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

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.

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.

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

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.

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.

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.

[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.

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.

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

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.

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.

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

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.

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

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.

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

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

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.

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.

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…

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

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.

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.

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

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

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

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.

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.

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.

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.

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

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.

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.

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.

[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.

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.

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

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.

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.

A temporary local energy pool coupled to neuronal activity: fluctuations of extracellular lactate levels in rat brain monitored with rapid-response enzyme-based sensor.

PubMed

Hu, Y; Wilson, G S

1997-10-01

A successfully developed enzyme-based lactate microsensor with rapid response time allows the direct and continuous in vivo measurement of lactic acid concentration with high temporal resolution in brain extracellular fluid. The fluctuations coupled to neuronal activity in extracellular lactate concentration were explored in the dentate gyrus of the hippocampus of the rat brain after electrical stimulation of the perforant pathway. Extracellular glucose and oxygen levels were also detected simultaneously by coimplantation of a fast-response glucose sensor and an oxygen electrode, to provide novel information of trafficking of energy substances in real time related to local neuronal activity. The results first give a comprehensive picture of complementary energy supply and use of lactate and glucose in the intact brain tissue. In response to acute neuronal activation, the brain tissue shifts immediately to significant energy supply by lactate. A local temporary fuel "reservoir" is established behind the blood-brain barrier, evidenced by increased extracellular lactate concentration. The pool can be depleted rapidly, up to 28% in 10-12 s, by massive, acute neuronal use after stimulation and can be replenished in approximately 20 s. Glutamate-stimulated astrocytic glycolysis and the increase of regional blood flow may regulate the lactate concentration of the pool in different time scales to maintain local energy homeostasis.

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.

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

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

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

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.

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

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.

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.

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

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…

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.

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

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.

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.

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.

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.

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.

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.

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.

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

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.

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

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

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.

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.

Combining parasite lactate dehydrogenase-based and histidine-rich protein 2-based rapid tests to improve specificity for diagnosis of malaria Due to Plasmodium knowlesi and other Plasmodium species in Sabah, Malaysia.

PubMed

Grigg, Matthew J; William, Timothy; Barber, Bridget E; Parameswaran, Uma; Bird, Elspeth; Piera, Kim; Aziz, Ammar; Dhanaraj, Prabakaran; Yeo, Tsin W; Anstey, Nicholas M

2014-06-01

Plasmodium knowlesi causes severe and fatal malaria in Malaysia. Microscopic misdiagnosis is common and may delay appropriate treatment. P. knowlesi can cross-react with "species-specific" parasite lactate dehydrogenase (pLDH) monoclonal antibodies used in rapid diagnostic tests (RDTs) to detect P. falciparum and P. vivax. At one tertiary-care hospital and two district hospitals in Sabah, we prospectively evaluated two combination RDTs for malaria diagnosis by using both a pan-Plasmodium-pLDH (pan-pLDH)/P. falciparum-specific-pLDH (Pf-pLDH) RDT (OptiMAL-IT) and a non-P. falciparum VOM-pLDH/Pf-HRP2 RDT (CareStart). Differential cross-reactivity among these combinations was hypothesized to differentiate P. knowlesi from other Plasmodium monoinfections. Among 323 patients with PCR-confirmed P. knowlesi (n = 193), P. falciparum (n = 93), and P. vivax (n = 37) monoinfections, the VOM-pLDH individual component had the highest sensitivity for nonsevere (35%; 95% confidence interval [CI], 27 to 43%) and severe (92%; CI, 81 to 100%) P. knowlesi malaria. CareStart demonstrated a P. knowlesi sensitivity of 42% (CI, 34 to 49%) and specificity of 74% (CI, 65 to 82%), a P. vivax sensitivity of 83% (CI, 66 to 93%) and specificity of 71% (CI, 65 to 76%), and a P. falciparum sensitivity of 97% (CI, 90 to 99%) and specificity of 99% (CI, 97 to 100%). OptiMAL-IT demonstrated a P. knowlesi sensitivity of 32% (CI, 25 to 39%) and specificity of 21% (CI, 15 to 29%), a P. vivax sensitivity of 60% (CI, 42 to 75%) and specificity of 97% (CI, 94 to 99%), and a P. falciparum sensitivity of 82% (CI, 72 to 89%) and specificity of 39% (CI, 33 to 46%). The combination of CareStart plus OptiMAL-IT for P. knowlesi using predefined criteria gave a sensitivity of 25% (CI, 19 to 32%) and specificity of 97% (CI, 92 to 99%). Combining two RDT combinations was highly specific for P. knowlesi malaria diagnosis; however, sensitivity was poor. The specificity of pLDH RDTs was decreased for P. vivax and P

Combining Parasite Lactate Dehydrogenase-Based and Histidine-Rich Protein 2-Based Rapid Tests To Improve Specificity for Diagnosis of Malaria Due to Plasmodium knowlesi and Other Plasmodium Species in Sabah, Malaysia

PubMed Central

William, Timothy; Barber, Bridget E.; Parameswaran, Uma; Bird, Elspeth; Piera, Kim; Aziz, Ammar; Dhanaraj, Prabakaran; Yeo, Tsin W.; Anstey, Nicholas M.

2014-01-01

Plasmodium knowlesi causes severe and fatal malaria in Malaysia. Microscopic misdiagnosis is common and may delay appropriate treatment. P. knowlesi can cross-react with “species-specific” parasite lactate dehydrogenase (pLDH) monoclonal antibodies used in rapid diagnostic tests (RDTs) to detect P. falciparum and P. vivax. At one tertiary-care hospital and two district hospitals in Sabah, we prospectively evaluated two combination RDTs for malaria diagnosis by using both a pan-Plasmodium-pLDH (pan-pLDH)/P. falciparum-specific-pLDH (Pf-pLDH) RDT (OptiMAL-IT) and a non-P. falciparum VOM-pLDH/Pf-HRP2 RDT (CareStart). Differential cross-reactivity among these combinations was hypothesized to differentiate P. knowlesi from other Plasmodium monoinfections. Among 323 patients with PCR-confirmed P. knowlesi (n = 193), P. falciparum (n = 93), and P. vivax (n = 37) monoinfections, the VOM-pLDH individual component had the highest sensitivity for nonsevere (35%; 95% confidence interval [CI], 27 to 43%) and severe (92%; CI, 81 to 100%) P. knowlesi malaria. CareStart demonstrated a P. knowlesi sensitivity of 42% (CI, 34 to 49%) and specificity of 74% (CI, 65 to 82%), a P. vivax sensitivity of 83% (CI, 66 to 93%) and specificity of 71% (CI, 65 to 76%), and a P. falciparum sensitivity of 97% (CI, 90 to 99%) and specificity of 99% (CI, 97 to 100%). OptiMAL-IT demonstrated a P. knowlesi sensitivity of 32% (CI, 25 to 39%) and specificity of 21% (CI, 15 to 29%), a P. vivax sensitivity of 60% (CI, 42 to 75%) and specificity of 97% (CI, 94 to 99%), and a P. falciparum sensitivity of 82% (CI, 72 to 89%) and specificity of 39% (CI, 33 to 46%). The combination of CareStart plus OptiMAL-IT for P. knowlesi using predefined criteria gave a sensitivity of 25% (CI, 19 to 32%) and specificity of 97% (CI, 92 to 99%). Combining two RDT combinations was highly specific for P. knowlesi malaria diagnosis; however, sensitivity was poor. The specificity of pLDH RDTs was decreased for P. vivax and

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

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.

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.

Base excess is an accurate predictor of elevated lactate in ED septic patients.

PubMed

Montassier, Emmanuel; Batard, Eric; Segard, Julien; Hardouin, Jean-Benoît; Martinage, Arnaud; Le Conte, Philippe; Potel, Gille

2012-01-01

Prior studies showed that lactate is a useful marker in sepsis. However, lactate is often not routinely drawn or rapidly available in the emergency department (ED). The study aimed to determine if base excess (BE), widely and rapidly available in the ED, could be used as a surrogate marker for elevated lactate in ED septic patients. This was a prospective and observational cohort study. From March 2009 to March 2010, consecutive patients 18 years or older who presented to the ED with a suspected severe sepsis were enrolled in the study. Lactate and BE measurements were performed. We defined, a priori, a clinically significant lactate to be greater than 3 mmol/L and BE less than -4 mmol/L. A total of 224 patients were enrolled in the study. The average BE was -4.5 mmol/L (SD, 4.9) and the average lactate was 3.5 mmol/L (SD, 2.9). The sensitivity of a BE less than -4 mmol/L in predicting elevated lactate greater than 3 mmol/L was 91.1% (95% confidence interval, 85.5%-96.6%) and the specificity was 88.6% (95% confidence interval, 83.0%-94.2%). The area under the curve was 0.95. Base excess is an accurate marker for the prediction of elevated lactate in the ED. The measurement of BE, obtained in a few minutes in the ED, provides a secure and quick method, similar to the electrocardiogram at triage for patients with chest pain, to determine the patients with sepsis who need an early aggressive resuscitation. Copyright © 2012 Elsevier Inc. All rights reserved.

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.

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.

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.

Cupriavidus necator JMP134 rapidly reduces furfural with a Zn-dependent alcohol dehydrogenase.

PubMed

Li, Qunrui; Metthew Lam, L K; Xun, Luying

2011-11-01

Ethanol is a renewable biofuel, and it can be produced from lignocellulosic biomass. The biomass is usually converted to hydrolysates that consist of sugar and sugar derivatives, such as furfural. Yeast ferments sugar to ethanol, but furfural higher than 3 mM is inhibitory. It can take several days for yeast cells to reduce furfural to non-inhibitory furfuryl alcohol before producing ethanol. Bioreduction of furfural to furfuryl alcohol before fermentation may relieve yeast from furfural toxicity. We observed that Cupriavidus necator JMP134, a strict aerobe, rapidly reduced 17 mM furfural to less than 3 mM within 14 min with cell turbidity of 1.0 at 600 nm at 50°C. The rapid reduction consumed ethanol. The "furfural reductase" (FurX) was purified, and it oxidized ethanol to acetaldehyde and reduced furfural to furfuryl alcohol with NAD(+) as the cofactor. The protein was identified with mass spectrometry fingerprinting to be a hypothetical protein belonging to Zn-dependent alcohol dehydrogenase family. The furX-inactivation mutant of C. necator JMP134 lost the ability to rapidly reduce furfural, and Escherichia coli producing recombinant FurX gained the ability. Thus, an alcohol dehydrogenase enabled bacteria to rapidly reduce furfural with ethanol as the reducing power.

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

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

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.

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...

APTEC: aptamer-tethered enzyme capture as a novel rapid diagnostic test for malaria.

PubMed

Dirkzwager, Roderick M; Kinghorn, Andrew B; Richards, Jack S; Tanner, Julian A

2015-03-18

We report the rapid diagnosis of malaria by aptamer-tethered enzyme capture (APTEC) whereby an aptamer captures biomarker Plasmodium falciparum lactate dehydrogenase (PfLDH) then activity is measured colorimetrically. The robust test was sensitive (limit of detection = 4.9 ng mL(-1)) and could reliably diagnose malaria in clinical blood samples.

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

Rapid synthesis of triazine inhibitors of inosine monophosphate dehydrogenase.

PubMed

Pitts, William J; Guo, Junqing; Dhar, T G Murali; Shen, Zhongqi; Gu, Henry H; Watterson, Scott H; Bednarz, Mark S; Chen, Bang Chi; Barrish, Joel C; Bassolino, Donna; Cheney, Daniel; Fleener, Catherine A; Rouleau, Katherine A; Hollenbaugh, Diane L; Iwanowicz, Edwin J

2002-08-19

A series of novel triazine-based small molecule inhibitors (IV) of inosine monophosphate dehydrogenase was prepared. The synthesis and the structure-activity relationships (SAR) derived from in vitro studies are described.

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.

Development and validation of a rapid, aldehyde dehydrogenase bright–based cord blood potency assay

PubMed Central

Noldner, Pamela; Troy, Jesse D.; Cheatham, Lynn; Parrish, Amanda; Page, Kristin; Gentry, Tracy; Balber, Andrew E.; Kurtzberg, Joanne

2016-01-01

Banked, unrelated umbilical cord blood provides access to hematopoietic stem cell transplantation for patients lacking matched bone marrow donors, yet 10% to 15% of patients experience graft failure or delayed engraftment. This may be due, at least in part, to inadequate potency of the selected cord blood unit (CBU). CBU potency is typically assessed before cryopreservation, neglecting changes in potency occurring during freezing and thawing. Colony-forming units (CFUs) have been previously shown to predict CBU potency, defined as the ability to engraft in patients by day 42 posttransplant. However, the CFU assay is difficult to standardize and requires 2 weeks to perform. Consequently, we developed a rapid multiparameter flow cytometric CBU potency assay that enumerates cells expressing high levels of the enzyme aldehyde dehydrogenase (ALDH bright [ALDHbr]), along with viable CD45+ or CD34+ cell content. These measurements are made on a segment that was attached to a cryopreserved CBU. We validated the assay with prespecified criteria testing accuracy, specificity, repeatability, intermediate precision, and linearity. We then prospectively examined the correlations among ALDHbr, CD34+, and CFU content of 3908 segments over a 5-year period. ALDHbr (r = 0.78; 95% confidence interval [CI], 0.76-0.79), but not CD34+ (r = 0.25; 95% CI, 0.22-0.28), was strongly correlated with CFU content as well as ALDHbr content of the CBU. These results suggest that the ALDHbr segment assay (based on unit characteristics measured before release) is a reliable assessment of potency that allows rapid selection and release of CBUs from the cord blood bank to the transplant center for transplantation. PMID:26968535

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.

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.

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.

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.

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

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

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.

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.

Development and validation of a rapid, aldehyde dehydrogenase bright-based cord blood potency assay.

PubMed

Shoulars, Kevin; Noldner, Pamela; Troy, Jesse D; Cheatham, Lynn; Parrish, Amanda; Page, Kristin; Gentry, Tracy; Balber, Andrew E; Kurtzberg, Joanne

2016-05-12

Banked, unrelated umbilical cord blood provides access to hematopoietic stem cell transplantation for patients lacking matched bone marrow donors, yet 10% to 15% of patients experience graft failure or delayed engraftment. This may be due, at least in part, to inadequate potency of the selected cord blood unit (CBU). CBU potency is typically assessed before cryopreservation, neglecting changes in potency occurring during freezing and thawing. Colony-forming units (CFUs) have been previously shown to predict CBU potency, defined as the ability to engraft in patients by day 42 posttransplant. However, the CFU assay is difficult to standardize and requires 2 weeks to perform. Consequently, we developed a rapid multiparameter flow cytometric CBU potency assay that enumerates cells expressing high levels of the enzyme aldehyde dehydrogenase (ALDH bright [ALDH(br)]), along with viable CD45(+) or CD34(+) cell content. These measurements are made on a segment that was attached to a cryopreserved CBU. We validated the assay with prespecified criteria testing accuracy, specificity, repeatability, intermediate precision, and linearity. We then prospectively examined the correlations among ALDH(br), CD34(+), and CFU content of 3908 segments over a 5-year period. ALDH(br) (r = 0.78; 95% confidence interval [CI], 0.76-0.79), but not CD34(+) (r = 0.25; 95% CI, 0.22-0.28), was strongly correlated with CFU content as well as ALDH(br) content of the CBU. These results suggest that the ALDH(br) segment assay (based on unit characteristics measured before release) is a reliable assessment of potency that allows rapid selection and release of CBUs from the cord blood bank to the transplant center for transplantation. © 2016 by The American Society of Hematology.

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

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

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.

[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.

Cotton fabric-based electrochemical device for lactate measurement in saliva.

PubMed

Malon, Radha S P; Chua, K Y; Wicaksono, Dedy H B; Córcoles, Emma P

2014-06-21

Lactate measurement is vital in clinical diagnostics especially among trauma and sepsis patients. In recent years, it has been shown that saliva samples are an excellent applicable alternative for non-invasive measurement of lactate. In this study, we describe a method for the determination of lactate concentration in saliva samples by using a simple and low-cost cotton fabric-based electrochemical device (FED). The device was fabricated using template method for patterning the electrodes and wax-patterning technique for creating the sample placement/reaction zone. Lactate oxidase (LOx) enzyme was immobilised at the reaction zone using a simple entrapment method. The LOx enzymatic reaction product, hydrogen peroxide (H2O2) was measured using chronoamperometric measurements at the optimal detection potential (-0.2 V vs. Ag/AgCl), in which the device exhibited a linear working range between 0.1 to 5 mM, sensitivity (slope) of 0.3169 μA mM(-1) and detection limit of 0.3 mM. The low detection limit and wide linear range were suitable to measure salivary lactate (SL) concentration, thus saliva samples obtained under fasting conditions and after meals were evaluated using the FED. The measured SL varied among subjects and increased after meals randomly. The proposed device provides a suitable analytical alternative for rapid and non-invasive determination of lactate in saliva samples. The device can also be adapted to a variety of other assays that requires simplicity, low-cost, portability and flexibility.

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

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.

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

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.

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

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

A wire-based dual-analyte sensor for glucose and lactate: in vitro and in vivo evaluation.

PubMed

Ward, W Kenneth; House, Jody L; Birck, Jonathan; Anderson, Ellen M; Jansen, Lawrence B

2004-06-01

Continuous measurement of lactate is potentially useful for detecting physical exhaustion and for monitoring critical care conditions characterized by hypoperfusion, such as heart failure. In some conditions, it may be desirable to monitor more than one metabolic parameter concurrently. For this reason, we designed and fabricated twisted wire-based microelectrodes that can measure both lactate and glucose. These dual-analyte sensors were characterized in vitro by measuring their response to the analyte of interest and to assess whether they were susceptible to interference from the other analyte. When measured in stirred aqueous buffer, lactate sensors detected a very small amount of crosstalk from glucose in vitro, although this signal was less than 3% of the response to lactate. Glucose sensors did not detect crosstalk from lactate. Sensors were implanted subcutaneously in rats and tested during infusions of lactate and glucose. Each sensing electrode responded rapidly to changes in its analyte concentration, and there was no evidence of in vivo crosstalk. This study constitutes proof of the concept that oxidase-based, amperometric wire microsensors can detect changes in glucose and lactate during subcutaneous implantation in rats.

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.

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

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.

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.

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

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.

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

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.

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.

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

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.

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.

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

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

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.

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

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.

Paper-Based Device for Rapid Visualization of NADH Based on Dissolution of Gold Nanoparticles.

PubMed

Liang, Pingping; Yu, Haixiang; Guntupalli, Bhargav; Xiao, Yi

2015-07-15

We describe a paper-based device that enables rapid and sensitive room-temperature detection of dihydronicotinamide adenine dinucleotide (NADH) via a colorimetric readout and demonstrate its value for monitoring NAD+-driven enzymatic reactions. Our system is based on NADH-mediated inhibition of gold nanoparticle (AuNPs) dissolution in a Au3+-cetyltrimethylammonium bromide (CTAB) solution. We fabricated a device consisting of a mixed cellulose ester paper featuring a wax-encircled, AuNP-coated film atop a cotton absorbent layer sandwiched between two plastic cover layers. In the absence of NADH, the Au3+-CTAB complex dissolves the AuNP layer completely, generating a white color in the test zone. In the presence of NADH, Au3+ is rapidly reduced to Au+, greatly decreasing the dissolution of AuNPs and yielding a red color that becomes stronger at increasing concentrations of NADH. This device exploits capillary force-assisted vertical diffusion, allowing us to apply a 25 μL sample to a surface-confined test zone to achieve a detection limit of 12.5 μM NADH. We used the enzyme glucose dehydrogenase as a model to demonstrate that our paper-based device can monitor NAD+-driven biochemical processes with and without selective dehydrogenase inhibitors by naked-eye observation within 4 min at room temperature in a small sample volume. We believe that our paper-based device could offer a valuable and low-cost analytical tool for monitoring NAD+-associated enzymatic reactions and screening for dehydrogenase inhibitors in a variety of testing contexts.

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.

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.

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.

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

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

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.

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.

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

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.

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

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.

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...

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

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.

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.

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

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.

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

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.

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.

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.

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

Venous glucose, serum lactate and base deficit as biochemical predictors of mortality in patients with polytrauma.

PubMed

Saad, Sameh; Mohamed, Naglaa; Moghazy, Amr; Ellabban, Gouda; El-Kamash, Soliman

2016-01-01

The trauma and injury severity score (TRISS) and Acute Physiology and Chronic Health Evaluation IV (APACHE IV) are accurate but complex. This study aimed to compare venous glucose, levels of serum lactate, and base deficit in polytraumatized patients as simple parameters to predict the mortality in these patients versus (TRISS) and (APACHE IV). This was a comparative cross-sectional study of 282 patients with polytrauma presented to the Emergency Department (ED). The best cut off value of TRISS probability of survival score for prediction of mortality among poly-traumatized patients was ≤90. APACHE IV demonstrated 67% sensitivity and 95% specificity at 95% CI at cut off point 99. The best cutoff value of Random Blood Sugar was >140 mg/dl, with 89% sensitivity, 49% specificity; base deficit was less than -5.6 with 64% sensitivity, 93% specificity; lactate was >2.6 mmol/L with 92%, sensitivity, 42% specificity. Venous glucose, serum lactate and base deficit are easy and rapid biochemical predictors of mortality in patients with polytrauma. These predictors could be used as TRISS and APACHE IV in predicting mortality.

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.

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.

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.

