest that lactate may induce intramuscular TG storage and mitochondrial maintenance in myotubes through inhibition of the cAMP pathway by activation of GPR81 in a positive feedback manner. PMID:27645401

  20. Efficient reduction of the formation of by-products and improvement of production yield of 2,3-butanediol by a combined deletion of alcohol dehydrogenase, acetate kinase-phosphotransacetylase, and lactate dehydrogenase genes in metabolically engineered Klebsiella oxytoca in mineral salts medium.

    PubMed

    Jantama, Kaemwich; Polyiam, Pattharasedthi; Khunnonkwao, Panwana; Chan, Sitha; Sangproo, Maytawadee; Khor, Kirin; Jantama, Sirima Suvarnakuta; Kanchanatawee, Sunthorn

    2015-07-01

    Klebsiella oxytoca KMS005 (∆adhE∆ackA-pta∆ldhA) was metabolically engineered to improve 2,3-butanediol (BDO) yield. Elimination of alcohol dehydrogenase E (adhE), acetate kinase A-phosphotransacetylase (ackA-pta), and lactate dehydrogenase A (ldhA) enzymes allowed BDO production as a primary pathway for NADH re-oxidation, and significantly reduced by-products. KMS005 was screened for the efficient glucose utilization by metabolic evolution. KMS005-73T improved BDO production at a concentration of 23.5±0.5 g/L with yield of 0.46±0.02 g/g in mineral salts medium containing 50 g/L glucose in a shake flask. KMS005-73T also exhibited BDO yields of about 0.40-0.42 g/g from sugarcane molasses, cassava starch, and maltodextrin. During fed-batch fermentation, KMS005-73T produced BDO at a concentration, yield, and overall and specific productivities of 117.4±4.5 g/L, 0.49±0.02 g/g, 1.20±0.05 g/Lh, and 27.2±1.1 g/gCDW, respectively. No acetoin, lactate, and formate were detected, and only trace amounts of acetate and ethanol were formed. The strain also produced the least by-products and the highest BDO yield among other Klebsiella strains previously developed. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  1. Carbon Flux Trapping: Highly Efficient Production of Polymer-Grade d-Lactic Acid with a Thermophilic d-Lactate Dehydrogenase.

    PubMed

    Li, Chao; Tao, Fei; Xu, Ping

    2016-08-17

    High production of polymer-grade d-lactic acid is urgently required, particularly for the synthesis of polylactic acid. High-temperature fermentation has multiple advantages, such as lower equipment requirement and energy consumption, which are essential for lowering operating costs. We identified and introduced a unique d-lactate dehydrogenase into a thermotolerant butane-2,3-diol-producing strain. Carbon flux "trapping" was achieved by a "trapping point" created by combination of the introduced enzyme and the host efflux pump, which afforded irreversible transport of d-lactic acid. The overall carbon flux of the engineered strain was significantly enhanced and was redistributed predominantly to d-lactic acid. Under optimized conditions at 50 °C, d-lactic acid reached the highest titer (226.6 g L(-1) ) reported to date. This discovery allows us to extend the carbon flux trapping strategy to engineering complex metabolic networks. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Global sequence diversity of the lactate dehydrogenase gene in Plasmodium falciparum.

    PubMed

    Simpalipan, Phumin; Pattaradilokrat, Sittiporn; Harnyuttanakorn, Pongchai

    2018-01-09

    Antigen-detecting rapid diagnostic tests (RDTs) have been recommended by the World Health Organization for use in remote areas to improve malaria case management. Lactate dehydrogenase (LDH) of Plasmodium falciparum is one of the main parasite antigens employed by various commercial RDTs. It has been hypothesized that the poor detection of LDH-based RDTs is attributed in part to the sequence diversity of the gene. To test this, the present study aimed to investigate the genetic diversity of the P. falciparum ldh gene in Thailand and to construct the map of LDH sequence diversity in P. falciparum populations worldwide. The ldh gene was sequenced for 50 P. falciparum isolates in Thailand and compared with hundreds of sequences from P. falciparum populations worldwide. Several indices of molecular variation were calculated, including the proportion of polymorphic sites, the average nucleotide diversity index (π), and the haplotype diversity index (H). Tests of positive selection and neutrality tests were performed to determine signatures of natural selection on the gene. Mean genetic distance within and between species of Plasmodium ldh was analysed to infer evolutionary relationships. Nucleotide sequences of P. falciparum ldh could be classified into 9 alleles, encoding 5 isoforms of LDH. L1a was the most common allelic type and was distributed in P. falciparum populations worldwide. Plasmodium falciparum ldh sequences were highly conserved, with haplotype and nucleotide diversity values of 0.203 and 0.0004, respectively. The extremely low genetic diversity was maintained by purifying selection, likely due to functional constraints. Phylogenetic analysis inferred the close genetic relationship of P. falciparum to malaria parasites of great apes, rather than to other human malaria parasites. This study revealed the global genetic variation of the ldh gene in P. falciparum, providing knowledge for improving detection of LDH-based RDTs and supporting the candidacy of

  3. Expression, purification, crystallization and preliminary X-ray crystallographic analysis of L-lactate dehydrogenase and its H171C mutant from Bacillus subtilis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhang, Yanfeng; Gao, Xiaoli

    2012-08-31

    L-Lactate dehydrogenase (LDH) is an important enzyme involved in the last step of glycolysis that catalyzes the reversible conversion of pyruvate to L-lactate with the simultaneous oxidation of NADH to NAD{sup +}. In this study, wild-type LDH from Bacillus subtilis (BsLDH-WT) and the H171C mutant (BsLDH-H171C) were expressed in Escherichia coli and purified to near-homogeneity. BsLDH-WT was crystallized in the presence of fructose 1,6-bisphosphate (FBP) and NAD{sup +} and the crystal diffracted to 2.38 {angstrom} resolution. The crystal belonged to space group P3, with unit-cell parameters a = b = 171.04, c = 96.27 {angstrom}. BsLDH-H171C was also crystallized asmore » the apoenzyme and in complex with NAD{sup +}, and data sets were collected to 2.20 and 2.49 {angstrom} resolution, respectively. Both BsLDH-H171C crystals belonged to space group P3, with unit-cell parameters a = b = 133.41, c = 99.34 {angstrom} and a = b = 133.43, c = 99.09 {angstrom}, respectively. Tetramers were observed in the asymmetric units of all three crystals.« less

  4. Blocking Lactate Export by Inhibiting the Myc Target MCT1 Disables Glycolysis and Glutathione Synthesis

    PubMed Central

    Doherty, Joanne R.; Yang, Chunying; Scott, Kristen E. N.; Cameron, Michael D.; Fallahi, Mohammad; Li, Weimin; Hall, Mark A.; Amelio, Antonio L.; Mishra, Jitendra K.; Li, Fangzheng; Tortosa, Mariola; Genau, Heide Marika; Rounbehler, Robert J.; Lu, Yunqi; Dang, Chi. V.; Kumar, K. Ganesh; Butler, Andrew A.; Bannister, Thomas D.; Hooper, Andrea T.; Unsal-Kacmaz, Keziban; Roush, William R.; Cleveland, John L.

    2014-01-01

    Myc oncoproteins induce genes driving aerobic glycolysis, including lactate dehydrogenase-A that generates lactate. Here we report that Myc controls transcription of the lactate transporter SLC16A1/MCT1, and that elevated MCT1 levels are manifest in premalignant and neoplastic Eμ-Myc transgenic B cells and in human malignancies with MYC or MYCN involvement. Notably, disrupting MCT1 function leads to an accumulation of intracellular lactate that rapidly disables tumor cell growth and glycolysis, provoking marked alterations in glycolytic intermediates, and reductions in glucose transport, and in levels of ATP, NADPH and glutathione. Reductions in glutathione then lead to increases in hydrogen peroxide, mitochondrial damage and, ultimately, cell death. Finally, forcing glycolysis by metformin treatment augments this response and the efficacy of MCT1 inhibitors, suggesting an attractive combination therapy for MYC/MCT1-expressing malignancies. PMID:24285728

  5. Blocking lactate export by inhibiting the Myc target MCT1 Disables glycolysis and glutathione synthesis.

    PubMed

    Doherty, Joanne R; Yang, Chunying; Scott, Kristen E N; Cameron, Michael D; Fallahi, Mohammad; Li, Weimin; Hall, Mark A; Amelio, Antonio L; Mishra, Jitendra K; Li, Fangzheng; Tortosa, Mariola; Genau, Heide Marika; Rounbehler, Robert J; Lu, Yunqi; Dang, Chi V; Kumar, K Ganesh; Butler, Andrew A; Bannister, Thomas D; Hooper, Andrea T; Unsal-Kacmaz, Keziban; Roush, William R; Cleveland, John L

    2014-02-01

    Myc oncoproteins induce genes driving aerobic glycolysis, including lactate dehydrogenase-A that generates lactate. Here, we report that Myc controls transcription of the lactate transporter SLC16A1/MCT1 and that elevated MCT1 levels are manifest in premalignant and neoplastic Eμ-Myc transgenic B cells and in human malignancies with MYC or MYCN involvement. Notably, disrupting MCT1 function leads to an accumulation of intracellular lactate that rapidly disables tumor cell growth and glycolysis, provoking marked alterations in glycolytic intermediates, reductions in glucose transport, and in levels of ATP, NADPH, and ultimately, glutathione (GSH). Reductions in GSH then lead to increases in hydrogen peroxide, mitochondrial damage, and ultimately, cell death. Finally, forcing glycolysis by metformin treatment augments this response and the efficacy of MCT1 inhibitors, suggesting an attractive combination therapy for MYC/MCT1-expressing malignancies.

  6. Major Role of NAD-Dependent Lactate Dehydrogenases in the Production of l-Lactic Acid with High Optical Purity by the Thermophile Bacillus coagulans

    PubMed Central

    Wang, Limin; Cai, Yumeng; Zhu, Lingfeng; Guo, Honglian

    2014-01-01

    Bacillus coagulans 2-6 is an excellent producer of optically pure l-lactic acid. However, little is known about the mechanism of synthesis of the highly optically pure l-lactic acid produced by this strain. Three enzymes responsible for lactic acid production—NAD-dependent l-lactate dehydrogenase (l-nLDH; encoded by ldhL), NAD-dependent d-lactate dehydrogenase (d-nLDH; encoded by ldhD), and glycolate oxidase (GOX)—were systematically investigated in order to study the relationship between these enzymes and the optical purity of lactic acid. Lactobacillus delbrueckii subsp. bulgaricus DSM 20081 (a d-lactic acid producer) and Lactobacillus plantarum subsp. plantarum DSM 20174 (a dl-lactic acid producer) were also examined in this study as comparative strains, in addition to B. coagulans. The specific activities of key enzymes for lactic acid production in the three strains were characterized in vivo and in vitro, and the levels of transcription of the ldhL, ldhD, and GOX genes during fermentation were also analyzed. The catalytic activities of l-nLDH and d-nLDH were different in l-, d-, and dl-lactic acid producers. Only l-nLDH activity was detected in B. coagulans 2-6 under native conditions, and the level of transcription of ldhL in B. coagulans 2-6 was much higher than that of ldhD or the GOX gene at all growth phases. However, for the two Lactobacillus strains used in this study, ldhD transcription levels were higher than those of ldhL. The high catalytic efficiency of l-nLDH toward pyruvate and the high transcription ratios of ldhL to ldhD and ldhL to the GOX gene provide the key explanations for the high optical purity of l-lactic acid produced by B. coagulans 2-6. PMID:25217009

  7. Major Role of NAD-Dependent Lactate Dehydrogenases in the Production of l-Lactic Acid with High Optical Purity by the Thermophile Bacillus coagulans.

    PubMed

    Wang, Limin; Cai, Yumeng; Zhu, Lingfeng; Guo, Honglian; Yu, Bo

    2014-12-01

    Bacillus coagulans 2-6 is an excellent producer of optically pure l-lactic acid. However, little is known about the mechanism of synthesis of the highly optically pure l-lactic acid produced by this strain. Three enzymes responsible for lactic acid production-NAD-dependent l-lactate dehydrogenase (l-nLDH; encoded by ldhL), NAD-dependent d-lactate dehydrogenase (d-nLDH; encoded by ldhD), and glycolate oxidase (GOX)-were systematically investigated in order to study the relationship between these enzymes and the optical purity of lactic acid. Lactobacillus delbrueckii subsp. bulgaricus DSM 20081 (a d-lactic acid producer) and Lactobacillus plantarum subsp. plantarum DSM 20174 (a dl-lactic acid producer) were also examined in this study as comparative strains, in addition to B. coagulans. The specific activities of key enzymes for lactic acid production in the three strains were characterized in vivo and in vitro, and the levels of transcription of the ldhL, ldhD, and GOX genes during fermentation were also analyzed. The catalytic activities of l-nLDH and d-nLDH were different in l-, d-, and dl-lactic acid producers. Only l-nLDH activity was detected in B. coagulans 2-6 under native conditions, and the level of transcription of ldhL in B. coagulans 2-6 was much higher than that of ldhD or the GOX gene at all growth phases. However, for the two Lactobacillus strains used in this study, ldhD transcription levels were higher than those of ldhL. The high catalytic efficiency of l-nLDH toward pyruvate and the high transcription ratios of ldhL to ldhD and ldhL to the GOX gene provide the key explanations for the high optical purity of l-lactic acid produced by B. coagulans 2-6. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  8. Activity of select dehydrogenases with sepharose-immobilized N(6)-carboxymethyl-NAD.

    PubMed

    Beauchamp, Justin; Vieille, Claire

    2015-01-01

    N(6)-carboxymethyl-NAD (N(6)-CM-NAD) can be used to immobilize NAD onto a substrate containing terminal primary amines. We previously immobilized N(6)-CM-NAD onto sepharose beads and showed that Thermotoga maritima glycerol dehydrogenase could use the immobilized cofactor with cofactor recycling. We now show that Saccharomyces cerevisiae alcohol dehydrogenase, rabbit muscle L-lactate dehydrogenase (type XI), bovine liver L-glutamic dehydrogenase (type III), Leuconostoc mesenteroides glucose-6-phosphate dehydro-genase, and Thermotoga maritima mannitol dehydrogenase are active with soluble N(6)-CM-NAD. The products of all enzymes but 6-phospho-D-glucono-1,5-lactone were formed when sepharose-immobilized N(6)-CM-NAD was recycled by T. maritima glycerol dehydrogenase, indicating that N(6)-immobilized NAD is suitable for use by a variety of different dehydrogenases. Observations of the enzyme active sites suggest that steric hindrance plays a greater role in limiting or allowing activity with the modified cofactor than do polarity and charge of the residues surrounding the N(6)-amine group on NAD.

  9. Foetal and lactational exposure to alcohol increases oxidative capacity of brown adipose tissue in the rat. A possible relationship to cot death.

    PubMed Central

    Huttunen, P.; Kortelainen, M. L.; Hirvonen, J.

    1989-01-01

    The effect was studied of chronic alcohol intake in the rat during pregnancy and lactation on the brown adipose tissue (BAT) in pups. The idea was to find a possible relationship to cot death since in some cot death victims increased amounts of BAT have been observed. Exposure to ethanol increased the relative weight of the brown adipose tissue in pups and enhanced both its total protein content and the activities of the oxidative enzymes, succinate dehydrogenase and cytochrome oxidase. In the BAT of pups sympathetic activity, as demonstrated by noradrenaline, was also increased by long-term exposure to alcohol. In theory, an increased thermogenic capacity of the BAT in the newborn together with other factors such as emotional stress and infections could lead to death from hyperthermia, in which case only non-specific morphological signs would be found in the cadaver. PMID:2605116

  10. Highly elevated serum lactate dehydrogenase is associated with central nervous system relapse in patients with diffuse large B-cell lymphoma: Results of a multicenter prospective cohort study.

    PubMed

    Kim, Seok Jin; Hong, Jun Sik; Chang, Myung Hee; Kim, Jeong-A; Kwak, Jae-Yong; Kim, Jin Seok; Yoon, Dok Hyun; Lee, Won Sik; Do, Young Rok; Kang, Hye Jin; Eom, Hyeon-Seok; Park, Yong; Won, Jong-Ho; Mun, Yeung-Chul; Kim, Hyo Jung; Kwon, Jung Hye; Kong, Jee Hyun; Oh, Sung Yong; Lee, Sunah; Bae, Sung Hwa; Yang, Deok-Hwan; Jun, Hyun Jung; Kim, Yang Soo; Yun, Hwan Jung; Lee, Soon Il; Kim, Min Kyoung; Park, Eun Kyung; Kim, Won Seog; Suh, Cheolwon

    2016-11-01

    Central nervous system involvement remains a challenging issue in the treatment of patients with diffuse large B-cell lymphoma. We conducted a prospective cohort study with newly diagnosed diffuse large B-cell lymphoma patients receiving rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone to identify incidence and risk factors for central nervous system involvement. Among 595 patients, 279 patients received pre-treatment central nervous system evaluation, and 14 patients had central nervous system involvement at diagnosis (2.3% out of entire patients and 5.0% out of the 279 patients). For those patients, median follow-up duration was 38.2 months and some of them achieved long-term survival. Out of 581 patients who did not have central nervous system involvement at diagnosis, 26 patients underwent secondary central nervous system relapse with a median follow-up of 35 months, and the median time to central nervous system involvement was 10.4 months (range: 3.4-29.2). Serum lactate dehydrogenase > ×3 upper limit of normal range, the Eastern Cooperative Oncology Group performance status ≥ 2, and involvement of sinonasal tract or testis, were independent risk factors for central nervous system relapse in multivariate analysis. Our study suggests that enhanced stratification of serum lactate dehydrogenase according to the National Comprehensive Cancer Network-International Prognostic Index may contribute to better prediction for central nervous system relapse in patients with diffuse large B-cell lymphoma. This trial was registered at clinicaltrials.gov identifier: 01202448.

  11. Highly elevated serum lactate dehydrogenase is associated with central nervous system relapse in patients with diffuse large B-cell lymphoma: Results of a multicenter prospective cohort study

    PubMed Central

    Kim, Seok Jin; Hong, Jun Sik; Chang, Myung Hee; Kim, Jeong-A; Kwak, Jae-Yong; Kim, Jin Seok; Yoon, Dok Hyun; Lee, Won Sik; Do, Young Rok; Kang, Hye Jin; Eom, Hyeon-Seok; Park, Yong; Won, Jong-Ho; Mun, Yeung-Chul; Kim, Hyo Jung; Kwon, Jung Hye; Kong, Jee Hyun; Oh, Sung Yong; Lee, Sunah; Bae, Sung Hwa; Yang, Deok-Hwan; Jun, Hyun Jung; Kim, Yang Soo; Yun, Hwan Jung; Il Lee, Soon; Kim, Min Kyoung; Park, Eun Kyung; Kim, Won Seog; Suh, Cheolwon

    2016-01-01

    Central nervous system involvement remains a challenging issue in the treatment of patients with diffuse large B-cell lymphoma. We conducted a prospective cohort study with newly diagnosed diffuse large B-cell lymphoma patients receiving rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone to identify incidence and risk factors for central nervous system involvement. Among 595 patients, 279 patients received pre-treatment central nervous system evaluation, and 14 patients had central nervous system involvement at diagnosis (2.3% out of entire patients and 5.0% out of the 279 patients). For those patients, median follow-up duration was 38.2 months and some of them achieved long-term survival. Out of 581 patients who did not have central nervous system involvement at diagnosis, 26 patients underwent secondary central nervous system relapse with a median follow-up of 35 months, and the median time to central nervous system involvement was 10.4 months (range: 3.4–29.2). Serum lactate dehydrogenase > ×3 upper limit of normal range, the Eastern Cooperative Oncology Group performance status ≥ 2, and involvement of sinonasal tract or testis, were independent risk factors for central nervous system relapse in multivariate analysis. Our study suggests that enhanced stratification of serum lactate dehydrogenase according to the National Comprehensive Cancer Network-International Prognostic Index may contribute to better prediction for central nervous system relapse in patients with diffuse large B-cell lymphoma. This trial was registered at clinicaltrials.gov identifier: 01202448. PMID:27713132

  12. Contributory roles of two l-lactate dehydrogenases for l-lactic acid production in thermotolerant Bacillus coagulans

    PubMed Central

    Sun, Lifan; Zhang, Caili; Lyu, Pengcheng; Wang, Yanping; Wang, Limin; Yu, Bo

    2016-01-01

    Thermotolerant Bacillus coagulans is considered to be a more promising producer for bio-chemicals, due to its capacity to withstand harsh conditions. Two L-lactate dehydrogenase (LDH) encoding genes (ldhL1 and ldhL2) and one D-LDH encoding gene (ldhD) were annotated from the B. coagulans DSM1 genome. Transcriptional analysis revealed that the expression of ldhL2 was undetectable while the ldhL1 transcription level was much higher than that of ldhD at all growth phases. Deletion of the ldhL2 gene revealed no difference in fermentation profile compared to the wild-type strain, while ldhL1 single deletion or ldhL1ldhL2 double deletion completely blocked L-lactic acid production. Complementation of ldhL1 in the above knockout strains restored fermentation profiles to those observed in the wild-type strain. This study demonstrates ldhL1 is crucial for L-lactic acid production and NADH balance in B. coagulans DSM1 and lays the fundamental for engineering the thermotolerant B. coagulans strain as a platform chemicals producer. PMID:27885267

  13. Impact of critical process and formulation parameters affecting in-process stability of lactate dehydrogenase during the secondary drying stage of lyophilization: a mini freeze dryer study.

    PubMed

    Luthra, Sumit; Obert, Jean-Philippe; Kalonia, Devendra S; Pikal, Michael J

    2007-09-01

    The stresses during the secondary-drying stage of lyophilization were investigated using a controlled humidity mini-freeze-dryer [Luthra S, Obert J-P, Kalonia DS, Pikal MJ. 2007. Investigation of drying stresses on proteins during lyophilization: Differentiation between primary and secondary-drying stresses on lactate dehydrogenase using a humidity controlled mini freeze-dryer. J Pharm Sci 96: 61-70.]. Lactate dehydrogenase (LDH), was formulated in: (1) Tween 80, (2) citrate buffer, and (3) both Tween 80 and citrate buffer. Protein activity recovery was measured as a function of relative humidity (RH), product temperature, and drying duration. Studies were also conducted with different concentrations of sucrose, sorbitol, and poly (vinyl pyrrolidone) (PVP). LDH stability was affected to a small extent by RH and significantly by drying temperature and duration. Complete stabilization of LDH was observed when lyophilized with sucrose and PVP but only a partial stabilization was observed with sorbitol. The mini-freeze-dryer enabled studying the process parameters independently, unlike a conventional study where these effects are generally convoluted. The results suggest that the stability of the protein is a function of the dynamics of the system during lyophilization. The origin of the stabilization effect of sucrose, which could, in principle, be attributed both to direct interaction with the protein or vitrification of the protein was elucidated using lyoprotectants that can either hydrogen bond well with the protein (sorbitol) or form a good glass (PVP). It appears both effects are required for complete stabilization of the protein. (c) 2007 Wiley-Liss, Inc. and the American Pharmacists Association.

  14. Type II flavohemoglobin of Mycobacterium smegmatis oxidizes d-lactate and mediate electron transfer.

    PubMed

    Thakur, Naveen; Kumar, Ashwani; Dikshit, Kanak L

    2018-06-01

    Two distantly related flavohemoglobins (FHbs), MsFHbI and MsFHbII, having crucial differences in their heme and reductase domains, co-exist in Mycobacterium smegmatis. Function of MsFHbI is associated with nitric-oxide detoxification but physiological relevance of MsFHbII remains unknown. This study unravels some unique spectral and functional characteristics of MsFHbII. Unlike conventional type I FHbs, MsFHbII lacks nitric-oxide dioxygenase and NADH oxidase activities but utilizes d-lactate as an electron donor to mediate electron transfer. MsFHbII carries a d-lactate dehydrogenase type FAD binding motif in its reductase domain and oxidizes d-lactate in a FAD dependent manner to reduce the heme iron, suggesting that the globin is acting as an electron acceptor. Importantly, expression of MsFHbII in Escherichia coli imparted protection under oxidative stress, suggesting its important role in stress management of its host. Since M. smegmatis lacks the gene encoding for d-lactate dehydrogenase and d-lactate is produced during aerobic metabolism and also as a by-product of lipid peroxidation, the ability of MsFHbII to metabolize d-lactate may provide it a unique ability to balance the oxidative stress generated due to accumulation of d-lactate in the cell and at the same time sequester electrons and pass it to the respiratory apparatus. Copyright © 2018 Elsevier B.V. All rights reserved.

  15. Structure and Function of Plasmodium falciparum malate dehydrogenase: Role of Critical Amino Acids in C-substrate Binding Procket

    USDA-ARS?s Scientific Manuscript database

    Malaria parasite thrives on anaerobic fermentation of glucose for energy. Earlier studies from our lab have demonstrated that a cytosolic malate dehydrogenase (PfMDH) with striking similarity to lactate dehydrogenase (PfLDH) might complement PfLDH function in Plasmodium falciparum. The N-terminal g...

  16. A metabolic switch in brain: glucose and lactate metabolism modulation by ascorbic acid.

    PubMed

    Castro, Maite A; Beltrán, Felipe A; Brauchi, Sebastián; Concha, Ilona I

    2009-07-01

    In this review, we discuss a novel function of ascorbic acid in brain energetics. It has been proposed that during glutamatergic synaptic activity neurons preferably consume lactate released from glia. The key to this energetic coupling is the metabolic activation that occurs in astrocytes by glutamate and an increase in extracellular [K(+)]. Neurons are cells well equipped to consume glucose because they express glucose transporters and glycolytic and tricarboxylic acid cycle enzymes. Moreover, neuronal cells express monocarboxylate transporters and lactate dehydrogenase isoenzyme 1, which is inhibited by pyruvate. As glycolysis produces an increase in pyruvate concentration and a decrease in NAD(+)/NADH, lactate and glucose consumption are not viable at the same time. In this context, we discuss ascorbic acid participation as a metabolic switch modulating neuronal metabolism between rest and activation periods. Ascorbic acid is highly concentrated in CNS. Glutamate stimulates ascorbic acid release from astrocytes. Ascorbic acid entry into neurons and within the cell can inhibit glucose consumption and stimulate lactate transport. For this switch to occur, an ascorbic acid flow is necessary between astrocytes and neurons, which is driven by neural activity and is part of vitamin C recycling. Here, we review the role of glucose and lactate as metabolic substrates and the modulation of neuronal metabolism by ascorbic acid.

  17. Improved production of homo-D-lactic acid via xylose fermentation by introduction of xylose assimilation genes and redirection of the phosphoketolase pathway to the pentose phosphate pathway in L-Lactate dehydrogenase gene-deficient Lactobacillus plantarum.

    PubMed

    Okano, Kenji; Yoshida, Shogo; Yamada, Ryosuke; Tanaka, Tsutomu; Ogino, Chiaki; Fukuda, Hideki; Kondo, Akihiko

    2009-12-01

    The production of optically pure d-lactic acid via xylose fermentation was achieved by using a Lactobacillus plantarum NCIMB 8826 strain whose l-lactate dehydrogenase gene was deficient and whose phosphoketolase genes were replaced with a heterologous transketolase gene. After 60 h of fermentation, 41.2 g/liter of d-lactic acid was produced from 50 g/liter of xylose.

  18. Measurements of C-reactive protein in serum and lactate dehydrogenase in serum and synovial fluid of patients with osteoarthritis.

    PubMed

    Hurter, K; Spreng, D; Rytz, U; Schawalder, P; Ott-Knüsel, F; Schmökel, H

    2005-03-01

    Diagnosis of osteoarthritis (OA) is based upon the clinical orthopaedic examination and the radiographic assessment, both of which can be non-specific and insensitive in early joint disease. The aim of our study was to investigate if there is an increase in serum levels of C-reactive protein (CRP) in degenerative joint disease (DJD) and if CRP could be used to help diagnose OA. We also wished to investigate whether it was possible to distinguish a joint with clinically and radiographically confirmed OA from a healthy joint by comparing lactate dehydrogenase (LDH) levels within the synovial fluid and the serum. We have shown a difference in synovial LDH levels between diseased and healthy joints (P<0.0001). There was also a significant difference between LDH in arthritic synovial fluid and serum, with no correlation between the values. Despite the fact that the values of our clinical patients tended to be higher than the values of our control group (P=0.05) all measured values were within the normal limits of previous publications. From these data, we conclude that single measurements of serum CRP do not permit detection of OA in clinical patients and that serum LDH is not a reliable marker for osteoarthritis. LDH levels in the synovial fluid could be of diagnostic value for identifying osteoarthritis.

  19. Molecular cloning, characterization, and immunolocalization of two lactate dehydrogenase homologous genes from Taenia solium.

    PubMed

    Du, Wuying; Hu, Fengyu; Yang, Yabo; Hu, Dong; Hu, Xuchu; Yu, Xinbing; Xu, Jin; Dai, Jialin; Liao, Xinjiang; Huang, Jiang

    2011-09-01

    Two novel genes encoding lactate dehydrogenase A (LDHA) and B (LDHB) homologues, respectively, were identified from the cDNA libraries of adult Taenia solium (T. solium). The two deduced amino acid sequences both show more than 50% identity to the homologues for Danio rerio, Xenopus laevis, Schistosoma japonicum, Sus scrofa, Homo sapiens, et al. The identity of the amino acid sequence between TsLDHA and TsLDHB is 57.4%, and that of the nucleotide sequence is 61.5%. Recombinant TsLDHA homologue (rTsLDHA) and TsLDHB homologue (rTsLDHB) were expressed in Escherichia coli BL21/DE3 and purified. Though there were some differences in the sequence, the two LDH isozyme homologues show similarity in the conserved LDH domain, topological structure, primary immunological traits, localization on the tegument of T. solium adult, and partial physicochemical properties. The linear B-cell epitope analysis of TsLDHA and TsLDHB discovered a TsLDHA specific epitope. The purified rTsLDHA and rTsLDHB could be recognized by rat immuno-sera, serum from swine, or a patient infected with T. solium, respectively, but Western blot analysis showed cross-reactions, not only between these two LDH members but also with other common human tapeworms or helminths. The results suggested that the two LDH homologues are similar in the characteristics of LDH family, and they are not specific antigens for immunodiagnosis.

  20. Acute and chronic effects of clofibrate and clofibric acid on the enzymes acetylcholinesterase, lactate dehydrogenase and catalase of the mosquitofish, Gambusia holbrooki.

    PubMed

    Nunes, B; Carvalho, F; Guilhermino, L

    2004-12-01

    The objective of this study was to investigate both acute and chronic effects of clofibrate and clofibric acid on the enzymes acetylcholinesterase (AChE), lactate dehydrogenase (LDH) and catalase (CAT) of the mosquitofish (Gambusia holbrooki). AChE, commonly used as a biomarker of neurotoxicity, was determined in the total head. LDH, an important enzyme of anaerobic metabolism, was quantified in dorsal muscle, and CAT, enzyme which has been used as indicative parameter of peroxisome proliferation, was determined in the liver. Furthermore, alterations of body and liver weight were also determined, through the calculation of the ratios final body weight/initial body weight, liver weight/final body weight, liver weight/gills weight and liver weight/head weight. Acute exposure of G. holbrooki to both clofibrate and clofibric acid induced a decrease in liver CAT activity, an increase in muscle LDH activity, while no effects were observed on AChE activity. However, chronic exposure did not alter significantly the enzymatic activities, suggesting reduced or null effects over these pathways, relative to effects reported in other species. No effects were observed for the calculated ratios, except a significant weight reduction for males chronically exposed to clofibrate.

  1. Stable Suppression of Lactate Dehydrogenase Activity during Anoxia in the Foot Muscle of Littorina littorea and the Potential Role of Acetylation as a Novel Posttranslational Regulatory Mechanism

    PubMed Central

    Shahriari, Ali; Dawson, Neal J.; Bell, Ryan A. V.; Storey, Kenneth B.

    2013-01-01

    The intertidal marine snail, Littorina littorea, has evolved to withstand extended bouts of oxygen deprivation brought about by changing tides or other potentially harmful environmental conditions. Survival is dependent on a strong suppression of its metabolic rate and a drastic reorganization of its cellular biochemistry in order to maintain energy balance under fixed fuel reserves. Lactate dehydrogenase (LDH) is a crucial enzyme of anaerobic metabolism as it is typically responsible for the regeneration of NAD+, which allows for the continued functioning of glycolysis in the absence of oxygen. This study compared the kinetic and structural characteristics of the D-lactate specific LDH (E.C. 1.1.1.28) from foot muscle of aerobic control versus 24 h anoxia-exposed L. littorea. Anoxic LDH displayed a near 50% decrease in V max (pyruvate-reducing direction) as compared to control LDH. These kinetic differences suggest that there may be a stable modification and regulation of LDH during anoxia, and indeed, subsequent dot-blot analyses identified anoxic LDH as being significantly less acetylated than the corresponding control enzyme. Therefore, acetylation may be the regulatory mechanism that is responsible for the suppression of LDH activity during anoxia, which could allow for the production of alternative glycolytic end products that in turn would increase the ATP yield under fixed fuel reserves. PMID:24233354

  2. Oxygen-Inducible Conversion of Lactate to Acetate in Heterofermentative Lactobacillus brevis ATCC 367.

    PubMed

    Guo, Tingting; Zhang, Li; Xin, Yongping; Xu, ZhenShang; He, Huiying; Kong, Jian

    2017-11-01

    Lactobacillus brevis is an obligatory heterofermentative lactic acid bacterium that produces high levels of acetate, which improve the aerobic stability of silages against deterioration caused by yeasts and molds. However, the mechanism involved in acetate accumulation has yet to be elucidated. Here, experimental evidence indicated that aerobiosis resulted in the conversion of lactate to acetate after glucose exhaustion in L. brevis ATCC 367 (GenBank accession number NC_008497). To elucidate the conversion pathway, in silico analysis showed that lactate was first converted to pyruvate by the reverse catalytic reaction of lactate dehydrogenase (LDH); subsequently, pyruvate conversion to acetate might be mediated by pyruvate dehydrogenase (PDH) or pyruvate oxidase (POX). Transcriptional analysis indicated that the pdh and pox genes of L. brevis ATCC 367 were upregulated 37.92- and 18.32-fold, respectively, by oxygen and glucose exhaustion, corresponding to 5.32- and 2.35-fold increases in the respective enzyme activities. Compared with the wild-type strain, the transcription and enzymatic activity of PDH remained stable in the Δ pox mutant, while those of POX increased significantly in the Δ pdh mutant. More lactate but less acetate was produced in the Δ pdh mutant than in the wild-type and Δ pox mutant strains, and more H 2 O 2 (a product of the POX pathway) was produced in the Δ pdh mutant. We speculated that the high levels of aerobic acetate accumulation in L. brevis ATCC 367 originated mainly from the reuse of lactate to produce pyruvate, which was further converted to acetate by the predominant and secondary functions of PDH and POX, respectively. IMPORTANCE PDH and POX are two possible key enzymes involved in aerobic acetate accumulation in lactic acid bacteria (LAB). It is currently thought that POX plays the major role in aerobic growth in homofermentative LAB and some heterofermentative LAB, while the impact of PDH remains unclear. In this study, we

  3. Oxygen-Inducible Conversion of Lactate to Acetate in Heterofermentative Lactobacillus brevis ATCC 367

    PubMed Central

    Guo, Tingting; Zhang, Li; Xin, Yongping; Xu, ZhenShang; He, Huiying

    2017-01-01

    ABSTRACT Lactobacillus brevis is an obligatory heterofermentative lactic acid bacterium that produces high levels of acetate, which improve the aerobic stability of silages against deterioration caused by yeasts and molds. However, the mechanism involved in acetate accumulation has yet to be elucidated. Here, experimental evidence indicated that aerobiosis resulted in the conversion of lactate to acetate after glucose exhaustion in L. brevis ATCC 367 (GenBank accession number NC_008497). To elucidate the conversion pathway, in silico analysis showed that lactate was first converted to pyruvate by the reverse catalytic reaction of lactate dehydrogenase (LDH); subsequently, pyruvate conversion to acetate might be mediated by pyruvate dehydrogenase (PDH) or pyruvate oxidase (POX). Transcriptional analysis indicated that the pdh and pox genes of L. brevis ATCC 367 were upregulated 37.92- and 18.32-fold, respectively, by oxygen and glucose exhaustion, corresponding to 5.32- and 2.35-fold increases in the respective enzyme activities. Compared with the wild-type strain, the transcription and enzymatic activity of PDH remained stable in the Δpox mutant, while those of POX increased significantly in the Δpdh mutant. More lactate but less acetate was produced in the Δpdh mutant than in the wild-type and Δpox mutant strains, and more H2O2 (a product of the POX pathway) was produced in the Δpdh mutant. We speculated that the high levels of aerobic acetate accumulation in L. brevis ATCC 367 originated mainly from the reuse of lactate to produce pyruvate, which was further converted to acetate by the predominant and secondary functions of PDH and POX, respectively. IMPORTANCE PDH and POX are two possible key enzymes involved in aerobic acetate accumulation in lactic acid bacteria (LAB). It is currently thought that POX plays the major role in aerobic growth in homofermentative LAB and some heterofermentative LAB, while the impact of PDH remains unclear. In this study, we

  4. Myoclonic epilepsy with ragged-red fibers without increased lactate levels.

    PubMed

    Kimura, Shigemi; Ozasa, Shiro; Nakamura, Kyoko; Nomura, Keiko; Kosuge, Hirofumi

    2009-07-01

    Myoclonic epilepsy associated with ragged-red fibers is one of the mitochondrial encephalomyopathies. Pathogenic mitochondrial DNA mutations have been identified in the mitochondrial transfer RNA (tRNA)(Lys) at positions 8344 and 8356. Characteristics of myoclonic epilepsy associated with ragged-red fibers include myoclonic epilepsy, generalized epilepsy, hearing loss, exercise intolerance, lactic acidosis, and ragged-red fibers. The elevated lactate level is one of the most important symptoms needed to make a diagnosis of mitochondrial encephalomyopathy. In the present case, however, myoclonic epilepsy was associated with ragged-red fibers but without increased lactate levels. Therefore, myoclonic epilepsy associated with ragged-red fibers should be suspected in a patient who has myoclonic epilepsy that is difficult to control with antiepileptic medications and who has other symptoms of mitochondrial disease, such as mental retardation, even if the patient's lactate level is normal.

  5. Effects of interaction with gene carrier polyethyleneimines on conformation and enzymatic activity of pig heart lactate dehydrogenase.

    PubMed

    Wang, Fan; Mo, Junyong; Huang, Aimin; Zhang, Min; Ma, Lin

    2018-06-15

    Polyethyleneimine (PEI) has long been considered as "golden standard" for polymeric gene delivery carrier, however also induces cytotoxicity. To make a further insight into the molecular basis of PEI cytotoxicity, fluorescence, absorption and circular dichroism spectroscopy were conducted to investigate the influence of PEI (average molecular weight 25,000 and 1800 Da) on the conformation of pig heart lactate dehydrogenase (LDH) and its catalytic efficiency. Zeta-potential measurement and isothermal titration calorimetry were used to reveal the interaction between PEI and LDH. PEI was found to bind onto the surface of LDH predominantly via hydrophobic interaction, inducing a more compact conformation and an increased surface hydrophobicity of the enzyme. The conformational change of LDH induced by PEI binding had little influence on the complex formation between LDH and reduced nicotinamide adenine dinucleotide (NADH, the co-enzyme). However, the nonspecific binding of PEI on the surface of LDH retarded the turnover of the enzyme. Meanwhile, the large quantity of amine groups on the polymer chain made PEI subject to form complexes with NADH and pyruvate (the substrate) via hydrogen bond and electrostatic interaction, which greatly reduced the binding efficient of LDH. The polymer size played an important role in PEI-LDH interaction. The smaller size of lower molecular weight PEI facilitated the close contact with LDH and consequential reduction of the turnover number of the enzyme. However, higher molecular weight PEI was more favorable for competitive binding with NADH and pyruvate and generally decreased the catalytic efficient of LDH. Copyright © 2018. Published by Elsevier B.V.

  6. Identification of proteins interacting with lactate dehydrogenase in claw muscle of the porcelain crab Petrolisthes cinctipes

    PubMed Central

    Cayenne, Andrea P.; Gabert, Beverly; Stillman, Jonathon H.

    2011-01-01

    Biochemical adaptation of enzymes involves conservation of activity, stability and affinity across a wide range of intracellular and environmental conditions. Enzyme adaptation by alteration of primary structure is well known, but the roles of protein-protein interactions in enzyme adaptation are less well understood. Interspecific differences in thermal stability of lactate dehydrogenase (LDH) in porcelain crabs (genus Petrolisthes) are related to intrinsic differences among LDH molecules and by interactions with other stabilizing proteins. Here, we identified proteins that interact with LDH in porcelain crab claw muscle tissue using co-immunoprecipitation, and showed LDH exists in high molecular weight complexes using size exclusion chromatography and Western blot analyses. Co-immunoprecipitated proteins were separated using 2D SDS PAGE and analyzed by LC/ESI using peptide MS/MS. Peptide MS/MS ions were compared to an EST database for Petrolisthes cinctipes to identify proteins. Identified proteins included cytoskeletal elements, glycolytic enzymes, a phosphagen kinase, and the respiratory protein hemocyanin. Our results support the hypothesis that LDH interacts with glycolytic enzymes in a metabolon structured by cytoskeletal elements that may also include the enzyme for transfer of the adenylate charge in glycolytically produced ATP. Those interactions may play specific roles in biochemical adaptation of glycolytic enzymes. PMID:21968246

  7. Human mesenchymal stromal cell-secreted lactate induces M2-macrophage differentiation by metabolic reprogramming

    PubMed Central

    Civini, Sara; Pacelli, Consiglia; Dieng, Mame Massar; Lemieux, William; Jin, Ping; Bazin, Renée; Patey, Natacha; Marincola, Francesco M.; Moldovan, Florina; Zaouter, Charlotte; Trudeau, Louis-Eric; Benabdhalla, Basma; Louis, Isabelle; Beauséjour, Christian; Stroncek, David; Le Deist, Françoise; Haddad, Elie

    2016-01-01

    Human mesenchymal stromal cells (MSC) have been shown to dampen immune response and promote tissue repair, but the underlying mechanisms are still under investigation. Herein, we demonstrate that umbilical cord-derived MSC (UC-MSC) alter the phenotype and function of monocyte-derived dendritic cells (DC) through lactate-mediated metabolic reprogramming. UC-MSC can secrete large quantities of lactate and, when present during monocyte-to-DC differentiation, induce instead the acquisition of M2-macrophage features in terms of morphology, surface markers, migratory properties and antigen presentation capacity. Microarray expression profiling indicates that UC-MSC modify the expression of metabolic-related genes and induce a M2-macrophage expression signature. Importantly, monocyte-derived DC obtained in presence of UC-MSC, polarize naïve allogeneic CD4+ T-cells into Th2 cells. Treatment of UC-MSC with an inhibitor of lactate dehydrogenase strongly decreases lactate concentration in culture supernatant and abrogates the effect on monocyte-to-DC differentiation. Metabolic analysis further revealed that UC-MSC decrease oxidative phosphorylation in differentiating monocytes while strongly increasing the spare respiratory capacity proportional to the amount of secreted lactate. Because both MSC and monocytes are recruited in vivo at the site of tissue damage and inflammation, we propose the local increase of lactate concentration induced by UC-MSC and the consequent enrichment in M2-macrophage generation as a mechanism to achieve immunomodulation. PMID:27070086

  8. Lactation and appetite-regulating hormones: increased maternal plasma peptide YY concentrations 3-6 months postpartum.

    PubMed

    Vila, Greisa; Hopfgartner, Judith; Grimm, Gabriele; Baumgartner-Parzer, Sabina M; Kautzky-Willer, Alexandra; Clodi, Martin; Luger, Anton

    2015-10-28

    Breast-feeding is associated with maternal hormonal and metabolic changes ensuring adequate milk production. In this study, we investigate the impact of breast-feeding on the profile of changes in maternal appetite-regulating hormones 3-6 months postpartum. Study participants were age- and BMI-matched lactating mothers (n 10), non-lactating mothers (n 9) and women without any history of pregnancy or breast-feeding in the previous 12 months (control group, n 10). During study sessions, young mothers breast-fed or bottle-fed their babies, and maternal blood samples were collected at five time points during 90 min: before, during and after feeding the babies. Outcome parameters were plasma concentrations of ghrelin, peptide YY (PYY), leptin, adiponectin, prolactin, cortisol, insulin, glucose and lipid values. At baseline, circulating PYY concentrations were significantly increased in lactating mothers (100·3 (se 6·7) pg/ml) v. non-lactating mothers (73·6 (se 4·9) pg/ml, P=0·008) and v. the control group (70·2 (se 9) pg/ml, P=0·021). We found no differences in ghrelin, leptin and adiponectin values. Baseline prolactin concentrations were over 4-fold higher in lactating mothers (P<0·001). Lactating women had reduced TAG levels and LDL-cholesterol:HDL-cholesterol ratio, but increased waist circumference, when compared with non-lactating women. Breast-feeding sessions further elevated circulating prolactin (P<0·001), but induced no acute effects on appetite-regulating hormones. In summary, one single breast-feeding session did not acutely modulate circulating appetite-regulating hormones, but increased baseline PYY concentrations are associated with prolonged lactation. PYY might play a role in the coordination of energy balance during lactation, increasing fat mobilisation from maternal depots and ensuring adequate milk production for the demands of the growing infant.

  9. Ectoparasite Caligus rogercresseyi modifies the lactate response in Atlantic salmon (Salmo salar) and Coho salmon (Oncorhynchus kisutch).

    PubMed

    Vargas-Chacoff, L; Muñoz, J L P; Hawes, C; Oyarzún, R; Pontigo, J P; Saravia, J; González, M P; Mardones, O; Labbé, B S; Morera, F J; Bertrán, C; Pino, J; Wadsworth, S; Yáñez, A

    2017-08-30

    Although Caligus rogercresseyi negatively impacts Chilean salmon farming, the metabolic effects of infection by this sea louse have never been completely characterized. Therefore, this study analyzed lactate responses in the plasma, as well as the liver/muscle lactate dehydrogenase (LDH) activity and gene expression, in Salmo salar and Oncorhynchus kisutch infested by C. rogercresseyi. The lactate responses of Atlantic and Coho salmon were modified by the ectoparasite. Both salmon species showed increasing in plasma levels, whereas enzymatic activity increased in the muscle but decreased in the liver. Gene expression was overexpressed in both Coho salmon tissues but only in the liver for Atlantic salmon. These results suggest that salmonids need more energy to adapt to infection, resulting in increased gene expression, plasma levels, and enzyme activity in the muscles. The responses differed between both salmon species and over the course of infection, suggesting potential species-specific responses to sea-lice infection. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Partial reconstruction of in vitro gluconeogenesis arising from mitochondrial l-lactate uptake/metabolism and oxaloacetate export via novel L-lactate translocators.

    PubMed

    De Bari, Lidia; Atlante, Anna; Valenti, Daniela; Passarella, Salvatore

    2004-05-15

    In the light of the occurrence of L-lactate dehydrogenase inside the mitochondrial matrix, we looked at whether isolated rat liver mitochondria can take up and metabolize L-lactate, and provide oxaloacetate outside mitochondria, thus contributing to a partial reconstruction of gluconeogenesis in vitro. We found that: (1) L-lactate (10 mM), added to mitochondria in the presence of a cocktail of glycolysis/gluconeogenesis enzymes and cofactors, can lead to synthesis of glyceraldehyde-3-phosphate at a rate of about 7 nmol/min per mg mitochondrial protein. (2) Three novel translocators exist to mediate L-lactate traffic across the inner mitochondrial membrane. An L-lactate/H+ symporter was identified by measuring fluorimetrically the rate of endogenous pyridine nucleotide reduction. Consistently, L-lactate oxidation was found to occur with P/O ratio=3 (where P/O ratio is the ratio of mol of ATP synthesized to mol of oxygen atoms reduced to water during oxidative phosphorylation) and with generation of membrane potential. Proton uptake, which occurred as a result of addition of L-lactate to RLM together with electron flow inhibitors, and mitochondrial swelling in ammonium L-lactate solutions were also monitored. L-Lactate/oxaloacetate and L-lactate/pyruvate anti-porters were identified by monitoring photometrically the appearance of L-lactate counter-anions outside mitochondria. These L-lactate translocators, which are distinct from the monocarboxylate carrier, were found to differ from each other in V(max) values and in inhibition and pH profiles, and proved to regulate mitochondrial L-lactate metabolism in vitro. The role of lactate/mitochondria interactions in gluconeogenesis is discussed.

  11. Lactate Utilization Is Regulated by the FadR-Type Regulator LldR in Pseudomonas aeruginosa

    PubMed Central

    Gao, Chao; Hu, Chunhui; Zheng, Zhaojuan; Jiang, Tianyi; Dou, Peipei; Zhang, Wen; Che, Bin; Wang, Yujiao; Lv, Min

    2012-01-01

    NAD-independent l-lactate dehydrogenase (l-iLDH) and NAD-independent d-lactate dehydrogenase (d-iLDH) activities are induced coordinately by either enantiomer of lactate in Pseudomonas strains. Inspection of the genomic sequences of different Pseudomonas strains revealed that the lldPDE operon comprises 3 genes, lldP (encoding a lactate permease), lldD (encoding an l-iLDH), and lldE (encoding a d-iLDH). Cotranscription of lldP, lldD, and lldE in Pseudomonas aeruginosa strain XMG starts with the base, C, that is located 138 bp upstream of the lldP ATG start codon. The lldPDE operon is located adjacent to lldR (encoding an FadR-type regulator, LldR). The gel mobility shift assays revealed that the purified His-tagged LldR binds to the upstream region of lldP. An XMG mutant strain that constitutively expresses d-iLDH and l-iLDH was found to contain a mutation in lldR that leads to an Ile23-to-serine substitution in the LldR protein. The mutated protein, LldRM, lost its DNA-binding activity. A motif with a hyphenated dyad symmetry (TGGTCTTACCA) was identified as essential for the binding of LldR to the upstream region of lldP by using site-directed mutagenesis. l-Lactate and d-lactate interfered with the DNA-binding activity of LldR. Thus, l-iLDH and d-iLDH were expressed when the operon was induced in the presence of l-lactate or d-lactate. PMID:22408166

  12. Lactate oxidation coupled to energy production in mitochondria like particles from Setaria digitata, a filarial parasite.

    PubMed

    Sivan, V M; Raj, R K

    1994-10-14

    In the filarial parasite, Setaria digitata, the mitochondria like particles (MLP) show NAD reduction with sodium lactate. The MLP also reduces dye and ferricyanide with lactate. The ferricyanide reduction by lactate is found to be sensitive to the cytochrome o inhibitor orthohydroxy diphenyl (OHD) and complex I inhibitor rotenone, modulated by ADP (+) and ATP (-) and inhibited by pyruvate and oxaloacetate. MLP shows lactate oxidation sensitive to OHD, rotenone and sodium malonate. Thus, the lactate utilizing complex system, consisting of an NADH generating MLP bound lactate dehydrogenase and a lactate flavocytochrome reductase tightly linked to complex I and cytochrome o, produces ATP in functional association with fumarate reductase complex and other enzyme systems. Hence, this study provides new dimensions to the study of metabolism in filarial parasites.

  13. DNA Sequence Polymorphism of the Lactate Dehydrogenase Genefrom Iranian Plasmodium vivax and Plasmodium falciparum Isolates.

    PubMed

    Getacher Feleke, Daniel; Nateghpour, Mehdi; Motevalli Haghi, Afsaneh; Hajjaran, Homa; Farivar, Leila; Mohebali, Mehdi; Raoofian, Reza

    2015-01-01

    Parasite lactate dehydrogenase (pLDH) is extensively employed as malaria rapid diagnostic tests (RDTs). Moreover, it is a well-known drug target candidate. However, the genetic diversity of this gene might influence performance of RDT kits and its drug target candidacy. This study aimed to determine polymorphism of pLDH gene from Iranian isolates of P. vivax and P. falciparum. Genomic DNA was extracted from whole blood of microscopically confirmed P. vivax and P. falciparum infected patients. pLDH gene of P. falciparum and P. vivax was amplified using conventional PCR from 43 symptomatic malaria patients from Sistan and Baluchistan Province, Southeast Iran from 2012 to 2013. Sequence analysis of 15 P. vivax LDH showed fourteen had 100% identity with P. vivax Sal-1 and Belem strains. Two nucleotide substitutions were detected with only one resulted in amino acid change. Analysis of P. falciparum LDH sequences showed six of the seven sequences had 100% homology with P. falciparum 3D7 and Mzr-1. Moreover, PfLDH displayed three nucleotide changes that resulted in changing only one amino acid. PvLDH and PfLDH showed 75%-76% nucleotide and 90.4%-90.76% amino acid homology. pLDH gene from Iranian P. falciparum and P. vivax isolates displayed 98.8-100% homology with 1-3 nucleotide substitutions. This indicated this gene was relatively conserved. Additional studies can be done weather this genetic variation can influence the performance of pLDH based RDTs or not.

  14. Enhancement of Astroglial Aerobic Glycolysis by Extracellular Lactate-Mediated Increase in cAMP

    PubMed Central

    Vardjan, Nina; Chowdhury, Helena H.; Horvat, Anemari; Velebit, Jelena; Malnar, Maja; Muhič, Marko; Kreft, Marko; Krivec, Špela G.; Bobnar, Saša T.; Miš, Katarina; Pirkmajer, Sergej; Offermanns, Stefan; Henriksen, Gjermund; Storm-Mathisen, Jon; Bergersen, Linda H.; Zorec, Robert

    2018-01-01

    Besides being a neuronal fuel, L-lactate is also a signal in the brain. Whether extracellular L-lactate affects brain metabolism, in particular astrocytes, abundant neuroglial cells, which produce L-lactate in aerobic glycolysis, is unclear. Recent studies suggested that astrocytes express low levels of the L-lactate GPR81 receptor (EC50 ≈ 5 mM) that is in fat cells part of an autocrine loop, in which the Gi-protein mediates reduction of cytosolic cyclic adenosine monophosphate (cAMP). To study whether a similar signaling loop is present in astrocytes, affecting aerobic glycolysis, we measured the cytosolic levels of cAMP, D-glucose and L-lactate in single astrocytes using fluorescence resonance energy transfer (FRET)-based nanosensors. In contrast to the situation in fat cells, stimulation by extracellular L-lactate and the selective GPR81 agonists, 3-chloro-5-hydroxybenzoic acid (3Cl-5OH-BA) or 4-methyl-N-(5-(2-(4-methylpiperazin-1-yl)-2-oxoethyl)-4-(2-thienyl)-1,3-thiazol-2-yl)cyclohexanecarboxamide (Compound 2), like adrenergic stimulation, elevated intracellular cAMP and L-lactate in astrocytes, which was reduced by the inhibition of adenylate cyclase. Surprisingly, 3Cl-5OH-BA and Compound 2 increased cytosolic cAMP also in GPR81-knock out astrocytes, indicating that the effect is GPR81-independent and mediated by a novel, yet unidentified, excitatory L-lactate receptor-like mechanism in astrocytes that enhances aerobic glycolysis and L-lactate production via a positive feedback mechanism. PMID:29867342

  15. Changes in lactate dehydrogenase are associated with central gray matter lesions in newborns with hypoxic-ischemic encephalopathy.

    PubMed

    Yum, Sook Kyung; Moon, Cheong-Jun; Youn, Young-Ah; Sung, In Kyung

    2017-05-01

    Biomarkers may predict neurological prognosis in infants with hypoxic-ischemic encephalopathy (HIE). We evaluated the relationship between serum lactate dehydrogenase (LDH) and brain magnetic resonance imaging (MRI), which predicts neurodevelopmental outcomes, in order to assess whether LDH levels are similarly predictive. Medical records were reviewed for infants with HIE and LDH levels were assessed on the first (LDH 1 ) and third (LDH 3 ) days following birth. Receiver operating characteristic curves were obtained in relation to central gray matter hypoxic-ischemic lesions. Of 92 patients, 52 (56.5%) had hypoxic-ischemic lesions on brain MRI, and 21 of these infants (40.4%) had central gray matter lesions. LDH 1 and LDH 3 did not differ; however, the percentage change (ΔLDH%) was significantly higher in infants with central gray matter lesions (36.9% versus 6.6%, p = 0.006). With cutoffs of 187 (IU/L, ΔLDH) and 19.4 (%, ΔLDH%), the sensitivity, specificity, positive predictive value and negative predictive value were 71.4, 69.0, 40.5 and 89.1%, respectively. The relative risk was 5.57 (p = 0.001). Changes in serum LDH may be a useful biomarker for predicting future neurodevelopmental prognosis in infants with HIE.

  16. Novel strategy for phenyllactic acid biosynthesis from phenylalanine by whole cell recombinant Escherichia coli coexpressing L-phenylalanine oxidase and L-lactate dehydrogenase.

    PubMed

    Zhang, Jianzhi; Li, Xi

    2018-01-01

    To enhance the efficiency of phenyllactic acid (PLA) production from L-phenylalanine (L-Phe) by introducing a novel artificial pathway into Escherichia coli RESULTS: The production of PLA from L-Phe by recombinant E. coli (ldh-lpox) coexpressing L-phenylalanine oxidase and L-lactate dehydrogenase was studied. The new PLA synthesis pathway was confirmed to be efficient in recombinant E. coli. Subsequently, two different biocatalyst processes were carried out and optimized for PLA production. In the whole cell biosynthesis process at high cell density using collected recombinant cells as catalyst, at optimal conditions (L-Phe 6 g/l, pH 7.5, 35 °C, CDW 24.5 g/l and 200 rpm), the recombinant E. coli (ldh-lpox) produced 1.62 g PLA/l with a conversion of 28% from L-Phe. Similarly, during the two-temperature-stage fermentation process in flasks using IPTG-induced cells, the temperature in the second stage was increased to 35 °C to benefit the biocatalyst process, and comparable phenyllactic acid production of 1.47 g/l was obtained from 12 g L-Phe/l. Recombinant E. coli (ldh-lpox) was efficient in PLA production realizing a high titer of several folds compared with studies using L-Phe as substrate.

  17. Increasing anaerobic acetate consumption and ethanol yields in Saccharomyces cerevisiae with NADPH-specific alcohol dehydrogenase.

    PubMed

    Henningsen, Brooks M; Hon, Shuen; Covalla, Sean F; Sonu, Carolina; Argyros, D Aaron; Barrett, Trisha F; Wiswall, Erin; Froehlich, Allan C; Zelle, Rintze M

    2015-12-01

    Saccharomyces cerevisiae has recently been engineered to use acetate, a primary inhibitor in lignocellulosic hydrolysates, as a cosubstrate during anaerobic ethanolic fermentation. However, the original metabolic pathway devised to convert acetate to ethanol uses NADH-specific acetylating acetaldehyde dehydrogenase and alcohol dehydrogenase and quickly becomes constrained by limited NADH availability, even when glycerol formation is abolished. We present alcohol dehydrogenase as a novel target for anaerobic redox engineering of S. cerevisiae. Introduction of an NADPH-specific alcohol dehydrogenase (NADPH-ADH) not only reduces the NADH demand of the acetate-to-ethanol pathway but also allows the cell to effectively exchange NADPH for NADH during sugar fermentation. Unlike NADH, NADPH can be freely generated under anoxic conditions, via the oxidative pentose phosphate pathway. We show that an industrial bioethanol strain engineered with the original pathway (expressing acetylating acetaldehyde dehydrogenase from Bifidobacterium adolescentis and with deletions of glycerol-3-phosphate dehydrogenase genes GPD1 and GPD2) consumed 1.9 g liter(-1) acetate during fermentation of 114 g liter(-1) glucose. Combined with a decrease in glycerol production from 4.0 to 0.1 g liter(-1), this increased the ethanol yield by 4% over that for the wild type. We provide evidence that acetate consumption in this strain is indeed limited by NADH availability. By introducing an NADPH-ADH from Entamoeba histolytica and with overexpression of ACS2 and ZWF1, we increased acetate consumption to 5.3 g liter(-1) and raised the ethanol yield to 7% above the wild-type level. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  18. Lactation persistency as a component trait of the selection index and increase in reliability by using single nucleotide polymorphism in net merit defined as the first five lactation milk yields and herd life.

    PubMed

    Togashi, K; Hagiya, K; Osawa, T; Nakanishi, T; Yamazaki, T; Nagamine, Y; Lin, C Y; Matsumoto, S; Aihara, M; Hayasaka, K

    2012-08-01

    We first sought to clarify the effects of discounted rate, survival rate, and lactation persistency as a component trait of the selection index on net merit, defined as the first five lactation milks and herd life (HL) weighted by 1 and 0.389 (currently used in Japan), respectively, in units of genetic standard deviation. Survival rate increased the relative economic importance of later lactation traits and the first five lactation milk yields during the first 120 months from the start of the breeding scheme. In contrast, reliabilities of the estimated breeding value (EBV) in later lactation traits are lower than those of earlier lactation traits. We then sought to clarify the effects of applying single nucleotide polymorphism (SNP) on net merit to improve the reliability of EBV of later lactation traits to maximize their increased economic importance due to increase in survival rate. Net merit, selection accuracy, and HL increased by adding lactation persistency to the selection index whose component traits were only milk yields. Lactation persistency of the second and (especially) third parities contributed to increasing HL while maintaining the first five lactation milk yields compared with the selection index whose only component traits were milk yields. A selection index comprising the first three lactation milk yields and persistency accounted for 99.4% of net merit derived from a selection index whose components were identical to those for net merit. We consider that the selection index comprising the first three lactation milk yields and persistency is a practical method for increasing lifetime milk yield in the absence of data regarding HL. Applying SNP to the second- and third-lactation traits and HL increased net merit and HL by maximizing the increased economic importance of later lactation traits, reducing the effect of first-lactation milk yield on HL (genetic correlation (rG) = -0.006), and by augmenting the effects of the second- and third-lactation

  19. Oligosaccharide-based Surfactant/Citric Acid Buffer System Stabilizes Lactate Dehydrogenase during Freeze-drying and Storage without the Addition of Natural Sugar.

    PubMed

    Ogawa, Shigesaburo; Kawai, Ryuichiro; Koga, Maito; Asakura, Kouichi; Takahashi, Isao; Osanai, Shuichi

    2016-06-01

    Experiments were conducted to assess the maintenance effects of oligosaccharide-based surfactants on the enzymatic activity of a model protein, lactate dehydrogenase (LDH), during freeze-drying and room temperature storage using the citric acid buffer system. Oligosaccharide-based surfactants, which exhibit a high glass transition temperature (Tg), promoted the eminent retention of enzymatic activity during these protocols, whereas monosaccharide-based surfactants with a low Tg displayed poor performance at high concentration, albeit much better than that of Tween 80 at middle concentration. The increase in the alkyl chain length did not exert positive effects as observed for the maintenance effect during freeze-thawing, but an amphiphilic nature and a glass forming ability were crucial for the effective stabilization at a low excipient concentration during freeze-drying. Even a low oligosaccharide-based surfactant content (0.1 mg mL(-1)) could maintain LDH activity during freeze-drying, but a high surfactant content (1.0 mg mL(-1)) was required to prevent buffer precipitation and retain high LDH activity on storage. Regarding storage, glass formation restricted molecular mobility in the lyophilized matrix, and LDH activity was effectively retained. The present results describe a strategy based on the glass-forming ability of surfactant-type excipients that affords a natural sugar-free formulation or an alternative use for polysorbate-type surfactants.

  20. Feeding 5-hydroxy-l-tryptophan during the transition from pregnancy to lactation increases calcium mobilization from bone in rats.

    PubMed

    Laporta, J; Peters, T L; Weaver, S R; Merriman, K E; Hernandez, L L

    2013-05-01

    An increasing demand for calcium during pregnancy and lactation can result in both clinical and subclinical hypocalcemia during the early lactation period in several mammalian species, in particular the dairy cow. Serotonin (5-HT) was recently identified as a regulator of lactation and bone turnover. The purpose of this study was to determine whether supplementation of the maternal diet with a 5-HT precursor would increase maternal bone turnover and calcium mobilization to maintain appropriate circulating maternal concentrations of ionized calcium during lactation. Female Sprague-Dawley rats (n = 30) were fed either a control diet (n = 15) or a diet supplemented with the 5-HT precursor 5-hydroxytryptophan (5-HTP, 0.2%; n = 15) from day 13 of pregnancy through day 9 of lactation. Maternal serum and plasma (day 1 and day 9 of lactation), milk and pup weight (daily), mammary gland and bone tissue (day 9 of lactation) were collected for analysis. The 5-HTP diet elevated circulating maternal concentrations of 5-HT on day 1 and day 9 of lactation and parathyroid hormone related-protein (PTHrP) on day 9 of lactation (P < 0.033). In addition, 5-HTP supplementation increased total serum calcium concentrations on day 1 of lactation and total milk calcium concentration on day 9 of lactation (P < 0.032). Supplemental 5-HTP did not alter milk yield, maternal body weight, mammary gland structure, or pup litter weights (P > 0.05). Supplemental 5-HTP also resulted in increased concentrations of mammary 5-HT and PTHrP, as well as increased mRNA expression of rate-limiting enzyme in 5-HT synthesis, tryptophan hydroxylase 1, and Pthrp mRNA on day 9 of lactation (P < 0.028). In addition, supplementation of 5-HTP resulted in increased mRNA expression of maternal mammary calcium transporters and resorption of bone in the femur, indicated by increase osteoclast number and diameter as well as mRNA expression of classical markers of bone resorption on day 9 of lactation (P < 0

  1. Carnitine status and lactate increase in patients with type I juvenile diabetes.

    PubMed

    Evangeliou, A; Gourgiotis, D; Karagianni, C; Markouri, M; Anogianaki, N; Mamoulakis, D; Maropoulos, G; Tsakalidis, C; Frentzayias, A; Nicolaidou, P

    2010-12-01

    In 32 juvenile patients suffering from insulin dependent diabetes we observed a carnitine imbalance (increase in acylcarnitine and reduction of free carnitine), which was higher in patients with the highest levels of glycosylated hemoglobin. Parallel to that, in patients with the most prominent carnitine imbalance, there was the highest increase in the postprandial lactic acid level and the highest increase in the lactate/pyruvate ratio, without relating to ketosis. In addition, we observed a decrease in free carnitine related to the length of time after appearance of diabetes. This was a prospective study of a cohort of 32 children and young adolescents with insulin dependent diabetes mellitus. All patients were on insulin treatment. Plasma concentrations of total, free and acyl-Carnitine were evaluated in 12 hours fasting blood samples and before the morning administration of insulin. Blood glucose, cholesterol, triglycerides, and lactate, pyruvate, beta-hydroxybutyrate and free fatty acid levels were measured. The postprandial highest increase of the lactate and lactate/pyruvate ratio observed in patients with the highest degree of carnitine imbalance, namely with poorliest regulated diabetes, raises the question of a coincidental mitochondrial dysfunction. On the ground of our own data, such a claim cannot be substantiated for our patients. In contrast we suggest that the role of other factors like increased gluconeogenesis, degree of ketosis need to be sought. In order to clarify the role of carnitine in the pathophysiology of disease we need also data from other tissues. Carnitine in the peripheral blood reflects only the 1% of the total body carnitine ; furthermore, patients with diabetes exhibit changes in carnitine status not only in the peripheral blood but also in other body tissues, mainly in muscles.

  2. Cariogenicity of a lactate dehydrogenase-deficient mutant of Streptococcus mutans serotype c in gnotobiotic rats.

    PubMed

    Fitzgerald, R J; Adams, B O; Sandham, H J; Abhyankar, S

    1989-03-01

    A lactate dehydrogenase-deficient (Ldh-) mutant of a human isolate of Streptococcus mutans serotype c was tested in a gnotobiotic rat caries model. Compared with the wild-type Ldh-positive (Ldh+) strains, it was significantly (alpha less than or equal to 0.005) less cariogenic in experiments with two different sublines of Sprague-Dawley rats. The Ldh- mutant strain 044 colonized the oral cavity of the test animals to the same extent as its parent strain 041, although its initial implantation was slightly but not significantly (P greater than or equal to 0.2) less. Multiple oral or fecal samples plated on 2,3,5-triphenyltetrazolium indicator medium revealed no evidence of back mutation from Ldh- to Ldh+ in vivo. Both Ldh+ strain 041 and Ldh- strain 044 demonstrated bacteriocinlike activity in vitro against a number of human strains of mutans streptococci representing serotype a (S. cricetus) and serotypes c and e (S. mutans). Serotypes b (S. rattus) and f (S. mutans) and strains of S. mitior, S. sanguis, and S. salivarius were not inhibited. Thus, Ldh mutant strain 044 possesses a number of desirable traits that suggest it should be investigated further as a possible effector strain for replacement therapy of dental caries. These traits include its stability and low cariogenicity in the sensitive gnotobiotic rat caries model, its bacteriocinlike activity against certain other cariogenic S. mutans (but not against more inocuous indigenous oral streptococci), and the fact that it is a member of the most prevalent human serotype of cariogenic streptococci.

  3. A comparative proteomic analysis of Bacillus coagulans in response to lactate stress during the production of L-lactic acid.

    PubMed

    Wang, Xiuwen; Qin, Jiayang; Wang, Landong; Xu, Ping

    2014-12-01

    The growth rate and maximum biomass of Bacillus coagulans 2-6 were inhibited by lactate; inhibition by sodium lactate was stronger than by calcium lactate. The differences of protein expressions by B. coagulans 2-6 under the lactate stress were determined using two-dimensional electrophoresis coupled with mass spectrometric identification. Under the non-stress condition, calcium lactate stress and sodium lactate stress, the number of detected protein spots was 1,571 ± 117, 1,281 ± 231 and 904 ± 127, respectively. Four proteins with high expression under lactate stress were identified: lactate dehydrogenase, cysteine synthase A, aldo/keto reductase and ribosomal protein L7/L12. These proteins are thus potential targets for the reconstruction of B. coagulans to promote its resistance to lactate stress.

