Physiological serum copper concentrations found in malignancies cause unfolding induced aggregation of human serum albumin in vitro.

PubMed

Rizvi, Asim; Furkan, Mohd; Naseem, Imrana

2017-12-15

Malignancies are characterized by several drastic metabolic changes, one of which is a progressive rise in the levels of serum copper. This rise in serum copper is documented across all malignancies and across malignancies in several species. This study aims to explore in vitro the effect of increased copper levels on the structure of the blood protein human serum albumin. Exposure of human serum albumin to physiologically relevant copper concentrations for 21 days resulted in structural modifications in the protein which were evident by changes in the intrinsic florescence. A loss of the predominantly alpha helical structure of human serum albumin was recorded along with a tendency to form protein aggregates. This aggregation was characterized by Thioflavin T and Congo Red assays. Rayleigh light scattering and turbidity assays confirmed aggregation. The aggregates were visually confirmed using transmission electron microscopy. This is the first report implicating increased copper levels as a cause of aggregation of blood proteins in malignancies. The physiological and biochemical implications of this phenomenon are discussed. Copyright © 2017. Published by Elsevier Inc.

Human serum activates CIDEB-mediated lipid droplet enlargement in hepatoma cells

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

Singaravelu, Ragunath; National Research Council of Canada, Ottawa, Ontario K1A 0R6; Lyn, Rodney K.

Highlights: •Human serum induced differentiation of hepatoma cells increases cellular lipid droplet (LD) size. •The observed increase in LD size correlates with increased PGC-1α and CIDEB expression. •Induction of CIDEB expression correlates with rescue of VLDL secretion and loss of ADRP. •siRNA knockdown of CIDEB impairs the human serum mediated increase in LD size. •This system represents a cost-efficient model to study CIDEB’s role in lipid biology. -- Abstract: Human hepatocytes constitutively express the lipid droplet (LD) associated protein cell death-inducing DFFA-like effector B (CIDEB). CIDEB mediates LD fusion, as well as very-low-density lipoprotein (VLDL) maturation. However, there are limitedmore » cell culture models readily available to study CIDEB’s role in these biological processes, as hepatoma cell lines express negligible levels of CIDEB. Recent work has highlighted the ability of human serum to differentiate hepatoma cells. Herein, we demonstrate that culturing Huh7.5 cells in media supplemented with human serum activates CIDEB expression. This activation occurs through the induced expression of PGC-1α, a positive transcriptional regulator of CIDEB. Coherent anti-Stokes Raman scattering (CARS) microscopy revealed a correlation between CIDEB levels and LD size in human serum treated Huh7.5 cells. Human serum treatment also resulted in a rapid decrease in the levels of adipose differentiation-related protein (ADRP). Furthermore, individual overexpression of CIDEB was sufficient to down-regulate ADRP protein levels. siRNA knockdown of CIDEB revealed that the human serum mediated increase in LD size was CIDEB-dependent. Overall, our work highlights CIDEB’s role in LD fusion, and presents a new model system to study the PGC-1α/CIDEB pathway’s role in LD dynamics and the VLDL pathway.« less

Generation of immunosuppressive mesenchymal stem cells in allogeneic human serum.

PubMed

Le Blanc, Katarina; Samuelsson, Håkan; Lönnies, Lena; Sundin, Mikael; Ringdén, Olle

2007-10-27

Mesenchymal stem cells (MSC) may be used to treat acute graft-versus-host disease and for tissue repair. In vitro expansion of MSC has been achieved in the presence of fetal calf serum (FCS). For safety and regulatory reasons, we explored if FCS could be replaced by human blood group AB serum. Proliferation and fold increase of MSC was higher in the presence of AB-serum, compared to FCS. Similar to cells generated in FCS media, MSC from AB-serum media were more than 95% positive for CD90, CD105 and human leukocyte antigen (HLA) class I, and negative for hematopoietic and endothelial markers CD14, CD31, CD34, CD45, and CD80. HLA class II expression was higher in MSC generated in AB-serum, but decreased with higher passage numbers. MSC generated in AB-serum suppressed lymphocyte proliferation in mixed lymphocyte cultures and after stimulation with phytohemagglutinin. MSC expanded in AB-serum and FCS have similar in vitro properties.