Catalytic Mechanism of Short Ethoxy Chain Nonylphenol Dehydrogenase Belonging to a Polyethylene Glycol Dehydrogenase Group in the GMC Oxidoreductase Family

PubMed Central

Liu, Xin; Ohta, Takeshi; Kawabata, Takeshi; Kawai, Fusako

2013-01-01

Ethoxy (EO) chain nonylphenol dehydrogenase (NPEO-DH) from Ensifer sp. AS08 and EO chain octylphenol dehydrogenase from Pseudomonas putida share common molecular characteristics with polyethylene glycol (PEG) dehydrogenases (PEG-DH) and comprise a PEG-DH subgroup in the family of glucose-methanol-choline (GMC) oxidoreductases that includes glucose/alcohol oxidase and glucose/choline dehydrogenase. Three-dimensional (3D) molecular modeling suggested that differences in the size, secondary structure and hydropathy in the active site caused differences in their substrate specificities toward EO chain alkylphenols and free PEGs. Based on 3D molecular modeling, site-directed mutagenesis was utilized to introduce mutations into potential catalytic residues of NPEO-DH. From steady state and rapid kinetic characterization of wild type and mutant NPEO-DHs, we can conclude that His465 and Asn507 are directly involved in the catalysis. Asn507 mediates the transfer of proton from a substrate to FAD and His465 transfers the same proton from the reduced flavin to an electron acceptor. PMID:23306149

Catalytic mechanism of short ethoxy chain nonylphenol dehydrogenase belonging to a polyethylene glycol dehydrogenase group in the GMC oxidoreductase family.

PubMed

Liu, Xin; Ohta, Takeshi; Kawabata, Takeshi; Kawai, Fusako

2013-01-10

Ethoxy (EO) chain nonylphenol dehydrogenase (NPEO-DH) from Ensifer sp. AS08 and EO chain octylphenol dehydrogenase from Pseudomonas putida share common molecular characteristics with polyethylene glycol (PEG) dehydrogenases (PEG-DH) and comprise a PEG-DH subgroup in the family of glucose-methanol-choline (GMC) oxidoreductases that includes glucose/alcohol oxidase and glucose/choline dehydrogenase. Three-dimensional (3D) molecular modeling suggested that differences in the size, secondary structure and hydropathy in the active site caused differences in their substrate specificities toward EO chain alkylphenols and free PEGs. Based on 3D molecular modeling, site-directed mutagenesis was utilized to introduce mutations into potential catalytic residues of NPEO-DH. From steady state and rapid kinetic characterization of wild type and mutant NPEO-DHs, we can conclude that His465 and Asn507 are directly involved in the catalysis. Asn507 mediates the transfer of proton from a substrate to FAD and His465 transfers the same proton from the reduced flavin to an electron acceptor.

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

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.

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.

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.

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

Performance of the CareStart Glucose-6-Phosphate Dehydrogenase (G6PD) Rapid Diagnostic Test in Gressier, Haiti

PubMed Central

von Fricken, Michael E.; Weppelmann, Thomas A.; Eaton, Will T.; Masse, Roseline; Beau de Rochars, Madsen V. E.; Okech, Bernard A.

2014-01-01

Administering primaquine (PQ) to treat malaria patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency can pose a serious risk of drug-induced hemolysis (DIH). New easy to use point-of-care rapid diagnostic tests are being developed as an alternative to labor-intensive spectrophotometric methods, but they require field testing before they can be used at scale. This study screened 456 participants in Gressier, Haiti using the Access Bio CareStart qualitative G6PD rapid detection test compared with the laboratory-based Trinity Biotech quantitative spectrophotometric assay. Findings suggest that the CareStart test was 90% sensitive for detecting individuals with severe deficiency and 84.8% sensitive for detecting individuals with moderate and severe deficiency compared with the Trinity Biotech assay. A high negative predictive value of 98.2% indicates excellent performance in determining those patients able to take PQ safely. The CareStart G6PD test holds much value for screening malaria patients to determine eligibility for PQ therapy. PMID:24778197

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

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.

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

Cerebrospinal fluid acid-base status and lactate and pyruvate concentrations after convulsions of varied duration and aetiology in children.

PubMed Central

Simpson, H; Habel, A H; George, E L

1977-01-01

Twenty-two infants and children were studied after convulsions of varied cause and duration. Arterial and CSF acid-base variables, lactate and pyruvate concentrations, and lactate/pyruvate ratios were measured between 3 and 18 hours after convulsive episodes. Biochemical signs of cerebral hypoxia were found in 7 patients with prolonged (greater than 30 minutes) or recurrent short convulsions. These signs were absent in patients with single short convulsions. These findings indicate that cerebral hypoxia and possible brain damage is a hazard of prolonged or rapidly recurring short convulsions. PMID:23078

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.

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.

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.

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.

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.

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

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.

[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.

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.

Performance of the CareStart glucose-6-phosphate dehydrogenase (G6PD) rapid diagnostic test in Gressier, Haiti.

PubMed

von Fricken, Michael E; Weppelmann, Thomas A; Eaton, Will T; Masse, Roseline; Beau de Rochars, Madsen V E; Okech, Bernard A

2014-07-01

Administering primaquine (PQ) to treat malaria patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency can pose a serious risk of drug-induced hemolysis (DIH). New easy to use point-of-care rapid diagnostic tests are being developed as an alternative to labor-intensive spectrophotometric methods, but they require field testing before they can be used at scale. This study screened 456 participants in Gressier, Haiti using the Access Bio CareStart qualitative G6PD rapid detection test compared with the laboratory-based Trinity Biotech quantitative spectrophotometric assay. Findings suggest that the CareStart test was 90% sensitive for detecting individuals with severe deficiency and 84.8% sensitive for detecting individuals with moderate and severe deficiency compared with the Trinity Biotech assay. A high negative predictive value of 98.2% indicates excellent performance in determining those patients able to take PQ safely. The CareStart G6PD test holds much value for screening malaria patients to determine eligibility for PQ therapy. © The American Society of Tropical Medicine and Hygiene.

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.

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

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.

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.

Novel amide-based inhibitors of inosine 5'-monophosphate dehydrogenase.

PubMed

Watterson, Scott H; Liu, Chunjian; Dhar, T G Murali; Gu, Henry H; Pitts, William J; Barrish, Joel C; Fleener, Catherine A; Rouleau, Katherine; Sherbina, N Z; Hollenbaugh, Diane L; Iwanowicz, Edwin J

2002-10-21

A series of novel amide-based small molecule inhibitors of inosine monophosphate dehydrogenase (IMPDH) was explored. The synthesis and the structure-activity relationships (SARs) derived from in vitro studies are described.

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.

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

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

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

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

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

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

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

Effect of water-based recovery on blood lactate removal after high-intensity exercise.

PubMed

Lucertini, Francesco; Gervasi, Marco; D'Amen, Giancarlo; Sisti, Davide; Rocchi, Marco Bruno Luigi; Stocchi, Vilberto; Benelli, Piero

2017-01-01

This study assessed the effectiveness of water immersion to the shoulders in enhancing blood lactate removal during active and passive recovery after short-duration high-intensity exercise. Seventeen cyclists underwent active water- and land-based recoveries and passive water and land-based recoveries. The recovery conditions lasted 31 minutes each and started after the identification of each cyclist's blood lactate accumulation peak, induced by a 30-second all-out sprint on a cycle ergometer. Active recoveries were performed on a cycle ergometer at 70% of the oxygen consumption corresponding to the lactate threshold (the control for the intensity was oxygen consumption), while passive recoveries were performed with subjects at rest and seated on the cycle ergometer. Blood lactate concentration was measured 8 times during each recovery condition and lactate clearance was modeled over a negative exponential function using non-linear regression. Actual active recovery intensity was compared to the target intensity (one sample t-test) and passive recovery intensities were compared between environments (paired sample t-tests). Non-linear regression parameters (coefficients of the exponential decay of lactate; predicted resting lactates; predicted delta decreases in lactate) were compared between environments (linear mixed model analyses for repeated measures) separately for the active and passive recovery modes. Active recovery intensities did not differ significantly from the target oxygen consumption, whereas passive recovery resulted in a slightly lower oxygen consumption when performed while immersed in water rather than on land. The exponential decay of blood lactate was not significantly different in water- or land-based recoveries in either active or passive recovery conditions. In conclusion, water immersion at 29°C would not appear to be an effective practice for improving post-exercise lactate removal in either the active or passive recovery modes.

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.

A Portable Luminometer with a Disposable Electrochemiluminescent Biosensor for Lactate Determination

PubMed Central

Martínez-Olmos, Antonio; Ballesta-Claver, Julio; Palma, Alberto J.; Valencia-Mirón, Maria del Carmen; Capitán-Vallvey, Luis Fermin

2009-01-01

A hand-held luminometer for measuring electrochemiluminescence (ECL) for lactate determination and based on one-shot biosensors fabricated using screen-printed electrodes is described. The lactate recognition system is based on lactate oxidase and the transduction system consists of electro-oxidation of luminol, with all the reagents immobilized in a Methocel membrane. The membrane composition and reaction conditions have been optimized to obtain adequate sensitivity. The luminometer is based on a large silicon photodiode as detector and includes a programmable potentiostat to initialize the chemical reaction and signal processing circuitry, designed to acquire a low level photocurrent with offset cancelation, low pass filtering for noise attenuation and adjustable gain up to 1012 V/A. The one-shot biosensor responds to lactate rapidly, with an acquisition time of 2.5 min, obtaining a linear dependence from 8 × 10−6 to 2 × 10−4 M, a detection limit of 2.4 × 10−6 M and a sensor-to-sensor reproducibility (relative standard deviation, RSD) of around 7–10 % at the medium level of the range. PMID:22408475

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/

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.

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.

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.

Structural basis for cellobiose dehydrogenase action during oxidative cellulose degradation

NASA Astrophysics Data System (ADS)

Tan, Tien-Chye; Kracher, Daniel; Gandini, Rosaria; Sygmund, Christoph; Kittl, Roman; Haltrich, Dietmar; Hällberg, B. Martin; Ludwig, Roland; Divne, Christina

2015-07-01

A new paradigm for cellulose depolymerization by fungi focuses on an oxidative mechanism involving cellobiose dehydrogenases (CDH) and copper-dependent lytic polysaccharide monooxygenases (LPMO); however, mechanistic studies have been hampered by the lack of structural information regarding CDH. CDH contains a haem-binding cytochrome (CYT) connected via a flexible linker to a flavin-dependent dehydrogenase (DH). Electrons are generated from cellobiose oxidation catalysed by DH and shuttled via CYT to LPMO. Here we present structural analyses that provide a comprehensive picture of CDH conformers, which govern the electron transfer between redox centres. Using structure-based site-directed mutagenesis, rapid kinetics analysis and molecular docking, we demonstrate that flavin-to-haem interdomain electron transfer (IET) is enabled by a haem propionate group and that rapid IET requires a closed CDH state in which the propionate is tightly enfolded by DH. Following haem reduction, CYT reduces LPMO to initiate oxygen activation at the copper centre and subsequent cellulose depolymerization.

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.

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

Electrochemical lactate biosensor based upon chitosan/carbon nanotubes modified screen-printed graphite electrodes for the determination of lactate in embryonic cell cultures.

PubMed

Hernández-Ibáñez, Naiara; García-Cruz, Leticia; Montiel, Vicente; Foster, Christopher W; Banks, Craig E; Iniesta, Jesús

2016-03-15

l-lactate is an essential metabolite present in embryonic cell culture. Changes of this important metabolite during the growth of human embryo reflect the quality and viability of the embryo. In this study, we report a sensitive, stable, and easily manufactured electrochemical biosensor for the detection of lactate within embryonic cell cultures media. Screen-printed disposable electrodes are used as electrochemical sensing platforms for the miniaturization of the lactate biosensor. Chitosan/multi walled carbon nanotubes composite have been employed for the enzymatic immobilization of the lactate oxidase enzyme. This novel electrochemical lactate biosensor analytical efficacy is explored towards the sensing of lactate in model (buffer) solutions and is found to exhibit a linear response towards lactate over the concentration range of 30.4 and 243.9 µM in phosphate buffer solution, with a corresponding limit of detection (based on 3-sigma) of 22.6 µM and exhibits a sensitivity of 3417 ± 131 µAM(-1) according to the reproducibility study. These novel electrochemical lactate biosensors exhibit a high reproducibility, with a relative standard deviation of less than 3.8% and an enzymatic response over 82% after 5 months stored at 4 °C. Furthermore, high performance liquid chromatography technique has been utilized to independently validate the electrochemical lactate biosensor for the determination of lactate in a commercial embryonic cell culture medium providing excellent agreement between the two analytical protocols. Copyright © 2015 Elsevier B.V. All rights reserved.

Novel guanidine-based inhibitors of inosine monophosphate dehydrogenase.

PubMed

Iwanowicz, Edwin J; Watterson, Scott H; Liu, Chunjian; Gu, Henry H; Mitt, Toomas; Leftheris, Katerina; Barrish, Joel C; Fleener, Catherine A; Rouleau, Katherine; Sherbina, N Z; Hollenbaugh, Diane L

2002-10-21

A series of novel guanidine-based small molecule inhibitors of inosine monophosphate dehydrogenase (IMPDH) was explored. IMPDH catalyzes the rate determining step in guanine nucleotide biosynthesis and is a target for anticancer, immunosuppressive and antiviral therapy. The synthesis and the structure-activity relationships (SARs), derived from in vitro studies, for this new series of inhibitors is given.

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.

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

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.

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.

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.

Binding, hydration, and decarboxylation of the reaction intermediate glutaconyl-coenzyme A by human glutaryl-CoA dehydrogenase.

PubMed

Westover, J B; Goodman, S I; Frerman, F E

2001-11-20

Glutaconyl-coenzyme A (CoA) is the presumed enzyme-bound intermediate in the oxidative decarboxylation of glutaryl-CoA that is catalyzed by glutaryl-CoA dehydrogenase. We demonstrated glutaconyl-CoA bound to glutaryl-CoA dehydrogenase after anaerobic reduction of the dehydrogenase with glutaryl-CoA. Glutaryl-CoA dehydrogenase also has intrinsic enoyl-CoA hydratase activity, a property of other members of the acyl-CoA dehydrogenase family. The enzyme rapidly hydrates glutaconyl-CoA at pH 7.6 with a k(cat) of 2.7 s(-1). The k(cat) in the overall oxidation-decarboxylation reaction at pH 7.6 is about 9 s(-1). The binding of glutaconyl-CoA was quantitatively assessed from the K(m) in the hydratase reaction, 3 microM, and the K(i), 1.0 microM, as a competitive inhibitor of the dehydrogenase. These values compare with K(m) and K(i) of 4.0 and 12.9 microM, respectively, for crotonyl-CoA. Glu370 is the general base catalyst in the dehydrogenase that abstracts an alpha-proton of the substrate to initiate the catalytic pathway. The mutant dehydrogenase, Glu370Gln, is inactive in the dehydrogenation and the hydratase reactions. However, this mutant dehydrogenase decarboxylates glutaconyl-CoA to crotonyl-CoA without oxidation-reduction reactions of the dehydrogenase flavin. Addition of glutaconyl-CoA to this mutant dehydrogenase results in a rapid, transient increase in long-wavelength absorbance (lambda(max) approximately 725 nm), and crotonyl-CoA is found as the sole product. We propose that this 725 nm-absorbing species is the delocalized crotonyl-CoA anion that follows decarboxylation and that the decay is the result of slow protonation of the anion in the absence of the general acid catalyst, Glu370(H(+)). In the absence of detectable oxidation-reduction, the data indicate that oxidation-reduction of the dehydrogenase flavin is not essential for decarboxylation of glutaconyl-CoA.

Outcomes of a hospital-based employee lactation program.

PubMed

Spatz, Diane L; Kim, Gabriella S; Froh, Elizabeth B

2014-12-01

Little has been published about employee lactation support in hospitals and other healthcare facilities. The Children's Hospital of Philadelphia (CHOP), Philadelphia, PA, has a comprehensive employee lactation program. The objective of this study was to describe the breastfeeding practices of our employees and compare these results with national Centers for Disease Control and Prevention (CDC) data. The human resources department generated a list of all employees who filed for maternity leave between 2007 and 2011. These employees were contacted confidentially via e-mail to complete an electronic-based (SurveyMonkey.com) questionnaire. An initial message and three reminder messages were sent over a 3-month period during the last quarter of 2012, with 545 women completing the survey (response rate, 40%). Women who responded to the survey had significantly higher breastfeeding initiation rates compared with national CDC data (94.5% vs. 76.9%; p<0.0001). At 6 months, significantly more CHOP employees were breastfeeding (78.6% vs. 47.2%; p<0.0001). At 12 months 32.4% of CHOP employees were still breastfeeding compared with CDC data of 25.5% (p=0.0003). Additionally, over 20% of CHOP employees breastfed their infants for over 12 months (no national data for comparison). Within CHOP's comprehensive employee lactation program, women achieved breastfeeding milestones that well exceeded national data and the Healthy People 2020 targets for breastfeeding initiation and duration. CHOP's employee lactation program can serve as a model for other institutions.

Treatment of mastitis during lactation.

PubMed

Pyörälä, S

2009-04-01

Treatment of mastitis should be based on bacteriological diagnosis and take national and international guidelines on prudent use of antimicrobials into account. In acute mastitis, where bacteriological diagnosis is not available, treatment should be initiated based on herd data and personal experience. Rapid bacteriological diagnosis would facilitate the proper selection of the antimicrobial. Treating subclinical mastitis with antimicrobials during lactation is seldom economical, because of high treatment costs and generally poor efficacy. All mastitis treatment should be evidence-based, i.e., the efficacy of each product and treatment length should be demonstrated by scientific studies. Use of on-farm written protocols for mastitis treatment promotes a judicious use of antimicrobials and reduces the use of antimicrobials.

Treatment of mastitis during lactation

PubMed Central

2009-01-01

Treatment of mastitis should be based on bacteriological diagnosis and take national and international guidelines on prudent use of antimicrobials into account. In acute mastitis, where bacteriological diagnosis is not available, treatment should be initiated based on herd data and personal experience. Rapid bacteriological diagnosis would facilitate the proper selection of the antimicrobial. Treating subclinical mastitis with antimicrobials during lactation is seldom economical, because of high treatment costs and generally poor efficacy. All mastitis treatment should be evidence-based, i.e., the efficacy of each product and treatment length should be demonstrated by scientific studies. Use of on-farm written protocols for mastitis treatment promotes a judicious use of antimicrobials and reduces the use of antimicrobials. PMID:22081939

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

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

Microfluidics for rapid detection of isocitrate dehydrogenase 1 mutation for intraoperative application.

PubMed

Aibaidula, Abudumijiti; Zhao, Wang; Wu, Jin-Song; Chen, Hong; Shi, Zhi-Feng; Zheng, Lu-Lu; Mao, Ying; Zhou, Liang-Fu; Sui, Guo-Dong

2016-06-01

OBJECT Conventional methods for isocitrate dehydrogenase 1 (IDH1) detection, such as DNA sequencing and immunohistochemistry, are time- and labor-consuming and cannot be applied for intraoperative analysis. To develop a new approach for rapid analysis of IDH1 mutation from tiny tumor samples, this study used microfluidics as a method for IDH1 mutation detection. METHODS Forty-seven glioma tumor samples were used; IDH1 mutation status was investigated by immunohistochemistry and DNA sequencing. The microfluidic device was fabricated from polydimethylsiloxane following standard soft lithography. The immunoanalysis was conducted in the microfluidic chip. Fluorescence images of the on-chip microcolumn taken by the charge-coupled device camera were collected as the analytical results readout. Fluorescence signals were analyzed by NIS-Elements software to gather detailed information about the IDH1 concentration in the tissue samples. RESULTS DNA sequencing identified IDH1 R132H mutation in 33 of 47 tumor samples. The fluorescence signal for IDH1-mutant samples was 5.49 ± 1.87 compared with 3.90 ± 1.33 for wild type (p = 0.005). Thus, microfluidics was capable of distinguishing IDH1-mutant tumor samples from wild-type samples. When the cutoff value was 4.11, the sensitivity of microfluidics was 87.9% and the specificity was 64.3%. CONCLUSIONS This new approach was capable of analyzing IDH1 mutation status of tiny tissue samples within 30 minutes using intraoperative microsampling. This approach might also be applied for rapid pathological diagnosis of diffuse gliomas, thus guiding personalized resection.

Rapid purification of the over-expressed membrane 3 β-hydroxysteroid dehydrogenase in the presence of detergents

NASA Astrophysics Data System (ADS)

Zhou, Ming; Breton, Rock; Azzi, Arezki; Lin, Sheng-Xiang

1996-10-01

Three-beta hydroxysteroid dehydrogenase / Δ 5-Δ 4 isomerase catalyses a key step in the transformation of all 5-prognen-3β-ol and 5-androsten-3β-ol steroids into the corresponding Δ 4-3-keto-steroids. Human type I 3β-HSD can be found in the subcellular fractions of mitochondria and microsome. A 1.5 kbp cDNA encoding human type I 3β-HSD was inserted into the transfer vector pBlueBac to form plasmid pBB / 3β-HSD. The recombinant baculovirus was obtained by co-transfection of wild type AcNPV genomic DNA and PBB / 3β-HSD in Sf9 cells, then used to infect Sf9 cells to over-express human 3β-HSD protein. The 3β-HSD sample was purified to homogeneity by a rapid procedure, consisting of an anion-exchange and an adsorbance chromatographies, based on FPLC and some detergents application. The whole process was successful with a purification rate of 90 fold and a high recovery (70%). The kinetic study showed a Vmax of 500 nmol/min · mg and a Km of 2.8 μM, being much more active than those reported.

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.

Effect of different levels of rapidly degradable carbohydrates calculated by a simple rumen model on performance of lactating dairy cows.