  4. Effect of a marathon run on serum lipoproteins, creatine kinase, and lactate dehydrogenase in recreational runners.

    PubMed

    Kobayashi, Yoshio; Takeuchi, Toshiko; Hosoi, Teruo; Yoshizaki, Hidekiyo; Loeppky, Jack A

    2005-12-01

    The objective of this study was to determine the effect of a marathon run on serum lipid and lipoprotein concentrations and serum muscle enzyme activities and follow their recovery after the run. These blood concentrations were measured before, immediately after, and serially after a marathon run in 15 male recreational runners. The triglyceride level was significantly elevated postrace, then fell 30% below baseline 1 day after the run, and returned to baseline after 1 week. Total cholesterol responded less dramatically but with a similar pattern. High-density lipoprotein cholesterol remained significantly elevated and low-density lipoprotein cholesterol was transiently reduced for 3 days after the run. The total cholesterol/high-density cholesterol ratio was significantly lowered for 3 days. Serum lactate dehydrogenase activity significantly doubled postrace and then declined but remained elevated for 2 weeks. Serum creatine kinase activity peaked 24 hr after the run, with a 15-fold rise, and returned to baseline after 1 week. The rise of these enzymes reflects mechanically damaged muscle cells leaking contents into the interstitial fluid. It is concluded that a prolonged strenuous exercise bout in recreational runners, such as a marathon, produces beneficial changes in lipid blood profiles that are significant for only 3 days. However, muscle damage is also evident for 1 week or more from the dramatic and long-lasting effect on enzyme levels. Laboratory values for these runners were outside normal ranges for some days after the race.

  5. Total lactate dehydrogenase activity of tail muscle is not cold-adapted in nocturnal lizards from cool-temperate habitats.

    PubMed

    Hare, K M; Miller, J H; Clark, A G; Daugherty, C H

    2005-12-01

    The dependence of metabolic processes on temperature constrains the behavior, physiology and ecology of many ectothermic animals. The evolution of nocturnality in lizards, especially in temperate regions, requires adaptations for activity at low temperatures when optimal body temperatures are unlikely to be obtained. We examined whether nocturnal lizards have cold-adapted lactate dehydrogenase (LDH). LDH was chosen as a representative metabolic enzyme. We measured LDH activity of tail muscle in six lizard species (n=123: three nocturnal, two diurnal and one crepuscular) between 5 and 35 degrees C and found no differences in LDH-specific activity or thermal sensitivity among the species. Similarly, the specific activity and thermal sensitivity of LDH were similar between skinks and geckos. Similar enzyme activities among nocturnal and diurnal lizards indicate that there is no selection of temperature specific LDH enzyme activity at any temperature. As many nocturnal lizards actively thermoregulate during the day, LDH may be adapted for a broad range of temperatures rather than adapted specifically for the low temperatures encountered when the animals are active. The total activity of LDH in tropical and temperate lizards is not cold-adapted. More data are required on biochemical adaptations and whole animal thermal preferences before trends can be established.

  6. Characterization of lactate utilization and its implication on the physiology of Haemophilus influenzae.

    PubMed

    Lichtenegger, Sabine; Bina, Isabelle; Roier, Sandro; Bauernfeind, Stilla; Keidel, Kristina; Schild, Stefan; Anthony, Mark; Reidl, Joachim

    2014-05-01

    Haemophilus influenzae is a Gram-negative bacillus and a frequent commensal of the human nasopharynx. Earlier work demonstrated that in H. influenzae type b, l-lactate metabolism is associated with serum resistance and in vivo survival of the organism. To further gain insight into lactate utilization of the non-typeable (NTHi) isolate 2019 and laboratory prototype strain Rd KW20, deletion mutants of the l-lactate dehydrogenase (lctD) and permease (lctP) were generated and characterized. It is shown, that the apparent KM of l-lactate uptake is 20.1μM as determined for strain Rd KW20. Comparison of the COPD isolate NTHi 2019-R with the corresponding lctP knockout strain for survival in human serum revealed no lactate dependent serum resistance. In contrast, we observed a 4-fold attenuation of the mutant strain in a murine model of nasopharyngeal colonization. Characterization of lctP transcriptional control shows that the lactate utilization system in H. influenzae is not an inductor inducible system. Rather negative feedback regulation was observed in the presence of l-lactate and this is dependent on the ArcAB regulatory system. Additionally, for 2019 it was found that lactate may have signaling function leading to increased cell growth in late log phase under conditions where no l-lactate is metabolized. This effect seems to be ArcA independent and was not observed in strain Rd KW20. We conclude that l-lactate is an important carbon-source and may act as host specific signal substrate which fine tunes the globally acting ArcAB regulon and may additionally affect a yet unknown signaling system and thus may contribute to enhanced in vivo survival. Copyright © 2014 Elsevier GmbH. All rights reserved.

  7. Advanced Stage, Increased Lactate Dehydrogenase, and Primary Site, but Not Adolescent Age (≥ 15 Years), Are Associated With an Increased Risk of Treatment Failure in Children and Adolescents With Mature B-Cell Non-Hodgkin's Lymphoma: Results of the FAB LMB 96 Study

    PubMed Central

    Cairo, Mitchell S.; Sposto, Richard; Gerrard, Mary; Auperin, Anne; Goldman, Stanton C.; Harrison, Lauren; Pinkerton, Ross; Raphael, Martine; McCarthy, Keith; Perkins, Sherrie L.; Patte, Catherine

    2012-01-01

    Purpose Adolescents (age 15 to 21 years) compared with younger children with mature B-cell non-Hodgkin's lymphoma (NHL) have been historically considered to have an inferior prognosis. We therefore analyzed the impact of age and other diagnostic factors on the risk of treatment failure in children and adolescents treated on the French-American-British Mature B-Cell Lymphoma 96 (FAB LMB 96) trial. Patients and Methods Patients were divided by risk: group A (limited), group B (intermediate), and group C (advanced), as previously described. Prognostic factors analyzed for event-free survival (EFS) included age (< 15 v ≥ 15 years), stage (I/II v III/IV), primary site, lactate dehydrogenase (LDH), bone marrow/CNS (BM/CNS) involvement, and histology (diffuse large B-cell lymphoma v mediastinal B-cell lymphoma v Burkitt lymphoma or Burkitt-like lymphoma). Results The 3-year EFS for the whole cohort was 88% ± 1%. Age was not associated as a risk factor for increased treatment failure in either univariate analysis (P = .15) or multivariate analysis (P = .58). Increased LDH (≥ 2 × upper limit of normal [ULN] v < 2 × ULN), primary site, and BM-positive/CNS-positive disease were all independent risk factors associated with a significant increase in treatment failure rate (relative risk, 2.0; P < .001, P < .012, and P < .001, respectively). Conclusion LDH level at diagnosis, mediastinal disease, and combined BM-positive/CNS-positive involvement are independent risk factors in children with mature B-cell NHL. Future studies should be developed to identify specific therapeutic strategies (immunotherapy) to overcome these risk factors and to identify the biologic basis associated with these prognostic factors in children with mature B-cell NHL. PMID:22215753

  8. Salivary lactate dehydrogenase levels can provide early diagnosis of hypoxic-ischaemic encephalopathy in neonates with birth asphyxia.

    PubMed

    Mehta, Akshay; Chawla, Deepak; Kaur, Jasbinder; Mahajan, Vidushi; Guglani, Vishal

    2015-06-01

    Timely detection of hypoxic-ischaemic encephalopathy (HIE) is crucial for selecting neonates who are likely to benefit from neuroprotective therapy. This study evaluated the efficacy of salivary lactate dehydrogenase (LDH) in the early diagnosis of HIE among neonates with perinatal asphyxia. We prospectively enrolled 30 neonates who needed resuscitation at birth or had a history of delayed cry into the HIE group if they developed HIE within 12 h of birth. The control group comprised 30 neonates who had no evidence of HIE, but had intrapartum foetal distress or needed resuscitation at birth. LDH was measured using saliva samples collected within 12 h of birth. Salivary LDH was significantly higher in the HIE group, with a median of 2578 and an interquartile range (IQR) of 1379-3408 international units per litre (IU/L), than in the control group (median 558.5, IQR: 348-924 IU/L, p < 0.001). The test demonstrated excellent discriminating ability: the area under the curve was 0.92 and the levels of 893 IU/L showed a sensitivity of 90% and a specificity of 73.3%. Measuring salivary LDH among neonates with birth asphyxia provided an early and accurate diagnosis of HIE and could be used as a triage tool. ©2015 Foundation Acta Paediatrica. Published by John Wiley & Sons Ltd.

  9. Lactation counseling increases exclusive breast-feeding rates in Ghana.

    PubMed

    Aidam, Bridget A; Pérez-Escamilla, Rafael; Lartey, Anna

    2005-07-01

    Exclusive breast-feeding (EBF) rates remain low despite numerous health benefits associated with this behavior. We conducted a randomized trial on the effect of lactation counseling on EBF, which controlled for the Hawthorne effect while also varying the timing of the intervention. Pregnant women attending prenatal clinics in Tema were randomly assigned to 1 of 2 intervention groups (IG) or to a control group (C), as follows: 1) EBF support given pre-, peri-, and postnatally (IG1; n = 43); 2) EBF support given only peri- and postnatally (IG2; n = 44); or 3) nonbreast-feeding health educational support (C; n = 49) that had an equal amount of contact with lactation counselors. Two educational sessions were provided prenatally, and 9 home follow-up visits were provided in the 6-mo postpartum period. Infant feeding data were collected monthly at the participant's home. The 3 groups did not differ in sociodemographic characteristics. At 6 mo postpartum, 90.0% in IG1 and 74.4% in IG2 had exclusively breast-fed during the previous month. By contrast, only 47.7% in C were doing so (P = 0.008). Similarly, the percentage of EBF during the 6 mo was significantly higher (P = 0.02) among IG1 and IG2 (39.5%) than among C (19.6%). The 100% increase in EBF rates can be attributed to the lactation counseling provided. Additional prenatal EBF support may not be needed within a context of strong routine prenatal EBF education.

  10. Moderate hyperventilation during intravenous anesthesia increases net cerebral lactate efflux.

    PubMed

    Grüne, Frank; Kazmaier, Stephan; Sonntag, Hans; Stolker, Robert Jan; Weyland, Andreas

    2014-02-01

    Hyperventilation is known to decrease cerebral blood flow (CBF) and to impair cerebral metabolism, but the threshold in patients undergoing intravenous anesthesia is unknown. The authors hypothesized that reduced CBF associated with moderate hyperventilation might impair cerebral aerobic metabolism in patients undergoing intravenous anesthesia. Thirty male patients scheduled for coronary surgery were included in a prospective, controlled crossover trial. Measurements were performed under fentanyl-midazolam anesthesia in a randomized sequence aiming at partial pressures of carbon dioxide of 30 and 50 mmHg. Endpoints were CBF, blood flow velocity in the middle cerebral artery, and cerebral metabolic rates for oxygen, glucose, and lactate. Global CBF was measured using a modified Kety-Schmidt technique with argon as inert gas tracer. CBF velocity of the middle cerebral artery was recorded by transcranial Doppler sonography. Data were presented as mean (SD). Two-sided paired t tests and one-way ANOVA for repeated measures were used for statistical analysis. Moderate hyperventilation significantly decreased CBF by 60%, blood flow velocity by 41%, cerebral oxygen delivery by 58%, and partial pressure of oxygen of the jugular venous bulb by 45%. Cerebral metabolic rates for oxygen and glucose remained unchanged; however, net cerebral lactate efflux significantly increased from -0.38 (2.18) to -2.41(2.43) µmol min 100 g. Moderate hyperventilation, when compared with moderate hypoventilation, in patients with cardiovascular disease undergoing intravenous anesthesia increased net cerebral lactate efflux and markedly reduced CBF and partial pressure of oxygen of the jugular venous bulb, suggesting partial impairment of cerebral aerobic metabolism at clinically relevant levels of hypocapnia.

  11. Genetic polymorphism and isoenzyme patterns of lactate dehydrogenase in tench (Tinca tinca), crucian carp (Carassius carassius) and carp (Cyprinus carpio).

    PubMed

    Valenta, M; Slechta, V; Slechtová, V; Kálal, L

    1977-01-01

    Isoenzyme patterns and the polymorphism of lactate dehydrogenase (LDH) were investigated in 3 fish species of family Cyprinidae, i.e. tench (Tinca tinca), crucian carp (Carassius carassius) and carp (Cyprinus carpio). The isoenzyme patterns were tissue and species specific. In crucian carp subunits with different electrophoretic mobility are present, which are genetically controlled from the B1, B2, A1, A2 and C loci, while the set of loci in carp is B1, B2, A, C1 and C2 and in tench B, A, C. The locus B of LDH in tench, the locus B2 in crucian carp, and the loci B1, C1 and C2 in carp are polymorphic and have two different alleles in each case. The polymorphism did not affect the total LDH activity in the tissues. All the populations investigated were in Hardy-Weinberg equilibrium. The genetic control of the polymorphism in B1 and C1 loci in carp was proved by test matings. The polymorphism in B loci tested in erythrocytes may be utilized as genetic markers in the fish breeding.

  12. Ischaemic Priapism and Glucose-6-Phosphate Dehydrogenase Deficiency: A Mechanism of Increased Oxidative Stress?

    PubMed

    Morrison, B F; Thompson, E B; Shah, S D; Wharfe, G H

    2014-07-03

    Ischaemic priapism is a devastating urological condition that has the potential to cause permanent erectile dysfunction. The disorder has been associated with numerous medical conditions and the use of pharmacotherapeutic agents. The aetiology is idiopathic in a number of cases. There are two prior case reports of the association of ischaemic priapism and glucose-6-phosphate dehydrogenase (G6PD) deficiency. We report on a third case of priapism associated with G6PD deficiency and review recently described molecular mechanisms of increased oxidative stress in the pathophysiology of ischaemic priapism. The case report of a 32-year old Afro-Caribbean male with his first episode of major ischaemic priapism is described. Screening for common causes of ischaemic priapism, including sickle cell disease was negative. Glucose-6-phosphate dehydrogenase deficiency was discovered on evaluation for priapism. Penile aspiration was performed and erectile function was good post treatment.Glucose-6-phosphate dehydrogenase deficiency is a cause for ischaemic priapism and should be a part of the screening process in idiopathic causes of the disorder. Increased oxidative stress occurs in G6PD deficiency and may lead to priapism.

  13. Effects of Sesame (Sesamum indicum L.) Supplementation on Creatine Kinase, Lactate Dehydrogenase, Oxidative Stress Markers, and Aerobic Capacity in Semi-Professional Soccer Players

    PubMed Central

    Barbosa, Carlos V. da Silva; Silva, Alexandre S.; de Oliveira, Caio V. C.; Massa, Nayara M. L.; de Sousa, Yasmim R. F.; da Costa, Whyara K. A.; Silva, Ayice C.; Delatorre, Plínio; Carvalho, Rhayane; Braga, Valdir de Andrade; Magnani, Marciane

    2017-01-01

    Nutritional intervention with antioxidants rich foods has been considered a strategy to minimize the effects of overtraining in athletes. This experimental, randomized, and placebo-controlled study evaluated the effects of consumption of sesame (Sesamum indicum L.) on muscle damage markers, oxidative stress, systemic inflammation, and aerobic performance in male semi-professional soccer players. Twenty athletes were randomly assigned to groups that received 40 g (two tablespoons) per day of sesame or a placebo during 28 days of regular training (exposed to routine training that includes loads of heavy training in the final half of the season). Before and after intervention, creatine kinase (CK), lactate dehydrogenase (LDH), malondialdehyde (MDA), superoxide dismutase (SOD), C-reactive protein (hs-CRP), and aerobic capacity were evaluated. Before intervention, a physiologic imbalance was noted in both groups related to CK and LDH levels. Sesame intake caused a reduction of CK (19%, p < 0.05), LDH (37%, p < 0.05), MDA (55%, p < 0.05) and hs-CRP (53%, p < 0.05) and increased SOD (14%, p < 0.05), vitamin A (25%, p < 0.05), and vitamin E (65%, p < 0.05) in the experimental group. These phenomena were accompanied by increased aerobic capacity (17%, p < 0.05). The placebo group showed an increase in CK (5%, p < 0.05) and no significant change in LDH, SOD or vitamin A. MDA levels decreased (21%, p < 0.05) and vitamin E increased (14%, p < 0.05) in the placebo group, but to a much lesser extent than in the experimental group. These results show that sesame consumption may reduce muscle damage and oxidative stress while improving the aerobic capacity in soccer players. PMID:28408889

  14. Saccharomyces cerevisiae Forms d-2-Hydroxyglutarate and Couples Its Degradation to d-Lactate Formation via a Cytosolic Transhydrogenase*♦

    PubMed Central

    Becker-Kettern, Julia; Paczia, Nicole; Conrotte, Jean-François; Kay, Daniel P.; Guignard, Cédric; Jung, Paul P.; Linster, Carole L.

    2016-01-01

    The d or l form of 2-hydroxyglutarate (2HG) accumulates in certain rare neurometabolic disorders, and high d-2-hydroxyglutarate (d-2HG) levels are also found in several types of cancer. Although 2HG has been detected in Saccharomyces cerevisiae, its metabolism in yeast has remained largely unexplored. Here, we show that S. cerevisiae actively forms the d enantiomer of 2HG. Accordingly, the S. cerevisiae genome encodes two homologs of the human d-2HG dehydrogenase: Dld2, which, as its human homolog, is a mitochondrial protein, and the cytosolic protein Dld3. Intriguingly, we found that a dld3Δ knock-out strain accumulates millimolar levels of d-2HG, whereas a dld2Δ knock-out strain displayed only very moderate increases in d-2HG. Recombinant Dld2 and Dld3, both currently annotated as d-lactate dehydrogenases, efficiently oxidized d-2HG to α-ketoglutarate. Depletion of d-lactate levels in the dld3Δ, but not in the dld2Δ mutant, led to the discovery of a new type of enzymatic activity, carried by Dld3, to convert d-2HG to α-ketoglutarate, namely an FAD-dependent transhydrogenase activity using pyruvate as a hydrogen acceptor. We also provide evidence that Ser3 and Ser33, which are primarily known for oxidizing 3-phosphoglycerate in the main serine biosynthesis pathway, in addition reduce α-ketoglutarate to d-2HG using NADH and represent major intracellular sources of d-2HG in yeast. Based on our observations, we propose that d-2HG is mainly formed and degraded in the cytosol of S. cerevisiae cells in a process that couples d-2HG metabolism to the shuttling of reducing equivalents from cytosolic NADH to the mitochondrial respiratory chain via the d-lactate dehydrogenase Dld1. PMID:26774271

  15. Systematic Engineering of Escherichia coli for d-Lactate Production from Crude Glycerol.

    PubMed

    Wang, Zei Wen; Saini, Mukesh; Lin, Li-Jen; Chiang, Chung-Jen; Chao, Yun-Peng

    2015-11-04

    Crude glycerol resulting from biodiesel production is an abundant and renewable resource. However, the impurities in crude glycerol usually make microbial fermentation problematic. This issue was addressed by systematic engineering of Escherichia coli for the production of d-lactate from crude glycerol. First, mgsA and the synthetic pathways of undesired products were eliminated in E. coli, rendering the strain capable of homofermentative production of optically pure d-lactate. To direct carbon flux toward d-lactate, the resulting strain was endowed with an enhanced expression of glpD-glpK in the glycerol catabolism and of a heterologous gene encoding d-lactate dehydrogenase. Moreover, the strain was evolved to improve its utilization of cruder glycerol and subsequently equipped with the FocA channel to export intracellular d-lactate. Finally, the fed-batch fermentation with two-phase culturing was carried out with a bioreactor. As a result, the engineered strain enabled production of 105 g/L d-lactate (99.9% optical purity) from 121 g/L crude glycerol at 40 h. The result indicates the feasibility of our approach to engineering E. coli for the crude glycerol-based fermentation.

  16. Dichloroacetate effects on glucose and lactate oxidation by neurons and astroglia in vitro and on glucose utilization by brain in vivo.

    PubMed

    Itoh, Yoshiaki; Esaki, Takanori; Shimoji, Kazuaki; Cook, Michelle; Law, Mona J; Kaufman, Elaine; Sokoloff, Louis

    2003-04-15

    Neuronal cultures in vitro readily oxidized both D-[(14)C]glucose and l-[(14)C]lactate to (14)CO(2), whereas astroglial cultures oxidized both substrates sparingly and metabolized glucose predominantly to lactate and released it into the medium. [(14)C]Glucose oxidation to (14)CO(2) varied inversely with unlabeled lactate concentration in the medium, particularly in neurons, and increased progressively with decreasing lactate concentration. Adding unlabeled glucose to the medium inhibited [(14)C]lactate oxidation to (14)CO(2) only in astroglia but not in neurons, indicating a kinetic preference in neurons for oxidation of extracellular lactate over intracellular pyruvatelactate produced by glycolysis. Protein kinase-catalyzed phosphorylation inactivates pyruvate dehydrogenase (PDH), which regulates pyruvate entry into the tricarboxylic acid cycle. Dichloroacetate inhibits this kinase, thus enhancing PDH activity. In vitro dichloroacetate stimulated glucose and lactate oxidation to CO(2) and reduced lactate release mainly in astroglia, indicating that limitations in glucose and lactate oxidation by astroglia may be due to a greater balance of PDH toward the inactive form. To assess the significance of astroglial export of lactate to neurons in vivo, we attempted to diminish this traffic in rats by administering dichloroacetate (50 mgkg) intravenously to stimulate astroglial lactate oxidation and then examined the effects on baseline and functionally activated local cerebral glucose utilization (lCMR(glc)). Dichloroacetate raised baseline lCMR(glc) throughout the brain and decreased the percent increases in lCMR(glc) evoked by functional activation. These studies provide evidence in support of the compartmentalization of glucose metabolism between astroglia and neurons but indicate that the compartmentalization may be neither complete nor entirely obligatory.

  17. Rapamycin (mTORC1 inhibitor) reduces the production of lactate and 2-hydroxyglutarate oncometabolites in IDH1 mutant fibrosarcoma cells.

    PubMed

    Hujber, Zoltán; Petővári, Gábor; Szoboszlai, Norbert; Dankó, Titanilla; Nagy, Noémi; Kriston, Csilla; Krencz, Ildikó; Paku, Sándor; Ozohanics, Olivér; Drahos, László; Jeney, András; Sebestyén, Anna

    2017-06-02

    Multiple studies concluded that oncometabolites (e.g. D-2-hydroxyglutarate (2-HG) related to mutant isocitrate dehydrogenase 1/2 (IDH1/2) and lactate) have tumour promoting potential. Regulatory mechanisms implicated in the maintenance of oncometabolite production have great interest. mTOR (mammalian target of rapamycin) orchestrates different pathways, influences cellular growth and metabolism. Considering hyperactivation of mTOR in several malignancies, the question has been addressed whether mTOR operates through controlling of oncometabolite accumulation in metabolic reprogramming. HT-1080 cells - carrying originally endogenous IDH1 mutation - were used in vitro and in vivo. Anti-tumour effects of rapamycin were studied using different assays. The main sources and productions of the oncometabolites (2-HG and lactate) were analysed by 13 C-labeled substrates. Alterations at protein and metabolite levels were followed by Western blot, flow cytometry, immunohistochemistry and liquid chromatography mass spectrometry using rapamycin, PP242 and different glutaminase inhibitors, as well. Rapamycin (mTORC1 inhibitor) inhibited proliferation, migration and altered the metabolic activity of IDH1 mutant HT-1080 cells. Rapamycin reduced the level of 2-HG sourced mainly from glutamine and glucose derived lactate which correlated to the decreased incorporation of 13 C atoms from 13 C-substrates. Additionally, decreased expressions of lactate dehydrogenase A and glutaminase were also observed both in vitro and in vivo. Considering the role of lactate and 2-HG in regulatory network and in metabolic symbiosis it could be assumed that mTOR inhibitors have additional effects besides their anti-proliferative effects in tumours with glycolytic phenotype, especially in case of IDH1 mutation (e.g. acute myeloid leukemias, gliomas, chondrosarcomas). Based on our new results, we suggest targeting mTOR activity depending on the metabolic and besides molecular genetic phenotype of

  18. Low anaerobic threshold and increased skeletal muscle lactate production in subjects with Huntington's disease.

    PubMed

    Ciammola, Andrea; Sassone, Jenny; Sciacco, Monica; Mencacci, Niccolò E; Ripolone, Michela; Bizzi, Caterina; Colciago, Clarissa; Moggio, Maurizio; Parati, Gianfranco; Silani, Vincenzo; Malfatto, Gabriella

    2011-01-01

    Mitochondrial defects that affect cellular energy metabolism have long been implicated in the etiology of Huntington's disease (HD). Indeed, several studies have found defects in the mitochondrial functions of the central nervous system and peripheral tissues of HD patients. In this study, we investigated the in vivo oxidative metabolism of exercising muscle in HD patients. Ventilatory and cardiometabolic parameters and plasma lactate concentrations were monitored during incremental cardiopulmonary exercise in twenty-five HD subjects and twenty-five healthy subjects. The total exercise capacity was normal in HD subjects but notably the HD patients and presymptomatic mutation carriers had a lower anaerobic threshold than the control subjects. The low anaerobic threshold of HD patients was associated with an increase in the concentration of plasma lactate. We also analyzed in vitro muscular cell cultures and found that HD cells produce more lactate than the cells of healthy subjects. Finally, we analyzed skeletal muscle samples by electron microscopy and we observed striking mitochondrial structural abnormalities in two out of seven HD subjects. Our findings confirm mitochondrial abnormalities in HD patients' skeletal muscle and suggest that the mitochondrial dysfunction is reflected functionally in a low anaerobic threshold and an increased lactate synthesis during intense physical exercise. Copyright © 2010 Movement Disorder Society.

  19. The effect of extracellular alkalinization on lactate metabolism of breast cancer stem cells: Overview of LDH-A, LDH-B, MCT1 and MCT4 gene expression

    NASA Astrophysics Data System (ADS)

    Neolaka, G. M. G.; Yustisia, I.; Sadikin, M.; Wanandi, S. I.

    2017-08-01

    Changes in the metabolic status of cancer cells are presumed to be correlated with the adjustment of these cells to extracellular changes. Cell glycolysis increases the production of intracellular lactate catalyzed by the lactate dehydrogenases, both LDH-A and LDH-B. An increase in intracellular lactate can affect extracellular pH balance through monocarboxylate transporters, particularly MCT1 and MCT4. This study aimed to analyze the effects of extracellular alkalinization on the lactate metabolism of human breast cancer stem cells (BCSCs). In this study, human primary BCSCs (CD24-/CD44+ cells) were treated with 100 mM sodium bicarbonate for 0.5, 24, and 48 h in DMEM F12/HEPES. After incubation, extracellular pH was measured and cells were harvested to extract the total RNA and protein. The expression of LDH-A, LDH-B, MCT1, and MCT4 mRNA genes were analyzed using qRT-PCR method. Our study shows that administration of sodium bicarbonate in the BCSC culture medium could increase extracellular pH. To balance the increase of extracellular pH, BCSCs regulated the expression of LDH-A, LDH-B, MCT1, and MCT4 genes. As the extracellular pH increases, the expression of LDH-A that converts pyruvate to lactate increased along with the increase of MCT 4 and MCT 1 expression, which act as lactate transporters. As the incubation time increases, the pH decreases, leading to the suppression of LDH-A and increase of LDH-B expression that converts lactate into pyruvate. Therefore, we suggest that the extracellular alkalinization by sodium bicarbonate in BCSCs affected the genes that regulate lactate metabolism.

  20. Bioconversion of methane to lactate by an obligate methanotrophic bacterium

    DOE PAGES

    Henard, Calvin A.; Smith, Holly; Dowe, Nancy; ...

    2016-02-23

    Methane is the second most abundant greenhouse gas (GHG), with nearly 60% of emissions derived from anthropogenic sources. Microbial conversion of methane to fuels and value-added chemicals offers a means to reduce GHG emissions, while also valorizing this otherwise squandered high-volume, high-energy gas. However, to date, advances in methane biocatalysis have been constrained by the low-productivity and limited genetic tractability of natural methane-consuming microbes. Here, leveraging recent identification of a novel, tractable methanotrophic bacterium, Methylomicrobium buryatense, we demonstrate microbial biocatalysis of methane to lactate, an industrial platform chemical. Heterologous overexpression of a Lactobacillus helveticus L-lactate dehydrogenase in M. buryatense resultedmore » in an initial titer of 0.06 g lactate/L from methane. Cultivation in a 5 L continuously stirred tank bioreactor enabled production of 0.8 g lactate/L, representing a 13-fold improvement compared to the initial titer. The yields (0.05 g lactate/g methane) and productivity (0.008 g lactate/L/h) indicate the need and opportunity for future strain improvement. Additionally, real-time analysis of methane utilization implicated gas-to-liquid transfer and/or microbial methane consumption as process limitations. This work opens the door to develop an array of methanotrophic bacterial strain-engineering strategies currently employed for biocatalytic sugar upgrading to “green” chemicals and fuels.« less

  1. Bioconversion of methane to lactate by an obligate methanotrophic bacterium

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Henard, Calvin A.; Smith, Holly; Dowe, Nancy

    Methane is the second most abundant greenhouse gas (GHG), with nearly 60% of emissions derived from anthropogenic sources. Microbial conversion of methane to fuels and value-added chemicals offers a means to reduce GHG emissions, while also valorizing this otherwise squandered high-volume, high-energy gas. However, to date, advances in methane biocatalysis have been constrained by the low-productivity and limited genetic tractability of natural methane-consuming microbes. Here, leveraging recent identification of a novel, tractable methanotrophic bacterium, Methylomicrobium buryatense, we demonstrate microbial biocatalysis of methane to lactate, an industrial platform chemical. Heterologous overexpression of a Lactobacillus helveticus L-lactate dehydrogenase in M. buryatense resultedmore » in an initial titer of 0.06 g lactate/L from methane. Cultivation in a 5 L continuously stirred tank bioreactor enabled production of 0.8 g lactate/L, representing a 13-fold improvement compared to the initial titer. The yields (0.05 g lactate/g methane) and productivity (0.008 g lactate/L/h) indicate the need and opportunity for future strain improvement. Additionally, real-time analysis of methane utilization implicated gas-to-liquid transfer and/or microbial methane consumption as process limitations. This work opens the door to develop an array of methanotrophic bacterial strain-engineering strategies currently employed for biocatalytic sugar upgrading to “green” chemicals and fuels.« less

  2. Bioconversion of methane to lactate by an obligate methanotrophic bacterium

    PubMed Central

    Henard, Calvin A.; Smith, Holly; Dowe, Nancy; Kalyuzhnaya, Marina G.; Pienkos, Philip T.; Guarnieri, Michael T.

    2016-01-01

    Methane is the second most abundant greenhouse gas (GHG), with nearly 60% of emissions derived from anthropogenic sources. Microbial conversion of methane to fuels and value-added chemicals offers a means to reduce GHG emissions, while also valorizing this otherwise squandered high-volume, high-energy gas. However, to date, advances in methane biocatalysis have been constrained by the low-productivity and limited genetic tractability of natural methane-consuming microbes. Here, leveraging recent identification of a novel, tractable methanotrophic bacterium, Methylomicrobium buryatense, we demonstrate microbial biocatalysis of methane to lactate, an industrial platform chemical. Heterologous overexpression of a Lactobacillus helveticus L-lactate dehydrogenase in M. buryatense resulted in an initial titer of 0.06 g lactate/L from methane. Cultivation in a 5 L continuously stirred tank bioreactor enabled production of 0.8 g lactate/L, representing a 13-fold improvement compared to the initial titer. The yields (0.05 g lactate/g methane) and productivity (0.008 g lactate/L/h) indicate the need and opportunity for future strain improvement. Additionally, real-time analysis of methane utilization implicated gas-to-liquid transfer and/or microbial methane consumption as process limitations. This work opens the door to develop an array of methanotrophic bacterial strain-engineering strategies currently employed for biocatalytic sugar upgrading to “green” chemicals and fuels. PMID:26902345

  3. Antimalarial Activity of Potential Inhibitors of Plasmodium falciparum Lactate Dehydrogenase Enzyme Selected by Docking Studies

    PubMed Central

    Penna-Coutinho, Julia; Cortopassi, Wilian Augusto; Oliveira, Aline Alves; França, Tanos Celmar Costa; Krettli, Antoniana Ursine

    2011-01-01

    The Plasmodium falciparum lactate dehydrogenase enzyme (PfLDH) has been considered as a potential molecular target for antimalarials due to this parasite's dependence on glycolysis for energy production. Because the LDH enzymes found in P. vivax, P. malariae and P. ovale (pLDH) all exhibit ∼90% identity to PfLDH, it would be desirable to have new anti-pLDH drugs, particularly ones that are effective against P. falciparum, the most virulent species of human malaria. Our present work used docking studies to select potential inhibitors of pLDH, which were then tested for antimalarial activity against P. falciparum in vitro and P. berghei malaria in mice. A virtual screening in DrugBank for analogs of NADH (an essential cofactor to pLDH) and computational studies were undertaken, and the potential binding of the selected compounds to the PfLDH active site was analyzed using Molegro Virtual Docker software. Fifty compounds were selected based on their similarity to NADH. The compounds with the best binding energies (itraconazole, atorvastatin and posaconazole) were tested against P. falciparum chloroquine-resistant blood parasites. All three compounds proved to be active in two immunoenzymatic assays performed in parallel using monoclonals specific to PfLDH or a histidine rich protein (HRP2). The IC50 values for each drug in both tests were similar, were lowest for posaconazole (<5 µM) and were 40- and 100-fold less active than chloroquine. The compounds reduced P. berghei parasitemia in treated mice, in comparison to untreated controls; itraconazole was the least active compound. The results of these activity trials confirmed that molecular docking studies are an important strategy for discovering new antimalarial drugs. This approach is more practical and less expensive than discovering novel compounds that require studies on human toxicology, since these compounds are already commercially available and thus approved for human use. PMID:21779323

  4. Increased circulating D-lactate levels predict risk of mortality after hemorrhage and surgical trauma in baboons.

    PubMed

    Sobhian, Babak; Kröpfl, Albert; Hölzenbein, Thomas; Khadem, Anna; Redl, Heinz; Bahrami, Soheyl

    2012-05-01

    Patients with hemorrhagic shock and/or trauma are at risk of developing colonic ischemia associated with bacterial translocation that may lead to multiple organ failure and death. Intestinal ischemia is difficult to diagnose noninvasively. The present retrospective study was designed to determine whether circulating plasma D-lactate is associated with mortality in a clinically relevant two-hit model in baboons. Hemorrhagic shock was induced in anesthetized baboons (n = 24) by controlled bleeding (mean arterial pressure, 40 mmHg), base excess (maximum -5 mmol/L), and time (maximum 3 h). To mimic clinical setting more closely, all animals underwent a surgical trauma after resuscitation including midshaft osteotomy stabilized with reamed femoral interlocking nailing and were followed for 7 days. Hemorrhagic shock/surgical trauma resulted in 66% mortality by day 7. In nonsurvivor (n = 16) hemorrhagic shock/surgical trauma baboons, circulating D-lactate levels were significantly increased (2-fold) at 24 h compared with survivors (n = 8), whereas the early increase during hemorrhage and resuscitation declined during the early postresuscitation phase with no difference between survivors and nonsurvivors. Moreover, D-lactate levels remained elevated in the nonsurvival group until death, whereas it decreased to baseline in survivors. Prediction of death (receiver operating characteristic test) by D-lactate was accurate with an area under the curve (days 1-3 after trauma) of 0.85 (95% confidence interval, 0.72-0.93). The optimal D-lactate cutoff value of 25.34 μg/mL produced sensitivity of 73% to 99% and specificity of 50% to 83%. Our data suggest that elevation of plasma D-lactate after 24 h predicts an increased risk of mortality after hemorrhage and trauma.

  5. Transport of pyruvate and lactate in yeast mitochondria.

    PubMed

    Briquet, M

    1977-02-07

    Evidence for the existence of mediated transport of pyruvate and lactate in isolated mitochondria of Saccharomyces cerevisiae is presented. 1. The mitochondrial oxidation of pyruvate is specifically inhibited by the monocarboxylic oxoacids alpha-ketoisocaproate and by alpha-cyano-3-hydroxycinnamate, while pyruvate and malate dehydrogenases activities are not inhibited. 2. The stimulation of the mitochondrial oxidations of succinate, alpha-ketoglutarate and citrate by pyruvate are also inhibited by alpha-cyano-3-hydroxycinnamate. 3. The [14C]pyruvate uptake by yeast mitochondria follows saturation kinetics and is completely inhibited by alpha-cyano-3-hydroxycinnamate. 4. Large amplitude passive swellings of mitochondria of the wild type and of cytoplasmic rho- and rho-n mutants are induced by isoosmotic ammonium pyruvate and lactate. These pH-dependent swellings are inhibited by alpha-cyano-3-hydroxycinnamate suggesting that the carrier system is not coded by mitochondrial DNA.

  6. Increased brain lactate is central to the development of brain edema in rats with chronic liver disease.

    PubMed

    Bosoi, Cristina R; Zwingmann, Claudia; Marin, Helen; Parent-Robitaille, Christian; Huynh, Jimmy; Tremblay, Mélanie; Rose, Christopher F

    2014-03-01

    The pathogenesis of brain edema in patients with chronic liver disease (CLD) and minimal hepatic encephalopathy (HE) remains undefined. This study evaluated the role of brain lactate, glutamine and organic osmolytes, including myo-inositol and taurine, in the development of brain edema in a rat model of cirrhosis. Six-week bile-duct ligated (BDL) rats were injected with (13)C-glucose and de novo synthesis of lactate, and glutamine in the brain was quantified using (13)C nuclear magnetic resonance spectroscopy (NMR). Total brain lactate, glutamine, and osmolytes were measured using (1)H NMR or high performance liquid chromatography. To further define the interplay between lactate, glutamine and brain edema, BDL rats were treated with AST-120 (engineered activated carbon microspheres) and dichloroacetate (DCA: lactate synthesis inhibitor). Significant increases in de novo synthesis of lactate (1.6-fold, p<0.001) and glutamine (2.2-fold, p<0.01) were demonstrated in the brains of BDL rats vs. SHAM-operated controls. Moreover, a decrease in cerebral myo-inositol (p<0.001), with no change in taurine, was found in the presence of brain edema in BDL rats vs. controls. BDL rats treated with either AST-120 or DCA showed attenuation in brain edema and brain lactate. These two treatments did not lead to similar reductions in brain glutamine. Increased brain lactate, and not glutamine, is a primary player in the pathogenesis of brain edema in CLD. In addition, alterations in the osmoregulatory response may also be contributing factors. Our results suggest that inhibiting lactate synthesis is a new potential target for the treatment of HE. Copyright © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

  7. Lactate dehydrogenase downregulation mediates the inhibitory effect of diallyl trisulfide on proliferation, metastasis, and invasion in triple-negative breast cancer.

    PubMed

    Cheng, Shi-Yann; Yang, Yao-Chih; Ting, Kuan-Lun; Wen, Su-Ying; Viswanadha, Vijaya Padma; Huang, Chih-Yang; Kuo, Wei-Wen

    2017-04-01

    The Warburg effect plays a critical role in tumorigenesis, suggesting that specific agents targeting Warburg effect key proteins may be a promising strategy for cancer therapy. Previous studies have shown that diallyl trisulfide (DATS) inhibits proliferation of breast cancer cells by inducing apoptosis in vitro and in vivo. However, whether the Warburg effect is involved with the apoptosis-promoting action of DATS is unclear. Here, we show that the action of DATS is associated with downregulation of lactate dehydrogenase A (LDHA), an essential protein of the Warburg effect whose upregulation is closely related to tumorigenesis. Interestingly, inhibition of the Warburg effect by DATS in breast cancer cells did not greatly affect normal cells. Furthermore, DATS inhibited growth of breast cancer cells, particularly in MDA-MB-231, a triple-negative breast cancer (TNBC) cell, and reduced proliferation and migration; invasion was reversed by over-expression of LDHA. These data suggest that DATS inhibits breast cancer growth and aggressiveness through a novel pathway targeting the key enzyme of the Warburg effect. Our study shows that LDHA downregulation is involved in the apoptotic effect of DATS on TNBC. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1390-1398, 2017. © 2016 Wiley Periodicals, Inc.

  8. Characterization of Plasmodium Lactate Dehydrogenase and Histidine-Rich Protein 2 Clearance Patterns via Rapid On-Bead Detection from a Single Dried Blood Spot

    PubMed Central

    Markwalter, Christine F.; Gibson, Lauren E.; Mudenda, Lwiindi; Kimmel, Danielle W.; Mbambara, Saidon; Thuma, Philip E.; Wright, David W.

    2018-01-01

    Abstract. A rapid, on-bead enzyme-linked immunosorbent assay for Plasmodium lactate dehydrogenase (pLDH) and Plasmodium falciparum histidine-rich protein 2 (HRP2) was adapted for use with dried blood spot (DBS) samples. This assay detected both biomarkers from a single DBS sample with only 45 minutes of total incubation time and detection limits of 600 ± 500 pM (pLDH) and 69 ± 30 pM (HRP2), corresponding to 150 and 24 parasites/μL, respectively. This sensitive and reproducible on-bead detection method was used to quantify pLDH and HRP2 in patient DBS samples from rural Zambia collected at multiple time points after treatment. Biomarker clearance patterns relative to parasite clearance were determined; pLDH clearance followed closely with parasite clearance, whereas most patients maintained detectable levels of HRP2 for 35–52 days after treatment. Furthermore, weak-to-moderate correlations between biomarker concentration and parasite densities were found for both biomarkers. This work demonstrates the utility of the developed assay for epidemiological study and surveillance of malaria. PMID:29557342

  9. Basal levels of metabolic activity are elevated in Genetic Absence Epilepsy Rats from Strasbourg (GAERS): measurement of regional activity of cytochrome oxidase and lactate dehydrogenase by histochemistry.

    PubMed

    Dufour, Franck; Koning, Estelle; Nehlig, Astrid

    2003-08-01

    The Genetic Absence Epilepsy Rats from Strasbourg (GAERS) are considered an isomorphic, predictive, and homologous model of human generalized absence epilepsy. It is characterized by the expression of spike-and-wave discharges in the thalamus and cortex. In this strain, basal regional rates of cerebral glucose utilization measured by the quantitative autoradiographic [(14)C]2-deoxyglucose technique display a widespread consistent increase compared to a selected strain of genetically nonepileptic rats (NE). In order to verify whether these high rates of glucose metabolism are paralleled by elevated activities of the enzymes of the glycolytic and tricarboxylic acid cycle pathways, we measured by histochemistry the regional activity of the two key enzymes of glucose metabolism, lactate dehydrogenase (LDH) for the anaerobic pathway and cytochrome oxidase (CO) for the aerobic pathway coupled to oxidative phosphorylation. CO and LDH activities were significantly higher in GAERS than in NE rats in 24 and 28 of the 30 brain regions studied, respectively. The differences in CO and LDH activity between both strains were widespread, affected all brain systems studied, and ranged from 12 to 63%. The data of the present study confirm the generalized increase in cerebral glucose metabolism in GAERS, occurring both at the glycolytic and at the oxidative step. However, they still do not allow us to understand why the ubiquitous mutation(s) generates spike-and-wave discharges only in the thalamocortical circuit.

  10. Functional and structural characterization of the pentapeptide insertion of Theileria annulata lactate dehydrogenase by site-directed mutagenesis, comparative modeling and molecular dynamics simulations.

    PubMed

    Erdemir, Aysegul; Mutlu, Ozal

    2017-06-01

    Lactate dehydrogenase (LDH) is an important metabolic enzyme in glycolysis and it has been considered as the main energy source in many organisms including apicomplexan parasites. Differences at the active site loop of the host and parasite LDH's makes this enzyme an attractive target for drug inhibitors. In this study, five amino acid insertions in the active site pocket of Theileria annulata LDH (TaLDH) were deleted by PCR-based site-directed mutagenesis, expression and activity analysis of mutant and wild type TaLDH enzymes were performed. Removal of the insertion at the active site loop caused production of an inactive enzyme. Furthermore, structures of wild and mutant enzymes were predicted by comparative modeling and the importance of the insertions at the active site loop were also assigned by molecular docking and dynamics simulations in order to evaluate essential role of this loop for the enzymatic activity. Pentapeptide insertion removal resulted in loss of LDH activity due to deletion of Trp96 and conformational change of Arg98 because of loop instability. Analysis of wild type and mutant enzymes with comparative molecular dynamics simulations showed that the fluctuations of the loop residues increase in mutant enzyme. Together with in silico studies, in vitro results revealed that active site loop has a vital role in the enzyme activity and our findings promise hope for the further drug design studies against theileriosis and other apicomplexan parasite diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. A novel mode of lactate metabolism in strictly anaerobic bacteria.

    PubMed

    Weghoff, Marie Charlotte; Bertsch, Johannes; Müller, Volker

    2015-03-01

    Lactate is a common substrate for major groups of strictly anaerobic bacteria, but the biochemistry and bioenergetics of lactate oxidation is obscure. The high redox potential of the pyruvate/lactate pair of E0 ' = -190 mV excludes direct NAD(+) reduction (E0 ' = -320 mV). To identify the hitherto unknown electron acceptor, we have purified the lactate dehydrogenase (LDH) from the strictly anaerobic, acetogenic bacterium Acetobacterium woodii. The LDH forms a stable complex with an electron-transferring flavoprotein (Etf) that exhibited NAD(+) reduction only when reduced ferredoxin (Fd(2-) ) was present. Biochemical analyses revealed that the LDH/Etf complex of A. woodii uses flavin-based electron confurcation to drive endergonic lactate oxidation with NAD(+) as oxidant at the expense of simultaneous exergonic electron flow from reduced ferredoxin (E0 ' ≈ -500 mV) to NAD(+) according to: lactate + Fd(2-)  + 2 NAD(+)  → pyruvate + Fd + 2 NADH. The reduced Fd(2-) is regenerated from NADH by a sequence of events that involves conversion of chemical (ATP) to electrochemical ( Δ μ ˜ Na + ) and finally redox energy (Fd(2-) from NADH) via reversed electron transport catalysed by the Rnf complex. Inspection of genomes revealed that this metabolic scenario for lactate oxidation may also apply to many other anaerobes. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

  12. Saccharomyces cerevisiae Forms D-2-Hydroxyglutarate and Couples Its Degradation to D-Lactate Formation via a Cytosolic Transhydrogenase.

    PubMed

    Becker-Kettern, Julia; Paczia, Nicole; Conrotte, Jean-François; Kay, Daniel P; Guignard, Cédric; Jung, Paul P; Linster, Carole L

    2016-03-18

    The D or L form of 2-hydroxyglutarate (2HG) accumulates in certain rare neurometabolic disorders, and high D-2-hydroxyglutarate (D-2HG) levels are also found in several types of cancer. Although 2HG has been detected in Saccharomyces cerevisiae, its metabolism in yeast has remained largely unexplored. Here, we show that S. cerevisiae actively forms the D enantiomer of 2HG. Accordingly, the S. cerevisiae genome encodes two homologs of the human D-2HG dehydrogenase: Dld2, which, as its human homolog, is a mitochondrial protein, and the cytosolic protein Dld3. Intriguingly, we found that a dld3Δ knock-out strain accumulates millimolar levels of D-2HG, whereas a dld2Δ knock-out strain displayed only very moderate increases in D-2HG. Recombinant Dld2 and Dld3, both currently annotated as D-lactate dehydrogenases, efficiently oxidized D-2HG to α-ketoglutarate. Depletion of D-lactate levels in the dld3Δ, but not in the dld2Δ mutant, led to the discovery of a new type of enzymatic activity, carried by Dld3, to convert D-2HG to α-ketoglutarate, namely an FAD-dependent transhydrogenase activity using pyruvate as a hydrogen acceptor. We also provide evidence that Ser3 and Ser33, which are primarily known for oxidizing 3-phosphoglycerate in the main serine biosynthesis pathway, in addition reduce α-ketoglutarate to D-2HG using NADH and represent major intracellular sources of D-2HG in yeast. Based on our observations, we propose that D-2HG is mainly formed and degraded in the cytosol of S. cerevisiae cells in a process that couples D-2HG metabolism to the shuttling of reducing equivalents from cytosolic NADH to the mitochondrial respiratory chain via the D-lactate dehydrogenase Dld1. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Metabolic profiling indicates impaired pyruvate dehydrogenase function in myalgic encephalopathy/chronic fatigue syndrome

    PubMed Central

    Mella, Olav; Bruland, Ove; Risa, Kristin; Dyrstad, Sissel E.; Alme, Kine; Rekeland, Ingrid G.; Sapkota, Dipak; Røsland, Gro V.; Fosså, Alexander; Ktoridou-Valen, Irini; Lunde, Sigrid; Sørland, Kari; Lien, Katarina; Herder, Ingrid; Thürmer, Hanne; Gotaas, Merete E.; Baranowska, Katarzyna A.; Bohnen, Louis M.L.J.; Schäfer, Christoph; McCann, Adrian; Sommerfelt, Kristian; Helgeland, Lars; Ueland, Per M.; Dahl, Olav

    2016-01-01

    Myalgic encephalopathy/chronic fatigue syndrome (ME/CFS) is a debilitating disease of unknown etiology, with hallmark symptoms including postexertional malaise and poor recovery. Metabolic dysfunction is a plausible contributing factor. We hypothesized that changes in serum amino acids may disclose specific defects in energy metabolism in ME/CFS. Analysis in 200 ME/CFS patients and 102 healthy individuals showed a specific reduction of amino acids that fuel oxidative metabolism via the TCA cycle, mainly in female ME/CFS patients. Serum 3-methylhistidine, a marker of endogenous protein catabolism, was significantly increased in male patients. The amino acid pattern suggested functional impairment of pyruvate dehydrogenase (PDH), supported by increased mRNA expression of the inhibitory PDH kinases 1, 2, and 4; sirtuin 4; and PPARδ in peripheral blood mononuclear cells from both sexes. Myoblasts grown in presence of serum from patients with severe ME/CFS showed metabolic adaptations, including increased mitochondrial respiration and excessive lactate secretion. The amino acid changes could not be explained by symptom severity, disease duration, age, BMI, or physical activity level among patients. These findings are in agreement with the clinical disease presentation of ME/CFS, with inadequate ATP generation by oxidative phosphorylation and excessive lactate generation upon exertion. PMID:28018972

  14. Hypoxia-induced IL-32β increases glycolysis in breast cancer cells.

    PubMed

    Park, Jeong Su; Lee, Sunyi; Jeong, Ae Lee; Han, Sora; Ka, Hye In; Lim, Jong-Seok; Lee, Myung Sok; Yoon, Do-Young; Lee, Jeong-Hyung; Yang, Young

    2015-01-28

    IL-32β is highly expressed and increases the migration and invasion of gastric, lung, and breast cancer cells. Since IL-32 enhances VEGF production under hypoxic conditions, whether IL-32β is regulated by hypoxia was examined. Hypoxic conditions and a mimetic chemical CoCl2 enhanced IL-32β production. When cells were treated with various inhibitors of ROS generation to prevent hypoxia-induced ROS function, IL-32β production was suppressed by both NADPH oxidase and mitochondrial ROS inhibitors. IL-32β translocated to the mitochondria under hypoxic conditions, where it was associated with mitochondrial biogenesis. Thus, whether hypoxia-induced IL-32β is associated with oxidative phosphorylation (OXPHOS) or glycolysis was examined. Glycolysis under aerobic and anaerobic conditions is impaired in IL-32β-depleted cells, and the hypoxia-induced IL-32β increased glycolysis through activation of lactate dehydrogenase. Src is also known to increase lactate dehydrogenase activity, and the hypoxia-induced IL-32β was found to stimulate Src activation by inhibiting the dephosphorylation of Src. These findings revealed that a hypoxia-ROS-IL-32β-Src-glycolysis pathway is associated with the regulation of cancer cell metabolism. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  15. Mildly elevated lactate levels are associated with microcirculatory flow abnormalities and increased mortality: a microSOAP post hoc analysis.

    PubMed

    Vellinga, Namkje A R; Boerma, E Christiaan; Koopmans, Matty; Donati, Abele; Dubin, Arnaldo; Shapiro, Nathan I; Pearse, Rupert M; van der Voort, Peter H J; Dondorp, Arjen M; Bafi, Tony; Fries, Michael; Akarsu-Ayazoglu, Tulin; Pranskunas, Andrius; Hollenberg, Steven; Balestra, Gianmarco; van Iterson, Mat; Sadaka, Farid; Minto, Gary; Aypar, Ulku; Hurtado, F Javier; Martinelli, Giampaolo; Payen, Didier; van Haren, Frank; Holley, Anthony; Gomez, Hernando; Mehta, Ravindra L; Rodriguez, Alejandro H; Ruiz, Carolina; Canales, Héctor S; Duranteau, Jacques; Spronk, Peter E; Jhanji, Shaman; Hubble, Sheena; Chierego, Marialuisa; Jung, Christian; Martin, Daniel; Sorbara, Carlo; Bakker, Jan; Ince, Can

    2017-10-18

    Mildly elevated lactate levels (i.e., 1-2 mmol/L) are increasingly recognized as a prognostic finding in critically ill patients. One of several possible underlying mechanisms, microcirculatory dysfunction, can be assessed at the bedside using sublingual direct in vivo microscopy. We aimed to evaluate the association between relative hyperlactatemia, microcirculatory flow, and outcome. This study was a predefined subanalysis of a multicenter international point prevalence study on microcirculatory flow abnormalities, the Microcirculatory Shock Occurrence in Acutely ill Patients (microSOAP). Microcirculatory flow abnormalities were assessed with sidestream dark-field imaging. Abnormal microcirculatory flow was defined as a microvascular flow index (MFI) < 2.6. MFI is a semiquantitative score ranging from 0 (no flow) to 3 (continuous flow). Associations between microcirculatory flow abnormalities, single-spot lactate measurements, and outcome were analyzed. In 338 of 501 patients, lactate levels were available. For this substudy, all 257 patients with lactate levels ≤ 2 mmol/L (median [IQR] 1.04 [0.80-1.40] mmol/L) were included. Crude ICU mortality increased with each lactate quartile. In a multivariable analysis, a lactate level > 1.5 mmol/L was independently associated with a MFI < 2.6 (OR 2.5, 95% CI 1.1-5.7, P = 0.027). In a heterogeneous ICU population, a single-spot mildly elevated lactate level (even within the reference range) was independently associated with increased mortality and microvascular flow abnormalities. In vivo microscopy of the microcirculation may be helpful in discriminating between flow- and non-flow-related causes of mildly elevated lactate levels. ClinicalTrials.gov, NCT01179243 . Registered on August 3, 2010.

  16. SIRT3 deacetylates and increases pyruvate dehydrogenase activity in cancer cells.

    PubMed

    Ozden, Ozkan; Park, Seong-Hoon; Wagner, Brett A; Song, Ha Yong; Zhu, Yueming; Vassilopoulos, Athanassios; Jung, Barbara; Buettner, Garry R; Gius, David

    2014-11-01

    Pyruvate dehydrogenase E1α (PDHA1) is the first component enzyme of the pyruvate dehydrogenase (PDH) complex that transforms pyruvate, via pyruvate decarboxylation, into acetyl-CoA that is subsequently used by both the citric acid cycle and oxidative phosphorylation to generate ATP. As such, PDH links glycolysis and oxidative phosphorylation in normal as well as cancer cells. Herein we report that SIRT3 interacts with PDHA1 and directs its enzymatic activity via changes in protein acetylation. SIRT3 deacetylates PDHA1 lysine 321 (K321), and a PDHA1 mutant mimicking a deacetylated lysine (PDHA1(K321R)) increases PDH activity, compared to the K321 acetylation mimic (PDHA1(K321Q)) or wild-type PDHA1. Finally, PDHA1(K321Q) exhibited a more transformed in vitro cellular phenotype compared to PDHA1(K321R). These results suggest that the acetylation of PDHA1 provides another layer of enzymatic regulation, in addition to phosphorylation, involving a reversible acetyllysine, suggesting that the acetylome, as well as the kinome, links glycolysis to respiration. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Decreases in activation energy and substrate affinity in cold-adapted A4-lactate dehydrogenase: evidence from the Antarctic notothenioid fish Chaenocephalus aceratus.

    PubMed

    Fields, Peter A; Houseman, Daniel E

    2004-12-01

    Enzyme function is strongly affected by temperature, and orthologs from species adapted to different thermal environments often show temperature compensation in kinetic properties. Antarctic notothenioid fishes live in a habitat of constant, extreme cold (-1.86 +/- 2 degrees C), and orthologs of the enzyme A4-lactate dehydrogenase (A4-LDH) in these species have adapted to this environment through higher catalytic rates, lower Arrhenius activation energies (Ea), and increases in the apparent Michaelis constant for the substrate pyruvate (Km(PYR)). Here, site-directed mutagenesis was used to determine which amino acid substitutions found in A4-LDH of the notothenioid Chaenocephalus aceratus, with respect to orthologs from warm-adapted teleosts, are responsible for these adaptive changes in enzyme function. Km(PYR) was measured in eight single and two double mutants, and Ea was tested in five single and two double mutants in the temperature range 0 degrees C-20 degrees C. Of the four mutants that had an effect on these parameters, two increased Ea but did not affect Km(PYR) (Gly224Ser, Ala310Pro), and two increased both Ea and Km(PYR) (Glu233Met, Gln317Val). The double mutants Glu233Met/Ala310Pro and Glu233Met/Gln317Val increased Km(PYR) and Ea to levels not significantly different from the A4-LDH of a warm temperate fish (Gillichthys mirabilis, habitat temperature 10 degrees C-35 degrees C). The four single mutants are associated with two alpha-helices that move during the catalytic cycle; those that affect Ea but not Km(PYR) are further from the active site than those that affect both parameters. These results provide evidence that (1) cold adaptation in A4-LDH involves changes in mobility of catalytically important molecular structures; (2) these changes may alter activation energy alone or activation energy and substrate affinity together; and (3) the extent to which these parameters are affected may depend on the location of the substitutions within the mobile

  18. Secondary Increase of Lactate Levels in Asphyxiated Newborns during Hypothermia Treatment: Reflect of Suboptimal Hemodynamics (A Case Series and Review of the Literature)

    PubMed Central

    Al Balushi, Asim; Guilbault, Marie-Pier; Wintermark, Pia

    2015-01-01

    Objective To evaluate whether a secondary increase of serum lactate levels in asphyxiated newborns during hypothermia treatment may reflect suboptimal dynamics. Methods–Retrospective case series and review of the literature. We present the clinical course of four asphyxiated newborns treated with hypothermia who presented with hypotension requiring inotropic support, and who displayed a secondary increase of serum lactate levels during hypothermia treatment. Serial serum lactate levels are correlated with blood pressure and inotropic support within the first 96 hours of life. Results Lactate levels initially decreased in the four patients. However, each of them started to present lower blood pressure, and lactate levels started to increase again. Inotropic support was started to raise blood pressure. The introduction of an epinephrine drip consistently worsened the increase of lactate levels in these newborns, whereas dopamine and dobutamine enabled the clearance of lactate in addition to raising the blood pressure. Rewarming was associated with hemodynamics perturbations (a decrease of blood pressure and/or an increase of lactate levels) in the three newborns who survived. Conclusions Lactate levels during the first 4 days of life should be followed as a potential marker for suboptimal hemodynamic status in term asphyxiated newborns treated with hypothermia, for whom the maintenance of homeostasis during hypothermia treatment is of utmost importance to alleviate brain injury. PMID:26929870

  19. Maternal Exposure to Ethanol During Pregnancy and Lactation Affects Glutamatergic System and Induces Oxidative Stress in Offspring Hippocampus.

    PubMed

    Cesconetto, Patricia A; Andrade, Camila M; Cattani, Daiane; Domingues, Juliana T; Parisotto, Eduardo B; Filho, Danilo W; Zamoner, Ariane

    2016-01-01

    Alcohol abuse during pregnancy leads to intellectual disability and morphological defects in the offspring. The aim of this study was to determine the effect of chronic maternal ethanol (EtOH) consumption during pregnancy and lactation on glutamatergic transmission regulation, energy deficit, and oxidative stress in the hippocampus of the offspring. EtOH was administered to dams in drinking water at increasing doses (2 to 20%) from the gestation day 5 to lactation day 21. EtOH and tap water intake by treated and control groups, respectively, were measured daily. Results showed that EtOH exposure does not affect fluid intake over the course of pregnancy and lactation. The toxicity of maternal exposure to EtOH was demonstrated by decreased offspring body weight at experimental age, on postnatal day 21. Moreover, maternal EtOH exposure decreased (45) Ca(2+) influx in the offspring's hippocampus. Corroborating this finding, EtOH increased both Na(+) -dependent and Na(+) -independent glial [(14) C]-glutamate uptake in hippocampus of immature rats. Also, maternal EtOH exposure decreased glutamine synthetase activity and induced aspartate aminotransferase enzymatic activity, suggesting that in EtOH-exposed offspring hippocampus, glutamate is preferentially used as a fuel in tricarboxylic acid cycle instead of being converted into glutamine. In addition, EtOH exposure decreased [U-14C]-2-deoxy-D-glucose uptake in offspring hippocampus. The decline in glucose transport coincided with increased lactate dehydrogenase activity, suggesting an adaptative response in EtOH-exposed offspring hippocampus, using lactate as an alternative fuel. These events were associated with oxidative damage, as demonstrated by changes in the enzymatic antioxidant defense system and lipid peroxidation. Taken together, the results demonstrate that maternal exposure to EtOH during pregnancy and lactation impairs glutamatergic transmission, as well as inducing oxidative stress and energy deficit in

  20. Choline intakes exceeding recommendations during human lactation improve breast milk choline content by increasing PEMT pathway metabolites.

    PubMed

    Davenport, Crystal; Yan, Jian; Taesuwan, Siraphat; Shields, Kelsey; West, Allyson A; Jiang, Xinyin; Perry, Cydne A; Malysheva, Olga V; Stabler, Sally P; Allen, Robert H; Caudill, Marie A

    2015-09-01

    Demand for the vital nutrient choline is high during lactation; however, few studies have examined choline metabolism and requirements in this reproductive state. The present study sought to discern the effects of lactation and varied choline intake on maternal biomarkers of choline metabolism and breast milk choline content. Lactating (n=28) and control (n=21) women were randomized to 480 or 930 mg choline/day for 10-12 weeks as part of a controlled feeding study. During the last 4-6 weeks, 20% of the total choline intake was provided as an isotopically labeled choline tracer (methyl-d9-choline). Blood, urine and breast milk samples were collected for choline metabolite quantification, enrichment measurements, and gene expression analysis of choline metabolic genes. Lactating (vs. control) women exhibited higher (P < .001) plasma choline concentrations but lower (P ≤ .002) urinary excretion of choline metabolites, decreased use of choline as a methyl donor (e.g., lower enrichment of d6-dimethylglycine, P ≤ .08) and lower (P ≤ .02) leukocyte expression of most choline-metabolizing genes. A higher choline intake during lactation differentially influenced breast milk d9- vs. d3-choline metabolite enrichment. Increases (P ≤ .03) were detected among the d3-metabolites, which are generated endogenously via the hepatic phosphatidylethanolamine N-methyltransferase (PEMT), but not among the d9-metabolites generated from intact exogenous choline. These data suggest that lactation induces metabolic adaptations that increase the supply of intact choline to the mammary epithelium, and that extra maternal choline enhances breast milk choline content by increasing supply of PEMT-derived choline metabolites. This trial was registered at clinicaltrials.gov as NCT01127022. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Glucose uptake and lactate production in cells exposed to CoCl(2) and in cells overexpressing the Glut-1 glucose transporter.

    PubMed

    Hwang, Daw-Yang; Ismail-Beigi, Faramarz

    2002-03-15

    Glut-1-mediated glucose transport is augmented in response to a variety of conditions and stimuli. In this study we examined the metabolic fate of glucose in cells in which glucose transport is stimulated by exposure to CoCl(2), an agent that stimulates the expression of a set of hypoxia-responsive genes including several glycolytic enzymes and the Glut-1 glucose transporter. Similarly, we determined the metabolic fate of glucose in stably transfected cells overexpressing Glut-1. Exposure of Clone 9 liver cell line, 3T3-L1 fibroblasts, and C(2)C(12) myoblasts to CoCl(2) resulted in an increase glucose uptake and in the activity of glucose phosphorylation ("hexokinase") and lactate dehydrogenase. In cells treated with CoCl(2), the net increase in glucose taken up was accounted for by its near-complete conversion to lactate. Cells stably transfected to overexpress Glut-1 also exhibited enhanced net uptake of glucose with the near-complete conversion of the increased glucose taken up to lactate; however, the effect in these cells was observed in the absence of any change in the activity of two glycolytic enzymes examined. These findings suggest that in cells in which glucose transport is rate-limiting for glucose metabolism, enhancement of the glucose entry step per se results in a near-complete conversion of the extra glucose to lactate.

  2. Exogenous lactate supply affects lactate kinetics of rainbow trout, not swimming performance

    PubMed Central

    Omlin, Teye; Langevin, Karolanne

    2014-01-01

    Intense swimming causes circulatory lactate accumulation in rainbow trout because lactate disposal (Rd) is not stimulated as strongly as lactate appearance (Ra). This mismatch suggests that maximal Rd is limited by tissue capacity to metabolize lactate. This study uses exogenous lactate to investigate what constrains maximal Rd and minimal Ra. Our goals were to determine how exogenous lactate affects: 1) Ra and Rd of lactate under baseline conditions or during graded swimming, and 2) exercise performance (critical swimming speed, Ucrit) and energetics (cost of transport, COT). Results show that exogenous lactate allows swimming trout to boost maximal Rd lactate by 40% and reach impressive rates of 56 μmol·kg−1·min−1. This shows that the metabolic capacity of tissues for lactate disposal is not responsible for setting the highest Rd normally observed after intense swimming. Baseline endogenous Ra (resting in normoxic water) is not significantly reduced by exogenous lactate supply. Therefore, trout have an obligatory need to produce lactate, either as a fuel for oxidative tissues and/or from organs relying on glycolysis. Exogenous lactate does not affect Ucrit or COT, probably because it acts as a substitute for glucose and lipids rather than extra fuel. We conclude that the observed 40% increase in Rd lactate is made possible by accelerating lactate entry into oxidative tissues via monocarboxylate transporters (MCTs). This observation together with the weak expression of MCTs and the phenomenon of white muscle lactate retention show that lactate metabolism of rainbow trout is significantly constrained by transmembrane transport. PMID:25121611

  3. Higher thermostability of l-lactate dehydrogenases is a key factor in decreasing the optical purity of d-lactic acid produced from Lactobacillus coryniformis.

    PubMed

    Gu, Sol-A; Jun, Chanha; Joo, Jeong Chan; Kim, Seil; Lee, Seung Hwan; Kim, Yong Hwan

    2014-05-10

    Lactobacillus coryniformis is known to produce d-lactic acid as a dominant fermentation product at a cultivation temperature of approximately 30°C. However, the considerable production of l-lactic acid is observed when the fermentation temperature is greater than 40°C. Because optically pure lactates are synthesized from pyruvate by the catalysis of chiral-specific d- or l-lactate dehydrogenase, the higher thermostability of l-LDHs is assumed to be one of the key factors decreasing the optical purity of d-lactic acid produced from L. coryniformis at high temperature. To verify this hypothesis, two types of d-ldh genes and six types of l-ldh genes based on the genomic information of L. coryniformis were synthesized and expressed in Escherichia coli. Among the LDHs tested, five LDHs showed activity and were used to construct polyclonal antibodies. d-LDH1, l-LDH2, and l-LDH3 were found to be expressed in L. coryniformis by Western blotting analysis. The half-life values (t1/2) of the LDHs at 40°C were estimated to be 10.50, 41.76, and 2311min, and the T50(10) values were 39.50, 39.90, and 58.60°C, respectively. In addition, the Tm values were 36.0, 41.0, and 62.4°C, respectively, which indicates that l-LDH has greater thermostability than d-LDH. The higher thermostability of l-LDHs compared with that of d-LDH1 may be a major reason why the enantiopurity of d-lactic acid is decreased at high fermentation temperatures. The key enzymes characterized will suggest a direction for the design of genetically modified lactic acid bacteria to produce optically pure d-lactic acid. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. Effectiveness of potassium carbonate sesquihydrate to increase dietary cation-anion difference in early lactation cows.

    PubMed

    Harrison, J; White, R; Kincaid, R; Block, E; Jenkins, T; St-Pierre, N

    2012-07-01

    The effect of additional dietary potassium in early lactation dairy cows was evaluated with the addition of potassium carbonate sesquihydrate, which increased dietary K from 1.3 to 2.1% of dry matter (DM) from wk 3 to 12 of lactation. Cows fed potassium carbonate sesquihydrate in the form of DCAD Plus (Church & Dwight Co. Inc., Princeton, NJ) had increased DM intake, milk fat percentage and yield, energy-corrected milk, and efficiency of milk production per unit of DM intake. Milk fat of cows fed higher dietary K had a lower concentration of trans fatty acids, suggesting a role for potassium carbonate sesquihydrate in the rumen in the biohydrogenation processes converting linoleic to stearic acid. Cows fed the diet with 2.1% K had greater apparent balance of K, and no effects were noted on the concentration of blood Mg or amount of fecal Mg. The data support the feeding of greater amounts of K in the early lactation cow. Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  5. Brain lactate kinetics: Modeling evidence for neuronal lactate uptake upon activation.