STUDY OF THE ADSORPTION ON AND DESORPTION FROM POLYSTYRENE-HUMAN SERUM ALBUMIN CONJUGATES OF RABBIT ANTI-HUMAN SERUM ALBUMIN ANTIBODIES HAVING DIFFERENT SPECIFICITIES

PubMed Central

Webb, Tom; Lapresle, Claude

1961-01-01

An insoluble specific adsorbent for anti-human serum albumin antibodies was prepared by coupling human serum albumin (H.S.A.) to polystyrene by azo bonds. Rabbit anti-H.S.A. immune serum was passed through a column of the adsorbent. It was shown that different volumes of the immune serum were required for the saturation of the different determinant groups of H.S.A. by their corresponding antibodies. The elution of the anti-H.S.A. antibodies adsorbed on the column was achieved by passing successively through the column an acetate buffer pH 3.0 and a solution of 0.1 N HCl in 0.15 M NaCl. The antibodies were eluted in three different fractions, each of which was composed of γ-globulins only. These three fractions contained different proportions of antibodies of different specificities. PMID:13783579

Serum human chorionic gonadotropin levels and thyroid hormone levels in gestational transient thyrotoxicosis: Is the serum hCG level useful for differentiating between active Graves' disease and GTT?

PubMed

Yoshihara, Ai; Noh, Jaeduk Yoshimura; Mukasa, Koji; Suzuki, Miho; Ohye, Hidemi; Matsumoto, Masako; Kunii, Yo; Watanabe, Natsuko; Suzuki, Nami; Kameda, Toshiaki; Sugino, Kiminori; Ito, Koichi

2015-01-01

Gestational transient thyrotoxicosis (GTT) is defined as transient thyrotoxicosis caused by the stimulating effect of human chorionic gonadotropin (hCG) during pregnancy. We attempted to identify the serum hCG level that causes GTT, and we compared the serum hCG levels and thyroid hormone levels of GTT patients according to whether they had a background of thyroid disease. We also evaluated serum hCG as a parameter for differentiating between active Graves' disease (GD) and GTT. We reviewed the 135 cases of pregnant women who came to our hospital to be evaluated for thyrotoxicosis during their 7th to 14th week of pregnancy, and their serum hCG level was measured at that time. Among the 135 pregnant women with thyrotoxicosis; 103 of the women had GTT, and the other 32 women had active GD. There were no correlations between their serum hCG levels and free thyroid hormone levels. There were no significant differences in thyroid hormone levels or hCG levels among the GTT groups with different thyroid disease backgrounds; i.e., the GTT group without thyroid disease, GTT group with chronic thyroiditis, GTT group with non-functioning thyroid nodules, and GTT group with GD in remission. The serum hCG level of the GTT group was significantly higher than in the active GD group, but it was not a good parameter for differentiating between the two groups. The FT3/FT4 ratio of the active GD was significantly higher than in GTT group, and was a better parameter for differentiation.

Human serum-derived hydroxy long-chain fatty acids exhibit anti-inflammatory and anti-proliferative activity

PubMed Central

2011-01-01

Background Circulating levels of novel long-chain hydroxy fatty acids (called GTAs) were recently discovered in the serum of healthy subjects which were shown to be reduced in subjects with colorectal cancer (CRC), independent of tumor burden or disease stage. The levels of GTAs were subsequently observed to exhibit an inverse association with age in the general population. The current work investigates the biological activity of these fatty acids by evaluating the effects of enriched human serum extracts on cell growth and inflammation. Methods GTAs were extracted from commercially available bulk human serum and then chromatographically separated into enriched (GTA-positive) and depleted (GTA-negative) fractions. SW620, MCF7 and LPS stimulated RAW264.7 cells were treated with various concentrations of the GTA-positive and GTA-negative extracts, and the effects on cell growth and inflammation determined. Results Enriched fractions resulted in poly-ADP ribose polymerase (PARP) cleavage, suppression of NFκB, induction of IκBα, and reduction in NOS2 mRNA transcript levels. In RAW264.7 mouse macrophage cells, incubation with enriched fractions prior to treatment with LPS blocked the induction of several pro-inflammatory markers including nitric oxide, TNFα, IL-1β, NOS2 and COX2. Conclusions Our results show that human serum extracts enriched with endogenous long-chain hydroxy fatty acids possess anti-inflammatory and anti-proliferative activity. These findings support a hypothesis that the reduction of these metabolites with age may result in a compromised ability to defend against uncontrolled cell growth and inflammation, and could therefore represent a significant risk for the development of CRC. PMID:21586136

Serum factors and clinical characteristics associated with serum E-Screen activity

PubMed Central

Wang, Jue; Trentham-Dietz, Amy; Hemming, Jocelyn D. C.; Hedman, Curtis J.; Sprague, Brian L.