PubMed

Doorenbos, J; Martín-Tereso, J; Dijkstra, J; van Laar, H

2017-07-01

Aggregating rumen degradation characteristics of different carbohydrate components into the term modeled rapidly degradable carbohydrates (mRDC) can simplify diet formulation by accounting for differences in rate and extent of carbohydrate degradation within and between feedstuffs. This study sought to evaluate responses of lactating dairy cows to diets formulated with increasing levels of mRDC, keeping the supply of other nutrients as constant as possible. The mRDC content of feedstuffs was calculated based on a simple rumen model including soluble, washable, and nonwashable but potentially degradable fractions, as well as the fractional degradation and passage rates, of sugar, starch, neutral detergent fiber, and other carbohydrates. The mRDC term effectively represents the total amount of carbohydrates degraded in the rumen within 2 h after ingestion. Fifty-two lactating Holstein cows (of which 4 were rumen fistulated) were assigned to 4 treatments in a 4 × 4 Latin square design. Treatments were fed as a total mixed ration consisting of 25.4% corn silage, 23.1% grass silage, 11.6% grass hay, and 39.9% concentrate on a dry matter basis. Differences in mRDC were created by exchanging nonforage neutral detergent fiber-rich ingredients (mainly sugar beet pulp) with starch-rich ingredients (mainly wheat) and by exchanging corn (slowly degradable starch) with wheat (rapidly degradable starch) in the concentrate, resulting in 4 treatments that varied in dietary mRDC level of 167, 181, 194, or 208 g/kg of dry matter. Level of mRDC did not affect dry matter intake. Fat- and protein-corrected milk production and milk fat and lactose yield were greatest at 181 mRDC and decreased with further increases in mRDC. Milk protein yield and concentration increased with increasing mRDC level. Mean rumen pH and diurnal variation in ruminal pH did not differ between treatments. Total daily meal time and number of visits per meal were smaller at 181 and 194 mRDC. Despite milk

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.

Flexible electrochemical biosensors based on graphene nanowalls for the real-time measurement of lactate

NASA Astrophysics Data System (ADS)

Chen, Qianwei; Sun, Tai; Song, Xuefen; Ran, Qincui; Yu, Chongsheng; Yang, Jun; Feng, Hua; Yu, Leyong; Wei, Dapeng

2017-08-01

We demonstrate a flexible biosensor for lactate detection based on l-lactate oxidase immobilized by chitosan film cross-linked with glutaraldehyde on the surface of a graphene nanowall (GNW) electrode. The oxygen-plasma technique was developed to enhance the wettability of the GNWs, and the strength of the sensor’s oxidation response depended on the concentration of lactate. First, in order to eliminate interference from other substances, biosensors were primarily tested in deionized water and displayed good electrochemical reversibility at different scan rates (20-100 mV s-1), a large index range (1.0 μM to 10.0 mM) and a low detection limit (1.0 μM) for lactate. Next, these sensors were further examined in phosphate buffer solution (to mimick human body fluids), and still exhibited high sensitivity, stability and flexibility. These results show that the GNW-based lactate biosensors possess important potential for application in clinical analysis, sports medicine and the food industry.

Flexible electrochemical biosensors based on graphene nanowalls for the real-time measurement of lactate.

PubMed

Chen, Qianwei; Sun, Tai; Song, Xuefen; Ran, Qincui; Yu, Chongsheng; Yang, Jun; Feng, Hua; Yu, Leyong; Wei, Dapeng

2017-08-04

We demonstrate a flexible biosensor for lactate detection based on l-lactate oxidase immobilized by chitosan film cross-linked with glutaraldehyde on the surface of a graphene nanowall (GNW) electrode. The oxygen-plasma technique was developed to enhance the wettability of the GNWs, and the strength of the sensor's oxidation response depended on the concentration of lactate. First, in order to eliminate interference from other substances, biosensors were primarily tested in deionized water and displayed good electrochemical reversibility at different scan rates (20-100 mV s -1 ), a large index range (1.0 μM to 10.0 mM) and a low detection limit (1.0 μM) for lactate. Next, these sensors were further examined in phosphate buffer solution (to mimick human body fluids), and still exhibited high sensitivity, stability and flexibility. These results show that the GNW-based lactate biosensors possess important potential for application in clinical analysis, sports medicine and the food industry.

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.

Relayed 13C magnetization transfer: Detection of malate dehydrogenase reaction in vivo

NASA Astrophysics Data System (ADS)

Yang, Jehoon; Shen, Jun

2007-02-01

Malate dehydrogenase catalyzes rapid interconversion between dilute metabolites oxaloacetate and malate. Both oxaloacetate and malate are below the detection threshold of in vivo MRS. Oxaloacetate is also in rapid exchange with aspartate catalyzed by aspartate aminotransferase, the latter metabolite is observable in vivo using 13C MRS. We hypothesized that the rapid turnover of oxaloacetate can effectively relay perturbation of magnetization between malate and aspartate. Here, we report indirect observation of the malate dehydrogenase reaction by saturating malate C2 resonance at 71.2 ppm and detecting a reduced aspartate C2 signal at 53.2 ppm due to relayed magnetization transfer via oxaloacetate C2 at 201.3 ppm. Using this strategy the rate of the cerebral malate dehydrogenase reaction was determined to be 9 ± 2 μmol/g wet weight/min (means ± SD, n = 5) at 11.7 Tesla in anesthetized adult rats infused with [1,6- 13C 2]glucose.

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.

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.

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.

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.

(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.

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.

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.

Amperometric L-lactate biosensor based on screen-printed carbon electrode containing cobalt phthalocyanine, coated with lactate oxidase-mesoporous silica conjugate layer.

PubMed

Shimomura, Takeshi; Sumiya, Touru; Ono, Masatoshi; Ito, Tetsuji; Hanaoka, Taka-aki

2012-02-10

A novel amperometric biosensor for the measurement of L-lactate has been developed. The device comprises a screen-printed carbon electrode containing cobalt phthalocyanine (CoPC-SPCE), coated with lactate oxidase (LOD) that is immobilized in mesoporous silica (FSM8.0) using a polymer matrix of denatured polyvinyl alcohol; a Nafion layer on the electrode surface acts as a barrier to interferents. The sampling unit attached to the SPCE requires only a small sample volume of 100 μL for each measurement. The measurement of l-lactate is based on the signal produced by hydrogen peroxide, the product of the enzymatic reaction. The behavior of the biosensor, LOD-FSM8.0/Naf/CoPC-SPCE, was examined in terms of pH, applied potential, sensitivity and operational range, selectivity, and storage stability. The sensor showed an optimum response at a pH of 7.4 and an applied potential of +450 mV. The determination range and the response time for L-lactate were 18.3 μM to 1.5 mM and approximately 90s, respectively. In addition, the sensor exhibited high selectivity for L-lactate and was quite stable in storage, showing no noticeable change in its initial response after being stored for over 9 months. These results indicate that our method provides a simple, cost-effective, high-performance biosensor for l-lactate. Copyright © 2011 Elsevier B.V. All rights reserved.

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.

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.

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.

Disposable L-lactate biosensor based on a screen-printed carbon electrode enhanced by graphene

NASA Astrophysics Data System (ADS)

Tu, Dandan; He, Yu; Rong, Yuanzhen; Wang, You; Li, Guang

2016-04-01

In this work, an amperometric L-lactate biosensor based on a graphene-modified screen-printed carbon electrode (SPCE) was constructed. First, the electrocatalytic performance of the SPCE modified with graphene by a one-step electrodeposition process (OerGO/SPCE) was investigated. The cyclic voltammogram of OerGO/SPCE, which showed a well-defined redox peak, had a smaller peak potential separation than that of SPCE, revealing the improvement in electron transfer speed brought about by modifying with graphene. Next, lactate oxidase and potassium ferricyanide were dropped on the OerGO/SPCE to construct a graphene-modified L-lactate biosensor (LOD/K3[Fe(CN)6]/OerGO/SPCE). The proposed biosensor, with a detection limit of 60 μM, had a high sensitivity (42.42 μA mM-1 cm-2) when working at a low working potential (0.15 V). The linear range was 0.5 mM-15 mM, covering the detecting range of L-lactate in clinical applications. The L-lactate biosensor had a short response time (10 s) and required only 10 μl of the sample. This L-lactate sensor modified with electrodeposited graphene had a larger sensitivity than that based on the bare SPCE. Thus, our low-cost and disposable L-lactate biosensor enhanced by graphene can perform as an attractive electrochemical device that can be manufactured for point-of-care testing (POCT) devices and be employed in POCT applications.

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

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.

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

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.

Potential Biomarkers and Their Applications for Rapid and Reliable Detection of Malaria

PubMed Central

Jain, Priyamvada; Chakma, Babina; Patra, Sanjukta; Goswami, Pranab

2014-01-01

Malaria has been responsible for the highest mortality in most malaria endemic countries. Even after decades of malaria control campaigns, it still persists as a disease of high mortality due to improper diagnosis and rapidly evolving drug resistant malarial parasites. For efficient and economical malaria management, WHO recommends that all malaria suspected patients should receive proper diagnosis before administering drugs. It is thus imperative to develop fast, economical, and accurate techniques for diagnosis of malaria. In this regard an in-depth knowledge on malaria biomarkers is important to identify an appropriate biorecognition element and utilize it prudently to develop a reliable detection technique for diagnosis of the disease. Among the various biomarkers, plasmodial lactate dehydrogenase and histidine-rich protein II (HRP II) have received increasing attention for developing rapid and reliable detection techniques for malaria. The widely used rapid detection tests (RDTs) for malaria succumb to many drawbacks which promotes exploration of more efficient economical detection techniques. This paper provides an overview on the current status of malaria biomarkers, along with their potential utilization for developing different malaria diagnostic techniques and advanced biosensors. PMID:24804253

Implications of Parasites Lacking Plasmodium falciparum Histidine-Rich Protein 2 on Malaria Morbidity and Control When Rapid Diagnostic Tests Are Used for Diagnosis.

PubMed

Gatton, Michelle L; Dunn, Jessica; Chaudhry, Alisha; Ciketic, Sadmir; Cunningham, Jane; Cheng, Qin

2017-04-01

Rapid diagnostic tests (RDTs) are an important tool for malaria diagnosis, with most using antibodies against Plasmodium falciparum histidine-rich protein 2 (PfHRP2). Reports of P. falciparum lacking this protein are increasing, creating a problem for diagnosis of falciparum malaria in locations without quality-assured microscopy. An agent-based stochastic simulation model of P. falciparum transmission was used to investigate the selective pressure exerted on parasite populations by use of RDTs for diagnosis of symptomatic cases. The model considered parasites with normal, reduced, or no PfHRP2, and diagnosis using PfHRP2-only or combination RDTs. Use of PfHRP2-only RDTs in communities where a PfHRP2-negative parasite was introduced during the simulation resulted in transmission of the parasite in >80% of cases, compared with <30% for normal or PfHRP2-reduced parasites. Using PfHRP2-only RDTs in the presence of PfHRP2-negative parasites caused an increase in prevalence, reduced RDT positivity within symptomatic patients but no change in the number of antimalarial treatments due to false-negative RDT results. Diagnosis with PfHRP2/Pf-Plasmodium lactate dehydrogenase combination RDTs did not select for PfHRP2-negative parasites. The use of PfHRP2-only RDTs is sufficient to select P. falciparum parasites lacking this protein, thus posing a significant public health problem, which could be moderated by using PfHRP2/Pf-Plasmodium lactate dehydrogenase combination RDTs. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

Validation of caffeine dehydrogenase from Pseudomonas sp. strain CBB1 as a suitable enzyme for a rapid caffeine detection and potential diagnostic test.

PubMed

Mohanty, Sujit K; Yu, Chi Li; Gopishetty, Sridhar; Subramanian, Mani

2014-08-06

Excess consumption of caffeine (>400 mg/day/adult) can lead to adverse health effects. Recent introduction of caffeinated products (gums, jelly beans, energy drinks) might lead to excessive consumption, especially among children and nursing mothers, hence attracting the Food and Drug Administration's attention and product withdrawals. An "in-home" test will aid vigilant consumers in detecting caffeine in beverages and milk easily and quickly, thereby restricting its consumption. Known diagnostic methods lack speed and sensitivity. We report a caffeine dehydrogenase (Cdh)-based test which is highly sensitive (1-5 ppm) and detects caffeine in beverages and mother's milk in 1 min. Other components in these complex test samples do not interfere with the detection. Caffeine-dependent reduction of the dye iodonitrotetrazolium chloride results in shades of pink proportional to the levels in test samples. This test also estimates caffeine levels in pharmaceuticals, comparable to high-performance liquid chromatography. The Cdh-based test is the first with the desired attributes of a rapid and robust caffeine diagnostic kit.

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

Lactate metabolism and cytosolic NADH reducing equivalents in ovine adipocytes.

PubMed

Yang, Y T; White, L S; Muir, L A

1982-01-01

1. Isolated ovine adipocytes, unlike rat adipose tissue, could utilize lactate at a high rate. 2. When the rate of fatty acid synthesis was attenuated with 5-(tetradecyloxy)-2-furoic acid, a fatty acid synthesis inhibitor, there was a good positive correlation between the rates of lactate oxidation to CO2 and lactate incorporation into fatty acids. 3. Addition of 2,4-dinitrophenol enhanced lactate oxidation to CO2 independent of fatty acid synthesis. Under this condition, estimated cytosolic NADH formation from lactate dehydrogenation exceeded the need of NADH for cytosolic oxaloacetate reduction and for glyceride glycerol formation. 4. Mitochondria isolated from ovine adipocytes oxidized added NADH rapidly in a reconstituted alpha-glycerophosphate shuttle system. 5. It is possible that the ability of ovine adipocytes to utilize lactate may be related to the active alpha-glycerophosphate shuttle for cytosolic NADH reoxidation.

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.

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

Accuracy of Two Malaria Rapid Diagnostic Tests (RDTS) for Initial Diagnosis and Treatment Monitoring in a High Transmission Setting in Uganda

PubMed Central

Mbabazi, Phoebe; Hopkins, Heidi; Osilo, Emmanuel; Kalungu, Michael; Byakika-Kibwika, Pauline; Kamya, Moses R.

2015-01-01

Malaria rapid diagnostic tests (RDTs) may improve fever management in areas without microscopy. We compared the accuracy of histidine-rich protein 2 (HRP2) and Plasmodium lactate dehydrogenase (pLDH)-based RDTs, using expert microscopy as a gold standard, for initial diagnosis, treatment monitoring, and diagnosis of recurrent malaria in a cohort of children followed longitudinally in a high-transmission area in Uganda. For 305 initial fever episodes, sensitivity was 98% for HRP2 and 87% for pLDH, whereas specificity was 55% and 96%, respectively. The HRP2 gave 51% false-positive results on Day 28, whereas pLDH gave no false positives after Day 7. For 59 recurrent fever episodes during follow-up, sensitivity was 100% for HRP2 and 91% for pLDH, whereas specificity was 33% and 100%, respectively. The HRP2-based RDTs are useful for initial diagnosis of malaria caused by superior sensitivity; however, as a result of superior specificity, pLDH-based RDTs are more appropriate to monitor treatment and diagnose recurrent malaria. PMID:25624399

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.

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.

Importance of measuring lactate levels in children with sepsis.

PubMed

Anil, Nisha

2017-10-10

Sepsis is a major public health problem as well as one of the leading causes of preventable death in children because of failure to recognise the early signs and symptoms and to resuscitate rapidly. Blood lactate levels are used to assess the severity of sepsis and the effectiveness of resuscitation. Lactate levels are easily obtainable and should be checked in all patients admitted with suspected sepsis within six hours of presentation. The test should be repeated four and eight-hours post-diagnosis of sepsis. For the diagnosis of sepsis, patients' clinical symptoms, along with the combined analysis of partial pressure of oxygen, carbon dioxide and lactate levels, should be used. A multitude of factors can cause elevated lactate levels and so clinicians should use elevated levels cautiously by considering all other aetiologies. This article, which focuses on practice in Australia but makes reference to the UK, discusses the importance of measuring lactate levels in sepsis, the pathophysiology of lactate production, causes of elevated lactate levels, lactate measurement, nursing management of patients with elevated lactate levels, limitations of using lactate as a biomarker for diagnosing sepsis and implications for practice. ©2012 RCN Publishing Company Ltd. All rights reserved. Not to be copied, transmitted or recorded in any way, in whole or part, without prior permission of the publishers.

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.

Evaluation of a PfHRP2 and a pLDH-based Rapid Diagnostic Test for the Diagnosis of Severe Malaria in 2 Populations of African Children

PubMed Central

Hendriksen, Ilse C. E.; Mtove, George; Pedro, Alínia José; Gomes, Ermelinda; Silamut, Kamolrat; Lee, Sue J.; Mwambuli, Abraham; Gesase, Samwel; Reyburn, Hugh; Day, Nicholas P. J.; White, Nicholas J.; von Seidlein, Lorenz

2011-01-01

Background. Rapid diagnostic tests (RDTs) now play an important role in the diagnosis of falciparum malaria in many countries where the disease is endemic. Although these tests have been extensively evaluated in uncomplicated falciparum malaria, reliable data on their performance for diagnosing potentially lethal severe malaria is lacking. Methods. We compared a Plasmodium falciparum histidine-rich-protein2 (PfHRP2)–based RDT and a Plasmodium lactate dehydrogenase (pLDH)–based RDT with routine microscopy of a peripheral blood slide and expert microscopy as a reference standard for the diagnosis of severe malaria in 1898 children who presented with severe febrile illness at 2 centers in Mozambique and Tanzania. Results. The overall sensitivity, specificity, positive predictive value, and negative predictive values of the PfHRP2-based test were 94.0%, 70.9%, 85.4%, and 86.8%, respectively, and for the pLDH-based test, the values were 88.0%, 88.3%, 93.2%, and 80.3%, respectively. At parasite counts <1000 parasites/μL (n = 173), sensitivity of the pLDH-based test was low (45.7%), compared with that of the PfHRP2-based test (69.9%). Both RDTs performed better than did the routine slide reading in a clinical laboratory as assessed in 1 of the centers. Conclusion. The evaluated PfHRP2-based RDT is an acceptable alternative to routine microscopy for diagnosing severe malaria in African children and performed better than did the evaluated pLDH-based RDT. PMID:21467015

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.

[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.

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.

Determination of ammonium ion using a reagentless amperometric biosensor based on immobilized alanine dehydrogenase.

PubMed

Tan, Ling Ling; Musa, Ahmad; Lee, Yook Heng

2011-01-01

The use of the enzyme alanine dehydrogenase (AlaDH) for the determination of ammonium ion (NH(4)(+)) usually requires the addition of pyruvate substrate and reduced nicotinamide adenine dinucleotide (NADH) simultaneously to effect the reaction. This addition of reagents is inconvenient when an enzyme biosensor based on AlaDH is used. To resolve the problem, a novel reagentless amperometric biosensor using a stacked methacrylic membrane system coated onto a screen-printed carbon paste electrode (SPE) for NH(4)(+) ion determination is described. A mixture of pyruvate and NADH was immobilized in low molecular weight poly(2-hydroxyethyl methacrylate) (pHEMA) membrane, which was then deposited over a photocured pHEMA membrane (photoHEMA) containing alanine dehydrogenase (AlaDH) enzyme. Due to the enzymatic reaction of AlaDH and the pyruvate substrate, NH(4)(+) was consumed in the process and thus the signal from the electrocatalytic oxidation of NADH at an applied potential of +0.55 V was proportional to the NH(4)(+) ion concentration under optimal conditions. The stacked methacrylate membranes responded rapidly and linearly to changes in NH(4)(+) ion concentrations between 10-100 mM, with a detection limit of 0.18 mM NH(4)(+) ion. The reproducibility of the amperometrical NH(4)(+) biosensor yielded low relative standard deviations between 1.4-4.9%. The stacked membrane biosensor has been successfully applied to the determination of NH(4)(+) ion in spiked river water samples without pretreatment. A good correlation was found between the analytical results for NH(4)(+) obtained from the biosensor and the Nessler spectrophotometric method.

Efficient calcium lactate production by fermentation coupled with crystallization-based in situ product removal.

PubMed

Xu, Ke; Xu, Ping

2014-07-01

Lactic acid is a platform chemical with various industrial applications, and its derivative, calcium lactate, is an important food additive. Fermentation coupled with in situ product removal (ISPR) can provide more outputs with high productivity. The method used in this study was based on calcium lactate crystallization. Three cycles of crystallization were performed during the fermentation course using a Bacillus coagulans strain H-1. As compared to fed-batch fermentation, this method showed 1.7 times higher average productivity considering seed culture, with 74.4% more L-lactic acid produced in the fermentation with ISPR. Thus, fermentation coupled with crystallization-based ISPR may be a biotechnological alternative that provides an efficient system for production of calcium lactate or lactic acid. Copyright © 2014 Elsevier Ltd. All rights reserved.

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.

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

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

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.

The Eyes Have It: A Problem-Based Learning Exercise in Molecular Evolution

ERIC Educational Resources Information Center

White, Harold B.

2007-01-01

Molecular evolution provides an interesting context in which to use problem-based learning because it integrates a variety of topics in biology, biochemistry, and molecular biology. This three-stage problem for advanced students deals with the structure, multiple functions, and properties of lactate dehydrogenase isozymes, and the related…

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

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.

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

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.

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.

Evaluation of the sensitivity of a pLDH-based and an aldolase-based rapid diagnostic test for diagnosis of uncomplicated and severe malaria caused by PCR-confirmed Plasmodium knowlesi, Plasmodium falciparum, and Plasmodium vivax.