    PubMed

    Aubert, Agnès; Costalat, Robert; Magistretti, Pierre J; Pellerin, Luc

    2005-11-08

    A critical issue in brain energy metabolism is whether lactate produced within the brain by astrocytes is taken up and metabolized by neurons upon activation. Although there is ample evidence that neurons can efficiently use lactate as an energy substrate, at least in vitro, few experimental data exist to indicate that it is indeed the case in vivo. To address this question, we used a modeling approach to determine which mechanisms are necessary to explain typical brain lactate kinetics observed upon activation. On the basis of a previously validated model that takes into account the compartmentalization of energy metabolism, we developed a mathematical model of brain lactate kinetics, which was applied to published data describing the changes in extracellular lactate levels upon activation. Results show that the initial dip in the extracellular lactate concentration observed at the onset of stimulation can only be satisfactorily explained by a rapid uptake within an intraparenchymal cellular compartment. In contrast, neither blood flow increase, nor extracellular pH variation can be major causes of the lactate initial dip, whereas tissue lactate diffusion only tends to reduce its amplitude. The kinetic properties of monocarboxylate transporter isoforms strongly suggest that neurons represent the most likely compartment for activation-induced lactate uptake and that neuronal lactate utilization occurring early after activation onset is responsible for the initial dip in brain lactate levels observed in both animals and humans.

  6. Macromolecular crowding effect upon in vitro enzyme kinetics: mixed activation-diffusion control of the oxidation of NADH by pyruvate catalyzed by lactate dehydrogenase.

    PubMed

    Balcells, Cristina; Pastor, Isabel; Vilaseca, Eudald; Madurga, Sergio; Cascante, Marta; Mas, Francesc

    2014-04-17

    Enzyme kinetics studies have been usually designed as dilute solution experiments, which differ substantially from in vivo conditions. However, cell cytosol is crowded with a high concentration of molecules having different shapes and sizes. The consequences of such crowding in enzymatic reactions remain unclear. The aim of the present study is to understand the effect of macromolecular crowding produced by dextran of different sizes and at diverse concentrations in the well-known reaction of oxidation of NADH by pyruvate catalyzed by L-lactate dehydrogenase (LDH). Our results indicate that the reaction rate is determined by both the occupied volume and the relative size of dextran obstacles with respect to the enzyme present in the reaction. Moreover, we analyzed the influence of macromolecular crowding on the Michaelis-Menten constants, vmax and Km. The obtained results show that only high concentrations and large sizes of dextran reduce both constants suggesting a mixed activation-diffusion control of this enzymatic reaction due to the dextran crowding action. From our knowledge, this is the first experimental study that depicts mixed activation-diffusion control in an enzymatic reaction due to the effect of crowding.

  7. Increase in ethanol yield via elimination of lactate production in an ethanol-tolerant mutant of Clostridium thermocellum

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Biswas, Ranjita; Prabhu, Sandeep; Lynd, Lee R

    2014-01-01

    Large-scale production of lignocellulosic biofuel is a potential solution to sustainably meet global energy needs. One-step consolidated bioprocessing (CBP) is a potentially advantageous approach for the production of biofuels, but requires an organism capable of hydrolyzing biomass to sugars and fermenting the sugars to ethanol at commercially viable titers and yields. Clostridium thermocellum, a thermophilic anaerobe, can ferment cellulosic biomass to ethanol and organic acids, but low yield, low titer, and ethanol sensitivity remain barriers to industrial production. Here, we deleted the hypoxanthine phosphoribosyltransferase gene in ethanol tolerant strain of C. thermocellum adhE*(EA) in order to allow use of previouslymore » developed gene deletion tools, then deleted lactate dehydrogenase (ldh) to redirect carbon flux towards ethanol. Upon deletion of ldh, the adhE*(EA) ldh strain produced 30% more ethanol than wild type on minimal medium. The adhE*(EA) ldh strain retained tolerance to 5% v/v ethanol, resulting in an ethanol tolerant platform strain of C. thermocellum for future metabolic engineering efforts.« less

  8. A Lactate Kinetics Method for Assessing the Maximal Lactate Steady State Workload

    PubMed Central

    Hering, Gernot O.; Hennig, Ewald M.; Riehle, Hartmut J.; Stepan, Jens

    2018-01-01

    During a continuously increasing exercise workload (WL) a point will be reached at which arterial lactate accumulates rapidly. This so-called lactate threshold (LT) is associated with the maximal lactate steady state workload (MLSSW), the highest WL, at which arterial lactate concentration [LA] does not change. However, the physiological range in which the LT and the MLSSW occur has not been demonstrated directly. We used minor WL variations in the MLSSW range to assess arterial lactate kinetics in 278 treadmill and 148 bicycle ergometer exercise tests. At a certain workload, minimal further increment of running speed (0.1–0.15 m/s) or cycling power (7–10 W) caused a steep elevation of [LA] (0.9 ± 0.43 mM, maximum increase 2.4 mM), indicating LT achievement. This sharp [LA] increase was more pronounced when higher WL increments were used (0.1 vs. 0.30 m/s, P = 0.02; 0.15 vs. 0.30 m/s, P < 0.001; 7 vs. 15 W, P = 0.002; 10 vs. 15 W, P = 0.001). A subsequent workload reduction (0.1 m/s/7 W) stopped the [LA] increase indicating MLSSW realization. LT based determination of running speed (MLSSW) was highly reproducible on a day-to-day basis (r = 0.996, P < 0.001), valid in a 10 km constant velocity setting (r = 0.981, P < 0.001) and a half marathon race (r = 0.969, P < 0.001). These results demonstrate a fine-tuned regulation of exercise-related lactate metabolism, which can be reliably captured by assessing lactate kinetics at the MLSSW. PMID:29651253

  9. Absence of the Birt-Hogg-Dubé gene product is associated with increased hypoxia-inducible factor transcriptional activity and a loss of metabolic flexibility.

    PubMed

    Preston, R S; Philp, A; Claessens, T; Gijezen, L; Dydensborg, A B; Dunlop, E A; Harper, K T; Brinkhuizen, T; Menko, F H; Davies, D M; Land, S C; Pause, A; Baar, K; van Steensel, M A M; Tee, A R

    2011-03-10

    Under conditions of reduced tissue oxygenation, hypoxia-inducible factor (HIF) controls many processes, including angiogenesis and cellular metabolism, and also influences cell proliferation and survival decisions. HIF is centrally involved in tumour growth in inherited diseases that give rise to renal cell carcinoma (RCC), such as Von Hippel-Lindau syndrome and tuberous sclerosis complex. In this study, we examined whether HIF is involved in tumour formation of RCC in Birt-Hogg-Dubé syndrome. For this, we analysed a Birt-Hogg-Dubé patient-derived renal tumour cell line (UOK257) that is devoid of the Birt-Hogg-Dubé protein (BHD) and observed high levels of HIF activity. Knockdown of BHD expression also caused a threefold activation of HIF, which was not as a consequence of more HIF1α or HIF2α protein. Transcription of HIF target genes VEGF, BNIP3 and CCND1 was also increased. We found nuclear localization of HIF1α and increased expression of VEGF, BNIP3 and GLUT1 in a chromophobe carcinoma from a Birt-Hogg-Dubé patient. Our data also reveal that UOK257 cells have high lactate dehydrogenase, pyruvate kinase and 3-hydroxyacyl-CoA dehydrogenase activity. We observed increased expression of pyruvate dehydrogenase kinase 1 (a HIF gene target), which in turn leads to increased phosphorylation and inhibition of pyruvate dehydrogenase. Together with increased protein levels of GLUT1, our data reveal that UOK257 cells favour glycolytic rather than lipid metabolism (a cancer phenomenon termed the 'Warburg effect'). UOK257 cells also possessed a higher expression level of the L-lactate influx monocarboxylate transporter 1 and consequently utilized L-lactate as a metabolic fuel. As a result of their higher dependency on glycolysis, we were able to selectively inhibit the growth of these UOK257 cells by treatment with 2-deoxyglucose. This work suggests that targeting glycolytic metabolism may be used therapeutically to treat Birt-Hogg-Dubé-associated renal lesions.

  10. Brain lactate kinetics: Modeling evidence for neuronal lactate uptake upon activation

    PubMed Central

    Aubert, Agnès; Costalat, Robert; Magistretti, Pierre J.; Pellerin, Luc

    2005-01-01

    A critical issue in brain energy metabolism is whether lactate produced within the brain by astrocytes is taken up and metabolized by neurons upon activation. Although there is ample evidence that neurons can efficiently use lactate as an energy substrate, at least in vitro, few experimental data exist to indicate that it is indeed the case in vivo. To address this question, we used a modeling approach to determine which mechanisms are necessary to explain typical brain lactate kinetics observed upon activation. On the basis of a previously validated model that takes into account the compartmentalization of energy metabolism, we developed a mathematical model of brain lactate kinetics, which was applied to published data describing the changes in extracellular lactate levels upon activation. Results show that the initial dip in the extracellular lactate concentration observed at the onset of stimulation can only be satisfactorily explained by a rapid uptake within an intraparenchymal cellular compartment. In contrast, neither blood flow increase, nor extracellular pH variation can be major causes of the lactate initial dip, whereas tissue lactate diffusion only tends to reduce its amplitude. The kinetic properties of monocarboxylate transporter isoforms strongly suggest that neurons represent the most likely compartment for activation-induced lactate uptake and that neuronal lactate utilization occurring early after activation onset is responsible for the initial dip in brain lactate levels observed in both animals and humans. PMID:16260743

  11. Lack of utility of arteriojugular venous differences of lactate as a reliable indicator of increased brain anaerobic metabolism in traumatic brain injury.

    PubMed

    Poca, Maria A; Sahuquillo, Juan; Vilalta, Anna; Garnacho, Angel

    2007-04-01

    Ischemic lesions are highly prevalent in patients with traumatic brain injuries (TBIs) and are the single most important cause of secondary brain damage. The prevention and early treatment of these lesions is the primary aim in the modem treatment of these patients. One of the most widely used monitoring techniques at the bedside is quantification of brain extracellular level of lactate by using arteriojugular venous differences of lactate (AVDL). The purpose of this study was to determine the sensitivity, specificity, and predictive value of AVDL as an indicator of increases in brain lactate production in patients with TBIs. Arteriojugular venous differences of lactate were calculated every 6 hours using samples obtained though a catheter placed in the jugular bulb in 45 patients with diffuse head injuries (57.8%) or evacuated brain lesions (42.2%). Cerebral lactate concentration obtained with a 20-kD microdialysis catheter implanted in undamaged tissue was used as the de facto gold standard. Six hundred seventy-three AVDL determinations and cerebral microdialysis samples were obtained simultaneously; 543 microdialysis samples (81%) showed lactate values greater than 2 mmol/L, but only 21 AVDL determinations (3.1%) showed an increase in brain lactate. No correlation was found between AVDL and cerebral lactate concentration (p = 0.014, p = 0.719). Arteriojugular venous differences of lactate had a sensitivity and specificity of 3.3 and 97.7%, respectively, with a false-negative rate of 96.7% and a false-positive rate of 2.3%. Arteriojugular venous differences of lactate do not reliably reflect increased cerebral lactate production and consequently are not reliable in ruling out brain ischemia in patients with TBIs. The clinical use of this monitoring method in neurocritical care should be reconsidered.

  12. Characterization of the major dehydrogenase related to d-lactic acid synthesis in Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293.

    PubMed

    Li, Ling; Eom, Hyun-Ju; Park, Jung-Mi; Seo, Eunyoung; Ahn, Ji Eun; Kim, Tae-Jip; Kim, Jeong Hwan; Han, Nam Soo

    2012-10-10

    Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293 is a lactic acid bacterium that converts pyruvate mainly to d-(-)-lactic acid by using d-(-)-lactate dehydrogenase (ldhD). The aim of this study was to identify the gene responsible for d-lactic acid formation in this organism and to characterize the enzyme to facilitate the production of optically pure d-lactic acid. A genomic analysis of L. mesenteroides ATCC 8293 revealed that 7 genes encode lactate-related dehydrogenase. According to transcriptomic, proteomic, and phylogenetic analyses, LEUM_1756 was the major gene responsible for the production of d-lactic acid. The LEUM_1756 gene, of 996bp and encoding 332 amino acids (36.5kDa), was cloned and overexpressed in Escherichia coli BL21(DE3) Star from an inducible pET-21a(+) vector. The enzyme was purified by Ni-NTA column chromatography and showed a specific activity of 4450U/mg, significantly higher than those of other previously reported ldhDs. The gel permeation chromatography analysis showed that the purified enzyme exists as tetramers in solution and this was the first report among lactic acid bacteria. The pH and temperature optima were pH 8.0 and 30°C, respectively, for the pyruvate reduction reaction, and pH 11.0 and 20°C, respectively, for the lactate oxidation reaction. The K(m) kinetic parameters for pyruvate and lactate were 0.58mM and 260mM, respectively. In addition, the k(cat) values for pyruvate and lactate were 2900s(-1) and 2280s(-1), respectively. The enzyme was not inhibited by Ca(2+), Co(2+), Cu(2+), Mg(2+), Mn(2+), Na(+), or urea, but was inhibited by 1mM Zn(2+) and 1mM SDS. Copyright © 2012 Elsevier Inc. All rights reserved.

  13. Glucose, Lactate and Glutamine but not Glutamate Support Depolarization-Induced Increased Respiration in Isolated Nerve Terminals.

    PubMed

    Hohnholt, Michaela C; Andersen, Vibe H; Bak, Lasse K; Waagepetersen, Helle S

    2017-01-01

    Synaptosomes prepared from various aged and gene modified experimental animals constitute a valuable model system to study pre-synaptic mechanisms. Synaptosomes were isolated from whole brain and the XFe96 extracellular flux analyzer (Seahorse Bioscience) was used to study mitochondrial respiration and glycolytic rate in presence of different substrates. Mitochondrial function was tested by sequentially exposure of the synaptosomes to the ATP synthase inhibitor, oligomycin, the uncoupler FCCP (carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone) and the electron transport chain inhibitors rotenone and antimycin A. The synaptosomes exhibited intense respiratory activity using glucose as substrate. The FCCP-dependent respiration was significantly higher with 10 mM glucose compared to 1 mM glucose. Synaptosomes also readily used pyruvate as substrate, which elevated basal respiration, activity-dependent respiration induced by veratridine and the respiratory response to uncoupling compared to that obtained with glucose as substrate. Also lactate was used as substrate by synaptosomes but in contrast to pyruvate, mitochondrial lactate mediated respiration was comparable to respiration using glucose as substrate. Synaptosomal respiration using glutamate and glutamine as substrates was significantly higher compared to basal respiration, whereas oligomycin-dependent and FCCP-induced respiration was lower compared to the responses obtained in the presence of glucose as substrate. We provide evidence that synaptosomes are able to use besides glucose and pyruvate also the substrates lactate, glutamate and glutamine to support their basal respiration. Veratridine was found to increase respiration supported by glucose, pyruvate, lactate and glutamine and FCCP was found to increase respiration supported by glucose, pyruvate and lactate. This was not the case when glutamate was the only energy substrate.

  14. Novel model of neuronal bioenergetics: postsynaptic utilization of glucose but not lactate correlates positively with Ca2+ signalling in cultured mouse glutamatergic neurons.

    PubMed

    Bak, Lasse K; Obel, Linea F; Walls, Anne B; Schousboe, Arne; Faek, Sevan A A; Jajo, Farah S; Waagepetersen, Helle S

    2012-04-05

    We have previously investigated the relative roles of extracellular glucose and lactate as fuels for glutamatergic neurons during synaptic activity. The conclusion from these studies was that cultured glutamatergic neurons utilize glucose rather than lactate during NMDA (N-methyl-d-aspartate)-induced synaptic activity and that lactate alone is not able to support neurotransmitter glutamate homoeostasis. Subsequently, a model was proposed to explain these results at the cellular level. In brief, the intermittent rises in intracellular Ca2+ during activation cause influx of Ca2+ into the mitochondrial matrix thus activating the tricarboxylic acid cycle dehydrogenases. This will lead to a lower activity of the MASH (malate-aspartate shuttle), which in turn will result in anaerobic glycolysis and lactate production rather than lactate utilization. In the present work, we have investigated the effect of an ionomycin-induced increase in intracellular Ca2+ (i.e. independent of synaptic activity) on neuronal energy metabolism employing 13C-labelled glucose and lactate and subsequent mass spectrometric analysis of labelling in glutamate, alanine and lactate. The results demonstrate that glucose utilization is positively correlated with intracellular Ca2+ whereas lactate utilization is not. This result lends further support for a significant role of glucose in neuronal bioenergetics and that Ca2+ signalling may control the switch between glucose and lactate utilization during synaptic activity. Based on the results, we propose a compartmentalized CiMASH (Ca2+-induced limitation of the MASH) model that includes intracellular compartmentation of glucose and lactate metabolism. We define pre- and post-synaptic compartments metabolizing glucose and glucose plus lactate respectively in which the latter displays a positive correlation between oxidative metabolism of glucose and Ca2+ signalling.

  15. Mixed lactate and caffeine compound increases satellite cell activity and anabolic signals for muscle hypertrophy.

    PubMed

    Oishi, Yoshimi; Tsukamoto, Hayato; Yokokawa, Takumi; Hirotsu, Keisuke; Shimazu, Mariko; Uchida, Kenji; Tomi, Hironori; Higashida, Kazuhiko; Iwanaka, Nobumasa; Hashimoto, Takeshi

    2015-03-15

    We examined whether a mixed lactate and caffeine compound (LC) could effectively elicit proliferation and differentiation of satellite cells or activate anabolic signals in skeletal muscles. We cultured C2C12 cells with either lactate or LC for 6 h. We found that lactate significantly increased myogenin and follistatin protein levels and phosphorylation of P70S6K while decreasing the levels of myostatin relative to the control. LC significantly increased protein levels of Pax7, MyoD, and Ki67 in addition to myogenin, relative to control. LC also significantly increased follistatin expression relative to control and stimulated phosphorylation of mTOR and P70S6K. In an in vivo study, male F344/DuCrlCrlj rats were assigned to control (Sed, n = 10), exercise (Ex, n = 12), and LC supplementation (LCEx, n = 13) groups. LC was orally administered daily. The LCEx and Ex groups were exercised on a treadmill, running for 30 min at low intensity every other day for 4 wk. The LCEx group experienced a significant increase in the mass of the gastrocnemius (GA) and tibialis anterior (TA) relative to both the Sed and Ex groups. Furthermore, the LCEx group showed a significant increase in the total DNA content of TA compared with the Sed group. The LCEx group experienced a significant increase in myogenin and follistatin expression of GA relative to the Ex group. These results suggest that administration of LC can effectively increase muscle mass concomitant with elevated numbers of myonuclei, even with low-intensity exercise training, via activated satellite cells and anabolic signals. Copyright © 2015 the American Physiological Society.

  16. Impaired hippocampal glucose metabolism during and after flurothyl-induced seizures in mice: Reduced phosphorylation coincides with reduced activity of pyruvate dehydrogenase.

    PubMed

    McDonald, Tanya S; Borges, Karin

    2017-07-01

    To determine changes in glucose metabolism and the enzymes involved in the hippocampus ictally and postictally in the acute mouse flurothyl seizure model. [U- 13 C]-Glucose was injected (i.p.) prior to, or following a 5 min flurothyl-induced seizure. Fifteen minutes later, mice were killed and the total metabolite levels and % 13 C enrichment were analyzed in the hippocampal formation using gas chromatography-mass spectrometry. Activities of key metabolic and antioxidant enzymes and the phosphorylation status of pyruvate dehydrogenase were measured, along with lipid peroxidation. During seizures, total lactate levels increased 1.7-fold; however, [M + 3] enrichment of both lactate and alanine were reduced by 30% and 43%, respectively, along with a 28% decrease in phosphofructokinase activity. Postictally the % 13 C enrichments of all measured tricarboxylic acid (TCA) cycle intermediates and the amino acids were reduced by 46-93%. At this time, pyruvate dehydrogenase (PDH) activity was 56% of that measured in controls, and there was a 1.9-fold increase in the phosphorylation of PDH at ser232. Phosphorylation of PDH is known to decrease its activity. Here, we show that the increase of lactate levels during flurothyl seizures is from a source other than [U- 13 C]-glucose, such as glycogen. Surprisingly, although we saw a reduction in phosphofructokinase activity during the seizure, metabolism of [U- 13 C]-glucose into the TCA cycle seemed unaffected. Similar to our recent findings in the chronic phase of the pilocarpine model, postictally the metabolism of glucose by glycolysis and the TCA cycle was impaired along with reduced PDH activity. Although this decrease in activity may be a protective mechanism to reduce oxidative stress, which is observed in the flurothyl model, ATP is critical to the recovery of ion and neurotransmitter balance and return to normal brain function. Thus we identified promising novel strategies to enhance energy metabolism and recovery from

  17. Hepatic lipidosis in anorectic, lactating holstein cattle: a retrospective study of serum biochemical abnormalities.

    PubMed

    Cebra, C K; Garry, F B; Getzy, D M; Fettman, M J

    1997-01-01

    The association between hepatic lipidosis (HL) and disease in 59 anorectic, ketotic, lactating Holstein heifers and cows was investigated. Severe HL, as determined by histologic evaluation of liver tissue, was present in 46 animals; only half of these animals required intensive treatment for ketosis, and only half had serum biochemical evidence of liver disease, as determined by the presence of a last value of 2-fold or greater than the upper limit of the reference ranges for at least 2 of the 4 serum tests: gamma-glutamyl transferase, aspartate aminotransferase, and sorbitol dehydrogenase activities and bile acid concentrations. Most cattle with biochemical evidence of liver disease and severe HL had been lactating for 14 or more days. Cows that required intensive treatment inconsistently had serum biochemical evidence of liver disease. Although cattle with severe HL had significantly higher serum bilirubin concentrations and aspartate aminotransferase and sorbitol dehydrogenase activities than cattle with less severe lipidosis, the specificity of abnormally high serum sorbitol dehydrogenase activity or bilirubin concentration for severe lipidosis was only 8%. Abnormally high serum aspartate aminotransferase activity was 83% sensitive and 62% specific for severe lipidosis. Serum glucose and total carbon dioxide concentrations were significantly lower in cattle with severe lipidosis than in those with mild or moderate lipidosis, and low serum glucose or total carbon dioxide concentrations were rare in cattle without severe lipidosis. From these data, we conclude that the use of a single biochemical or histopathologic criterion to define severity of disease or degree of liver compromise in anorectic, ketotic cows results in the misidentification of many animals.

  18. The intrinsically disordered protein LEA7 from Arabidopsis thaliana protects the isolated enzyme lactate dehydrogenase and enzymes in a soluble leaf proteome during freezing and drying.

    PubMed

    Popova, Antoaneta V; Rausch, Saskia; Hundertmark, Michaela; Gibon, Yves; Hincha, Dirk K

    2015-10-01

    The accumulation of Late Embryogenesis Abundant (LEA) proteins in plants is associated with tolerance against stresses such as freezing and desiccation. Two main functions have been attributed to LEA proteins: membrane stabilization and enzyme protection. We have hypothesized previously that LEA7 from Arabidopsis thaliana may stabilize membranes because it interacts with liposomes in the dry state. Here we show that LEA7, contrary to this expectation, did not stabilize liposomes during drying and rehydration. Instead, it partially preserved the activity of the enzyme lactate dehydrogenase (LDH) during drying and freezing. Fourier-transform infrared (FTIR) spectroscopy showed no evidence of aggregation of LDH in the dry or rehydrated state under conditions that lead to complete loss of activity. To approximate the complex influence of intracellular conditions on the protective effects of a LEA protein in a convenient in-vitro assay, we measured the activity of two Arabidopsis enzymes (glucose-6-P dehydrogenase and ADP-glucose pyrophosphorylase) in total soluble leaf protein extract (Arabidopsis soluble proteome, ASP) after drying and rehydration or freezing and thawing. LEA7 partially preserved the activity of both enzymes under these conditions, suggesting its role as an enzyme protectant in vivo. Further FTIR analyses indicated the partial reversibility of protein aggregation in the dry ASP during rehydration. Similarly, aggregation in the dry ASP was strongly reduced by LEA7. In addition, mixtures of LEA7 with sucrose or verbascose reduced aggregation more than the single additives, presumably through the effects of the protein on the H-bonding network of the sugar glasses. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Multiple e-pharmacophore modelling pooled with high-throughput virtual screening, docking and molecular dynamics simulations to discover potential inhibitors of Plasmodium falciparum lactate dehydrogenase (PfLDH).

    PubMed

    Saxena, Shalini; Durgam, Laxman; Guruprasad, Lalitha

    2018-05-14

    Development of new antimalarial drugs continues to be of huge importance because of the resistance of malarial parasite towards currently used drugs. Due to the reliance of parasite on glycolysis for energy generation, glycolytic enzymes have played important role as potential targets for the development of new drugs. Plasmodium falciparum lactate dehydrogenase (PfLDH) is a key enzyme for energy generation of malarial parasites and is considered to be a potential antimalarial target. Presently, there are nearly 15 crystal structures bound with inhibitors and substrate that are available in the protein data bank (PDB). In the present work, we attempted to consider multiple crystal structures with bound inhibitors showing affinity in the range of 1.4 × 10 2 -1.3 × 10 6  nM efficacy and optimized the pharmacophore based on the energy involved in binding termed as e-pharmacophore mapping. A high throughput virtual screening (HTVS) combined with molecular docking, ADME predictions and molecular dynamics simulation led to the identification of 20 potential compounds which could be further developed as novel inhibitors for PfLDH.

  20. Increasing milk solids production across lactation through genetic selection and intensive pasture-based feed system.

    PubMed

    Coleman, J; Pierce, K M; Berry, D P; Brennan, A; Horan, B

    2010-09-01

    The objective of the study was to quantify the effect of genetic improvement using the Irish total merit index, the Economic Breeding Index (EBI), on overall performance and lactation profiles for milk, milk solids, body weight (BW), and body condition score (BCS) within 2 pasture-based systems of milk production likely to be used in the future, following abolition of the European Union's milk quota system. Three genotypes of Holstein-Friesian dairy cattle were established from within the Moorepark dairy research herd: LowNA, indicative of animals with North American origin and average or lower genetic merit at the time of the study; HighNA, North American Holstein-Friesians of high genetic merit; and HighNZ, New Zealand Holstein-Friesians of high genetic merit. Animals from within each genotype were randomly allocated to 1 of 2 possible pasture-based feeding systems (FS): 1) The Moorepark pasture (MP) system (2.64 cows/ha and 344 kg of concentrate supplement per cow per lactation) and 2) a high output per hectare (HC) system (2.85 cows/ha and 1,056 kg of concentrate supplement per cow per lactation). Pasture was allocated to achieve similar postgrazing residual sward heights for both treatments. A total of 126, 128, and 140 spring-calving dairy cows were used during the years 2006, 2007, and 2008, respectively. Each group had an individual farmlet of 17 paddocks and all groups were managed similarly throughout the study. The effects of genotype, FS, and the interaction between genotype and FS on milk production, BW, and BCS across lactation were studied using mixed models with factorial arrangements of genotype and FS accounting for the repeated cow records across years. No significant genotype by FS interaction was observed for any of the variables measured. Results show that milk solids production of the national average dairy cow can be increased across lactation through increased EBI. High EBI genotypes (HighNA and HighNZ) produced more milk solids per cow and

  1. Diminution in energy expenditure during lactation.

    PubMed Central

    Illingworth, P J; Jung, R T; Howie, P W; Leslie, P; Isles, T E

    1986-01-01

    Energy expenditure at rest and in response to a meal and to an infusion of noradrenaline was measured in 12 lactating women and compared with that in seven bottle feeding women and seven non-pregnant, non-lactating controls. The energy response of the lactating women was remeasured after lactation stopped. During lactation the resting metabolic rate was unaltered but there was a reduced response to infusion of noradrenaline and to a meal, which increased to normal control values after lactation stopped. Such reductions in expenditure were not found in women who had been bottle feeding and were tested at a similar six to eight weeks post partum. These findings suggest that metabolic efficiency is enhanced in lactating women, who may not need to increase energy intake to the extent suggested by current recommended dietary allowances. PMID:3081114

  2. Effectiveness of administration of gonadotropin-releasing hormone at Days 11, 14 or 15 after anticipated ovulation for increasing fertility of lactating dairy cows and non-lactating heifers.

    PubMed

    Franco, M; Thompson, P M; Brad, A M; Hansen, P J

    2006-09-01

    One strategy for improving fertility in cattle is mid-cycle administration of GnRH to increase progesterone secretion and delay luteolysis. This strategy might be especially useful during hot weather because heat stress increases uterine prostaglandin release and reduces development of the elongating embryo. A series of experiments was conducted to test the efficacy of GnRH for increasing fertility. There was no effect of administration of 100 microg GnRH at Day 11 after anticipated ovulation on pregnancy rates in virgin heifers subjected to timed artificial insemination (TAI) during the summer. Similarly, there was no beneficial effect of administration of GnRH at Day 11 after anticipated ovulation on pregnancy rates of lactating cows subjected to TAI in summer and winter. Three experiments tested effects of injection of GnRH at Days 14 or 15 after anticipated ovulation on pregnancy rates of lactating cows. The first experiment used 477 lactating cows subjected to TAI. Cows receiving GnRH at Day 14 had higher pregnancy rates in both summer and winter than cows receiving vehicle (20.3 versus 12.7%, P<0.02). When this experiment was repeated during summer with 137 cows, there was a negative effect of GnRH treatment at Day 14 on pregnancy rate. In the third experiment, lactating cows during summer were inseminated at detected estrus and cows were assigned to treatment with either GnRH or vehicle at Days 14 or 15 after insemination. Pregnancy rates were 25.6% (32/125) for cows receiving vehicle, 20.7% (19/92) for cows receiving GnRH at Day 14, and 20.3% (16/79) for cows receiving GnRH at Day 15. In conclusion, GnRH administration at Days 11-15 after anticipated ovulation or estrus did not consistently increase pregnancy rates in either cool or warm seasons.

  3. Inhibition of Growth by Combined Treatment with Inhibitors of Lactate Dehydrogenase and either Phenformin or Inhibitors of 6-Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase 3.

    PubMed

    Lea, Michael A; Guzman, Yolanda; Desbordes, Charles

    2016-04-01

    Enhanced glycolysis in cancer cells presents a target for chemotherapy. Previous studies have indicated that proliferation of cancer cells can be inhibited by treatment with phenformin and with an inhibitor of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB) namely 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO). In the present work, the action of two inhibitors that are effective at lower concentrations than 3PO, namely 1-(3-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one (PQP) and 1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one (PFK15) were investigated. The inhibitors of lactate dehydrogenase (LDHA) studied in order of half-maximal inhibitory concentrations were methyl 1-hydroxy-6-phenyl-4-(trifluoromethyl)-1H-indole-2-carboxylate (NHI-2) < isosafrole < oxamate. In colonic and bladder cancer cells, additive growth inhibitory effects were seen with the LDHA inhibitors, of which NHI-2 was effective at the lowest concentrations. Growth inhibition was generally greater with PFK15 than with PQP. The increased acidification of the culture medium and glucose uptake caused by phenformin was blocked by combined treatment with PFKFB3 or LDHA inhibitors. The results suggest that combined treatment with phenformin and inhibitors of glycolysis can cause additive inhibition of cell proliferation and may mitigate lactic acidosis caused by phenformin when used as a single agent. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  4. Mutant E. coli strain with increased succinic acid production

    DOEpatents

    Donnelly, Mark; Millard, Cynthia S.; Stols, Lucy

    1998-01-01

    A method for isolating succinic acid producing bacteria is provided comprising increasing the biomass of an organism which lacks the ability to catabolize pyruvate, and then subjecting the biomass to glucose-rich medium in an anaerobic environment to enable pyruvate-catabolizing mutants to grow. The invention also provides for a mutant that produces high amounts of succinic acid, which as been derived from a parent which lacked the genes for pyruvate formate lyase and lactate dehydrogenase, and which belongs to the E.coli Group of Bacteria.

  5. Mutant E. coli strain with increased succinic acid production

    DOEpatents

    Donnelly, Mark; Millard, Cynthia S.; Stols, Lucy

    2001-09-25

    A method for isolating succinic acid producing bacteria is provided comprising increasing the biomass of an organism which lacks the ability to catabolize pyruvate, and then subjecting the biomass to glucose-rich medium in an anaerobic environment to enable pyruvate-catabolizing mutants to grow. The invention also provides for a mutant that produces high amounts of succinic acid, which has been derived from a parent which lacked the genes for pyruvate formate lyase and lactate dehydrogenase, and which belongs to the E.coli Group of Bacteria.

  6. Mutant E. coli strain with increased succinic acid production

    DOEpatents

    Donnelly, Mark; Millard, Cynthia S.; Stols, Lucy

    2002-01-01

    A method for isolating succinic acid producing bacteria is provided comprising increasing the biomass of an organism which lacks the ability to catabolize pyruvate, and then subjecting the biomass to glucose-rich medium in an anaerobic environment to enable pyruvate-catabolizing mutants to grow. The invention also provides for a mutant that produces high amounts of succinic acid, which has been derived from a parent which lacked the genes for pyruvate formate lyase and lactate dehydrogenase, and which belongs to the E.coli Group of Bacteria.

  7. Phosphorylation status of pyruvate dehydrogenase distinguishes metabolic phenotypes of cultured rat brain astrocytes and neurons.

    PubMed

    Halim, Nader D; Mcfate, Thomas; Mohyeldin, Ahmed; Okagaki, Peter; Korotchkina, Lioubov G; Patel, Mulchand S; Jeoung, Nam Ho; Harris, Robert A; Schell, Michael J; Verma, Ajay

    2010-08-01

    Glucose metabolism in nervous tissue has been proposed to occur in a compartmentalized manner with astrocytes contributing largely to glycolysis and neurons being the primary site of glucose oxidation. However, mammalian astrocytes and neurons both contain mitochondria, and it remains unclear why in culture neurons oxidize glucose, lactate, and pyruvate to a much larger extent than astrocytes. The objective of this study was to determine whether pyruvate metabolism is differentially regulated in cultured neurons versus astrocytes. Expression of all components of the pyruvate dehydrogenase complex (PDC), the rate-limiting step for pyruvate entry into the Krebs cycle, was determined in cultured astrocytes and neurons. In addition, regulation of PDC enzymatic activity in the two cell types via protein phosphorylation was examined. We show that all components of the PDC are expressed in both cell types in culture, but that PDC activity is kept strongly inhibited in astrocytes through phosphorylation of the pyruvate dehydrogenase alpha subunit (PDH alpha). In contrast, neuronal PDC operates close to maximal levels with much lower levels of phosphorylated PDH alpha. Dephosphorylation of astrocytic PDH alpha restores PDC activity and lowers lactate production. Our findings suggest that the glucose metabolism of astrocytes and neurons may be far more flexible than previously believed. (c) 2010 Wiley-Liss, Inc.

  8. Plasma Lactate Dehydrogenase Levels Predict Mortality in Acute Aortic Syndromes: A Diagnostic Accuracy and Observational Outcome Study.

    PubMed

    Morello, Fulvio; Ravetti, Anna; Nazerian, Peiman; Liedl, Giovanni; Veglio, Maria Grazia; Battista, Stefania; Vanni, Simone; Pivetta, Emanuele; Montrucchio, Giuseppe; Mengozzi, Giulio; Rinaldi, Mauro; Moiraghi, Corrado; Lupia, Enrico

    2016-02-01

    In acute aortic syndromes (AAS), organ malperfusion represents a key event impacting both on diagnosis and outcome. Increased levels of plasma lactate dehydrogenase (LDH), a biomarker of malperfusion, have been reported in AAS, but the performance of LDH for the diagnosis of AAS and the relation of LDH with outcome in AAS have not been evaluated so far.This was a bi-centric prospective diagnostic accuracy study and a cohort outcome study. From 2008 to 2014, patients from 2 Emergency Departments suspected of having AAS underwent LDH assay at presentation. A final diagnosis was obtained by aortic imaging. Patients diagnosed with AAS were followed-up for in-hospital mortality.One thousand five hundred seventy-eight consecutive patients were clinically eligible, and 999 patients were included in the study. The final diagnosis was AAS in 201 (20.1%) patients. Median LDH was 424 U/L (interquartile range [IQR] 367-557) in patients with AAS and 383 U/L (IQR 331-460) in patients with alternative diagnoses (P < 0.001). Using a cutoff of 450 U/L, the sensitivity of LDH for AAS was 44% (95% confidence interval [CI] 37-51) and the specificity was 73% (95% CI 69-76). Overall in-hospital mortality for AAS was 23.8%. Mortality was 32.6% in patients with LDH ≥ 450 U/L and 16.8% in patients with LDH < 450 U/L (P = 0.006). Following stratification according to LDH quartiles, in-hospital mortality was 12% in the first (lowest) quartile, 18.4% in the second quartile, 23.5% in the third quartile, and 38% in the fourth (highest) quartile (P = 0.01). LDH ≥ 450 U/L was further identified as an independent predictor of death in AAS both in univariate and in stepwise logistic regression analyses (odds ratio 2.28, 95% CI 1.11-4.66; P = 0.025), in addition to well-established risk markers such as advanced age and hypotension. Subgroup analysis showed excess mortality in association with LDH ≥ 450 U/L in elderly, hemodynamically stable and in nonsurgically

  9. Novel model of neuronal bioenergetics: postsynaptic utilization of glucose but not lactate correlates positively with Ca2+ signalling in cultured mouse glutamatergic neurons

    PubMed Central

    Bak, Lasse K.; Obel, Linea F.; Walls, Anne B.; Schousboe, Arne; Faek, Sevan A.A.; Jajo, Farah S.; Waagepetersen, Helle S.

    2012-01-01

    We have previously investigated the relative roles of extracellular glucose and lactate as fuels for glutamatergic neurons during synaptic activity. The conclusion from these studies was that cultured glutamatergic neurons utilize glucose rather than lactate during NMDA (N-methyl-d-aspartate)-induced synaptic activity and that lactate alone is not able to support neurotransmitter glutamate homoeostasis. Subsequently, a model was proposed to explain these results at the cellular level. In brief, the intermittent rises in intracellular Ca2+ during activation cause influx of Ca2+ into the mitochondrial matrix thus activating the tricarboxylic acid cycle dehydrogenases. This will lead to a lower activity of the MASH (malate–aspartate shuttle), which in turn will result in anaerobic glycolysis and lactate production rather than lactate utilization. In the present work, we have investigated the effect of an ionomycin-induced increase in intracellular Ca2+ (i.e. independent of synaptic activity) on neuronal energy metabolism employing 13C-labelled glucose and lactate and subsequent mass spectrometric analysis of labelling in glutamate, alanine and lactate. The results demonstrate that glucose utilization is positively correlated with intracellular Ca2+ whereas lactate utilization is not. This result lends further support for a significant role of glucose in neuronal bioenergetics and that Ca2+ signalling may control the switch between glucose and lactate utilization during synaptic activity. Based on the results, we propose a compartmentalized CiMASH (Ca2+-induced limitation of the MASH) model that includes intracellular compartmentation of glucose and lactate metabolism. We define pre- and post-synaptic compartments metabolizing glucose and glucose plus lactate respectively in which the latter displays a positive correlation between oxidative metabolism of glucose and Ca2+ signalling. PMID:22385215

  10. Ketones and lactate increase cancer cell “stemness”, driving recurrence, metastasis and poor clinical outcome in breast cancer

    PubMed Central

    Tsirigos, Aristotelis; Lin, Zhao; Pavlides, Stephanos; Wang, Chengwang; Flomenberg, Neal; Knudsen, Erik S; Howell, Anthony; Pestell, Richard G

    2011-01-01

    Previously, we showed that high-energy metabolites (lactate and ketones) “fuel” tumor growth and experimental metastasis in an in vivo xenograft model, most likely by driving oxidative mitochondrial metabolism in breast cancer cells. To mechanistically understand how these metabolites affect tumor cell behavior, here we used genome-wide transcriptional profiling. Human breast cancer cells (MCF7) were cultured with lactate or ketones, and then subjected to transcriptional analysis (exon-array). Interestingly, our results show that treatment with these high-energy metabolites increases the transcriptional expression of gene profiles normally associated with “stemness”, including genes upregulated in embryonic stem (ES) cells. Similarly, we observe that lactate and ketones promote the growth of bonafide ES cells, providing functional validation. The lactate- and ketone-induced “gene signatures” were able to predict poor clinical outcome (including recurrence and metastasis) in human breast cancer patients. Taken together, our results are consistent with the idea that lactate and ketone utilization in cancer cells promotes the “cancer stem cell” phenotype, resulting in significant decreases in patient survival. One possible mechanism by which high-energy metabolites might induce stemness is by increasing the pool of Acetyl-CoA, leading to increased histone acetylation and elevated gene expression. Thus, our results mechanistically imply that clinical outcome in breast cancer could simply be determined by epigenetics and energy metabolism, rather than by the accumulation of specific “classical” gene mutations. We also suggest that high-risk cancer patients (identified by the lactate/ketone gene signatures) could be treated with new therapeutics that target oxidative mitochondrial metabolism, such as the anti-oxidant and “mitochondrial poison” metformin. Finally, we propose that this new approach to personalized cancer medicine be termed

  11. Efficient production of optically pure D-lactic acid from raw corn starch by using a genetically modified L-lactate dehydrogenase gene-deficient and alpha-amylase-secreting Lactobacillus plantarum strain.

    PubMed

    Okano, Kenji; Zhang, Qiao; Shinkawa, Satoru; Yoshida, Shogo; Tanaka, Tsutomu; Fukuda, Hideki; Kondo, Akihiko

    2009-01-01

    In order to achieve direct and efficient fermentation of optically pure D-lactic acid from raw corn starch, we constructed L-lactate dehydrogenase gene (ldhL1)-deficient Lactobacillus plantarum and introduced a plasmid encoding Streptococcus bovis 148 alpha-amylase (AmyA). The resulting strain produced only D-lactic acid from glucose and successfully expressed amyA. With the aid of secreting AmyA, direct D-lactic acid fermentation from raw corn starch was accomplished. After 48 h of fermentation, 73.2 g/liter of lactic acid was produced with a high yield (0.85 g per g of consumed sugar) and an optical purity of 99.6%. Moreover, a strain replacing the ldhL1 gene with an amyA-secreting expression cassette was constructed. Using this strain, direct D-lactic acid fermentation from raw corn starch was accomplished in the absence of selective pressure by antibiotics. This is the first report of direct D-lactic acid fermentation from raw starch.

  12. Efficient Production of Optically Pure d-Lactic Acid from Raw Corn Starch by Using a Genetically Modified l-Lactate Dehydrogenase Gene-Deficient and α-Amylase-Secreting Lactobacillus plantarum Strain▿

    PubMed Central

    Okano, Kenji; Zhang, Qiao; Shinkawa, Satoru; Yoshida, Shogo; Tanaka, Tsutomu; Fukuda, Hideki; Kondo, Akihiko

    2009-01-01

    In order to achieve direct and efficient fermentation of optically pure d-lactic acid from raw corn starch, we constructed l-lactate dehydrogenase gene (ldhL1)-deficient Lactobacillus plantarum and introduced a plasmid encoding Streptococcus bovis 148 α-amylase (AmyA). The resulting strain produced only d-lactic acid from glucose and successfully expressed amyA. With the aid of secreting AmyA, direct d-lactic acid fermentation from raw corn starch was accomplished. After 48 h of fermentation, 73.2 g/liter of lactic acid was produced with a high yield (0.85 g per g of consumed sugar) and an optical purity of 99.6%. Moreover, a strain replacing the ldhL1 gene with an amyA-secreting expression cassette was constructed. Using this strain, direct d-lactic acid fermentation from raw corn starch was accomplished in the absence of selective pressure by antibiotics. This is the first report of direct d-lactic acid fermentation from raw starch. PMID:19011066

  13. Procalcitonin, C-reactive protein and serum lactate dehydrogenase in the diagnosis of bacterial sepsis, SIRS and systemic candidiasis.

    PubMed

    Miglietta, Fabio; Faneschi, Maria Letizia; Lobreglio, Giambattista; Palumbo, Claudio; Rizzo, Adriana; Cucurachi, Marco; Portaccio, Gerolamo; Guerra, Francesco; Pizzolante, Maria

    2015-09-01

    The aim of this study was to evaluate procalcitonin (PCT), C-reactive protein (CRP), platelet count (PLT) and serum lactate dehydrogenase (LDH) as early markers for diagnosis of SIRS, bacterial sepsis and systemic candidiasis in intensive care unit (ICU) patients. Based on blood culture results, the patients were divided into a sepsis group (70 patients), a SIRS group (42 patients) and a systemic candidiasis group (33 patients). PCT, CRP, LDH and PLT levels were measured on day 0 and on day 2 from the sepsis symptom onset. PCT levels were higher in Gram negative sepsis than those in Gram positive sepsis, although the P value between the two subgroups is not significant (P=0.095). Bacterial sepsis group had higher PCT and CRP levels compared with the systemic candidiasis group, whereas PLT and LDH levels showed similar levels in these two subgroups. The AUC for PCT (AUC: 0.892, P <0.001) was larger than for CRP (AUC: 0.738, P <0.001). The best cut-off values for PCT and CRP were 0.99 ng/mL and 76.2 mg/L, respectively. Diagnostic sensitivity and specificity for PCT were 84.3% and 81.8% whereas CRP showed a sensitivity of 77.2% and a specificity of 63.6%. However, PCT was unable to discriminate between SIRS and systemic candidiasis groups (P=0.093 N.S.). In conclusion, PCT can be used as a preliminary marker in the event of clinical suspicion of systemic candidiasis; however, low PCT levels (<0.99 ng/mL) necessarily require the use of other specific markers of candidaemia to confirm the diagnosis, due to great uniformity of PCT levels in systemic candidiasis and SIRS groups.

  14. [Experience in using the latent activity of leukocytic lactate dehydrogenase isoenzymes for the integral estimate of the level of free radical oxidation in patients with neurotic disorders].

    PubMed

    Kuskov, M V

    2006-06-01

    The aggregatory properties of a leukocytic homogenate were studied by analyzing the activity of its lactate dehydrogenase (LDH) isoenzymes from patients with neurotic disorders on admission and during treatment. As a parameter reflecting the aggregatory properties of the leukocytic homogenate, the latent activity of LDH isoenzymes was studied. On admission, the patients were shown to have a lower latent activity, which restored during treatment to the control values, than in the control group. There was also a synchronous pattern of a change in the osmotic stability of red blood cells with the latent activity of leukocytic LDH isoenzymes in the treated patients. It is obvious that latent activity values reflect the level of free radical oxidation in the body. For detailed testing of the aggregatory properties of a cellular lysate, the trends in the latent activity of LDH isoenzymes were examined, which failed to reveal an unambiguous recovery of the observed parameters during therapy. Based on the findings, the author discusses whether this method can be used to analyze the time course of changes in a psychopathological process and to predict its outcome.

  15. Poliomyelitis in MuLV-infected ICR-SCID mice after injection of basement membrane matrix contaminated with lactate dehydrogenase-elevating virus.

    PubMed

    Carlson Scholz, Jodi A; Garg, Rohit; Compton, Susan R; Allore, Heather G; Zeiss, Caroline J; Uchio, Edward M

    2011-10-01

    The arterivirus lactate dehydrogenase-elevating virus (LDV) causes life-long viremia in mice. Although LDV infection generally does not cause disease, infected mice that are homozygous for the Fv1(n) allele are prone to develop poliomyelitis when immunosuppressed, a condition known as age-dependent poliomyelitis. The development of age-dependent poliomyelitis requires coinfection with endogenous murine leukemia virus. Even though LDV is a common contaminant of transplantable tumors, clinical signs of poliomyelitis after inadvertent exposure to LDV have not been described in recent literature. In addition, LDV-induced poliomyelitis has not been reported in SCID or ICR mice. Here we describe the occurrence of poliomyelitis in ICR-SCID mice resulting from injection of LDV-contaminated basement membrane matrix. After exposure to LDV, a subset of mice presented with clinical signs including paresis, which was associated with atrophy of the hindlimb musculature, and tachypnea; in addition, some mice died suddenly with or without premonitory signs. Mice presenting within the first 6 mo after infection had regions of spongiosis, neuronal necrosis and astrocytosis of the ventral spinal cord, and less commonly, brainstem. Axonal degeneration of ventral roots prevailed in more chronically infected mice. LDV was identified by RT-PCR in 12 of 15 mice with typical neuropathology; positive antiLDV immunolabeling was identified in all PCR-positive animals (n = 7) tested. Three of 8 mice with neuropathology but no clinical signs were LDV negative by RT-PCR. RT-PCR yielded murine leukemia virus in spinal cords of all mice tested, regardless of clinical presentation or neuropathology.

  16. MCT1 and MCT4 Expression and Lactate Flux Activity Increase During White and Brown Adipogenesis and Impact Adipocyte Metabolism.

    PubMed

    Petersen, Charlotte; Nielsen, Mette D; Andersen, Elise S; Basse, Astrid L; Isidor, Marie S; Markussen, Lasse K; Viuff, Birgitte M; Lambert, Ian H; Hansen, Jacob B; Pedersen, Stine F

    2017-10-12

    Adipose tissue takes up glucose and releases lactate, thereby contributing significantly to systemic glucose and lactate homeostasis. This implies the necessity of upregulation of net acid and lactate flux capacity during adipocyte differentiation and function. However, the regulation of lactate- and acid/base transporters in adipocytes is poorly understood. Here, we tested the hypothesis that adipocyte thermogenesis, browning and differentiation are associated with an upregulation of plasma membrane lactate and acid/base transport capacity that in turn is important for adipocyte metabolism. The mRNA and protein levels of the lactate-H + transporter MCT1 and the Na + ,HCO 3 - cotransporter NBCe1 were upregulated in mouse interscapular brown and inguinal white adipose tissue upon cold induction of thermogenesis and browning. MCT1, MCT4, and NBCe1 were furthermore strongly upregulated at the mRNA and protein level upon differentiation of cultured pre-adipocytes. Adipocyte differentiation was accompanied by increased plasma membrane lactate flux capacity, which was reduced by MCT inhibition and by MCT1 knockdown. Finally, in differentiated brown adipocytes, glycolysis (assessed as ECAR), and after noradrenergic stimulation also oxidative metabolism (OCR), was decreased by MCT inhibition. We suggest that upregulation of MCT1- and MCT4-mediated lactate flux capacity and NBCe1-mediated HCO 3 - /pH homeostasis are important for the physiological function of mature adipocytes.

  17. Mutant E. coli strain with increased succinic acid production

    DOEpatents

    Donnelly, M.; Millard, C.S.; Stols, L.

    1998-06-23

    A method for isolating succinic acid producing bacteria is provided comprising increasing the biomass of an organism which lacks the ability to catabolize pyruvate, and then subjecting the biomass to glucose-rich medium in an anaerobic environment to enable pyruvate-catabolizing mutants to grow. The invention also provides for a mutant that produces high amounts of succinic acid, which as been derived from a parent which lacked the genes for pyruvate formate lyase and lactate dehydrogenase, and which belongs to the E.coli Group of Bacteria. 2 figs.

  18. Are arterial, muscle and working limb lactate exchange data obtained on men at altitude consistent with the hypothesis of an intracellular lactate shuttle?

    PubMed

    Brooks, G A

    1999-01-01

    The "Lactate Shuttle" Hypothesis posits that lactate removal requires exchange among producing and consuming cells. The "Intra-cellular Lactate Shuttle" hypothesis posits that lactate exchange occurs among compartments within cells, and that mitochondria are the major sites of cellular lactate disposal. Thus, cells with high mitochondrial densities (cardiocytes, myocytes, hepatocytes) are those which participate in lactate clearance. The model of an Intracellular Lactate Shuttle recognizes that the Keq for LDH is 3.6 x 10(4) M-1; thus, glycolysis results in cytosolic lactate production regardless of the intracellular PO2. The model also requires presence of a mitochondrial monocarboxylate transporter (MCT) that allows uptake of lactate as well as pyruvate, and intra-mitochondrial LDH whose function is linked to the ETC, and which permits lactate-->pyruvate conversion and oxidation. Recently, we have shown that liver, heart and muscle mitochondria readily oxidize lactate and contain LDH and MCT1. Accordingly, we have concluded that lactate is the predominant monocarboxylate oxidized by mitochondria in vivo. The model of an "Intra-cellular Lactate Shuttle" is consistent with many of the observations on men at sea level and altitude. The observations include: oxidation is the primary fate of lactate disposal during rest and exercise; lactate production and oxidation occur simultaneously within resting and working muscle; increasing [lactate]a increases muscle lactate extraction, and that by increasing SaO2 acclimatization reduces blood [lactate].

  19. Dinosaur lactation?

    PubMed

    Else, Paul L

    2013-02-01

    Lactation is a process associated with mammals, yet a number of birds feed their newly hatched young on secretions analogous to the milk of mammals. These secretions are produced from various sections (crop organ, oesophageal lining and proventriculus) of the upper digestive tract and possess similar levels of fat and protein, as well as added carotenoids, antibodies and, in the case of pigeons and doves, epidermal growth factor. Parental care in avian species has been proposed to originate from dinosaurs. This study examines the possibility that some dinosaurs used secretory feeding to increase the rate of growth of their young, estimated to be similar to that of present day birds and mammals. Dinosaur 'lactation' could also have facilitated immune responses as well as extending parental protection as a result of feeding newly hatched young in nest environments. While the arguments for dinosaur lactation are somewhat generic, a case study for lactation in herbivorous site-nesting dinosaurs is presented. It is proposes that secretory feeding could have been used to bridge the gap between hatching and establishment of the normal diet in some dinosaurs.

  20. Alcohol Dehydrogenase and Ethanol in the Stems of Trees 1

    PubMed Central

    Kimmerer, Thomas W.; Stringer, Mary A.

    1988-01-01

    Anaerobic fermentation in plants is usually thought to be a transient phenomenon, brought about by environmental limitations to oxygen availability, or by structural constraints to oxygen transport. The vascular cambium of trees is separated from the air by the outer bark and secondary phloem, and we hypothesized that the cambium may experience sufficient hypoxia to induce anaerobic fermentation. We found high alcohol dehydrogenase activity in the cambium of several tree species. Mean activity of alcohol dehydrogenase in Populus deltoides was 165 micromoles NADH oxidized per minute per gram fresh weight in May. Pyruvate decarboxylase activity was also present in the cambium of P. deltoides, with mean activity of 26 micromoles NADH oxidized per minute per gram fresh weight in May. Lactate dehydrogenase activity was not present in any tree species we examined. Contrary to our expectation, alcohol dehydrogenase activity was inversely related to bark thickness in Acer saccharum and unrelated to bark thickness in two Populus species. Bark thickness may be less important in limiting oxygen availability to the cambium than is oxygen consumption by rapidly respiring phloem and cambium in actively growing trees. Ethanol was present in the vascular cambium of all species examined, with mean concentrations of 35 to 143 nanomoles per gram fresh weight, depending on species. Ethanol was also present in xylem sap and may have been released from the cambium into the transpiration stream. The presence in the cambium of the enzymes necessary for fermentation as well as the products of fermentation is evidence that respiration in the vascular cambium of trees may be oxygen-limited, but other biosynthetic origins of ethanol have not been ruled out. PMID:16666209

  1. Elevated lactate during psychogenic hyperventilation.

    PubMed

    ter Avest, E; Patist, F M; Ter Maaten, J C; Nijsten, M W N

    2011-04-01

    Elevated arterial lactate levels are closely related to morbidity and mortality in various patient categories. In the present retrospective study, the relation between arterial lactate, partial pressure of carbon dioxide (Pco(2)) and pH was systematically investigated in patients who visited the emergency department (ED) with psychogenic hyperventilation. Over a 5-month period, all the patients who visited the ED of a university hospital with presumed psychogenic hyperventilation were evaluated. Psychogenic hyperventilation was presumed to be present when an increased respiratory rate (>20 min) was documented at or before the ED visit and when somatic causes explaining the hyperventilation were absent. Arterial blood gas and lactate levels (reference values 0.5-1.5 mmol/l) were immediately measured by a point-of-care analyser that was managed and calibrated by the central laboratory. During the study period, 46 patients were diagnosed as having psychogenic hyperventilation. The median (range) Pco(2) for this group was 4.3 (2.0-5.5) kPa, the pH was 7.47 (7.40-7.68) and the lactate level was 1.2 (0.5-4.4) mmol/l. 14 participants (30%) had a lactate level above the reference value of 1.5 mmol/l. Pco(2) was the most important predictor of lactate in multivariate analysis. None of the participants underwent any medical treatment other than observation at the ED or had been hospitalised after their ED visit. In patients with psychogenic hyperventilation, lactate levels are frequently elevated. Whereas high lactates are usually associated with acidosis and an increased risk of poor outcome, in patients with psychogenic hyperventilation, high lactates are associated with hypocapnia and alkalosis. In this context, elevated arterial lactate levels should not be regarded as an adverse sign.

  2. [Bone histomorphometry of lactating and no lactating hyperthyroid rats].

    PubMed

    Serakides, Rogéria; Ocarino, Natália de Melo; Magalhães, Fernanda do Carmo; Souza, Cíntia de Almeida; Leite, Eveline Dias; Freitas, Edmilson Santos de

    2008-06-01

    The objective of this study was to verify if hyperthyroidism potentiates the osteopenia lactational. 24 adult female rats were distributed in four groups: euthyroid no lactating (control), euthyroid lactating, hyperthyroid no lactating and hyperthyroid lactating. 20 days after gestation, all the animals were necropsied. The thoracic and lumbar vertebrae, the femur and tibia were decalcified and processed for histomorphometric analysis. The euthyroid lactating group presented intense osteopenia in the studied bones. In the hyperthyroid no lactating group, there was not any change in trabecular bone percentage in none of the analyzed bone. In the hyperthyroid lactating group, there was osteopenia in the tibia and femur, similar to the one in the euthyroid lactating group. But the trabecular bone percentage in all the vertebral bodies was significantly larger in comparison with the euthyroid lactating group. It was concluded that the hyperthyroidism does not potentiate the osteopenia lactational in female rats, but it minimizes the vertebral osteopenia once it stimulates the osteoblastic activity.

  3. The BRAF inhibitor vemurafenib activates mitochondrial metabolism and inhibits hyperpolarized pyruvate-lactate exchange in BRAF mutant human melanoma cells

    PubMed Central

    Delgado-Goni, Teresa; Falck Miniotis, Maria; Wantuch, Slawomir; Parkes, Harold G.; Marais, Richard; Workman, Paul; Leach, Martin O.; Beloueche-Babari, Mounia

    2016-01-01

    Understanding the impact of BRAF signaling inhibition in human melanoma on key disease mechanisms is important for developing biomarkers of therapeutic response and combination strategies to improve long term disease control. This work investigates the downstream metabolic consequences of BRAF inhibition with vemurafenib, the molecular and biochemical processes that underpin them, their significance for antineoplastic activity and potential as non-invasive imaging response biomarkers.1H NMR spectroscopy showed that vemurafenib decreases the glycolytic activity of BRAF mutant (WM266.4 and SKMEL28) but not BRAFWT (CHL-1 and D04) human melanoma cells. In WM266.4 cells, this was associated with increased acetate, glycine and myo-inositol levels and decreased fatty acyl signals, while the bioenergetic status was maintained. 13C NMR metabolic flux analysis of treated WM266.4 cells revealed inhibition of de novo lactate synthesis and glucose utilization, associated with increased oxidative and anaplerotic pyruvate carboxylase mitochondrial metabolism and decreased lipid synthesis. This metabolic shift was associated with depletion of HKII, acyl-CoA dehydrogenase 9, 3-phosphoglycerate dehydrogenase and monocarboxylate transporter (MCT) 1 and 4 in BRAF mutant but not BRAFWT cells and, interestingly, decreased BRAF mutant cell dependency on glucose and glutamine for growth. Further, the reduction in MCT1 expression observed led to inhibition of hyperpolarized 13C-pyruvate-lactate exchange, a parameter that is translatable to in vivo imaging studies, in live WM266.4 cells. In conclusion, our data provide new insights into the molecular and metabolic consequences of BRAF inhibition in BRAF-driven human melanoma cells that may have potential for combinatorial therapeutic targeting as well as non-invasive imaging of response. PMID:27765851

  4. Calcium signaling in brain mitochondria: interplay of malate aspartate NADH shuttle and calcium uniporter/mitochondrial dehydrogenase pathways.

    PubMed

    Contreras, Laura; Satrústegui, Jorgina

    2009-03-13

    Ca2+ signaling in mitochondria has been mainly attributed to Ca2+ entry to the matrix through the Ca2+ uniporter and activation of mitochondrial matrix dehydrogenases. However, mitochondria can also sense increases in cytosolic Ca2+ through a mechanism that involves the aspartate-glutamate carriers, extramitochondrial Ca2+ activation of the NADH malate-aspartate shuttle (MAS). Both pathways are linked through the shared substrate alpha-ketoglutarate (alphaKG). Here we have studied the interplay between the two pathways under conditions of Ca2+ activation. We show that alphaKG becomes limiting when Ca2+ enters in brain or heart mitochondria, but not liver mitochondria, resulting in a drop in alphaKG efflux through the oxoglutarate carrier and in a drop in MAS activity. Inhibition of alphaKG efflux and MAS activity by matrix Ca2+ in brain mitochondria was fully reversible upon Ca2+ efflux. Because of their differences in cytosolic calcium concentration requirements, the MAS and Ca2+ uniporter-mitochondrial dehydrogenase pathways are probably sequentially activated during a Ca2+ transient, and the inhibition of MAS at the center of the transient may provide an explanation for part of the increase in lactate observed in the stimulated brain in vivo.

  5. A Review of Worksite Lactation Accommodations.

    PubMed

    Hilliard, Elizabeth Dianne

    2017-01-01

    The purpose of this review was to examine workplace lactation accommodations, and their association with breastfeeding duration, and identify strategies occupational health professionals can use to promote lactation improvements. This study included literature published from 1985 through 2015 and listed in PubMed and CINAHL. Using Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA), 11 articles were identified for review. Presence of a corporate lactation program, on-site child care, and return to work/telephone lactation consultation were consistently associated with breastfeeding at 6 months. Other breastfeeding accommodations (i.e., lactation spaces, lactation breaks, worksite lactation policies, and supervisor/coworker support) were not consistently associated with breastfeeding duration. Occupational health professionals can play key roles in improving the effectiveness of lactation accommodations. Assuring adequate implementation of accommodations, increasing communication and marketing of accommodations, and promoting supervisor and coworker support are areas that occupational health professionals should explore for improving lactation duration.

  6. The functional readthrough extension of malate dehydrogenase reveals a modification of the genetic code

    PubMed Central

    Hofhuis, Julia; Schueren, Fabian; Nötzel, Christopher; Lingner, Thomas; Gärtner, Jutta; Jahn, Olaf

    2016-01-01

    Translational readthrough gives rise to C-terminally extended proteins, thereby providing the cell with new protein isoforms. These may have different properties from the parental proteins if the extensions contain functional domains. While for most genes amino acid incorporation at the stop codon is far lower than 0.1%, about 4% of malate dehydrogenase (MDH1) is physiologically extended by translational readthrough and the actual ratio of MDH1x (extended protein) to ‘normal' MDH1 is dependent on the cell type. In human cells, arginine and tryptophan are co-encoded by the MDH1x UGA stop codon. Readthrough is controlled by the 7-nucleotide high-readthrough stop codon context without contribution of the subsequent 50 nucleotides encoding the extension. All vertebrate MDH1x is directed to peroxisomes via a hidden peroxisomal targeting signal (PTS) in the readthrough extension, which is more highly conserved than the extension of lactate dehydrogenase B. The hidden PTS of non-mammalian MDH1x evolved to be more efficient than the PTS of mammalian MDH1x. These results provide insight into the genetic and functional co-evolution of these dually localized dehydrogenases. PMID:27881739

  7. Comparative performance of aldolase and lactate dehydrogenase rapid diagnostic tests in Plasmodium vivax detection

    PubMed Central

    2014-01-01

    Background Misdiagnosis of malaria by commercial rapid diagnostic tests (RDTs) is a major cause of concern in the diagnosis of malaria. This retrospective study was aimed at assessing the relative performance of four RDTs with emphasis on the detection of two Plasmodium vivax antigens: aldolase and lactate dehydrogenase (LDH). Methods Three commercially available Plasmodium LDH or aldolase antigen detection kits (One Step Malaria P.f/P.v, ParaHit Total ver. 1.0, SD Bioline Malaria) and an anti-P. vivax aldolase-specific monoclonal antibody (mAb) pair 1C3-12 F10 were evaluated with P. vivax positive as well as non-P. vivax samples and healthy samples using blood smear examination as standard. Each test was read according to the manufacturer’s instructions. Results MAb 1C3-12 F10 pair targeting P. vivax-specific aldolase exhibited very good specificity and sensitivity of 100 and 97.4%, respectively. Positive predictive value (PPV) and negative predictive value (NPV) of 100 and 99.5%, respectively, were also observed. The anti-P. vivax LDH in the One-Step Malaria P.f/P.v test showed sensitivity, specificity, PPV and NPV of 93.5, 98.0, 88.9 and 98.8%, respectively. ParaHit Total ver. 1.0 targeting the pan-aldolase antigen showed sensitivity, specificity of 97.4 and 99.6%, respectively. PPV and NPV were both 99.5%. SD Bioline had sensitivity, specificity, PPV and NPV of 93.5, 100, 100 and 98.8%, respectively. The overall sensitivity and specificity of all four RDTs were acceptable, especially for the aldolase detection tests. Five (6.5%) of the P. vivax-positive samples (n = 77) that were confirmed by microscopic examination as well as the two aldolase detection RDTs (mAb 1C3-12 F10 and ParaHit Total ver.1.0) were undetected by the two LDH detection RDTs (One Step Malaria P.f/P.v and SD Bioline). Similarly, two positive samples (2.6%) that were positively confirmed by the LDH detection RDTs were also undetected by the aldolase detection test kits. Conclusion

  8. Brain lactate metabolism: the discoveries and the controversies

    PubMed Central

    Dienel, Gerald A

    2012-01-01

    Potential roles for lactate in the energetics of brain activation have changed radically during the past three decades, shifting from waste product to supplemental fuel and signaling molecule. Current models for lactate transport and metabolism involving cellular responses to excitatory neurotransmission are highly debated, owing, in part, to discordant results obtained in different experimental systems and conditions. Major conclusions drawn from tabular data summarizing results obtained in many laboratories are as follows: Glutamate-stimulated glycolysis is not an inherent property of all astrocyte cultures. Synaptosomes from the adult brain and many preparations of cultured neurons have high capacities to increase glucose transport, glycolysis, and glucose-supported respiration, and pathway rates are stimulated by glutamate and compounds that enhance metabolic demand. Lactate accumulation in activated tissue is a minor fraction of glucose metabolized and does not reflect pathway fluxes. Brain activation in subjects with low plasma lactate causes outward, brain-to-blood lactate gradients, and lactate is quickly released in substantial amounts. Lactate utilization by the adult brain increases during lactate infusions and strenuous exercise that markedly increase blood lactate levels. Lactate can be an ‘opportunistic', glucose-sparing substrate when present in high amounts, but most evidence supports glucose as the major fuel for normal, activated brain. PMID:22186669

  9. Function of muscle-type lactate dehydrogenase and citrate synthase of the Galápagos marine iguana, Amblyrhynchus cristatus, in relation to temperature.

    PubMed

    Fields, Peter A; Strothers, Chad M; Mitchell, Mark A

    2008-05-01

    The Galápagos marine iguana, Amblyrhynchus cristatus, is unique among lizards in foraging subtidally, leading to activity across a broad range of ambient temperatures ( approximately 14-40 degrees C). To determine whether the marine iguana shows any biochemical changes consistent with maintaining enzyme function at both warm and cold body temperatures, we examined the function of the aerobic enzyme citrate synthase (CS) and the muscle isoform of the anaerobic enzyme lactate dehydrogenase (A(4)-LDH) in A. cristatus and a confamilial species, Iguana iguana, from 14 to 46 degrees C. We also deduced amino acid sequences from cDNA of each enzyme. In CS, despite two amino acid substitutions, we found no difference in the apparent Michaelis-Menten constant K(m) of oxaloacetate at any temperature, indicating that the substrate affinity of CS in A. cristatus has not adapted to changes in thermal environment. In A(4)-LDH, we used site-directed mutagenesis to show that the substitutions T9A and I283V (A. cristatus --> I. iguana) individually have no effect on kinetics, but together significantly decrease the K(m) of pyruvate and catalytic rate constant (k(cat)) of the A. cristatus ortholog. Thus, our data show that A. cristatus A(4)-LDH has not become cold adapted in response to this species' aquatic foraging behavior, and instead may be consistent with moderate warm adaptation with respect to the I. iguana ortholog.