2013-01-01

Background The E-Screen bioassay can measure the mitogenicity of human serum and thus may be useful as a biomarker in epidemiologic studies of breast cancer. While the assay’s MCF-7 cells are known to proliferate in response to estrogen, the specific determinants of variation in E-Screen activity in human serum samples are poorly understood. We sought to identify serum molecules and patient characteristics associated with serum E-Screen activity among postmenopausal women. Methods Postmenopausal women (N=219) aged 55–70 with no history of postmenopausal hormone use or breast cancer completed a questionnaire and provided a blood sample. Serum was analyzed for E-Screen activity and a variety of molecules including sex hormones, growth factors, and environmental chemicals. Stepwise selection procedures were used to identify correlates of E-Screen activity. Results Serum samples from all women had detectable E-Screen activity, with a median estradiol equivalents value of 0.027 ng/mL and interquartile range of 0.018–0.036 ng/mL. In the final multivariable-adjusted model, serum E-Screen activity was positively associated with serum estradiol, estrone, IGFBP-3, and testosterone levels (p<0.05), as well as body mass index (p=0.03). Serum E-Screen activity was lower among women with higher SHBG (p<0.0001) and progesterone levels (p=0.03). Conclusion Serum E-Screen activity varies according to levels of endogenous estrogens and other serum molecules. Obesity appears to confer additional serum mitogenicity beyond its impact on the measured hormones and growth factors. Impact By capturing mitogenicity due to a variety of patient and serum factors, the E-Screen may provide advantages for use as a biomarker in breast cancer studies. PMID:23588007

Analysis of human serum phosphopeptidome by a focused database searching strategy.

PubMed

Zhu, Jun; Wang, Fangjun; Cheng, Kai; Song, Chunxia; Qin, Hongqiang; Hu, Lianghai; Figeys, Daniel; Ye, Mingliang; Zou, Hanfa

2013-01-14

As human serum is an important source for early diagnosis of many serious diseases, analysis of serum proteome and peptidome has been extensively performed. However, the serum phosphopeptidome was less explored probably because the effective method for database searching is lacking. Conventional database searching strategy always uses the whole proteome database, which is very time-consuming for phosphopeptidome search due to the huge searching space resulted from the high redundancy of the database and the setting of dynamic modifications during searching. In this work, a focused database searching strategy using an in-house collected human serum pro-peptidome target/decoy database (HuSPep) was established. It was found that the searching time was significantly decreased without compromising the identification sensitivity. By combining size-selective Ti (IV)-MCM-41 enrichment, RP-RP off-line separation, and complementary CID and ETD fragmentation with the new searching strategy, 143 unique endogenous phosphopeptides and 133 phosphorylation sites (109 novel sites) were identified from human serum with high reliability. Copyright © 2012 Elsevier B.V. All rights reserved.

Isolation of human anti-serum albumin Fab antibodies with an extended serum-half life.

PubMed

Kang, Hyeon-Ju; Kim, Hye-Jin; Cha, Sang-Hoon

2016-01-01

The serum albumin (SA) has been exploited to generate long-acting biotherapeutics by taking advantage of the FcRn-mediated recycling mechanism in a direct or an indirect way. Since Fab fragments have been proven to be clinically safe for human usage, we assumed that human anti-SA Fab antibodies could have a great potential as a carrier molecule to extend the serum half-life of therapeutic proteins. We, herein, had attempted to isolate anti-SA Fab antibodies from HuDVFab-8L antibody library via a phage display technology, and identified eight discrete human Fab antibodies. One of the Fab antibodies, SL335, showed the strongest binding reactivity to human SA with nM range of affinity at both pH 6 and pH 7.4, and cross-reacted to SAs from various species including rat, mouse, canine and monkey. The in vivo pharmacokinetic assay using a rat model indicated that SL335 has approximately 10 fold longer serum half-life and 26 to 44-fold increase in AUC0 → ∞ compared to the negative control Fab molecule in both intravenous and subcutaneous administrations. Knowing that Fabs have proven to be safe in clinics for a long time, SL335 seems to have a great potential in generating long-acting protein drugs by tagging effector molecules with either chemical conjugation or genetic fusion. Copyright © 2015 Elsevier B.V. All rights reserved.

Serum thymulin in human zinc deficiency.