PubMed

Barber, Bridget E; William, Timothy; Grigg, Matthew J; Piera, Kim; Yeo, Tsin W; Anstey, Nicholas M

2013-04-01

Plasmodium knowlesi can cause severe and fatal human malaria in Southeast Asia. Rapid diagnosis of all Plasmodium species is essential for initiation of effective treatment. Rapid diagnostic tests (RDTs) are sensitive for detection of uncomplicated and severe falciparum malaria but have not been systematically evaluated in knowlesi malaria. At a tertiary referral hospital in Sabah, Malaysia, we prospectively evaluated the sensitivity of two combination RDTs for the diagnosis of uncomplicated and severe malaria from all three potentially fatal Plasmodium species, using a pan-Plasmodium lactate dehydrogenase (pLDH)-P. falciparum histidine-rich protein 2 (PfHRP2) RDT (First Response) and a pan-Plasmodium aldolase-PfHRP2 RDT (ParaHIT). Among 293 hospitalized adults with PCR-confirmed Plasmodium monoinfection, the sensitivity of the pLDH component of the pLDH-PfHRP2 RDT was 74% (95/129; 95% confidence interval [CI], 65 to 80%), 91% (110/121; 95% CI, 84 to 95%), and 95% (41/43; 95% CI, 85 to 99%) for PCR-confirmed P. knowlesi, P. falciparum, and P. vivax infections, respectively, and 88% (30/34; 95% CI, 73 to 95%), 90% (38/42; 95% CI, 78 to 96%), and 100% (12/12; 95% CI, 76 to 100%) among patients tested before antimalarial treatment was begun. Sensitivity in severe malaria was 95% (36/38; 95% CI, 83 to 99), 100% (13/13; 95% CI, 77 to 100), and 100% (7/7; 95% CI, 65 to 100%), respectively. The aldolase component of the aldolase-PfHRP2 RDT performed poorly in all Plasmodium species. The pLDH-based RDT was highly sensitive for the diagnosis of severe malaria from all species; however, neither the pLDH- nor aldolase-based RDT demonstrated sufficiently high overall sensitivity for P. knowlesi. More sensitive RDTs are needed in regions of P. knowlesi endemicity.

A method for simultaneous echo planar imaging of hyperpolarized 13C pyruvate and 13C lactate

NASA Astrophysics Data System (ADS)

Reed, Galen D.; Larson, Peder E. Z.; von Morze, Cornelius; Bok, Robert; Lustig, Michael; Kerr, Adam B.; Pauly, John M.; Kurhanewicz, John; Vigneron, Daniel B.

2012-04-01

A rapid echo planar imaging sequence for dynamic imaging of [1-13C] lactate and [1-13C] pyruvate simultaneously was developed. Frequency-based separation of these metabolites was achieved by spatial shifting in the phase-encoded direction with the appropriate choice of echo spacing. Suppression of the pyruvate-hydrate and alanine resonances is achieved through an optimized spectral-spatial RF waveform. Signal sampling efficiency as a function of pyruvate and lactate excitation angle was simulated using two site exchange models. Dynamic imaging is demonstrated in a transgenic mouse model, and phantom validations of the RF pulse frequency selectivity were performed.

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

Analytical performance of three whole blood point-of-care lactate devices compared to plasma lactate comparison methods and a flow-injection mass spectrometry method.

PubMed

Tolan, Nicole V; Wockenfus, Amy M; Koch, Christopher D; Crews, Bridgit O; Dietzen, Dennis J; Karon, Brad S

2017-03-01

Point of care (POC) whole blood lactate testing may facilitate rapid detection of sepsis. We evaluated three POC methods against both plasma lactate comparison methods and a flow-injection mass spectrometric (MS) method. Nova StatStrip, Abbott i-STAT CG4+ and Radiometer ABL90 POC lactate methods were evaluated against the mean of Cobas Integra 400 and Vitros 350 plasma lactate. POC methods were also compared to a flow-injection mass spectrometric assay measuring lactate in ZnSO 4 -precipitated whole blood extracts. Intra- and inter-assay precision was determined using quality control material. Method comparison included specimens from normal donors at rest, after exertion, and after spiking with lactic acid. Intra- and inter-assay coefficient of variation was <5% for i-STAT and ABL90; but ranged from 3.1-8.2% on two StatStrip meters. Mean (±SD) bias between POC and plasma lactate ranged from -0.2±0.9 (i-STAT and ABL90) to -0.4±1.2 (StatStrip) mmol/L. At concentrations >6mmol/L, all POC methods showed proportional negative bias compared to plasma methods; but this bias was not observed when compared to the MS method. Despite proportional negative bias, all POC methods demonstrated acceptable concordance (94-100%) with plasma lactate within the reference interval (<2.3mmol/L) and >4mmol/L, commonly used clinical cut-offs for detection of sepsis. POC lactate methods demonstrate acceptable concordance with plasma lactate across commonly used clinical cut-offs for detection of sepsis. Due to systematic negative bias at higher lactate concentrations, POC and plasma lactate should not be used interchangeably to monitor patients with elevated lactate concentrations. Copyright © 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

A microbial factory for lactate-based polyesters using a lactate-polymerizing enzyme

PubMed Central

Taguchi, Seiichi; Yamada, Miwa; Matsumoto, Ken'ichiro; Tajima, Kenji; Satoh, Yasuharu; Munekata, Masanobu; Ohno, Katsuhiro; Kohda, Katsunori; Shimamura, Takashi; Kambe, Hiromi; Obata, Shusei

2008-01-01

Polylactate (PLA) is synthesized as a representative bio-based polyester by the chemo-bio process on the basis of metal catalyst-mediated chemical polymerization of lactate (LA) supplied by microbial fermentation. To establish the one-step microbial process for synthesis of LA-based polyesters, we explored whether polyhydroxyalkanoate (PHA) synthase would exhibit polymerizing activity toward a LA-coenzyme A (CoA), based on the fact that PHA monomeric constituents, especially 3-hydroxybutyrate (3HB), are structurally analogous to LA. An engineered PHA synthase was discovered as a candidate by a two-phase in vitro polymerization system previously developed. An LA-CoA producing Escherichia coli strain with a CoA transferase gene was constructed, and the generation of LA-CoA was demonstrated by capillary electrophoresis/MS analysis. Next, when the engineered PHA synthase gene was introduced into the resultant recombinant strain, we confirmed the one-step biosynthesis of the LA-incorporated copolyester, P(6 mol% LA-co-94 mol% 3HB), with a number-average molecular weight of 1.9 × 105, as revealed by gel permeation chromatography, gas chromatography/MS, and NMR. PMID:18978031

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.

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.

DFT-based prediction of reactivity of short-chain alcohol dehydrogenase

NASA Astrophysics Data System (ADS)

Stawoska, I.; Dudzik, A.; Wasylewski, M.; Jemioła-Rzemińska, M.; Skoczowski, A.; Strzałka, K.; Szaleniec, M.

2017-06-01

The reaction mechanism of ketone reduction by short chain dehydrogenase/reductase, ( S)-1-phenylethanol dehydrogenase from Aromatoleum aromaticum, was studied with DFT methods using cluster model approach. The characteristics of the hydride transfer process were investigated based on reaction of acetophenone and its eight structural analogues. The results confirmed previously suggested concomitant transfer of hydride from NADH to carbonyl C atom of the substrate with proton transfer from Tyr to carbonyl O atom. However, additional coupled motion of the next proton in the proton-relay system, between O2' ribose hydroxyl and Tyr154 was observed. The protonation of Lys158 seems not to affect the pKa of Tyr154, as the stable tyrosyl anion was observed only for a neutral Lys158 in the high pH model. The calculated reaction energies and reaction barriers were calibrated by calorimetric and kinetic methods. This allowed an excellent prediction of the reaction enthalpies (R2 = 0.93) and a good prediction of the reaction kinetics (R2 = 0.89). The observed relations were validated in prediction of log K eq obtained for real whole-cell reactor systems that modelled industrial synthesis of S-alcohols.

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.

An insert-based enzymatic cell culture system to rapidly and reversibly induce hypoxia: investigations of hypoxia-induced cell damage, protein expression and phosphorylation in neuronal IMR-32 cells

PubMed Central

Huang, Ying; Zitta, Karina; Bein, Berthold; Steinfath, Markus; Albrecht, Martin

2013-01-01

SUMMARY Ischemia-reperfusion injury and tissue hypoxia are of high clinical relevance because they are associated with various pathophysiological conditions such as myocardial infarction and stroke. Nevertheless, the underlying mechanisms causing cell damage are still not fully understood, which is at least partially due to the lack of cell culture systems for the induction of rapid and transient hypoxic conditions. The aim of the study was to establish a model that is suitable for the investigation of cellular and molecular effects associated with transient and long-term hypoxia and to gain insights into hypoxia-mediated mechanisms employing a neuronal culture system. A semipermeable membrane insert system in combination with the hypoxia-inducing enzymes glucose oxidase and catalase was employed to rapidly and reversibly generate hypoxic conditions in the culture medium. Hydrogen peroxide assays, glucose measurements and western blotting were performed to validate the system and to evaluate the effects of the generated hypoxia on neuronal IMR-32 cells. Using the insert-based two-enzyme model, hypoxic conditions were rapidly induced in the culture medium. Glucose concentrations gradually decreased, whereas levels of hydrogen peroxide were not altered. Moreover, a rapid and reversible (onoff) generation of hypoxia could be performed by the addition and subsequent removal of the enzyme-containing inserts. Employing neuronal IMR-32 cells, we showed that 3 hours of hypoxia led to morphological signs of cellular damage and significantly increased levels of lactate dehydrogenase (a biochemical marker of cell damage). Hypoxic conditions also increased the amounts of cellular procaspase-3 and catalase as well as phosphorylation of the pro-survival kinase Akt, but not Erk1/2 or STAT5. In summary, we present a novel framework for investigating hypoxia-mediated mechanisms at the cellular level. We claim that the model, the first of its kind, enables researchers to rapidly and

pLDH level of clinically isolated Plasmodium vivax and detection limit of pLDH based malaria rapid diagnostic test.

PubMed

Jang, Jin Woo; Cho, Chi Hyun; Han, Eun Taek; An, Seong Soo A; Lim, Chae Seung

2013-06-03

The malaria rapid diagnostic tests (RDTs) are now widely used in the world. Compared to Plasmodium falciparum, a poor sensitivity of RDTs was reported against Plasmodium vivax based on the adopted antibody against pan-Plasmodium antigen lactate dehydrogenase (pLDH) or aldolase. Levels of pLDH were measured from patient with P. vivax, and the correlations between the levels of pLDH and the sensitivities of RDTs were analysed among Republic of Korea (ROK) isolates. Three RDTs, OptiMAL test, SD BIOLINE Malaria Ag P.f/Pan test, Humasis Malaria Pf/Pan antigen test, and the Genedia pLDH antigen ELISA were performed with blood samples from 152 febrile patients and 100 healthy controls. Three malaria RDTs revealed sensitivities between 85.5 (131/152) and 86.8% (132/152) with highest sensitivity for the detection of P.vivax by pLDH antigen ELISA test (145/152, 95.4%) in comparison to traditional microscopy using Giemsa-stained slides. None of the healthy control tested positive by three RDTs or ELISA, indicating 100% specificity in their respective test. Levels of pLDH among Korean P. vivax isolates ranged between 0 ng/mL and 22,387.2 ng/mL (mean ± standard deviation 3,917.5 ± 6,120.9 ng/mL). The lower detection limits of three RDTs were between 25 and 50 ng/mL with artificially diluted samples. The moderate degree of correlation was observed between parasitaemia and concentrations of pLDH (r = 0.4, p < 0.05). The pLDH levels of P. vivax are the main explanation for the variations in the performance of pLDH-based RDTs. Therefore, comparing sensitivities of RDT may need to include targeted biomarker value of patients.

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

Convenient microtiter plate-based, oxygen-independent activity assays for flavin-dependent oxidoreductases based on different redox dyes

PubMed Central

Brugger, Dagmar; Krondorfer, Iris; Zahma, Kawah; Stoisser, Thomas; Bolivar, Juan M; Nidetzky, Bernd; Peterbauer, Clemens K; Haltrich, Dietmar

2014-01-01

Flavin-dependent oxidoreductases are increasingly recognized as important biocatalysts for various industrial applications. In order to identify novel activities and to improve these enzymes in engineering approaches, suitable screening methods are necessary. We developed novel microtiter-plate-based assays for flavin-dependent oxidases and dehydrogenases using redox dyes as electron acceptors for these enzymes. 2,6-dichlorophenol-indophenol, methylene green, and thionine show absorption changes between their oxidized and reduced forms in the visible range, making it easy to judge visually changes in activity. A sample set of enzymes containing both flavoprotein oxidases and dehydrogenases – pyranose 2-oxidase, pyranose dehydrogenase, cellobiose dehydrogenase, d-amino acid oxidase, and l-lactate oxidase – was selected. Assays for these enzymes are based on a direct enzymatic reduction of the redox dyes and not on the coupled detection of a reaction product as in the frequently used assays based on hydrogen peroxide formation. The different flavoproteins show low Michaelis constants with these electron acceptor substrates, and therefore these dyes need to be added in only low concentrations to assure substrate saturation. In conclusion, these electron acceptors are useful in selective, reliable and cheap MTP-based screening assays for a range of flavin-dependent oxidoreductases, and offer a robust method for library screening, which could find applications in enzyme engineering programs. PMID:24376171

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

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.

Technical performance of lactate biosensors and a test-strip device during labour.

PubMed

Luttkus, A K; Fotopoulou, C; Sehouli, J; Stupin, J; Dudenhausen, J W

2010-04-01

Lactate in fetal blood has a high diagnostic power to detect fetal compromise due to hypoxia, as lactate allows an estimation of duration and intensity of metabolic acidemia. Biosensor technology allows an instantaneous diagnosis of fetal compromise in the delivery room. The goal of the current investigation is to define the preanalytical and analytical biases of this technology under routine conditions in a labour ward in comparison to test-strip technology, which allows measurement of lactate alone. Three lactate biosensors (RapidLab 865, Siemens Medical Solutions Diagnostics, Bad Nauheim, Germany; Radiometer ABL625 and ABL 700, Radiometer Copenhagen, Denmark) and one test-strip device (Lactate Pro, Oxford Instruments, UK) were evaluated regarding precision in serial and repetitive measurements in over 1350 samples of fetal whole blood. The coefficient of variation (CV) and the standard deviation (SD) were calculated. The average value of all three biosensors was defined as an artificial reference value (refval). Blood tonometry was performed in order to test the quality of respiratory parameters and to simulate conditions of fetal hypoxia (pO (2): 10 and 20 mmHg). The precision of serial measurements of all biosensors indicated a coefficient of variation (CV) between 1.55 and 3.16% with an SD from 0.042 to 0.053 mmol/L. The test-strip device (Lactate Pro) mounted to 0.117 mmol/L and 3.99% (SD, CV). When compared to our reference value (refval) ABL 625 showed the closest correlation of -0.1%, while Siemens RapidLab 865 showed an overestimation of +8.9%, ABL700 an underestimation of -6.2% and Lactate Pro of -3.7%. For routine use all tested biosensors show sufficient precision. The test-strip device shows a slightly higher standard deviation. A direct comparison of measured lactate values from the various devices needs to be interpreted with caution as each method detects different lactate concentrations. Furthermore, the 40 min process of tonometry led to an

Pregnancy development from day 28 to 42 of gestation in postpartum Holstein cows that were either milked (lactating) or not milked (not lactating) after calving.

PubMed

Green, Jacob C; Meyer, Joseph P; Williams, Amanda M; Newsom, Emily M; Keisler, Duane H; Lucy, Matthew C

2012-05-01

The objective was to determine if lactation affects fetal and placental development from day 28 to 42 of gestation. Bos taurus Holstein cows were assigned to one of the two treatments immediately after parturition (lactating (n=23) or nonlactating (dried off immediately after calving; n=20)). Cows were inseminated at ~60 days postpartum with semen from a single ejaculate. Pregnant cows were slaughtered at 1 of 3 days of gestation (day 28, 35, or 42) and tissues were collected. The interval to first insemination, services per conception, and days to pregnancy were similar for lactating and nonlactating cows. Lactating cows had greater plasma GH and nonesterified fatty acids. Nonlactating cows had greater plasma glucose, insulin, and IGF1. There was no effect of lactation on plasma progesterone or estradiol concentrations. Lactation had a negative effect on the weight of the fetus and placenta (weights were less in lactating cows). Fetuses collected from cows that became pregnant after first insemination were heavier than fetuses collected from cows that became pregnant after second or third insemination. Pregnancy after first insemination was associated with greater blood glucose and IGF1 during the first 30 days postpartum. The conclusions were that lactation negatively affects the growth of fetal and placental tissues perhaps through a mechanism that involves hormones and metabolites that are affected by lactation. Fetal growth within cows conceiving at first insemination compared to second or third insemination was more rapid and was associated with greater blood glucose and IGF1 early postpartum (before day 30).

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.

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 ...

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.

Comparative evaluation of a new lactation curve model for pasture-based Holstein-Friesian dairy cows.

PubMed

Adediran, S A; Ratkowsky, D A; Donaghy, D J; Malau-Aduli, A E O

2012-09-01

Fourteen lactation models were fitted to average and individual cow lactation data from pasture-based dairy systems in the Australian states of Victoria and Tasmania. The models included a new "log-quadratic" model, and a major objective was to evaluate and compare the performance of this model with the other models. Nine empirical and 5 mechanistic models were first fitted to average test-day milk yield of Holstein-Friesian dairy cows using the nonlinear procedure in SAS. Two additional semiparametric models were fitted using a linear model in ASReml. To investigate the influence of days to first test-day and the number of test-days, 5 of the best-fitting models were then fitted to individual cow lactation data. Model goodness of fit was evaluated using criteria such as the residual mean square, the distribution of residuals, the correlation between actual and predicted values, and the Wald-Wolfowitz runs test. Goodness of fit was similar in all but one of the models in terms of fitting average lactation but they differed in their ability to predict individual lactations. In particular, the widely used incomplete gamma model most displayed this failing. The new log-quadratic model was robust in fitting average and individual lactations, and was less affected by sampled data and more parsimonious in having only 3 parameters, each of which lends itself to biological interpretation. Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Lactate and acid base changes during laparoscopic cholecystectomy.

PubMed

Ibraheim, Osama A; Samarkandi, Abdulhamid H; Alshehry, Hassan; Faden, Awatif; Farouk, Eltinay Omar

2006-02-01

The observation of hemodynamic and metabolic impairment related to CO2 pneumoperitoneum and postoperative mesenteric ischemia reports following laparoscopic procedures have raised concern about local and systemic effects of increase intraabdominal pressure during laparoscopic procedures. The present study aims to evaluate the metabolic and acid base responses of using high pressure versus low pressure pneumoperitonium in patients undergoing laparoscopic cholecystectomy in a prospective randomized clinical trial. 20 ASA I-II patients scheduled for elective laparoscopic cholecystectomy were randomly allocated to one of two study groups; high pressure pneumoperitoneum 12-14mmHg (HPP, n=10) versus low pressure pneumoperitoneum 6-8mmHg (LPP, n=10) undergoing laparoscopic cholecystectomy. Arterial blood gases and lactate levels were determined after induction of anesthesia (before pneumoperitonium), then after 10 min, then 30 min after insufflations and at the end of surgery and 1 hour postoperatively. Nurses in recovery unit reported pain assessment starting postoperatively until 3 hours on a 10mm VAS (0-10). Statistical significant was established at P<0.05. Bicarbonate was significantly (P>0.0412) lower in high pressure group at 30 min and 60 min after insufflations. In high pressure group lactate levels increased significantly as compared to low pressure group, (at 30 minutes after the establishment of abdominal pneumatic inflation P<0.006 and remained significantly increased (P<0.001) until the end of surgery and one hour thereafter) (P<0.001). The mean postoperative pain score during second hour (VAS) at HPP group was 7.4 +/- 1.17 which is significantly (P < or = 0.006) higher than pain score in LPP group 5.0 +/- 1.886. Shoulder tip pain was reported in 3 patients in the high pressure group and only one patient in the lower pressure group. High-pressure pneumoperitonium causes statistically significant elevation in the arterial lactate level intraoperatively until

A rare cause of metabolic acidosis: ketoacidosis in a non-diabetic lactating woman

PubMed Central

Ali, Amjad; Webster, Jonathan

2017-01-01

Ketoacidosis occurring during lactation has been described infrequently. The condition is incompletely understood, but it appears to be associated with a combination of increased metabolic demands during lactation, reduction in carbohydrate intake and acute illness. We present a case of a 27-year-old woman, 8 weeks post-partum, who was exclusively breastfeeding her child whilst following a low carbohydrate diet. She developed gastroenteritis and was unable to tolerate an oral diet for several days. She presented with severe metabolic acidosis on admission with a blood 3-hydroxybutyrate of 5.4 mmol/L. She was treated with intravenous dextrose and intravenous sodium bicarbonate, and given dietary advice to increase her carbohydrate intake. She made a rapid and full recovery. We provide a summary of the common causes of ketoacidosis and compare our case with other presentations of lactation ketoacidosis. Learning points: Ketoacidosis in the lactating woman is a rare cause of raised anion gap metabolic acidosis. Low carbohydrate intake, starvation, intercurrent illness or a combination of these factors could put breastfeeding women at risk of ketoacidosis. Ketoacidosis in the lactating woman has been shown to resolve rapidly with sufficient carbohydrate intake and intravenous dextrose. Early diagnosis and prompt treatment are essential because the condition is reported to be reversible with a low chance of recurrence with appropriate dietary advice. PMID:28924478

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

IMP Dehydrogenase: Structural Schizophrenia and an Unusual Base

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

Hedstrom,L.; Gan, L.

2006-01-01

Textbooks describe enzymes as relatively rigid templates for the transition state of a chemical reaction, and indeed an enzyme such as chymotrypsin, which catalyzes a relatively simple hydrolysis reaction, is reasonably well described by this model. Inosine monophosphate dehydrogenase (IMPDH) undergoes a remarkable array of conformational transitions in the course of a complicated catalytic cycle, offering a dramatic counterexample to this view. IMPDH displays several other unusual mechanistic features, including an Arg residue that may act as a general base catalyst and a dynamic monovalent cation site. Further, IMPDH appears to be involved in 'moon-lighting' functions that may require additionalmore » conformational states. How the balance between conformational states is maintained and how the various conformational states interconvert is only beginning to be understood.« less

[Cloning,expression and functional identification of secoisolariciresinol dehydrogenase gene from Dysosma versipellis callus].