  10. Effect of HX108-CS supplementation on exercise capacity and lactate accumulation after high-intensity exercise.

    PubMed

    Oh, Seung-Lyul; Chang, Hyukki; Kim, Hee-Jae; Kim, Yong-An; Kim, Dong-Sik; Ho, Seong-Hyun; Kim, Seon-Hee; Song, Wook

    2013-04-15

    In the present study, we determined the effects of HX108-CS (mixed extract of Schisandra chinensis and Chaenomeles sinensis) supplementation on lactate accumulation and endurance capacity. Furthermore, we examined CK (creatine kinase), LDH (lactate dehydrogenase) activity to determine whether the HX108-CS affected markers of skeletal muscle injury in vivo and in vitro. Exercise capacity was measured by an exhaustive swimming test using ICR mice divided into four groups; one group received distilled water (DW) (Control group, n = 10), and the other groups received three different dosages of HX108-CS (10, 50 and 100 mg/kg, n = 10 per group) solution in water orally. Then, for the time-dependent measurements of blood lactate, CK, and LDH, Sprague-Dawley rats were divided into two groups; one received DW (Control group, n = 10), and the other group received HX108-CS (100 mg/kg, n = 10) solution in the same way as mice. Before the exercise test, the animals were given either DW or HX108-CS for 2 weeks. High-intensity treadmill exercise was performed for 30 minutes. Blood samples were collected and analyzed during and after exercise. For the in vitro experiment, C2C12 cells were treated with HX108-CS to examine its effect on lactate production, CK, and LDH activity. Blood lactate concentration was significantly lowered immediately after treadmill exercise in HX108-CS group; however, there were no significant differences in activities of CK and LDH between HX108-CS and control during treadmill exercise and recovery phase. Furthermore, treatment with 100 mg/kg of HX108-CS led to a significant increase in the time to exhaustion in swimming test, and concurrently blood lactate concentration was significantly decreased in 50 and 100 mg/kg treated group. Moreover, our results of in vitro experiment showed that HX108-CS suppressed lactate production, CK, and LDH activity in a dose-dependent manner. These results suggest that supplementation with HX

  11. Effect of HX108-CS supplementation on exercise capacity and lactate accumulation after high-intensity exercise

    PubMed Central

    2013-01-01

    Background In the present study, we determined the effects of HX108-CS (mixed extract of Schisandra chinensis and Chaenomeles sinensis) supplementation on lactate accumulation and endurance capacity. Furthermore, we examined CK (creatine kinase), LDH (lactate dehydrogenase) activity to determine whether the HX108-CS affected markers of skeletal muscle injury in vivo and in vitro. Methods Exercise capacity was measured by an exhaustive swimming test using ICR mice divided into four groups; one group received distilled water (DW) (Control group, n = 10), and the other groups received three different dosages of HX108-CS (10, 50 and 100 mg/kg, n = 10 per group) solution in water orally. Then, for the time-dependent measurements of blood lactate, CK, and LDH, Sprague–Dawley rats were divided into two groups; one received DW (Control group, n = 10), and the other group received HX108-CS (100 mg/kg, n = 10) solution in the same way as mice. Before the exercise test, the animals were given either DW or HX108-CS for 2 weeks. High-intensity treadmill exercise was performed for 30 minutes. Blood samples were collected and analyzed during and after exercise. For the in vitro experiment, C2C12 cells were treated with HX108-CS to examine its effect on lactate production, CK, and LDH activity. Results Blood lactate concentration was significantly lowered immediately after treadmill exercise in HX108-CS group; however, there were no significant differences in activities of CK and LDH between HX108-CS and control during treadmill exercise and recovery phase. Furthermore, treatment with 100 mg/kg of HX108-CS led to a significant increase in the time to exhaustion in swimming test, and concurrently blood lactate concentration was significantly decreased in 50 and 100 mg/kg treated group. Moreover, our results of in vitro experiment showed that HX108-CS suppressed lactate production, CK, and LDH activity in a dose-dependent manner. Conclusions These

  12. Silent information regulator 1 modulator resveratrol increases brain lactate production and inhibits mitochondrial metabolism, whereas SRT1720 increases oxidative metabolism.

    PubMed

    Rowlands, Benjamin D; Lau, Chew Ling; Ryall, James G; Thomas, Donald S; Klugmann, Matthias; Beart, Philip M; Rae, Caroline D

    2015-07-01

    Silent information regulators (SIRTs) have been shown to deacetylate a range of metabolic enzymes, including those in glycolysis and the Krebs cycle, and thus alter their activity. SIRTs require NAD(+) for their activity, linking cellular energy status to enzyme activity. To examine the impact of SIRT1 modulation on oxidative metabolism, this study tests the effect of ligands that are either SIRT-activating compounds (resveratrol and SRT1720) or SIRT inhibitors (EX527) on the metabolism of (13)C-enriched substrates by guinea pig brain cortical tissue slices with (13)C and (1)H nuclear magnetic resonance spectroscopy. Resveratrol increased lactate labeling but decreased incorporation of (13)C into Krebs cycle intermediates, consistent with effects on AMPK and inhibition of the F0/F1-ATPase. By testing with resveratrol that was directly applied to astrocytes with a Seahorse analyzer, increased glycolytic shift and increased mitochondrial proton leak resulting from interactions of resveratrol with the mitochondrial electron transport chain were revealed. SRT1720, by contrast, stimulated incorporation of (13)C into Krebs cycle intermediates and reduced incorporation into lactate, although the inhibitor EX527 paradoxically also increased Krebs cycle (13)C incorporation. In summary, the various SIRT1 modulators show distinct acute effects on oxidative metabolism. The strong effects of resveratrol on the mitochondrial respiratory chain and on glycolysis suggest that caution should be used in attempts to increase bioavailability of this compound in the CNS. © 2015 Wiley Periodicals, Inc.

  13. Tungsten and Molybdenum Regulation of Formate Dehydrogenase Expression in Desulfovibrio vulgaris Hildenborough ▿

    PubMed Central

    da Silva, Sofia M.; Pimentel, Catarina; Valente, Filipa M. A.; Rodrigues-Pousada, Claudina; Pereira, Inês A. C.

    2011-01-01

    Formate is an important energy substrate for sulfate-reducing bacteria in natural environments, and both molybdenum- and tungsten-containing formate dehydrogenases have been reported in these organisms. In this work, we studied the effect of both metals on the levels of the three formate dehydrogenases encoded in the genome of Desulfovibrio vulgaris Hildenborough, with lactate, formate, or hydrogen as electron donors. Using Western blot analysis, quantitative real-time PCR, activity-stained gels, and protein purification, we show that a metal-dependent regulatory mechanism is present, resulting in the dimeric FdhAB protein being the main enzyme present in cells grown in the presence of tungsten and the trimeric FdhABC3 protein being the main enzyme in cells grown in the presence of molybdenum. The putatively membrane-associated formate dehydrogenase is detected only at low levels after growth with tungsten. Purification of the three enzymes and metal analysis shows that FdhABC3 specifically incorporates Mo, whereas FdhAB can incorporate both metals. The FdhAB enzyme has a much higher catalytic efficiency than the other two. Since sulfate reducers are likely to experience high sulfide concentrations that may result in low Mo bioavailability, the ability to use W is likely to constitute a selective advantage. PMID:21498650

  14. Fructose intake during gestation and lactation differentially affects the expression of hippocampal neurosteroidogenic enzymes in rat offspring.

    PubMed

    Mizuno, Genki; Munetsuna, Eiji; Yamada, Hiroya; Ando, Yoshitaka; Yamazaki, Mirai; Murase, Yuri; Kondo, Kanako; Ishikawa, Hiroaki; Teradaira, Ryoji; Suzuki, Koji; Ohashi, Koji

    2017-02-01

    Neurosteroids, steroidal hormones synthesized de novo from cholesterol within the brain, stimulate hippocampal functions such as neuron protection and synapse formation. Previously, we examined the effect of maternal fructose on the transcriptional regulation of neurosteroidogenic enzymes. We found that the mRNA expression level of the steroidogenic acute regulatory protein (StAR), peripheral benzodiazepine receptor (PBR), cytochrome P450(11β), 11β-hydroxysteroid dehydrogenase (HSD), and 17β-HSD was altered. However, we could not determine whether maternal fructose intake played a role in the gestation or lactation period because the dam rats were fed fructose solution during both periods. Thus, in this study, we analyzed the hippocampi of the offspring of dams fed fructose during the gestation or lactation period. Maternal fructose consumption during either the gestation or lactation period did not affect the mRNA levels of StAR, P450(17α), 11β-HSD-2, and 17β-HSD-1. PBR expression was down-regulated, even when rats consumed fructose during the lactation period only, while fructose consumption during gestation tended to activate the expression of P450(11β)-2. We found that maternal fructose intake during gestation and lactation differentially affected the expression of hippocampal neurosteroidogenic enzymes in the offspring.

  15. Fortified tuna bone powder supplementation increases bone mineral density of lactating rats and their offspring.

    PubMed

    Suntornsaratoon, Panan; Charoenphandhu, Narattaphol; Krishnamra, Nateetip

    2018-03-01

    Breastfeeding leads to bone calcium loss for milk production, resulting in progressive maternal osteopenia. Calcium supplement from natural sources has been postulated to be more beneficial to bone health than purified CaCO 3 because natural sources also contain other nutrients such as certain amino acids that might enhance calcium metabolism. Herein, we examined the effect of calcium supplementation from tuna bone powder and CaCO 3 on bones of dams and the offspring. Both forms of calcium supplement, i.e. tuna bone powder and CaCO 3 , increased maternal bone mineral density (BMD). However, bone histomorphometry revealed that only tuna bone had beneficial effect on maternal bone microstructure, i.e. increased bone formation, decreased bone resorption and increased in bone volume. Regarding the mechanical properties, the decreased ultimate load in non-supplement lactating mothers was restored to the load seen in nulliparous animals by calcium supplementation. Moreover, both tuna bone and CaCO 3 supplementation in mothers led to increased milk calcium concentration and consequently increased BMD in the growing offspring. Calcium supplement from tuna bone powder was effective in preventing maternal osteopenia. Tuna bone, which is a readily available fishing industrial waste, is a good alternative source of calcium supplement that increases BMD in both lactating mothers and the neonates. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  16. Mitochondrial Respiratory Defect Causes Dysfunctional Lactate Turnover via AMP-activated Protein Kinase Activation in Human-induced Pluripotent Stem Cell-derived Hepatocytes*

    PubMed Central

    Im, Ilkyun; Jang, Mi-jin; Park, Seung Ju; Lee, Sang-Hee; Choi, Jin-Ho; Yoo, Han-Wook; Kim, Seyun; Han, Yong-Mahn

    2015-01-01

    A defective mitochondrial respiratory chain complex (DMRC) causes various metabolic disorders in humans. However, the pathophysiology of DMRC in the liver remains unclear. To understand DMRC pathophysiology in vitro, DMRC-induced pluripotent stem cells were generated from dermal fibroblasts of a DMRC patient who had a homoplasmic mutation (m.3398T→C) in the mitochondrion-encoded NADH dehydrogenase 1 (MTND1) gene and that differentiated into hepatocytes (DMRC hepatocytes) in vitro. DMRC hepatocytes showed abnormalities in mitochondrial characteristics, the NAD+/NADH ratio, the glycogen storage level, the lactate turnover rate, and AMPK activity. Intriguingly, low glycogen storage and transcription of lactate turnover-related genes in DMRC hepatocytes were recovered by inhibition of AMPK activity. Thus, AMPK activation led to metabolic changes in terms of glycogen storage and lactate turnover in DMRC hepatocytes. These data demonstrate for the first time that energy depletion may lead to lactic acidosis in the DMRC patient by reduction of lactate uptake via AMPK in liver. PMID:26491018

  17. The effect of trinitrobenzene sulfonic acid (TNB) on colonocyte arachidonic acid metabolism.

    PubMed

    Stratton, M D; Sexe, R; Peterson, B; Kaminski, D L; Li, A P; Longo, W E

    1996-02-01

    In previous studies we found that luminal perfusion of the isolated left colon of the rabbit with the hapten, trinitrobenzene, resulted in the production of an acute inflammatory process associated with alterations in eicosanoid metabolism. As the colitis was attenuated by cyclooxygenase inhibitors it is possible that the inflammation was mediated by arachidonic acid metabolites. In the present study it was intended to evaluate the effect of trinitrobenzene on eicosanoid metabolism in transformed human colonic cells by exposing Caco-2++ cells to various doses of trinitrobenzene. Cell injury was evaluated by measuring lactate dehydrogenase levels and cyclooxygenase and lipoxygenase activity was evaluated by measuring prostanoid and leukotriene production. In separate experiments resting and trinitrobenzene stimulated cells were treated with indomethacin and dexamethasone. Trinitrobenzene produced increased prostaglandin E2 and 6-keto prostaglandin F1alpha++ and increased lactate dehydrogenase levels. Leukotriene B4 was significantly increased compared to control values at the highest TNB concentration administered. Indomethacin inhibited the lactate dehydrogenase and prostanoid changes, suggesting that the inflammatory changes produced were mediated by the prostanoids. Dexamethasone administered for 1 hr prior to trinitrobenzene decreased the 6-keto prostaglandin F1alpha but did not alter trinitrobenzene produced changes in lactate dehydrogenase concentrations. Exposure of Caco-2 cells to dexamethasone for 24 hr decreased the trinitrobenzene produced lactate dehydrogenase and eicosanoid changes. The results suggest that trinitrobenzene produces an acute injury to Caco-2 cells that may be mediated by the cyclooxygenase enzymes.

  18. Expression of Rice Mature Carbonic Anhydrase Gene Increase E. coli Tolerance to Heat Stress.

    PubMed

    Tianpei, Xiuzi; Mao, Zhinang; Zhu, Yingguo; Li, Shaoqing

    2015-05-01

    Carbonic anhydrate is a zinc-containing metalloenzyme and involved in plant abiotic stress tolerance. In this study, we found that heat stress could induce rice mature carbonic anhydrate gene over-expression in rice plants. An Escherichia coli heterologous expression system was performed to identify the function of rice mature carbonic anhydrate in vitro. By sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), mature OsCA fusion protein was identified and proved to be soluble. The results of spot, survival rate, and growth curve assay demonstrated that the expression of the mature OsCA could enhance the thermo-tolerance of the induced mature OsCA recombinants in comparison with controls under heat stress. Meanwhile, compared with controls, the levels of reactive oxygen species in induced mature OsCA recombinants were apparently low under heat stress, and correspondingly, activities of the critical antioxidant enzymes including superoxide dismutase, catalase, and peroxidase in the induced mature OsCA recombinants were significantly increased. Additionally, relative to controls, the activity of the lactate dehydrogenase decreased in the induced mature OsCA recombinants under heat stress. Based on these results, we suggest that mature OsCA protein could confer the E. coli recombinants' tolerance to heat stress by a synergistic fashion of increasing the antioxidant enzymes' activities to reduce the oxidative damage and maintaining the lactate dehydrogenase (LDH) activity of E. coli.

  19. Evaluation of Milk Trace Elements, Lactate Dehydrogenase, Alkaline Phosphatase and Aspartate Aminotransferase Activity of Subclinical Mastitis as and Indicator of Subclinical Mastitis in Riverine Buffalo (Bubalus bubalis).

    PubMed

    Guha, Anirban; Gera, Sandeep; Sharma, Anshu

    2012-03-01

    Mastitis is a highly morbid disease that requires detection at the subclinical stage. Tropical countries like India mainly depend on milch buffaloes for milk. The present study was conducted to investigate whether the trace minerals viz. copper (Cu), iron (Fe), zinc (Zn), cobalt (Co) and manganese (Mn) and enzyme activity of lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and aspartate aminotransferase (AST) in riverine buffalo milk can be used as an indicator of subclinical mastitis (SCM) with the aim of developing suitable diagnostic kit for SCM. Trace elements and enzyme activity in milk were estimated with Atomic absorption Spectrophotometer, GBC 932 plus and biochemical methods, respectively. Somatic cell count (SCC) was done microscopically. The cultural examination revealed Gram positive bacteria as the most prevalent etiological agent. A statistically significant (p<0.01) increase in SCC, Fe, Zn, Co and LDH occurred in SCM milk containing gram positive bacterial agents only. ALP was found to be elevated in milk infected by both gram positive and negative bacteria. The percent sensitivity, specificity and accuracy, predictive values and likelihood ratios were calculated taking bacterial culture examination and SCC≥2×10(5) cells/ml of milk as the benchmark. Only ALP and Zn, the former being superior, were found to be suitable for diagnosis of SCM irrespective of etiological agents. LDH, Co and Fe can be introduced in the screening programs where Gram positive bacteria are omnipresent. It is recommended that both ALP and Zn be measured together in milk to diagnose buffalo SCM, irrespective of etiology.

  20. DB Dehydrogenase: an online integrated structural database on enzyme dehydrogenase.

    PubMed

    Nandy, Suman Kumar; Bhuyan, Rajabrata; Seal, Alpana

    2012-01-01

    Dehydrogenase enzymes are almost inevitable for metabolic processes. Shortage or malfunctioning of dehydrogenases often leads to several acute diseases like cancers, retinal diseases, diabetes mellitus, Alzheimer, hepatitis B & C etc. With advancement in modern-day research, huge amount of sequential, structural and functional data are generated everyday and widens the gap between structural attributes and its functional understanding. DB Dehydrogenase is an effort to relate the functionalities of dehydrogenase with its structures. It is a completely web-based structural database, covering almost all dehydrogenases [~150 enzyme classes, ~1200 entries from ~160 organisms] whose structures are known. It is created by extracting and integrating various online resources to provide the true and reliable data and implemented by MySQL relational database through user friendly web interfaces using CGI Perl. Flexible search options are there for data extraction and exploration. To summarize, sequence, structure, function of all dehydrogenases in one place along with the necessary option of cross-referencing; this database will be utile for researchers to carry out further work in this field. The database is available for free at http://www.bifku.in/DBD/

  1. Relationship between season, lactation number and incidence of clinical mastitis in different stages of lactation in a Holstein dairy farm.

    PubMed

    Moosavi, Maede; Mirzaei, Abdolah; Ghavami, Mohsen; Tamadon, Amin

    2014-01-01

    The aim of the present study was to compare the occurrence and duration of clinical mastitis in different seasons, stages of lactation period and parities in a Holstein dairy farm in Iran. A retrospective epidemiological survey from April 2005 to March 2008 was conducted on 884 clinical mastitis cases of 7437 lactations. Data of each case including calendar-date of mastitis onset, days in milk (DIM) of mastitis onset (early: 0-74 DIM; middle: 75-150 DIM, and late ≥ 150 DIM), duration of mastitis, and parity (1, 2, and ≥ 3) were recorded. Based on date of mastitis onset, cases were classified into stages of lactation. Moreover, beginning of mastitis was seasonally categorized. Duration of clinical mastitis after treatment in early lactation was less than late lactation in the first-parity cows (p = 0.005). In early lactation period, the first-parity cows suffered clinical mastitis in days earlier than two other parity groups (p < 0.001). Moreover, in late lactation period, the first-parity cows had clinical mastitis in days later than cows in the third and more parities (p = 0.002). Occurrence of clinical mastitis in summer increased in late lactation period but in winter increased in early lactation period (p = 0.001). In addition, occurrence time of clinical mastitis in summer were in days later than in spring (p = 0.02) and winter (p = 0.03) in early lactation period. In conclusion, occurrence of mastitis in winter and spring during early lactation and in summer during late lactation period were more prevalent especially in lower parities.

  2. Relationship between season, lactation number and incidence of clinical mastitis in different stages of lactation in a Holstein dairy farm

    PubMed Central

    Moosavi, Maede; Mirzaei, Abdolah; Ghavami, Mohsen; Tamadon, Amin

    2014-01-01

    The aim of the present study was to compare the occurrence and duration of clinical mastitis in different seasons, stages of lactation period and parities in a Holstein dairy farm in Iran. A retrospective epidemiological survey from April 2005 to March 2008 was conducted on 884 clinical mastitis cases of 7437 lactations. Data of each case including calendar-date of mastitis onset, days in milk (DIM) of mastitis onset (early: 0-74 DIM; middle: 75-150 DIM, and late ≥ 150 DIM), duration of mastitis, and parity (1, 2, and ≥ 3) were recorded. Based on date of mastitis onset, cases were classified into stages of lactation. Moreover, beginning of mastitis was seasonally categorized. Duration of clinical mastitis after treatment in early lactation was less than late lactation in the first-parity cows (p = 0.005). In early lactation period, the first-parity cows suffered clinical mastitis in days earlier than two other parity groups (p < 0.001). Moreover, in late lactation period, the first-parity cows had clinical mastitis in days later than cows in the third and more parities (p = 0.002). Occurrence of clinical mastitis in summer increased in late lactation period but in winter increased in early lactation period (p = 0.001). In addition, occurrence time of clinical mastitis in summer were in days later than in spring (p = 0.02) and winter (p = 0.03) in early lactation period. In conclusion, occurrence of mastitis in winter and spring during early lactation and in summer during late lactation period were more prevalent especially in lower parities. PMID:25568687

  3. Bioactivity-Guided Identification and Cell Signaling Technology to Delineate the Lactate Dehydrogenase A Inhibition Effects of Spatholobus suberectus on Breast Cancer

    PubMed Central

    Wang, Zhiyu; Wang, Dongmei; Han, Shouwei; Wang, Neng; Mo, Feizhi; Loo, Tjing Yung; Shen, Jiangang; Huang, Hui; Chen, Jianping

    2013-01-01

    Aerobic glycolysis is an important feature of cancer cells. In recent years, lactate dehydrogenase A (LDH-A) is emerging as a novel therapeutic target for cancer treatment. Seeking LDH-A inhibitors from natural resources has been paid much attention for drug discovery. Spatholobus suberectus (SS) is a common herbal medicine used in China for treating blood-stasis related diseases such as cancer. This study aims to explore the potential medicinal application of SS for LDH-A inhibition on breast cancer and to determine its bioactive compounds. We found that SS manifested apoptosis-inducing, cell cycle arresting and anti-LDH-A activities in both estrogen-dependent human MCF-7 cells and estrogen-independent MDA-MB-231 cell. Oral herbal extracts (1 g/kg/d) administration attenuated tumor growth and LDH-A expression in both breast cancer xenografts. Bioactivity-guided fractionation finally identified epigallocatechin as a key compound in SS inhibiting LDH-A activity. Further studies revealed that LDH-A plays a critical role in mediating the apoptosis-induction effects of epigallocatechin. The inhibited LDH-A activities by epigallocatechin is attributed to disassociation of Hsp90 from HIF-1α and subsequent accelerated HIF-1α proteasome degradation. In vivo study also demonstrated that epigallocatechin could significantly inhibit breast cancer growth, HIF-1α/LDH-A expression and trigger apoptosis without bringing toxic effects. The preclinical study thus suggests that the potential medicinal application of SS for inhibiting cancer LDH-A activity and the possibility to consider epigallocatechin as a lead compound to develop LDH-A inhibitors. Future studies of SS for chemoprevention or chemosensitization against breast cancer are thus warranted. PMID:23457597

  4. Development of an enzymatic assay to measure lactate in perchloric acid-precipitated cerebrospinal fluid.

    PubMed

    Lu, Jun; Genzen, Jonathan R; Grenache, David G

    2018-04-27

    Individuals with inherited deficiencies of the pyruvate dehydrogenase complex or the respiratory chain complex can have increased concentrations of cerebrospinal fluid (CSF) lactate. Such measurements are clinical useful when measured in conjunction with pyruvate in order to calculate the lactate:pyruvate (L:P) ratio, a useful surrogate of cytosolic redox status. CSF pyruvate is measured in a protein-free supernatant prepared by the addition of CSF to perchloric acid while lactate is measured in untreated CSF. Utilizing the same sample for both lactate and pyruvate measurements is desirable. To develop a method to measure lactate in perchloric-acid precipitated CSF and validate the L:P ratio as calculated from the analysis of both analytes in the same sample. Samples were prepared by the addition of 1 mL CSF to 2 mL 8% (w/v) cold perchloric acid, incubated on ice for 10 min, then centrifuged to obtain a protein-free supernatant. Lactate was measured by its oxidation to pyruvate and hydrogen peroxide using lactate oxidase and the absorbance of the resulting chromogen determined at 540 nm on a Roche cobas c501 chemistry analyzer. Method accuracy, linearity, imprecision and sensitivity were determined and a reference interval was verified. To assess accuracy, this method was compared to lactate determined in unaltered CSF at another laboratory using 41 specimens with lactate concentrations from 0.6-11.9 mmol/L. Linear regression produced a slope of 1.09 and y-intercept of 0.26 (R 2  = 1.00). Recovery was performed by ad-mixes of a high lactate standard and a CSF pool in different ratios to create a set of 19 samples prior to preparing protein-free supernatants. Recovery was 94.6-100% (mean ± SD was 97.4 ± 1.4%) at lactate concentrations of 2.68 to 12.63 mmol/L. Linearity was determined by combining two supernatants with low and high lactate concentrations in different ratios to create a set of six samples (0.15-12.70 mmol/L) that were

  5. Metabolic engineering of mannitol production in Lactococcus lactis: influence of overexpression of mannitol 1-phosphate dehydrogenase in different genetic backgrounds.

    PubMed

    Wisselink, H Wouter; Mars, Astrid E; van der Meer, Pieter; Eggink, Gerrit; Hugenholtz, Jeroen

    2004-07-01

    To obtain a mannitol-producing Lactococcus lactis strain, the mannitol 1-phosphate dehydrogenase gene (mtlD) from Lactobacillus plantarum was overexpressed in a wild-type strain, a lactate dehydrogenase(LDH)-deficient strain, and a strain with reduced phosphofructokinase activity. High-performance liquid chromatography and (13)C nuclear magnetic resonance analysis revealed that small amounts (<1%) of mannitol were formed by growing cells of mtlD-overexpressing LDH-deficient and phosphofructokinase-reduced strains, whereas resting cells of the LDH-deficient transformant converted 25% of glucose into mannitol. Moreover, the formed mannitol was not reutilized upon glucose depletion. Of the metabolic-engineering strategies investigated in this work, mtlD-overexpressing LDH-deficient L. lactis seemed to be the most promising strain for mannitol production.

  6. Maternal Nicotine Exposure During Late Gestation and Lactation Increases Anxiety-Like and Impulsive Decision-Making Behavior in Adolescent Offspring of Rat.

    PubMed

    Lee, Hyunchan; Chung, Sooyeon; Noh, Jihyun

    2016-10-01

    Prenatal nicotine exposure over an entire pregnancy has been associated with an increased prevalence of hyperactivity, anxiety-like behavior and depression-like behavior in mature rats. However, the effects of maternal nicotine exposure in late gestation and lactation on the psychology and behavior of adolescent rat offspring are unclear. Thus, we investigated the effect of nicotine exposure during late gestation and lactation on anxiety-like and impulsive decision-making behavior in adolescent offspring of rat. Female rats were orally exposed to nicotine which is within range of plasma level of human chronic smokers during the period of third last period of gestation and lactation. When the offspring were weaned, we observed alterations in the anxiety-like behavior and decision-making ability of adolescent rat offspring using light/dark box test and T-maze delay-based cost-benefit decision-making task. The maternal consumption of nicotine reduced both the time spent in the light compartment and the number of transitions compared to nicotine-free rats. Moreover, such nicotine exposed adolescent offspring rats showed impulsive decision making which chose the instant reward in a decision-making situation. We found that nicotine exposure during late gestation and lactation induces an increase in anxiety-like and impulsive decision-making behavior at this developmental stage. These findings suggest that maternal nicotine-exposed offspring are at an increased risk of developing anxious and impulsive behavior.

  7. A novel amperometric biosensor based on gold nanoparticles anchored on reduced graphene oxide for sensitive detection of l-lactate tumor biomarker.

    PubMed

    Azzouzi, Sawsen; Rotariu, Lucian; Benito, Ana M; Maser, Wolfgang K; Ben Ali, Mounir; Bala, Camelia

    2015-07-15

    In this work, a novel amperometric biosensor based on gold nanoparticles anchored on reduced graphene oxide (RGO-AuNPs) and l-lactate dehydrogenase (LDH) was developed for the sensing of l-lactate. Firstly, the RGO-AuNPs modified screen printed electrodes were tested for NADH detection showing a wide dynamic range and a low detection limit. Next, the biosensor was constructed by incorporating both enzyme and RGO-AuNPs in a sol gel matrix derived from tetrametoxysilane and methyltrimetoxysilane. The enzyme loading, working pH, and coenzyme concentration were optimized. The biosensor linearly responded to l-lactate in the range of 10µM-5mM and showed a good specific sensitivity of 154µA/mMcm(2) with a detection limit of 0.13µM. This was accompanied by good reproducibility and operational stability. Tests on artificial serum proved that l-lactate can be determined practically without interferences from commonly interfering compounds such as urate, paracetamol and l-ascorbate. Our LDH/RGO-AuNPs/SPCE based biosensor thus performs as electrochemical device for the detection of l-lactate as a viable early cancer bio-marker. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. [The effect of subchronic inhalations of nitric oxide on metabolic processes in blood of experimental animals].

    PubMed

    Soloveva, A G; Peretyagin, S P

    2016-01-01

    Metabolic processes were investigated in plasma and erythrocytes of Wistar rats exposed to daily 10-min sessions of NO inhalation for 30 days. These included determination of glucose and lactate, catalase activity, and activities of aldehyde dehydrogenase (ALDH), lactate dehydrogenase (LDH), and catalase. NO inhalation in a concentration of 20 ppm, 50 ppm and 100 ppm caused an increase in glucose and lactate. Inhalation of 100 ppm NO also increased catalase activity. Inhalation of all NO concentrations resulted in a decrease of ALDH activity, while the decrease in LDH activity was observed at NO concentrations of 50-100 ppm.

  9. Investigation of drying stresses on proteins during lyophilization: differentiation between primary and secondary-drying stresses on lactate dehydrogenase using a humidity controlled mini freeze-dryer.

    PubMed

    Luthra, Sumit; Obert, Jean-Philippe; Kalonia, Devendra S; Pikal, Michael J

    2007-01-01

    This article describes the design, performance testing, and application of a controlled humidity mini-freeze-dryer in studying the physical stability of lactate dehydrogenase during lyophilization. Performance evaluation of the mini-freeze-dryer was conducted with tests, namely water sublimation, radiation heat exchange, lowest achievable temperature, and leak testing. Protein stability studies were conducted by comparing protein activity at various stages of lyophilization with the initial activity. The shelf and condenser temperature were stable at <-40 degrees C, wall temperature was within 2 degrees C of the shelf temperature, and the leak rate was small. The chamber pressure was controlled by the ice on the condenser and the product temperature during sublimation was equal to the shelf temperature. Addition of Tween 80 prevented activity loss in solution and after freeze-thaw. No activity loss was observed after primary-drying even in absence of lyoprotectants and with collapse of cake structure. Five percent (w/w) sucrose concentration was required to achieve full stabilization. In conclusion, performance testing established that the mini-freeze-dryer was suitable for mechanistic freeze-drying studies. Secondary-drying was the critical step for protein stability. The concentration of sucrose required to stabilize the protein completely was several orders of magnitude higher than that required to satisfy the direct interaction requirement of the protein. (c) 2006 Wiley-Liss, Inc. and the American Pharmacists Association.

  10. Serum lactate dehydrogenase profile as a retrospective indicator of uterine preparedness for labor: a prospective, observational study

    PubMed Central

    2013-01-01

    Background Lactate dehydrogenase (LDH) isoenzymes are required for adenosine triphosphate production, with each of five different isoenzymes having varying proficiencies in anaerobic versus aerobic environments. With advancing pregnancy, the isoenzyme profile in uterine muscle shifts toward a more anaerobic profile, speculatively to facilitate uterine efficiency during periods of low oxygen that accompany labor contractions. Profile shifting may even occur throughout labor. Maternal serum LDH levels between 24–48 hours following delivery predominantly originate from uterine muscle, reflecting the enzymatic state of the myometrium during labor. Our purpose was to describe serum LDH isoenzymes 24–30 hours post-delivery to determine if cervical dilation rates following labor admission were associated with a particular LDH profile. We also compared differences in post-delivery LDH isoenzyme profiles between women admitted in pre-active versus established active labor. Methods Low-risk, nulliparous women with spontaneous labor onset were sampled (n = 91). Maternal serum LDH was measured at labor admission and 24–30 hours post-vaginal delivery. Rates of cervical dilation during the first four hours after admission were also measured. Spearman’s rho coefficients were used for association testing and t tests evaluated for group and paired-sample differences. Results More efficient dilation following admission was associated with decreased LDH1 (p = 0.029) and increased LDH3 and LDH4 (p = 0.017 and p = 0.017, respectively) in the post-delivery period. Women admitted in established active labor had higher relative serum levels of LDH3 (t = 2.373; p = 0.023) and LDH4 (t = 2.268; p = 0.029) and lower levels of LDH1 (t = 2.073; p = 0.045) and LDH5 (t = 2.041; p = 0.048) when compared to women admitted in pre-active labor. Despite having similar dilatations at admission (3.4 ± 0.5 and 3.7 ± 0.6 cm, respectively

  11. On-farm feeding interventions to increase milk production in lactating dairy cows.

    PubMed

    Wanapat, Metha; Foiklang, Suban; Phesatcha, Kampanat; Paoinn, Chainarong; Ampapon, Thiwakorn; Norrapoke, Thitima; Kang, Sungchhang

    2017-04-01

    The objective of this study was to investigate the effect of tropical legume (Phaseolus calcaratus) mixed with ruzi grass feeding on the performance of lactating dairy cows. Eighty-eight lactating dairy cows from 22 smallholder dairy farms northeast of Thailand were assigned to respective dietary treatments according to a Randomized Completely Block Design (RCBD). Four cows were selected from each farm and were allocated into two different feeding groups as follows: ruzi grass and P. calcaratus mixed with ruzi grass (1:1 ratio), respectively. All cows were fed with roughage ad libitum with 1:2 ratio of concentrate diet to milk yield. The results revealed that total dry matter intake, ruminal volatile fatty acids, and ammonia nitrogen concentration were enhanced when cows were fed with P. calcaratus mixed with ruzi grass (P < 0.05). Moreover, feeding tropical legume mixed with ruzi grass could increase milk production and milk protein in this study. Importantly, an economical assessment showed that milk income and the profit from milk sale were significantly greater in cows fed the mixture of roughage than those from the non-mixed group. This study concluded that high-quality roughage as tropical legume mixed with ruzi grass at the ratio of 1:1 brought out the remarkable and practical implementation for smallholder dairy farms, and the intervention was practical and deserving of more on-farm intervention.

  12. Blockade of P-Glycoprotein Decreased the Disposition of Phenformin and Increased Plasma Lactate Level.

    PubMed

    Choi, Min-Koo; Song, Im-Sook

    2016-03-01

    This study aimed to investigate the in vivo relevance of P-glycoprotein (P-gp) in the pharmacokinetics and adverse effect of phenformin. To investigate the involvement of P-gp in the transport of phenformin, a bi-directional transport of phenformin was carried out in LLC-PK1 cells overexpressing P-gp, LLC-PK1-Pgp. Basal to apical transport of phenformin was 3.9-fold greater than apical to basal transport and became saturated with increasing phenformin concentration (2-75 μM) in LLC-PK1-Pgp, suggesting the involvement of P-gp in phenformin transport. Intrinsic clearance mediated by P-gp was 1.9 μL/min while passive diffusion clearance was 0.31 μL/min. Thus, P-gp contributed more to phenformin transport than passive diffusion. To investigate the contribution of P-gp on the pharmacokinetics and adverse effect of phenformin, the effects of verapamil, a P-gp inhibitor, on the pharmacokinetics of phenformin were also examined in rats. The plasma concentrations of phenformin were increased following oral administration of phenformin and intravenous verapamil infusion compared with those administerd phenformin alone. Pharmacokinetic parameters such as Cmax and AUC of phenformin increased and CL/F and Vss/F decreased as a consequence of verapamil treatment. These results suggested that P-gp blockade by verapamil may decrease the phenformin disposition and increase plasma phenformin concentrations. P-gp inhibition by verapamil treatment also increased plasma lactate concentration, which is a crucial adverse event of phenformin. In conclusion, P-gp may play an important role in phenformin transport process and, therefore, contribute to the modulation of pharmacokinetics of phenformin and onset of plasma lactate level.

  13. Blockade of P-Glycoprotein Decreased the Disposition of Phenformin and Increased Plasma Lactate Level

    PubMed Central

    Choi, Min-Koo; Song, Im-Sook

    2016-01-01

    This study aimed to investigate the in vivo relevance of P-glycoprotein (P-gp) in the pharmacokinetics and adverse effect of phenformin. To investigate the involvement of P-gp in the transport of phenformin, a bi-directional transport of phenformin was carried out in LLC-PK1 cells overexpressing P-gp, LLC-PK1-Pgp. Basal to apical transport of phenformin was 3.9-fold greater than apical to basal transport and became saturated with increasing phenformin concentration (2–75 μM) in LLC-PK1-Pgp, suggesting the involvement of P-gp in phenformin transport. Intrinsic clearance mediated by P-gp was 1.9 μL/min while passive diffusion clearance was 0.31 μL/min. Thus, P-gp contributed more to phenformin transport than passive diffusion. To investigate the contribution of P-gp on the pharmacokinetics and adverse effect of phenformin, the effects of verapamil, a P-gp inhibitor, on the pharmacokinetics of phenformin were also examined in rats. The plasma concentrations of phenformin were increased following oral administration of phenformin and intravenous verapamil infusion compared with those administerd phenformin alone. Pharmacokinetic parameters such as Cmax and AUC of phenformin increased and CL/F and Vss/F decreased as a consequence of verapamil treatment. These results suggested that P-gp blockade by verapamil may decrease the phenformin disposition and increase plasma phenformin concentrations. P-gp inhibition by verapamil treatment also increased plasma lactate concentration, which is a crucial adverse event of phenformin. In conclusion, P-gp may play an important role in phenformin transport process and, therefore, contribute to the modulation of pharmacokinetics of phenformin and onset of plasma lactate level. PMID:26797108

  14. Expression of the yeast NADH dehydrogenase Ndi1 in Drosophila confers increased lifespan independently of dietary restriction

    PubMed Central

    Sanz, Alberto; Soikkeli, Mikko; Portero-Otín, Manuel; Wilson, Angela; Kemppainen, Esko; McIlroy, George; Ellilä, Simo; Kemppainen, Kia K.; Tuomela, Tea; Lakanmaa, Matti; Kiviranta, Essi; Stefanatos, Rhoda; Dufour, Eric; Hutz, Bettina; Naudí, Alba; Jové, Mariona; Zeb, Akbar; Vartiainen, Suvi; Matsuno-Yagi, Akemi; Yagi, Takao; Rustin, Pierre; Pamplona, Reinald; Jacobs, Howard T.

    2010-01-01

    Mutations in mitochondrial oxidative phosphorylation complex I are associated with multiple pathologies, and complex I has been proposed as a crucial regulator of animal longevity. In yeast, the single-subunit NADH dehydrogenase Ndi1 serves as a non-proton-translocating alternative enzyme that replaces complex I, bringing about the reoxidation of intramitochondrial NADH. We have created transgenic strains of Drosophila that express yeast NDI1 ubiquitously. Mitochondrial extracts from NDI1-expressing flies displayed a rotenone-insensitive NADH dehydrogenase activity, and functionality of the enzyme in vivo was confirmed by the rescue of lethality resulting from RNAi knockdown of complex I. NDI1 expression increased median, mean, and maximum lifespan independently of dietary restriction, and with no change in sirtuin activity. NDI1 expression mitigated the aging associated decline in respiratory capacity and the accompanying increase in mitochondrial reactive oxygen species production, and resulted in decreased accumulation of markers of oxidative damage in aged flies. Our results support a central role of mitochondrial oxidative phosphorylation complex I in influencing longevity via oxidative stress, independently of pathways connected to nutrition and growth signaling. PMID:20435911

  15. Capacitive malaria aptasensor using Plasmodium falciparum glutamate dehydrogenase as target antigen in undiluted human serum.

    PubMed

    Singh, Naveen K; Arya, Sunil K; Estrela, Pedro; Goswami, Pranab

    2018-06-08

    A capacitive aptasensor for detecting the malaria biomarker, Plasmodium falciparum glutamate dehydrogenase (PfGDH), directly in human serum samples developed. A thiolated ssDNA aptamer (NG3) that binds specifically to PfGDH antigen with high affinity (K d = 79 nM) was used to develop the aptasensor. The aptasensor produced capacitance response at an optimized frequency of 2 Hz in a non-Faradaic electrochemical impedance based signal transduction platform. The aptasensor exhibited a wide dynamic range of 100 fM-100 nM with a limits of detection of 0.77 pM in serum samples. The interference from other predominant malarial biomarkers, namely, Plasmodium falciparum -lactate dehydrogenase and -histidine rich protein-II on the aptasensor was negligible. This PfGDH aptasensor with highly sensitive and label free detection capability has great application potential for diagnosis of asymptotic malaria and monitoring the regression of malaria during treatment regime with antimalarial drugs. Copyright © 2018 Elsevier B.V. All rights reserved.

  16. Influence of disposable, concentric needle electrodes on muscle enzyme and lactate serum levels.

    PubMed

    Finsterer, Josef; Mittendorfer, Bettina; Neuhuber, Werner; Löscher, Wolfgang N

    2002-08-01

    Several studies addressed the question whether needle-EMG causes elevation of muscle enzymes [aspartate-aminotransferase, alanine-aminotransferase, lactate-dehydrogenase, creatine-phosphokinase (CPK), isoenzyme-MB, aldolase] and lactate with conflicting results. However, these studies used sterilizable needle electrodes and different protocols and methods to record EMGs and determine muscle enzymes. This study examined if muscle enzymes are elevated immediately after or 24 h following EMGs with disposable, concentric needle-electrodes, and if they are dependent on age, sex, muscle, number of investigated sites and previous CPK-elevation. In 53 subjects, 24 woman, 29 men, aged 17-88 years, muscle enzymes were determined before, immediately after and 24 h following EMG with disposable, concentric needle-electrodes. Muscle enzymes were not different before, immediately after and 24 h following the EMG. Muscle enzymes were not different between patients 60 years of age. Apart from higher CPK in men than women, muscle enzymes were not different between the genders. Apart from CPK, muscle enzymes were not different between the brachial biceps and anterior tibial muscle. Muscle enzymes were not different if 20 sites were investigated and were independent on pre-EMG CPK-levels. In conclusion this study shows that muscle enzymes do not increase immediately or 24 h following EMG with disposable, concentric needle-electrodes, irrespective of age, gender, muscle, number of investigated sites and pre-EMG CK-levels.

  17. Glucocorticoid regulation in rat brain cell cultures. Hydrocortisone increases the rate of synthesis of glycerol phosphate dehydrogenase in C6 glioma cells. [Tritium tracer technique

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    McGinnis, J.F.; de Vellis, J.

    Cytoplasmic glycerol phosphate dehydrogenase (sn-glycerol-3-phosphate: NAD/sup +/ 2-oxidoreductase, EC 1.1.1.8) was rapidly purified from rat skeletal muscle in high yield using a combination of classical and affinity techniques. A single band of protein having a molecular weight of 30,000 was found using dodecyl sulfate-polyacrylamide gel electrophoresis. Antisera were generated in rabbits against the purified enzyme and demonstrated to be monospecific by Ouchterlony immunodiffusion against crude homogenates from hydrocortisone-induced and uninduced C6 cells. All of the radioactivity in immunoprecipitates from (/sup 3/H)leucine-labeled cells co-migrated with purified glycerol phosphate dehydrogenase. The amount of radioactivity precipitated was directly proportional to the amount ofmore » labeled glycerol phosphate dehydrogenase present, indicating that the assay could be used to quantitate newly synthesized glycerol phosphate dehydrogenase molecules. Using these techniques, the induction of glycerol phosphate dehydrogenase activity by hydrocortisone in the C6 glioma cell line was shown to be due to an increase in the rate of synthesis of the enzyme. Analysis of the kinetics of induction and deinduction supports the above conclusion and suggests that there is essentially no change in the rate of degradation of glycerol phosphate dehydrogenase in the presence and absence of hormone.« less

  18. Consumption of sucrose, but not high fructose corn syrup, leads to increased adiposity and dyslipidaemia in the pregnant and lactating rat.

    PubMed

    Toop, C R; Muhlhausler, B S; O'Dea, K; Gentili, S

    2015-02-01

    Excess consumption of added sugars, including sucrose and high fructose corn syrup (HFCS-55), have been implicated in the global epidemics of obesity and type 2 diabetes. This study aimed to investigate and compare the impact of maternal consumption of sucrose or HFCS-55 during pregnancy and lactation on the metabolic health of the dam and her offspring at birth. Female Albino Wistar rats were given access to chow and water, in addition to a sucrose or HFCS-55 beverage (10% w/v) before, and during pregnancy and lactation. Maternal glucose tolerance was determined throughout the study, and a postmortem was conducted on dams following lactation, and on offspring within 24 h of birth. Sucrose and HFCS-55 consumption resulted in increased total energy intake compared with controls, however the increase from sucrose consumption was accompanied by a compensatory decrease in chow consumption. There was no effect of sucrose or HFCS-55 consumption on body weight, however sucrose consumption resulted in increased adiposity and elevated total plasma cholesterol in the dam, while HFCS-55 consumption resulted in increased plasma insulin and decreased plasma non-esterified fatty acids (NEFA). Maternal HFCS-55 consumption was associated with decreased relative liver weight and plasma NEFA in the offspring at birth. There was no effect of either treatment on pup weight at birth. These findings suggest that both sucrose and HFCS-55 consumption during pregnancy and lactation have the potential to impact negatively on maternal metabolic health, which may have adverse consequences for the long-term health of the offspring.

  19. Regional analyses of CNS microdialysate glucose and lactate in seizure patients.

    PubMed

    Cornford, Eain M; Shamsa, Kamran; Zeitzer, Jamie M; Enriquez, Cathleen M; Wilson, Charles L; Behnke, Eric J; Fried, Itzhak; Engel, Jerome

    2002-11-01

    To correlate glucose (and lactate) results obtained from microdialysate to recent studies suggesting that glucose transporter activity may be significantly altered in seizures. We used a fluorometric technique to quantify glucose and lactate levels in microdialysates collected from two to four depth electrodes implanted per patient in the temporal and frontal lobes of a series of four patients. Hour-by-hour and day-to-day changes in brain glucose and lactate levels at the same site were recorded. Additionally we compared regional variations in lactate/glucose ratios around the predicted epileptogenic region. Lactate/glucose ratios in the range of 1-2:1 were the most commonly seen. When the lactate/glucose ratio was <1:1, we typically observed a relative increase in local glucose concentration (rather than decreased lactate), suggesting increased transport, perhaps without increased glycolysis. In some sites, lactate/glucose ratios of 3:1-15:1 were seen, suggesting that a circumscribed zone of inhibition of tricarboxylic acid cycle activity may have been locally induced. In these dialysates, collected from probes closer to the epileptogenic region, the large increase in lactate/glucose ratios was a result of both increased lactate and reduced glucose levels. We conclude that regional variations in brain extracellular glucose concentrations may be of greater magnitude than previously believed and become even more accentuated in partial seizure patients. Data from concomitant assays of microdialysate lactate and glucose may aid in understanding cerebral metabolism.

  20. A lactate dehydrogenase ELISA-based assay for the in vitro determination of Plasmodium berghei sensitivity to anti-malarial drugs.

    PubMed

    Orjuela-Sánchez, Pamela; Duggan, Erika; Nolan, John; Frangos, John A; Carvalho, Leonardo Jm

    2012-11-05

    Plasmodium berghei rodent malaria is a well-known model for the investigation of anti-malarial drug efficacy in vivo. However, the availability of drug in vitro assays in P. berghei is reduced when compared with the spectrum of techniques existing for Plasmodium falciparum. New alternatives to the current manual or automated methods described for P. berghei are attractive. The present study reports a new ELISA drug in vitro assay for P. berghei using two monoclonal antibodies against the parasite lactate dehydrogenase (pLDH). This procedure includes a short-in vitro culture, the purification of schizonts and the further generation of synchronized mice infections. Early stages of the parasite are then incubated against different concentrations of anti-malarial drugs using micro-plates. The novelty of this procedure in P. berghei relies on the quantification of the drug activity derived from the amount of pLDH estimated by an ELISA assay using two monoclonal antibodies: 14C1 and 19G7. The IC₅₀s obtained through the ELISA assay were compared with those from the micro-test. The initial parameters of the synchronized samples used in the in vitro assays were a parasitaemia of 0.5% and haematocrit of 1%, with an incubation period of 22 hours at 36.5°C. pLDH detection using a 14C1 coating at 10 μg/ml and 19G7 at 2.5 × 10⁻³ μg/ml provided good readouts of optical densities with low background in negative controls and specific detection levels for all parasite stages. IC₅₀s values derived from the ELISA assay for artesunate, chloroquine, amodiaquine and quinine were: 15, 7, 2, and 144 nM, respectively. When artesunate and chloroquine IC₅₀s were evaluated using the micro-test similar values were obtained. This ELISA-based in vitro drug assay is easy to implement, fast, and avoids the use radioisotopes or expensive equipment. The utility of this simple assay for screening anti-malarial drug activity against P. berghei in vitro is demonstrated.

  1. Plasmodium glyceraldehyde-3-phosphate dehydrogenase: A potential malaria diagnostic target.

    PubMed

    Krause, Robert G E; Hurdayal, Ramona; Choveaux, David; Przyborski, Jude M; Coetzer, Theresa H T; Goldring, J P Dean

    2017-08-01

    Malaria rapid diagnostic tests (RDTs) are immunochromatographic tests detecting Plasmodial histidine-rich protein 2 (HRP2), lactate dehydrogenase (LDH) and aldolase. HRP2 is only expressed by Plasmodium falciparum parasites and the protein is not expressed in several geographic isolates. LDH-based tests lack sensitivity compared to HRP2 tests. This study explored the potential of the Plasmodial glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as a new malaria diagnostic biomarker. The P. falciparum and P. yoelii proteins were recombinantly expressed in BL21(DE3) Escherischia coli host cells and affinity purified. Two epitopes (CADGFLLIGEKKVSVFA and CAEKDPSQIPWGKCQV) specific to P. falciparum GAPDH and one common to all mammalian malaria species (CKDDTPIYVMGINH) were identified. Antibodies were raised in chickens against the two recombinant proteins and the three epitopes and affinity purified. The antibodies detected the native protein in parasite lysates as a 38 kDa protein and immunofluorescence verified a parasite cytosolic localization for the native protein. The antibodies suggested a 4-6 fold higher concentration of native PfGAPDH compared to PfLDH in immunoprecipitation and ELISA formats, consistent with published proteomic data. PfGAPDH shows interesting potential as a malaria diagnostic biomarker. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Adrenal 11-beta hydroxysteroid dehydrogenase activity in response to stress.

    PubMed

    Zallocchi, Marisa; Matković, Laura; Damasco, María C

    2004-06-01

    This work studied the effect of stresses produced by simulated gavage or gavage with 200 mmol/L HCl two hours before adrenal extraction, on the activities of the 11beta-hydroxysteroid dehydrogenase 1 and 11beta-hydroxysteroid dehydrogenase 2 isoforms present in the rat adrenal gland. These activities were determined on immediately prepared adrenal microsomes following incubations with 3H-corticosterone and NAD+ or NADP+. 11-dehydrocorticosterone was measured as an end-product by TLC, and controls were adrenal microsomes from rats kept under basal (unstressed) conditions. 11beta-hydroxysteroid dehydrogenase 1 activity, but not 11beta-hydroxysteroid dehydrogenase 2 activity, was increased under both stress-conditions. Homeostatically, the stimulation of 11beta-hydroxysteroid dehydrogenase 1 activity would increase the supply of glucocorticoids. These, in turn, would activate the enzyme phenylethanolamine N-methyl transferase, thereby improving the synthesis of epinephrine as part of the stress-response.

  3. Lactation and reproduction*

    PubMed Central

    Thomson, A. M.; Hytten, F. E.; Black, A. E.

    1975-01-01

    The authors review the literature on the effect of lactation on fertility in the absence of contraception and on the effects of contraceptive measures on lactation. They examine data from several countries on the intervals between births and on the return of menstruation and ovulation after childbirth, comparing lactating with nonlactating women. They conclude that lactation is an inefficient contraceptive for the individual, but that in populations sustained lactation is associated with reduced fertility. Possible physiological mechanisms causing lactation amenorrhoea are discussed. Though much of the literature on the effect of contraceptives on lactation is inadequate, there is general agreement that the estrogen component of hormonal preparations has an adverse effect on lactation, but that progestins alone do not. Many questions remain. Is this effect seen in established lactation, or only in the puerperal period? Is it a direct pharmacological effect, or are pill-users the mothers least motivated to maintain breast-feeding? Does a close relationship exist between hormones given and lactation performance? The authors comment on some of the technical deficiencies of previous studies in this field and discuss practical possibilities of, and limitations to, obtaining adequate scientific information in the future. PMID:1084804

  4. Glymphatic clearance controls state-dependent changes in brain lactate concentration.

    PubMed

    Lundgaard, Iben; Lu, Minh Lon; Yang, Ezra; Peng, Weiguo; Mestre, Humberto; Hitomi, Emi; Deane, Rashid; Nedergaard, Maiken

    2017-06-01

    Brain lactate concentration is higher during wakefulness than in sleep. However, it is unknown why arousal is linked to an increase in brain lactate and why lactate declines within minutes of sleep. Here, we show that the glymphatic system is responsible for state-dependent changes in brain lactate concentration. Suppression of glymphatic function via acetazolamide treatment, cisterna magna puncture, aquaporin 4 deletion, or changes in body position reduced the decline in brain lactate normally observed when awake mice transition into sleep or anesthesia. Concurrently, the same manipulations diminished accumulation of lactate in cervical, but not in inguinal lymph nodes when mice were anesthetized. Thus, our study suggests that brain lactate is an excellent biomarker of the sleep-wake cycle and increases further during sleep deprivation, because brain lactate is inversely correlated with glymphatic-lymphatic clearance. This analysis provides fundamental new insight into brain energy metabolism by demonstrating that glucose that is not fully oxidized can be exported as lactate via glymphatic-lymphatic fluid transport.

  5. Glymphatic clearance controls state-dependent changes in brain lactate concentration

    PubMed Central

    Lu, Minh Lon; Yang, Ezra; Peng, Weiguo; Mestre, Humberto; Hitomi, Emi; Deane, Rashid; Nedergaard, Maiken

    2016-01-01

    Brain lactate concentration is higher during wakefulness than in sleep. However, it is unknown why arousal is linked to an increase in brain lactate and why lactate declines within minutes of sleep. Here, we show that the glymphatic system is responsible for state-dependent changes in brain lactate concentration. Suppression of glymphatic function via acetazolamide treatment, cisterna magna puncture, aquaporin 4 deletion, or changes in body position reduced the decline in brain lactate normally observed when awake mice transition into sleep or anesthesia. Concurrently, the same manipulations diminished accumulation of lactate in cervical, but not in inguinal lymph nodes when mice were anesthetized. Thus, our study suggests that brain lactate is an excellent biomarker of the sleep–wake cycle and increases further during sleep deprivation, because brain lactate is inversely correlated with glymphatic-lymphatic clearance. This analysis provides fundamental new insight into brain energy metabolism by demonstrating that glucose that is not fully oxidized can be exported as lactate via glymphatic-lymphatic fluid transport. PMID:27481936

  6. Prognostic significance of blood lactate and lactate clearance in trauma patients.

    PubMed

    Régnier, Marie-Alix; Raux, Mathieu; Le Manach, Yannick; Asencio, Yves; Gaillard, Johann; Devilliers, Catherine; Langeron, Olivier; Riou, Bruno

    2012-12-01

    Lactate has been shown to be a prognostic biomarker in trauma. Although lactate clearance has already been proposed as an intermediate endpoint in randomized trials, its precise role in trauma patients remains to be determined. Blood lactate levels and lactate clearance (LC) were calculated at admission and 2 and 4 h later in trauma patients. The association of initial blood lactate level and lactate clearance with mortality was tested using receiver-operating characteristics curve, logistic regression using triage scores, Trauma Related Injury Severity Score as a reference standard, and reclassification method. The authors evaluated 586 trauma patients (mean age 38±16 yr, 84% blunt and 16% penetrating, mortality 13%). Blood lactate levels at admission were elevated in 327 (56%) patients. The lactate clearance should be calculated within the first 2 h after admission as LC0-2 h was correlated with LC0-4 h (R=0.55, P<0.001) but not with LC2-4 h (R=0.04, not significant). The lactate clearance provides additional predictive information to initial blood lactate levels and triage scores and the reference score. This additional information may be summarized using a categorical approach (i.e., less than or equal to -20 %/h) in contrast to initial blood lactate. The results were comparable in patients with high (5 mM/l or more) initial blood lactate. Early (0-2 h) lactate clearance is an important and independent prognostic variable that should probably be incorporated in future decision schemes for the resuscitation of trauma patients.

  7. Immobilization Increases the Stability and Reusability of Pigeon Pea NADP+ Linked Glucose-6-Phosphate Dehydrogenase.

    PubMed

    Singh, Siddhartha; Singh, Amit Kumar; Singh, M Chandrakumar; Pandey, Pramod Kumar

    2017-02-01

    Immobilization of enzymes is valuably important as it improves the stability and hence increases the reusability of enzymes. The present investigation is an attempt for immobilization of purified glucose-6-phosphate dehydrogenase from pigeon pea on different matrix. Maximum immobilization was achieved when alginate was used as immobilization matrix. As compared to soluble enzyme the alginate immobilized enzyme exhibited enhanced optimum pH and temperature. The alginate immobilized enzyme displayed more than 80% activity up to 7 continuous reactions and more than 50% activity up to 11 continuous reactions.

  8. [The effects of increased dietary calcium intake on bone mineral density in long-term lactating women, and recovery of bone loss caused by long-term lactation with low calcium diet].

    PubMed

    Yoneyama, Kyoko; Ikeda, Junko

    2004-12-01

    The purpose of this study was to examine the efficacy of an increased calcium (Ca) diet for preventing bone mineral loss in long-term lactating women, considering bone metabolism, and recovery of bone loss caused by long-term lactation with low dietary Ca intake. Two groups of long-term (> 12 mon.) lactating women ... one with an enhanced Ca intake (Group M, n = 22) and the other with diet feeding no cow's milk and no milk products (Group N, n = 16) ... and a control group of 21 non-lactating postpartum women (Group C) were studied. Bone mineral density (BMD) was measured by ultrasonic bone densitometry. Stiffness calculated from the combined value of speed of sound and broadband ultrasound attenuation was used as an index of BMD. BMD and bone metabolic markers in urine and serum (only M and C groups) were assessed from 1 approximately 12 weeks postpartum (initial) at six-month intervals for a maximum of two years and changes were compared among the groups. 1. The mean (+/- SD) dietary Ca intake was 1032 (209) mg/day in the M group. 2. After lactating for one year, the N group demonstrated significant decrease in BMD, with both 1 and 2 babies, whereas the M group had no significant change. 3. The BMD in the N group returned to initial levels at 0.5 approximately 1 year post-weaning, 4. In the N group, compared with the M group, the urinary Hydroxyproline/creatinine ratio was significantly higher at the initial measurement and half a year thereafter, while urinary Ca/ creatinine ratio was significantly lower after a year. However, there were no significant differences between the M and C groups. 5. Serum bone alkaline phosphatase was significantly higher in the M group compared with the C group. Bone loss during long-term lactation can be prevented with adequate dietary Ca intake. Once lost, recovery to initial levels occurs 0.5 approximately 1 year post-weaning.

  9. Increased male offspring's risk of metabolic-neuroendocrine dysfunction and overweight after fructose-rich diet intake by the lactating mother.

    PubMed

    Alzamendi, Ana; Castrogiovanni, Daniel; Gaillard, Rolf C; Spinedi, Eduardo; Giovambattista, Andrés

    2010-09-01

    An adverse endogenous environment during early life predisposes the organism to develop metabolic disorders. We evaluated the impact of intake of an iso-caloric fructose rich diet (FRD) by lactating mothers (LM) on several metabolic functions of their male offspring. On postnatal d 1, ad libitum eating, lactating Sprague-Dawley rats received either 10% F (wt/vol; FRD-LM) or tap water (controls, CTR-LM) to drink throughout lactation. Weaned male offspring were fed ad libitum a normal diet, and body weight (BW) and food intake were registered until experimentation (60 d of age). Basal circulating levels of metabolic markers were evaluated. Both iv glucose tolerance and hypothalamic leptin sensitivity tests were performed. The hypothalamus was dissected for isolation of total RNA and Western blot analysis. Retroperitoneal (RP) adipose tissue was dissected and either kept frozen for gene analysis or digested to isolate adipocytes or for histological studies. FRD rats showed increased BW and decreased hypothalamic sensitivity to exogenous leptin, enhanced food intake (between 49-60 d), and decreased hypothalamic expression of several anorexigenic signals. FRD rats developed increased insulin and leptin peripheral levels and decreased adiponectinemia; although FRD rats normally tolerated glucose excess, it was associated with enhanced insulin secretion. FRD RP adipocytes were enlarged and spontaneously released high leptin, although they were less sensitive to insulin-induced leptin release. Accordingly, RP fat leptin gene expression was high in FRD rats. Excessive fructose consumption by lactating mothers resulted in deep neuroendocrine-metabolic disorders of their male offspring, probably enhancing the susceptibility to develop overweight/obesity during adult life.

  10. In vitro effects of bicarbonate and bicarbonate-lactate buffered peritoneal dialysis solutions on mesothelial and neutrophil function.

    PubMed

    Topley, N; Kaur, D; Petersen, M M; Jörres, A; Williams, J D; Faict, D; Holmes, C J

    1996-02-01

    The inclusion of bicarbonate in the formulation of peritoneal dialysis solutions may avoid the in vitro impairment of certain cell functions seen with acidic lactate-based fluids. The supranormal physiological levels of HCO3- and PCO2 inherent in such formulations may, however, not be biocompatible. This study compared the in vitro biocompatibility of a pH 5.2 lactate-based formulation with formulations containing either 40 mM lactate at pH 7.4, 38 mM HCO3- at pH 6.8 (PCO2 at approximately 240 mm Hg) or 7.4 (PCO2 at approximately 60 mm Hg), and 25 mM HCO3- plus 15 mM lactate at pH 6.8 (PCO2 at approximately 160 mm Hg) or 7.4 (PCO2 at approximately 40 mm Hg). Significant release of lactate dehydrogenase or decreases in ATP content by human peritoneal mesothelial cells (HPMC) and human peripheral polymorphonuclear leukocytes (PMN) after a 30-min exposure to each test solution was only seen with the pH 5.2 lactate-based fluid. The ATP content of HPMC exposed to this fluid returned to control levels after 30 min of recovery in M199 control medium but showed a trend toward decreasing ATP content at 240 min. Similarly, interleukin (IL)-1 beta-induced IL-6 synthesis by HPMC was also only significantly reduced by the pH 5.2 lactate solution. PMN chemiluminescence was unaffected by 30-min exposure to all test solutions except for the pH 5.2 lactate formulation. Staphylococcus epidermidis phagocytosis was reduced to between 46 to 57% of control with all test solutions except the pH 5.2 lactate solution, which further suppressed the chemiluminescence response to 17% of control. These data suggest that short exposure to supranormal physiological levels of HCO3- and PCO2 does not impair HPMC or PMN viability and function. Furthermore, neutral pH lactate-containing solutions show equivalent biocompatibility to bicarbonate-based ones.

  11. Stimulation of d- and l-lactate dehydrogenases transcriptional levels in presence of diammonium hydrogen phosphate resulting to enhanced lactic acid production by Lactobacillus strain.

    PubMed

    Singhvi, Mamata; Zendo, Takeshi; Iida, Hiroshi; Gokhale, Digambar; Sonomoto, Kenji

    2017-12-01

    The present study revealed the effect of nitrogen sources on lactic acid production and stimulation of d- and l-lactate dehydrogenases (LDH) of parent Lactobacillus lactis NCIM 2368 and its mutant RM2-24 generated after UV mutagenesis. Both the parent and mutant strains were evaluated for d-lactic acid production in control and modified media. The modified media did not show remarkable effect on lactic acid production in case of parent whereas mutant exhibited significant enhancement in d-lactic acid production along with the appearance of l-lactic acid in the broth. Both LDH activities and specific activities were found to be higher in mutant than the parent strain. These results suggested that the diammonium hydrogen phosphate in modified media triggered the expression of LDH genes leading to enhanced lactic acid production. This observation has been proved by studying the expression levels of d- and l-LDH genes of parent and mutant in control and modified media using quantitative RT-PCR technique. In case of mutant, the transcriptional levels of d-LDH and l-LDH increased ∼17 fold and ∼1.38 fold respectively in modified medium compared to the values obtained with control medium. In case of parent, no significant change in transcriptional levels of d- and l-LDH was found when the cells were grown in either control medium or modified medium. This study suggested that the mutant, RM2-24 has l-LDH gene which is expressed in presence of (NH 4 ) 2 HPO 4 resulting in l-lactic acid production. Co-production of l-lactic acid in d-lactic acid fermentation may be detrimental in the PLA production. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  12. Inhibition of lactation.

    PubMed

    Llewellyn-Jones, D

    1975-01-01

    The mechanism and hormonal regulation of lactation is explained and illustrated with a schematic representation. Circulating estrogen above a critical amount seems to be the inhibitory factor controlling lactation during pregnancy. Once delivery occurs, the level of estrogen falls, that of prolactin rises, and lactation begins. Nonsuckling can be used to inhibit lactation. Estrogens can also be used to inhibit lactation more quickly and with less pain. The reported association between estrogens and puerperal thromboembolism cannot be considered conclusive due to defects in the reporting studies. There is no reason not to use estrogens in lactation inhibition except for women over 35 who experienced a surgical delivery. Alternative therapy is available for these women. The recently-developed drug, brom-ergocryptine, may replace other methods of lactation inhibition.

  13. Lactate dehydrogenase test

    MedlinePlus

    Normal value range is 105 to 333 international units per liter (IU/L). Normal value ranges may vary slightly among different laboratories. Some labs use different measurements or test different samples. Talk to your provider about ...

  14. (1-3)-beta-D-glucan in association with lactate dehydrogenase as biomarkers of Pneumocystis pneumonia (PcP) in HIV-infected patients.

    PubMed

    Esteves, F; Lee, C-H; de Sousa, B; Badura, R; Seringa, M; Fernandes, C; Gaspar, J F; Antunes, F; Matos, O

    2014-07-01

    Pneumocystis pneumonia (PcP) is a major HIV-related illness caused by Pneumocystis jirovecii. Definitive diagnosis of PcP requires microscopic detection of P. jirovecii in pulmonary specimens. The objective of this study was to evaluate the usefulness of two serum markers in the diagnosis of PcP. Serum levels of (1-3)-beta-d-glucan (BG) and lactate dehydrogenase (LDH) were investigated in 100 HIV-positive adult patients and 50 healthy blood donors. PcP cases were confirmed using indirect immunofluorescence with monoclonal anti-Pneumocystis antibodies and nested-PCR to amplify the large subunit mitochondrial rRNA gene of P. jirovecii in pulmonary specimens. BG and LDH levels in serum were measured using quantitative microplate-based assays. BG and LDH positive sera were statistically associated with PcP cases (P ≤ 0.001). Sensitivity, specificity, positive/negative predictive values (PPV/NPV), and positive/negative likelihood ratios (PLR/NLR) were 91.3 %, 61.3 %, 85.1 %, 79.2 %, 2.359, and 0.142, respectively, for the BG kit assay, and 91.3 %, 35.5 %, 75.9 %, 64.7 %, 1.415 and 0.245, respectively, for the LDH test. Serologic markers levels combined with the clinical diagnostic criteria for PcP were evaluated for their usefulness in diagnosis of PcP. The most promising cutoff levels for diagnosis of PcP were determined to be 400 pg/ml of BG and 350 U/l of LDH, which combined with clinical data presented 92.8 % sensitivity, 83.9 % specificity, 92.8 % PPV, 83.9 % NPV, 5.764 PLR and 0.086 NLR (P < 0.001). This study confirmed that BG is a reliable indicator for detecting P. jirovecii infection. The combination between BG/LDH levels and clinical data is a promising alternative approach for PcP diagnosis.

  15. Lactate dehydrogenase predicts combined progression-free survival after sequential therapy with abiraterone and enzalutamide for patients with castration-resistant prostate cancer.

    PubMed

    Mori, Keiichiro; Kimura, Takahiro; Onuma, Hajime; Kimura, Shoji; Yamamoto, Toshihiro; Sasaki, Hiroshi; Miki, Jun; Miki, Kenta; Egawa, Shin

    2017-07-01

    An array of clinical issues remains to be resolved for castration-resistant prostate cancer (CRPC), including the sequence of drug use and drug cross-resistance. At present, no clear guidelines are available for the optimal sequence of use of novel agents like androgen-receptor axis-targeted (ARAT) agents, particularly enzalutamide, and abiraterone. This study retrospectively analyzed a total of 69 patients with CRPC treated with sequential therapy using enzalutamide followed by abiraterone or vice versa. The primary outcome measure was the comparative combined progression-free survival (PFS) comprising symptomatic and/or radiographic PFS. Patients were also compared for total prostate-specific antigen (PSA)-PFS, overall survival (OS), and PSA response. The predictors of combined PFS and OS were analyzed with a backward-stepwise multivariate Cox model. Of the 69 patients, 46 received enzalutamide first, followed by abiraterone (E-A group), and 23 received abiraterone, followed by enzalutamide (A-E group). The two groups were not significantly different with regard to basic data, except for hemoglobin values. In a comparison with the E-A group, the A-E group was shown to be associated with better combined PFS in Kaplan-Meier analysis (P = 0.043). Similar results were obtained for total PSA-PFS (P = 0.049), while OS did not differ between groups (P = 0.62). Multivariate analysis demonstrated that pretreatment lactate dehydrogenase (LDH) values and age were significant predictors of longer combined PFS (P < 0.05). Likewise, multivariate analysis demonstrated that pretreatment hemoglobin values and performance status were significant predictors of longer OS (P < 0.05). The results of this study suggested the A-E sequence had longer combined PSA and total PSA-PFS compared to the E-A sequence in patients with CRPC. LDH values in sequential therapy may serve as a predictor of longer combined PFS. © 2017 Wiley Periodicals, Inc.

  16. Regulation of bone mineral loss during lactation

    NASA Technical Reports Server (NTRS)

    Brommage, R.; Deluca, H. F.

    1985-01-01

    The effects of varyng dietary calcium and phosphorous levels, vitamin D deficiency, oophorectomy, adrenalectomy, and simultaneous pregnancy on bone mineral loss during lactation in rats are studied. The experimental procedures and evaluations are described. The femur ash weight of lactating and nonlactating rats are calculated. The data reveals that a decrease in dietary calcium of 0.02 percent results in an increased loss of bone mineral, an increase in calcium to 1.4 percent does not lessen bone mineral loss, and bone mineral loss in vitamin D deficient rats is independent of calcium levels. It is observed that changes in dietary phosphorous level, oophorectomy, adrenalectomy, and simultaneous pragnancy do not reduce bone mineral loss during lactation. The analysis of various hormones to determine the mechanism that triggers bone mineral loss during lactation is presented.

  17. Increase in accumulation of strontium-90 in the maternal skeleton during pregnancy and lactation: analysis of the Techa River data.