PubMed

Shen, Yun; Chen, Ri-Dao; Xie, Ke-Bo; Zou, Jian-Hua; Dai, Jun-Gui

2016-12-01

Secoisolariciresinol dehydrogenase (SDH) is a key enzyme involved in the biosynthetic pathway of podophyllotoxin.In this study, two SDH candidate genes,SO282 and SO1223, were cloned from callus of Dysosma versipellis by homology-based PCR and rapid amplification of cDNA end (RACE).The SDH candidate genes were expressed in Escherichia coli and the subsequent enzyme assay in vitro showed that recombinant SO282 had the SDH activity. These results pave the way to the follow-up investigation of the biosynthetic of podophyllotoxin. Copyright© by the Chinese Pharmaceutical Association.

A physiologically based pharmacokinetic model for lactational transfer of Na-131I

NASA Astrophysics Data System (ADS)

Turner, Anita Loretta

The excretion of radionuclides in human breast milk after administration of radiopharmaceuticals is a concern as a radiation risk to nursing infants. It is not uncommon to administer radiopharmaceuticals to lactating patients due to emergency nuclear medicine investigations such as thyroid complications, kidney failure, and pulmonary embolism. There is a need to quantify the amount of radioactivity translocated into breast milk in cases of ingestion by a breast-fed infant. A physiologically based pharmacokinetic model (PBPK) and a modified International Commission on Radiological Protection (ICRP) model have been developed to predict iodine concentrations in breast milk after ingestion of radioiodine by the mother. In the PBPK model, all compartments are interconnected by blood flow and represent real anatomic tissue regions in the body. All parameters involved are measurable values with physiological or physiochemical meaning such as tissue masses, blood flow rates, partition coefficients and cardiac output. However, some of the parameters such as the partition coefficients and metabolic constants are not available for iodine and had to be inferred from other information. The structure of the PBPK model for the mother consists of the following tissue compartments: gastrointestinal tract, blood, kidney, thyroid, milk, and other tissues. With the exception of the milk compartment, the model for the nursing infant is structured similarly to the mother. The ICRP model describing iodine metabolism in a standard 70-kg man was modified to represent iodine metabolism in a lactating woman and nursing infant. The parameters involved in this model are transfer rates and biological half-lives which are based on experimental observations. The results of the PBPK model and the modified ICRP model describing the lactational transfer of iodine were compared. When administering 1 mCi of Na131I to the lactating mother, the concentration reaches a maximum of 0.1 mCi/liter in 24

Rapid epidemiologic assessment of glucose-6-phosphate dehydrogenase deficiency in malaria-endemic areas in Southeast Asia using a novel diagnostic kit.

PubMed

Jalloh, A; Tantular, I S; Pusarawati, S; Kawilarang, A P; Kerong, H; Lin, K; Ferreira, M U; Matsuoka, H; Arai, M; Kita, K; Kawamoto, F

2004-05-01

We recently reported a new rapid screening method for glucose-6-phosphate dehydrogenase (G6PD) deficiency. This method incorporates a new formazan substrate (WST-8) and is capable of detecting heterozygous females both qualitatively and quantitatively. Here, we report its evaluation during field surveys at three malaria centres and in malaria-endemic villages of Myanmar and Indonesia, either alone or in combination with a rapid on-site diagnosis of malaria. A total of 57 severe (45 males and 12 females) and 34 mild (five males and 29 females) cases of G6PD deficiency were detected among 855 subjects in Myanmar whilst 30 severe (25 males and five females) and 23 mild (six males and 17 females) cases were found among 1286 subjects in Indonesia. In all cases, severe deficiency was confirmed with another formazan method but due to limitations in its detection threshold, mild cases were misdiagnosed as G6PD-normal by this latter method. Our results indicate that the novel method can qualitatively detect both severely deficient subjects as well as heterozygous females in the field. The antimalarial drug, primaquine, was safely prescribed to Plasmodium vivax-infected patients in Myanmar. Our new, rapid screening method may be essential for the diagnosis of G6PD deficiency particularly in rural areas without electricity, and can be recommended for use in malaria control programmes.

Lactate clearance as the predictor of outcome in pediatric septic shock.

PubMed

Choudhary, Richa; Sitaraman, Sadasivan; Choudhary, Anita

2017-01-01

Septic shock can rapidly evolve into multiple system organ failure and death. In the recent years, hyperlactatemia has been found to be a risk factor for mortality in critically ill adults. To evaluate the predictive value of lactate clearance and to determine the optimal cut-off value for predicting outcome in children with septic shock. A prospective observational study was performed on children with septic shock admitted to pediatric Intensive Care Unit (PICU). Serial lactate levels were measured at PICU admission, 24 and 48 h later. Lactate clearance, percent decrease in lactate level in 24 h, was calculated. The primary outcome measure was survival or nonsurvival at the end of hospital stay. We performed receiver operating characteristic analyses to calculate optimal cut-off values. The mean lactate levels at admission were significantly higher in the nonsurvivors than survivors, 5.12 ± 3.51 versus 3.13 ± 1.71 mmol/L ( P = 0.0001). The cut-off for lactate level at admission for the best prediction of mortality was determined as ≥4 mmol/L (odds ratio 5.4; 95% confidence interval [CI] =2.45-12.09). Mean lactate clearance was significantly higher in survivors than nonsurvivors (17.9 ± 39.9 vs. -23.2 ± 62.7; P < 0.0001). A lactate clearance rate of <10% at 24 h had a sensitivity and specificity of 78.7% and 72.2%, respectively and a positive predictive value of 83.1% for death. Failure to achieve a lactate clearance of more than 10% was associated with greater risk of mortality (likelihood ratio + 2.83; 95% CI = 1.82-4.41). Serial lactate levels can be used to predict outcome in pediatric septic shock. A 24 h lactate clearance cut-off of <10% is a predictor of in-hospital mortality in such patients.

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

Effect of capture stress on plasma enzyme activities in rainbow trout (Salmo gairdneri)

USGS Publications Warehouse

Bouck, G.R.; Cairns, M. A.; Christian, A. R.

1978-01-01

Four capture methods were used to collect domesticated rainbow trout (Salmo gairdneri): angling, electroshocking, seining, and direct netting (control). Blood was sampled rapidly upon capture, usually within 2 min. No significant differences were noted within the time frame of the experiment between the four capture groups for plasma protein concentration, lactate dehydrogenase activity, or leucine aminonaphthylamidase activity. Creatine phosphokinase activity was elevated among electroshocked fish. Acid phosphatase activity was too low for accurate measurement. Hematocrits were significantly elevated by capture struggles. These results indicate that these capture methods do not preclude the use of plasma enzyme levels for investigating the health of wild fish. Key words: plasma enzyme, capture stress, physiology, plasma protein, rainbow trout, lactate dehydrogenase, leucine aminonaphthylamidase, creatine phosphokinase

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.

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.

[Isolation and identification of a lactate-utilizing, butyrate-producing bacterium and its primary metabolic characteristics].

PubMed

Liu, Wei; Zhu, Wei-yun; Yao, Wen; Mao, Sheng-yong

2007-06-01

The distal mammalian gut harbors prodigiously abundant microbes, which provide unique metabolic traits to host. A lactate-utilizing, butyrate-producing bacterium, strain LB01, was isolated from adult swine feces by utilizing modified Hungate technique with rumen liquid-independent YCFA medium supplemented with lactate as the single carbon source. It was an obligate anaerobic, Gram positive bacterium, and could utilize glucose, fructose, maltose and lactate with a large amount of gas products. 16S rRNA sequence analysis revealed that it had the high similarity with members of the genus Megasphaera. The metabolic characteristics of strain LB01 was investigated by using in vitro fermentation system. Lactate at the concentration of 65 mmol/L in YCFA medium was rapidly consumed within 9 hours and was mainly converted to propionate and butyrate after 24h. As the level of acetate declined, the concentration of butyrate rose only in the presence of glucose, suggesting that butyrate could possibly be synthesized by the acetyl CoA: butyryl CoA transferase. When co-cultured with lactic acid bacteria strain K9, strain LB01 evidently reduced the concentration of lactate produced by strain K9 and decelerated the rapid pH drop, finally producing 12.11 mmol/L butyrate and 4.06 mmol/L propionate. The metabolic characteristics that strain LB01 efficiently converts toxic lactate and excessive acetate to butyrate can prevent lactate and acetate accumulation in the large intestine and maintain the slightly acidic environment of the large intestine, consequently revealing that stain LB01 could act as a potential probiotics.

Electrochemical l-Lactic Acid Sensor Based on Immobilized ZnO Nanorods with Lactate Oxidase

PubMed Central

Ibupoto, Zafar Hussain; Ali Shah, Syed Muhammad Usman; Khun, Kimleang; Willander, Magnus

2012-01-01

In this work, fabrication of gold coated glass substrate, growth of ZnO nanorods and potentiometric response of lactic acid are explained. The biosensor was developed by immobilizing the lactate oxidase on the ZnO nanorods in combination with glutaraldehyde as a cross linker for lactate oxidase enzyme. The potentiometric technique was applied for the measuring the output (EMF) response of l-lactic acid biosensor. We noticed that the present biosensor has wide linear detection range of concentration from 1 × 10−4–1 × 100 mM with acceptable sensitivity about 41.33 ± 1.58 mV/decade. In addition, the proposed biosensor showed fast response time less than 10 s, a good selectivity towards l-lactic acid in presence of common interfering substances such as ascorbic acid, urea, glucose, galactose, magnesium ions and calcium ions. The present biosensor based on immobilized ZnO nanorods with lactate oxidase sustained its stability for more than three weeks. PMID:22736960

Electrochemical L-lactic acid sensor based on immobilized ZnO nanorods with lactate oxidase.

PubMed

Ibupoto, Zafar Hussain; Shah, Syed Muhammad Usman Ali; Khun, Kimleang; Willander, Magnus

2012-01-01

In this work, fabrication of gold coated glass substrate, growth of ZnO nanorods and potentiometric response of lactic acid are explained. The biosensor was developed by immobilizing the lactate oxidase on the ZnO nanorods in combination with glutaraldehyde as a cross linker for lactate oxidase enzyme. The potentiometric technique was applied for the measuring the output (EMF) response of l-lactic acid biosensor. We noticed that the present biosensor has wide linear detection range of concentration from 1 × 10(-4)-1 × 10(0) mM with acceptable sensitivity about 41.33 ± 1.58 mV/decade. In addition, the proposed biosensor showed fast response time less than 10 s, a good selectivity towards l-lactic acid in presence of common interfering substances such as ascorbic acid, urea, glucose, galactose, magnesium ions and calcium ions. The present biosensor based on immobilized ZnO nanorods with lactate oxidase sustained its stability for more than three weeks.

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.

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

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.

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.

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.

T85C polymorphisms of the dihydropyrimidine dehydrogenase gene detected in gastric cancer tissues by high-resolution melting curve analysis.

PubMed

Fang, Weijia; Xu, Nong; Jin, Dazhi; Chen, Yu; Chen, Xiaogang; Zheng, Yi; Shen, Hong; Yuan, Ying; Zheng, Shusen

2012-01-01

Dihydropyrimidine dehydrogenase is a key enzyme acting on the metabolic pathway of medications for gastric cancer. High-resolution melting curve technology, which was developed recently, can distinguish the wild-type dihydropyrimidine dehydrogenase gene from multiple polymorphisms by fluorescent quantitative polymerase chain reaction products in a direct and effective manner. T85C polymorphisms of dihydropyrimidine dehydrogenase in the peripheral blood of 112 Chinese gastric cancer patients were detected by real-time polymerase chain reaction combined with high-resolution melting curve technology. Primer design, along with the reaction system and conditions, was optimized based on the GenBank sequence. Seventy nine cases of wild-type (TT, [70.5%]), 29 cases of heterozygous (TC, [25.9%]), and 4 cases of homozygous mutant (CC, [3.6%]) were observed. The result was completely consistent with the results of the sequencing. Real-time polymerase chain reaction combined with high-resolution melting curve technology is a rapid, simple, reliable, direct-viewing, and convenient method for the detection and screening of polymorphisms.

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.

Hypoxia-induced carbonic anhydrase IX facilitates lactate flux in human breast cancer cells by non-catalytic function.

PubMed

Jamali, Somayeh; Klier, Michael; Ames, Samantha; Barros, L Felipe; McKenna, Robert; Deitmer, Joachim W; Becker, Holger M

2015-09-04

The most aggressive tumour cells, which often reside in hypoxic environments, rely on glycolysis for energy production. Thereby they release vast amounts of lactate and protons via monocarboxylate transporters (MCTs), which exacerbates extracellular acidification and supports the formation of a hostile environment. We have studied the mechanisms of regulated lactate transport in MCF-7 human breast cancer cells. Under hypoxia, expression of MCT1 and MCT4 remained unchanged, while expression of carbonic anhydrase IX (CAIX) was greatly enhanced. Our results show that CAIX augments MCT1 transport activity by a non-catalytic interaction. Mutation studies in Xenopus oocytes indicate that CAIX, via its intramolecular H(+)-shuttle His200, functions as a "proton-collecting/distributing antenna" to facilitate rapid lactate flux via MCT1. Knockdown of CAIX significantly reduced proliferation of cancer cells, suggesting that rapid efflux of lactate and H(+), as enhanced by CAIX, contributes to cancer cell survival under hypoxic conditions.

Hypoxia-induced carbonic anhydrase IX facilitates lactate flux in human breast cancer cells by non-catalytic function

PubMed Central

Jamali, Somayeh; Klier, Michael; Ames, Samantha; Felipe Barros, L.; McKenna, Robert; Deitmer, Joachim W.; Becker, Holger M.

2015-01-01

The most aggressive tumour cells, which often reside in hypoxic environments, rely on glycolysis for energy production. Thereby they release vast amounts of lactate and protons via monocarboxylate transporters (MCTs), which exacerbates extracellular acidification and supports the formation of a hostile environment. We have studied the mechanisms of regulated lactate transport in MCF-7 human breast cancer cells. Under hypoxia, expression of MCT1 and MCT4 remained unchanged, while expression of carbonic anhydrase IX (CAIX) was greatly enhanced. Our results show that CAIX augments MCT1 transport activity by a non-catalytic interaction. Mutation studies in Xenopus oocytes indicate that CAIX, via its intramolecular H+-shuttle His200, functions as a “proton-collecting/distributing antenna” to facilitate rapid lactate flux via MCT1. Knockdown of CAIX significantly reduced proliferation of cancer cells, suggesting that rapid efflux of lactate and H+, as enhanced by CAIX, contributes to cancer cell survival under hypoxic conditions. PMID:26337752

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.

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

Pharmacophore Models and Pharmacophore-Based Virtual Screening: Concepts and Applications Exemplified on Hydroxysteroid Dehydrogenases.

PubMed

Kaserer, Teresa; Beck, Katharina R; Akram, Muhammad; Odermatt, Alex; Schuster, Daniela

2015-12-19

Computational methods are well-established tools in the drug discovery process and can be employed for a variety of tasks. Common applications include lead identification and scaffold hopping, as well as lead optimization by structure-activity relationship analysis and selectivity profiling. In addition, compound-target interactions associated with potentially harmful effects can be identified and investigated. This review focuses on pharmacophore-based virtual screening campaigns specifically addressing the target class of hydroxysteroid dehydrogenases. Many members of this enzyme family are associated with specific pathological conditions, and pharmacological modulation of their activity may represent promising therapeutic strategies. On the other hand, unintended interference with their biological functions, e.g., upon inhibition by xenobiotics, can disrupt steroid hormone-mediated effects, thereby contributing to the development and progression of major diseases. Besides a general introduction to pharmacophore modeling and pharmacophore-based virtual screening, exemplary case studies from the field of short-chain dehydrogenase/reductase (SDR) research are presented. These success stories highlight the suitability of pharmacophore modeling for the various application fields and suggest its application also in futures studies.

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.

Milk production during the colostral period is not related to the later lactational performance in dairy cows.

PubMed

Kessler, E C; Bruckmaier, R M; Gross, J J

2014-01-01

In dairy cows, milk yield increases rapidly after parturition until a peak at around wk 6 of lactation. However, the description of the shape of the lactation curve is commonly based on weekly average milk yields. For a more detailed analysis of the milk production curve from the very beginning of lactation including the colostral period and the effect of colostrum yield on further lactational performance, the first 10 milkings after parturition, daily milk yields from d 1 to 28 of lactation, and the cumulative milk production on d 100 to 305 of lactation were investigated in 17 primiparous and 39 multiparous cows milked twice daily. Milk yield at the first milking after parturition (colostrum) ranged from 1.3 to 20.7kg (Δ=19.4kg) in multiparous and from 1.8 to 10.9kg in primiparous animals (Δ=9.1kg). At the tenth milking, milk production ranged from 9.2 to 21.5kg (Δ=12.3kg) in multiparous and from 7.0 to 15.2kg (Δ=8.2kg) in primiparous animals. Immediately after parturition, daily milk production increased rapidly, but after approximately 1wk in lactation, the slope of the daily milk production curve flattened and continued more linear. A nonlinear regression equation was used to determine this timely change, which occurred earlier in primiparous (d 6.9±0.3) than in multiparous cows (d 8.2±0.2). The correlation between the amount of first colostrum and milk production during further lactation decreased already from 0.47 on d 5 to 0.32 on d 14. In multiparous cows, the correlation between total milk production of the previous 305d standard lactation and the amount of first colostrum was not significant (correlation=0.29), whereas the correlation with the daily production increased from 0.45 on d 5 to 0.69 on d 14. However, in primiparous animals, correlations between first-colostrum yield and daily milk yields up to d 28 of lactation were not significant, possibly due to the smaller sample size compared with multiparous animals. First-colostrum yield and

Response of saliva Na/K ratio to changing Na supply of lactating cows under tropical conditions.

PubMed

Thiangtum, Wandee; Schonewille, J Thomas; Verstegen, Martin Wa; Arsawakulsudhi, Supot; Rukkwamsuk, Theera; Hendriks, Wouter H

2017-06-01

Factorial determination of the sodium (Na) requirement of heat-stressed lactating cows is hindered by accurate estimates of the Na losses through sweat. Direct studies, therefore, may be needed requiring information on the time course of healthy animals to become Na depleted and the subsequent rate of repletion. The rate of Na depletion and subsequent rate of Na repletion with two levels of dietary Na to lactating dairy cows housed under tropical conditions were investigated using the salivary Na/K. The 12 lactating cows (salivary Na/K ratio 14.6) rapidly developed clinical signs of Na deficiency, including pica, polyuria and polydipsia, reduced body weight and reduced milk yield when fed a low-Na ration (0.33 g kg -1 dry matter (DM)) for 3 weeks. Deficiency symptoms were associated with a rapid decrease in salivary Na/K ratio to <4.3 from 7 to 21 days. Subsequent repletion of the cows with NaCl to a ration concentration of 1.1 or 1.6 g Na kg -1 DM for 5 weeks did not restore salivary Na/K ratio to values of >6. A daily Na intake of heat-stressed lactating cows to a ration intake of 1.6 g Na kg -1 DM was insufficient to restore Na deficiency. One week was sufficient to deplete heat-stressed lactating cows of Na, allowing for rapid dose-response studies utilizing the salivary Na/K ratio as a parameter for Na status of cows under tropical conditions. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

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

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.

Lactate flux in astrocytes is enhanced by a non-catalytic action of carbonic anhydrase II

PubMed Central

Stridh, Malin H; Alt, Marco D; Wittmann, Sarah; Heidtmann, Hella; Aggarwal, Mayank; Riederer, Brigitte; Seidler, Ursula; Wennemuth, Gunther; McKenna, Robert; Deitmer, Joachim W; Becker, Holger M

2012-01-01

Rapid exchange of metabolites between different cell types is crucial for energy homeostasis of the brain. Besides glucose, lactate is a major metabolite in the brain and is primarily produced in astrocytes. In the present study, we report that carbonic anhydrase 2 (CAII) enhances both influx and efflux of lactate in mouse cerebellar astrocytes. The augmentation of lactate transport is independent of the enzyme's catalytic activity, but requires direct binding of CAII to the C-terminal of the monocarboxylate transporter MCT1, one of the major lactate/proton cotransporters in astrocytes and most tissues. By employing its intramolecular proton shuttle, CAII, bound to MCT1, can act as a ‘proton collecting antenna’ for the transporter, suppressing the formation of proton microdomains at the transporter-pore and thereby enhancing lactate flux. By this mechanism CAII could enhance transfer of lactate between astrocytes and neurons and thus provide the neurons with an increased supply of energy substrate. PMID:22451434

Analytical sensitivity of current best-in-class malaria rapid diagnostic tests.