    PubMed

    Tolstykh, Evgenia I; Shagina, Natalia B; Degteva, Marina O

    2014-08-01

    The unique contamination of the Techa River (Southern Urals, Russia) in the 1950s by long-lived (90)Sr allows investigation of the accumulation of bone-seeking elements in humans. This study is based on information compiled at the Urals Research Center for Radiation Medicine (Chelyabinsk, Russia) over a long period of time. It includes the results of in vivo measurements of (90)Sr-body burden with a whole body counter (WBC), data on personal medical examinations and residence and family histories. Data on 185 women from two Techa riverside villages Muslyumovo and Brodokalmak were selected. The settlements differ in terms of (90)Sr diet intake (higher in Muslyumovo than in Brodokalmak) and ethnicity (residents were mainly Slavs in Brodokalmak and Turkic in Muslyumovo). Results of a total of 555 WBC measurements performed in 1974-1997 were available for the women studied; maximum measured values reached 40 kBq/body. The women from each settlement were subdivided into three groups according to their childbearing history: pregnancy and lactation occurred (1) during the period of maximal (90)Sr intake (1950-1951); (2) after the period of maximal intake and (3) before this period or women who were childless. An increase was found in accumulation of (90)Sr in maternal skeleton during pregnancy and lactation (group 1) by a factor of 1.5-2 in comparison with non-pregnant, non-lactating women. This result was found in both Muslyumovo and Brodokalmak samples. An increase in accumulation of toxic elements in pregnant/lactating women is associated with increased radiation/toxic doses and risk for the women's health.

  18. CD147 Required for Corneal Endothelial Lactate Transport

    PubMed Central

    Li, Shimin; Nguyen, Tracy T.; Bonanno, Joseph A.

    2014-01-01

    Purpose. CD147/basigin is a chaperone for lactate:H+ cotransporters (monocarboxylate transporters) MCT1 and MCT4. We tested the hypothesis that MCT1 and -4 in corneal endothelium contribute to lactate efflux from stroma to anterior chamber and that silencing CD147 expression would cause corneal edema. Methods. CD147 was silenced via small interfering ribonucleic acid (siRNA) transfection of rabbit corneas ex vivo and anterior chamber lenti-small hairpin RNA (shRNA) pseudovirus in vivo. CD147 and MCT expression was examined by Western blot, RT-PCR, and immunofluorescence. Functional effects were examined by measuring lactate-induced cell acidification, corneal lactate efflux, [lactate], central cornea thickness (CCT), and Azopt (a carbonic anhydrase inhibitor) sensitivity. Results. In ex vivo corneas, 100 nM CD147 siRNA reduced CD147, MCT1, and MCT4 expression by 85%, 79%, and 73%, respectively, while MCT2 expression was unaffected. CD147 siRNA decreased lactate efflux from 3.9 ± 0.81 to 1.5 ± 0.37 nmol/min, increased corneal [lactate] from 19.28 ± 7.15 to 56.73 ± 8.97 nmol/mg, acidified endothelial cells (pHi = 6.83 ± 0.07 vs. 7.19 ± 0.09 in control), and slowed basolateral lactate-induced acidification from 0.0034 ± 0.0005 to 0.0012 ± 0.0005 pH/s, whereas apical acidification was unchanged. In vivo, CD147 shRNA increased CCT by 28.1 ± 0.9 μm at 28 days; Azopt increased CCT to 24.4 ± 3.12 vs. 12.0 ± 0.48 μm in control, and corneal [lactate] was 47.63 ± 6.29 nmol/mg in shCD147 corneas and 17.82 ± 4.93 nmol/mg in paired controls. Conclusions. CD147 is required for the expression of MCT1 and MCT4 in the corneal endothelium. Silencing CD147 slows lactate efflux, resulting in stromal lactate accumulation and corneal edema, consistent with lactate efflux as a significant component of the corneal endothelial pump. PMID:24970254

  19. CD147 required for corneal endothelial lactate transport.

    PubMed

    Li, Shimin; Nguyen, Tracy T; Bonanno, Joseph A

    2014-06-26

    CD147/basigin is a chaperone for lactate:H(+) cotransporters (monocarboxylate transporters) MCT1 and MCT4. We tested the hypothesis that MCT1 and -4 in corneal endothelium contribute to lactate efflux from stroma to anterior chamber and that silencing CD147 expression would cause corneal edema. CD147 was silenced via small interfering ribonucleic acid (siRNA) transfection of rabbit corneas ex vivo and anterior chamber lenti-small hairpin RNA (shRNA) pseudovirus in vivo. CD147 and MCT expression was examined by Western blot, RT-PCR, and immunofluorescence. Functional effects were examined by measuring lactate-induced cell acidification, corneal lactate efflux, [lactate], central cornea thickness (CCT), and Azopt (a carbonic anhydrase inhibitor) sensitivity. In ex vivo corneas, 100 nM CD147 siRNA reduced CD147, MCT1, and MCT4 expression by 85%, 79%, and 73%, respectively, while MCT2 expression was unaffected. CD147 siRNA decreased lactate efflux from 3.9 ± 0.81 to 1.5 ± 0.37 nmol/min, increased corneal [lactate] from 19.28 ± 7.15 to 56.73 ± 8.97 nmol/mg, acidified endothelial cells (pHi = 6.83 ± 0.07 vs. 7.19 ± 0.09 in control), and slowed basolateral lactate-induced acidification from 0.0034 ± 0.0005 to 0.0012 ± 0.0005 pH/s, whereas apical acidification was unchanged. In vivo, CD147 shRNA increased CCT by 28.1 ± 0.9 μm at 28 days; Azopt increased CCT to 24.4 ± 3.12 vs. 12.0 ± 0.48 μm in control, and corneal [lactate] was 47.63 ± 6.29 nmol/mg in shCD147 corneas and 17.82 ± 4.93 nmol/mg in paired controls. CD147 is required for the expression of MCT1 and MCT4 in the corneal endothelium. Silencing CD147 slows lactate efflux, resulting in stromal lactate accumulation and corneal edema, consistent with lactate efflux as a significant component of the corneal endothelial pump. Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.

  20. Novel homologous lactate transporter improves L-lactic acid production from glycerol in recombinant strains of Pichia pastoris.

    PubMed

    de Lima, Pollyne Borborema Almeida; Mulder, Kelly Cristina Leite; Melo, Nadiele Tamires Moreira; Carvalho, Lucas Silva; Menino, Gisele Soares; Mulinari, Eduardo; de Castro, Virgilio H; Dos Reis, Thaila F; Goldman, Gustavo Henrique; Magalhães, Beatriz Simas; Parachin, Nádia Skorupa

    2016-09-15

    Crude glycerol is the main byproduct of the biodiesel industry. Although it can have different applications, its purification is costly. Therefore, in this study a biotechnological route has been proposed for further utilization of crude glycerol in the fermentative production of lactic acid. This acid is largely utilized in food, pharmaceutical, textile, and chemical industries, making it the hydroxycarboxylic acid with the highest market potential worldwide. Currently, industrial production of lactic acid is done mainly using sugar as the substrate. Thus here, for the first time, Pichia pastoris has been engineered for heterologous L-lactic acid production using glycerol as a single carbon source. For that, the Bos taurus lactate dehydrogenase gene was introduced into P. pastoris. Moreover, a heterologous and a novel homologous lactate transporter have been evaluated for L-lactic acid production. Batch fermentation of the P. pastoris X-33 strain producing LDHb allowed for lactic acid production in this yeast. Although P. pastoris is known for its respiratory metabolism, batch fermentations were performed with different oxygenation levels, indicating that lower oxygen availability increased lactic acid production by 20 %, pushing the yeast towards a fermentative metabolism. Furthermore, a newly putative lactate transporter from P. pastoris named PAS has been identified by search similarity with the lactate transporter from Saccharomyces cerevisiae Jen1p. Both heterologous and homologous transporters, Jen1p and PAS, were evaluated in one strain already containing LDH activity. Fed-batch experiments of P. pastoris strains carrying the lactate transporter were performed with the batch phase at aerobic conditions followed by an aerobic oxygen-limited phase where production of lactic acid was favored. The results showed that the strain containing PAS presented the highest lactic acid titer, reaching a yield of approximately 0.7 g/g. We showed that P. pastoris has a

  1. Aqueous solubility of calcium L-lactate, calcium D-gluconate, and calcium D-lactobionate: importance of complex formation for solubility increase by hydroxycarboxylate mixtures.

    PubMed

    Vavrusova, Martina; Munk, Merete Bøgelund; Skibsted, Leif H

    2013-08-28

    Among the calcium hydroxycarboxylates important for cheese quality, D-lactobionate [Ksp = (7.0 ± 0.3) × 10(-3) mol(3) L(-3)] and L-lactate [Ksp = (5.8 ± 0.2) × 10(-3) mol(3) L(-3)] were found more soluble than D-gluconate [Ksp = (7.1 ± 0.2) × 10(-4) mol(3) L(-3)], as indicated by the solubility products determined electrochemically for aqueous 1.0 M NaCl at 25.0 °C. Still, solubility of calcium L-lactate increases by 45% in the presence of 0.50 M sodium D-gluconate and by 37% in the presence of 0.50 M sodium D-lactobionate, while solubility of calcium D-gluconate increases by 66 and 85% in the presence of 0.50 M sodium L-lactate and 0.50 M sodium D-lactobionate, respectively, as determined by complexometric titration. Sodium L-lactate and sodium D-gluconate have only little influence on solubility of calcium D-lactobionate. The increased solubility is described quantitatively by calcium binding to D-gluconate (K1 = 14 ± 3 mol(-1) L) in 1.0 M NaCl at 25 °C, D-lactobionate (K1 = 11 ± 2 mol(-1) L), and L-lactate (K1 = 8 ± 2 mol(-1) L), as indicated by the association constants determined electrochemically. In mixed hydroxycarboxylate solutions, calcium binding is quantitatively described by the geometric mean of the individual association constants for both aqueous 1.0 and 0.20 M NaCl, indicating a 1:1 stoichiometry for complex formation.

  2. Lactate storm marks cerebral metabolism following brain trauma.

    PubMed

    Lama, Sanju; Auer, Roland N; Tyson, Randy; Gallagher, Clare N; Tomanek, Boguslaw; Sutherland, Garnette R

    2014-07-18

    Brain metabolism is thought to be maintained by neuronal-glial metabolic coupling. Glia take up glutamate from the synaptic cleft for conversion into glutamine, triggering glial glycolysis and lactate production. This lactate is shuttled into neurons and further metabolized. The origin and role of lactate in severe traumatic brain injury (TBI) remains controversial. Using a modified weight drop model of severe TBI and magnetic resonance (MR) spectroscopy with infusion of (13)C-labeled glucose, lactate, and acetate, the present study investigated the possibility that neuronal-glial metabolism is uncoupled following severe TBI. Histopathology of the model showed severe brain injury with subarachnoid and hemorrhage together with glial cell activation and positive staining for Tau at 90 min post-trauma. High resolution MR spectroscopy of brain metabolites revealed significant labeling of lactate at C-3 and C-2 irrespective of the infused substrates. Increased (13)C-labeled lactate in all study groups in the absence of ischemia implied activated astrocytic glycolysis and production of lactate with failure of neuronal uptake (i.e. a loss of glial sensing for glutamate). The early increase in extracellular lactate in severe TBI with the injured neurons rendered unable to pick it up probably contributes to a rapid progression toward irreversible injury and pan-necrosis. Hence, a method to detect and scavenge the excess extracellular lactate on site or early following severe TBI may be a potential primary therapeutic measure. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. In Vivo Evidence for a Lactate Gradient from Astrocytes to Neurons.

    PubMed

    Mächler, Philipp; Wyss, Matthias T; Elsayed, Maha; Stobart, Jillian; Gutierrez, Robin; von Faber-Castell, Alexandra; Kaelin, Vincens; Zuend, Marc; San Martín, Alejandro; Romero-Gómez, Ignacio; Baeza-Lehnert, Felipe; Lengacher, Sylvain; Schneider, Bernard L; Aebischer, Patrick; Magistretti, Pierre J; Barros, L Felipe; Weber, Bruno

    2016-01-12

    Investigating lactate dynamics in brain tissue is challenging, partly because in vivo data at cellular resolution are not available. We monitored lactate in cortical astrocytes and neurons of mice using the genetically encoded FRET sensor Laconic in combination with two-photon microscopy. An intravenous lactate injection rapidly increased the Laconic signal in both astrocytes and neurons, demonstrating high lactate permeability across tissue. The signal increase was significantly smaller in astrocytes, pointing to higher basal lactate levels in these cells, confirmed by a one-point calibration protocol. Trans-acceleration of the monocarboxylate transporter with pyruvate was able to reduce intracellular lactate in astrocytes but not in neurons. Collectively, these data provide in vivo evidence for a lactate gradient from astrocytes to neurons. This gradient is a prerequisite for a carrier-mediated lactate flux from astrocytes to neurons and thus supports the astrocyte-neuron lactate shuttle model, in which astrocyte-derived lactate acts as an energy substrate for neurons. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. An exploratory analysis of alkaline phosphatase, lactate dehydrogenase, and prostate-specific antigen dynamics in the phase 3 ALSYMPCA trial with radium-223

    PubMed Central

    Sartor, O.; Coleman, R. E.; Nilsson, S.; Heinrich, D.; Helle, S. I.; O’Sullivan, J. M.; Vogelzang, N. J.; Bruland, Ø.; Kobina, S.; Wilhelm, S.; Xu, L.; Shan, M.; Kattan, M. W.; Parker, C.

    2017-01-01

    Background Baseline clinical variables are prognostic for overall survival (OS) in patients with castration-resistant prostate cancer (CRPC). Their prognostic and predictive value with agents targeting bone metastases, such as radium-223, is not established. Patients and methods The radium-223 ALSYMPCA trial enrolled patients with CRPC and symptomatic bone metastases. Prognostic potential of baseline variables was assessed using Cox models. Percentage changes in biomarker levels from baseline were evaluated during the trial period; changes from baseline to week 12 were evaluated for association with OS and surrogacy. Results Eastern Cooperative Oncology Group performance status, total alkaline phosphatase (tALP), lactate dehydrogenase (LDH), and prostate-specific antigen (PSA) at baseline were associated with OS (P ≤ 0.0003) in the intent-to-treat population (radium-223, N = 614; placebo, N = 307). tALP declined from baseline within 4 weeks after beginning radium-223, by week 12 declining in 87% of radium-223 and 23% of placebo patients (P < 0.001). LDH declined in 51% and 34% (P = 0.003), whereas PSA declined in 27% and 14% (P = 0.160). Mean tALP change from baseline was 32.2% decrease with radium-223 and 37.2% increase with placebo. Radium-223 patients with tALP decline from baseline to week 12 (confirmed ≥3 weeks from week 12) had 55% lower risk of death (hazard ratio = 0.45; 95% CI 0.34–0.61) versus those with no confirmed tALP decline. Proportional treatment effect (PTE) values for tALP, LDH, and PSA changes from baseline at week 12 as OS surrogate markers were 0.34 (95% CI: 0–0.746), 0.07 (95% CI: 0–0.211), and 0 (95% CI: 0–0.082), respectively. Conclusions Significant tALP declines (versus placebo) occurred as early as 4 weeks after beginning radium-223 therapy. tALP or LDH declines at 12 weeks correlated with longer OS, but did not meet statistical surrogacy requirements. Dynamic changes in tALP and LDH during

  5. The Deletion of the Succinate Dehydrogenase Gene KlSDH1 in Kluyveromyces lactis Does Not Lead to Respiratory Deficiency

    PubMed Central

    Saliola, Michele; Bartoccioni, Paola Chiara; De Maria, Ilaria; Lodi, Tiziana; Falcone, Claudio

    2004-01-01

    We have isolated a Kluyveromyces lactis mutant unable to grow on all respiratory carbon sources with the exception of lactate. Functional complementation of this mutant led to the isolation of KlSDH1, the gene encoding the flavoprotein subunit of the succinate dehydrogenase (SDH) complex, which is essential for the aerobic utilization of carbon sources. Despite the high sequence conservation of the SDH genes in Saccharomyces cerevisiae and K. lactis, they do not have the same relevance in the metabolism of the two yeasts. In fact, unlike SDH1, KlSDH1 was highly expressed under both fermentative and nonfermentative conditions. In addition to this, but in contrast with S. cerevisiae, K. lactis strains lacking KlSDH1 were still able to grow in the presence of lactate. In these mutants, oxygen consumption was one-eighth that of the wild type in the presence of lactate and was normal with glucose and ethanol, indicating that the respiratory chain was fully functional. Northern analysis suggested that alternative pathway(s), which involves pyruvate decarboxylase and the glyoxylate cycle, could overcome the absence of SDH and allow (i) lactate utilization and (ii) the accumulation of succinate instead of ethanol during growth on glucose. PMID:15189981

  6. Histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase.

    PubMed

    Van Noorden, C J

    1984-01-01

    Histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase has found many applications in biomedical research. However, up to several years ago, the methods used often appeared to be unreliable because many artefacts occurred during processing and staining of tissue sections or cells. The development of histochemical methods preventing loss or redistribution of the enzyme by using either polyvinyl alcohol as a stabilizer or a semipermeable membrane interposed between tissue section and incubation medium, has lead to progress in the topochemical localization of glucose-6-phosphate dehydrogenase. Optimization of incubation conditions has further increased the precision of histochemical methods. Precise cytochemical methods have been developed either by the use of a polyacrylamide carrier in which individual cells have been incorporated before staining or by including polyvinyl alcohol in the incubation medium. In the present text, these methods for the histochemical and cytochemical localization of glucose-6-phosphate dehydrogenase for light microscopical and electron microscopical purposes are extensively discussed along with immunocytochemical techniques. Moreover, the validity of the staining methods is considered both for the localization of glucose-6-phosphate dehydrogenase activity in cells and tissues and for cytophotometric analysis. Finally, many applications of the methods are reviewed in the fields of functional heterogeneity of tissues, early diagnosis of carcinoma, effects of xenobiotics on cellular metabolism, diagnosis of inherited glucose-6-phosphate dehydrogenase deficiency, analysis of steroid-production in reproductive organs, and quality control of oocytes of mammals. It is concluded that the use of histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase is of highly significant value in the study of diseased tissues. In many cases, the first pathological change is an increase in glucose-6-phosphate dehydrogenase activity

  7. Chronically increased glucose uptake by adipose tissue leads to lactate production and improved insulin sensitivity rather than obesity in the mouse.

    PubMed

    Muñoz, S; Franckhauser, S; Elias, I; Ferré, T; Hidalgo, A; Monteys, A M; Molas, M; Cerdán, S; Pujol, A; Ruberte, J; Bosch, F

    2010-11-01

    In adipocytes, triacylglycerol synthesis depends on the formation of glycerol 3-phosphate, which originates either from glucose, through glycolysis, or from lactate, through glyceroneogenesis. However, glucose is traditionally viewed as the main precursor of the glycerol backbone and thus, enhanced glucose uptake would be expected to result in increased triacylglycerol synthesis and contribute to obesity. To further explore this issue, we generated a mouse model with chronically increased glucose uptake in adipose tissue by expressing Gck, which encodes the glucokinase enzyme. Here we show that the production of high levels of glucokinase led to increased adipose tissue glucose uptake and lactate production, improved glucose tolerance and higher whole-body and skeletal muscle insulin sensitivity. There was no parallel increase in glycerol 3-phosphate synthesis in vivo, fat accumulation or obesity. Moreover, at high glucose concentrations, in cultured fat cells overproducing glucokinase, glycerol 3-phosphate synthesis from pyruvate decreased, while glyceroneogenesis increased in fat cells overproducing hexokinase II. These findings indicate that the absence of glucokinase inhibition by glucose 6-phosphate probably led to increased glycolysis and blocked glyceroneogenesis in the mouse model. Furthermore, this study suggests that under physiological conditions, when blood glucose increases, glyceroneogenesis may prevail over glycolysis for triacylglycerol formation because of the inhibition of hexokinase II by glucose 6-phosphate. Together these results point to the indirect pathway (glucose to lactate to glycerol 3-phosphate) being key for fat deposition in adipose tissue.

  8. Effects of nutrition on pregnant and lactating sows.

    PubMed

    Einarsson, S; Rojkittikhun, T

    1993-01-01

    It has been suggested that the long-term reproduction of the sow is best served by minimizing weight and fat loss in lactation. Such a strategy would require only a minimal restoration of weight in the following pregnancy, which would be beneficial, since the greater feed intake and weight gain in pregnancy, the greater the weight loss in lactation. Feeding ad libitum should be practised during lactation while gestation feed intake must be held low. A relationship between feed intake and embryo survival has been demonstrated in several studies, but the data are sometimes difficult to interpret. High energy feeding during the premating period and during early pregnancy, however, are often associated with increased embryo mortality. A short-term starvation in lactation decreased prolactin to post-weaning concentrations, and insulin and glucose to very low concentrations. Prolactin increased very rapidly after refeeding indicating that a neural mechanism might be involved. The increasing levels of cholecystokinin after refeeding and the neural reflex triggered might be related to this increase in prolactin. No changes in LH release were observed during the periods of starvation or refeeding. The catabolic rate during the first week of lactation is higher in sows with higher backfat thickness than in late gestation. As lactation progresses a more balanced metabolism is achieved regardless of backfat thickness before parturition. High-weight-loss primiparous sows need a longer recovery period from their negative energy balance during lactation than do low-weight-loss primiparous sows or multiparous sows. Several investigations have demonstrated that sows losing excessive amounts of body weight have extended weaning to oestrous intervals and an increase in anoestrus. Sows with low body-weight loss during lactation have higher plasma insulin and lower cortisol around weaning than do sows with high body-weight loss. What remains undefined is the degree of weight or

  9. Serum lactate dehydrogenase with a systemic inflammation score is useful for predicting response and survival in patients with newly diagnosed diffuse large B-cell lymphoma.

    PubMed

    Jung, Sung-Hoon; Yang, Deok-Hwan; Ahn, Jae-Sook; Kim, Yeo-Kyeoung; Kim, Hyeoung-Joon; Lee, Je-Jung

    2015-01-01

    We evaluated the relationship between serum lactate dehydrogenase (LDH) level with systemic inflammation score and survival in 213 patients with diffuse large B-cell lymphoma (DLBCL) receiving R-CHOP chemotherapy. The patients were classified into 3 groups based on LDH with the Glasgow Prognostic Score (L-GPS). A score of 2 was assigned to patients with elevated C-reactive protein, hypoalbuminemia and elevated LDH, a score of 1 to those with one or two abnormalities and a score of 0 to those with no abnormality. In multivariate analysis, independent poor prognostic factors for progression-free survival were L-GPS 2 [hazard ratio (HR) 5.415, p = 0.001], Eastern Cooperative Oncology Group performance status (ECOG PS) ≥2 (HR 3.504, p = 0.001) and bulky lesion (HR 2.030, p = 0.039). Independent poor prognostic factors for overall survival were L-GPS 2 (HR 5.898, p = 0.001) and ECOG PS ≥2 (HR 3.525, p = 0.001). The overall response rate for the R-CHOP chemotherapy decreased according to the L-GPS; it was 96.7% at L-GPS 0, 87% at L-GPS 1 and 75% at L-GPS 2 (p = 0.009). L-GPS based on systemic inflammatory indicators may be a useful clinical prognostic indicator for survival, and predicts the response for R-CHOP chemotherapy in patients with newly diagnosed DLBCL. © 2014 S. Karger AG, Basel.

  10. Reduced nursing frequency during prolonged lactation in the mouse decreases milk production and increases mammary expression of tryptophan hydroxylase 1 (TPH1), but does not accelerate mammary gland remodeling

    USDA-ARS?s Scientific Manuscript database

    We have observed that lactating mouse dams nursed 4 times per day (4X) maintained lactation, but had lower milk yields by the weigh-suckle-weigh method, than dams nursed ad libitum (AL). Therefore, we hypothesized that decreased nursing frequency would also decrease lactation persistence, increase m...

  11. Effects of high and low blood lactate concentrations on sweat lactate response.

    PubMed

    Green, J M; Bishop, P A; Muir, I H; McLester, J R; Heath, H E

    2000-11-01

    Sweat lactate results from eccrine gland metabolism, however, the possible clearance of blood lactate through sweat has not been resolved. On separate days in an environmental chamber (32 +/- 1 C) 12 subjects completed a constant load (CON) (30 min at 40% VO2 max) and an interval cycling trial (INT) (15 one-min intervals at 80% VO2 max, each separated by one min rest) each designed to elicit different blood lactate responses. Each 30 min cycling trial was preceded by 15 min warm-up (30 watts) and followed by 15 min passive rest. Sweat and blood were analyzed for lactate concentration at 15, 25, 35, 45, and 60 min during CON and INT. Total body water loss was used to calculate sweat rate (ml/hr). Blood lactate was significantly greater (p < or = 0.05) at 25, 35, 45, and 60 min during INT compared to CON (approximately 5 mmol/L vs 1.5 mmol/L). Sweat lactate was not significantly different (p>0.05) between trials at any time (approximately 10 mmol/L). Sweat rates (approximately 600ml/hr) and estimated total lactate secretion were not significantly different (CON vs. INT) (p > 0.05). Elevated blood lactate was not associated with changes in sweat lactate concentration. Sweat lactate seems to originate in eccrine glands independent of blood lactate.

  12. Improvement of L(+)-Lactic Acid Production of Rhizopus Oryzae by Low-Energy Ions and Analysis of Its Mechanism

    NASA Astrophysics Data System (ADS)

    Ge, Chunmei; Yang, Yingge; Fan, Yonghong; Li, Wen; Pan, Renrui; Zheng, Zhiming; Yu, Zengliang

    2008-02-01

    The wild type strain Rhizopus oryzae PW352 was mutated by means of nitrogen ion implantation (15 keV, 7.8 × 1014 ~ 2.08 × 1015 ions/cm2) to find an industrial strain with a higher L(+)-lactic acid yield, and two mutants RE3303 and RF9052 were isolated. In order to discuss the mechanism primarily, Lactate Dehydrogenase of Rhizopus oryzae was studied. While the two mutants produced L(+)-lactic acid by 75% more than the wild strain did, their specific activity of Lactate Dehydrogenase was found to be higher than that in the wild strain. The optimum temperature of Lactate Dehydrogenase in Rhizopus oryzae RF9052 was higher. Compared to the wild strain, the Michaelis constant (Km) value of Lactate Dehydrogenase in the mutants was changed. All these changes show that L(+)-lactic acid production has a correlation with the specific activity of Lactate Dehydrogenase. The low-energy ions, implanted into the strain, may improve the specific activity of Lactate Dehydrogenase by influencing its gene structure and protein structure.

  13. Effect of Exercise-Induced Lactate Elevation on Brain Lactate Levels During Hypoglycemia in Patients With Type 1 Diabetes and Impaired Awareness of Hypoglycemia.

    PubMed

    Wiegers, Evita C; Rooijackers, Hanne M; Tack, Cees J; Groenewoud, Hans J M M; Heerschap, Arend; de Galan, Bastiaan E; van der Graaf, Marinette

    2017-12-01

    Since altered brain lactate handling has been implicated in the development of impaired awareness of hypoglycemia (IAH) in type 1 diabetes, the capacity to transport lactate into the brain during hypoglycemia may be relevant in its pathogenesis. High-intensity interval training (HIIT) increases plasma lactate levels. We compared the effect of HIIT-induced hyperlacticacidemia on brain lactate during hypoglycemia between 1 ) patients with type 1 diabetes and IAH, 2 ) patients with type 1 diabetes and normal awareness of hypoglycemia, and 3 ) healthy participants without diabetes ( n = 6 per group). All participants underwent a hypoglycemic (2.8 mmol/L) clamp after performing a bout of HIIT on a cycle ergometer. Before HIIT (baseline) and during hypoglycemia, brain lactate levels were determined continuously with J-difference-editing 1 H-MRS, and time curves were analyzed using nonlinear mixed-effects modeling. At the beginning of hypoglycemia (after HIIT), brain lactate levels were elevated in all groups but most pronounced in patients with IAH. During hypoglycemia, brain lactate decreased ∼30% below baseline in patients with IAH but returned to baseline levels and remained there in the other two groups. Our results support the concept of enhanced lactate transport as well as increased lactate oxidation in patients with type 1 diabetes and IAH. © 2017 by the American Diabetes Association.

  14. Bifunctional isocitrate-homoisocitrate dehydrogenase: a missing link in the evolution of beta-decarboxylating dehydrogenase.

    PubMed

    Miyazaki, Kentaro

    2005-05-27

    Beta-decarboxylating dehydrogenases comprise 3-isopropylmalate dehydrogenase, isocitrate dehydrogenase, and homoisocitrate dehydrogenase. They share a high degree of amino acid sequence identity and occupy equivalent positions in the amino acid biosynthetic pathways for leucine, glutamate, and lysine, respectively. Therefore, not only the enzymes but also the whole pathways should have evolved from a common ancestral pathway. In Pyrococcus horikoshii, only one pathway of the three has been identified in the genomic sequence, and PH1722 is the sole beta-decarboxylating dehydrogenase gene. The organism does not require leucine, glutamate, or lysine for growth; the single pathway might play multiple (i.e., ancestral) roles in amino acid biosynthesis. The PH1722 gene was cloned and expressed in Escherichia coli and the substrate specificity of the recombinant enzyme was investigated. It exhibited activities on isocitrate and homoisocitrate at near equal efficiency, but not on 3-isopropylmalate. PH1722 is thus a novel, bifunctional beta-decarboxylating dehydrogenase, which likely plays a dual role in glutamate and lysine biosynthesis in vivo.

  15. Testicular lactate content is compromised in men with Klinefelter Syndrome.

    PubMed

    Alves, Marco G; Martins, Ana D; Jarak, Ivana; Barros, Alberto; Silva, Joaquina; Sousa, Mário; Oliveira, Pedro F

    2016-03-01

    Klinefelter syndrome (KS) is the most common genetic cause of human infertility, but the mechanism(s) responsible for its phenotype remain largely unknown. KS is associated with alterations in body composition and with a higher risk of developing metabolic diseases. We therefore hypothesized that KS men seeking fertility treatment possess an altered testicular metabolism profile that may hamper the nutritional support of spermatogenesis. Testicular biopsies from control (46, XY) (n = 6) and KS (47, XXY) (n = 6) individuals were collected and analyzed by proton high-resolution magic-angle spinning nuclear magnetic resonance spectroscopy. The mRNA and protein expression of crucial glycolysis-associated enzymes and transporters were evaluated in parallel by quantitative PCR and Western blot, respectively. Our data revealed altered regulation of glucose transporters (GLUT1 and GLUT3); phosphofructokinase 1, liver isoform (PFKL); and lactate dehydrogenase A (LDHA) expression in the testis of KS patients. Moreover, we detected a severe reduction in lactate and creatine accumulation within testicular tissue from KS men. The aberrant levels of the biomarkers detected in testicular biopsies of KS men may therefore be associated with the infertility phenotypes presented by these men. Mol. Reprod. Dev. 83: 208-216, 2016. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

  16. Proteomic analysis of physiological function response to hot summer in liver from lactating dairy cows.

    PubMed

    Wang, Qiangjun; Zhao, Xiaowei; Zhang, Zijun; Zhao, Huiling; Huang, Dongwei; Cheng, Guanglong; Yang, Yongxin

    2017-04-01

    Lactation performance of dairy cattle is susceptible to heat stress. The liver is one of the most crucial organs affected by high temperature in dairy cows. However, the physiological adaption by the liver to hot summer conditions has not been well elucidated in lactating dairy cows. In the present study, proteomic analysis of the liver in dairy cows in spring and hot summer was performed using a label-free method. In total, 127 differentially expressed proteins were identified; most of the upregulated proteins were involved in protein metabolic processes and responses to stimuli, whereas most of the downregulated proteins were related to oxidation-reduction. Pathway analysis indicated that 3 upregulated heat stress proteins (HSP90α, HSP90β, and endoplasmin) were enriched in the NOD-like receptor signaling pathway, whereas several downregulated NADH dehydrogenase proteins were involved in the oxidative phosphorylation pathway. The protein-protein interaction network indicated that several upregulated HSPs (HSP90α, HSP90β, and GRP78) were involved in more interactions than other proteins and were thus considered as central hub nodes. Our findings provide novel insights into the physiological adaption of liver function in lactating dairy cows to natural high temperature. Copyright © 2017. Published by Elsevier Ltd.

  17. Digitalis metabolism and human liver alcohol dehydrogenase.

    PubMed Central

    Frey, W A; Vallee, B L

    1980-01-01

    Human liver alcohol dehydrogenase (alcohol: NAD" oxidoreductase, EC 1.1.1.1) catalyzes the oxidation of the 3 beta-OH group of digitoxigenin, digoxigenin, and gitoxigenin to their 3-keto derivatives, which have been characterized by high performance liquid chromatography and mass spectrometry. These studies have identified human liver alcohol dehydrogenase as the unknown NAD(H)-dependent liver enzyme specific for the free hydroxyl group at C3 of the cardiac genins; this hydroxyl is the critical site of the genins' enzymatic oxidation and concomitant pharmacological inactivation in humans. Several kinetic approaches have demonstrated that ethanol and the pharmacologically active components of the digitalis glycosides are oxidized with closely similar kcat/Km values at the same site on human liver alcohol dehydrogenase, for which they compete. Human liver alcohol dehydrogenase thereby becomes an important biochemical link in the metabolism, pharmacology, and toxicology of ethanol and these glycosides, structurally unrelated agents that are both used widely. Both the competition of ethanol with these cardiac sterols and the narrow margin of safety in the therapeutic use of digitalis derivatives would seem to place at increased risk those individuals who receive digitalis and simultaneously consume large amounts of ethanol or whose alcohol dehydrogenase function is impaired. PMID:6987673

  18. The Activity of Class I-IV Alcohol Dehydrogenase Isoenzymes and Aldehyde Dehydrogenase in Bladder Cancer Cells.

    PubMed

    Orywal, Karolina; Jelski, Wojciech; Werel, Tadeusz; Szmitkowski, Maciej

    2018-01-02

    The aim of this study was to determine the differences in the activity of Alcohol Dehydrogenase (ADH) isoenzymes and Aldehyde Dehydrogenase (ALDH) in normal and cancerous bladder cells. Class III, IV of ADH and total ADH activity were measured by the photometric method and class I, II ADH and ALDH activity by the fluorometric method. Significantly higher total activity of ADH was found in both, low-grade and high-grade bladder cancer, in comparison to healthy tissues. The increased activity of total ADH in bladder cancer cells may be the cause of metabolic disorders in cancer cells, which may intensify carcinogenesis.

  19. Activation of mitochondrial calpain and increased cardiac injury: beyond AIF release

    PubMed Central

    Thompson, Jeremy; Hu, Ying; Lesnefsky, Edward J.

    2015-01-01

    Calpain 1 (CPN1) is a ubiquitous cysteine protease that exists in both cytosol and cardiac mitochondria. Mitochondrial CPN1 (mit-CPN1) is located in the intermembrane space and matrix. Activation of mit-CPN1 within the intermembrane space increases cardiac injury by releasing apoptosis-inducing factor from mitochondria during ischemia-reperfusion (IR). We asked if activation of mit-CPN1 is involved in mitochondrial injury during IR. MDL-28170 (MDL) was used to inhibit CPN1 in buffer-perfused hearts following 25-min ischemia and 30-min reperfusion. MDL treatment decreased the release of lactate dehydrogenase into coronary effluent compared with untreated hearts, indicating that inhibition of CPN1 decreases cardiac injury. MDL also prevented the cleavage of spectrin (a substrate of CPN1) in cytosol during IR, supporting that MDL treatment decreased cytosolic calpain activation. In addition, MDL markedly improved calcium retention capacity compared with untreated heart, suggesting that MDL treatment decreases mitochondrial permeability transition pore opening. In addition, we found that IR led to decreased complex I activity, whereas inhibition of mit-CPN1 using MDL protected complex I. Pyruvate dehydrogenase content was decreased following IR. However, pyruvate dehydrogenase content was preserved in MDL-treated mitochondria. Taken together, MDL treatment decreased cardiac injury during IR by inhibiting both cytosolic and mit-CPN1. Activation of mit-CPN1 increases cardiac injury during IR by sensitizing mitochondrial permeability transition pore opening and impairing mitochondrial metabolism through damage of complex I. PMID:26637561

  20. Single mutation in Shine-Dalgarno-like sequence present in the amino terminal of lactate dehydrogenase of Plasmodium effects the production of an eukaryotic protein expressed in a prokaryotic system.

    PubMed

    Cicek, Mustafa; Mutlu, Ozal; Erdemir, Aysegul; Ozkan, Ebru; Saricay, Yunus; Turgut-Balik, Dilek

    2013-06-01

    One of the most important step in structure-based drug design studies is obtaining the protein in active form after cloning the target gene. In one of our previous study, it was determined that an internal Shine-Dalgarno-like sequence present just before the third methionine at N-terminus of wild type lactate dehydrogenase enzyme of Plasmodium falciparum prevent the translation of full length protein. Inspection of the same region in P. vivax LDH, which was overproduced as an active enzyme, indicated that the codon preference in the same region was slightly different than the codon preference of wild type PfLDH. In this study, 5'-GGAGGC-3' sequence of P. vivax that codes for two glycine residues just before the third methionine was exchanged to 5'-GGAGGA-3', by mimicking P. falciparum LDH, to prove the possible effects of having an internal SD-like sequence when expressing an eukaryotic protein in a prokaryotic system. Exchange was made by site-directed mutagenesis. Results indicated that having two glycine residues with an internal SD-like sequence (GGAGGA) just before the third methionine abolishes the enzyme activity due to the preference of the prokaryotic system used for the expression. This study emphasizes the awareness of use of a prokaryotic system to overproduce an eukaryotic protein.

  1. The Effects of Direct Oxygen Supply During Static Cold Preservation of Rat Livers: An Experimental Study.

    PubMed

    Zumrutdal, Emin; Karateke, Faruk; Eser, Pınar Eylem; Turan, Umit; Ozyazici, Sefa; Sozutek, Alper; Gulkaya, Mustafa; Kunt, Mevlut

    2016-12-01

    We aimed to determine the biochemical and histopathologic effects of direct oxygen supply to the preservation fluid of static cold storage system with a simple method on rat livers. Sixteen rats were randomly divided into 2 groups: the control group, which contained Ringer's lactate as preservation fluid; and the oxygen group, which contained oxygen and Ringer's lactate for preservation. Each liver was placed in a bag containing 50 mL Ringer's lactate and placed in ice-filled storage containers. One hundred percent oxygen supplies were given via a simple, inexpensive system created in our laboratory, to the livers in oxygen group. We obtained samples for histopathologic evaluation in the 12th hour. In addition, 3 mL of preservation fluid was subjected to biochemical analysis at 0, sixth, and twelfth hours. Aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, and pH levels were measured from the preservation fluid. In oxygen-supplemented group, the acceleration speed of increase in alanine aminotransferase and lactate dehydrogenase levels at sixth hour and lactate dehydrogenase, alanine aminotransferase, and lactate dehydrogenase levels at 12th hour were statistically significantly reduced. In histopathologic examination, all parameters except ballooning were statistically significantly better in the oxygen-supplemented group. This simple system for oxygenation of liver tissues during static cold storage was shown to be effective with good results in biochemical and histopathologic assessments. Because this is a simple, inexpensive, and easily available method, larger studies are warranted to evaluate its effects (especially in humans).

  2. Lactate metabolism and its effects on glucose metabolism in an excised neural tissue.

    PubMed

    Larrabee, M G

    1995-04-01

    Chains of lumbar sympathetic ganglia, excised from 15-day-old chicken embryos, were incubated for 4 h at 36 degrees C in a bicarbonate-buffered physiological salt solution containing 5.5 mM glucose and equilibrated with 5% CO2-95% O2. [U-14C]Glucose and [U-14C]lactate were used as tracers to measure the products of glucose and lactate metabolism, respectively, including CO2, lactate, and constituents of the tissue. When 5 mM lactate was added to bathing solution containing 5.5 mM glucose, lactate carbon displaced 50-70% of the glucose carbon otherwise used for CO2 production and provided about three times as much carbon for CO2 as did glucose. The lactate addition increased the total carbon incorporated into CO2 and into constituents of the tissue above those observed with glucose alone and also increased the lactate released to the bathing solution from [U-14C]-glucose. The latter increase was evidently due to an interference with reuptake of the lactate released from the ganglion cells, not to an increase in the cellular release itself. When the volume of bathing solution was increased 10-fold relative to that of the tissue, the average output of CO2 from [U-14C]glucose during a 4-h incubation was decreased by 50% when 5 mM lactate was present but was not affected significantly in the absence of added lactate. It is concluded that the effect of changing volume in the presence of lactate was due to the effects of lactate on glucose metabolism described above and resulted from a lower average lactate concentration in the smaller volume than in the larger one, due to metabolic depletion of the added lactate.(ABSTRACT TRUNCATED AT 250 WORDS)

  3. Upregulation of Zinc Absorption Matches Increases in Physiologic Requirements for Zinc in Women Consuming High- or Moderate-Phytate Diets during Late Pregnancy and Early Lactation.

    PubMed

    Hambidge, K Michael; Miller, Leland V; Mazariegos, Manolo; Westcott, Jamie; Solomons, Noel W; Raboy, Victor; Kemp, Jennifer F; Das, Abhik; Goco, Norman; Hartwell, Ty; Wright, Linda; Krebs, Nancy F

    2017-06-01

    Background: Estimated physiologic requirements (PRs) for zinc increase in late pregnancy and early lactation, but the effect on dietary zinc requirements is uncertain. Objective: The aim of this study was to determine changes in daily fractional absorbed zinc and total absorbed zinc (TAZ) from ad libitum diets of differing phytate contents in relation to physiologic zinc requirements during pregnancy and lactation. Methods: This was a prospective observational study of zinc absorption at 8 (phase 1) and 34 (phase 2) wk of gestation and 2 (phase 3) and 6 (phase 4) mo of lactation. Participants were indigenous Guatemalan women of childbearing age whose major food staple was maize and who had been randomly assigned in a larger study to either of 2 ad libitum feeding groups: low-phytate maize (LP; 1.6 mg/g; n = 14) or control maize (C; 7.1 mg/g; n = 8). Total dietary zinc (milligrams per day, TDZ) and phytate (milligrams per day) were determined from duplicate diets and fractional absorption (FAZ) by dual isotope ratio technique (TAZ = TDZ × FAZ). All variables were examined longitudinally and by group and compared with PRs. TAZ values at later phases were compared with phase 1. Measured TAZ was compared with predicted TAZ for nonpregnant, nonlactating (NPNL) women. Results: TAZ was greater in the LP group than in the C group at all phases. All variables increased from phase 1 to phases 2 and 3 and declined at phase 4. TAZ increased by 1.25 mg/d ( P = 0.045) in the C group and by 0.81 mg/d ( P = 0.058) in the LP group at phase 2. At phase 3, the increases were 2.66 mg/d ( P = 0.002) in the C group and 2.28 mg/d ( P = 0.0004) in the LP group, compared with a 1.37-mg/d increase in PR. Measured TAZ was greater than predicted values in phases 2-4. Conclusions: Upregulation of zinc absorption in late pregnancy and early lactation matches increases in PRs of pregnant and lactating women, regardless of dietary phytate, which has implications for dietary zinc requirements of

  4. A single amino acid change (Y318F) in the L-arabitol dehydrogenase (LadA) from Aspergillus niger results in a significant increase in affinity for D-sorbitol

    PubMed Central

    2009-01-01

    Background L-arabitol dehydrogenase (LAD) and xylitol dehydrogenase (XDH) are involved in the degradation of L-arabinose and D-xylose, which are among the most abundant monosaccharides on earth. Previous data demonstrated that LAD and XDH not only differ in the activity on their biological substrate, but also that only XDH has significant activity on D-sorbitol and may therefore be more closely related to D-sorbitol dehydrogenases (SDH). In this study we aimed to identify residues involved in the difference in substrate specificity. Results Phylogenetic analysis demonstrated that LAD, XDH and SDH form 3 distinct groups of the family of dehydrogenases containing an Alcohol dehydrogenase GroES-like domain (pfam08240) and likely have evolved from a common ancestor. Modelling of LadA and XdhA of the saprobic fungus Aspergillus niger on human SDH identified two residues in LadA (M70 and Y318), that may explain the absence of activity on D-sorbitol. While introduction of the mutation M70F in LadA of A. niger resulted in a nearly complete enzyme inactivation, the Y318F resulted in increased activity for L-arabitol and xylitol. Moreover, the affinity for D-sorbitol was increased in this mutant. Conclusion These data demonstrates that Y318 of LadA contributes significantly to the substrate specificity difference between LAD and XDH/SDH. PMID:19674460

  5. Higher Accuracy of the Lactate Minimum Test Compared to Established Threshold Concepts to Determine Maximal Lactate Steady State in Running.

    PubMed

    Wahl, Patrick; Zwingmann, Lukas; Manunzio, Christian; Wolf, Jacob; Bloch, Wilhelm

    2018-05-18

    This study evaluated the accuracy of the lactate minimum test, in comparison to a graded-exercise test and established threshold concepts (OBLA and mDmax) to determine running speed at maximal lactate steady state. Eighteen subjects performed a lactate minimum test, a graded-exercise test (2.4 m·s -1 start,+0.4 m·s -1 every 5 min) and 2 or more constant-speed tests of 30 min to determine running speed at maximal lactate steady state. The lactate minimum test consisted of an initial lactate priming segment, followed by a short recovery phase. Afterwards, the initial load of the subsequent incremental segment was individually determined and was increased by 0.1 m·s -1 every 120 s. Lactate minimum was determined by the lowest measured value (LM abs ) and by a third-order polynomial (LM pol ). The mean difference to maximal lactate steady state was+0.01±0.14 m·s -1 (LM abs ), 0.04±0.15 m·s -1 (LM pol ), -0.06±0.31 m·s 1 (OBLA) and -0.08±0.21 m·s 1 (mDmax). The intraclass correlation coefficient (ICC) between running velocity at maximal lactate steady state and LM abs was highest (ICC=0.964), followed by LM pol (ICC=0.956), mDmax (ICC=0.916) and OBLA (ICC=0.885). Due to the higher accuracy of the lactate minimum test to determine maximal lactate steady state compared to OBLA and mDmax, we suggest the lactate minimum test as a valid and meaningful concept to estimate running velocity at maximal lactate steady state in a single session for moderately up to well-trained athletes. © Georg Thieme Verlag KG Stuttgart · New York.

  6. Lactate response to different volume patterns of power clean.

    PubMed

    Date, Anand S; Simonson, Shawn R; Ransdell, Lynda B; Gao, Yong

    2013-03-01

    The ability to metabolize or tolerate lactate and produce power simultaneously can be an important determinant of performance. Current training practices for improving lactate use include high-intensity aerobic activities or a combination of aerobic and resistance training. Excessive aerobic training may have undesired physiological adaptations (e.g., muscle loss, change in fiber types). The role of explosive power training in lactate production and use needs further clarification. We hypothesized that high-volume explosive power movements such as Olympic lifts can increase lactate production and overload lactate clearance. Hence, the purpose of this study was to assess lactate accumulation after the completion of 3 different volume patterns of power cleans. Ten male recreational athletes (age 24.22 ± 1.39 years) volunteered. Volume patterns consisted of 3 sets × 3 repetition maximum (3RM) (low volume [LV]), 3 sets × 6 reps at 80-85% of 3RM (midvolume [MV]), and 3 sets × 9 reps at 70-75% of 3RM (high volume [HV]). Rest period was identical at 2 minutes. Blood samples were collected immediately before and after each volume pattern. The HV resulted in the greatest lactate accumulation (7.43 ± 2.94 mmol·L) vs. (5.27 ± 2.48 and 4.03 ± 1.78 mmol·L in MV and LV, respectively). Mean relative increase in lactate was the highest in HV (356.34%). The findings indicate that lactate production in power cleans is largely associated with volume, determined by number of repetitions, load, and rest interval. High-volume explosive training may impose greater metabolic demands than low-volume explosive training and may improve ability to produce power in the presence of lactate. The role of explosive power training in overloading the lactate clearance mechanism should be examined further, especially for athletes of intermittent sport.

  7. Effect of ornithine and lactate on urea synthesis in isolated hepatocytes.

    PubMed Central

    Briggs, S; Freedland, R A

    1976-01-01

    1. In hepatocytes isolated from 24 h-starved rats, urea production from ammonia was stimulated by addition of lactate, in both the presence and the absence of ornithine. The relationship of lactate concentration to the rate of urea synthesis was hyperbolic. 2. Other glucose precursors also stimulated urea production to varying degrees, but none more than lactate. Added oleate and butyrate did not stimulate urea synthesis. 3. Citrulline accumulation was largely dependent on ornithine concentration. As ornithine was increased from 0 to 40 mM, the rate of citrulline accumulation increased hyperbolically, and was half-maximal when ornithine was 8-12 mM. 4. The rate of citrulline accumulation was independent of the presence of lactate, but with pyruvate the rate increased. 5. The rate of urea production continued to increase as ornithine was varied from 0 to 40 mM. 6. It was concluded that intermediates provided by both ornithine and lactate are limiting for urea production from ammonia in isolated liver cells. It was suggested that the stimulatory effect of lactate lies in increased availability of cytosolic aspartate for condensation with citrulline. PMID:1008850

  8. Identification of a dehydrogenase acting on D-2-hydroxyglutarate

    PubMed Central

    2004-01-01

    Extracts of frozen rat liver were found to catalyse the formation of 3H2O from DL-2-hydroxy[2-3H]glutarate. Three peaks of enzyme activities were observed on separation by chromatography on DEAE-Sepharose. The first and second peaks corresponded to an enzyme acting on L-2-hydroxyglutarate and the third peak corresponded to an enzyme acting on D-2-hydroxyglutarate, as indicated by competitive inhibition of the detritiation of the racemic radioactive compound by the unlabelled L- and D-isomers respectively. The enzyme acting on the D-form was further characterized. It was independent of NAD or NADP and it converted D-2-hydroxyglutarate into α-ketoglutarate, transferring electrons to artificial electron acceptors. It also oxidized D-lactate, D-malate and meso-tartrate and was stimulated by Zn2+, Co2+ and Mn2+, but not by Mg2+ or Ca2+. Subcellular fractionation indicated that it was present in the mitochondrial fraction. The enzyme was further purified by chromatography on Blue Trisacryl and phenyl-Sepharose, up to a stage where only a few bands were still visible by SDS/ PAGE. Among the four candidate polypeptides that were identified by MS, one corresponded to a predicted mitochondrial protein homologous with FAD-dependent D-lactate dehydrogenase. The corresponding human protein was expressed in HEK-293 cells and it was shown to catalyse the detritiation of DL-2-hydroxy[2-3H]glutarate with similar properties as the purified rat enzyme. PMID:15070399

  9. Identification of a dehydrogenase acting on D-2-hydroxyglutarate.

    PubMed

    Achouri, Younes; Noël, Gaëtane; Vertommen, Didier; Rider, Mark H; Veiga-Da-Cunha, Maria; Van Schaftingen, Emile

    2004-07-01

    Extracts of frozen rat liver were found to catalyse the formation of 3H2O from DL-2-hydroxy[2-3H]glutarate. Three peaks of enzyme activities were observed on separation by chromatography on DEAE-Sepharose. The first and second peaks corresponded to an enzyme acting on L-2-hydroxyglutarate and the third peak corresponded to an enzyme acting on D-2-hydroxyglutarate, as indicated by competitive inhibition of the detritiation of the racemic radioactive compound by the unlabelled L- and D-isomers respectively. The enzyme acting on the D-form was further characterized. It was independent of NAD or NADP and it converted D-2-hydroxyglutarate into a-ketoglutarate, transferring electrons to artificial electron acceptors. It also oxidized D-lactate, D-malate and meso-tartrate and was stimulated by Zn2+, Co2+ and Mn2+, but not by Mg2+ or Ca2+. Subcellular fractionation indicated that it was present in the mitochondrial fraction. The enzyme was further purified by chromatography on Blue Trisacryl and phenyl-Sepharose, up to a stage where only a few bands were still visible by SDS/PAGE. Among the four candidate polypeptides that were identified by MS, one corresponded to a predicted mitochondrial protein homologous with FAD-dependent D-lactate dehydrogenase. The corresponding human protein was expressed in HEK-293 cells and it was shown to catalyse the detritiation of DL-2-hydroxy[2-3H]glutarate with similar properties as the purified rat enzyme.

  10. Lactation and changes in maternal metabolic risk factors.

    PubMed

    Gunderson, Erica P; Lewis, Cora E; Wei, Gina S; Whitmer, Rachel A; Quesenberry, Charles P; Sidney, Steve

    2007-03-01

    To examine the relationship between duration of lactation and changes in maternal metabolic risk factors. This 3-year prospective study examined changes in metabolic risk factors among lactating women from preconception to postweaning and among nonlactating women from preconception to postdelivery, in comparison with nongravid women. Of 1,051 (490 black, 561 white) women who attended two consecutive study visits in years 7 (1992-1993) and 10 (1995-1996), 942 were nongravid and 109 had one interim birth. Of parous women, 48 (45%) did not lactate, and 61 (55%) lactated and weaned before year 10. The lactated and weaned women were subdivided by duration of lactation into less than 3 months and 3 months or more. Multiple linear regression models estimated mean 3-year changes in metabolic risk factors adjusted for age, race, parity, education, and behavioral covariates. Both parous women who did not lactate and parous women who lactated and weaned gained more weight (+5.6, +4.4 kg) and waist girth (+5.3, +4.9 cm) than nongravid women over the 3-year interval; P<.001. Low-density lipoprotein cholesterol (+6.7 mg/dL, P<.05) and fasting insulin (+2.6 microunits, P=.06) increased more for parous women who did not lactate than for nongravid and parous women who lactated and weaned. High-density lipoprotein cholesterol decrements for both parous women who did not lactate and parous women who lactated and weaned were 4.0 mg/dL greater than for nongravid women (P<.001). Among parous, lactated and weaned women, lactation for 3 months or longer was associated with a smaller decrement in high-density lipoprotein cholesterol (-1.3 mg/dL versus -7.3 mg/dL for less than 3 months; P<.01). Lactation may attenuate unfavorable metabolic risk factor changes that occur with pregnancy, with effects apparent after weaning. As a modifiable behavior, lactation may affect women's future risk of cardiovascular and metabolic diseases. II.

  11. Directed modification of L-LcLDH1, an L-lactate dehydrogenase from Lactobacillus casei, to improve its specific activity and catalytic efficiency towards phenylpyruvic acid.

    PubMed

    Li, Jian-Fang; Li, Xue-Qing; Liu, Yan; Yuan, Feng-Jiao; Zhang, Ting; Wu, Min-Chen; Zhang, Ji-Ru

    2018-05-22

    To improve the specific activity and catalytic efficiency of L-LcLDH1, an NADH-dependent allosteric L-lactate dehydrogenase from L. casei, towards phenylpyruvic acid (PPA), its directed modification was conducted based on the semi-rational design. The three variant genes, Lcldh1 Q88R , Lcldh1 I229A and Lcldh1 T235G , were constructed by whole-plasmid PCR as designed theoretically, and expressed in E. coli BL21(DE3), respectively. The purified mutant, L-LcLDH1 Q88R or L-LcLDH1 I229A , displayed the specific activity of 451.5 or 512.4 U/mg towards PPA, by which the asymmetric reduction of PPA afforded L-phenyllactic acid (PLA) with an enantiomeric excess (ee p ) more than 99%. Their catalytic efficiencies (k cat /K m ) without D-fructose-1,6-diphosphate (D-FDP) were 4.8- and 5.2-fold that of L-LcLDH1. Additionally, the k cat /K m values of L-LcLDH1 Q88R and L-LcLDH1 I229A with D-FDP were 168.4- and 8.5-fold higher than those of the same enzymes without D-FDP, respectively. The analysis of catalytic mechanisms by molecular docking (MD) simulation indicated that substituting I229 in L-LcLDH1 with Ala enlarges the space of substrate-binding pocket, and that the replacement of Q88 with Arg makes the inlet of pocket larger than that of L-LcLDH1. Copyright © 2018 Elsevier B.V. All rights reserved.

  12. Karnofsky Performance Status and Lactate Dehydrogenase Predict the Benefit of Palliative Whole-Brain Irradiation in Patients With Advanced Intra- and Extracranial Metastases From Malignant Melanoma

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Partl, Richard, E-mail: richard.partl@medunigraz.at; Richtig, Erika; Avian, Alexander

    2013-03-01

    Purpose: To determine prognostic factors that allow the selection of melanoma patients with advanced intra- and extracerebral metastatic disease for palliative whole-brain radiation therapy (WBRT) or best supportive care. Methods and Materials: This was a retrospective study of 87 patients who underwent palliative WBRT between 1988 and 2009 for progressive or multiple cerebral metastases at presentation. Uni- and multivariate analysis took into account the following patient- and tumor-associated factors: gender and age, Karnofsky performance status (KPS), neurologic symptoms, serum lactate dehydrogenase (LDH) level, number of intracranial metastases, previous resection or stereotactic radiosurgery of brain metastases, number of extracranial metastasis sites,more » and local recurrences as well as regional lymph node metastases at the time of WBRT. Results: In univariate analysis, KPS, LDH, number of intracranial metastases, and neurologic symptoms had a significant influence on overall survival. In multivariate survival analysis, KPS and LDH remained as significant prognostic factors, with hazard ratios of 3.3 (95% confidence interval [CI] 1.6-6.5) and 2.8 (95% CI 1.6-4.9), respectively. Patients with KPS ≥70 and LDH ≤240 U/L had a median survival of 191 days; patients with KPS ≥70 and LDH >240 U/L, 96 days; patients with KPS <70 and LDH ≤240 U/L, 47 days; and patients with KPS <70 and LDH >240 U/L, only 34 days. Conclusions: Karnofsky performance status and serum LDH values indicate whether patients with advanced intra- and extracranial tumor manifestations are candidates for palliative WBRT or best supportive care.« less

  13. 1H-NMR and Hyperpolarized 13C-NMR Assays of Pyruvate-Lactate Exhange: a comparative study

    PubMed Central

    Orton, Matthew R.; Tardif, Nicolas; Parkes, Harold G.; Robinson, Simon P.; Leach, Martin O.; Chung, Yuen-Li; Eykyn, Thomas R.

    2015-01-01

    Pyruvate-lactate exchange is mediated by the enzyme lactate dehydrogenase (LDH) and is central to the altered energy metabolism in cancer cells. Measurement of exchange kinetics using hyperpolarized 13C NMR has provided a biomarker of response to novel therapeutics. In this study we investigated an alternative in vitro 1H assay, using [3-13C]pyruvate, and compared the measured kinetics with a hyperpolarized 13C-NMR assay, using [1-13C]pyruvate, under the same conditions in human colorectal carcinoma SW1222 cells. The apparent forward reaction rate constants (kPL) derived from the two assays showed no significant difference, and both assays had similar reproducibility (kPL = 0.506 ± 0.054 and kPL = 0.441 ± 0.090 nmol/s/106 cells, (mean ± standard deviation, n = 3); 1H, 13C assays respectively). The apparent backward reaction rate constant (kLP) could only be measured with good reproducibility using the 1H-NMR assay (kLP = 0.376 ± 0.091 nmol/s/106 cells, (mean ± standard deviation, n = 3)). The 1H-NMR assay has adequate sensitivity to measure real-time pyruvate-lactate exchange kinetics in vitro, offering a complementary and accessible assay of apparent LDH activity. PMID:23712817

  14. Droplet Microfluidic Platform for the Determination of Single-Cell Lactate Release.

    PubMed

    Mongersun, Amy; Smeenk, Ian; Pratx, Guillem; Asuri, Prashanth; Abbyad, Paul

    2016-03-15

    Cancer cells release high levels of lactate that has been correlated to increased metastasis and tumor recurrence. Single-cell measurements of lactate release can identify malignant cells and help decipher metabolic cancer pathways. We present here a novel droplet microfluidic method that allows the fast and quantitative determination of lactate release in many single cells. Using passive forces, droplets encapsulated cells are positioned in an array. The single-cell lactate release rate is determined from the increase in droplet fluorescence as the lactate is enzymatically converted to a fluorescent product. The method is used to measure the cell-to-cell variance of lactate release in K562 leukemia and U87 glioblastoma cancer cell lines and under the chemical inhibition of lactate efflux. The technique can be used in the study of cancer biology, but more broadly in cell biology, to capture the full range of stochastic variations in glycolysis activity in heterogeneous cell populations in a repeatable and high-throughput manner.

  15. Lactate shuttling and lactate use as fuel after traumatic brain injury: metabolic considerations

    PubMed Central

    Dienel, Gerald A

    2014-01-01

    Lactate is proposed to be generated by astrocytes during glutamatergic neurotransmission and shuttled to neurons as ‘preferred' oxidative fuel. However, a large body of evidence demonstrates that metabolic changes during activation of living brain disprove essential components of the astrocyte–neuron lactate shuttle model. For example, some glutamate is oxidized to generate ATP after its uptake into astrocytes and neuronal glucose phosphorylation rises during activation and provides pyruvate for oxidation. Extension of the notion that lactate is a preferential fuel into the traumatic brain injury (TBI) field has important clinical implications, and the concept must, therefore, be carefully evaluated before implementation into patient care. Microdialysis studies in TBI patients demonstrate that lactate and pyruvate levels and lactate/pyruvate ratios, along with other data, have important diagnostic value to distinguish between ischemia and mitochondrial dysfunction. Results show that lactate release from human brain to blood predominates over its uptake after TBI, and strong evidence for lactate metabolism is lacking; mitochondrial dysfunction may inhibit lactate oxidation. Claims that exogenous lactate infusion is energetically beneficial for TBI patients are not based on metabolic assays and data are incorrectly interpreted. PMID:25204393

  16. Analysis of lactate concentrations in canine synovial fluid.

    PubMed

    Proot, J L J; de Vicente, F; Sheahan, D E

    2015-01-01

    To report synovial fluid lactate concentrations in normal and pathological canine joints. Controlled, prospective study. Lactate was measured in synovial fluid using a hand-held meter and the rest of the fluid was sent to a commercial laboratory for analysis. Samples were divided into four groups; group 1: control, group 2: osteoarthritis, group 3: immune-mediated inflammatory arthritis, and group 4: septic arthritis. Statistical analysis was performed to compare lactate concentrations between the four groups and to examine the predictive value of lactate in the diagnosis of septic arthritis. A correlation was sought between synovial fluid lactate and synovial fluid total nucleated cell count and total protein. Seventy-four samples were investigated from 55 dogs. Statistical analysis found that lactate concentrations were significantly higher in the septic arthritis group than in each of the other three groups. No significant correlation could be found between synovial fluid lactate concentrations and synovial fluid total nucleated cell count or synovial fluid total protein. Lactate concentration was found to be a useful predictor of septic arthritis, with a low concentration pointing towards exclusion rather than a high concentration to the diagnosis of septic arthritis. Synovial fluid lactate concentration is not a good marker for osteoarthritis or immune-mediated inflammatory arthritis, but it is significantly increased in septic arthritis and could help the clinician in ruling out this condition in a quick and cost-effective way.

  17. Arterial cord blood lactate at birth correlates with duration of pushing efforts.

    PubMed

    Dessolle, Lionel; Lebrec, Jeremie; Daraï, Emile

    2010-01-01

    To evaluate the impact of the duration of pushing efforts on arterial cord blood lactate values. This was a prospective observational study of 124 consecutive normal vaginal deliveries in a tertiary teaching hospital. Arterial cord blood lactate was determined immediately at birth with a test strip method. Univariate and multivariate analyses were performed to check for clinical determinants of lactate levels. The main measure was lactate according to the duration of pushing efforts. Arterial cord lactates increased significantly and were strongly correlated with the duration of pushing efforts, independent of gestational age and birthweight. Women pushing for more than 20 min had higher arterial cord blood lactates (4.9 +/- 1.4 vs. 3.3 +/- 1.16 mM, respectively) and a higher rate of lactates >6 mM (18 vs. 3%) than those pushing for less than 20 min. At normal delivery, arterial cord blood lactates increase significantly with the duration of pushing efforts. Pushing for more than 20 min is associated with an increased risk of metabolic acidosis in the neonate. Further studies are required to evaluate the clinical significance of these observations. 2009 S. Karger AG, Basel.

  18. Glucose-6-phosphate dehydrogenase

    MedlinePlus

    ... page: //medlineplus.gov/ency/article/003671.htm Glucose-6-phosphate dehydrogenase test To use the sharing features on this page, please enable JavaScript. Glucose-6-phosphate dehydrogenase (G6PD) is a protein that helps ...

  19. Fabrication of Flexible Arrayed Lactate Biosensor Based on Immobilizing LDH-NAD+ on NiO Film Modified by GO and MBs

    PubMed Central

    Yan, Siao-Jie; Liao, Yi-Hung; Lai, Chih-Hsien; Wu, You-Xiang; Wu, Cian-Yi; Chen, Hsiang-Yi; Huang, Hong-Yu; Wu, Tong-Yu

    2017-01-01

    We proposed the flexible arrayed lactate biosensor based on immobilizing l-lactate dehydrogenase (LDH) and nicotinamide adenine dinucleotide (NAD+) on nickel oxide (NiO) film, and which the average sensitivity could be enhanced by using graphene oxide (GO) and magnetic beads (MBs). By using GO and MBs, it exhibits excellent sensitivity (45.397 mV/mM) with a linearity of 0.992 in a range of 0.2 mM to 3 mM. According to the results of electrochemical impedance spectroscopy (EIS), the electron transfer resistance of LDH-NAD+-MBs/GPTS/GO/NiO film was smaller than those of LDH-NAD+/GPTS/GO/NiO film and LDH-NAD+/GPTS/NiO film, and it presented the outstanding electron transfer ability. After that, the limit of detection, anti-interference effect and bending test were also investigated. PMID:28704960

  20. Peroxisome proliferator-activated receptor γ agonism attenuates endotoxaemia-induced muscle protein loss and lactate accumulation in rats.

    PubMed

    Crossland, Hannah; Constantin-Teodosiu, Dumitru; Gardiner, Sheila M; Greenhaff, Paul L

    2017-07-01

    The peroxisome proliferator-activated receptor γ (PPARγ) agonist rosiglitazone (Rosi) appears to provide protection against organ dysfunction during endotoxaemia. We examined the potential benefits of Rosi on skeletal muscle protein maintenance and carbohydrate metabolism during lipopolysaccharide (LPS)-induced endotoxaemia. Sprague-Dawley rats were fed either standard chow (control) or standard chow containing Rosi (8.5 ± 0.1 mg·kg -1 ·day -1 ) for 2 weeks before and during 24 h continuous intravenous infusion of LPS (15 μg·kg -1 ·h -1 ) or saline. Rosi blunted LPS-induced increases in muscle tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) mRNA by 70% ( P <0.05) and 64% ( P <0.01) respectively. Furthermore, Rosi suppressed the LPS-induced reduction in phosphorylated AKT and phosphorylated Forkhead box O (FOXO) 1 protein, as well as the up-regulation of muscle RING finger 1 (MuRF1; P <0.01) mRNA and the LPS-induced increase in 20S proteasome activity ( P <0.05). Accordingly, LPS reduced the muscle protein:DNA ratio (∼30%, P <0.001), which Rosi offset. Increased muscle pyruvate dehydrogenase kinase 4 (PDK4) mRNA ( P <0.001) and muscle lactate accumulation ( P <0.001) during endotoxaemia were suppressed by Rosi. Thus, pre-treatment with Rosi reduced muscle cytokine accumulation and blunted muscle protein loss and lactate accumulation during endotoxaemia, and at least in part by reducing activation of molecular events known to increase muscle protein breakdown and mitochondrial pyruvate use. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  1. [Bone loss in lactating women and post-pregnancy osteoporosis].

    PubMed

    Hirata, Go; Chaki, Osamu

    2011-09-01

    Measurement of the bone mineral density have shown that lactating women had 1 to 3% decrease in bone mineral density. Post pregnancy osteoporosis is rare condition that causes fragile fracture mostly in vertebrae. The bone loss in lactating women is caused by calcium loss, decrease in estrogen level, and increase in PTHrP (parathyroid hormone related protein) level. Some data have shown that extended lactation and amenorrhea had an association with the degree of bone loss. Mostly, the bone loss of the lactating women recovers to the baseline level, soon after the weaning, and there is no long term effect. Post pregnancy osteoporosis should be concerned, when we see a lactating woman with fragile fracture of the vertebrae.

  2. The importance of alcohol dehydrogenase in regulation of ethanol metabolism in rat liver cells.

    PubMed Central

    Page, R A; Kitson, K E; Hardman, M J

    1991-01-01

    We used titration with the inhibitors tetramethylene sulphoxide and isobutyramide to assess quantitatively the importance of alcohol dehydrogenase in regulation of ethanol oxidation in rat hepatocytes. In hepatocytes isolated from starved rats the apparent Flux Control Coefficient (calculated assuming a single-substrate irreversible reaction with non-competitive inhibition) of alcohol dehydrogenase is 0.3-0.5. Adjustment of this coefficient to allow for alcohol dehydrogenase being a two-substrate reversible enzyme increases the value by 1.3-1.4-fold. The final value of the Flux Control Coefficient of 0.5-0.7 indicates that alcohol dehydrogenase is a major rate-determining enzyme, but that other factors also have a regulatory role. In hepatocytes from fed rats the Flux Control Coefficient for alcohol dehydrogenase decreases with increasing acetaldehyde concentration. This suggests that, as acetaldehyde concentrations rise, control of the pathway shifts from alcohol dehydrogenase to other enzymes, particularly aldehyde dehydrogenase. There is not a single rate-determining step for the ethanol metabolism pathway and control is shared among several steps. PMID:1898355

  3. Proteome analysis of Aspergillus niger: Lactate added in starch-containing medium can increase production of the mycotoxin fumonisin B2 by modifying acetyl-CoA metabolism

    PubMed Central

    2009-01-01

    Background Aspergillus niger is a filamentous fungus found in the environment, on foods and feeds and is used as host for production of organic acids, enzymes and proteins. The mycotoxin fumonisin B2 was recently found to be produced by A. niger and hence very little is known about production and regulation of this metabolite. Proteome analysis was used with the purpose to reveal how fumonisin B2 production by A. niger is influenced by starch and lactate in the medium. Results Fumonisin B2 production by A. niger was significantly increased when lactate and starch were combined in the medium. Production of a few other A. niger secondary metabolites was affected similarly by lactate and starch (fumonisin B4, orlandin, desmethylkotanin and pyranonigrin A), while production of others was not (ochratoxin A, ochratoxin alpha, malformin A, malformin C, kotanin, aurasperone B and tensidol B). The proteome of A. niger was clearly different during growth on media containing 3% starch, 3% starch + 3% lactate or 3% lactate. The identity of 59 spots was obtained, mainly those showing higher or lower expression levels on medium with starch and lactate. Many of them were enzymes in primary metabolism and other processes that affect the intracellular level of acetyl-CoA or NADPH. This included enzymes in the pentose phosphate pathway, pyruvate metabolism, the tricarboxylic acid cycle, ammonium assimilation, fatty acid biosynthesis and oxidative stress protection. Conclusions Lactate added in a medium containing nitrate and starch can increase fumonisin B2 production by A. niger as well as production of some other secondary metabolites. Changes in the balance of intracellular metabolites towards a higher level of carbon passing through acetyl-CoA and a high capacity to regenerate NADPH during growth on medium with starch and lactate were found to be the likely cause of this effect. The results lead to the hypothesis that fumonisin production by A. niger is regulated by acetyl

  4. Effect of various kinds of beverages on stress oxidative, F2 isoprostane, serum lipid and blood glucose of elite taekwondo players.

    PubMed

    Maghsoudi, Zahra; Shiranian, Ashfin; Askai, Gholamreza; Ghaisvand, Reza

    2016-01-01

    Athletes' recovery is important in improving their performance. Nutritional strategies can be effective in enhancing recovery rate. Choosing the best food items in appropriate intervals can play effective roles in resynthesis of fuels and recovery of muscle injury. Beverage micro and macronutrient content are helpful in fuel restoration. In this study, we assess the effects of various kinds of beverages on oxidative stress, muscle injury, and metabolic risk factors in taekwondo players. This quasi-experimental study was performed on 21 taekwondo players of Isfahan. After collecting fasting blood, they performed runningbased anaerobic sprint test (RAST). Blood lactate was tested again and participants were divided into 3 intervention groups, that is, receiving 500 cc dough, non-alcoholic beer, and chocolate milk at 4 day intervals. After a 2-h recovery period, blood sampling was repeated. Elites consumed other beverages in later phases. Dietary intake and fasting triglyceride, cholesterol, blood sugar, lactate dehydrogenase, and F 2 -isoprostane concentrations were determined. Data were analyzed with a simple repeated-measures test and post-hoc tests using the Statistical Package for the Social Sciences software. Data showed that cholesterol levels non-significantly decreased after intervention. Triglyceride level was lower after taking dough and carbohydrate replacement drink. Blood glucose concentration increased after intervention periods, however, this increase was significant only after non-alcoholic beverage consumption. Lactate dehydrogenase levels reduced after all cycles, however, F 2 -isoprostane level showed no significant change. There was not significant change in lactate dehydrogenase and F 2 -isoprostane levels. Non-alcoholic beer consumption can reduce lactate dehydrogenase concentration; however, it leads to blood sugar increase. Moreover, dough consumption significantly reduced triglyceride level in taekwondo players.

  5. Lactate Profile During Greco-Roman Wrestling Matchx

    PubMed Central

    Karnincic, Hrvoje; Tocilj, Zoran; Uljevic, Ognjen; Erceg, Marko

    2009-01-01

    The objective of this study was to determine and compare lactate profile of two groups of Greco-Roman wrestlers with different competences and training experience. Study was conducted on 10 wrestles that were members of Croatian national team and 10 wrestlers that were members of Wrestling club Split. Lactate samples were collected at four intervals during control fights that were held according to international wrestling rules of World wrestling federation FILA. Values of lactate increased as competition progressed, and they were highest at the end of the match for both groups of wrestlers. According to this study there were no significant differences in lactate between two groups at the end of the match, while significant differences were noted during the match. The information about lactate profile presented in this study can be used by coaches and wrestlers to develop condition programs. Key Points There were no significant differences in lactate concentrations at the end of the match between two proficiency levels of wrestlers. More proficient (elite) wrestlers raise lactates gradually through the wrestling match while less proficient (club) wrestlers raise it abruptly at the end of the first bout. Both groups of wrestlers are unable to sustain same level of activity through the match suggesting that they are utilizing too much energy from anaerobic glycolysis. PMID:24474881

  6. Associations of insulin resistance later in lactation on fertility of dairy cows.

    PubMed

    Baruselli, P S; Vieira, L M; Sá Filho, M F; Mingoti, R D; Ferreira, R M; Chiaratti, M R; Oliveira, L H; Sales, J N; Sartori, R

    2016-07-01

    The challenge of getting dairy cows pregnant during early lactation is a well-described, worldwide problem. However, specifically in farms with poor reproductive, nutritional, and environmental conditions/management, a low pregnancy rate during early lactation is followed inevitably by an increased number of nonpregnant cows after 150 days in milk, with even more difficulties to achieve pregnancy. Therefore, several studies were designed to understand and develop strategies to mitigate reduced fertility of cows during late lactation. Experiments were performed under tropical regions to determine metabolic status during lactation and association of stage of lactation on oocyte quality and fertility. Lactating cows with extended days not pregnant (e.g.,>150 days in milk) often had systemic metabolic alterations, including development of peripheral insulin resistance and various oocyte alterations, including reduced expression of genes encoding glucose transport proteins, reduced amounts of mtDNA, increased expression of mitochondria-related genes, and increased expression of apoptosis-related genes. Additionally, in vitro embryo production and pregnancy per AI were lower in late- versus early-lactation cows in some but not all studies. Notwithstanding, when a normal embryo was transferred to a cow in late lactation, the pregnancy per transfer was reasonable, reinforcing the assertion that fertility problems in late-lactation cows may be associated with oocyte quality, fertilization, and/or failure of early embryo development. In conclusion, insulin resistance may reduce oocyte competence and consequently fertility in late-lactation dairy cows. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Prevalence and incidence of intramammary infections in lactating dairy goats.

    PubMed

    McDougall, S; Malcolm, D; Prosser, Cg

    2014-05-01

    To determine the prevalence of intramammary infection (IMI) of lactating dairy goats between 0 and 4 days postpartum, the prevalence and incidence rate of new IMI at Weeks 2, 14 and 27 of lactation, and the relationship between herd-level prevalence of IMI and bulk tank somatic cell count (BTSCC). Milk samples were collected from 8% of a herd (total 624 does) from 18 dairy goat herds in the Waikato region of New Zealand, for bacteriology and somatic cell count (SCC) determination, from both glands within 4 days of kidding (Week 0) and again at Weeks 2, 14 and 27. Prevalence of IMI was determined at each time point and incidence rate calculated for Weeks 0-2, 2-14, and 14-27. Greenwood and Reed-Frost models were compared for estimation of the transmission parameter for all pathogens, and for Staphylococcus aureus, coagulase negative staphylococci (CNS) and Corynebacterium spp. separately. Bacteria were isolated from 1,122/4,814 (23.3%) glands, with CNS (13.4%) and Corynebacterium spp. (7.3%) being the most common isolates. Prevalence of any IMI increased with stage of lactation, varied among herds, and increased with age (all p<0.05). Incidence rate was 80, 24 and 7 new IMI/10,000 gland days for Weeks 0-2, 2-14 and 14-27, respectively. Incidence rate for any IMI increased with age and with the presence of an IMI in the contralateral gland, and varied among herds (p<0.001). The transmission of each pathogen was better modelled assuming contagiousness (Reed-Frost models), than not (Greenwood models). At gland level, IMI increased SCC at all stages of lactation (p<0.001). The gland prevalence of IMI within herds was positively associated with ln BTSCC at Week 2 (p=0.02), but not Weeks 14 or 27 (p>0.05). Prevalence of IMI increased with stage of lactation, but the highest incidence rate of new IMI occurred in early lactation. Models accounting for the contagious nature of infection fitted better than those not accounting for contagiousness. BTSCC was only associated

  8. Multiple alcohol dehydrogenases but no functional acetaldehyde dehydrogenase causing excessive acetaldehyde production from ethanol by oral streptococci.

    PubMed

    Pavlova, Sylvia I; Jin, Ling; Gasparovich, Stephen R; Tao, Lin

    2013-07-01

    Ethanol consumption and poor oral hygiene are risk factors for oral and oesophageal cancers. Although oral streptococci have been found to produce excessive acetaldehyde from ethanol, little is known about the mechanism by which this carcinogen is produced. By screening 52 strains of diverse oral streptococcal species, we identified Streptococcus gordonii V2016 that produced the most acetaldehyde from ethanol. We then constructed gene deletion mutants in this strain and analysed them for alcohol and acetaldehyde dehydrogenases by zymograms. The results showed that S. gordonii V2016 expressed three primary alcohol dehydrogenases, AdhA, AdhB and AdhE, which all oxidize ethanol to acetaldehyde, but their preferred substrates were 1-propanol, 1-butanol and ethanol, respectively. Two additional dehydrogenases, S-AdhA and TdhA, were identified with specificities to the secondary alcohol 2-propanol and threonine, respectively, but not to ethanol. S. gordonii V2016 did not show a detectable acetaldehyde dehydrogenase even though its adhE gene encodes a putative bifunctional acetaldehyde/alcohol dehydrogenase. Mutants with adhE deletion showed greater tolerance to ethanol in comparison with the wild-type and mutant with adhA or adhB deletion, indicating that AdhE is the major alcohol dehydrogenase in S. gordonii. Analysis of 19 additional strains of S. gordonii, S. mitis, S. oralis, S. salivarius and S. sanguinis showed expressions of up to three alcohol dehydrogenases, but none showed detectable acetaldehyde dehydrogenase, except one strain that showed a novel ALDH. Therefore, expression of multiple alcohol dehydrogenases but no functional acetaldehyde dehydrogenase may contribute to excessive production of acetaldehyde from ethanol by certain oral streptococci.

  9. Multiple alcohol dehydrogenases but no functional acetaldehyde dehydrogenase causing excessive acetaldehyde production from ethanol by oral streptococci

    PubMed Central

    Pavlova, Sylvia I.; Jin, Ling; Gasparovich, Stephen R.

    2013-01-01

    Ethanol consumption and poor oral hygiene are risk factors for oral and oesophageal cancers. Although oral streptococci have been found to produce excessive acetaldehyde from ethanol, little is known about the mechanism by which this carcinogen is produced. By screening 52 strains of diverse oral streptococcal species, we identified Streptococcus gordonii V2016 that produced the most acetaldehyde from ethanol. We then constructed gene deletion mutants in this strain and analysed them for alcohol and acetaldehyde dehydrogenases by zymograms. The results showed that S. gordonii V2016 expressed three primary alcohol dehydrogenases, AdhA, AdhB and AdhE, which all oxidize ethanol to acetaldehyde, but their preferred substrates were 1-propanol, 1-butanol and ethanol, respectively. Two additional dehydrogenases, S-AdhA and TdhA, were identified with specificities to the secondary alcohol 2-propanol and threonine, respectively, but not to ethanol. S. gordonii V2016 did not show a detectable acetaldehyde dehydrogenase even though its adhE gene encodes a putative bifunctional acetaldehyde/alcohol dehydrogenase. Mutants with adhE deletion showed greater tolerance to ethanol in comparison with the wild-type and mutant with adhA or adhB deletion, indicating that AdhE is the major alcohol dehydrogenase in S. gordonii. Analysis of 19 additional strains of S. gordonii, S. mitis, S. oralis, S. salivarius and S. sanguinis showed expressions of up to three alcohol dehydrogenases, but none showed detectable acetaldehyde dehydrogenase, except one strain that showed a novel ALDH. Therefore, expression of multiple alcohol dehydrogenases but no functional acetaldehyde dehydrogenase may contribute to excessive production of acetaldehyde from ethanol by certain oral streptococci. PMID:23637459

  10. Channel-mediated lactate release by K⁺-stimulated astrocytes.

    PubMed

    Sotelo-Hitschfeld, Tamara; Niemeyer, María I; Mächler, Philipp; Ruminot, Iván; Lerchundi, Rodrigo; Wyss, Matthias T; Stobart, Jillian; Fernández-Moncada, Ignacio; Valdebenito, Rocío; Garrido-Gerter, Pamela; Contreras-Baeza, Yasna; Schneider, Bernard L; Aebischer, Patrick; Lengacher, Sylvain; San Martín, Alejandro; Le Douce, Juliette; Bonvento, Gilles; Magistretti, Pierre J; Sepúlveda, Francisco V; Weber, Bruno; Barros, L Felipe

    2015-03-11

    Excitatory synaptic transmission is accompanied by a local surge in interstitial lactate that occurs despite adequate oxygen availability, a puzzling phenomenon termed aerobic glycolysis. In addition to its role as an energy substrate, recent studies have shown that lactate modulates neuronal excitability acting through various targets, including NMDA receptors and G-protein-coupled receptors specific for lactate, but little is known about the cellular and molecular mechanisms responsible for the increase in interstitial lactate. Using a panel of genetically encoded fluorescence nanosensors for energy metabolites, we show here that mouse astrocytes in culture, in cortical slices, and in vivo maintain a steady-state reservoir of lactate. The reservoir was released to the extracellular space immediately after exposure of astrocytes to a physiological rise in extracellular K(+) or cell depolarization. Cell-attached patch-clamp analysis of cultured astrocytes revealed a 37 pS lactate-permeable ion channel activated by cell depolarization. The channel was modulated by lactate itself, resulting in a positive feedback loop for lactate release. A rapid fall in intracellular lactate levels was also observed in cortical astrocytes of anesthetized mice in response to local field stimulation. The existence of an astrocytic lactate reservoir and its quick mobilization via an ion channel in response to a neuronal cue provides fresh support to lactate roles in neuronal fueling and in gliotransmission. Copyright © 2015 the authors 0270-6474/15/354168-11$15.00/0.

  11. Fabrication of Flexible Arrayed Lactate Biosensor Based on Immobilizing LDH-NAD⁺ on NiO Film Modified by GO and MBs.

    PubMed

    Chou, Jung-Chuan; Yan, Siao-Jie; Liao, Yi-Hung; Lai, Chih-Hsien; Wu, You-Xiang; Wu, Cian-Yi; Chen, Hsiang-Yi; Huang, Hong-Yu; Wu, Tong-Yu

    2017-07-12

    We proposed the flexible arrayed lactate biosensor based on immobilizing l-lactate dehydrogenase (LDH) and nicotinamide adenine dinucleotide ( NAD + ) on nickel oxide (NiO) film, and which the average sensitivity could be enhanced by using graphene oxide (GO) and magnetic beads (MBs). By using GO and MBs, it exhibits excellent sensitivity (45.397 mV/mM) with a linearity of 0.992 in a range of 0.2 mM to 3 mM. According to the results of electrochemical impedance spectroscopy (EIS), the electron transfer resistance of LDH- NAD + -MBs/GPTS/GO/NiO film was smaller than those of LDH-NAD⁺/GPTS/GO/NiO film and LDH- NAD + /GPTS/NiO film, and it presented the outstanding electron transfer ability. After that, the limit of detection, anti-interference effect and bending test were also investigated.

  12. FGF-21 and skeletal remodeling during and after lactation in C57BL/6J mice.

    PubMed

    Bornstein, Sheila; Brown, Sue A; Le, Phuong T; Wang, Xunde; DeMambro, Victoria; Horowitz, Mark C; MacDougald, Ormond; Baron, Roland; Lotinun, Sutada; Karsenty, Gerard; Wei, Wei; Ferron, Mathieu; Kovacs, Christopher S; Clemmons, David; Wan, Yihong; Rosen, Clifford J

    2014-09-01

    Lactation is associated with significant alterations in both body composition and bone mass. Systemic and local skeletal factors such as receptor activator of nuclear factor κ-B ligand (RANKL), PTHrP, calcitonin, and estrogen are known to regulate bone remodeling during and after lactation. Fibroblast growth factor 21 (FGF-21) may function as an endocrine factor to regulate body composition changes during lactation by inducing gluconeogenesis and fatty acid oxidation. In this study, we hypothesized that the metabolic changes during lactation were due in part to increased circulating FGF-21, which in turn could accentuate bone loss. We longitudinally characterized body composition in C57BL/6J (B6) mice during (day 7 and day 21 of lactation) and after normal lactation (day 21 postlactation). At day 7 of lactation, areal bone density declined by 10% (P < .001), bone resorption increased (P < .0001), percent fat decreased by 20%, energy expenditure increased (P < .01), and markers of brown-like adipogenesis were suppressed in the inguinal depot and in preformed brown adipose tissue. At day 7 of lactation there was a 2.4-fold increase in serum FGF-21 vs baseline (P < .0001), a 8-fold increase in hepatic FGF-21 mRNA (P < .03), a 2-fold increase in undercarboxylated osteocalcin (Glu13 OCn) (P < .01), and enhanced insulin sensitivity. Recovery of total areal bone density was noted at day 21 of lactation, whereas the femoral trabecular bone volume fraction was still reduced (P < .01). Because FGF-21 levels rose rapidly at day 7 of lactation in B6 lactating mice, we next examined lactating mice with a deletion in the Fgf21 gene. Trabecular and cortical bone masses were maintained throughout lactation in FGF-21(-/-) mice, and pup growth was normal. Compared with lactating control mice, lactating FGF-21(-/-) mice exhibited an increase in bone formation, but no change in bone resorption. In conclusion, in addition to changes in calciotropic hormones, systemic FGF-21 plays a

  13. Inhibition effects of furfural on alcohol dehydrogenase, aldehyde dehydrogenase and pyruvate dehydrogenase.

    PubMed Central

    Modig, Tobias; Lidén, Gunnar; Taherzadeh, Mohammad J

    2002-01-01

    The kinetics of furfural inhibition of the enzymes alcohol dehydrogenase (ADH; EC 1.1.1.1), aldehyde dehydrogenase (AlDH; EC 1.2.1.5) and the pyruvate dehydrogenase (PDH) complex were studied in vitro. At a concentration of less than 2 mM furfural was found to decrease the activity of both PDH and AlDH by more than 90%, whereas the ADH activity decreased by less than 20% at the same concentration. Furfural inhibition of ADH and AlDH activities could be described well by a competitive inhibition model, whereas the inhibition of PDH was best described as non-competitive. The estimated K(m) value of AlDH for furfural was found to be about 5 microM, which was lower than that for acetaldehyde (10 microM). For ADH, however, the estimated K(m) value for furfural (1.2 mM) was higher than that for acetaldehyde (0.4 mM). The inhibition of the three enzymes by 5-hydroxymethylfurfural (HMF) was also measured. The inhibition caused by HMF of ADH was very similar to that caused by furfural. However, HMF did not inhibit either AlDH or PDH as severely as furfural. The inhibition effects on the three enzymes could well explain previously reported in vivo effects caused by furfural and HMF on the overall metabolism of Saccharomyces cerevisiae, suggesting a critical role of these enzymes in the observed inhibition. PMID:11964178

  14. Residual hemothorax after chest tube placement correlates with increased risk of empyema following traumatic injury

    PubMed Central

    Karmy-Jones, Riyad; Holevar, Michele; Sullivan, Ryan J; Fleisig, Ani; Jurkovich, Gregory J

    2008-01-01

    BACKGROUND: Empyema complicates tube thoracostomy following trauma in up to 10% of cases. Studies of potential risk factors of empyema have included use of antibiotics, site of injury and technique of chest tube placement. Residual fluid has also been cited as a risk factor for empyema, although the imaging technique to identify this varies. OBJECTIVE: To determine whether residual hemothorax detected by chest x-ray (CXR) after one or more initial chest tubes predicts an increased risk of empyema. METHODS: A study of patients admitted to two level I trauma centres between January 7, 2004, and December 31, 2004, was conducted. All patients who received a chest tube in the emergency department, did not undergo thoracotomy within 24 h, and survived more than two days were followed. Empyema was defined as a pleural effusion with positive cultures, and a ratio of pleural fluid lactate dehydrogenase to serum lactate dehydrogenase greater than 0.6 in the setting of elevated leukocyte count and fever. Factors analyzed included the presence of retained hemothorax on CXR after the most recent tube placement in the emergency room, age, mechanism of injury and injury severity score. RESULTS: A total of 102 patients met the criteria. Nine patients (9%) developed empyema: seven of 21 patients (33%) with residual hemothorax developed empyema versus two of 81 patients (2%) without residual hemothorax developed empyema (P=0.001). Injury severity score was significantly higher in those who developed empyema (31.4±26) versus those who did not (22.6±13; P=0.03). CONCLUSIONS: The presence of residual hemothorax detected by CXR after tube thoracostomy should prompt further efforts, including thoracoscopy, to drain it. With increasing injury severity, there may be increased benefit in terms of reducing empyema with this approach. PMID:18716687

  15. No change in energy efficiency in lactation: Insights from a longitudinal study.

    PubMed

    Bender, Richard L; Williams, Heather S; Dufour, Darna L

    2017-11-01

    Lactation is the most energy-demanding phase of reproduction for human females, but it is still unclear how women in different environments are able to meet this additional energy demand. Previous studies have investigated whether changes in metabolism could have an energy-sparing effect in lactation, with conflicting results. Here, we asked whether increased energy efficiency in physical activity serves as an energy-sparing mechanism in lactation. We used a longitudinal design with a control group. Participants were 33 well-nourished, exclusively breastfeeding women and 29 non-pregnant, non-lactating (NPNL) controls aged 32 ± 4 years. Lactating women were measured at peak- and post-lactation. NPNL controls completed a baseline measurement and a follow-up visit. Energy efficiency in physical activity was assessed using a graded submaximal exercise test and calculated as delta efficiency (change in work accomplished over change in energy expended) and gross efficiency (work accomplished over energy expended). There was no significant change in either delta efficiency or gross efficiency from peak to post lactation in lactating women, and no significant difference in delta efficiency between lactating women and NPNL controls at any time period. However, lactating women showed greater between-visit variation in delta efficiency than the NPNL controls. Additionally, 79% of lactating participants lost weight between visits (mean weight loss -3.6 ± 2.3kg), consistent with a mobilization of body tissues to support lactation. We found no support for the idea that lactating women undergo an increase in energy efficiency to support the energy costs of lactation. © 2017 Wiley Periodicals, Inc.

  16. Increased resistance to oxidative stress in normal and glucose-6-phosphate dehydrogenase-deficient hemolysates in the presence of enzyme substrates.

    PubMed

    Yücel, G; Yeşilkaya, A; Aksu, T A; Yeğin, A; Alicigüzel, Y

    1997-01-01

    Erythrocytes and hemolysates from 10 normal and 10 glucose-6-phosphate dehydrogenase-deficient individuals were incubated with cumene hydroperoxide, and free radical-induced lipid peroxidation was monitored by chemiluminescence. Chemiluminescence intensities in erythrocytes of normal and deficient subjects were similar in the presence or absence of glucose-6-phosphate dehydrogenase substrates. Hemolysates of normal and deficient subjects also showed similar chemiluminescence in the absence of substrates. However, with the addition of substrates to the incubation medium, deficient hemolysates reached maximum chemiluminescence intensity within a shorter period, and maximum values were higher than in normal hemolysates. We believe this offers a new means of detection of glucose-6-phosphate dehydrogenase-deficient patients.

  17. Maternal Zinc Intakes and Homeostatic Adjustments during Pregnancy and Lactation

    PubMed Central

    Donangelo, Carmen Marino; King, Janet C.

    2012-01-01

    Zinc plays critical roles during embryogenesis, fetal growth, and milk secretion, which increase the zinc need for pregnancy and lactation. Increased needs can be met by increasing the dietary zinc intake, along with making homeostatic adjustments in zinc utilization. Potential homeostatic adjustments include changes in circulating zinc, increased zinc absorption, decreased zinc losses, and changes in whole body zinc kinetics. Although severe zinc deficiency during pregnancy has devastating effects, systematic reviews and meta-analysis of the effect of maternal zinc supplementation on pregnancy outcomes have consistently shown a limited benefit. We hypothesize, therefore, that zinc homeostatic adjustments during pregnancy and lactation improve zinc utilization sufficiently to provide the increased zinc needs in these stages and, therefore, mitigate immediate detrimental effects due to a low zinc intake. The specific questions addressed are the following: How is zinc utilization altered during pregnancy and lactation? Are those homeostatic adjustments influenced by maternal zinc status, dietary zinc, or zinc supplementation? These questions are addressed by critically reviewing results from published human studies on zinc homeostasis during pregnancy and lactation carried out in different populations worldwide. PMID:22852063

  18. Rhabdomyolysis and Artifactual Increase in Plasma Bicarbonate Concentration in an Amazon Parrot (Amazona species).

    PubMed

    Leissinger, Mary K; Johnson, James G; Tully, Thomas N; Gaunt, Stephen D

    2017-09-01

    A 7-year-old male Amazon parrot housed outdoors presented with acute collapse, marked lethargy, and open-mouth breathing. The patient had stiffness of the pectoral muscles, and petechiation and ecchymosis noted around the eyes and beneath the mandible. Laboratory data revealed markedly increased aspartate aminotransferase, creatine kinase, and lactate dehydrogenase activity consistent with rhabdomyolysis, as well as markedly increased plasma bicarbonate concentration. Marked clinical improvement and resolution of laboratory abnormalities occurred with fluid therapy, administration of a nonsteroidal anti-inflammatory medication, and husbandry modifications, including indoor housing and dietary alteration. A spurious increase in bicarbonate measurement as documented in equine and bovine cases of rhabdomyolysis also occurred in this avian patient and must be considered for accurate interpretation of acid-base status in exotic species presenting with consistent clinical signs.

  19. Clinical value of jointly detection serum lactate dehydrogenase/pleural fluid adenosine deaminase and pleural fluid carcinoembryonic antigen in the identification of malignant pleural effusion.

    PubMed

    Zhang, Fan; Hu, Lijuan; Wang, Junjun; Chen, Jian; Chen, Jie; Wang, Yumin

    2017-09-01

    Limited data are available for the diagnostic value, and for the diagnostic sensitivity and specificity of joint detection of serum lactate dehydrogenase (sLDH)/pleural fluid adenosine deaminase (pADA) and pleural fluid carcinoembryonic antigen (pCEA) in malignant pleural effusion (MPE). We collected 987 pleural effusion specimens (of which 318 were malignant pleural effusion, 374 were tubercular pleural effusion, and 295 were parapneumonic effusion specimens) from the First Affiliated Hospital of Wenzhou Medical University from July 2012 to March 2016. The pADA, sLDH, pleural fluid LDH (pLDH), serum C-reactive protein (sCRP), pleural fluid protein, pCEA, white blood cell (WBC), and red blood cell (RBC) were analyzed, and the clinical data of each group were collected for statistical analysis. The level of sLDH/pADA, pCEA, and RBC from the MPE group was markedly higher than the tuberculosis pleural effusion (TB) group (Mann-Whitney U=28422.000, 9278.000, 30518, P=.000, .000, .000) and the parapneumonic pleural fluid group (Mann-Whitney U=5972.500, 7113.000, 36750.500, P=.000, .000, .000). The receiver operating characteristic curve ROC showed that the area under the ROC curve (AUC) (=0.924, 0.841) of pCEA and sLDH/pADA (cutoff=4.9, 10.6) were significantly higher than other markers for the diagnosis of MPE. Thus, joint detection of pCEA and sLDH/pADA suggested that the sensitivity, specificity, and AUC was 0.94, 81.70, and 94.32 at the cutoff 0.16 and diagnostic performance was higher than pCEA or sLDH/pADA. Joint detection of sLDH/pADA and pCEA can be used as a good indicator for the identification of benign and MPE with higher sensitivity and specificity than pCEA or sLDH/pADA. © 2016 Wiley Periodicals, Inc.

  20. From gene to structure: Lactobacillus bulgaricus D-lactate dehydrogenase from yogurt as an integrated curriculum model for undergraduate molecular biology and biochemistry laboratory courses.

    PubMed

    Lawton, Jeffrey A; Prescott, Noelle A; Lawton, Ping X

    2018-05-01

    We have developed an integrated, project-oriented curriculum for undergraduate molecular biology and biochemistry laboratory courses spanning two semesters that is organized around the ldhA gene from the yogurt-fermenting bacterium Lactobacillus bulgaricus, which encodes the enzyme d-lactate dehydrogenase. The molecular biology module, which consists of nine experiments carried out over eleven sessions, begins with the isolation of genomic DNA from L. bulgaricus in yogurt and guides students through the process of cloning the ldhA gene into a prokaryotic expression vector, followed by mRNA isolation and characterization of recombinant gene expression levels using RT-PCR. The biochemistry module, which consists of nine experiments carried out over eight sessions, begins with overexpression of the cloned ldhA gene and guides students through the process of affinity purification, biochemical characterization of the purified LdhA protein, and analysis of enzyme kinetics using various substrates and an inhibitor, concluding with a guided inquiry investigation of structure-function relationships in the three-dimensional structure of LdhA using molecular visualization software. Students conclude by writing a paper describing their work on the project, formatted as a manuscript to be submitted for publication in a scientific journal. Overall, this curriculum, with its emphasis on experiential learning, provides hands-on training with a variety of common laboratory techniques in molecular biology and biochemistry and builds experience with the process of scientific reasoning, along with reinforcement of essential transferrable skills such as critical thinking, information literacy, and written communication, all within the framework of an extended project having the look and feel of a research experience. © 2018 by The International Union of Biochemistry and Molecular Biology, 46(3):270-278, 2018. © 2018 The International Union of Biochemistry and Molecular Biology.

  1. Conformational heterogeneity within the Michaelis complex of lactate dehydrogenase†

    PubMed Central

    Deng, Hua; Vu, Dung V.; Clinch, Keith; Desamero, Ruel; Dyer, R. Brian; Callender, Robert

    2011-01-01

    A series of isotope edited IR measurements, both static as well as temperature jump relaxation spectroscopy, are performed on lactate dehydrogenase (LDH) to determine the ensemble of structures available to its Michaelis complex. There clearly has been a substantial reduction in the number of states available to the pyruvate substrate (as modeled by the substrate mimic, oxamate) and NADH when bound to protein compared to dissolved in solution, as determined by the bandwidths and positions of the critical C2=O band of bound substrate mimic and the C4-H stretch of NADH reduced nicotinamide group. Moreover, it is found that a strong ionic bond (characterized by a signature IR band discovered in this study) is formed between the carboxyl group of bound pyruvate with (presumably) Arg171, forming a strong ‘anchor’ within the protein matrix. However, conformational heterogeneity within the Michaelis complex is found that has an impact on both catalytic efficiency and thermodynamics of the enzyme. PMID:21568287

  2. Guinea-pig liver testosterone 17 beta-dehydrogenase (NADP+) and aldehyde reductase exhibit benzene dihydrodiol dehydrogenase activity.

    PubMed Central

    Hara, A; Hayashibara, M; Nakayama, T; Hasebe, K; Usui, S; Sawada, H

    1985-01-01

    We have kinetically and immunologically demonstrated that testosterone 17 beta-dehydrogenase (NADP+) isoenzymes (EC 1.1.1.64) and aldehyde reductase (EC 1.1.1.2) from guinea-pig liver catalyse the oxidation of benzene dihydrodiol (trans-1,2-dihydroxycyclohexa-3,5-diene) to catechol. One isoenzyme of testosterone 17 beta-dehydrogenase, which has specificity for 5 beta-androstanes, oxidized benzene dihydrodiol at a 3-fold higher rate than 5 beta-dihydrotestosterone, and showed a more than 4-fold higher affinity for benzene dihydrodiol and Vmax. value than did another isoenzyme, which exhibits specificity for 5 alpha-androstanes, and aldehyde reductase. Immunoprecipitation of guinea-pig liver cytosol with antisera against the testosterone 17 beta-dehydrogenase isoenzymes and aldehyde reductase indicated that most of the benzene dihydrodiol dehydrogenase activity in the tissue is due to testosterone 17 beta-dehydrogenase. PMID:2983661

  3. Amylase addition increases starch ruminal digestion in first-lactation cows fed high and low starch diets.

    PubMed

    Nozière, P; Steinberg, W; Silberberg, M; Morgavi, D P

    2014-01-01

    The objective of this study was to evaluate the effect of an exogenous amylase preparation on digestion of low- and high-starch diets in dairy cattle. Rumen and total-tract nutrient digestibility were measured in a 4×4 Latin square design with 28-d periods using 4 first-lactation cows cannulated at the rumen and duodenum. Corn silage-based diets had 20 or 30% starch, attained by changing the composition of concentrate, with or without addition of an exogenous amylase preparation. Effects of the enzyme additive were observed on ruminal digestibility but not at the total-tract level. Ruminal digestibility of starch increased from 75% in control to 81% with amylase supplementation. This difference in ruminal starch digestion was compensated postruminally, so that the total-tract digestibility of starch was almost complete and did not differ between treatments. The amylase supplement also increased the true ruminal digestibility of organic matter but did not affect microbial N flow to the duodenum. Amylase supplement reduced the proportion of acetate and butyrate and increased that of propionate, particularly in the high-starch diet, where it tended to increase the concentration of total volatile fatty acids in the rumen. Other effects were a higher amylase activity in the solid-associated microbial community and a tendency for lower numbers of protozoa. In contrast, we observed no changes in intake, production, dry matter and fiber (neutral detergent fiber and acid detergent fiber) digestibility, or ruminal digestion, and no or small changes on selected fibrolytic and amylolytic bacteria and on the microbial community in general. We conclude that the exogenous amylase improved starch digestion in the rumen in first-lactation cows with moderate intake and production levels. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  4. Temporal dynamics of lactate concentration in the human brain during acute inspiratory hypoxia

    PubMed Central

    Harris, Ashley D; Roberton, Victoria H; Huckle, Danielle L; Saxena, Neeraj; Evans, C John; Murphy, Kevin; Hall, Judith E; Bailey, Damian M; Mitsis, Georgios; Edden, Richard A E; Wise, Richard G

    2012-01-01

    Purpose To demonstrate the feasibility of measuring the temporal dynamics of cerebral lactate concentration and examine these dynamics in human subjects using MRS during hypoxia. Methods A respiratory protocol consisting of 10 min baseline normoxia, 20 min inspiratory hypoxia and ending with 10 min normoxic recovery was used, throughout which lactate-edited MRS was performed. This was repeated four times in three subjects. A separate session was performed to measure blood lactate. Impulse response functions using end-tidal oxygen and blood lactate as system inputs and cerebral lactate as the system output were examined to describe the dynamics of the cerebral lactate response to a hypoxic challenge. Results The average lactate increase was 20%±15% during the last half of the hypoxic challenge. Significant changes in cerebral lactate concentration were observed after 400s. The average relative increase in blood lactate was 188%±95%. The temporal dynamics of cerebral lactate concentration was reproducibly demonstrated with 200s time bins of MRS data (coefficient of variation 0.063±0.035 between time bins in normoxia). The across subject coefficient of variation was 0.333. Conclusions The methods for measuring the dynamics of the cerebral lactate response developed here would be useful to further investigate the brain’s response to hypoxia. PMID:23197421

  5. Rise in plasma lactate concentrations with psychosocial stress: a possible sign of cerebral energy demand.

    PubMed

    Kubera, Britta; Hubold, Christian; Otte, Saskia; Lindenberg, Ann-Sophie; Zeiss, Irena; Krause, Regina; Steinkamp, Mirja; Klement, Johanna; Entringer, Sonja; Pellerin, Luc; Peters, Achim

    2012-01-01

    It is known that exogenous lactate given as an i.v. energy infusion is able to counteract a neuroglycopenic state that developed during psychosocial stress. It is unknown, however, whether the brain under stressful conditions can induce a rise in plasma lactate to satisfy its increased needs during stress. Since lactate is i) an alternative cerebral energy substrate to glucose and ii) its plasmatic concentration is influenced by the sympathetic nervous system, the present study aimed at investigating whether plasma lactate concentrations increase with psychosocial stress in humans. 30 healthy young men participated in two sessions (stress induced by the Trier Social Stress Test and a non-stress control session). Blood samples were frequently taken to assess plasma lactate concentrations and stress hormone profiles. Plasma lactate increased 47% during psychosocial stress (from 0.9 ± 0.05 to 1.4 ± 0.1 mmol/l; interaction time × stress intervention: F = 19.7, p < 0.001). This increase in lactate concentrations during stress was associated with an increase in epinephrine (R(2) = 0.221, p = 0.02) and ACTH concentrations (R(2) = 0.460, p < 0.001). Plasma lactate concentrations increase during acute psychosocial stress in humans. This finding suggests the existence of a demand mechanism that functions to allocate an additional source of energy from the body towards the brain, which we refer to as 'cerebral lactate demand'.

  6. Evaluation of three parasite lactate dehydrogenase-based rapid diagnostic tests for the diagnosis of falciparum and vivax malaria

    PubMed Central

    Ashley, Elizabeth A; Touabi, Malek; Ahrer, Margareta; Hutagalung, Robert; Htun, Khayae; Luchavez, Jennifer; Dureza, Christine; Proux, Stephane; Leimanis, Mara; Lwin, Myo Min; Koscalova, Alena; Comte, Eric; Hamade, Prudence; Page, Anne-Laure; Nosten, François; Guerin, Philippe J

    2009-01-01

    Background In areas where non-falciparum malaria is common rapid diagnostic tests (RDTs) capable of distinguishing malaria species reliably are needed. Such tests are often based on the detection of parasite lactate dehydrogenase (pLDH). Methods In Dawei, southern Myanmar, three pLDH based RDTs (CareStart™ Malaria pLDH (Pan), CareStart™ Malaria pLDH (Pan, Pf) and OptiMAL-IT®)were evaluated in patients presenting with clinically suspected malaria. Each RDT was read independently by two readers. A subset of patients with microscopically confirmed malaria had their RDTs repeated on days 2, 7 and then weekly until negative. At the end of the study, samples of study batches were sent for heat stability testing. Results Between August and November 2007, 1004 patients aged between 1 and 93 years were enrolled in the study. Slide microscopy (the reference standard) diagnosed 213 Plasmodium vivax (Pv) monoinfections, 98 Plasmodium falciparum (Pf) mono-infections and no malaria in 650 cases. The sensitivities (sens) and specificities (spec), of the RDTs for the detection of malaria were- CareStart Malaria™ pLDH (Pan) test: sens 89.1% [CI95 84.2-92.6], spec 97.6% [CI95 96.5-98.4] OptiMal-IT®: Pf+/- other species detection: sens 95.2% [CI95 87.5-98.2], spec 94.7% [CI95 93.3-95.8]; non-Pf detection alone: sens 89.6% [CI95 83.6-93.6], spec 96.5% [CI95 94.8-97.7] CareStart Malaria™ pLDH (Pan, Pf): Pf+/- other species: sens 93.5% [CI9585.4-97.3], spec 97.4% [95.9-98.3]; non-Pf: sens 78.5% [CI9571.1-84.4], spec 97.8% [CI95 96.3-98.7] Inter-observer agreement was excellent for all tests (kappa > 0.9). The median time for the RDTs to become negative was two days for the CareStart™ Malaria tests and seven days for OptiMAL-IT®. Tests were heat stable up to 90 days except for OptiMAL-IT® (Pf specific pLDH stable to day 20 at 35°C). Conclusion None of the pLDH-based RDTs evaluated was able to detect non-falciparum malaria with high sensitivity, particularly at low

  7. Evaluation of three parasite lactate dehydrogenase-based rapid diagnostic tests for the diagnosis of falciparum and vivax malaria.

    PubMed

    Ashley, Elizabeth A; Touabi, Malek; Ahrer, Margareta; Hutagalung, Robert; Htun, Khayae; Luchavez, Jennifer; Dureza, Christine; Proux, Stephane; Leimanis, Mara; Lwin, Myo Min; Koscalova, Alena; Comte, Eric; Hamade, Prudence; Page, Anne-Laure; Nosten, François; Guerin, Philippe J

    2009-10-27

    In areas where non-falciparum malaria is common rapid diagnostic tests (RDTs) capable of distinguishing malaria species reliably are needed. Such tests are often based on the detection of parasite lactate dehydrogenase (pLDH). In Dawei, southern Myanmar, three pLDH based RDTs (CareStart Malaria pLDH (Pan), CareStart Malaria pLDH (Pan, Pf) and OptiMAL-IT)were evaluated in patients presenting with clinically suspected malaria. Each RDT was read independently by two readers. A subset of patients with microscopically confirmed malaria had their RDTs repeated on days 2, 7 and then weekly until negative. At the end of the study, samples of study batches were sent for heat stability testing. Between August and November 2007, 1004 patients aged between 1 and 93 years were enrolled in the study. Slide microscopy (the reference standard) diagnosed 213 Plasmodium vivax (Pv) monoinfections, 98 Plasmodium falciparum (Pf) mono-infections and no malaria in 650 cases. The sensitivities (sens) and specificities (spec), of the RDTs for the detection of malaria were- CareStart Malaria pLDH (Pan) test: sens 89.1% [CI95 84.2-92.6], spec 97.6% [CI95 96.5-98.4]. OptiMal-IT: Pf+/- other species detection: sens 95.2% [CI95 87.5-98.2], spec 94.7% [CI95 93.3-95.8]; non-Pf detection alone: sens 89.6% [CI95 83.6-93.6], spec 96.5% [CI95 94.8-97.7]. CareStart Malaria pLDH (Pan, Pf): Pf+/- other species: sens 93.5% [CI95 85.4-97.3], spec 97.4% [95.9-98.3]; non-Pf: sens 78.5% [CI95 71.1-84.4], spec 97.8% [CI95 96.3-98.7]. Inter-observer agreement was excellent for all tests (kappa > 0.9). The median time for the RDTs to become negative was two days for the CareStart Malaria tests and seven days for OptiMAL-IT. Tests were heat stable up to 90 days except for OptiMAL-IT (Pf specific pLDH stable to day 20 at 35 degrees C). None of the pLDH-based RDTs evaluated was able to detect non-falciparum malaria with high sensitivity, particularly at low parasitaemias. OptiMAL-IT performed best overall and

  8. Prolactin regulation of oxytocin neurone activity in pregnancy and lactation.

    PubMed

    Augustine, Rachael A; Ladyman, Sharon R; Bouwer, Gregory T; Alyousif, Yousif; Sapsford, Tony J; Scott, Victoria; Kokay, Ilona C; Grattan, David R; Brown, Colin H

    2017-06-01

    During lactation, prolactin promotes milk synthesis and oxytocin stimulates milk ejection. In virgin rats, prolactin inhibits the activity of oxytocin-secreting neurones. We found that prolactin inhibition of oxytocin neurone activity is lost in lactation, and that some oxytocin neurones were excited by prolactin in lactating rats. The change in prolactin regulation of oxytocin neurone activity was not associated with a change in activation of intracellular signalling pathways known to couple to prolactin receptors. The change in prolactin regulation of oxytocin neurone activity in lactation might allow coordinated activation of both populations of neurones when required for successful lactation. Secretion of prolactin for milk synthesis and oxytocin for milk secretion is required for successful lactation. In virgin rats, prolactin inhibits oxytocin neurones but this effect would be counterproductive during lactation when secretion of both hormones is required for synthesis and delivery of milk to the newborn. Hence, we determined the effects of intracerebroventricular (i.c.v.) prolactin on oxytocin neurones in urethane-anaesthetised virgin, pregnant and lactating rats. Prolactin (2 μg) consistently inhibited oxytocin neurones in virgin and pregnant rats (by 1.9 ± 0.4 and 1.8 ± 0.5 spikes s -1 , respectively), but not in lactating rats; indeed, prolactin excited six of 27 oxytocin neurones by >1 spike s -1 in lactating rats but excited none in virgin or pregnant rats (χ 2 2  = 7.2, P = 0.03). Vasopressin neurones were unaffected by prolactin (2 μg) in virgin rats but were inhibited by 1.1 ± 0.2 spikes s -1 in lactating rats. Immunohistochemistry showed that i.c.v. prolactin increased oxytocin expression in virgin and lactating rats and increased signal transducer and activator of transcription 5 phosphorylation to a similar extent in oxytocin neurones of virgin and lactating rats. Western blotting showed that i.c.v. prolactin did not affect

  9. Regulation of NAD+-linked isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase by Ca2+ ions within toluene-permeabilized rat heart mitochondria. Interactions with regulation by adenine nucleotides and NADH/NAD+ ratios.

    PubMed Central

    Rutter, G A; Denton, R M

    1988-01-01

    1. Toluene-permeabilized rat heart mitochondria have been used to study the regulation of NAD+-linked isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase by Ca2+, adenine and nicotinamide nucleotides, and to compare the properties of the enzymes in situ, with those in mitochondrial extracts. 2. Although K0.5 values (concn. giving half-maximal effect) for Ca2+ of 2-oxoglutarate dehydrogenase were around 1 microM under all conditions, corresponding values for NAD+-linked isocitrate dehydrogenase were in the range 5-43 microM. 3. For both enzymes, K0.5 values for Ca2+ observed in the presence of ATP were 3-10-fold higher than those in the presence of ADP, with values increasing over the ADP/ATP range 0.0-1.0. 4. 2-Oxoglutarate dehydrogenase was less sensitive to inhibition by NADH when assayed in permeabilized mitochondria than in mitochondrial extracts. Similarly, the Km of NAD+-linked isocitrate dehydrogenase for threo-Ds-isocitrate was lower in permeabilized mitochondria than in extracts under all the conditions investigated. 5. It is concluded that in the intact heart Ca2+ activation of NAD+-linked isocitrate dehydrogenase may not necessarily occur in parallel with that of the other mitochondrial Ca2+-sensitive enzymes, 2-oxoglutarate dehydrogenase and the pyruvate dehydrogenase system. PMID:3421900

  10. Alcohol consumption decreases lactate clearance in acutely injured patients☆

    PubMed Central

    Dezman, Zachary D.W.; Comer, Angela C.; Narayan, Mayur; Scalea, Thomas M.; Hirshon, Jon Mark; Smith, Gordon S.

    2017-01-01

    Introduction Alcohol, a common risk factor for injury, has direct toxic effects on the liver. The use of lactate clearance has been well described as an indicator of the adequacy of resuscitation in injured patients. We investigated whether acutely injured patients with positive blood alcohol content (+BAC) had less lactate clearance than sober patients. Methods We conducted a retrospective cohort study of acutely injured patients treated at an urban Level 1 trauma centre between January 2010 and December 2012. Blood alcohol and venous lactate levels were measured on all patients at the time of arrival. Study subjects were patients transported directly from the scene of injury, who had an elevated lactate concentration on arrival (≥3.0 mmol/L) and at least one subsequent lactate measurement within 24 h after admission. Lactate clearance ([Lactate1 − Lactate2]/Lactate1) was calculated for all patients. Chi-squared tests were used to compare values from sober and intoxicated subjects. Lactate clearance was plotted against alcohol levels and stratified by age and Injury Severity Score (ISS). Results Serial lactate concentration measurements were obtained in 3910 patients; 1674 of them had +BAC. Patients with +BAC were younger (mean age: 36.6 [SD 14.7] vs 41.0 [SD 19.9] years [p = 0.0001]), were more often male (83.4% vs 75.9% [p = 0.0001]), had more minor injuries (ISS < 9) (33.8% vs 27.1% [p = 0.0001]), had a lower in-hospital mortality rate (1.4% vs 3.9% [p = 0.0001]), but also had lower average lactate clearance (37.8% vs 47.6% [p = 0.0001]). The lactate clearance of the sober patients (47.6 [SD 33.5]) was twice that of those with +BAC >400 (23.5 [SD 6.5]). Lactate clearance decreased with increasing BAC irrespective of age and ISS. Conclusions In a large group of acutely injured patients, a dose-dependent decrease in lactate clearance was seen in those with elevated BAC. This relationship will cause a falsely elevated lactate reading or prolong lactate

  11. Workplace Lactation Support in Milwaukee County 5 Years After the Affordable Care Act.

    PubMed

    Lennon, Tyler; Willis, Earnestine

    2017-02-01

    Workplace lactation support has become increasingly important because returning to work is associated with discontinuing breastfeeding and women in the workforce are increasing. Research aim: This study examined workplace lactation support among Milwaukee County businesses 5 years after implementation of the Affordable Care Act's Break Time for Nursing Mothers provision. A cross-sectional survey of Milwaukee County businesses was conducted in the summer of 2015 that inquired about workplace policies, lactation spaces, and other lactation resources offered. Business supports were stratified based on employer sizes: large (> 500 employees), medium (50-499 employees), and small (20-49 employees). A lactation amenity score was calculated for each business based on lactation resources available. Three hundred surveys were distributed and 71 businesses voluntarily completed the survey. Small employers were excluded from statistical analysis due to fewer responses ( n = 8). Overall, 87.3% ( n = 55) of respondents reported providing access to a multiuser space for lactation and 65.1% ( n = 41) reported providing a designated lactation space. Large employers ( n = 30) were more likely than medium employers ( n = 33) to provide a designated lactation space for breastfeeding or expressing (86.7% vs. 45.5%, p < .001). Large employers' mean amenity score was significantly higher than that of medium employers (3.37 vs. 2.57, p = .014), and they were also more likely to offer additional supports including access to a lactation consultant, classes, and materials (46.7% vs. 12.1%, p < .01). Large employers provide more lactation support than medium employers in Milwaukee County. All employers, regardless of size, need to increase additional lactation support for women in the workplace.

  12. Blood lactate clearance after maximal exercise depends on active recovery intensity.

    PubMed

    Devlin, J; Paton, B; Poole, L; Sun, W; Ferguson, C; Wilson, J; Kemi, O J

    2014-06-01

    High-intensity exercise is time-limited by onset of fatigue, marked by accumulation of blood lactate. This is accentuated at maximal, all-out exercise that rapidly accumulates high blood lactate. The optimal active recovery intensity for clearing lactate after such maximal, all-out exercise remains unknown. Thus, we studied the intensity-dependence of lactate clearance during active recovery after maximal exercise. We constructed a standardized maximal, all-out treadmill exercise protocol that predictably lead to voluntary exhaustion and blood lactate concentration>10 mM. Next, subjects ran series of all-out bouts that increased blood lactate concentration to 11.5±0.2 mM, followed by recovery exercises ranging 0% (passive)-100% of the lactate threshold. Repeated measurements showed faster lactate clearance during active versus passive recovery (P<0.01), and that active recovery at 60-100% of lactate threshold was more efficient for lactate clearance than lower intensity recovery (P<0.05). Active recovery at 80% of lactate threshold had the highest rate of and shortest time constant for lactate clearance (P<0.05), whereas the response during the other intensities was graded (100%=60%>40%>passive recovery, P<0.05). Active recovery after maximal all-out exercise clears accumulated blood lactate faster than passive recovery in an intensity-dependent manner, with maximum clearance occurring at active recovery of 80% of lactate threshold.

  13. Overcoming cetuximab resistance in Ewing's sarcoma by inhibiting lactate dehydrogenase-A.

    PubMed

    Fu, Jiaxin; Jiang, Han; Wu, Chenxuan; Jiang, Yi; Xiao, Lianping; Tian, Yonggang

    2016-07-01

    Ewing's sarcoma, the second most common type of malignant bone tumor, generally occurs in children and young adults. The current treatment of Ewing's sarcoma comprises systemic anti‑cancer chemotherapy with complete surgical resection. However, the majority of patients with Ewing's sarcoma develop resistance to chemotherapy. The present study revealed an oncogenic role of lactate dehydrogenase‑A (LDHA) in the resistance of Ewing's sarcoma to cetuximab. LDHA was shown to be upregulated at the protein and mRNA level in cetuximab‑resistant Ewing's sarcoma tissues and a cell line. In addition, a link between LDHA‑induced glycolysis and cetuximab resistance in Ewing's sarcoma cells was revealed. Of note, inhibition of LDHA by either small interfering RNA or LDHA inhibitor oxamate significantly re‑sensitized cetuximab‑resistant cells to cetuximab. Combined treatment with LDHA inhibitor and cetuximab synergistically reduced the viability of cetuximab-resistant cells through the suppression of LDHA. The present study revealed a novel mechanism of cetuximab resistance from the perspective of cancer‑cell metabolism and provided a sensitization approach, which may aid in the development of anti-chemoresistance strategies for the treatment of cetuximab-resistant Ewing's sarcoma.

  14. Blood lactate levels in 31 female dogs with pyometra

    PubMed Central

    Hagman, Ragnvi; Reezigt, Bert Jan; Bergström Ledin, Hanna; Karlstam, Erika

    2009-01-01

    Background Canine pyometra is a life-threatening disease common in countries where spaying of dogs is not routinely performed. The disease is associated with endotoxemia, sepsis, systemic inflammatory response syndrome (SIRS) and a 3–4% mortality rate. Blood lactate analysis is clinically valuable in predicting prognosis and survival, evaluating tissue perfusion and treatment response in human and veterinary critical care settings. The aims of the present study were to investigate 1) the blood lactate levels of female dogs with pyometra by a hand-held analyser and 2) if these levels are related with the clinical status or other biochemical or hematological disorders. Methods In total 31 female dogs with pyometra admitted for surgical ovariohysterectomy and 16 healthy female control dogs were included in the present study. A complete physical examination including SIRS-status determination was performed. Blood samples for lactate concentrations, hematological and biochemical parameters, acid-base and blood gas analysis and other laboratory parameters were collected and subsequently analysed. The diagnosis pyometra was verified with histopathological examination of the uterus and ovaries. Increased hospitalisation length and presence of SIRS were used as indicators of outcome. Results In the pyometra group the median blood lactate level was 1,6 mmol l-1 (range <0.8–2.7 mmol l-1). In the control group the median lactate level was 1,2 mmol l-1 (range <0.8–2.1 mmol l-1). Of the 31 bitches 19 (61%) fulfilled 2 or more criteria for SIRS at inclusion, 10 bitches (32%) fulfilled 3 of the SIRS criteria whereas none accomplished more than 3 criteria. Lactate levels did not differ significantly between the pyometra and control group, or between the SIRS positive and SIRS negative dogs with pyometra. Increased lactate concentration (>2.5 mmol l-1) was demonstrated in one female dog with pyometra (3%), and was not associated with longer hospitalisation or presence of SIRS

  15. FGF-21 and Skeletal Remodeling During and After Lactation in C57BL/6J Mice

    PubMed Central

    Bornstein, Sheila; Brown, Sue A.; Le, Phuong T.; Wang, Xunde; DeMambro, Victoria; Horowitz, Mark C.; MacDougald, Ormond; Baron, Roland; Lotinun, Sutada; Karsenty, Gerard; Wei, Wei; Ferron, Mathieu; Kovacs, Christopher S.; Clemmons, David

    2014-01-01

    Lactation is associated with significant alterations in both body composition and bone mass. Systemic and local skeletal factors such as receptor activator of nuclear factor κ-B ligand (RANKL), PTHrP, calcitonin, and estrogen are known to regulate bone remodeling during and after lactation. Fibroblast growth factor 21 (FGF-21) may function as an endocrine factor to regulate body composition changes during lactation by inducing gluconeogenesis and fatty acid oxidation. In this study, we hypothesized that the metabolic changes during lactation were due in part to increased circulating FGF-21, which in turn could accentuate bone loss. We longitudinally characterized body composition in C57BL/6J (B6) mice during (day 7 and day 21 of lactation) and after normal lactation (day 21 postlactation). At day 7 of lactation, areal bone density declined by 10% (P < .001), bone resorption increased (P < .0001), percent fat decreased by 20%, energy expenditure increased (P < .01), and markers of brown-like adipogenesis were suppressed in the inguinal depot and in preformed brown adipose tissue. At day 7 of lactation there was a 2.4-fold increase in serum FGF-21 vs baseline (P < .0001), a 8-fold increase in hepatic FGF-21 mRNA (P < .03), a 2-fold increase in undercarboxylated osteocalcin (Glu13 OCn) (P < .01), and enhanced insulin sensitivity. Recovery of total areal bone density was noted at day 21 of lactation, whereas the femoral trabecular bone volume fraction was still reduced (P < .01). Because FGF-21 levels rose rapidly at day 7 of lactation in B6 lactating mice, we next examined lactating mice with a deletion in the Fgf21 gene. Trabecular and cortical bone masses were maintained throughout lactation in FGF-21−/− mice, and pup growth was normal. Compared with lactating control mice, lactating FGF-21−/− mice exhibited an increase in bone formation, but no change in bone resorption. In conclusion, in addition to changes in calciotropic hormones, systemic FGF-21 plays

  16. Daily energy expenditure across the course of lactation among urban Bangladeshi women.

    PubMed

    Rashid, M; Ulijaszek, S J

    1999-12-01

    Measures of energy intake of lactating women in developing countries show that intakes are often lower than those recommended by international bodies, while fat-mass losses are often substantially less than the 3-4 kg used in the calculations of recommendations, suggesting that physiological adaptation must be commonplace among such women. The cost of lactation may be met by reduction in energy expenditure, including reduced physical activity, as well as by mobilization of bodily soft tissue. However, daily energy expenditure of lactating women has been shown to increase across the course of lactation among women in a rural population in the Philippines and an urban population in India, with a decline in body weight across the course of lactation in both studies. In the present study, total daily energy expenditure and anthropometric body composition were measured longitudinally in 68 mothers from a poor urban area of Dhaka, Bangladesh, at 0, 1, 2, 4, and 8 months of lactation, to determine whether the increasing energy expenditure across lactation observed elsewhere also occurs in Bangladeshi women. In addition, the extent to which an extended period of lactation was accompanied by weight and body fat change in these women was determined. Energy expenditure by heart-rate monitoring and activity report, and body composition from anthropometry was carried out four times across the 8-month period of lactation. A small decline in body fat mass and a significant increase in total energy expenditure across this period were observed, confirming similar observations elsewhere in the developing world. Copyright 1999 Wiley-Liss, Inc.

  17. Flexible graphene bio-nanosensor for lactate.

    PubMed

    Labroo, Pratima; Cui, Yue

    2013-03-15

    The development of a flexible nanosensor for detecting lactate could expand opportunities for using graphene, both in fundamental studies for a variety of device platforms and in practical applications. Graphene is a delicate single-layer, two-dimensional network of carbon atoms with ultrasensitive sensing capabilities. Lactic acid is important for clinical analysis, sports medicine, and the food industry. Recently, wearable and flexible bioelectronics on plastics have attracted great interest for healthcare, sports and defense applications due to their advantages of being light-weight, bendable, or stretchable. Here, we demonstrate for the first time the development of a flexible graphene-based bio-nanosensor to detect lactate. Our results show that flexible lactate biosensors can be fabricated on a variety of plastic substrates. The sensor can detect lactate sensitively from 0.08 μM to 20 μM with a fast steady-state measuring time of 2s. The sensor can also detect lactate under different mechanical bending conditions, the sensor response decreased as the bending angle and number of bending repetitions increased. We anticipate that these results could open exciting opportunities for fundamental studies of flexible graphene bioelectronics by using other bioreceptors, as well as a variety of wearable, implantable, real-time, or on-site applications in fields ranging from clinical analysis to defense. Copyright © 2012 Elsevier B.V. All rights reserved.

  18. Targeting the lactate transporter MCT1 in endothelial cells inhibits lactate-induced HIF-1 activation and tumor angiogenesis.

    PubMed

    Sonveaux, Pierre; Copetti, Tamara; De Saedeleer, Christophe J; Végran, Frédérique; Verrax, Julien; Kennedy, Kelly M; Moon, Eui Jung; Dhup, Suveera; Danhier, Pierre; Frérart, Françoise; Gallez, Bernard; Ribeiro, Anthony; Michiels, Carine; Dewhirst, Mark W; Feron, Olivier

    2012-01-01

    Switching to a glycolytic metabolism is a rapid adaptation of tumor cells to hypoxia. Although this metabolic conversion may primarily represent a rescue pathway to meet the bioenergetic and biosynthetic demands of proliferating tumor cells, it also creates a gradient of lactate that mirrors the gradient of oxygen in tumors. More than a metabolic waste, the lactate anion is known to participate to cancer aggressiveness, in part through activation of the hypoxia-inducible factor-1 (HIF-1) pathway in tumor cells. Whether lactate may also directly favor HIF-1 activation in endothelial cells (ECs) thereby offering a new druggable option to block angiogenesis is however an unanswered question. In this study, we therefore focused on the role in ECs of monocarboxylate transporter 1 (MCT1) that we previously identified to be the main facilitator of lactate uptake in cancer cells. We found that blockade of lactate influx into ECs led to inhibition of HIF-1-dependent angiogenesis. Our demonstration is based on the unprecedented characterization of lactate-induced HIF-1 activation in normoxic ECs and the consecutive increase in vascular endothelial growth factor receptor 2 (VEGFR2) and basic fibroblast growth factor (bFGF) expression. Furthermore, using a variety of functional assays including endothelial cell migration and tubulogenesis together with in vivo imaging of tumor angiogenesis through intravital microscopy and immunohistochemistry, we documented that MCT1 blockers could act as bona fide HIF-1 inhibitors leading to anti-angiogenic effects. Together with the previous demonstration of MCT1 being a key regulator of lactate exchange between tumor cells, the current study identifies MCT1 inhibition as a therapeutic modality combining antimetabolic and anti-angiogenic activities.

  19. Targeting the Lactate Transporter MCT1 in Endothelial Cells Inhibits Lactate-Induced HIF-1 Activation and Tumor Angiogenesis

    PubMed Central

    Sonveaux, Pierre; Copetti, Tamara; De Saedeleer, Christophe J.; Végran, Frédérique; Verrax, Julien; Kennedy, Kelly M.; Moon, Eui Jung; Dhup, Suveera; Danhier, Pierre; Frérart, Françoise; Gallez, Bernard; Ribeiro, Anthony; Michiels, Carine

    2012-01-01

    Switching to a glycolytic metabolism is a rapid adaptation of tumor cells to hypoxia. Although this metabolic conversion may primarily represent a rescue pathway to meet the bioenergetic and biosynthetic demands of proliferating tumor cells, it also creates a gradient of lactate that mirrors the gradient of oxygen in tumors. More than a metabolic waste, the lactate anion is known to participate to cancer aggressiveness, in part through activation of the hypoxia-inducible factor-1 (HIF-1) pathway in tumor cells. Whether lactate may also directly favor HIF-1 activation in endothelial cells (ECs) thereby offering a new druggable option to block angiogenesis is however an unanswered question. In this study, we therefore focused on the role in ECs of monocarboxylate transporter 1 (MCT1) that we previously identified to be the main facilitator of lactate uptake in cancer cells. We found that blockade of lactate influx into ECs led to inhibition of HIF-1-dependent angiogenesis. Our demonstration is based on the unprecedented characterization of lactate-induced HIF-1 activation in normoxic ECs and the consecutive increase in vascular endothelial growth factor receptor 2 (VEGFR2) and basic fibroblast growth factor (bFGF) expression. Furthermore, using a variety of functional assays including endothelial cell migration and tubulogenesis together with in vivo imaging of tumor angiogenesis through intravital microscopy and immunohistochemistry, we documented that MCT1 blockers could act as bona fide HIF-1 inhibitors leading to anti-angiogenic effects. Together with the previous demonstration of MCT1 being a key regulator of lactate exchange between tumor cells, the current study identifies MCT1 inhibition as a therapeutic modality combining antimetabolic and anti-angiogenic activities. PMID:22428047

  20. Selective modification of the pyruvate dehydrogenase kinase isoform profile in skeletal muscle in hyperthyroidism: implications for the regulatory impact of glucose on fatty acid oxidation.

    PubMed

    Sugden, M C; Lall, H S; Harris, R A; Holness, M J

    2000-11-01

    The pyruvate dehydrogenase kinases (PDK1-4) regulate glucose oxidation through inhibitory phosphorylation of the pyruvate dehydrogenase complex (PDC). Immunoblot analysis with antibodies raised against recombinant PDK isoforms demonstrated changes in PDK isoform expression in response to experimental hyperthyroidism (100 microg/100 g body weight; 3 days) that was selective for fast-twitch vs slow-twitch skeletal muscle in that PDK2 expression was increased in the fast-twitch skeletal muscle (the anterior tibialis) (by 1. 6-fold; P<0.05) but not in the slow-twitch muscle (the soleus). PDK4 protein expression was increased by experimental hyperthyroidism in both muscle types, there being a greater response in the anterior tibialis (4.2-fold increase; P<0.05) than in the soleus (3.2-fold increase; P<0.05). The hyperthyroidism-associated up-regulation of PDK4 expression was observed in conjunction with suppression of skeletal-muscle PDC activity, but not suppression of glucose uptake/phosphorylation, as measured in vivo in conscious unrestrained rats (using the 2-[(3)H]deoxyglucose technique). We propose that increased PDK isoform expression contributes to the pathology of hyperthyroidism and to PDC inactivation by facilitating the operation of the glucose --> lactate --> glucose (Cori) and glucose --> alanine --> glucose cycles. We also propose that enhanced relative expression of the pyruvate-insensitive PDK isoform (PDK4) in skeletal muscle in hyperthyroidism uncouples glycolytic flux from pyruvate oxidation, sparing pyruvate for non-oxidative entry into the tricarboxylic acid (TCA) cycle, and thereby supporting entry of acetyl-CoA (derived from fatty acid oxidation) into the TCA cycle.

  1. CYTOCHEMICAL LOCALIZATION OF TWO GLYCOLYTIC DEHYDROGENASES IN WHITE SKELETAL MUSCLE

    PubMed Central

    Fahimi, H. Dariush; Karnovsky, Morris J.

    1966-01-01

    The cytochemical localization, by conventional methods, of lactate and glyceraldehyde-3-phosphate dehydrogenases is limited, firstly, by the solubility of these enzymes in aqueous media and, secondly, by the dependence of the final electron flow from reduced nicotinamide-adenine dinucleotide (NADH) to the tetrazolium on tissue diaphorase activity: localization is therefore that of the diaphorase, which in rabbit adductor magnus is mitochondrial. NADH has been found to have great affinity to bind in the sarcoplasmic reticulum, and, therefore, if it is generated freely in the incubation media containing 2,2',5,5'-tetra-p-nitrophenyl-3,3'-(3,3'-dimethoxy-4,4'-phenylene)-ditetrazolium chloride (TNBT) and N-methyl phenazonium methyl sulfate (PMS), it can bind there and cause a false staining. Since such a production of NADH can readily occur in the incubation media for glycolytic dehydrogenases due to diffusion of these soluble enzymes from tissue sections, the prevention of enzyme solubilization is extremely important. Fixation in formaldehyde prevented such enzyme diffusion, while at the same time sufficient activity persisted to allow for adequate staining. The incubation media contained PMS, so that the staining system was largely independent of tissue diaphorase activity. Application of these methods to adductor magnus of rabbit revealed by light microscopy, for both enzymes, a fine network which was shown by electron microscopy to represent staining of the sarcoplasmic reticulum. Mitochondria also reacted. These findings add further support for the notion that the sarcoplasmic reticulum is probably involved in glycolytic activity. PMID:4288329

  2. Early lactation production, health, and welfare characteristics of cows selected for extended lactation.

    PubMed

    Lehmann, J O; Mogensen, L; Kristensen, T

    2017-02-01

    Some cows are able to achieve relatively high milk yields during extended lactations beyond 305 d in milk, and farmers may be able to use this potential by selecting the most suitable cows for an extended lactation. However, the decision to postpone insemination has to rely on information available in early lactation. The main objectives of this study were, therefore, to assess the association between the information available in early lactation and the relative milk production of cows on extended lactation, and to investigate if this information can be used to differentiate time of first insemination between cows. Data came from 4 Danish private herds practicing extended lactation in which some cows are selected to have a delayed time of planned first insemination. Average herd size varied from 93 to 157 cows, and milk yield varied from 7,842 to 12,315 kg of energy-corrected milk (ECM) per cow per year across herds. The analysis was based on 422 completed extended lactations (427 ± 87 d), and each lactation was assigned to 1 of 3 (low, medium, and high) milk performance groups (MPG) within parity group within herd based on a standardized lactation yield. For cows in the high MPG, peak ECM yield, and ECM yield at dry off were significantly greater, the relative reduction in milk yield between 60 and 305 d in milk was significantly smaller, and a smaller proportion had a body condition score (scale: 1-5) at dry off of 3.5 or greater compared with cows in low MPG. Previous lactation days in milk at peak ECM yield and ECM yield at dry off were higher, the relative reduction in milk yield between 60 and 305 d in milk was smaller, and the number of inseminations per conception was higher for multiparous cows in high MPG compared with low. Current lactation ECM yield at second and third milk recording were greater for cows in high MPG compared with low. A principal component analysis indicated that variables related to fertility, diseases, and milk yield explained most

  3. Astrocyte-neuron lactate transport is required for long-term memory formation.

    PubMed

    Suzuki, Akinobu; Stern, Sarah A; Bozdagi, Ozlem; Huntley, George W; Walker, Ruth H; Magistretti, Pierre J; Alberini, Cristina M

    2011-03-04

    We report that, in the rat hippocampus, learning leads to a significant increase in extracellular lactate levels that derive from glycogen, an energy reserve selectively localized in astrocytes. Astrocytic glycogen breakdown and lactate release are essential for long-term but not short-term memory formation, and for the maintenance of long-term potentiation (LTP) of synaptic strength elicited in vivo. Disrupting the expression of the astrocytic lactate transporters monocarboxylate transporter 4 (MCT4) or MCT1 causes amnesia, which, like LTP impairment, is rescued by L-lactate but not equicaloric glucose. Disrupting the expression of the neuronal lactate transporter MCT2 also leads to amnesia that is unaffected by either L-lactate or glucose, suggesting that lactate import into neurons is necessary for long-term memory. Glycogenolysis and astrocytic lactate transporters are also critical for the induction of molecular changes required for memory formation, including the induction of phospho-CREB, Arc, and phospho-cofilin. We conclude that astrocyte-neuron lactate transport is required for long-term memory formation. Copyright © 2011 Elsevier Inc. All rights reserved.