PubMed

Jimenez, Alfons; Rees-Channer, Roxanne R; Perera, Rushini; Gamboa, Dionicia; Chiodini, Peter L; González, Iveth J; Mayor, Alfredo; Ding, Xavier C

2017-03-24

Rapid diagnostic tests (RDTs) are today the most widely used method for malaria diagnosis and are recommended, alongside microscopy, for the confirmation of suspected cases before the administration of anti-malarial treatment. The diagnostic performance of RDTs, as compared to microscopy or PCR is well described but the actual analytical sensitivity of current best-in-class tests is poorly documented. This value is however a key performance indicator and a benchmark value needed to developed new RDTs of improved sensitivity. Thirteen RDTs detecting either the Plasmodium falciparum histidine rich protein 2 (HRP2) or the plasmodial lactate dehydrogenase (pLDH) antigens were selected from the best performing RDTs according to the WHO-FIND product testing programme. The analytical sensitivity of these products was evaluated using a range of reference materials including P. falciparum and Plasmodium vivax whole parasite samples as well as recombinant proteins. The best performing HRP2-based RDTs could detect all P. falciparum cultured samples at concentrations as low as 0.8 ng/mL of HRP2. The limit of detection of the best performing pLDH-based RDT specifically detecting P. vivax was 25 ng/mL of pLDH. The analytical sensitivity of P. vivax and Pan pLDH-based RDTs appears to vary considerably from product to product, and improvement of the limit-of-detection for P. vivax detecting RDTs is needed to match the performance of HRP2 and Pf pLDH-based RDTs for P. falciparum. Different assays using different reference materials produce different values for antigen concentration in a given specimen, highlighting the need to establish universal reference assays.

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

Mixed acid-base disorders, hydroelectrolyte imbalance and lactate production in hypercapnic respiratory failure: the role of noninvasive ventilation.

PubMed

Terzano, Claudio; Di Stefano, Fabio; Conti, Vittoria; Di Nicola, Marta; Paone, Gregorino; Petroianni, Angelo; Ricci, Alberto

2012-01-01

Hypercapnic Chronic Obstructive Pulmonary Disease (COPD) exacerbation in patients with comorbidities and multidrug therapy is complicated by mixed acid-base, hydro-electrolyte and lactate disorders. Aim of this study was to determine the relationships of these disorders with the requirement for and duration of noninvasive ventilation (NIV) when treating hypercapnic respiratory failure. Sixty-seven consecutive patients who were hospitalized for hypercapnic COPD exacerbation had their clinical condition, respiratory function, blood chemistry, arterial blood gases, blood lactate and volemic state assessed. Heart and respiratory rates, pH, PaO(2) and PaCO(2) and blood lactate were checked at the 1st, 2nd, 6th and 24th hours after starting NIV. Nine patients were transferred to the intensive care unit. NIV was performed in 11/17 (64.7%) mixed respiratory acidosis-metabolic alkalosis, 10/36 (27.8%) respiratory acidosis and 3/5 (60%) mixed respiratory-metabolic acidosis patients (p = 0.026), with durations of 45.1 ± 9.8, 36.2 ± 8.9 and 53.3 ± 4.1 hours, respectively (p = 0.016). The duration of ventilation was associated with higher blood lactate (p<0.001), lower pH (p = 0.016), lower serum sodium (p = 0.014) and lower chloride (p = 0.038). Hyponatremia without hypervolemic hypochloremia occurred in 11 respiratory acidosis patients. Hypovolemic hyponatremia with hypochloremia and hypokalemia occurred in 10 mixed respiratory acidosis-metabolic alkalosis patients, and euvolemic hypochloremia occurred in the other 7 patients with this mixed acid-base disorder. Mixed acid-base and lactate disorders during hypercapnic COPD exacerbations predict the need for and longer duration of NIV. The combination of mixed acid-base disorders and hydro-electrolyte disturbances should be further investigated.

Mixed Acid-Base Disorders, Hydroelectrolyte Imbalance and Lactate Production in Hypercapnic Respiratory Failure: The Role of Noninvasive Ventilation

PubMed Central

Terzano, Claudio; Di Stefano, Fabio; Conti, Vittoria; Di Nicola, Marta; Paone, Gregorino; Petroianni, Angelo; Ricci, Alberto

2012-01-01

Background Hypercapnic Chronic Obstructive Pulmonary Disease (COPD) exacerbation in patients with comorbidities and multidrug therapy is complicated by mixed acid-base, hydro-electrolyte and lactate disorders. Aim of this study was to determine the relationships of these disorders with the requirement for and duration of noninvasive ventilation (NIV) when treating hypercapnic respiratory failure. Methods Sixty-seven consecutive patients who were hospitalized for hypercapnic COPD exacerbation had their clinical condition, respiratory function, blood chemistry, arterial blood gases, blood lactate and volemic state assessed. Heart and respiratory rates, pH, PaO2 and PaCO2 and blood lactate were checked at the 1st, 2nd, 6th and 24th hours after starting NIV. Results Nine patients were transferred to the intensive care unit. NIV was performed in 11/17 (64.7%) mixed respiratory acidosis–metabolic alkalosis, 10/36 (27.8%) respiratory acidosis and 3/5 (60%) mixed respiratory-metabolic acidosis patients (p = 0.026), with durations of 45.1±9.8, 36.2±8.9 and 53.3±4.1 hours, respectively (p = 0.016). The duration of ventilation was associated with higher blood lactate (p<0.001), lower pH (p = 0.016), lower serum sodium (p = 0.014) and lower chloride (p = 0.038). Hyponatremia without hypervolemic hypochloremia occurred in 11 respiratory acidosis patients. Hypovolemic hyponatremia with hypochloremia and hypokalemia occurred in 10 mixed respiratory acidosis–metabolic alkalosis patients, and euvolemic hypochloremia occurred in the other 7 patients with this mixed acid-base disorder. Conclusions Mixed acid-base and lactate disorders during hypercapnic COPD exacerbations predict the need for and longer duration of NIV. The combination of mixed acid-base disorders and hydro-electrolyte disturbances should be further investigated. PMID:22539963

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

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.

CSF lactate level: a useful diagnostic tool to differentiate acute bacterial and viral meningitis.

PubMed

Abro, Ali Hassan; Abdou, Ahmed Saheh; Ustadi, Abdulla M; Saleh, Ahmed Alhaj; Younis, Nadeem Javeed; Doleh, Wafa F

2009-08-01

To evaluate the potential role of CSF lactate level in the diagnosis of acute bacterial meningitis and in the differentiation between viral and bacterial meningitis. This was a hospital based observational study, conducted at Infectious Diseases Unit, Rashid Hospital Dubai, United Arab Emirates, from July 2004 to June 2007. The patients with clinical diagnosis of acute bacterial meningitis and who had CSF Gram stain/culture positive, CSF analysis suggestive of bacterial meningitis with negative Gram stain and culture but blood culture positive for bacteria and patients with clinical diagnosis suggestive of viral meningitis supported by CSF chemical analysis with negative Gram stain and culture as well as negative blood culture for bacteria were included in the study. CT scan brain was done for all patients before lumber puncture and CSF and blood samples were collected immediately after admission. CSF chemical analysis including lactate level was done on first spinal tap. The CSF lactate level was tested by Enzymatic Colorimetric method. A total 95 adult patients of acute meningitis (53 bacterial and 42 viral) fulfilled the inclusion criteria. Among 53 bacterial meningitis patients, Neisseria meningitides were isolated in 29 (54.7%), Strept. Pneumoniae in 18 (33.96%), Staph. Aureus in 2 (3.77%), Klebsiell Pneumoniae in 2 (3.77%), Strept. Agalactiae in 1 (1.8%) and E. Coli in 1 (1.8%). All the patients with bacterial meningitis had CSF lactate > 3.8 mmol/l except one, whereas none of the patients with viral meningitis had lactate level > 3.8 mmol/l. The mean CSF lactate level in bacterial meningitis cases amounted to 16.51 +/- 6.14 mmol/l, whereas it was significantly lower in viral group 2.36 +/- 0.6 mmol/l, p < .0001. CSF lactate level was significantly high in bacterial than viral meningitis and it can provide pertinent, rapid and reliable diagnostic information. Furthermore, CSF lactate level can also differentiate bacterial meningitis from viral one in a quick

Rapid Weight Loss Elicits Harmful Biochemical and Hormonal Responses in Mixed Martial Arts Athletes.

PubMed

Coswig, Victor Silveira; Fukuda, David Hideyoshi; Del Vecchio, Fabrício Boscolo

2015-10-01

The purpose of this study was to compare biochemical and hormonal responses between mixed martial arts (MMA) competitors with minimal prefight weight loss and those undergoing rapid weight loss (RWL). Blood samples were taken from 17 MMA athletes (Mean± SD; age: 27.4 ±5.3yr; body mass: 76.2 ± 12.4kg; height: 1.71 ± 0.05m and training experience: 39.4 ± 25 months) before and after each match, according to the official events rules. The no rapid weight loss (NWL, n = 12) group weighed in on the day of the event (~30 min prior fight) and athletes declared not having used RWL strategies, while the RWL group (n = 5) weighed in 24 hr before the event and the athletes claimed to have lost 7.4 ± 1.1kg, approximately 10% of their body mass in the week preceding the event. Results showed significant (p < .05) increases following fights, regardless of group, in lactate, glucose, lactate dehydrogenase (LDH), creatinine, and cortisol for all athletes. With regard to group differences, NWL had significantly (p < .05) greater creatinine levels (Mean± SD; pre to post) (NWL= 101.6 ± 15-142.3 ± 22.9μmol/L and RWL= 68.9 ± 10.6-79.5 ± 15.9μmol/L), while RWL had higher LDH (median [interquartile range]; pre to post) (NWL= 211.5[183-236] to 231[203-258]U/L and RWL= 390[370.5-443.5] to 488[463.5-540.5]U/L) and AST (NWL= 30[22-37] to 32[22-41]U/L and 39[32.5-76.5] to 72[38.5-112.5] U/L) values (NWL versus RWL, p < .05). Post hoc analysis showed that AST significantly increased in only the RWL group, while creatinine increased in only the NWL group. The practice of rapid weight loss showed a negative impact on energy availability and increased both muscle damage markers and catabolic expression in MMA fighters.

Comparative transcriptome analysis reveals different molecular mechanisms of Bacillus coagulans 2-6 response to sodium lactate and calcium lactate during lactic acid production.

PubMed

Qin, Jiayang; Wang, Xiuwen; Wang, Landong; Zhu, Beibei; Zhang, Xiaohua; Yao, Qingshou; Xu, Ping

2015-01-01

Lactate production is enhanced by adding calcium carbonate or sodium hydroxide during fermentation. However, Bacillus coagulans 2-6 can produce more than 180 g/L L-lactic acid when calcium lactate is accumulated, but less than 120 g/L L-lactic acid when sodium lactate is formed. The molecular mechanisms by which B. coagulans responds to calcium lactate and sodium lactate remain unclear. In this study, comparative transcriptomic methods based on high-throughput RNA sequencing were applied to study gene expression changes in B. coagulans 2-6 cultured in non-stress, sodium lactate stress and calcium lactate stress conditions. Gene expression profiling identified 712 and 1213 significantly regulated genes in response to calcium lactate stress and sodium lactate stress, respectively. Gene ontology assignments of the differentially expressed genes were performed. KEGG pathway enrichment analysis revealed that 'ATP-binding cassette transporters' were significantly affected by calcium lactate stress, and 'amino sugar and nucleotide sugar metabolism' was significantly affected by sodium lactate stress. It was also found that lactate fermentation was less affected by calcium lactate stress than by sodium lactate stress. Sodium lactate stress had negative effect on the expression of 'glycolysis/gluconeogenesis' genes but positive effect on the expression of 'citrate cycle (TCA cycle)' genes. However, calcium lactate stress had positive influence on the expression of 'glycolysis/gluconeogenesis' genes and had minor influence on 'citrate cycle (TCA cycle)' genes. Thus, our findings offer new insights into the responses of B. coagulans to different lactate stresses. Notably, our RNA-seq dataset constitute a robust database for investigating the functions of genes induced by lactate stress in the future and identify potential targets for genetic engineering to further improve L-lactic acid production by B. coagulans.

Comparative Transcriptome Analysis Reveals Different Molecular Mechanisms of Bacillus coagulans 2-6 Response to Sodium Lactate and Calcium Lactate during Lactic Acid Production

PubMed Central

Qin, Jiayang; Wang, Xiuwen; Wang, Landong; Zhu, Beibei; Zhang, Xiaohua; Yao, Qingshou; Xu, Ping

2015-01-01

Lactate production is enhanced by adding calcium carbonate or sodium hydroxide during fermentation. However, Bacillus coagulans 2-6 can produce more than 180 g/L L-lactic acid when calcium lactate is accumulated, but less than 120 g/L L-lactic acid when sodium lactate is formed. The molecular mechanisms by which B. coagulans responds to calcium lactate and sodium lactate remain unclear. In this study, comparative transcriptomic methods based on high-throughput RNA sequencing were applied to study gene expression changes in B. coagulans 2-6 cultured in non-stress, sodium lactate stress and calcium lactate stress conditions. Gene expression profiling identified 712 and 1213 significantly regulated genes in response to calcium lactate stress and sodium lactate stress, respectively. Gene ontology assignments of the differentially expressed genes were performed. KEGG pathway enrichment analysis revealed that ‘ATP-binding cassette transporters’ were significantly affected by calcium lactate stress, and ‘amino sugar and nucleotide sugar metabolism’ was significantly affected by sodium lactate stress. It was also found that lactate fermentation was less affected by calcium lactate stress than by sodium lactate stress. Sodium lactate stress had negative effect on the expression of ‘glycolysis/gluconeogenesis’ genes but positive effect on the expression of ‘citrate cycle (TCA cycle)’ genes. However, calcium lactate stress had positive influence on the expression of ‘glycolysis/gluconeogenesis’ genes and had minor influence on ‘citrate cycle (TCA cycle)’ genes. Thus, our findings offer new insights into the responses of B. coagulans to different lactate stresses. Notably, our RNA-seq dataset constitute a robust database for investigating the functions of genes induced by lactate stress in the future and identify potential targets for genetic engineering to further improve L-lactic acid production by B. coagulans. PMID:25875592

Smartphone-based enzymatic biosensor for oral fluid L-lactate detection in one minute using confined multilayer paper reflectometry.

PubMed

Calabria, Donato; Caliceti, Cristiana; Zangheri, Martina; Mirasoli, Mara; Simoni, Patrizia; Roda, Aldo

2017-08-15

The development of smartphone-based biosensors for point-of-care testing (POCT) applications allows realizing "all in one" instruments, with large potential distribution among the general population. With this respect, paper color-based detection performed by reflectance measurement is the most popular, simple, inexpensive and straightforward method. Despite the large number of scientific publications related to these biosensors, they still suffer from a poor detectability and reproducibility related to inhomogeneity of color development, which leads to low assay reproducibility. To overcome these problems, we propose a smartphone paper-based biosensor, in which all the reagents necessary to complete the analysis are co-entrapped on paper in a "wafer"-like bilayer film of polyelectrolytes (Poly (allyl amine hydrochloride/poly(sodium 4-styrene sulfonate)). Using a 3D printing low-cost technology we fabricated the smartphone-based device that consists in a cover accessory attached to the smartphone and incorporating a light diffuser over the flash to improve the image quality, a mini dark box and a disposable analytical cartridge containing all the reagents necessary for the complete analysis. The biosensor was developed exploiting coupled enzyme reactions for quantifying L-lactate in oral fluid, which is considered a biomarker of poor tissue perfusion, a key element in the management of severe sepsis, septic shock and in sports performance evaluation. The developed method is sensitive, rapid, and it allows detecting L-lactate in oral fluid in the relevant physiological range, with a limit of detection of 0.1mmolL -1 . The extreme simplicity of assay execution (no reagents need to be added) and flexibility of fabrication of the device, together with the high assay versatility (any oxidase can be coupled with HRP-based color change reaction) make our approach suitable for the realization of smartphone-based biosensors able to non-invasively detect a large variety of

3-cyanoindole-based inhibitors of inosine monophosphate dehydrogenase: synthesis and initial structure-activity relationships.

PubMed

Dhar, T G Murali; Shen, Zhongqi; Gu, Henry H; Chen, Ping; Norris, Derek; Watterson, Scott H; Ballentine, Shelley K; Fleener, Catherine A; Rouleau, Katherine A; Barrish, Joel C; Townsend, Robert; Hollenbaugh, Diane L; Iwanowicz, Edwin J

2003-10-20

A series of novel small molecule inhibitors of inosine monophosphate dehydrogenase (IMPDH), based upon a 3-cyanoindole core, were explored. IMPDH catalyzes the rate determining step in guanine nucleotide biosynthesis and is a target for anticancer, immunosuppressive and antiviral therapy. The synthesis and the structure-activity relationships (SAR), derived from in vitro studies, for this new series of inhibitors is given.

Evidence of In Vivo Absorption of Lactate and Modulation of Short Chain Fatty Acid Absorption from the Reticulorumen of Non-Lactating Cattle Fed High Concentrate Diets.

PubMed

Qumar, Muhammad; Khiaosa-Ard, Ratchaneewan; Pourazad, Poulad; Wetzels, Stefanie U; Klevenhusen, Fenja; Kandler, Wolfgang; Aschenbach, Jörg R; Zebeli, Qendrim

2016-01-01

Short-chain fatty acids (SCFAs) and lactate are endproducts of rumen fermentation and important energy sources for the host ruminant. Because their rapid accumulation results in ruminal acidosis, enhancement of the absorption of SCFA and lactate across reticuloruminal wall is instrumental in increasing energy supply and preventing ruminal acidosis in cattle. This study investigated whether the reticuloruminal absorption of SCFAs and lactate was altered by different strategies of high concentrate feeding. Eight rumen-cannulated, non-lactating Holstein cows were fed a forage-only diet (baseline) and then gradually adapted over 6 d to a 60% concentrate level. Thereafter, this concentrate-rich diet was fed for 4 wk either continuously (Con; n = 8) or interruptedly (Int; n = 8). Absorption of SCFAs and lactate was determined in vivo from the experimental buffer introduced into the washed reticulorumen. The buffer contained acetate, propionate, butyrate and lactate at a concentration of 60, 30, 10 and 5 mmol/L, respectively and Cr-EDTA as a marker for correcting ruminal water fluxes. The reticuloruminal absorption after 35 and 65 min of buffer incubation was measured at the baseline, after 1 wk of 60% concentrate feeding in the interrupted model (Int-1) and after 4 wk of concentrate feeding in both feeding models (Int-4 and Con-4). Data showed that the absorption rates of individual and total SCFAs during the first 35 min of incubation of Con-4 were highest (~1.7 times compared to baseline), while Int-1 and Int-4 were similar to respective baseline. Lactate was not absorbed during forage-only baseline and 1-wk concentrate feeding, but after 4-wk feeding of concentrates in both models. In conclusion, SCFAs absorption across the reticulorumen of non-lactating cattle was enhanced by the 4-wk continuous concentrate feeding, which seems to be more advantageous in terms of rumen acidosis prevention compared to the interrupted feeding model. The study provides evidence of

Evidence of In Vivo Absorption of Lactate and Modulation of Short Chain Fatty Acid Absorption from the Reticulorumen of Non-Lactating Cattle Fed High Concentrate Diets

PubMed Central

Qumar, Muhammad; Khiaosa-ard, Ratchaneewan; Pourazad, Poulad; Wetzels, Stefanie U.; Klevenhusen, Fenja; Kandler, Wolfgang; Aschenbach, Jörg R.; Zebeli, Qendrim

2016-01-01

Short-chain fatty acids (SCFAs) and lactate are endproducts of rumen fermentation and important energy sources for the host ruminant. Because their rapid accumulation results in ruminal acidosis, enhancement of the absorption of SCFA and lactate across reticuloruminal wall is instrumental in increasing energy supply and preventing ruminal acidosis in cattle. This study investigated whether the reticuloruminal absorption of SCFAs and lactate was altered by different strategies of high concentrate feeding. Eight rumen-cannulated, non-lactating Holstein cows were fed a forage-only diet (baseline) and then gradually adapted over 6 d to a 60% concentrate level. Thereafter, this concentrate-rich diet was fed for 4 wk either continuously (Con; n = 8) or interruptedly (Int; n = 8). Absorption of SCFAs and lactate was determined in vivo from the experimental buffer introduced into the washed reticulorumen. The buffer contained acetate, propionate, butyrate and lactate at a concentration of 60, 30, 10 and 5 mmol/L, respectively and Cr-EDTA as a marker for correcting ruminal water fluxes. The reticuloruminal absorption after 35 and 65 min of buffer incubation was measured at the baseline, after 1 wk of 60% concentrate feeding in the interrupted model (Int-1) and after 4 wk of concentrate feeding in both feeding models (Int-4 and Con-4). Data showed that the absorption rates of individual and total SCFAs during the first 35 min of incubation of Con-4 were highest (~1.7 times compared to baseline), while Int-1 and Int-4 were similar to respective baseline. Lactate was not absorbed during forage-only baseline and 1-wk concentrate feeding, but after 4-wk feeding of concentrates in both models. In conclusion, SCFAs absorption across the reticulorumen of non-lactating cattle was enhanced by the 4-wk continuous concentrate feeding, which seems to be more advantageous in terms of rumen acidosis prevention compared to the interrupted feeding model. The study provides evidence of

Chirped-Pulse and Cavity Based Fourier Transform Microwave Spectroscopy of the Methyl Lactate-Ammonia Adduct

NASA Astrophysics Data System (ADS)

Thomas, Javix; Sukhorukov, Oleksandr; Jaeger, Wolfgang; Xu, Yunjie

2012-06-01

The hydrogen bonded complex of ammonia with methyl lactate, a chiral alpha-hydroxyester, has been studied using rotational spectroscopy and high level ab initio calculations. Previous studies showed that methyl lactate can exist in a number of conformers. However, only the most stable one which has an intramolecular hydrogen bonded ring formed with its alcoholic hydroxyl and its carbonyl oxygen atom was detected experimentally An extensive ab initio search has been performed to locate all possible low energy conformers of the methyl lactate-ammonia contact pair. Five lowest energy conformers have been identified at the MP2/6-311++G(d,p) level. The lowest energy conformer favors an insertion arrangement, where ammonia is inserted into the existing intramolecular hydrogen bonded ring in the most stable methyl lactate conformer. Broadband scans for the rotational spectra of possible binary conformers have been carried out using a chirped-pulse Fourier transform microwave (FTMW) instrument. The most stable binary adduct was identified and assigned. The final frequency measurements have been done with a cavity based FTMW instrument. The spectrum observed shows complicated fine and hyperfine splitting patterns, likely due to the internal rotations of the methyl groups of methyl lactate and that of ammonia, as well as the 14N quadrupolar nucleus. The binary adduct with 15NH3 has also been studied to simplify the splitting pattern and to aid the assignments of the extensive splittings. The isotopic data and the fine and hyperfine structures will be discussed in terms of internal rotation dynamics and geometry of the hydrogen bonded adduct.