  4. Astrocyte-neuron lactate transport is required for long-term memory formation

    PubMed Central

    Suzuki, Akinobu; Stern, Sarah A.; Bozdagi, Ozlem; Huntley, George W.; Walker, Ruth H.; Magistretti, Pierre J.; Alberini, Cristina M.

    2011-01-01

    SUMMARY We report that in the rat hippocampus learning leads to a significant increase in extracellular lactate levels, which derive from glycogen, an energy reserve selectively localized in astrocytes. Astrocytic glycogen breakdown and lactate release are essential for long-term but not short-term memory formation, and for the maintenance of long-term potentiation (LTP) of synaptic strength elicited in-vivo. Disrupting the expression of the astrocytic lactate transporters monocarboxylate transporter 4 (MCT4) or MCT1 causes amnesia, which, like LTP impairment, is rescued by lactate but not equicaloric glucose. Disrupting the expression of the neuronal lactate transporter MCT2 also leads to amnesia that is unaffected by either L-lactate or glucose, suggesting that lactate import into neurons is necessary for long-term memory. Glycogenolysis and astrocytic lactate transporters are also critical for the induction of molecular changes required for memory formation, including the induction of phospho-CREB, Arc and phospho-cofilin. We conclude that astrocyte-neuron lactate transport is required for long-term memory formation. PMID:21376239

  5. Lactate promotes glutamine uptake and metabolism in oxidative cancer cells

    PubMed Central

    Pérez-Escuredo, Jhudit; Dadhich, Rajesh K; Dhup, Suveera; Cacace, Andrea; Van Hée, Vincent F; De Saedeleer, Christophe J; Sboarina, Martina; Rodriguez, Fabien; Fontenille, Marie-Joséphine; Brisson, Lucie; Porporato, Paolo E; Sonveaux, Pierre

    2016-01-01

    ABSTRACT Oxygenated cancer cells have a high metabolic plasticity as they can use glucose, glutamine and lactate as main substrates to support their bioenergetic and biosynthetic activities. Metabolic optimization requires integration. While glycolysis and glutaminolysis can cooperate to support cellular proliferation, oxidative lactate metabolism opposes glycolysis in oxidative cancer cells engaged in a symbiotic relation with their hypoxic/glycolytic neighbors. However, little is known concerning the relationship between oxidative lactate metabolism and glutamine metabolism. Using SiHa and HeLa human cancer cells, this study reports that intracellular lactate signaling promotes glutamine uptake and metabolism in oxidative cancer cells. It depends on the uptake of extracellular lactate by monocarboxylate transporter 1 (MCT1). Lactate first stabilizes hypoxia-inducible factor-2α (HIF-2α), and HIF-2α then transactivates c-Myc in a pathway that mimics a response to hypoxia. Consequently, lactate-induced c-Myc activation triggers the expression of glutamine transporter ASCT2 and of glutaminase 1 (GLS1), resulting in improved glutamine uptake and catabolism. Elucidation of this metabolic dependence could be of therapeutic interest. First, inhibitors of lactate uptake targeting MCT1 are currently entering clinical trials. They have the potential to indirectly repress glutaminolysis. Second, in oxidative cancer cells, resistance to glutaminolysis inhibition could arise from compensation by oxidative lactate metabolism and increased lactate signaling. PMID:26636483

  6. Reconstitution of the Escherichia coli pyruvate dehydrogenase complex.

    PubMed Central

    Reed, L J; Pettit, F H; Eley, M H; Hamilton, L; Collins, J H; Oliver, R M

    1975-01-01

    The binding of pyruvate dehydrogenase and dihydrolipoyl dehydrogenase (flavoprotein) to dihydrolipoyl transacetylase, the core enzyme of the E. coli pyruvate dehydrogenase complex [EC 1.2.4.1:pyruvate:lipoate oxidoreductase (decaryboxylating and acceptor-acetylating)], has been studied using sedimentation equilibrium analysis and radioactive enzymes in conjunction with gel filtration chromatography. The results show that the transacetylase, which consists of 24 apparently identical polypeptide chains organized into a cube-like structure, has the potential to bind 24 pyruvate dehydrogenase dimers in the absence of flavoprotein and 24 flavoprotein dimers in the absence of pyruvate dehydrogenase. The results of reconstitution experiments, utilizing binding and activity measurements, indicate that the transacetylase can accommodate a total of only about 12 pyruvate dehydrogenase dimers and six flavoprotein dimers and that this stoichiometry, which is the same as that of the native pyruvate dehydrogenase complex, produces maximum activity. It appears that steric hindrance between the relatively bulky pyruvate dehydrogenase and flavoprotein molecules prevents the transacetylase from binding 24 molecules of each ligand. A structural model for the native and reconstituted pyruvate dehydrogenase complexes is proposed in which the 12 pyruvate dehydrogenase dimers are distributed symmetrically on the 12 edges of the transacetylase cube and the six flavoprotein dimers are distributed in the six faces of the cube. Images PMID:1103138

  7. Isocitrate dehydrogenase 1 and 2 mutations in cholangiocarcinoma.

    PubMed

    Kipp, Benjamin R; Voss, Jesse S; Kerr, Sarah E; Barr Fritcher, Emily G; Graham, Rondell P; Zhang, Lizhi; Highsmith, W Edward; Zhang, Jun; Roberts, Lewis R; Gores, Gregory J; Halling, Kevin C

    2012-10-01

    Somatic mutations in isocitrate dehydrogenase 1 and 2 genes are common in gliomas and help stratify patients with brain cancer into histologic and molecular subtypes. However, these mutations are considered rare in other solid tumors. The aims of this study were to determine the frequency of isocitrate dehydrogenase 1 and 2 mutations in cholangiocarcinoma and to assess histopathologic differences between specimens with and without an isocitrate dehydrogenase mutation. We sequenced 94 formalin-fixed, paraffin-embedded cholangiocarcinoma (67 intrahepatic and 27 extrahepatic) assessing for isocitrate dehydrogenase 1 (codon 132) and isocitrate dehydrogenase 2 (codons 140 and 172) mutations. Multiple histopathologic characteristics were also evaluated and compared with isocitrate dehydrogenase 1/2 mutation status. Of the 94 evaluated specimens, 21 (22%) had a mutation including 14 isocitrate dehydrogenase 1 and 7 isocitrate dehydrogenase 2 mutations. Isocitrate dehydrogenase mutations were more frequently observed in intrahepatic cholangiocarcinoma than in extrahepatic cholangiocarcinoma (28% versus 7%, respectively; P = .030). The 14 isocitrate dehydrogenase 1 mutations were R132C (n = 9), R132S (n = 2), R132G (n = 2), and R132L (n = 1). The 7 isocitrate dehydrogenase 2 mutations were R172K (n = 5), R172M (n = 1), and R172G (n = 1). Isocitrate dehydrogenase mutations were more frequently observed in tumors with clear cell change (P < .001) and poorly differentiated histology (P = .012). The results of this study show for the first time that isocitrate dehydrogenase 1 and 2 genes are mutated in cholangiocarcinoma. The results of this study are encouraging because it identifies a new potential target for genotype-directed therapeutic trials and may represent a potential biomarker for earlier detection of cholangiocarcinoma in a subset of cases. Copyright © 2012 Elsevier Inc. All rights reserved.

  8. Postprandial plasma D-lactate concentrations after yogurt ingestion.

    PubMed

    de Vrese, M; Barth, C A

    1991-06-01

    The risk of D-lactic acidosis after consumption of yogurt was investigated in seven healthy volunteers. After ingestion of yogurt containing 1.06 mmol/kg body weight, D-lactic acid postprandial plasma D-lactate concentrations increased from 0.070 +/- 0.020 to a maximum of 0.200 +/- 0.010 mmol/l within 60 min. That was half the maximum concentration after the equivalent amount of D-lactate in the form of an aqueous solution of DL-lactate. The shape of the postprandial plasma D-lactate peak was flatter, but much broader after yogurt than after the aqueous solution, the peak areas being equal. When 0.64 mmol/kg body weight D-lactate were consumed as yogurt, plasma concentrations amounted to 0.086 +/- 0.030 mmol/l. Signs of a mild, transient, compensated metabolic acidosis, which was apparent in case of the aqueous lactic acid solution did not occur in case of yogurt. It is concluded that the consumption of foods containing D-lactic acid gives no reason for concern in healthy adults.

  9. Partial nucleotide sequences, and routine typing by polymerase chain reaction-restriction fragment length polymorphism, of the brown trout (Salmo trutta) lactate dehydrogenase, LDH-C1*90 and *100 alleles.

    PubMed

    McMeel, O M; Hoey, E M; Ferguson, A

    2001-01-01

    The cDNA nucleotide sequences of the lactate dehydrogenase alleles LDH-C1*90 and *100 of brown trout (Salmo trutta) were found to differ at position 308 where an A is present in the *100 allele but a G is present in the *90 allele. This base substitution results in an amino acid change from aspartic acid at position 82 in the LDH-C1 100 allozyme to a glycine in the 90 allozyme. Since aspartic acid has a net negative charge whilst glycine is uncharged, this is consistent with the electrophoretic observation that the LDH-C1 100 allozyme has a more anodal mobility relative to the LDH-C1 90 allozyme. Based on alignment of the cDNA sequence with the mouse genomic sequence, a local primer set was designed, incorporating the variable position, and was found to give very good amplification with brown trout genomic DNA. Sequencing of this fragment confirmed the difference in both homozygous and heterozygous individuals. Digestion of the polymerase chain reaction products with BslI, a restriction enzyme specific for the site difference, gave one, two and three fragments for the two homozygotes and the heterozygote, respectively, following electrophoretic separation. This provides a DNA-based means of routine screening of the highly informative LDH-C1* polymorphism in brown trout population genetic studies. Primer sets presented could be used to sequence cDNA of other LDH* genes of brown trout and other species.

  10. Milk losses associated with somatic cell counts by parity and stage of lactation.

    PubMed

    Gonçalves, Juliano L; Cue, Roger I; Botaro, Bruno G; Horst, José A; Valloto, Altair A; Santos, Marcos V

    2018-05-01

    The reduction of milk production caused by subclinical mastitis in dairy cows was evaluated through the regression of test-day milk yield on log-transformed somatic cell counts (LnSCC). Official test-day records (n = 1,688,054) of Holstein cows (n = 87,695) were obtained from 719 herds from January 2010 to December 2015. Editing was performed to ensure both reliability and consistency for the statistical analysis, and the final data set comprised 232,937 test-day records from 31,692 Holstein cows in 243 herds. A segmented regression was fitted to estimate the cutoff point in the LnSCC scale where milk yield started to be affected by mastitis. The statistical model used to explain daily milk yield included the effect of herd as a random effect and days in milk and LnSCC as fixed effects regressions, and analyses were performed by parity and stage of lactation. The cutoff point where milk yield starts to be affected by changes in LnSCC was estimated to be around 2.52 (the average of all estimates of approximately 12,400 cells/mL) for Holsteins cows from Brazilian herds. For first-lactation cows, milk losses per unit increase of LnSCC had estimates around 0.68 kg/d in the beginning of the lactation [5 to 19 d in milk (DIM)], 0.55 kg/d in mid-lactation (110 to 124 DIM), and 0.97 kg/d at the end of the lactation (289 to 304 DIM). For second-lactation cows, milk losses per unit increase of LnSCC had estimates around 1.47 kg/d in the beginning of the lactation (5 to 19 DIM), 1.09 kg/d in mid-lactation (110 to 124 DIM), and 2.45 kg/d at the end of the lactation (289 to 304 DIM). For third-lactation cows, milk losses per unit increase of LnSCC had estimates around 2.22 kg/d in the beginning of the lactation (5 to 19 DIM), 1.13 kg/d in mid-lactation (140 to 154 DIM), and 2.65 kg/d at the end of the lactation (289 to 304 DIM). Daily milk losses caused by increased LnSCC were dependent on parity and stage of lactation, and these factors should be considered when estimating

  11. 21 CFR 862.1670 - Sorbitol dehydrogenase test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Sorbitol dehydrogenase test system. 862.1670... Systems § 862.1670 Sorbitol dehydrogenase test system. (a) Identification. A sorbitol dehydrogenase test system is a device intended to measure the activity of the enzyme sorbitol dehydrogenase in serum...

  12. [Enzyme activity in the subcellular fractions of the liver of rats following a flight on board the Kosmos-1129 biosatellite].

    PubMed

    Tigranian, R A; Vetrova, E G; Abraham, S; Lin, C; Klein, H

    1983-01-01

    The activities of malate, isocitrate, and lactate dehydrogenases were measured in the liver mitochondrial and cytoplasmatic fractions of rats flown for 18.5 days onboard Cosmos-1129. The activities of the oxidative enzymes, malate and isocitrate dehydrogenases, in the mitochondrial fraction and those of the glycolytic enzyme, lactate dehydrogenase, in the cytoplasmatic fraction were found to decrease.

  13. Hypertonic Lactate to Improve Cerebral Perfusion and Glucose Availability After Acute Brain Injury.

    PubMed

    Carteron, Laurent; Solari, Daria; Patet, Camille; Quintard, Hervé; Miroz, John-Paul; Bloch, Jocelyne; Daniel, Roy T; Hirt, Lorenz; Eckert, Philippe; Magistretti, Pierre J; Oddo, Mauro

    2018-06-19

    Lactate promotes cerebral blood flow and is an efficient substrate for the brain, particularly at times of glucose shortage. Hypertonic lactate is neuroprotective after experimental brain injury; however, human data are limited. Prospective study (clinicaltrials.gov NCT01573507). Academic ICU. Twenty-three brain-injured subjects (13 traumatic brain injury/10 subarachnoid hemorrhage; median age, 59 yr [41-65 yr]; median Glasgow Coma Scale, 6 [3-7]). Three-hour IV infusion of hypertonic lactate (sodium lactate, 1,000 mmol/L; concentration, 30 µmol/kg/min) administered 39 hours (26-49 hr) from injury. We examined the effect of hypertonic lactate on cerebral perfusion (using transcranial Doppler) and brain energy metabolism (using cerebral microdialysis). The majority of subjects (13/23 = 57%) had reduced brain glucose availability (baseline pretreatment cerebral microdialysis glucose, < 1 mmol/L) despite normal baseline intracranial pressure (10 [7-15] mm Hg). Hypertonic lactate was associated with increased cerebral microdialysis lactate (+55% [31-80%]) that was paralleled by an increase in middle cerebral artery mean cerebral blood flow velocities (+36% [21-66%]) and a decrease in pulsatility index (-21% [13-26%]; all p < 0.001). Cerebral microdialysis glucose increased above normal range during hypertonic lactate (+42% [30-78%]; p < 0.05); reduced brain glucose availability correlated with a greater improvement of cerebral microdialysis glucose (Spearman r = -0.53; p = 0.009). No significant changes in cerebral perfusion pressure, mean arterial pressure, systemic carbon dioxide, and blood glucose were observed during hypertonic lactate (all p > 0.1). This is the first clinical demonstration that hypertonic lactate resuscitation improves both cerebral perfusion and brain glucose availability after brain injury. These cerebral vascular and metabolic effects appeared related to brain lactate supplementation rather than to systemic effects.

  14. Functional interaction between bicarbonate transporters and carbonic anhydrase modulates lactate uptake into mouse cardiomyocytes.

    PubMed

    Peetz, Jan; Barros, L Felipe; San Martín, Alejandro; Becker, Holger M

    2015-07-01

    Blood-derived lactate is a precious energy substrate for the heart muscle. Lactate is transported into cardiomyocytes via monocarboxylate transporters (MCTs) together with H(+), which couples lactate uptake to cellular pH regulation. In this study, we have investigated how the interplay between different acid/base transporters and carbonic anhydrases (CA), which catalyze the reversible hydration of CO2, modulates the uptake of lactate into isolated mouse cardiomyocytes. Lactate transport was estimated both as lactate-induced acidification and as changes in intracellular lactate levels measured with a newly developed Förster resonance energy transfer (FRET) nanosensor. Recordings of intracellular pH showed an increase in the rate of lactate-induced acidification when CA was inhibited by 6-ethoxy-2-benzothiazolesulfonamide (EZA), while direct measurements of lactate flux demonstrated a decrease in MCT transport activity, when CA was inhibited. The data indicate that catalytic activity of extracellular CA increases lactate uptake and counteracts intracellular lactate-induced acidification. We propose a hypothetical model, in which HCO3 (-), formed from cell-derived CO2 at the outer surface of the cardiomyocyte plasma membrane by membrane-anchored, extracellular CA, is transported into the cell via Na(+)/HCO3 (-) cotransport to counteract intracellular acidification, while the remaining H(+) stabilizes extracellular pH at the surface of the plasma membrane during MCT activity to enhance lactate influx into cardiomyocytes.

  15. The clinical diagnostic significance of cerebrospinal fluid D-lactate for bacterial meningitis.

    PubMed

    Chen, Zengqiang; Wang, Yumin; Zeng, Aibing; Chen, Lijiang; Wu, Ruihao; Chen, Bicheng; Chen, Mengquan; Bo, Jinshuang; Zhang, Hu; Peng, Qian; Lu, Jianxin; Meng, Qing H

    2012-10-09

    To study the clinical and laboratory significance of D‐lactate in the diagnosis of bacterial meningitis (BM). The levels of D‐lactate, L‐lactate, IL-6, IL-8, and other biochemical markers were determined in 83 CSF samples from different types of meningitis and the controls. The CSF values of D‐lactate, L‐lactate, IL-6, IL-8, erythrocytes, leukocytes, and protein were higher in patients with BM than those in the controls and patients with viral meningitis. The levels of D‐lactate, L‐lactate, IL-6, and erythrocytes in the BM group were higher than those in the tuberculous meningitis group. At the cutoff 12.8 μmol/l, D‐lactate showed the diagnostic sensitivity of 94.7%. D‐lactate gave the area under the curve (AUC) 0.905, which was higher than those of other markers. Using multiple marker detection, the AUC reached 0.956, which was the highest among all the parameters. Pearson correlation analysis revealed that D‐lactate was positively correlated to IL-6 and L‐lactate (r=0.727, 0.789 and P=0.000, 0.000, respectively). THE CSF concentrations of D‐lactate are significantly increased in the presence of BM. Measurement of D‐lactate provides a rapid diagnosis and differential diagnosis for BM. Combination of D‐lactate with other biochemical markers improves the specificity. Copyright © 2012 Elsevier B.V. All rights reserved.

  16. Lactate induces osteoblast differentiation by stabilization of HIF1α.

    PubMed

    Wu, Yu; Wang, Miaomiao; Feng, Haihua; Peng, Ying; Sun, Jieyun; Qu, Xiuxia; Li, Chunping

    2017-09-05

    Aerobic glycolysis is involved in osteoblast differentiation induced by Wnt signaling or PTH treatment. However, it is still unclear whether lactate, the end product of aerobic glycolysis, plays any role in osteoblast differentiation. Herein we report that in cultures of osteoblast-lineage cells, lactate promoted alkaline phosphatase-positive cell formation, increased the activity of alkaline phosphatase, and induced the expression of osteocalcin. This osteoblast differentiation-inducing effect of lactate can be inhibited by blocking its entry into cells with MCT1 siRNA or inhibitors, and by interfering with its metabolism by using specific siRNAs for LDHB and PDH. Moreover, lactate stabilized HIF1α expression and inhibited HIF1α activity, with BAY87-2243 lowering the osteoblast differentiation-inducing effect of lactate. Thus, these findings reveal an unrecognized role for aerobic glycolysis in osteoblast differentiation via its end product, lactate. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Mechanisms to conserve glucose in lactating women during a 42-h fast

    USDA-ARS?s Scientific Manuscript database

    Little is known about how lactating women accommodate for their increased glucose demands during fasting to avoid maternal hypoglycemia. The objective of this study was to determine whether lactating women conserve plasma glucose by reducing maternal glucose utilization by increasing utilization of ...

  18. Lactate threshold responses to a season of professional British youth soccer

    PubMed Central

    McMillan, K; Helgerud, J; Grant, S; Newell, J; Wilson, J; Macdonald, R; Hoff, J

    2005-01-01

    Objective: To examine the changes in aerobic endurance performance of professional youth soccer players throughout the soccer season. Methods: Nine youth soccer players were tested at six different time points throughout the soccer season by sub-maximal blood lactate assessment, using an incremental treadmill protocol. Whole blood lactate concentration and heart frequency (Hf) were determined at each exercise stage. Running velocities at the first lactate inflection point (v-Tlac) and at a blood lactate concentration of 4 mmol l–1 (v-4mM) were determined. Results: Running velocity at the two lactate thresholds increased from the start of pre-season training to the early weeks of the competitive season, from 11.67 (0.29) to 12.96 (0.28) km h–1 for v-Tlac, and from 13.62 (0.25) to 14.67 (0.24) km h–1 for v-4mM (p<0.001). However, v-Tlac and v-4mM when expressed relative to maximum heart frequency (Hfmax) remained unchanged. The Hf to blood lactate concentration relationship was unchanged after the pre-season training period. The two expressions of lactate threshold did not reveal differences between each other. Conclusion: Running velocity at v-Tlac and v-4mM increased significantly over the pre-season period, but v-Tlac and v-4mM were unchanged when expressed relative to Hfmax. This finding may indicate that increased endurance performance may be mainly attributable to alterations in Vo2max. Although lactate assessment of soccer players is useful for determining endurance training adaptations in soccer players, additional assessment of the other two determinants of endurance performance (Vo2max and running economy) may provide more useful information for determining physiological adaptations resulting from soccer training and training interventions. PMID:15976165

  19. Enzymatic characterization of a novel bovine liver dihydrodiol dehydrogenase--reaction mechanism and bile acid dehydrogenase activity.

    PubMed

    Nanjo, H; Adachi, H; Morihana, S; Mizoguchi, T; Nishihara, T; Terada, T

    1995-05-11

    Bovine liver cytosolic dihydrodiol dehydrogenase (DD3) has been characterized by its unique dihydrodiol dehydrogenase activity for trans-benzenedihydrodiol (trans-1,2-dihydrobenzene-1,2-diol) with the highest affinity and the greatest velocity among three multiple forms of dihydrodiol dehydrogenases (DD1-DD3). It is the first time that DD3 has shown a significant dehydrogenase activity for (S)-(+)-1-indanol with low Km value (0.33 +/- 0.022 mM) and high K(cat) value (25 +/- 0.79 min-1). The investigation of the product inhibition of (S)-(+)-1-indanol with NADP+ versus 1-indanone and NADPH clearly showed that the enzymatic reaction of DD3 may follow a typical ordered Bi Bi mechanism similar to many aldo/keto reductases. Additionally, DD3 was shown to catalyze the dehydrogenation of bile acids (lithocholic acid, taurolithocholic acid and taurochenodeoxycholic acid) having no 12-hydroxy groups with low Km values (17 +/- 0.65, 33 +/- 1.9 and 890 +/- 73 microM, respectively). In contrast, DD1, 3 alpha-hydroxysteroid dehydrogenase, shows a broad substrate specificity for many bile acids with higher affinity than those of DD3. Competitive inhibition of DD3 with androsterone against dehydrogenase activity for (S)-(+)-1-indanol, trans-benzenedihydrodiol or lithocholic acid suggests that these three substrates bind to the same substrate binding site of DD3, different from the case of human liver bile acid binder/dihydrodiol dehydrogenase (Takikawa, H., Stolz, A., Sugiyama, Y., Yoshida, H., Yamamoto, M. and Kaplowitz, N. (1990) J. Biol. Chem. 265, 2132-2136). Considering the reaction mechanism, DD3 may also play an important role in bile acids metabolism as well as the detoxication of aromatic hydrocarbons.

  20. Differences in the ribosomes prepared from lactating and non-lactating bovine mammary gland

    PubMed Central

    Herrington, M. D.; Hawtrey, A. O.

    1971-01-01

    1. Ribosomes prepared from bovine lactating mammary gland are able to synthesize protein, whereas similar preparations from non-lactating glands are not. Washing the ribosome suspensions through a medium containing 0.5m-ammonium chloride enhanced their ability to incorporate phenylalanine into polyphenylalanine. 2. Ribosomes isolated from non-lactating bovine mammary gland, in contrast with those from rat liver and lactating mammary gland, contained significant amounts of extraneous nucleases. These enzymes could be removed by washing with a medium A buffer containing 0.5m-ammonium chloride. 3. Only those ribosomes from functionally active tissues were able to bind polyuridylic acid and phenylalanyl-tRNA. PMID:5165653

  1. Plasma lactate concentration as a prognostic biomarker in dogs with gastric dilation and volvulus.

    PubMed

    Mooney, Erin; Raw, Cameron; Hughes, Dez

    2014-09-01

    Initial and serial plasma lactate concentrations can be used to guide decision making in individual dogs with GDV but care is necessary in phrasing conversations with owners. Published data suggests that survival is more likely and the chance of complications less in dogs with an initial plasma lactate of <4 mmol/L. An initial lactate >6 mmol/L makes gastric necrosis and greater expense more likely. However, because of the overlap between groups and the good overall survival rates, exploratory laparotomy should always be recommended irrespective of the plasma lactate concentration. Falls in plasma lactate of greater than ~40% after fluid resuscitation are likely to indicate better survival. If the initial plasma lactate concentration is moderately to severely increased (5->10 mmol/L) and a sustained increase in plasma lactate occurs after fluid resuscitation, the cause should be aggressively pursued. Many dogs with persistent hyperlactatemia over 24-48 hours do not survive. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. Influence of lactation and pregnancy + lactation on mechanical properties and mineral content of the rat femur.

    PubMed

    Peng, T C; Kusy, R P; Garner, S C; Hirsch, P F; De Blanco, M C

    1987-06-01

    The quality of bone was assessed from femurs of rats both during lactation and after pregnancy + lactation. Mechanical properties of stiffness, strength, toughness, and ductility were measured, along with standard measurements of dry weight, ash weight, and total bone mineral. No changes occurred during the first week of lactation. During the second and third weeks of lactation all bone parameters except ductility decreased significantly. These data are consistent with bone losing mineral in order to supplement the dietary calcium intake necessary for milk production. In other experiments, femurs were collected from nulliparous rats and from rats that had previously undergone 1-3 pregnancy + lactations. The largest changes in bone mineral and mechanical properties occurred after a single pregnancy + lactation period, although significant further decreases in stiffness and strength occurred after the second pregnancy + lactation. No additional losses occurred following the third pregnancy + lactation. Even 5 months after only one pregnancy + lactation period, the bone quality was still impaired as all bone properties were lower than in nulliparous controls. Because the changes, especially stiffness and strength, were relatively larger than the changes in dry and ash weights of bone, measurements of these mechanical properties provide a more sensitive method to evaluate the quality of bone.

  3. Effects of supplementation and stage of lactation on performance of grazing dairy ewes.

    PubMed

    Mikolayunas, C M; Thomas, D L; Albrecht, K A; Combs, D K; Berger, Y M; Eckerman, S R

    2008-04-01

    The majority of dairy sheep in the world are fed pasture and supplemental grain during lactation; however, no trials have reported the effects of supplementation of dairy ewes grazing improved pastures in North America. In trial 1, 56 three-year-old grazing dairy ewes in early [21 +/- 10 d in milk (DIM)] or late (136 +/- 9 DIM) lactation were fed 0 or 0.82 kg of dry matter/d per ewe of supplement (16.5% crude protein mixture of corn and a soybean meal-based high-protein pellet) in a 2 x 2 factorial arrangement of treatments. There were no significant interactions between stage of lactation and supplementation treatments. Average test-day milk production was higher in early-lactation ewes than in late-lactation ewes (1.74 vs. 1.21 kg/d, respectively). Although test-day milk protein percentage was higher in late-lactation ewes than in early-lactation ewes (5.02 vs. 4.86%, respectively), there was no difference in milk fat percentage between stages of lactation. Supplemented ewes had higher milk production (1.59 vs. 1.36 kg/d, respectively), lower milk fat percentage (5.75 vs. 6.00%, respectively), and lower milk protein percentage (4.84 vs. 5.04%, respectively) than unsupplemented ewes. Milk urea N levels were similar between the 2 stages of lactation and between the 2 supplementation treatments and were above recommended levels for dairy sheep, indicating an excess intake or inefficient utilization of protein for both supplementation treatments. In trial 2, 96 two-, three-, and four-year-old grazing dairy ewes in midlactation (112 +/- 21 DIM) were randomly assigned to 4 treatments of 0, 0.41, 0.82, or 1.24 kg of dry matter/d per ewe of whole corn. Average test-day milk production increased linearly and milk fat percentage decreased quadratically with increasing amounts of corn supplementation. Milk protein yield increased linearly, and milk urea N levels decreased quadratically with increasing amounts of corn supplementation, suggesting an improvement in the

  4. Attentive processes, blood lactate and CrossFit®.

    PubMed

    Perciavalle, Valentina; Marchetta, Nunzio Salvatore; Giustiniani, Salvatore; Borbone, Carlo; Perciavalle, Vincenzo; Petralia, Maria Cristina; Buscemi, Andrea; Coco, Marinella

    2016-11-01

    To analyze the influences of blood lactate produced during a specific session of CrossFit® on intensity and selectivity of attention. The first was evaluated by measuring the reaction time and the second by analyzing divided attention with a dual task. Fifteen male professionals of CrossFit® volunteered in the study. The training session was the Workout Of the Day (WOD) called 15.5, marked as: 27-21-15-9 repetitions (without recovery) in term of calories measured by using a rowing ergometer (e.g. 27 rowed calories) and in term of barbell full squats (raising a weight of 43 kg for men and of 29.5 kg for women). Blood lactate, blood glucose, reaction time, execution time of a dual task, number of errors and number of omissions were measured at rest, at the conclusion of the session and 15 minutes after its end. The levels of the blood lactate before the start of the session were considerably higher than those which normally occur at rest (<2 mmol /L), with a mean value of 4.5 mmol /l (± 1.99 SD). At the end of the workout session the blood lactate exhibited a significant increase, reaching a mean value of 13.8 mmol /l (± 1.18 SD) and then returning to values similar to the initial ones after 15 minutes. Blood glucose did not exhibit any statistically significant differences during the session. Reaction time, execution time, number of errors and number of omissions exhibited a significant worsening concomitantly with the increase in blood lactate. Athletes practicing CrossFit®, with high levels of blood lactate even at rest, should consequently have attentional performances somewhat limited.

  5. Vitamin A supplementation increases ratios of proinflammatory to anti-inflammatory cytokine responses in pregnancy and lactation

    PubMed Central

    Cox, S E; Arthur, P; Kirkwood, B R; Yeboah-Antwi, K; Riley, E M

    2006-01-01

    Vitamin A supplementation reduces child mortality in populations at risk of vitamin A deficiency and may also reduce maternal mortality. One possible explanation for this is that vitamin A deficiency is associated with altered immune function and cytokine dysregulation. Vitamin A deficiency in pregnancy may thus compound the pregnancy-associated bias of cellular immune responses towards Th-2-like responses and exacerbate susceptibility to intracellular pathogens. We assessed mitogen and antigen-induced cytokine responses during pregnancy and lactation in Ghanaian primigravidae receiving either vitamin A supplementation or placebo. This was a double-blind, randomized, placebo-controlled trial of weekly vitamin A supplementation in pregnant and lactating women. Pregnancy compared to postpartum was associated with a suppression of cytokine responses, in particular of the proinflammatory cytokines interferon (IFN)-γ and tumour necrosis factor (TNF)-α. Mitogen-induced TNF-α responses were associated with a decreased risk of peripheral parasitaemia during pregnancy. Furthermore, vitamin A supplementation was significantly associated with an increased ratio of mitogen-induced proinflammatory cytokine (IFN-γ) to anti-inflammatory cytokine (IL-10) during pregnancy and in the postpartum period. The results of this study indicate that suppression of proinflammatory type 1 immune responses and hence immunity to intracellular infections, resulting from the combined effects of pregnancy and vitamin A deficiency, might be ameliorated by vitamin A supplementation. PMID:16734607

  6. Lactate: link between glycolytic and oxidative metabolism.

    PubMed

    Brooks, George A

    2007-01-01

    Once thought to be the consequence of oxygen lack in contracting skeletal muscle, the glycolytic product lactate is formed and utilised continuously under fully aerobic conditions. 'Cell-cell' and 'intracellular lactate shuttle' concepts describe the roles of lactate in delivery of oxidative and gluconeogenic substrates as well as in cell signalling. Examples of cell-cell shuttles include lactate exchanges (i) between white-glycolytic and red-oxidative fibres within a working muscle bed; (ii) between working skeletal muscle and heart; and (iii) between tissues of net lactate release and gluconeogenesis. Lactate shuttles exist in diverse tissues including in the brain, where a shuttle between astrocytes and neurons is linked to glutamatergic signalling. Because lactate, the product of glycogenolysis and glycolysis, is disposed of by oxidative metabolism, lactate shuttling unites the two major processes of cellular energy transduction. Lactate disposal is mainly through oxidation, especially during exercise when oxidation accounts for 70-75% of removal and gluconeogenesis the remainder. Lactate flux occurs down proton and concentration gradients that are established by the mitochondrial lactate oxidation complex. Marathon running is a power activity requiring high glycolytic and oxidative fluxes; such activities require lactate shuttling. Knowledge of the lactate shuttle is yet to be imparted to the sport.

  7. Pretreatment Serum Lactate Dehydrogenase and N Classification Predict Long-Term Survival and Distant Metastasis in Patients With Nasopharyngeal Carcinoma Who Have A Positive Family History of Cancer

    PubMed Central

    Zhang, Wenna; Chen, Yupei; Zhou, Guanqun; Liu, Xu; Chen, Lei; Tang, Linglong; Mao, Yanping; Sun, Ying; Ma, Jun

    2015-01-01

    Abstract The purpose of the present study was to evaluate prognostic factors in patients with nasopharyngeal carcinoma (NPC) from the endemic area of southern China who have a positive family history (FH) of cancer. Retrospective analysis of 600 patients with nondisseminated NPC and a positive FH was conducted. The prognostic value of different factors for overall survival (OS), distant metastasis-free survival (DMFS), disease-free survival (DFS), and local relapse-free survival (LRFS) were assessed using Cox regression models. The 3-year OS, DMFS, DFS, and LRFS rates were 93.8%, 91.3%, 86.3%, and 93.8%, respectively. The FH tumor type was NPC for 226/600 (37.7%) patients and other cancers for 374/600 (62.3%) patients. The 3-year OS and DMFS rates for patients with an FH of NPC were 91.2% and 89.8%, respectively. Thirty of 600 (5.0%) patients had elevated pretreatment serum lactate dehydrogenase (LDH >245.0 IU/L). In multivariate analysis, N classification (HR 4.56, 95% CI 2.13–9.74, P < 0.0001) and elevated pretreatment serum LDH (HR 2.87, 95% CI 1.08–7.62, P = 0.034) were independent prognosticators for OS. Female patients (HR 0.42, 95% CI 0.19–0.95, P = 0.037) and patients with normal pretreatment serum LDH (HR 2.42, 95% CI 1.02–5.78, P = 0.046) had better DMFS. Elevated pretreatment serum LDH and N classification are independent prognostic factors for poorer survival in patients with NPC who have a positive FH of cancer. PMID:26376394

  8. Malate dehydrogenase isozymes in the longnose dace, Rhinichthys cataractae.

    PubMed

    Starzyk, R M; Merritt, R B

    1980-08-01

    The interspecies homology of dace supernatant (A2,AB,B2) and mitochondrial (C2) malate dehydrogenase isozymes has been established through cell fractionation and tissue distribution studies. Isolated supernatant malate dehydrogenase (s-MDH) isozymes show significant differences in Michaelis constants for oxaloacetate and in pH optima. Shifts in s-MDH isozyme pH optima with temperature may result in immediate compensation for increase in ectotherm body pH with decrease in temperature, but duplicate s-MDH isozymes are probably maintained through selection for tissue specific regulation of metabolism.

  9. Protein-protein interactions and substrate channeling in orthologous and chimeric aldolase-dehydrogenase complexes.

    PubMed

    Baker, Perrin; Hillis, Colleen; Carere, Jason; Seah, Stephen Y K

    2012-03-06

    Bacterial aldolase-dehydrogenase complexes catalyze the last steps in the meta cleavage pathway of aromatic hydrocarbon degradation. The aldolase (TTHB246) and dehydrogenase (TTHB247) from Thermus thermophilus were separately expressed and purified from recombinant Escherichia coli. The aldolase forms a dimer, while the dehydrogenase is a monomer; these enzymes can form a stable tetrameric complex in vitro, consisting of two aldolase and two dehydrogenase subunits. Upon complex formation, the K(m) value of 4-hydroxy-2-oxopentanoate, the substrate of TTHB246, is decreased 4-fold while the K(m) of acetaldehyde, the substrate of TTHB247, is increased 3-fold. The k(cat) values of each enzyme were reduced by ~2-fold when they were in a complex. The half-life of TTHB247 at 50 °C increased by ~4-fold when it was in a complex with TTHB246. The acetaldehyde product from TTHB246 could be efficiently channelled directly to TTHB247, but the channeling efficiency for the larger propionaldehyde was ~40% lower. A single A324G substitution in TTHB246 increased the channeling efficiency of propionaldehyde to a value comparable to that of acetaldehyde. Stable and catalytically competent chimeric complexes could be formed between the T. thermophilus enzymes and the orthologous aldolase (BphI) and dehydrogenase (BphJ) from the biphenyl degradation pathway of Burkholderia xenovorans LB400. However, channeling efficiencies for acetaldehyde in these chimeric complexes were ~10%. Structural and sequence analysis suggests that interacting residues in the interface of the aldolase-dehydrogenase complex are highly conserved among homologues, but coevolution of partner enzymes is required to fine-tune this interaction to allow for efficient substrate channeling.

  10. Redox Specificity of 2-Hydroxyacid-Coupled NAD+/NADH Dehydrogenases: A Study Exploiting “Reactive” Arginine as a Reporter of Protein Electrostatics

    PubMed Central

    Durani, Susheel

    2013-01-01

    With “reactive” arginine as a kinetic reporter, 2-hydroxyacid dehydrogenases are assessed in basis of their specialization as NAD+-reducing or NADH-oxidizing enzymes. Specifically, M4 and H4 lactate dehydrogenases (LDHs) and cytoplasmic and mitochondrial malate dehydrogenases (MDHs) are compared to assess if their coenzyme specificity may involve electrostatics of cationic or neutral nicotinamide structure as the basis. The enzymes from diverse eukaryote and prokaryote sources thus are assessed in “reactivity” of functionally-critical arginine as a function of salt concentration and pH. Electrostatic calculations were performed on “reactive” arginines and found good correspondence with experiment. The reductive and oxidative LDHs and MDHs are assessed in their count over ionizable residues and in placement details of the residues in their structures as proteins. The variants found to be high or low in ΔpKa of “reactive” arginine are found to be also strong or weak cations that preferentially oxidize NADH (neutral nicotinamide structure) or reduce NAD+ (cationic nicotinamide structure). The ionized groups of protein structure may thus be important to redox specificity of the enzyme on basis of electrostatic preference for the oxidized (cationic nicotinamide) or reduced (neutral nicotinamide) coenzyme. Detailed comparisons of isozymes establish that the residues contributing in their redox specificity are scrambled in structure of the reductive enzyme. PMID:24391777

  11. Zoledronate prevents lactation induced bone loss and results in additional post-lactation bone mass in mice.

    PubMed

    Wendelboe, Mette Høegh; Thomsen, Jesper Skovhus; Henriksen, Kim; Vegger, Jens Bay; Brüel, Annemarie

    2016-06-01

    In rodents, lactation is associated with a considerable and very rapid bone loss, which almost completely recovers after weaning. The aim of the present study was to investigate whether the bisphosphonate Zoledronate (Zln) can inhibit lactation induced bone loss, and if Zln interferes with recovery of bone mass after lactation has ceased. Seventy-six 10-weeks-old NMRI mice were divided into the following groups: Baseline, Pregnant, Lactation, Lactation+Zln, Recovery, Recovery+Zln, and Virgin Control (age-matched). The lactation period was 12days, then the pups were removed, and thereafter recovery took place for 28days. Zln, 100μg/kg, was given s.c. on the day of delivery, and again 4 and 8days later. Mechanical testing, μCT, and dynamic histomorphometry were performed. At L4, lactation resulted in a substantial loss of bone strength (-55% vs. Pregnant, p<0.01), BV/TV (-40% vs. Pregnant, p<0.01), and trabecular thickness (Tb.Th) (-29% vs. Pregnant, p<0.001). Treatment with Zln completely prevented lactation induced loss of bone strength, BV/TV, and Tb.Th at L4. Full recovery of micro-architectural and mechanical properties was found 28days after weaning in vehicle-treated mice. Interestingly, the recovery group treated with Zln during the lactation period had higher BV/TV (+45%, p<0.01) and Tb.Th (+16%, p<0.05) compared with virgin controls. Similar results were found at the proximal tibia and femur. This indicates that Zln did not interfere with the bone formation taking place after weaning. On this background, we conclude that post-lactation bone formation is not dependent on a preceding lactation induced bone loss. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Dacarbazine with or without oblimersen (a Bcl-2 antisense oligonucleotide) in chemotherapy-naive patients with advanced melanoma and low-normal serum lactate dehydrogenase: 'The AGENDA trial'.

    PubMed

    Bedikian, Agop Y; Garbe, Claus; Conry, Robert; Lebbe, Celeste; Grob, Jean J

    2014-06-01

    In a previous large randomized, open-label study, retrospective subset analysis revealed that the addition of the Bcl-2 antisense oligonucleotide oblimersen to dacarbazine (Dac) significantly improved overall survival, progression-free survival, and the response rate in chemotherapy-naive patients with advanced melanoma and normal baseline serum lactate dehydrogenase (LDH) levels. To confirm and expand on this observation, we conducted a prospective double-blind, placebo-controlled study to determine whether oblimersen augmented the efficacy of Dac in advanced melanoma patients with low-normal baseline LDH levels. A total of 314 chemotherapy-naive patients were randomly assigned to receive Dac (1000 mg/m(2)) preceded by a 5-day continuous intravenous infusion of either oblimersen sodium (7 mg/kg/day) or placebo every 21 days for less than eight cycles. Co-primary efficacy endpoints were overall survival and progression-free survival. Response and progression of the disease were assessed by independent blinded review of computed tomography scan images. No difference in overall nor progression-free survival was observed between the Dac-oblimersen and Dac-placebo groups. Although the overall (17.2 vs. 12.1%) and durable (10.8 vs. 7.6%) response rates numerically favored Dac-oblimersen over Dac-placebo, they did not differ significantly (P=0.19 and 0.32, respectively). The incidence of hematologic adverse events, particularly thrombocytopenia and neutropenia, was higher in the Dac-oblimersen group than in the Dac-placebo group. Withdrawals from the study because of treatment-related adverse events were low (i.e. <2.5%) in both groups. The addition of oblimersen to Dac did not significantly improve overall survival nor progression-free survival in patients with advanced melanoma and low-normal levels of LDH at baseline.

  13. Demonstration of 3 alpha(17 beta)-hydroxysteroid dehydrogenase distinct from 3 alpha-hydroxysteroid dehydrogenase in hamster liver.

    PubMed Central

    Ohmura, M; Hara, A; Nakagawa, M; Sawada, H

    1990-01-01

    NAD(+)-linked and NADP(+)-linked 3 alpha-hydroxysteroid dehydrogenases were purified to homogeneity from hamster liver cytosol. The two monomeric enzymes, although having similar molecular masses of 38,000, differed from each other in pI values, activation energy and heat stability. The two proteins also gave different fragmentation patterns by gel electrophoresis after digestion with protease. The NADP(+)-linked enzyme catalysed the oxidoreduction of various 3 alpha-hydroxysteroids, whereas the NAD(+)-linked enzyme oxidized the 3 alpha-hydroxy group of pregnanes and some bile acids, and the 17 beta-hydroxy group of testosterone and androstanes. The thermal stabilities of the 3 alpha- and 17 beta-hydroxysteroid dehydrogenase activities of the NAD(+)-linked enzyme were identical, and the two enzyme activities were inhibited by mixing 17 beta- and 3 alpha-hydroxysteroid substrates, respectively. Medroxyprogesterone acetate, hexoestrol and 3 beta-hydroxysteroids competitively inhibited 3 alpha- and 17 beta-hydroxysteroid dehydrogenase activities of the enzyme. These results show that hamster liver contains a 3 alpha(17 beta)-hydroxysteroid dehydrogenase structurally and functionally distinct from 3 alpha-hydroxysteroid dehydrogenase. Images Fig. 1. Fig. 2. PMID:2317205

  14. Changes in Plasma Progesterone Levels in the Caudal Vena Cava and the Jugular Vein and Luteinizing Hormone Secretion Pattern After Feeding in Lactating and Non-lactating Dairy Cows

    PubMed Central

    ENDO, Natsumi; NAGAI, Kiyosuke; TANAKA, Tomomi; KAMOMAE, Hideo

    2012-01-01

    Abstract The present study was designed to assess progesterone profiles at the secreted (caudal vena cava) and circulating levels (jugular vein) and luteinizing hormone (LH) secretion pattern in lactating and non-lactating cows with reference to feeding. Four lactating and four non-lactating cycling Holstein cows were examined. Blood samples were collected simultaneously from the caudal vena cava (via a catheter inserted from the coccygeal vein) and the jugular vein every 15 min for 12 h (0500–1700 h) during the functional luteal phase. Cows were fed 50% of the daily diet 6 h after the start of blood sampling. During the 12-h sampling period, mean progesterone concentrations in the caudal vena cava did not differ between lactating and non-lactating cows (49.0 ± 2.9 and 53.3 ± 3.7 ng/ml; mean ± SE), whereas mean progesterone concentrations in the jugular vein in lactating cows were higher than those in non-lactating cows (6.4 ± 0.1 and 5.6 ± 0.1 ng/ml, P < 0.001). Lactating cows had a higher frequency of LH pulses than non-lactating cows (7.0 ± 0.7 and 4.3 ± 0.9 pulses/12 h, P<0.05). The influence of feeding was not observed on LH profiles but was observed on progesterone profiles in both veins. Progesterone concentrations in the caudal vena cava increased after feeding in both groups. Progesterone concentrations in the jugular vein decreased after feeding in lactating cows but not in non-lactating cows. These results indicate the difference in feeding-related changes in progesterone dynamics between lactating and non-lactating cows. PMID:23171608

  15. Defining lactation acuity to improve patient safety and outcomes.

    PubMed

    Mannel, Rebecca

    2011-05-01

    While substantial evidence exists identifying risks factors associated with premature weaning from breastfeeding, there are no previously published definitions of patient acuity in the lactation field. This article defines evidence-based levels of lactation acuity based on maternal and infant characteristics. Patient acuity, matching severity of illness to intensity of care required, is an important determinant of patient safety and outcomes. It is often used as part of a patient classification system to determine staffing needs and acceptable workloads in health care settings. As acuity increases, more resources, including more skilled clinicians, are needed to provide optimal care. Developing an evidence-based definition of lactation acuity can help to standardize terminology, more effectively distribute health care staff resources, encourage research to verify the validity and reliability of lactation acuity, and potentially improve breastfeeding initiation and duration rates.

  16. Selenium and Antioxidant Status in Dairy Cows at Different Stages of Lactation.

    PubMed

    Gong, Jian; Xiao, Min

    2016-05-01

    Thirty-five multiparous Holstein cows averaging 550 ± 50 kg of body weight and in 2 to 4 parity were divided into three groups according to lactation stage (group A: nine cows from 4 to 1 weeks prepartum; group B: 11 cows from 1 to 30 days postpartum; group C: 15 cows from 30 to 100 days postpartum). Selenium concentration, malondialdehyde (MDA) level, glutathione peroxidase (GSH-Px) activity, thioredoxin reductase (TrxR) activity, and total antioxidant status (TAS) in serum were determined to evaluate selenium and antioxidant status in dairy cows at different stages of lactation. The results showed that mean serum selenium concentration, MDA level, and GSH-Px activity of cows in early lactation increased significantly (P < 0.05) when compared with cows in the dry period and peak lactation. Conversely, serum TrxR activity and TAS declined during this period (P < 0.05). The increase of serum MDA level during early lactation indicate that the reactive oxygen species, including lipid hydroperoxides, increase in this period, thus placing the cows at a greater risk of oxidative stress. The significant decrease in TrxR activity that is accompanied with a decrease in TAS during early lactation suggests that dairy cows have low antioxidant defense in this period and TrxR may be an important antioxidant defense mechanism in transition dairy cows.

  17. Base compositions of genes encoding alpha-actin and lactate dehydrogenase-A from differently adapted vertebrates show no temperature-adaptive variation in G + C content.

    PubMed

    Ream, Rachael A; Johns, Glenn C; Somero, George N

    2003-01-01

    There is a long-standing debate in molecular evolution concerning the putative importance of GC content in adapting the thermal stabilities of DNA and RNA. Most studies of this relationship have examined broad-scale compositional patterns, for example, total GC percentages in genomes and occurrence of GC-rich isochores. Few studies have systematically examined the GC contents of individual orthologous genes from differently thermally adapted species. When this has been done, the emphasis has been on comparing large numbers of genes in only a few species. We have approached the GC-adaptation temperature hypothesis in a different manner by examining patterns of base composition of genes encoding lactate dehydrogenase-A (ldh-a) and alpha-actin (alpha-actin) from 51 species of vertebrates whose adaptation temperatures ranged from -1.86 degrees C (Antarctic fishes) to approximately 45 degrees C (desert reptile). No significant positive correlation was found between any index of GC content (GC content of the entire sequence, GC content of the third codon position [GC(3)], and GC content at fourfold degenerate sites [GC(4)]) and any index of adaptation temperature (maximal, mean, or minimal body temperature). For alpha-actin, slopes of regression lines for all comparisons did not differ significantly from zero. For ldh-a, negative correlations between adaptation temperature and total GC content, GC(3), and GC(4) were observed but were shown to be due entirely to phylogenetic influences (as revealed by independent contrast analyses). This comparison of GC content across a wide range of ectothermic ("cold-blooded") and endothermic ("warm-blooded") vertebrates revealed that frogs of the genus Xenopus, which have commonly been used as a representative cold-blooded species, in fact are outliers among ectotherms for the alpha-actin analyses, raising concern about the appropriateness of choosing these amphibians as representative of ectothermic vertebrates in general. Our study

  18. Yeast surface display of dehydrogenases in microbial fuel-cells.

    PubMed

    Gal, Idan; Schlesinger, Orr; Amir, Liron; Alfonta, Lital

    2016-12-01

    Two dehydrogenases, cellobiose dehydrogenase from Corynascus thermophilus and pyranose dehydrogenase from Agaricus meleagris, were displayed for the first time on the surface of Saccharomyces cerevisiae using the yeast surface display system. Surface displayed dehydrogenases were used in a microbial fuel cell and generated high power outputs. Surface displayed cellobiose dehydrogenase has demonstrated a midpoint potential of -28mV (vs. Ag/AgCl) at pH=6.5 and was used in a mediator-less anode compartment of a microbial fuel cell producing a power output of 3.3μWcm(-2) using lactose as fuel. Surface-displayed pyranose dehydrogenase was used in a microbial fuel cell and generated high power outputs using different substrates, the highest power output that was achieved was 3.9μWcm(-2) using d-xylose. These results demonstrate that surface displayed cellobiose dehydrogenase and pyranose dehydrogenase may successfully be used in microbial bioelectrochemical systems. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Kinetics of lactate metabolism during acellular normothermic ex vivo lung perfusion.

    PubMed

    Koike, Terumoto; Yeung, Jonathan C; Cypel, Marcelo; Rubacha, Matthew; Matsuda, Yasushi; Sato, Masaaki; Waddell, Thomas K; Liu, Mingyao; Keshavjee, Shaf

    2011-12-01

    Plasma lactate has been used as a marker of poor prognosis in clinical conditions. However, the relationship between lactate production and lung function during acellular normothermic ex vivo lung perfusion (EVLP) is unclear. We investigated the kinetics of lactate metabolism during EVLP and the correlation of this marker with outcomes after transplant. Human donor lungs in our clinical EVLP trial (CLs; n = 28) and rejected donor lungs for experimental use (Els; n = 8) were perfused ex vivo using the Toronto technique. Lactate level, lactate/pyruvate (L/P) ratio, and glucose level in the perfusate were measured. In CLs, we examined the relationship between lactate metabolism during EVLP and early post-transplant outcomes. The hypoxia-inducible factor 1 sub-unit 1α (HIF-1α) level in lung tissue was examined in ELs. We performed double-lung EVLP in CLs and single-lung EVLP in ELs. In CLs, the lactate and L/P ratios at the end of EVLP had no correlation with early post-transplant outcomes despite lactate elevation during EVLP. Although lactate elevation was also present in all ELs, we were able to identify 2 groups based on L/P ratio at the end of EVLP. The group with the high L/P ratio had higher airway pressure during EVLP and higher HIF-1α in lung tissue at the end of EVLP. Lactate increases seen in the EVLP perfusate most often represent physiologic lactate production by the lung in a setting with reduced lactate clearance. Thus, patients who underwent transplantation after EVLP had good outcomes despite lactate elevation during EVLP. Copyright © 2011 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.

  20. Osteoporosis presenting in pregnancy, puerperium, and lactation.

    PubMed

    Kovacs, Christopher S

    2014-12-01

    To describe our current state of knowledge about the pathophysiology, incidence, and treatment of osteoporosis that presents during pregnancy, puerperium, and lactation. When vertebral fractures occur in pregnant or lactating women, it is usually unknown whether the skeleton was normal before pregnancy. Maternal adaptations increase bone resorption modestly during pregnancy but markedly during lactation. The net bone loss may occasionally precipitate fractures, especially in women who have underlying low bone mass or skeletal fragility prior to pregnancy. Bone mass and strength are normally restored postweaning. Transient osteoporosis of the hip is a sporadic disorder localized to one or both femoral heads; it is not due to generalized skeletal resorption. Anecdotal reports have used bisphosphonates, strontium ranelate, teriparatide, or vertebroplasty/kyphoplasty to treat postpartum vertebral fractures, but it is unclear whether these therapies had any added benefit over the spontaneous skeletal recovery that normally occurs after weaning. These relatively rare fragility fractures result from multifactorial causes, including skeletal disorders that precede pregnancy, and structural and metabolic stresses that can compromise skeletal strength during pregnancy and lactation. Further study is needed to determine when pharmacological or surgical therapy is warranted instead of conservative or expectant management.

  1. Dengue Virus NS1 Protein Modulates Cellular Energy Metabolism by Increasing Glyceraldehyde-3-Phosphate Dehydrogenase Activity

    PubMed Central

    Allonso, Diego; Andrade, Iamara S.; Conde, Jonas N.; Coelho, Diego R.; Rocha, Daniele C. P.; da Silva, Manuela L.; Ventura, Gustavo T.

    2015-01-01

    ABSTRACT Dengue is one of the main public health concerns worldwide. Recent estimates indicate that over 390 million people are infected annually with the dengue virus (DENV), resulting in thousands of deaths. Among the DENV nonstructural proteins, the NS1 protein is the only one whose function during replication is still unknown. NS1 is a 46- to 55-kDa glycoprotein commonly found as both a membrane-associated homodimer and a soluble hexameric barrel-shaped lipoprotein. Despite its role in the pathogenic process, NS1 is essential for proper RNA accumulation and virus production. In the present study, we identified that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) interacts with intracellular NS1. Molecular docking revealed that this interaction occurs through the hydrophobic protrusion of NS1 and the hydrophobic residues located at the opposite side of the catalytic site. Moreover, addition of purified recombinant NS1 enhanced the glycolytic activity of GAPDH in vitro. Interestingly, we observed that DENV infection promoted the relocalization of GAPDH to the perinuclear region, where NS1 is commonly found. Both DENV infection and expression of NS1 itself resulted in increased GAPDH activity. Our findings indicate that the NS1 protein acts to increase glycolytic flux and, consequently, energy production, which is consistent with the recent finding that DENV induces and requires glycolysis for proper replication. This is the first report to propose that NS1 is an important modulator of cellular energy metabolism. The data presented here provide new insights that may be useful for further drug design and the development of alternative antiviral therapies against DENV. IMPORTANCE Dengue represents a serious public health problem worldwide and is caused by infection with dengue virus (DENV). Estimates indicate that half of the global population is at risk of infection, with almost 400 million cases occurring per year. The NS1 glycoprotein is found in both the

  2. Dengue Virus NS1 Protein Modulates Cellular Energy Metabolism by Increasing Glyceraldehyde-3-Phosphate Dehydrogenase Activity.

    PubMed

    Allonso, Diego; Andrade, Iamara S; Conde, Jonas N; Coelho, Diego R; Rocha, Daniele C P; da Silva, Manuela L; Ventura, Gustavo T; Silva, Emiliana M; Mohana-Borges, Ronaldo

    2015-12-01

    Dengue is one of the main public health concerns worldwide. Recent estimates indicate that over 390 million people are infected annually with the dengue virus (DENV), resulting in thousands of deaths. Among the DENV nonstructural proteins, the NS1 protein is the only one whose function during replication is still unknown. NS1 is a 46- to 55-kDa glycoprotein commonly found as both a membrane-associated homodimer and a soluble hexameric barrel-shaped lipoprotein. Despite its role in the pathogenic process, NS1 is essential for proper RNA accumulation and virus production. In the present study, we identified that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) interacts with intracellular NS1. Molecular docking revealed that this interaction occurs through the hydrophobic protrusion of NS1 and the hydrophobic residues located at the opposite side of the catalytic site. Moreover, addition of purified recombinant NS1 enhanced the glycolytic activity of GAPDH in vitro. Interestingly, we observed that DENV infection promoted the relocalization of GAPDH to the perinuclear region, where NS1 is commonly found. Both DENV infection and expression of NS1 itself resulted in increased GAPDH activity. Our findings indicate that the NS1 protein acts to increase glycolytic flux and, consequently, energy production, which is consistent with the recent finding that DENV induces and requires glycolysis for proper replication. This is the first report to propose that NS1 is an important modulator of cellular energy metabolism. The data presented here provide new insights that may be useful for further drug design and the development of alternative antiviral therapies against DENV. Dengue represents a serious public health problem worldwide and is caused by infection with dengue virus (DENV). Estimates indicate that half of the global population is at risk of infection, with almost 400 million cases occurring per year. The NS1 glycoprotein is found in both the intracellular and the

  3. Influence of oxygen therapy on glucose-lactate metabolism after diffuse brain injury.

    PubMed

    Reinert, Michael; Schaller, Benoit; Widmer, Hans Rudolf; Seiler, Rolf; Bullock, Ross

    2004-08-01

    Severe traumatic brain injury (TBI) imposes a huge metabolic load on brain tissue, which can be summarized initially as a state of hypermetabolism and hyperglycolysis. In experiments O2 consumption has been shown to increase early after trauma, especially in the presence of high lactate levels and forced O2 availability. In recent clinical studies the effect of increasing O2 availability on brain metabolism has been analyzed. By their nature, however, clinical trauma models suffer from a heterogeneous injury distribution. The aim of this study was to analyze, in a standardized diffuse brain injury model, the effect of increasing the fraction of inspired O2 on brain glucose and lactate levels, and to compare this effect with the metabolism of the noninjured sham-operated brain. A diffuse severe TBI model developed by Foda and Maramarou, et al., in which a 420-g weight is dropped from a height of 2 m was used in this study. Forty-one male Wistar rats each weighing approximately 300 g were included. Anesthesized rats were monitored by placing a femoral arterial line for blood pressure and blood was drawn for a blood gas analysis. Two time periods were defined: Period A was defined as preinjury and Period B as postinjury. During Period B two levels of fraction of inspired oxygen (FiO2) were studied: air (FiO2 0.21) and oxygen (FiO2 1). Four groups were studied including sham-operated animals: air-air-sham (AAS); air-O2-sham (AOS); air-air-trauma (AAT); and air-O2-trauma (AOT). In six rats the effect of increasing the FiO2 on serum glucose and lactate was analyzed. During Period B lactate values in the brain determined using microdialysis were significantly lower (p < 0.05) in the AOT group than in the AAT group and glucose values in the brain determined using microdialysis were significantly higher (p < 0.04). No differences were demonstrated in the other groups. Increasing the FiO2 had no significant effect on the serum levels of glucose and lactate. Increasing the Fi

  4. 21 CFR 184.1311 - Ferrous lactate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... prepared by reacting calcium lactate or sodium lactate with ferrous sulfate, direct reaction of lactic acid with iron filings, reaction of ferrous chloride with sodium lactate, or reaction of ferrous sulfate...

  5. 21 CFR 184.1311 - Ferrous lactate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    .... It is prepared by reacting calcium lactate or sodium lactate with ferrous sulfate, direct reaction of lactic acid with iron filings, reaction of ferrous chloride with sodium lactate, or reaction of ferrous...

  6. Mechanism of Calcium Lactate Facilitating Phytic Acid Degradation in Soybean during Germination.

    PubMed

    Hui, Qianru; Yang, Runqiang; Shen, Chang; Zhou, Yulin; Gu, Zhenxin

    2016-07-13

    Calcium lactate facilitates the growth and phytic acid degradation of soybean sprouts, but the mechanism is unclear. In this study, calcium lactate (Ca) and calcium lactate with lanthanum chloride (Ca+La) were used to treat soybean sprouts to reveal the relevant mechanism. Results showed that the phytic acid content decreased and the availability of phosphorus increased under Ca treatment. This must be due to the enhancement of enzyme activity related to phytic acid degradation. In addition, the energy metabolism was accelerated by Ca treatment. The energy status and energy metabolism-associated enzyme activity also increased. However, the transmembrane transport of calcium was inhibited by La(3+) and concentrated in intercellular space or between the cell wall and cell membrane; thus, Ca+La treatment showed reverse results compared with those of Ca treatment. Interestingly, gene expression did not vary in accordance with their enzyme activity. These results demonstrated that calcium lactate increased the rate of phytic acid degradation by enhancing growth, phosphorus metabolism, and energy metabolism.

  7. Training-induced elevations in extracellular lactate in hippocampus and striatum: Dissociations by cognitive strategy and type of reward.

    PubMed

    Newman, Lori A; Scavuzzo, Claire J; Gold, Paul E; Korol, Donna L

    2017-01-01

    Recent evidence suggests that astrocytes convert glucose to lactate, which is released from the astrocytes and supports learning and memory. This report takes a multiple memory perspective to test the role of astrocytes in cognition using real-time lactate measurements during learning and memory. Extracellular lactate levels in the hippocampus or striatum were determined with lactate biosensors while rats were learning place (hippocampus-sensitive) or response (striatum-sensitive) versions of T-mazes. In the first experiment, rats were trained on the place and response tasks to locate a food reward. Extracellular lactate levels in the hippocampus increased beyond those of feeding controls during place training but not during response training. However, striatal lactate levels did not increase beyond those of controls when rats were trained on either the place or the response version of the maze. Because food ingestion itself increased blood glucose and brain lactate levels, the contribution of feeding may have confounded the brain lactate measures. Therefore, we conducted a second similar experiment using water as the reward. A very different pattern of lactate responses to training emerged when water was used as the task reward. First, provision of water itself did not result in large increases in either brain or blood lactate levels. Moreover, extracellular lactate levels increased in the striatum during response but not place learning, whereas extracellular lactate levels in the hippocampus did not differ across tasks. The findings from the two experiments suggest that the relative engagement of the hippocampus and striatum dissociates not only by task but also by reward type. The divergent lactate responses of the hippocampus and striatum in place and response tasks under different reward conditions may reflect ethological constraints tied to foraging for food and water. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Prognostic value of admission serum lactate concentrations in intensive care unit patients.

    PubMed

    Soliman, H M; Vincent, J-L

    2010-01-01

    Although blood lactate concentrations have an established prognostic value in circulatory shock or after cardiac arrest, their relationship with morbidity and length of stay in general intensive care unit (ICU) populations has not been well defined. This study included all 433 patients (246 surgical and 187 medical) consecutively admitted to the Department of medico-surgical intensive care. Hyperlactataemia was defined as a serum lactate concentration > or = 2 mEq/l. On admission, 195 patients (45%) had hyperlactataemia. Hyperlactataemic patients had higher Acute Physiology and Chronic Health Evaluation (APACHE) II (13.3 +/- 6.9 vs 10.0 +/- 5.2) and Sequential Organ Failure Assessment (SOFA) (5.3 +/- 3.3 vs 3.3 +/- 2.3) scores than patients with normal lactate concentrations (both p < 0.01). There was no overall difference in length of ICU stay (LOS) between the two groups but survivors in the hyperlactataemic group had a longer LOS than survivors in the normal lactate group, whereas hyperlactataemic non-survivors had a shorter LOS than normal lactate non-survivors. Mortality was 9% in patients with normal lactate concentrations and 23% in hyperlactataemic patients. The mortality rate increased with increasing lactate concentrations, from 17% in patients with lactate concentrations from 2-4 mEq/l to 64% in those with concentrations more than 8 mEq/l. Non-survivors had higher lactate concentrations than survivors on admission, and after 24 and 48 hours. Risk factors for developing hyperlactataemia that were present on admission were SOFA score > 5, mean arterial blood pressure less than 70 mmHg, blood sugar greater than 110 mg/dl, and current use of vasopressors. Our study documents a direct relationship between the serum lactate level on ICU admission and not only the risk of death in ICU but also the length of ICU stay. Hyperlactataemic survivors have a longer LOS and non-survivors a shorter LOS than normal lactate survivors and non-survivors, respectively.

  9. Lactating Mother and Psychotropic Drugs

    PubMed Central

    Tripathi, B. M.; Majumder, Pradipta

    2010-01-01

    Usage of psychotropics during pregnancy and lactation has always been a topic of debate and controversy. The debate stems from the potential adverse effects on the growing fetus or infants due to the transfer of psychotropic drugs through placenta or breast milk of mothers receiving them; and the problem of discontinuing psychotropics in lactating mother considering chances of relapse. However, most of the psychotropics are found to be relatively safe when used cautiously during the lactation phase. This article describes available data on the use of psychotropics in lactating mothers, in particular, in relation to the safety profile of infants. PMID:21327172

  10. 21 CFR 862.1420 - Isocitric dehydrogenase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... Systems § 862.1420 Isocitric dehydrogenase test system. (a) Identification. An isocitric dehydrogenase test system is a device intended to measure the activity of the enzyme isocitric dehydrogenase in serum... disease such as viral hepatitis, cirrhosis, or acute inflammation of the biliary tract; pulmonary disease...

  11. A mathematical model for lactate transport to red blood cells.

    PubMed

    Wahl, Patrick; Yue, Zengyuan; Zinner, Christoph; Bloch, Wilhelm; Mester, Joachim

    2011-03-01

    A simple mathematical model for the transport of lactate from plasma to red blood cells (RBCs) during and after exercise is proposed based on our experimental studies for the lactate concentrations in RBCs and in plasma. In addition to the influx associated with the plasma-to-RBC lactate concentration gradient, it is argued that an efflux must exist. The efflux rate is assumed to be proportional to the lactate concentration in RBCs. This simple model is justified by the comparison between the model-predicted results and observations: For all 33 cases (11 subjects and 3 different warm-up conditions), the model-predicted time courses of lactate concentrations in RBC are generally in good agreement with observations, and the model-predicted ratios between lactate concentrations in RBCs and in plasma at the peak of lactate concentration in RBCs are very close to the observed values. Two constants, the influx rate coefficient C (1) and the efflux rate coefficient C (2), are involved in the present model. They are determined by the best fit to observations. Although the exact electro-chemical mechanism for the efflux remains to be figured out in the future research, the good agreement of the present model with observations suggests that the efflux must get stronger as the lactate concentration in RBCs increases. The physiological meanings of C (1) and C (2) as well as their potential applications are discussed.

  12. Diaphorase Coupling Protocols for Red-Shifting Dehydrogenase Assays

    PubMed Central

    Davis, Mindy I.; Shen, Min; Simeonov, Anton

    2016-01-01

    Abstract Dehydrogenases are an important target for the development of cancer therapeutics. Dehydrogenases either produce or consume NAD(P)H, which is fluorescent but at a wavelength where many compounds found in chemical libraries are also fluorescent. By coupling dehydrogenases to diaphorase, which utilizes NAD(P)H to produce the fluorescent molecule resorufin from resazurin, the assay can be red-shifted into a spectral region that reduces interference from compound libraries. Dehydrogenases that produce NAD(P)H, such as isocitrate dehydrogenase 1 (IDH1), can be read in kinetic mode. Dehydrogenases that consume NAD(P)H, such as mutant IDH1 R132H, can be read in endpoint mode. Here, we report protocols for robust and miniaturized 1,536-well assays for WT IDH1 and IDH1 R132H coupled to diaphorase, and the counterassays used to further detect compound interference with the coupling reagents. This coupling technique is applicable to dehydrogenases that either produce or consume NAD(P)H, and the examples provided here can act as guidelines for the development of high-throughput screens against this enzyme class. PMID:27078681

  13. Protective effect of 20-hydroxyeicosatetraenoic acid (20-HETE) on adriamycin-induced toxicity of human renal tubular epithelial cell (HK-2).

    PubMed

    Tian, Ting; Li, Jin; Wang, Meng-Ying; Xie, Xian-Fei; Li, Qi-Xiong

    2012-05-15

    20-Hydroxyeicosatetraenoic acid is a cytochrome P4504A11 metabolite of arachidonic acid that plays an important role in the regulation of human renal functions. In the present study, we investigated the role of 20-hydroxyeicosatetraenoic acid on adriamycin induced toxicity in human renal tubular epithelial cells. Results showed that cell viability was decreased significantly and lactate dehydrogenase activity was increased significantly in a concentration-dependent manner when human renal tubular epithelial cells were incubated with adriamycin (10⁻⁷-10⁻³ mol/l) for 24h. In contrast, 20-hydroxyeicosatetraenoic acid (0.1, 1, 10, 50 μmol/l) increased cell survival and decreased lactate dehydrogenase activity concentration dependently in human renal tubular epithelial cells. When 20-hydroxyeicosatetraenoic acid (10, 50 μmol/l) was co-administered with adriamycin (10⁻³ mol/l), it significantly increased cell viability and decreased lactate dehydrogenase activity. On the other hand, N-hydroxy-N'-(4-butyl-2-methylphenyl)formamidine (HET-0016) (1 μM), a selective inhibitor of 20-hydroxyeicosatetraenoic acid synthesizing enzyme exaggerated cell viability reduction and lactate dehydrogenase activity augmentation induced by adriamycin. Adriamycin suppressed the expression of cytochrome P4504A11 gene and its protein production in human renal tubular epithelial cells. Furthermore, adriamycin was more effective than N-hydroxy-N'-(4-butyl-2-methylphenyl)formamidine at lowering the expression of cytochrome P4504A11 gene and its protein. These results suggest that 20-hydroxyeicosatetraenoic acid may protect adriamycin-induced toxicity of human renal tubular epithelial cells, meanwhile, adriamycin-induced toxicity of human renal tubular epithelial cells possibly involves inhibiting cytochrome P4504A11 expression. Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.

  14. Enhanced pyruvate dehydrogenase activity improves cardiac outcomes in a murine model of cardiac arrest.

    PubMed

    Piao, Lin; Fang, Yong-Hu; Kubler, Manfred M; Donnino, Michael W; Sharp, Willard W

    2017-01-01

    Post-ischemic changes in cellular metabolism alter myocardial and neurological function. Pyruvate dehydrogenase (PDH), the limiting step in mitochondrial glucose oxidation, is inhibited by increased expression of PDH kinase (PDK) during ischemia/reperfusion injury. This results in decreased utilization of glucose to generate cellular ATP. Post-cardiac arrest (CA) hypothermia improves outcomes and alters metabolism, but its influence on PDH and PDK activity following CA are unknown. We hypothesized that therapeutic hypothermia (TH) following CA is associated with the inhibition of PDK activity and increased PDH activity. We further hypothesized that an inhibitor of PDK activity, dichloroacetate (DCA), would improve PDH activity and post-CA outcomes. Anesthetized and ventilated adult female C57BL/6 wild-type mice underwent a 12-minute KCl-induced CA followed by cardiopulmonary resuscitation. Compared to normothermic (37°C) CA controls, administering TH (30°C) improved overall survival (72-hour survival rate: 62.5% vs. 28.6%, P<0.001), post-resuscitation myocardial function (ejection fraction: 50.9±3.1% vs. 27.2±2.0%, P<0.001; aorta systolic pressure: 132.7±7.3 vs. 72.3±3.0 mmHg, P<0.001), and neurological scores at 72-hour post CA (9.5±1.3 vs. 5.4±1.3, P<0.05). In both heart and brain, CA increased lactate concentrations (1.9-fold and 3.1-fold increase, respectively, P<0.01), decreased PDH enzyme activity (24% and 50% reduction, respectively, P<0.01), and increased PDK protein expressions (1.2-fold and 1.9-fold, respectively, P<0.01). In contrast, post-CA treatment with TH normalized lactate concentrations (P<0.01 and P<0.05) and PDK expressions (P<0.001 and P<0.05), while increasing PDH activity (P<0.01 and P<0.01) in both the heart and brain. Additionally, treatment with DCA (0.2 mg/g body weight) 30 min prior to CA improved both myocardial hemodynamics 2 hours post-CA (aortic systolic pressure: 123±3 vs. 96±4 mmHg, P<0.001) and 72-hour survival rates (50

  15. Bioavailability of seleno-compounds in the lactating rat

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Smith, A.M.; Picciano, M.F.

    Previously the authors reported an increased selenium (Se) requirement for lactating rats of at least 0.2 ppm dietary Se if provided as selenite (SEL). In the present study bioavailability of selenomethionine (SEM), Se yeast (SEY) and SEL was assessed. A casein-based diet (0.025 ppm Se) was fed to 9 groups of 8 rats each during pregnancy to produce a marginal Se deficiency. During lactation each group was fed the same diet containing either 0.1, 0.25, or 0.5 ppm Se as SEL, SEM, or SEY. On day 18 of lactation dams and pups were sacrificed and tissue Se and glutathione peroxidasemore » activities (GPx) determined. Although food intake of the dams and growth of the pups did not vary, selenium and GPx activities were dependent upon quantity and form of Se consumed. Using slope-ratio analysis linear increases in blood, tissue and milk Se content the bioavailabilities were SEM>SEY>SEL. Maximal GPx depended on the form of dietary Se with SEM>SEY>SEL. Maximal GPx occurred at 0.25 ppm dietary Se as SEM and SEY, but did not reach this activity when fed at 0.5 ppm Se as SEL. These results indicate that regardless of form, the NRC requirement for growing rats of 0.1 ppm Se, is not adequate during lactation to maintain maximum tissue GSH-Px in nursing pups. Based on higher tissue Se in dams and GPx in nursing pups the bioavailability of dietary Se to the lactating rat is greater when fed as SEM and SEY than as SEL.« less

  16. 21 CFR 862.1670 - Sorbitol dehydrogenase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... Systems § 862.1670 Sorbitol dehydrogenase test system. (a) Identification. A sorbitol dehydrogenase test system is a device intended to measure the activity of the enzyme sorbitol dehydrogenase in serum... cirrhosis or acute hepatitis. (b) Classification. Class I (general controls). The device is exempt from the...

  17. Economic weights for genetic improvement of lactation persistency and milk yield.

    PubMed

    Togashi, K; Lin, C Y

    2009-06-01

    This study aimed to establish a criterion for measuring the relative weight of lactation persistency (the ratio of yield at 280 d in milk to peak yield) in restricted selection index for the improvement of net merit comprising 3-parity total yield and total lactation persistency. The restricted selection index was compared with selection based on first-lactation total milk yield (I(1)), the first-two-lactation total yield (I(2)), and first-three-lactation total yield (I(3)). Results show that genetic response in net merit due to selection on restricted selection index could be greater than, equal to, or less than that due to the unrestricted index depending upon the relative weight of lactation persistency and the restriction level imposed. When the relative weight of total lactation persistency is equal to the criterion, the restricted selection index is equal to the selection method compared (I(1), I(2), or I(3)). The restricted selection index yielded a greater response when the relative weight of total lactation persistency was above the criterion, but a lower response when it was below the criterion. The criterion varied depending upon the restriction level (c) imposed and the selection criteria compared. A curvilinear relationship (concave curve) exists between the criterion and the restricted level. The criterion increases as the restriction level deviates in either direction from 1.5. Without prior information of the economic weight of lactation persistency, the imposition of the restriction level of 1.5 on lactation persistency would maximize change in net merit. The procedure presented allows for simultaneous modification of multi-parity lactation curves.

  18. Serum Lactate Predicts Adverse Outcomes in Emergency Department Patients With and Without Infection.

    PubMed

    Oedorf, Kimie; Day, Danielle E; Lior, Yotam; Novack, Victor; Sanchez, Leon D; Wolfe, Richard E; Kirkegaard, Hans; Shapiro, Nathan I; Henning, Daniel J

    2017-02-01

    Lactate levels are increasingly used to risk stratify emergency department (ED) patients with and without infection. Whether a serum lactate provides similar prognostic value across diseases is not fully elucidated. This study assesses the prognostic value of serum lactate in ED patients with and without infection to both report and compare relative predictive value across etiologies. We conducted a prospective, observational study of ED patients displaying abnormal vital signs (AVS) (heart rate ≥130 bpm, respiratory rate ≥24 bpm, shock index ≥1, and/or systolic blood pressure <90 mmHg). The primary outcome, deterioration, was a composite of acute renal failure, non-elective intubation, vasopressor administration or in-hospital mortality. Of the 1,152 patients with AVS who were screened, 488 patients met the current study criteria: 34% deteriorated and 12.5% died. The deterioration rate was 88/342 (26%, 95% CI: 21 - 30%) for lactate < 2.5 mmol/L, 47/90 (52%, 42 - 63%) for lactate 2.5 - 4.0 mmol/L, and 33/46 (72%, 59 - 85%) for lactate >4.0mmol/L. Trended stratified lactate levels were associated with deterioration for both infected (p<0.01) and non-infected (p<0.01) patients. In the logistic regression models, lactate > 4mmol/L was an independent predictor of deterioration for patients with infection (OR 4.8, 95% CI: 1.7 - 14.1) and without infection (OR 4.4, 1.7 - 11.5). Lactate levels can risk stratify patients with AVS who have increased risk of adverse outcomes regardless of infection status.

  19. Cumulative lactate and hospital mortality in ICU patients

    PubMed Central

    2013-01-01

    Background Both hyperlactatemia and persistence of hyperlactatemia have been associated with bad outcome. We compared lactate and lactate-derived variables in outcome prediction. Methods Retrospective observational study. Case records from 2,251 consecutive intensive care unit (ICU) patients admitted between 2001 and 2007 were analyzed. Baseline characteristics, all lactate measurements, and in-hospital mortality were recorded. The time integral of arterial blood lactate levels above the upper normal threshold of 2.2 mmol/L (lactate-time-integral), maximum lactate (max-lactate), and time-to-first-normalization were calculated. Survivors and nonsurvivors were compared and receiver operating characteristic (ROC) analysis were applied. Results A total of 20,755 lactate measurements were analyzed. Data are srpehown as median [interquartile range]. In nonsurvivors (n = 405) lactate-time-integral (192 [0–1881] min·mmol/L) and time-to-first normalization (44.0 [0–427] min) were higher than in hospital survivors (n = 1846; 0 [0–134] min·mmol/L and 0 [0–75] min, respectively; all p < 0.001). Normalization of lactate <6 hours after ICU admission revealed better survival compared with normalization of lactate >6 hours (mortality 16.6% vs. 24.4%; p < 0.001). AUC of ROC curves to predict in-hospital mortality was the largest for max-lactate, whereas it was not different among all other lactate derived variables (all p > 0.05). The area under the ROC curves for admission lactate and lactate-time-integral was not different (p = 0.36). Conclusions Hyperlactatemia is associated with in-hospital mortality in a heterogeneous ICU population. In our patients, lactate peak values predicted in-hospital mortality equally well as lactate-time-integral of arterial blood lactate levels above the upper normal threshold. PMID:23446002

  20. Reliability of the Lactate Scout point-of-care instrument for the determination of blood L-lactate concentration in sheep.

    PubMed

    Kaynar, Ozgur; Karapinar, Tolga; Hayirli, Armagan; Baydar, Ersoy

    2015-12-01

    Data on accuracy and precision of the Lactate Scout point-of-care (POC) analyzer in ovine medicine are lacking. The purpose of the study was to assess the reliability of the Lactate Scout in sheep. Fifty-seven sheep at varying ages with various diseases were included. Blood lactate concentration in samples collected from the jugular vein was measured immediately on the Lactate Scout. Plasma L-lactate concentration was measured by the Cobas autoanalyzer as the reference method. Data were subjected to Student's t-test, Passing-Bablok regression, and Bland-Altman plot analyses for comparison and assessment of accuracy, precision, and reliability. Plasma l-lactate concentration was consistently lower than blood L-lactate concentration (3.06 ± 0.24 vs 3.3 ± 0.3 mmol/L, P < .0001). There was a positive correlation between plasma and blood L-lactate concentrations (r = .98, P < .0001). The Lactate Scout had 99% accuracy and 98% precision with the reference method. Blood (Y) and plasma (X) L-lactate concentrations were fitted to Y = 0.28 + 1.00 · X, with a residual standard deviation of 0.31 and a negligible deviation from the identity line (P = .93). The bias was fitted to Y = 0.10 + 0.05 · X, with Sy.x of 0.44 (P < .07). The Lactate Scout has high accuracy and precision, with a negligible bias. It is a reliable POC analyzer to assess L-lactate concentration in ovine medicine. © 2015 American Society for Veterinary Clinical Pathology.

  1. Lactate Parameters Predict Clinical Outcomes in Patients with Nonvariceal Upper Gastrointestinal Bleeding.

    PubMed

    Lee, Seung Hoon; Min, Yang Won; Bae, Joohwan; Lee, Hyuk; Min, Byung Hoon; Lee, Jun Haeng; Rhee, Poong Lyul; Kim, Jae J

    2017-11-01

    The predictive role of lactate in patients with nonvariceal upper gastrointestinal bleeding (NVUGIB) has been suggested. This study evaluated several lactate parameters in terms of predicting outcomes of bleeding patients and sought to establish a new scoring model by combining lactate parameters and the AIMS65 score. A total of 114 patients with NVUGIB who underwent serum lactate level testing at least twice and endoscopic hemostasis within 24 hours after admission were retrospectively analyzed. The associations between five lactate parameters and clinical outcomes were evaluated and the predictive power of lactate parameter combined AIMS65s (L-AIMS65s) and AIMS56 scoring was compared. The most common cause of bleeding was gastric ulcer (48.2%). Lactate clearance rate (LCR) was associated with 30-day rebleeding (odds ratio [OR], 0.931; 95% confidence interval [CI], 0.872-0.994; P = 0.033). Initial lactate (OR, 1.313; 95% CI, 1.050-1.643; P = 0.017), maximal lactate (OR, 1.277; 95% CI, 1.037-1.573; P = 0.021), and average lactate (OR, 1.535; 95% CI, 1.137-2.072; P = 0.005) levels were associated with 30-day mortality. Initial lactate (OR, 1.213; 95% CI, 1.027-1.432; P = 0.023), maximal lactate (OR, 1.271; 95% CI, 1.074-1.504; P = 0.005), and average lactate (OR, 1.501; 95% CI, 1.150-1.959; P = 0.003) levels were associated with admission over 7 days. Although L-AIMS65s showed the highest area under the curve for prediction of each outcome, differences between L-AIMS65s and AIMS65 did not reach statistical significance. In conclusion, lactate parameters have a prognostic role in patients with NVUGIB. However, they do not increase the predictive power of AIMS65 when combined. © 2017 The Korean Academy of Medical Sciences.

  2. Calcitonin plays a critical role in regulating skeletal mineral metabolism during lactation.

    PubMed

    Woodrow, Janine P; Sharpe, Christopher J; Fudge, Neva J; Hoff, Ana O; Gagel, Robert F; Kovacs, Christopher S

    2006-09-01

    The maternal skeleton rapidly demineralizes during lactation to provide calcium to milk, responding to the stimuli of estrogen deficiency and mammary-secreted PTH-related protein. We used calcitonin/calcitonin gene-related peptide-alpha (Ctcgrp) null mice to determine whether calcitonin also modulates lactational mineral metabolism. During 21 d of lactation, spine bone mineral content dropped 53.6% in Ctcgrp nulls vs. 23.6% in wild-type (WT) siblings (P < 0.0002). After weaning, bone mineral content returned fully to baseline in 18.1 d in Ctcgrp null vs. 13.1 d in WT (P < 0.01) mice. Daily treatment with salmon calcitonin from the onset of lactation normalized the losses in Ctcgrp null mice, whereas calcitonin gene-related peptide-alpha or vehicle was without effect. Compared with WT, Ctcgrp null mice had increased circulating levels of PTH and up-regulation of mammary gland PTH-related protein mRNA. In addition, lactation caused the Ctcgrp null skeleton to undergo more trabecular thinning and increased trabecular separation compared with WT. Our studies confirm that an important physiological role of calcitonin is to protect the maternal skeleton against excessive resorption and attendant fragility during lactation and reveal that the postweaning skeleton has the remarkable ability to rapidly recover even from losses of over 50% of skeletal mineral content.

  3. Relationships between certain metabolic diseases and selected serum biochemical parameters in seropositive dairy cows against Neospora caninum infection in different stages of lactation

    PubMed

    Alekish, Myassar O.; Talafha, Abdelsalam Q; Alshehabat, Musa A; Ismail, Zuhair A Bani

    Neospora caninum is an important cause of abortion in dairy cattle. The general health of affected cows has not been investigated before. Therefore, the main objective of this study was to identify possible relationships between certain metabolic diseases and selected serum biochemical parameters in seropositive dairy cows against N. caninum antibodies in different stages of lactation. The study was carried out using 72 N. caninum seropositive cows and 61 seronegative dairy cows (control). Serum from all cows was tested to determine their N. caninum status (seropositive vs seronegative) using commercially available indirect enzyme-linked immunosorbent assay test kit (iELISA). In addition, serum biochemical parameters including beta-hydroxybutyrate (BHB), glucose, creatinine, blood urea nitrogen, total protein, albumin, alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine transaminase (ALT), lactate dehydrogenase (LDH) and gamma-glutamyltranspeptidase (GGT) were determined using routine laboratory methods. The stage of lactation was obtained at the time of sampling from farm records. Student independent t-test showed that there was a significant difference in the serum concentrations of BHB, AST, ALT, and LDH between seropositive and seronegative cows. There was no significant association between seropositivity and the stage of lactation. However, multivariable logistic regression analysis showed that there was a strong association between seropositivity and BHB concentrations. Results of this study indicate a possible relationship between N. caninum seropositivity and certain metabolic diseases such as ketosis and fatty liver syndrome in dairy cows.

  4. Correlation of low levels of nitrite and high levels of fetal hemoglobin in patients with sickle cell disease at baseline

    PubMed Central

    Elias, Darcielle Bruna Dias; Rocha, Lilianne Brito da Silva; Cavalcante, Maritza Barbosa; Pedrosa, Alano Martins; Justino, Izabel Cristina Bandeira; Gonçalves, Romélia Pinheiro

    2012-01-01

    Background Sickle cell disease is a hemoglobinopathy characterized by hemolytic anemia, increased susceptibility to infections and recurrent vaso-occlusive crises that reduces the quality of life of sufferers. Objective To evaluate the correlation of the levels of lactate dehydrogenase, malonaldehyde and nitrite to fetal hemoglobin in patients with sickle cell disease not under treatment with hydroxyurea in outpatients at a university hospital in Fortaleza, Ceará, Brazil. Methods Forty-four patients diagnosed with sickle cell disease were enrolled at baseline. Diagnosis was confirmed by evaluating the beta globin gene using polymerase chain reaction-restriction fragment length polymorphism. The concentration of fetal hemoglobin was obtained by high-performance liquid chromatography. Serum levels of nitrite, malonaldehyde and lactate dehydrogenase were measured by biochemical methods. Results Significantly higher levels of lactate dehydrogenase, nitrite and malonaldehyde were observed in patients with sickle cell disease compared to a control group. The study of the correlation between fetal hemoglobin levels and these variables showed a negative correlation with nitrite levels. No correlation was found between fetal hemoglobin and malonaldehyde or lactate dehydrogenase. When the study population was stratified according to fetal hemoglobin levels, a decrease in the levels of nitrite was observed with higher levels of fetal hemoglobin (p-value = 0.0415). Conclusion The results show that, similar to fetal hemoglobin levels, the concentration of nitrite can predict the clinical course of the disease, but should not be used alone as a modulator of prognosis in patients with sickle cell disease. PMID:23049438

  5. Glutamate Dehydrogenase from Apodachlya (Oomycetes) 1

    PubMed Central

    Price, Jeffrey S.; Gleason, Frank H.

    1972-01-01

    A glutamate dehydrogenase specific for nicotinamide-adenine-dinucleotide has been purified 50-fold from Apodachlya brachynema (Leptomitales). Certain physical, chemical, and kinetic properties of this enzyme have been studied, particularly specificity for coenzymes and substrates. With glucose as the sole carbon source, the synthesis of glutamate dehydrogenase was repressed, whereas glutamate, proline, alanine, or ornithine plus aspartate as sole carbon sources induced synthesis of the enzyme. These data indicate that the function of this enzyme is primarily degradative, although there is no evidence for a nicotinamide-adenine-dinucleotide-phosphate-specific biosynthetic glutamate dehydrogenase in Apodachlya. PMID:16657902

  6. Deletion of the Glucose-6-Phosphate Dehydrogenase Gene KlZWF1 Affects both Fermentative and Respiratory Metabolism in Kluyveromyces lactis▿

    PubMed Central

    Saliola, Michele; Scappucci, Gina; De Maria, Ilaria; Lodi, Tiziana; Mancini, Patrizia; Falcone, Claudio

    2007-01-01

    In Kluyveromyces lactis, the pentose phosphate pathway is an alternative route for the dissimilation of glucose. The first enzyme of the pathway is the glucose-6-phosphate dehydrogenase (G6PDH), encoded by KlZWF1. We isolated this gene and examined its role. Like ZWF1 of Saccharomyces cerevisiae, KlZWF1 was constitutively expressed, and its deletion led to increased sensitivity to hydrogen peroxide on glucose, but unlike the case for S. cerevisiae, the Klzwf1Δ strain had a reduced biomass yield on fermentative carbon sources as well as on lactate and glycerol. In addition, the reduced yield on glucose was associated with low ethanol production and decreased oxygen consumption, indicating that this gene is required for both fermentation and respiration. On ethanol, however, the mutant showed an increased biomass yield. Moreover, on this substrate, wild-type cells showed an additional band of activity that might correspond to a dimeric form of G6PDH. The partial dimerization of the G6PDH tetramer on ethanol suggested the production of an NADPH excess that was negative for biomass yield. PMID:17085636

  7. The Lactate/Albumin Ratio: A Valuable Tool for Risk Stratification in Septic Patients Admitted to ICU.

    PubMed

    Lichtenauer, Michael; Wernly, Bernhard; Ohnewein, Bernhard; Franz, Marcus; Kabisch, Bjoern; Muessig, Johanna; Masyuk, Maryna; Lauten, Alexander; Schulze, Paul Christian; Hoppe, Uta C; Kelm, Malte; Jung, Christian

    2017-09-02

    The lactate/albumin ratio has been reported to be associated with mortality in pediatric patients with sepsis. We aimed to evaluate the lactate/albumin ratio for its prognostic relevance in a larger collective of critically ill (adult) patients admitted to an intensive care unit (ICU). A total of 348 medical patients admitted to a German ICU for sepsis between 2004 and 2009 were included. Follow-up of patients was performed retrospectively between May 2013 and November 2013. The association of the lactate/albumin ratio (cut-off 0.15) and both in-hospital and post-discharge mortality was investigated. An optimal cut-off was calculated by means of Youden's index. The lactate/albumin ratio was elevated in non-survivors ( p < 0.001). Patients with an increased lactate/albumin ratio were of similar age, but clinically in a poorer condition and had more pronounced laboratory signs of multi-organ failure. An increased lactate/albumin ratio was associated with adverse in-hospital mortality. An optimal cut-off of 0.15 was calculated and was associated with adverse long-term outcome even after correction for APACHE2 and SAPS2. We matched 99 patients with a lactate/albumin ratio >0.15 to case-controls with a lactate/albumin ratio <0.15 corrected for APACHE2 scores: The group with a lactate/albumin ratio >0.15 evidenced adverse in-hospital outcome in a paired analysis with a difference of 27% (95%CI 10-43%; p < 0.01). Regarding long-term mortality, again, patients in the group with a lactate/albumin ratio >0.15 showed adverse outcomes ( p < 0.001). An increased lactate/albumin ratio was significantly associated with an adverse outcome in critically ill patients admitted to an ICU, even after correction for confounders. The lactate/albumin ratio might constitute an independent, readily available, and important parameter for risk stratification in the critically ill.

  8. Lactation stage-dependent expression of transporters in rat whole mammary gland and primary mammary epithelial organoids.

    PubMed

    Gilchrist, Samuel E; Alcorn, Jane

    2010-04-01

    Since solute carrier (SLC) and ATP-binding cassette (ABC) transporters play pivotal roles in the transport of both nutrients and drugs into breast milk, drug-nutrient transport interactions at the lactating mammary gland are possible. Our purpose was to characterize lactation stage-dependent changes in transporter expression in rat mammary gland and isolated mammary epithelial organoids (MEO) to provide additional insight for the safe use of maternal medications during breastfeeding. We used quantitative reverse transcription-polymerase chain reaction to assess the temporal expression patterns of SLC and ABC transporters in rat mammary gland and isolated MEO at different stages of lactation. In whole mammary gland five distinct patterns of expression emerged relative to late gestation: (i) decreasing throughout lactation (Mdr1a, Mdr1b, Mrp1, Octn2, Ent2, Ent3, Ncbt2, Mtx1); (ii) prominent increase in early lactation, which may remain elevated or decline with advancing lactation (Octn1, Cnt2, Cnt3, Ent1, Pept1, Pept2); (iii) constant but decreasing later in lactation (Octn3, Dmt1); (iv) increasing until mid-to-late lactation (Oct1, Cnt1); and (v) prominent increase late in lactation (Ncbt1). In isolated MEO (an enriched source of mammary epithelial cells) major differences in expression patterns were noted for Octn3, Ncbt1, and Mtx1, but otherwise were reasonably similar with the whole mammary gland. In conclusion our study augments existing data on transporter expression in the lactating mammary gland. These data should facilitate investigations into lactation-stage dependent changes in drug or nutrient milk-to-serum concentration ratios, the potential for drug- or disease-transporter interactions, and mechanistic studies of transporter function in the lactating mammary gland.

  9. BIOCHEMICAL EFFECTS IN NORMAL AND STONE FORMING RATS TREATED WITH THE RIPE KERNEL JUICE OF PLANTAIN (MUSA PARADISIACA)

    PubMed Central

    Devi, V. Kalpana; Baskar, R.; Varalakshmi, P.

    1993-01-01

    The effect of Musa paradisiaca stem kernel juice was investigated in experimental urolithiatic rats. Stone forming rats exhibited a significant elevation in the activities of two oxalate synthesizing enzymes - Glycollic acid oxidase and Lactate dehydrogenase. Deposition and excretion of stone forming constituents in kidney and urine were also increased in these rats. The enzyme activities and the level of crystalline components were lowered with the extract treatment. The extract also reduced the activities of urinary alkaline phosphatase, lactate dehydrogenase, r-glutamyl transferase, inorganic pyrophosphatase and β-glucuronidase in calculogenic rats. No appreciable changes were noticed with leucine amino peptidase activity in treated rats. PMID:22556626

  10. Genetic evaluation of lactation persistency for five breeds of dairy cattle.

    PubMed

    Cole, J B; Null, D J

    2009-05-01

    Cows with high lactation persistency tend to produce less milk than expected at the beginning of lactation and more than expected at the end. Best prediction of lactation persistency is calculated as a function of trait-specific standard lactation curves and linear regressions of test-day deviations on days in milk. Because regression coefficients are deviations from a tipping point selected to make yield and lactation persistency phenotypically uncorrelated it should be possible to use 305-d actual yield and lactation persistency to predict yield for lactations with later endpoints. The objectives of this study were to calculate (co)variance components and breeding values for best predictions of lactation persistency of milk (PM), fat (PF), protein (PP), and somatic cell score (PSCS) in breeds other than Holstein, and to demonstrate the calculation of prediction equations for 400-d actual milk yield. Data included lactations from Ayrshire, Brown Swiss, Guernsey (GU), Jersey (JE), and Milking Shorthorn (MS) cows calving since 1997. The number of sires evaluated ranged from 86 (MS) to 3,192 (JE), and mean sire estimated breeding value for PM ranged from 0.001 (Ayrshire) to 0.10 (Brown Swiss); mean estimated breeding value for PSCS ranged from -0.01 (MS) to -0.043 (JE). Heritabilities were generally highest for PM (0.09 to 0.15) and lowest for PSCS (0.03 to 0.06), with PF and PP having intermediate values (0.07 to 0.13). Repeatabilities varied considerably between breeds, ranging from 0.08 (PSCS in GU, JE, and MS) to 0.28 (PM in GU). Genetic correlations of PM, PF, and PP with PSCS were moderate and favorable (negative), indicating that increasing lactation persistency of yield traits is associated with decreases in lactation persistency of SCS, as expected. Genetic correlations among yield and lactation persistency were low to moderate and ranged from -0.55 (PP in GU) to 0.40 (PP in MS). Prediction equations for 400-d milk yield were calculated for each breed by

  11. Prevention of acute/severe hypoglycemia-induced neuron death by lactate administration.

    PubMed

    Won, Seok Joon; Jang, Bong Geom; Yoo, Byung Hoon; Sohn, Min; Lee, Min Woo; Choi, Bo Young; Kim, Jin Hee; Song, Hong Ki; Suh, Sang Won

    2012-06-01

    Hypoglycemia-induced cerebral neuropathy can occur in patients with diabetes who attempt tight control of blood glucose and may lead to cognitive dysfunction. Accumulating evidence from animal models suggests that hypoglycemia-induced neuronal death is not a simple result of glucose deprivation, but is instead the end result of a multifactorial process. In particular, the excessive activation of poly (ADP-ribose) polymerase-1 (PARP-1) consumes cytosolic nicotinamide adenine dinucleotide (NAD(+)), resulting in energy failure. In this study, we investigate whether lactate administration in the absence of cytosolic NAD(+) affords neuroprotection against hypoglycemia-induced neuronal death. Intraperitoneal injection of sodium L-lactate corrected arterial blood pH and blood lactate concentration after hypoglycemia. Lactate administered without glucose was not sufficient to promote electroencephalogram recovery from an isoelectric state during hypoglycemia. However, supplementation of glucose with lactate reduced neuronal death by ∼80% in the hippocampus. Hypoglycemia-induced superoxide production and microglia activation was also substantially reduced by administration of lactate. Taken together, these results suggest an intriguing possibility: that increasing brain lactate following hypoglycemia offsets the decrease in NAD(+) due to overactivation of PARP-1 by acting as an alternative energy substrate that can effectively bypass glycolysis and be fed directly to the citric acid cycle to maintain cellular ATP levels.

  12. Measurements of lactate in exhaled breath condensate at rest and after maximal exercise in young and healthy subjects.

    PubMed

    Marek, E M; Volke, J; Hawener, I; Platen, P; Mückenhoff, K; Marek, W

    2010-03-01

    Arterial lactate concentrations, taken as indicators of physical fitness, in athletes as well as in patients with cardio-respiratory or metabolic diseases, are measured invasively from arterialized ear lobe blood. Currently developed micro enzyme detectors permit a non-invasive measurement of hypoxia-related metabolites such as lactate in exhaled breath condensate (EBC). The aim of our study is to prove whether this technology will replace the traditional measurement of lactate in arterialized blood. Therefore, we determined the functional relation between lactate release in EBC and lactate concentration in blood in young and healthy subjects at rest and after exhausting bicycle exercise. During resting conditions as well as after exhausting bicycle exercise, 100 L of exhaled air along with blood samples from the ear lobe was collected after stationary load conditions in 16 healthy subjects. EBC was obtained by cooling the expired air volume with an ECoScreen I (FILT GmbH, Berlin) condenser. The analysis was performed within 90 min using an ECoCheck ampere meter (FILT GmbH, Berlin). Lactate measurements were performed using a bi-enzyme sensor after lactate oxidase-induced oxidation of lactate to pyruvate and H2O2. The rates of lactate release via the exhaled air were calculated from the lactate concentration, the volume and the collection time of the EBC. The functional relation of lactate release in exhaled air and lactate concentration of arterial blood was computed. At rest, the mean lactate concentration in arterialized blood was 0.93 ± 0.30 mmol L(-1). At a resting ventilation of 11.5 ± 3.4 L min(-1), the collection time for 100 L of exhaled air, Ts, was 8.4 ± 2.9 min, and 1.68 ± 0.40 mL EBC was obtained. In EBC, the lactate concentration was 21.4 ± 7.7 µmol L(-1), and the rate of lactate release rate in collected EBC was 4.5 ± 1.7 nmol min(-1). After maximal exercise load (220 ± 20 W), the blood lactate concentration increased to 10.9 ± 1.8 mmol L(-1

  13. Influence of Asymptomatic Pneumonia on the Response to Hemorrhage and Resuscitation in Swine

    DTIC Science & Technology

    2010-01-01

    and complete blood count (Pentra-120 Hemato- logy Analyzer, ABX Diagnostics, Irvine, CA); 3) total plasma protein, glucose, creatinine , lactate...dehydrogenase (LDH), aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatine kinase (CK), amylase and lactate (Vitros Chemistry System...CK. Creatinine increased at 15 min in both groups and remained elevated throughout the study. Mean total protein, amylase and ALT decreased similarly

  14. myo-Inositol metabolism during lactation and development in the rat. The prevention of lactation-induced fatty liver by dietary myo-inositol.

    PubMed

    Burton, L E; Wells, W W

    1976-11-01

    Effects of dietary myo-inositol deprivation were examined during prenatal and postnatal development and during lactation in the rat. The deficient diet contained no detectable myo-inositol while the supplemented diet contained 0.5% (w/w) myo-inositol while the supplemented diet ct contained 0.5% (w/w) myo-inositol at the expense of sucrose. Both diets contained 25% casein, adequate amounts of all known vitamins, choline, and essential fatty acids as well as 0.5% (w/w) phthalylsulfathiazole to depress myo-inositol contribution to the diet by microorganisms. Pregnant rats of the Holtzman strain were fed the respective diets during gestation and lactation, and pups were fed the corresponding diet after weaning until 3 months of age. There were no significant differen-es in body weight between experimental groups. Supplementation of the diet with myo-inositol significanly increased the levels of myo-inositol in plasma, liver, kidney, and intestine of pups at all ages examined, and significantly increased the levels of myo-inositol in the milk and mammary tissue during lactation. During lactation, the myo-inositol deprived dams developed severe fatty livers (31% w/w) characterized by diminished phosphatidyl-inositol (50%) and total phospholipid phosphorus (57%) levels as compared with controls. After weaning, the liver lipid content of the myo-inositol deprived dams returned to normal (4.5%). The data suggest that a possible threshold level of free myo-inositol (approximately 0.15 mumoles/g lipid-free tissue) was required to prevent fatty liver in lactating dams under these dietary conditions. Effects of the deficient diet on fertility were also examined. Based on sperm count and production of offspring, there were no differnences between the experimental and control males. Females of both groups showed equal ability to produce offspring.

  15. 21 CFR 866.5560 - Lactic dehydrogenase immunological test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5560 Lactic dehydrogenase immunological test system. (a) Identification. A lactic dehydrogenase... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Lactic dehydrogenase immunological test system...

  16. Renal Cortical Pyruvate Depletion during AKI

    PubMed Central

    Johnson, Ali C.M.; Becker, Kirsten

    2014-01-01

    Pyruvate is a key intermediary in energy metabolism and can exert antioxidant and anti-inflammatory effects. However, the fate of pyruvate during AKI remains unknown. Here, we assessed renal cortical pyruvate and its major determinants (glycolysis, gluconeogenesis, pyruvate dehydrogenase [PDH], and H2O2 levels) in mice subjected to unilateral ischemia (15–60 minutes; 0–18 hours of vascular reflow) or glycerol-induced ARF. The fate of postischemic lactate, which can be converted back to pyruvate by lactate dehydrogenase, was also addressed. Ischemia and glycerol each induced persistent pyruvate depletion. During ischemia, decreasing pyruvate levels correlated with increasing lactate levels. During early reperfusion, pyruvate levels remained depressed, but lactate levels fell below control levels, likely as a result of rapid renal lactate efflux. During late reperfusion and glycerol-induced AKI, pyruvate depletion corresponded with increased gluconeogenesis (pyruvate consumption). This finding was underscored by observations that pyruvate injection increased renal cortical glucose content in AKI but not normal kidneys. AKI decreased PDH levels, potentially limiting pyruvate to acetyl CoA conversion. Notably, pyruvate therapy mitigated the severity of AKI. This renoprotection corresponded with increases in cytoprotective heme oxygenase 1 and IL-10 mRNAs, selective reductions in proinflammatory mRNAs (e.g., MCP-1 and TNF-α), and improved tissue ATP levels. Paradoxically, pyruvate increased cortical H2O2 levels. We conclude that AKI induces a profound and persistent depletion of renal cortical pyruvate, which may induce additional injury. PMID:24385590

  17. The effect of lactational mastitis on the macronutrient content of breast milk.

    PubMed

    Say, Birgul; Dizdar, Evrim Alyamaç; Degirmencioglu, Halil; Uras, Nurdan; Sari, Fatma Nur; Oguz, Suna; Canpolat, Fuat Emre

    2016-07-01

    Mastitis in lactating mothers reduces milk production and alters the cellular composition of milk. Changes occurring in the mammary gland during the inflammatory response are believed to increase the permeability of the blood-milk barrier. This study examined the effect of mastitis during lactation on the macronutrient content of breast milk. The study was conducted at Zekai Tahir Burak Maternity Teaching Hospital. Transitional breast milk samples were obtained from term lactating mothers with or without mastitis. Milk protein, fat, carbohydrate, and energy levels were measured using a mid-infrared human milk analyzer. The study recruited 30 term lactating mothers: 15 mothers diagnosed with mastitis and 15 healthy mothers. The characteristics of the mothers in both groups were similar. Fat, carbohydrate, and energy levels were statistically lower in the milk samples of mothers with mastitis compared with the mothers without mastitis. Lactational mastitis was associated with lower breast milk fat, carbohydrate, and energy levels. The local inflammatory response induced by cytokines and increased blood-milk barrier permeability might account for the changes in the fat, carbohydrate, and energy levels of human milk. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  18. Relationship of creatine kinase, aspartate aminotransferase, lactate dehydrogenase, and proteinuria to cardiomyopathy in the owl monkey (Aotus vociferans)

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Gozalo, Alfonso S.; Chavera, Alfonso; Montoya, Enrique J.

    2008-02-01

    The purpose of this study was to determine serum reference values for crea- tine kinase (CK), aspartate aminotransferase (AST), and lactate dehydroge- nase (LDH) in captive-born and wild-caught owl monkeys to assess their usefulness for diagnosing myocardial disease. Urine samples were also collected and semi-quantitative tests performed. There was no statistically significant difference between CK, AST, and LDH when comparing both groups. However, when comparing monkeys with proteinuria to those without proteinuria, a statistically significant difference in CK value was observed (P = 0.021). In addition, the CK/AST ratio revealed that 29% of the animals included in this study hadmore » values suggesting cardiac infarction. Grossly, cardiac concentric hypertrophy of the left ventricle and small, pitted kidneys were the most common findings. Microscopically, myocardial fibrosis, contraction band necrosis, hypertrophy and hyperplasia of coronary arteries, medium-sized renal arteries, and afferent glomerular arteriolae were the most significant lesions, along with increased mesangial matrix and hypercellularity of glomeruli, Bowman’s capsule, and peritubular space fibroplasia. These findings suggest that CK, AST, and LDH along with urinalysis provide a reliable method for diagnosing cardiomyopathies in the owl monkey. In addition, CK/AST ratio, proteinuria, and the observed histological and ultrastructural changes suggest that Aotus vociferans suffer from arterial hypertension and chronic myocardial infarction.« less

  19. A Comparison of Oxidative Lactate Metabolism in Traumatically Injured Brain and Control Brain.

    PubMed

    Jalloh, Ibrahim; Helmy, Adel; Howe, Duncan J; Shannon, Richard J; Grice, Peter; Mason, Andrew; Gallagher, Clare N; Murphy, Michael P; Pickard, John D; Menon, David K; Carpenter, T Adrian; Hutchinson, Peter J; Carpenter, Keri L H

    2018-05-18

    Metabolic abnormalities occur after traumatic brain injury (TBI). Glucose is conventionally regarded as the major energy substrate, although lactate can also be an energy source. We compared 3- 13 C lactate metabolism in TBI with "normal" control brain and muscle, measuring 13 C-glutamine enrichment to assess tricarboxylic acid (TCA) cycle metabolism. Microdialysis catheters in brains of nine patients with severe TBI, five non-TBI brain surgical patients, and five resting muscle (non-TBI) patients were perfused (24 h in brain, 8 h in muscle) with 8 mmol/L sodium 3- 13 C lactate. Microdialysate analysis employed ISCUS and nuclear magnetic resonance. In TBI, with 3- 13 C lactate perfusion, microdialysate glucose concentration increased nonsignificantly (mean +11.9%, p = 0.463), with significant increases (p = 0.028) for lactate (+174%), pyruvate (+35.8%), and lactate/pyruvate ratio (+101.8%). Microdialysate 13 C-glutamine fractional enrichments (median, interquartile range) were: for C4 5.1 (0-11.1) % in TBI and 5.7 (4.6-6.8) % in control brain, for C3 0 (0-5.0) % in TBI and 0 (0-0) % in control brain, and for C2 2.9 (0-5.7) % in TBI and 1.8 (0-3.4) % in control brain. 13 C-enrichments were not statistically different between TBI and control brain, showing both metabolize 3- 13 C lactate via TCA cycle, in contrast to muscle. Several patients with TBI exhibited 13 C-glutamine enrichment above the non-TBI control range, suggesting lactate oxidative metabolism as a TBI "emergency option."

  20. 21 CFR 582.1207 - Calcium lactate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Calcium lactate. 582.1207 Section 582.1207 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Additives § 582.1207 Calcium lactate. (a) Product. Calcium lactate. (b) Conditions of use. This substance is...

  1. 21 CFR 582.1207 - Calcium lactate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Calcium lactate. 582.1207 Section 582.1207 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Additives § 582.1207 Calcium lactate. (a) Product. Calcium lactate. (b) Conditions of use. This substance is...

  2. Plasma lactate concentrations in free-ranging moose (Alces alces) immobilized with etorphine.

    PubMed

    Haga, Henning A; Wenger, Sandra; Hvarnes, Silje; Os, Oystein; Rolandsen, Christer M; Solberg, Erling J

    2009-11-01

    To investigate plasma lactate concentrations of etorphine-immobilized moose in relation to environmental, temporal and physiological parameters. Prospective clinical study. Fourteen female and five male moose (Alces alces), estimated age range 1-7 years. The moose were darted from a helicopter with 7.5 mg etorphine per animal using projectile syringes and a dart gun. Once immobilized, the moose were approached, a venous blood sample was obtained and vital signs including pulse oximetry were recorded. Diprenorphine was administered to reverse the effects of etorphine. Timing of events, ambient temperature and snow depth were recorded. Blood samples were cooled and centrifuged before plasma was harvested and frozen. The plasma was thawed later and lactate analysed. Data were analysed using descriptive statistics and regression analysis. All animals recovered uneventfully and were alive 12 weeks after immobilization. Mean +/- SD plasma lactate was found to be 9.2 +/- 2.1 mmol L(-1). Plasma lactate concentrations were related positively to snow depth and negatively to time from induction of immobilization to blood sampling. The model that best described the variability in plasma lactate concentrations used induction time (time from firing the dart to the moose being immobilized). The second best model included induction time and snow depth. Plasma lactate concentrations in these etorphine-immobilized moose were in the range reported for other immobilized wild ruminants. Decreasing induction time, which may be related to a more profound etorphine effect, and increasing snow depth possibly may increase plasma lactate concentrations in etorphine-immobilized moose.

  3. [The nutrition status of lactating women in China].

    PubMed

    Dong, C X; Yin, S A

    2016-12-06

    Nutritional status of lactating women is considered to be a quantitative indicator reflecting the status of reproductive health. To improve nutrition status of lactating women and promote breastfeeding through targeted intervention measures, their dietary and nutritional situations, and related problems, should be fully understood. Generally, energy and macronutrient intake of lactating women can reach or exceed recommended levels, especially during the first month postpartum. However, the intake of some micronutrients is difficult to meet the requirement. These include vitamin D and vitamin B 12 , iron and zinc, and calcium, if milk and dairy product consumption is very low, suggesting that extra supplementation should be considered. The percentage of obesity or postpartum weight retention showed an increasing trend in urban and rural areas and was related to decreased or delayed breastfeeding or early weaning. Common micronutrient deficiencies included in vitamin D, vitamin B 12 , iron and zinc. In this paper, we reviewed various characteristics of the lactating women, including nutritional status, postpartum weight retention and micronutrient deficiencies. We suggest that improved suggestions be developed for China, based on data from National Nutrition Survey, Chinese National Nutrition and Health Surveillance and related studies conducted over the past 10 years.

  4. GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE-S, A SPERM-SPECIFIC GLYCOLYTIC ENZYME, IS REQUIRED FOR SPERM MOTILITY AND MALE FERTILITY

    EPA Science Inventory

    While glycolysis is highly conserved, it is remarkable that several novel isozymes in this central metabolic pathway are found in mammalian sperm. Glyceraldehyde 3-phosphate dehydrogenase-S (GAPDS) is the product of a mouse gene expressed only during spermatogenesis and, like it...

  5. Kinetic mechanism of Escherichia coli isocitrate dehydrogenase and its inhibition by glyoxylate and oxaloacetate.

    PubMed Central

    Nimmo, H G

    1986-01-01

    The inhibition of Escherichia coli isocitrate dehydrogenase by glyoxylate and oxaloacetate was examined. The shapes of the progress curves in the presence of the inhibitors depended on the order of addition of the assay components. When isocitrate dehydrogenase or NADP+ was added last, the rate slowly decreased until a new, inhibited, steady state was obtained. When isocitrate was added last, the initial rate was almost zero, but the rate increased slowly until the same steady-state value was obtained. Glyoxylate and oxaloacetate gave competitive inhibition against isocitrate and uncompetitive inhibition against NADP+. Product-inhibition studies showed that isocitrate dehydrogenase obeys a compulsory-order mechanism, with coenzyme binding first. Glyoxylate and oxaloacetate bind to and dissociate from isocitrate dehydrogenase slowly. These observations can account for the shapes of the progress curves observed in the presence of the inhibitors. Condensation of glyoxylate and oxaloacetate produced an extremely potent inhibitor of isocitrate dehydrogenase. Analysis of the reaction by h.p.l.c. showed that this correlated with the formation of oxalomalate. This compound decomposed spontaneously in assay mixtures, giving 4-hydroxy-2-oxoglutarate, which was a much less potent inhibitor of the enzyme. Oxalomalate inhibited isocitrate dehydrogenase competitively with respect to isocitrate and was a very poor substrate for the enzyme. The data suggest that the inhibition of isocitrate dehydrogenase by glyoxylate and oxaloacetate is not physiologically significant. PMID:3521584

  6. Bisphenol S Alters the Lactating Mammary Gland and Nursing Behaviors in Mice Exposed During Pregnancy and Lactation.

    PubMed

    LaPlante, Charlotte D; Catanese, Mary C; Bansal, Ruby; Vandenberg, Laura N

    2017-10-01

    High doses of estrogenic pharmaceuticals were once prescribed to women to halt lactation. Yet, the effects of low-level xenoestrogens on lactation remain poorly studied. We investigated the effects of bisphenol S (BPS), an estrogen receptor (ER) agonist, on the lactating mammary gland; the arcuate nucleus, a region of the hypothalamus important for neuroendocrine control of lactational behaviors; and nursing behavior in CD-1 mice. Female mice were exposed to vehicle, 2 or 200 µg BPS/kg/d from pregnancy day 9 until lactational day (LD) 20, and tissues were collected on LD21. Tissues were also collected from a second group at LD2. BPS exposure significantly reduced the fraction of the mammary gland comprised of lobules, the milk-producing units, on LD21, but not LD2. BPS also altered expression of Esr1 and ERα in the mammary gland at LD21, consistent with early involution. In the arcuate nucleus, no changes were observed in expression of signal transducer and activator of transcription 5, a marker of prolactin signaling, or ERα, suggesting that BPS may act directly on the mammary gland. However, observations of nursing behavior collected during the lactational period revealed stage-specific effects on both pup and maternal nursing behaviors; BPS-treated dams spent significantly more time nursing later in the lactational period, and BPS-treated pups were less likely to initiate nursing. Pup growth and development were also stunted. These data indicate that low doses of BPS can alter lactational behaviors and the maternal mammary gland. Together, they support the hypothesis that pregnancy and lactation are sensitive to low-dose xenoestrogen exposures. Copyright © 2017 Endocrine Society.

  7. Exercise induces cerebral VEGF and angiogenesis via the lactate receptor HCAR1

    PubMed Central

    Morland, Cecilie; Andersson, Krister A.; Haugen, Øyvind P.; Hadzic, Alena; Kleppa, Liv; Gille, Andreas; Rinholm, Johanne E.; Palibrk, Vuk; Diget, Elisabeth H.; Kennedy, Lauritz H.; Stølen, Tomas; Hennestad, Eivind; Moldestad, Olve; Cai, Yiqing; Puchades, Maja; Offermanns, Stefan; Vervaeke, Koen; Bjørås, Magnar; Wisløff, Ulrik; Storm-Mathisen, Jon; Bergersen, Linda H.

    2017-01-01

    Physical exercise can improve brain function and delay neurodegeneration; however, the initial signal from muscle to brain is unknown. Here we show that the lactate receptor (HCAR1) is highly enriched in pial fibroblast-like cells that line the vessels supplying blood to the brain, and in pericyte-like cells along intracerebral microvessels. Activation of HCAR1 enhances cerebral vascular endothelial growth factor A (VEGFA) and cerebral angiogenesis. High-intensity interval exercise (5 days weekly for 7 weeks), as well as L-lactate subcutaneous injection that leads to an increase in blood lactate levels similar to exercise, increases brain VEGFA protein and capillary density in wild-type mice, but not in knockout mice lacking HCAR1. In contrast, skeletal muscle shows no vascular HCAR1 expression and no HCAR1-dependent change in vascularization induced by exercise or lactate. Thus, we demonstrate that a substance released by exercising skeletal muscle induces supportive effects in brain through an identified receptor. PMID:28534495

  8. Exercise induces cerebral VEGF and angiogenesis via the lactate receptor HCAR1.

    PubMed

    Morland, Cecilie; Andersson, Krister A; Haugen, Øyvind P; Hadzic, Alena; Kleppa, Liv; Gille, Andreas; Rinholm, Johanne E; Palibrk, Vuk; Diget, Elisabeth H; Kennedy, Lauritz H; Stølen, Tomas; Hennestad, Eivind; Moldestad, Olve; Cai, Yiqing; Puchades, Maja; Offermanns, Stefan; Vervaeke, Koen; Bjørås, Magnar; Wisløff, Ulrik; Storm-Mathisen, Jon; Bergersen, Linda H

    2017-05-23

    Physical exercise can improve brain function and delay neurodegeneration; however, the initial signal from muscle to brain is unknown. Here we show that the lactate receptor (HCAR1) is highly enriched in pial fibroblast-like cells that line the vessels supplying blood to the brain, and in pericyte-like cells along intracerebral microvessels. Activation of HCAR1 enhances cerebral vascular endothelial growth factor A (VEGFA) and cerebral angiogenesis. High-intensity interval exercise (5 days weekly for 7 weeks), as well as L-lactate subcutaneous injection that leads to an increase in blood lactate levels similar to exercise, increases brain VEGFA protein and capillary density in wild-type mice, but not in knockout mice lacking HCAR1. In contrast, skeletal muscle shows no vascular HCAR1 expression and no HCAR1-dependent change in vascularization induced by exercise or lactate. Thus, we demonstrate that a substance released by exercising skeletal muscle induces supportive effects in brain through an identified receptor.

  9. [Secondary osteoporosis UPDATE. Bone metabolic change and osteoporosis during pregnancy and lactation].

    PubMed

    Kurabayashi, Takumi; Tamura, Ryo; Hata, Yuki; Nishijima, Shota; Tsuneki, Ikunosuke; Tamura, Masaki; Yanase, Toru

    2010-05-01

    Calcium transfer from the mother to the infant during pregnancy and lactation plays an extremely important role in the bone health of the mother and neonate. Calcium aids in bone health through all ages but is especially crucial during pregnancy and lactation. Changes in the structure and metabolism of bone during pregnancy and the early stage of postpartum are evaluated by investigating bone mineral density (BMD), bone histomorphometry and bone markers of human or animal models. The bone resorption increased at the end of pregnancy and lactation, and the bone formation increases and the bone structure is almost recovered after cessation of lactating in postpartum. Puerperal BMD remained static over the subsequent 5-10 years. If the women have a low BMD at this stage of their reproductive life, it tends not to improve over this time. Perhaps identification of this at-risk group may lead to effective interventions to reduce fracture risk in later life.

  10. High-Fructose Corn-Syrup-Sweetened Beverage Intake Increases 5-Hour Breast Milk Fructose Concentrations in Lactating Women.

    PubMed

    Berger, Paige K; Fields, David A; Demerath, Ellen W; Fujiwara, Hideji; Goran, Michael I

    2018-05-24

    This study determined the effects of consuming a high-fructose corn syrup (HFCS)-sweetened beverage on breast milk fructose, glucose, and lactose concentrations in lactating women. At six weeks postpartum, lactating mothers ( n = 41) were randomized to a crossover study to consume a commercially available HFCS-sweetened beverage or artificially sweetened control beverage. At each session, mothers pumped a complete breast milk expression every hour for six consecutive hours. The baseline fasting concentrations of breast milk fructose, glucose, and lactose were 5.0 ± 1.3 µg/mL, 0.6 ± 0.3 mg/mL, and 6.8 ± 1.6 g/dL, respectively. The changes over time in breast milk sugars were significant only for fructose (treatment × time, p < 0.01). Post hoc comparisons showed the HFCS-sweetened beverage vs. control beverage increased breast milk fructose at 120 min (8.8 ± 2.1 vs. 5.3 ± 1.9 µg/mL), 180 min (9.4 ± 1.9 vs. 5.2 ± 2.2 µg/mL), 240 min (7.8 ± 1.7 vs. 5.1 ± 1.9 µg/mL), and 300 min (6.9 ± 1.4 vs. 4.9 ± 1.9 µg/mL) (all p < 0.05). The mean incremental area under the curve for breast milk fructose was also different between treatments (14.7 ± 1.2 vs. -2.60 ± 1.2 µg/mL × 360 min, p < 0.01). There was no treatment × time interaction for breast milk glucose or lactose. Our data suggest that the consumption of an HFCS-sweetened beverage increased breast milk fructose concentrations, which remained elevated up to five hours post-consumption.

  11. Knowledge about Iodine in Pregnant and Lactating Women in the Oslo Area, Norway.

    PubMed

    Garnweidner-Holme, Lisa; Aakre, Inger; Lilleengen, Anne Marie; Brantsæter, Anne Lise; Henjum, Sigrun

    2017-05-13

    Lack of knowledge about iodine may be a risk factor for iodine deficiency in pregnant and lactating women. The aim of this study was to assess knowledge about iodine and predictors of iodine knowledge scores among pregnant and lactating women. The study also examined whether iodine knowledge scores were associated with iodine status. A cross-sectional study was performed on 804 pregnant women and 175 lactating women from 18 to 44 years of age in 2016 in the Oslo area, Norway. Knowledge about iodine was collected through a self-administered, paper-based questionnaire. Iodine concentrations in urine and breast milk were measured using an inductively coupled plasma mass spectrometer (ICPMS). 74% of the pregnant women and 55% of the lactating women achieved none to low iodine knowledge scores. Higher educated pregnant women and those who had received information about iodine had significantly higher knowledge scores. In lactating women, increased age was associated with higher knowledge scores. Knowledge scores were not associated with participants' iodine status. This study revealed a lack of knowledge about the importance of iodine in pregnant and lactating women, as well as about the most important dietary sources. Public education initiatives are required to increase the awareness about iodine in these population groups.

  12. ARALAR/AGC1 deficiency, a neurodevelopmental disorder with severe impairment of neuronal mitochondrial respiration, does not produce a primary increase in brain lactate.

    PubMed

    Juaristi, Inés; García-Martín, María L; Rodrigues, Tiago B; Satrústegui, Jorgina; Llorente-Folch, Irene; Pardo, Beatriz

    2017-07-01

    ARALAR/AGC1 (aspartate-glutamate mitochondrial carrier 1) is an important component of the NADH malate-aspartate shuttle (MAS). AGC1-deficiency is a rare disease causing global cerebral hypomyelination, developmental arrest, hypotonia, and epilepsy (OMIM ID #612949); the aralar-KO mouse recapitulates the major findings in humans. This study was aimed at understanding the impact of ARALAR-deficiency in brain lactate levels as a biomarker. We report that lactate was equally abundant in wild-type and aralar-KO mouse brain in vivo at postnatal day 17. We find that lactate production upon mitochondrial blockade depends on up-regulation of lactate formation in astrocytes rather than in neurons. However, ARALAR-deficiency decreased cell respiration in neurons, not astrocytes, which maintained unchanged respiration and lactate production. As the primary site of ARALAR-deficiency is neuronal, this explains the lack of accumulation of brain lactate in ARALAR-deficiency in humans and mice. On the other hand, we find that the cytosolic and mitochondrial components of the glycerol phosphate shuttle are present in astrocytes with similar activities. This suggests that glycerol phosphate shuttle is the main NADH shuttle in astrocytes and explains the absence of effects of ARALAR-deficiency in these cells. © 2017 International Society for Neurochemistry.

  13. Hormonal regulation of the alpha-ketoglutarate dehydrogenase complex in the isolated perfused rat liver.

    PubMed

    Rashed, H M; Waller, F M; Patel, T B

    1988-04-25

    The metabolic flux through the alpha-ketoglutarate dehydrogenase reaction in perfused livers was monitored by measuring the rate of 14CO2 production from [1-14C]alpha-ketoglutarate. The rates of 14CO2 production and glucose production from [1-14C]alpha-ketoglutarate were increased with increasing perfusate alpha-ketoglutarate concentrations. Vasopressin, angiotensin II, and the alpha 1-adrenergic agonist phenylephrine stimulated transiently by 2.5-fold the metabolic flux through the alpha-ketoglutarate dehydrogenase reaction in the presence and absence of Ca2+ in the perfusion medium. High concentrations of glucagon (1 x 10(-8) M) and 8-p-chlorophenylthio-cAMP (100 microM) (data not shown) also stimulated transiently the metabolic flux through the alpha-ketoglutarate dehydrogenase reaction. However, lower glucagon concentrations (1 x 10(-9) M) stimulated the rate of 14CO2 production from [1-14C]alpha-ketoglutarate only under conditions optimized to fix the cellular oxidation-reduction state at an intermediate level, when glucagon (1 x 10(-9) M)-mediated elevation of cAMP content was greater than that observed under highly oxidizing and reducing conditions. These data indicate that agonists which increase cytosolic free Ca2+ levels stimulate the metabolic flux through the alpha-ketoglutarate dehydrogenase complex. Furthermore, the data presented here demonstrate for the first time that physiological glucagon concentrations stimulate the metabolic flux through the alpha-ketoglutarate dehydrogenase reaction only under conditions known to be optimal for glucagon-mediated Ca2+ mobilization in the isolated perfused rat liver.

  14. Structural analyses to identify selective inhibitors of glyceraldehyde 3-phosphate dehydrogenase-S, a sperm-specific glycolytic enzyme

    PubMed Central

    Danshina, Polina V.; Qu, Weidong; Temple, Brenda R.; Rojas, Rafael J.; Miley, Michael J.; Machius, Mischa; Betts, Laurie; O'Brien, Deborah A.

    2016-01-01

    STUDY HYPOTHESIS Detailed structural comparisons of sperm-specific glyceraldehyde 3-phosphate dehydrogenase, spermatogenic (GAPDHS) and the somatic glyceraldehyde 3-phosphate dehydrogenase (GAPDH) isozyme should facilitate the identification of selective GAPDHS inhibitors for contraceptive development. STUDY FINDING This study identified a small-molecule GAPDHS inhibitor with micromolar potency and >10-fold selectivity that exerts the expected inhibitory effects on sperm glycolysis and motility. WHAT IS KNOWN ALREADY Glycolytic ATP production is required for sperm motility and male fertility in many mammalian species. Selective inhibition of GAPDHS, one of the glycolytic isozymes with restricted expression during spermatogenesis, is a potential strategy for the development of a non-hormonal contraceptive that directly blocks sperm function. STUDY DESIGN, SAMPLES/MATERIALS, METHODS Homology modeling and x-ray crystallography were used to identify structural features that are conserved in GAPDHS orthologs in mouse and human sperm, but distinct from the GAPDH orthologs present in somatic tissues. We identified three binding pockets surrounding the substrate and cofactor in these isozymes and conducted a virtual screen to identify small-molecule compounds predicted to bind more tightly to GAPDHS than to GAPDH. Following the production of recombinant human and mouse GAPDHS, candidate compounds were tested in dose–response enzyme assays to identify inhibitors that blocked the activity of GAPDHS more effectively than GAPDH. The effects of a selective inhibitor on the motility of mouse and human sperm were monitored by computer-assisted sperm analysis, and sperm lactate production was measured to assess inhibition of glycolysis in the target cell. MAIN RESULTS AND THE ROLE OF CHANCE Our studies produced the first apoenzyme crystal structures for human and mouse GAPDHS and a 1.73 Å crystal structure for NAD+-bound human GAPDHS, facilitating the identification of unique

  15. Online Lactation Education for Healthcare Providers: A Theoretical Approach to Understanding Learning Outcomes.

    PubMed

    Watkins, Amanda L; Dodgson, Joan E; McClain, Darya Bonds

    2017-11-01

    Breastfeeding competencies are not standardized in healthcare education for any of the health professions. A few continuing education/professional development programs have been implemented, but research regarding the efficacy of these programs is scarce. Research aim: After a 45-hour lactation course, (a) Does breastfeeding knowledge increase? (b) Do beliefs and attitudes about infant feeding improve? (c) Does perceived behavioral control over performance of evidence-based lactation support practices increase? and (d) Do intentions to carry out evidence-based lactation support practices increase? A nonexperimental pretest-posttest self-report survey design was conducted with a nonprobability sample of participants ( N = 71) in a lactation course. Theory of Planned Behavior variables were measured and a before-after course analysis was completed. Significantly higher scores were found on the posttests for knowledge, beliefs about breastfeeding scale, and the perceived behavioral control scale. Participants' self-efficacy increased after the course; their beliefs about social norms and their ability to effect change in their workplaces did not change significantly. Participants' intention to perform actions that are consistent with the evidence-based breastfeeding supportive behaviors increased significantly. Positive beliefs about formula feeding significantly increased; this was unexpected. The Theory of Planned Behavior provided a useful approach for examining more meaningful learning outcomes than the traditional knowledge and/or satisfaction outcomes. This study was the first to suggest that more meaningful learning outcomes are needed to evaluate lactation programs. However, it is not enough to educate healthcare providers in evidence-based practice; the places they practice must have the infrastructure to support evidence-based practice.

  16. A Bacillus subtilis malate dehydrogenase gene.

    PubMed Central

    Jin, S; De Jesús-Berríos, M; Sonenshein, A L

    1996-01-01

    A Bacillus subtilis gene for malate dehydrogenase (citH) was found downstream of genes for citrate synthase and isocitrate dehydrogenase. Disruption of citH caused partial auxotrophy for aspartate and a requirement for aspartate during sporulation. In the absence of aspartate, citH mutant cells were blocked at a late stage of spore formation. PMID:8550482

  17. The Lactate/Albumin Ratio: A Valuable Tool for Risk Stratification in Septic Patients Admitted to ICU

    PubMed Central

    Lichtenauer, Michael; Wernly, Bernhard; Ohnewein, Bernhard; Kabisch, Bjoern; Masyuk, Maryna; Lauten, Alexander; Schulze, Paul Christian; Hoppe, Uta C.; Kelm, Malte; Jung, Christian

    2017-01-01

    The lactate/albumin ratio has been reported to be associated with mortality in pediatric patients with sepsis. We aimed to evaluate the lactate/albumin ratio for its prognostic relevance in a larger collective of critically ill (adult) patients admitted to an intensive care unit (ICU). A total of 348 medical patients admitted to a German ICU for sepsis between 2004 and 2009 were included. Follow-up of patients was performed retrospectively between May 2013 and November 2013. The association of the lactate/albumin ratio (cut-off 0.15) and both in-hospital and post-discharge mortality was investigated. An optimal cut-off was calculated by means of Youden’s index. The lactate/albumin ratio was elevated in non-survivors (p < 0.001). Patients with an increased lactate/albumin ratio were of similar age, but clinically in a poorer condition and had more pronounced laboratory signs of multi-organ failure. An increased lactate/albumin ratio was associated with adverse in-hospital mortality. An optimal cut-off of 0.15 was calculated and was associated with adverse long-term outcome even after correction for APACHE2 and SAPS2. We matched 99 patients with a lactate/albumin ratio >0.15 to case-controls with a lactate/albumin ratio <0.15 corrected for APACHE2 scores: The group with a lactate/albumin ratio >0.15 evidenced adverse in-hospital outcome in a paired analysis with a difference of 27% (95%CI 10–43%; p < 0.01). Regarding long-term mortality, again, patients in the group with a lactate/albumin ratio >0.15 showed adverse outcomes (p < 0.001). An increased lactate/albumin ratio was significantly associated with an adverse outcome in critically ill patients admitted to an ICU, even after correction for confounders. The lactate/albumin ratio might constitute an independent, readily available, and important parameter for risk stratification in the critically ill. PMID:28869492

  18. Breast pain in lactating mothers.

    PubMed

    Leung, S Sf

    2016-08-01

    The number of new mothers who breastfeed has increased dramatically over the last three decades. There is a concern that the present related medical service may be inadequate. Breast pain is the most common complaint among lactating mothers who seek medical help. This study aimed to investigate this problem. Medical records of women who presented with breast pain to a private clinic run by a doctor who was trained as an International Lactation Consultant were reviewed over a period of 6 months in 2015. Most patients were self-referred after chatting online. Assessment included characteristics and duration of pain, treatment prior to consultation, feeding practices, mother's diet, and breast examination. Any site of blockage was identified and relieved. Those with persistent pain were given antibiotics. When there were signs of abscess or abscess that could not be drained, they were referred to a breast surgeon. A total of 69 patients were seen of whom 45 had been breastfeeding for more than 1 month. Pain was experienced for longer than 7 days in 22 women. Antifungal or antibacterial treatment had been unsuccessful in 31 women prior to consultation. The diagnoses were engorgement in five women, blocked duct in 35, mastitis in 13, breast abscess in six, poor positioning and latch in seven, nipple cracks in two, and skin infection in one. Oral antibiotics were prescribed to 21 patients and local antifungal treatment was given to one patient only. Blocked duct was the most common cause of breast pain in lactating mothers. Without prompt relief it is possible that it will progress to mastitis/breast abscess or the mother may discontinue breastfeeding. This may be a suitable time for Hong Kong to set up one or more public full-time breastfeeding clinics to provide a better service to lactating mothers and to facilitate professional training and research.

  19. Urinary D-lactate excretion in infants receiving Lactobacillus johnsonii with formula.

    PubMed

    Haschke-Becher, Elisabeth; Brunser, Oscar; Cruchet, Sylvia; Gotteland, Martin; Haschke, Ferdinand; Bachmann, Claude

    2008-01-01

    Supplementation with certain probiotics can improve gut microbial flora and immune function but should not have adverse effects. This study aimed to assess the risk of D-lactate accumulation and subsequent metabolic acidosis in infants fed on formula containing Lactobacillus johnsonii (La1). In the framework of a double-blind, randomized controlled trial enrolling 71 infants aged 4-5 months, morning urine samples were collected before and 4 weeks after being fed formulas with or without La1 (1 x 10(8)/g powder) or being breastfed. Urinary D- and L-lactate concentrations were assayed by enzymatic, fluorimetric methods and excretion was normalized per mol creatinine. At baseline, no significant differences in urinary D-/L-lactate excretion among the formula-fed and breastfed groups were found. After 4 weeks, D-lactate excretion did not differ between the two formula groups, but was higher in both formula groups than in breastfed infants. In all infants receiving La1, urinary D-lactate concentrations remained within the concentration ranges of age-matched healthy infants which had been determined in an earlier study using the same analytical method. Urinary L-lactate also did not vary over time or among groups. Supplementation of La1 to formula did not affect urinary lactate excretion and there is no evidence of an increased risk of lactic acidosis. Copyright 2008 S. Karger AG, Basel.

  20. Comparison of lactate and bicarbonate buffered haemofiltration fluids: use in critically ill patients.

    PubMed

    Thomas, A N; Guy, J M; Kishen, R; Geraghty, I F; Bowles, B J; Vadgama, P

    1997-06-01

    To compare acid-base balance, lactate concentration, and haemodynamic and O2 transport variables during haemofiltration with replacement fluid containing 44.5 mmol/l Na+ lactate or 40 mmol/l Na+ HCO3- and 3 mmol/l lactic acid. A prospective, randomized trial. A multidisciplinary, adult intensive care unit in a university hospital. Forty acidotic patients who required haemofiltration, were dependent on mechanical ventilation, and had PA catheters in situ. During haemofiltration patients received lactate or bicarbonate replacement fluid at a mean rate of 1.7 l/h (SD 0.3). Arterial blood gases, plasma lactate, and haemodynamic and O2 transport variables were measured before and after 12 and 24 h haemofiltration. Ultrafiltrate was collected for lactate estimation. As means (SD). The net gain of lactate was 63 mmol/h (12 mmol) with Na+ lactate and 0 mmol/h (0.3 mmol) with Na+ HCO3-. There was a significant increase in pH and [lactate] in both groups, but [lactate] was higher in patients receiving lactate. Twenty-one patients survived to ICU discharge, these patients were significantly less acidotic after filtration (lactate group: 0 h: pH 7.23 (0.09), [lactate] 2.4 mmol/l (1.7); 12 h: pH 7.34 (0.09), [lactate] 4.7 mmol/l (2.4); 24 h: pH 7.36 (0.07), [lactate] 4.7 mmol (2.7). HCO3 group: 0 h: pH 7.23 (0.09), [lactate] 2.3 (1.3); 12 h: pH 7.32 (0.06), [lactate] 2.9 mmol/l (1.8); 24 h: pH 7.35 (0.08), [lactate] 2.8 mmol/l (2.0). Base deficit: survivors: 0 h: 9 mmol/l (4); 12 h: 2 mmol/l (3). Non-survivors: 0 h: 10 mmol/l (3); 12 h: 6 mmol/l (3)). Haemodynamic and O2 transport variables were not significantly affected by treatment group or outcome. The degree of correction of acidosis during the first 24 h of haemofiltration was determined by patients outcome but was not affected by the substitution of bicarbonate- for lactate-containing replacement fluids.