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 ...

Rapid diagnostic tests for malaria at sites of varying transmission intensity in Uganda.

PubMed

Hopkins, Heidi; Bebell, Lisa; Kambale, Wilson; Dokomajilar, Christian; Rosenthal, Philip J; Dorsey, Grant

2008-02-15

In Africa, fever is often treated presumptively as malaria, resulting in misdiagnosis and the overuse of antimalarial drugs. Rapid diagnostic tests (RDTs) for malaria may allow improved fever management. We compared RDTs based on histidine-rich protein 2 (HRP2) and RDTs based on Plasmodium lactate dehydrogenase (pLDH) with expert microscopy and PCR-corrected microscopy for 7000 patients at sites of varying malaria transmission intensity across Uganda. When all sites were considered, the sensitivity of the HRP2-based test was 97% when compared with microscopy and 98% when corrected by PCR; the sensitivity of the pLDH-based test was 88% when compared with microscopy and 77% when corrected by PCR. The specificity of the HRP2-based test was 71% when compared with microscopy and 88% when corrected by PCR; the specificity of the pLDH-based test was 92% when compared with microscopy and >98% when corrected by PCR. Based on Plasmodium falciparum PCR-corrected microscopy, the positive predictive value (PPV) of the HRP2-based test was high (93%) at all but the site with the lowest transmission rate; the pLDH-based test and expert microscopy offered excellent PPVs (98%) for all sites. The negative predictive value (NPV) of the HRP2-based test was consistently high (>97%); in contrast, the NPV for the pLDH-based test dropped significantly (from 98% to 66%) as transmission intensity increased, and the NPV for expert microscopy decreased significantly (99% to 54%) because of increasing failure to detect subpatent parasitemia. Based on the high PPV and NPV, HRP2-based RDTs are likely to be the best diagnostic choice for areas with medium-to-high malaria transmission rates in Africa.

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

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

A novel type of pathogen defense-related cinnamyl alcohol dehydrogenase.

PubMed

Logemann, E; Reinold, S; Somssich, I E; Hahlbrock, K

1997-08-01

We describe an aromatic alcohol dehydrogenase with properties indicating a novel type of function in the defense response of plants to pathogens. To obtain the enzyme free of contamination with possible isoforms, a parsley (Petroselinum crispum) cDNA comprising the entire coding region of the elicitor-responsive gene, ELI3, was expressed in Escherichia coli. In accord with large amino acid sequence similarities with established cinnamyl and benzyl alcohol dehydrogenases from other plants, the enzyme efficiently reduced various cinnamyl and benzyl aldehydes using NADPH as a co-substrate. Highest substrate affinities were observed for cinnamaldehyde, 4-coumaraldehyde and coniferaldehyde, whereas sinapaldehyde, one of the most efficient substrates of several previously analyzed cinnamyl alcohol dehydrogenases and a characteristic precursor molecule of angiosperm lignin, was not converted. A single form of ELI3 mRNA was strongly and rapidly induced in fungal elicitor-treated parsley cells. These results, together with earlier findings that the ELI3 gene is strongly activated both in elicitor-treated parsley cells and at fungal infection sites in parsley leaves, but not in lignifying tissue, suggest a specific role of this enzyme in pathogen defense-related phenylpropanoid metabolism.

The investigation of plasma glucose-6-phosphate dehydrogenase, 6-phoshogluconate dehydrogenase, glutathione reductase in premenauposal patients with iron deficiency anemia.

PubMed

Ozcicek, Fatih; Aktas, Mehmet; Türkmen, Kultigin; Coban, T Abdulkadir; Cankaya, Murat

2014-07-01

Iron is an essential element that is necessary for all cells in the body. Iron deficiency anemia (IDA) is one of the most common nutritional disorders in both developed and developing countries. The glutathione pathway is paramount to antioxidant defense and glucose-6-phosphate dehydrogenase (G6PD)-deficient cells do not cope well with oxidative damage. The goal of this study was to check the activities of G6PD, 6-phosphogluconate dehydrogenase, glutathione reductase in patients with IDA. We analyzed the plasma samples of 102 premenopausal women with IDA and 88 healthy control subjects. Glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activity as compared to the reduction of NADP +, glutathione reductase activity was performed based on the oxidation of NADPH. 2 ml of plasma were used in all analyzes. SPSS program was used for all of the statistical analysis. Diagnosis of iron deficiency in patients belonging to the analysis of blood were ferritin 3.60 ± 2.7 ng / mL, hemoglobin 9.4 ± 1.5 mg / dl and hematocrit 30.7 ± 4.1% ratio; in healthy subjects ferritin 53.5 ± 41.7 ng/ml, hemoglobin level 13.9 ± 1.3 mg / dl and hematocrit ratio 42 ± 3.53%. When compared to healthy subjects the glutathione reductase level (P<0.001) was found to be significantly higher in patients with IDA. IDA patients with moderate and severe anemia had lower GR activity when compared to IDA patients with mild anemia. But the plasma levels of glucose-6-phosphate dehydrogenase (P<0,600) and 6-phosphogluconate dehydrogenase (P<0,671) did not show any differences between healthy subjects and in patients with IDA. It was shown that Glucose-6-Phosphate Dehydrogenase and 6-Phosphogluconate Dehydrogenase have no effect on iron-deficiency anemia in patients. The plasma GR levels of premenopausal women with IDA were found to be higher compared to healthy subjects, which could be secondary to erythrocyte protection against oxidative stress being commonly seen in IDA.

The nutritive and immunoprotective quality of human milk beyond 1 year postpartum: are lactation-duration-based donor exclusions justified?

PubMed

Perrin, Maryanne Tigchelaar; Fogleman, April; Allen, Jonathan C

2013-08-01

Donor human milk is critical for the fragile preterm infant who does not have access to his or her mother's milk, improving survival rates and quality of survival and decreasing hospital stay. Despite the opening of donor milk banks around the world, shortages continue as demand for donor milk exceeds supply. One potential means of increasing supply is by reducing exclusion criteria that prohibit mothers from donating milk based on duration of lactation. Minimal research has been done on the composition of human milk during the second year of lactation, with most research focusing on the nutritive compounds and not the immunoprotective compounds. Several immunoprotective compounds, including lysozyme, lactoferrin, secretory immunoglobulin A, and oligosaccharides, are abundant in human milk compared to bovine-based infant formula and are partially or fully retained during Holder pasteurization, making them an important differentiating feature of donor milk. A PubMed search was conducted to review studies in human milk composition during the second year of lactation. Limitations of existing research include sample collection protocols, small study sizes, and use of populations that may have been at risk for nutritional deficiencies. Stable concentrations of several components were reported including protein, lactose, iron, copper, lactoferrin, and secretory immunoglobulin A. Lysozyme concentration increased during extended lactation, while zinc and calcium concentrations declined into the second year. Conflicting findings were reported on fat content, and no information was available regarding oligosaccharide content. More research is needed to create evidence-based guidelines regarding the nutritive and immunoprotective value of donor milk throughout the course of lactation.

Rapid detection of common Chinese glucose-6-phosphate dehydrogenase (G6PD) mutations by denaturing gradient gel electrophoresis (DGGE).

PubMed

Lam, V M; Huang, W; Lam, S T; Yeung, C Y; Johnson, P H

1996-03-01

We describe here the use of denaturing gradient gel electrophoresis (DGGE) to detect the most common Chinese glucose-6-phosphate dehydrogenase (G6PD) variants, which are the single point mutations: G-->T at nt 1376, G-->A at 1388 both in exon 12 and A-->G at nt 95 in exon 02. In each case, the mutant allele resolves well from the normal allele(s). The distinct heteroduplex bands are characteristic of a particular genotype suggesting that this feature is very useful for identifying all heterozygous carriers for this and other X-linked diseases. When the analysis is extended to other exons, DGGE scans the gene and coupled with direct sequencing, it leads to the identification of new G6PD variation(s). With this approach, we identified a mutation in exon 9 which had not been reported in Hong Kong. Since DGGE can rapidly screen many unknown samples in one gel, this approach could be used to diagnose these G6PD mutations and to identify the at-risk for counselling.

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

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

Disposable electrochemiluminescent biosensor for lactate determination in saliva.

PubMed

Ballesta Claver, J; Valencia Mirón, M C; Capitán-Vallvey, L F

2009-07-01

An electrochemiluminescence-based disposable biosensor for lactate is characterized. The lactate recognition system is based on lactate oxidase (LOx) and the transduction system consists of luminol. All the needed reagents, luminol, LOx, BSA, electrolyte and buffer have been immobilized by a Methocel membrane placed on the working electrode of the screen-printed electrochemical cell. The measurement of the electrochemiluminescence (ECL) is made possible via a photocounting head when 50 microl of sample is placed into the screen-printed cell with a circular container containing the disposable sensing membrane. The compositions of the membrane and reaction conditions have been optimized to obtain adequate sensitivity. The disposable biosensor responds to lactate after 20 s when two 1 s pulses at 0.5 V are applied to obtain the analytical parameter, the ECL initial rate. The linearized double logarithmic dependence for lactate shows a dynamic range from 10(-5) to 5 x 10(-4) M with a detection limit of 5 x 10(-6) M and a sensor-to-sensor repeatability, as relative standard deviation, RSD, of 3.30% at the medium level of the range. The ECL disposable biosensor was applied to the analysis of lactate in human saliva as an alternative procedure for obtaining the lactate level in a non-invasive way. Interferences coming from components of saliva were studied and eliminated in a simple way that was easy to handle. The procedure was validated for use in human saliva, comparing the results against an enzymatic reference procedure. The proposed method is quick, inexpensive, selective and sensitive and uses conventional ECL instrumentation.

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.

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.

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

Bendable Electro-chemical Lactate Sensor Printed with Silver Nano-particles

PubMed Central

Abrar, Md Abu; Dong, Yue; Lee, Paul Kyuheon; Kim, Woo Soo

2016-01-01

Here we report a flexible amperometric lactate biosensor using silver nanoparticle based conductive electrode. Mechanically bendable cross-serpentine-shaped silver electrode is generated on flexible substrate for the mechanical durability such as bending. The biosensor is designed and fabricated by modifying silver electrode with lactate oxidase immobilized by bovine serum albumin. The in-sensor pseudo Ag/AgCl reference electrode is fabricated by chloridization of silver electrode, which evinced its long-term potential stability against a standard commercial Ag/AgCl reference electrode. The amperometric response of the sensor shows linear dependence with lactate concentration of 1~25 mM/L. Anionic selectivity is achieved by using drop-casted Nafion coated on silver electrode against anionic interferences such as ascorbate. This non-invasive electrochemical lactate sensor also demonstrates excellent resiliency against mechanical deformation and temperature fluctuation which leads the possibility of using it on human epidermis for continuous measurement of lactate from sweat. Near field communication based wireless data transmission is demonstrated to reflect a practical approach of the sensor to measure lactate concentration portably using human perspiration. PMID:27465437

Bendable Electro-chemical Lactate Sensor Printed with Silver Nano-particles

NASA Astrophysics Data System (ADS)

Abrar, Md Abu; Dong, Yue; Lee, Paul Kyuheon; Kim, Woo Soo

2016-07-01

Here we report a flexible amperometric lactate biosensor using silver nanoparticle based conductive electrode. Mechanically bendable cross-serpentine-shaped silver electrode is generated on flexible substrate for the mechanical durability such as bending. The biosensor is designed and fabricated by modifying silver electrode with lactate oxidase immobilized by bovine serum albumin. The in-sensor pseudo Ag/AgCl reference electrode is fabricated by chloridization of silver electrode, which evinced its long-term potential stability against a standard commercial Ag/AgCl reference electrode. The amperometric response of the sensor shows linear dependence with lactate concentration of 1~25 mM/L. Anionic selectivity is achieved by using drop-casted Nafion coated on silver electrode against anionic interferences such as ascorbate. This non-invasive electrochemical lactate sensor also demonstrates excellent resiliency against mechanical deformation and temperature fluctuation which leads the possibility of using it on human epidermis for continuous measurement of lactate from sweat. Near field communication based wireless data transmission is demonstrated to reflect a practical approach of the sensor to measure lactate concentration portably using human perspiration.

The agreement between abnormal venous lactate and arterial lactate in the ED: a retrospective chart review.

PubMed

Bloom, B; Pott, J; Freund, Y; Grundlingh, J; Harris, T

2014-06-01

The evidence for prognostication using lactate is often based on arterial lactate (AL). Arterial sampling is painful and difficult, and carries risks. Studies comparing peripheral venous lactate (PVL) with AL showed little difference but predominantly included patients with normal lactate. The objective of this study was to measure agreement between PVL and AL in patients with elevated venous lactate. This is a retrospective cross-sectional study. ED patients age≥16, attending from October 2010 to June 2011 inclusive, with PVL≥2.0 mmol/L and AL taken within 1 hour. intravenous fluid prior to or between initial venous and arterial sampling. Primary endpoint: agreement between PVL and AL defined as mean difference±95% limits of agreement (LOA). The misclassification rate was assessed. N=232. VL median 3.50 mmol/L, range 2.00 to 15.00 mmol/L. AL median 2.45 mmol/L, range 1.0 to 13.2 mmol/L. The mean difference±SD between PVL and AL for all patients was 1.06±1.30 mmol/L (95%LOA -1.53 to 3.66 mmol/L). Using a cut-off of 2 mmol/L and 4 mmol/L, 36.2% and 17.9% of patients respectively were incorrectly classified as having elevated lactate. We report greater bias between VL and AL with broader LOA than previously documented. This may partly be due to the fact that we studied only patients with abnormal venous values, for whom close agreement would confer greatest clinical significance. The agreement between abnormal PVL and AL is poor and the high rate of misclassification may suggest that PVL is not a good substitute for AL if the venous lactate is abnormal. Copyright © 2014 Elsevier Inc. All rights reserved.

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

DIRECT BINDING OF GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE TO TELOMERIC DNA PROTECTS TELOMERES AGAINST CHEMOTHERAPY-INDUCED RAPID DEGRADATION

PubMed Central

Demarse, Neil A.; Ponnusamy, Suriyan; Spicer, Eleanor K.; Apohan, Elif; Baatz, John E.; Ogretmen, Besim; Davies, Christopher

2009-01-01

GAPDH (glyceraldehyde 3-phosphate dehydrogenase) is a glycolytic enzyme that displays several non-glycolytic activities, including the maintenance and/or protection of telomeres. In this study, we determined the molecular mechanism and biological role of the interaction between GAPDH and human telomeric DNA. Using gel shift assays, we show that recombinant GAPDH binds directly with high affinity (Kd = 45 nM) to a single-stranded oligonucleotide comprising three telomeric DNA repeats and that nucleotides T1, G5 and G6 of the TTAGGG repeat are essential for binding. The stoichiometry of the interaction is 2:1 (DNA: GAPDH), and GAPDH appears to form a high-molecular weight complex when bound to the oligonucleotide. Mutation of Asp32 and Cys149, which are localized to the NAD-binding site and the active site center of GAPDH, respectively, produced mutants that almost completely lost their telomere-binding functions both in vitro and in situ (in A549 human lung cancer cells). Treatment of A549 cells with the chemotherapeutic agents gemcitabine and doxorubicin resulted in increased nuclear localization of expressed wild-type GAPDH, where it protected telomeres against rapid degradation, concomitant with increased resistance to the growth inhibitory effects of these drugs. The non-DNA-binding mutants of GAPDH also localized to the nucleus when expressed in A549 cells, but did not confer any significant protection of telomeres against chemotherapy-induced degradation or growth inhibition, and this occurred without the involvement of caspase activation or apoptosis regulation. Overall, these data demonstrate that GAPDH binds telomeric DNA directly in vitro and may have a biological role in the protection of telomeres against rapid degradation in response to chemotherapeutic agents in A549 human lung cancer cells. PMID:19800890

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

Single Sodium Pyruvate Ingestion Modifies Blood Acid-Base Status and Post-Exercise Lactate Concentration in Humans

PubMed Central

Olek, Robert A.; Kujach, Sylwester; Wnuk, Damian; Laskowski, Radoslaw

2014-01-01

This study examined the effect of a single sodium pyruvate ingestion on a blood acid-base status and exercise metabolism markers. Nine active, but non-specifically trained, male subjects participated in the double-blind, placebo-controlled, crossover study. One hour prior to the exercise, subjects ingested either 0.1 g·kg−1 of body mass of a sodium pyruvate or placebo. The capillary blood samples were obtained at rest, 60 min after ingestion, and then three and 15 min after completing the workout protocol to analyze acid-base status and lactate, pyruvate, alanine, glucose concentrations. The pulmonary gas exchange, minute ventilation and the heart rate were measured during the exercise at a constant power output, corresponding to ~90% O2max. The blood pH, bicarbonate and the base excess were significantly higher after sodium pyruvate ingestion than in the placebo trial. The blood lactate concentration was not different after the ingestion, but the post-exercise was significantly higher in the pyruvate trial (12.9 ± 0.9 mM) than in the placebo trial (10.6 ± 0.3 mM, p < 0.05) and remained elevated (nonsignificant) after 15 min of recovery. The blood pyruvate, alanine and glucose concentrations, as well as the overall pulmonary gas exchange during the exercise were not affected by the pyruvate ingestion. In conclusion, the sodium pyruvate ingestion one hour before workout modified the blood acid-base status and the lactate production during the exercise. PMID:24841105

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

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

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

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.

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.

[Amperometric biosensor for lactate analysis in wines and grape must during fermentation].

PubMed

Shkotova, L V; Horiushkina, T B; Slast'ia, E A; Soldatkin, O P; Tranh-Minh, S; Chovelon, J M; Dziadevych, S V

2005-01-01

The amperometric biosensor based on lactate oxidase for determination of lactate has been developed, and two methods of immobilization of lactate oxidase on the surface of industrial screen-printed platinum electrodes SensLab were compared. A sensor with immobilized in the Resydrol polymer lactate oxidase by the method of physical adsorption is characterized of narrow dynamic range and greater response value in comparison with a biosensor based on immobilised in poly(3,4-ethylenedioxythiophene) lactate oxidase by the method of electrochemical polymerization. Operational stability of the biosensor developed was studied and it was shown, that the immobilization method does not influence their stability. The analysis of the lactate in wine and during wine fermentation has been conducted. High correlation of the data obtained by means of amperometric lactate biosensor and a standard method of an ionic chromatography has been shown. The developed biosensor could be applied in the food industry for the control and optimization of the wine fermentation process, and quality control of wine.

Mechanisms driving the lactate switch in Chinese hamster ovary cells.

PubMed

Hartley, Fiona; Walker, Tracy; Chung, Vicky; Morten, Karl

2018-03-31

The metabolism of Chinese Hamster Ovary (CHO) cells in a production environment has been extensively investigated. However, a key metabolic transition, the switch from lactate production to lactate consumption, remains enigmatic. Though commonly observed in CHO cultures, the mechanism(s) by which this metabolic shift is triggered is unknown. Despite this, efforts to control the switch have emerged due to the association of lactate consumption with improved cell growth and productivity. This review aims to consolidate current theories surrounding the lactate switch. The influence of pH, NAD + /NADH, pyruvate availability and mitochondrial function on lactate consumption are explored. A hypothesis based on the cellular redox state is put forward to explain the onset of lactate consumption. Various techniques implemented to control the lactate switch, including manipulation of the culture environment, genetic engineering, and cell line selection are also discussed. © 2018 Wiley Periodicals, Inc.

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

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

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

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.

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

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.

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.

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.

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

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.

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.

Safety of single low-dose primaquine in glucose-6-phosphate dehydrogenase deficient falciparum-infected African males: Two open-label, randomized, safety trials.

PubMed

Bastiaens, Guido J H; Tiono, Alfred B; Okebe, Joseph; Pett, Helmi E; Coulibaly, Sam A; Gonçalves, Bronner P; Affara, Muna; Ouédraogo, Alphonse; Bougouma, Edith C; Sanou, Guillaume S; Nébié, Issa; Bradley, John; Lanke, Kjerstin H W; Niemi, Mikko; Sirima, Sodiomon B; d'Alessandro, Umberto; Bousema, Teun; Drakeley, Chris

2018-01-01

Primaquine (PQ) actively clears mature Plasmodium falciparum gametocytes but in glucose-6-phosphate dehydrogenase deficient (G6PDd) individuals can cause hemolysis. We assessed the safety of low-dose PQ in combination with artemether-lumefantrine (AL) or dihydroartemisinin-piperaquine (DP) in G6PDd African males with asymptomatic P. falciparum malaria. In Burkina Faso, G6PDd adult males were randomized to treatment with AL alone (n = 10) or with PQ at 0.25 (n = 20) or 0.40 mg/kg (n = 20) dosage; G6PD-normal males received AL plus 0.25 (n = 10) or 0.40 mg/kg (n = 10) PQ. In The Gambia, G6PDd adult males and boys received DP alone (n = 10) or with 0.25 mg/kg PQ (n = 20); G6PD-normal males received DP plus 0.25 (n = 10) or 0.40 mg/kg (n = 10) PQ. The primary study endpoint was change in hemoglobin concentration during the 28-day follow-up. Cytochrome P-450 isoenzyme 2D6 (CYP2D6) metabolizer status, gametocyte carriage, haptoglobin, lactate dehydrogenase levels and reticulocyte counts were also determined. In Burkina Faso, the mean maximum absolute change in hemoglobin was -2.13 g/dL (95% confidence interval [CI], -2.78, -1.49) in G6PDd individuals randomized to 0.25 PQ mg/kg and -2.29 g/dL (95% CI, -2.79, -1.79) in those receiving 0.40 PQ mg/kg. In The Gambia, the mean maximum absolute change in hemoglobin concentration was -1.83 g/dL (95% CI, -2.19, -1.47) in G6PDd individuals receiving 0.25 PQ mg/kg. After adjustment for baseline concentrations, hemoglobin reductions in G6PDd individuals in Burkina Faso were more pronounced compared to those in G6PD-normal individuals receiving the same PQ doses (P = 0.062 and P = 0.022, respectively). Hemoglobin levels normalized during follow-up. Abnormal haptoglobin and lactate dehydrogenase levels provided additional evidence of mild transient hemolysis post-PQ. Single low-dose PQ in combination with AL and DP was associated with mild and transient reductions in hemoglobin. None of the study participants developed moderate or

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

Isolation and characterization of full-length putative alcohol dehydrogenase genes from polygonum minus

NASA Astrophysics Data System (ADS)

Hamid, Nur Athirah Abd; Ismail, Ismanizan

2013-11-01

Polygonum minus, locally named as Kesum is an aromatic herb which is high in secondary metabolite content. Alcohol dehydrogenase is an important enzyme that catalyzes the reversible oxidation of alcohol and aldehyde with the presence of NAD(P)(H) as co-factor. The main focus of this research is to identify the gene of ADH. The total RNA was extracted from leaves of P. minus which was treated with 150 μM Jasmonic acid. Full-length cDNA sequence of ADH was isolated via rapid amplification cDNA end (RACE). Subsequently, in silico analysis was conducted on the full-length cDNA sequence and PCR was done on genomic DNA to determine the exon and intron organization. Two sequences of ADH, designated as PmADH1 and PmADH2 were successfully isolated. Both sequences have ORF of 801 bp which encode 266 aa residues. Nucleotide sequence comparison of PmADH1 and PmADH2 indicated that both sequences are highly similar at the ORF region but divergent in the 3' untranslated regions (UTR). The amino acid is differ at the 107 residue; PmADH1 contains Gly (G) residue while PmADH2 contains Cys (C) residue. The intron-exon organization pattern of both sequences are also same, with 3 introns and 4 exons. Based on in silico analysis, both sequences contain "classical" short chain alcohol dehydrogenases/reductases ((c) SDRs) conserved domain. The results suggest that both sequences are the members of short chain alcohol dehydrogenase family.

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.

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

Leukoencephalopathy with thalamus and brainstem involvement and high lactate 'LTBL' caused by EARS2 mutations.

PubMed

Steenweg, Marjan E; Ghezzi, Daniele; Haack, Tobias; Abbink, Truus E M; Martinelli, Diego; van Berkel, Carola G M; Bley, Annette; Diogo, Luisa; Grillo, Eugenio; Te Water Naudé, Johann; Strom, Tim M; Bertini, Enrico; Prokisch, Holger; van der Knaap, Marjo S; Zeviani, Massimo

2012-05-01

In the large group of genetically undetermined infantile-onset mitochondrial encephalopathies, multiple defects of mitochondrial DNA-related respiratory-chain complexes constitute a frequent biochemical signature. In order to identify responsible genes, we used exome-next-generation sequencing in a selected cohort of patients with this biochemical signature. In an isolated patient, we found two mutant alleles for EARS2, the gene encoding mitochondrial glutamyl-tRNA synthetase. The brain magnetic resonance imaging of this patient was hallmarked by extensive symmetrical cerebral white matter abnormalities sparing the periventricular rim and symmetrical signal abnormalities of the thalami, midbrain, pons, medulla oblongata and cerebellar white matter. Proton magnetic resonance spectroscopy showed increased lactate. We matched this magnetic resonance imaging pattern with that of a cohort of 11 previously selected unrelated cases. We found mutations in the EARS2 gene in all. Subsequent detailed clinical and magnetic resonance imaging based phenotyping revealed two distinct groups: mild and severe. All 12 patients shared an infantile onset and rapidly progressive disease with severe magnetic resonance imaging abnormalities and increased lactate in body fluids and proton magnetic resonance spectroscopy. Patients in the 'mild' group partially recovered and regained milestones in the following years with striking magnetic resonance imaging improvement and declining lactate levels, whereas those of the 'severe' group were characterized by clinical stagnation, brain atrophy on magnetic resonance imaging and persistent lactate increases. This new neurological disease, early-onset leukoencephalopathy with thalamus and brainstem involvement and high lactate, is hallmarked by unique magnetic resonance imaging features, defined by a peculiar biphasic clinical course and caused by mutations in a single gene, EARS2, expanding the list of medically relevant defects of mitochondrial

Meta-analysis: accuracy of rapid tests for malaria in travelers returning from endemic areas.

PubMed

Marx, Arthur; Pewsner, Daniel; Egger, Matthias; Nüesch, Reto; Bucher, Heiner C; Genton, Blaise; Hatz, Christoph; Jüni, Peter

2005-05-17

Microscopic diagnosis of malaria is unreliable outside specialized centers. Rapid tests have become available in recent years, but their accuracy has not been assessed systematically. To determine the accuracy of rapid diagnostic tests for ruling out malaria in nonimmune travelers returning from malaria-endemic areas. The authors searched MEDLINE, EMBASE, CAB Health, and CINAHL (1988 to September 2004); hand-searched conference proceedings; checked reference lists; and contacted experts and manufacturers. Diagnostic accuracy studies in nonimmune individuals with suspected malaria were included if they compared rapid tests with expert microscopic examination or polymerase chain reaction tests. Data on study and patient characteristics and results were extracted in duplicate. The main outcome was the likelihood ratio for a negative test result (negative likelihood ratio) for Plasmodium falciparum malaria. Likelihood ratios were combined by using random-effects meta-analysis, stratified by the antigen targeted (histidine-rich protein-2 [HRP-2] or parasite lactate dehydrogenase [LDH]) and by test generation. Nomograms of post-test probabilities were constructed. The authors included 21 studies and 5747 individuals. For P. falciparum, HRP-2-based tests were more accurate than parasite LDH-based tests: Negative likelihood ratios were 0.08 and 0.13, respectively (P = 0.019 for difference). Three-band HRP-2 tests had similar negative likelihood ratios but higher positive likelihood ratios compared with 2-band tests (34.7 vs. 98.5; P = 0.003). For P. vivax, negative likelihood ratios tended to be closer to 1.0 for HRP-2-based tests than for parasite LDH-based tests (0.24 vs. 0.13; P = 0.22), but analyses were based on a few heterogeneous studies. Negative likelihood ratios for the diagnosis of P. malariae or P. ovale were close to 1.0 for both types of tests. In febrile travelers returning from sub-Saharan Africa, the typical probability of P. falciparum malaria is

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...

Structural Basis for "Flip-Flop" Action of Human Pyruvate Dehydrogenase

NASA Technical Reports Server (NTRS)

Ciszak, Ewa; Korotchkina, Lioubov; Dominiak, Paulina; Sidhu, Sukhdeep; Patel, Mulchand

2003-01-01

The derivative of vitamin B1, thiamin pyrophosphate is a cofactor of pyruvate dehydrogenase, a component enzyme of the mitochondrial pyruvate dehydrogenase multienzyme complex that plays a major role in directing energy metabolism in the cell. This cofactor is used to cleave the C(sup alpha)-C(=O) bond of pyruvate followed by reductive acetyl transfer to lipoyl-dihydrolipoamide acetyltransferase. In alpha(sub 2)beta(sub 2)-tetrameric human pyruvate dehydrogenase, there are two cofactor binding sites, each of them being a center of independently conducted, although highly coordinated enzymatic reactions. The dynamic nonequivalence of two, otherwise chemically equivalent, catalytic sites can now be understood based on the recently determined crystal structure of the holo-form of human pyruvate dehydrogenase at 1.95A resolution. The structure of pyruvate dehydrogenase was determined using a combination of MAD phasing and molecular replacement followed by rounds of torsion-angles molecular-dynamics simulated-annealing refinement. The final pyruvate dehydrogenase structure included coordinates for all protein amino acids two cofactor molecules, two magnesium and two potassium ions, and 742 water molecules. The structure was refined to R = 0.202 and R(sub free) = 0.244. Our structural analysis of the enzyme folding and domain assembly identified a simple mechanism of this protein motion required for the conduct of catalytic action.

[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.

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

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.

Central carbon metabolism in marine bacteria examined with a simplified assay for dehydrogenases.

PubMed

Wen, Weiwei; Wang, Shizhen; Zhou, Xiaofen; Fang, Baishan

2013-06-01

A simplified assay platform was developed to measure the activities of the key oxidoreductases in central carbon metabolism of various marine bacteria. Based on microplate assay, the platform was low-cost and simplified by unifying the reaction conditions of enzymes including temperature, buffers, and ionic strength. The central carbon metabolism of 16 marine bacteria, involving Pseudomonas, Exiguobacterium, Marinobacter, Citreicella, and Novosphingobium were studied. Six key oxidoreductases of central carbon metabolism, glucose-6-phosphate dehydrogenase, pyruvate dehydrogenase, 2-ketoglutarate dehydrogenase, malate dehydrogenase, malic enzyme, and isocitrate dehydrogenase were investigated by testing their activities in the pathway. High activity of malate dehydrogenase was found in Citreicella marina, and the specific activity achieved 22 U/mg in cell crude extract. The results also suggested that there was a considerable variability on key enzymes' activities of central carbon metabolism in some strains which have close evolutionary relationship while they adapted to the requirements of the niche they (try to) occupy.

Nanoparticle-based electrochemical sensors for the detection of lactate and hydrogen peroxide

NASA Astrophysics Data System (ADS)

Uzunoglu, Aytekin

In the present study, electrochemical sensors for the detection of lactate and hydrogen peroxide were constructed by exploiting the physicochemical properties of metal ad metal oxide nanoparticles. This study can be divided into two main sections. While chapter 2, 3, and 4 report on the construction of electrochemical lactate biosensors using CeO2 and CeO2-based mixed metal oxide nanoparticles, chapter 5 and 6 show the development of electrochemical hydrogen peroxide sensors by the decoration of the electrode surface with palladium-based nanoparticles. First generation oxidase enzyme-based sensors suffer from oxygen dependency which results in errors in the response current of the sensors in O2-lean environments. To address this challenge, the surface of the sensors must be modified with oxygen rich materials. In this regard, we developed a novel electrochemical lactate biosensor design by exploiting the oxygen storage capacity of CeO2 and CeO 2-CuO nanoparticles. By the introduction of CeO2 nanoparticles into the enzyme layer of the sensors, negative interference effect of ascorbate which resulted from the formation of oxygen-lean regions was eliminated successfully. When CeO2-based design was exposed to higher degree of O2 -depleted environments, however, the response current of the biosensors experienced an almost 21 % decrease, showing that the OSC of CeO2 was not high enough to sustain the enzymatic reactions. When CeO2-CuO nanoparticles, which have 5 times higher OSC than pristine CeO2, were used as an oxygen supply in the enzyme layer, the biosensors did not show any drop in the performance when moving from oxygen-rich to oxygen-lean conditions. In the second part of the study, PdCu/SPCE and PdAg/rGO-based electrochemical H2O2 sensors were designed and their performances were evaluated to determine their sensitivity, linear range, detection limit, and storage stability. In addition, practical applicability of the sensors was studied in human serum. The

Determination of the Subunit Molecular Mass and Composition of Alcohol Dehydrogenase by SDS-PAGE

ERIC Educational Resources Information Center

Nash, Barbara T.

2007-01-01

SDS-PAGE is a simple, rapid technique that has many uses in biochemistry and is readily adaptable to the undergraduate laboratory. It is, however, a technique prone to several types of procedural pitfalls. This article describes the use of SDS-PAGE to determine the subunit molecular mass and composition of yeast alcohol dehydrogenase employing…

Metabolic organization and effects of feeding on enzyme activities of the dogfish shark (Squalus acanthias) rectal gland.

PubMed

Walsh, Patrick J; Kajimura, Makiko; Mommsen, Thomas P; Wood, Chris M

2006-08-01

In order to investigate the metabolic poise of the elasmobranch rectal gland, we conducted two lines of experimentation. First, we examined the effects of feeding on plasma metabolites and enzyme activities from several metabolic pathways in several tissues of the dogfish shark, Squalus acanthias, after starvation and at 6, 20, 30 and 48 h post-feeding. We found a rapid and sustained ten-fold decrease in plasma beta-hydroxybutyrate at 6 h and beyond compared with starved dogfish, suggesting an upregulation in the use of this substrate, a decrease in production, or both. Plasma acetoacetate levels remain unchanged, whereas there was a slight and transient decrease in plasma glucose levels at 6 h. Several enzymes showed a large increase in activity post-feeding, including beta-hydroxybutyrate dehydrogenase in rectal gland and liver, and in rectal gland, isocitrate dehydrogenase, citrate synthase, lactate dehydrogenase, aspartate amino transferase, alanine amino transferase, glutamine synthetase and Na(+)/K(+) ATPase. Also notable in these enzyme measurements was the overall high level of activity in the rectal gland in general. For example, activity of the Krebs' TCA cycle enzyme citrate synthase (over 30 U g(-1)) was similar to activities in muscle from other species of highly active fish. Surprisingly, lactate dehydrogenase activity in the gland was also high (over 150 U g(-1)), suggesting either an ability to produce lactate anaerobically or use lactate as an aerobic fuel. Given these interesting observations, in the second aspect of the study we examined the ability of several metabolic substrates (alone and in combination) to support chloride secretion by the rectal gland. Among the substrates tested at physiological concentrations (glucose, beta-hydroxybutyrate, lactate, alanine, acetoacetate, and glutamate), only glucose could consistently maintain a viable preparation. Whereas beta-hydroxybutyrate could enhance gland activity when presented in combination

Direct Electron Transfer of Dehydrogenases for Development of 3rd Generation Biosensors and Enzymatic Fuel Cells.

PubMed

Bollella, Paolo; Gorton, Lo; Antiochia, Riccarda

2018-04-24

Dehydrogenase based bioelectrocatalysis has been increasingly exploited in recent years in order to develop new bioelectrochemical devices, such as biosensors and biofuel cells, with improved performances. In some cases, dehydrogeases are able to directly exchange electrons with an appropriately designed electrode surface, without the need for an added redox mediator, allowing bioelectrocatalysis based on a direct electron transfer process. In this review we briefly describe the electron transfer mechanism of dehydrogenase enzymes and some of the characteristics required for bioelectrocatalysis reactions via a direct electron transfer mechanism. Special attention is given to cellobiose dehydrogenase and fructose dehydrogenase, which showed efficient direct electron transfer reactions. An overview of the most recent biosensors and biofuel cells based on the two dehydrogenases will be presented. The various strategies to prepare modified electrodes in order to improve the electron transfer properties of the device will be carefully investigated and all analytical parameters will be presented, discussed and compared.

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.

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

The kinetics of lactate production and removal during whole-body exercise

PubMed Central

2012-01-01

Background Based on a literature review, the current study aimed to construct mathematical models of lactate production and removal in both muscles and blood during steady state and at varying intensities during whole-body exercise. In order to experimentally test the models in dynamic situations, a cross-country skier performed laboratory tests while treadmill roller skiing, from where work rate, aerobic power and blood lactate concentration were measured. A two-compartment simulation model for blood lactate production and removal was constructed. Results The simulated and experimental data differed less than 0.5 mmol/L both during steady state and varying sub-maximal intensities. However, the simulation model for lactate removal after high exercise intensities seems to require further examination. Conclusions Overall, the simulation models of lactate production and removal provide useful insight into the parameters that affect blood lactate response, and specifically how blood lactate concentration during practical training and testing in dynamical situations should be interpreted. PMID:22413898

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.

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.

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.

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.

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

Safety of oral tenofovir disoproxil fumarate-based HIV pre-exposure prophylaxis use in lactating HIV-uninfected women.

PubMed

Mugwanya, Kenneth K; John-Stewart, Grace; Baeten, Jared

2017-07-01

In settings where HIV is prevalent in heterosexual populations, pregnancy and postpartum breastfeeding periods can be associated with substantial HIV acquisition risk. Pre-exposure prophylaxis (PrEP) with daily oral tenofovir disoproxil fumarate (TDF)/emtricitabine is an attractive HIV prevention option for women who are lactating but data are limited on its safety during the lactation period. Areas covered: We provide a concise synthesis and summary of current evidence on the safety of TDF-based PrEP during breastfeeding. We conducted a review, searching Pubmed database and major PrEP conferences for primary studies with TDF-based PrEP exposure during postpartum breastfeeding. Expert opinion: TDF-based oral PrEP is an effective female-controlled HIV prevention option. There is evidence supporting the safety of TDF use for infant outcomes during breastfeeding in antiretroviral treatment regimens for HIV and hepatitis B virus, and more limited, but consistently safe, data from use of TDF as PrEP. The potential for risk is arguably outweighed for at-risk individuals by HIV prevention benefits, including indirect protection to the infant as a result of preventing HIV in the breastfeeding mother. As PrEP delivery is scaled up in heterosexual populations in high HIV prevalence settings and for at-risk persons in other settings, implementation science studies can provide a framework to increase the accrual of safety, acceptability, and use data related to PrEP during lactation.

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.

Structural and biochemical insights into 7β-hydroxysteroid dehydrogenase stereoselectivity.

PubMed

Savino, Simone; Ferrandi, Erica Elisa; Forneris, Federico; Rovida, Stefano; Riva, Sergio; Monti, Daniela; Mattevi, Andrea

2016-06-01

Hydroxysteroid dehydrogenases are of great interest as biocatalysts for transformations involving steroid substrates. They feature a high degree of stereo- and regio-selectivity, acting on a defined atom with a specific configuration of the steroid nucleus. The crystal structure of 7β-hydroxysteroid dehydrogenase from Collinsella aerofaciens reveals a loop gating active-site accessibility, the bases of the specificity for NADP(+) , and the general architecture of the steroid binding site. Comparison with 7α-hydroxysteroid dehydrogenase provides a rationale for the opposite stereoselectivity. The presence of a C-terminal extension reshapes the substrate site of the β-selective enzyme, possibly leading to an inverted orientation of the bound substrate. Proteins 2016; 84:859-865. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Fertilization and early embryonic development in heifers and lactating cows in summer and lactating and dry cows in winter.

PubMed

Sartori, R; Sartor-Bergfelt, R; Mertens, S A; Guenther, J N; Parrish, J J; Wiltbank, M C

2002-11-01

Two experiments in two seasons evaluated fertilization rate and embryonic development in dairy cattle. Experiment 1 (summer) compared lactating Holstein cows (n = 27; 97.3 +/- 4.1 d postpartum [dppl; 40.0 +/- 1.5 kg milk/d) to nulliparous heifers (n = 28; 11 to 17 mo old). Experiment 2 (winter) compared lactating cows (n = 27; 46.4 +/- 1.6 dpp; 45.9 +/- 1.4 kg milk/d) to dry cows (n = 26). Inseminations based on estrus included combined semen from four high-fertility bulls. Embryos and oocytes recovered 5 d after ovulation were evaluated for fertilization, embryo quality (1 = excellent to 5 = degenerate), nuclei/embryo, and accessory sperm. In experiment 1, 21 embryos and 17 unfertilized oocytes (UFO) were recovered from lactating cows versus 32 embryos and no UFO from heifers (55% vs. 100% fertilization). Embryos from lactating cows had inferior quality scores (3.8 +/- 0.4 vs. 2.2 +/- 0.3), fewer nuclei/embryo (19.3 +/- 3.7 vs. 36.8 +/- 3.0) but more accessory sperm (37.3 +/- 5.8 vs. 22.4 +/- 5.5/embryo) than embryos from heifers. Sperm were attached to 80% of UFO (17.8 +/- 12.1 sperm/UFO). In experiment 2, lactating cows yielded 36 embryos and 5 UFO versus 34 embryos and 4 UFO from dry cows (87.8 vs. 89.5% fertilization). Embryo quality from lactating cows was inferior to dry cows (3.1 +/- 0.3 vs. 2.2 +/- 0.3), but embryos had similar numbers of nuclei (27.2 +/- 2.7 vs. 30.6 +/- 2.1) and accessory sperm (42.0 +/- 9.4 vs. 36.5 +/- 6.3). From 53% of the flushings from lactating cows and 28% from dry cows, only nonviable embryos were collected. Thus, embryos of lactating dairy cows were detectably inferior to embryos from nonlactating females as early as 5 d after ovulation, with a surprisingly high percentage of nonviable embryos. In addition, fertilization rate was reduced only in summer, apparently due to an effect of heat stress on the oocyte.

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

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

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

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

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

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.

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.

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

Thiol-catalyzed formation of lactate and glycerate from glyceraldehyde. [significance in molecular evolution

NASA Technical Reports Server (NTRS)

Weber, A. L.

1983-01-01

The rate of lactate formation from glyceraldehyde, catalyzed by N-acetyl-cysteine at ambient temperature in aqueous sodium phosphate (pH 7.0), is more rapid at higher sodium phosphate concentrations and remains essentially the same in the presence and absence of oxygen. The dramatic increase in the rate of glycerate formation that is brought about by this thiol, N-acetylcysteine, is accompanied by commensurate decreases in the rates of glycolate and formate production. It is suggested that the thiol-dependent formation of lactate and glycerate occurs by way of their respective thioesters. Attention is given to the significance of these reactions in the context of molecular evolution.

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

Isozyme composition of lactate dehydrogenase of rat skeletal muscles after flight in Kosmos-690 biosatellite. [Effects of radiation on lactate dehydrogenase

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

Petrova, N.V.

1978-01-01