PubMed Central

Prasad, A S; Meftah, S; Abdallah, J; Kaplan, J; Brewer, G J; Bach, J F; Dardenne, M

1988-01-01

The activity of thymulin (a thymic hormone) is dependent on the presence of zinc in the molecule. We assayed serum thymulin activity in three models of mildly zinc-deficient (ZD) human subjects before and after zinc supplementation: (a) two human volunteers in whom a specific and mild zinc deficiency was induced by dietary means; (b) six mildly ZD adult sickle cell anemia (SCA) subjects; and (c) six mildly ZD adult non-SCA subjects. Their plasma zinc levels were normal and they showed no overt clinical manifestations of zinc deficiency. The diagnosis of mild zinc deficiency was based on the assay of zinc in lymphocytes, granulocytes, and platelets. Serum thymulin activity was decreased as a result of mild zinc deficiency and was corrected by in vivo and in vitro zinc supplementation, suggesting that this parameter was a sensitive indicator of zinc deficiency in humans. An increase in T101-, sIg-cells, decrease in T4+/T8+ ratio, and decreased IL 2 activity were observed in the experimental human model during the zinc depletion phase, all of which were corrected after repletion with zinc. Similar changes in lymphocyte subpopulation, correctable with zinc supplementation, were also observed in mildly ZD SCA subjects. Inasmuch as thymulin is known to induce intra- and extrathymic T cell differentiation, our studies provide a possible mechanism for the role of zinc on T cell functions. Images PMID:3262625

SEPARATION AND CHARACTERIZATION OF HUMAN SERUM CHYLOMICRONS

PubMed Central

Scanu, Angelo; Page, Irvine H.

1959-01-01

Chylomicrons were separated by low and high speed ultracentrifugation from lipemic sera of human subjects in the absorptive phase. The final chylomicron preparation was free from other serum components and contained a small constant amount of protein, approximately 2 per cent of the chylomicron fraction. Electrophoresis, immunochemical analysis, and absorption experiments identified the protein component as derived from a mixture of beta and alpha1 serum lipoproteins. Large aliquots of an emulsion of serum freed of chylomicrons and coconut oil were incubated at 37°C. for 2 hours and ultracentrifuged as in the preparation of chylomicrons. The fat particles now showed the presence of minute amounts of beta and alpha1 serum lipoproteins in almost the same proportion as found in chylomicrons. "Finger prints" of delipidized samples of chylomicrons and particles from serum-coconut oil emulsion gave similar, although not identical patterns. The data on "clearing factor" activity corroborated the finding that serum alpha1 lipoproteins are contained in chylomicrons and particles from serum-coconut oil emulsion. These two lipide particles, partially delipidized, were both able to activate a "clearing factor" system in vitro, a property exhibited only by intact or partially delipidized alpha1 serum lipoproteins. Clearing activity was satisfactorily determined by using an emulsion of coconut oil mixed in agar as a substrate to give an opaque gel, in which the diffusing enzyme showed its activity by areas of clearing. The results obtained by this technique were in agreement with those based on fall in optical density and non-esterified fatty acid production. Chemical analysis of serum chylomicrons showed a concentration of cholesterol and phospholipides higher than could be accounted for by the attached beta and alpha1 serum lipoproteins. On the basis of these results the assumption is made that in the blood stream small amounts of serum lipoproteins, by a process of

Unexpected extent of immunochemical cross-reactions between rabbit and human serum proteins

PubMed Central

Johnson, P. K.; Yoder, J. M.

1970-01-01

Precipitin experiments indicated an unexpected extent of immunochemical cross-reactions between rabbit and human serum proteins. Commerical goat or horse antisera to human or rabbit serum were used. Two of the proteins involved in the cross-reactions were lipoproteins. Imagesp294-ap296-a PMID:4991121

Certified reference materials (GBW09170 and 09171) of creatinine in human serum.

PubMed

Dai, Xinhua; Fang, Xiang; Shao, Mingwu; Li, Ming; Huang, Zejian; Li, Hongmei; Jiang, You; Song, Dewei; He, Yajuan

2011-02-15

Creatinine is the most widely used clinical marker for assessing renal function. Concentrations of creatinine in human serum need to be carefully checked in order to ensure accurate diagnosis of renal function. Therefore, development of certified reference materials (CRMs) of creatinine in serum is of increasing importance. In this study, two new CRMs (Nos. GBW09170 and 09171) for creatinine in human serum have been developed. They were prepared with mixtures of several dozens of healthy people's and kidney disease patient's serum, respectively. The certified values of 8.10, 34.1 mg/kg for these two CRMs have been assigned by liquid chromatography-isotope dilution mass spectrometry (LC-IDMS) method which was validated by using standard reference material (SRM) of SRM909b (a reference material obtained from National Institute of Standards and Technology, NIST). The expanded uncertainties of certified values for low and high concentrations were estimated to be 1.2 and 1.1%, respectively. The certified values were further confirmed by an international intercomparison for the determination of creatinine in human serum (Consultative Committee for Amount of Substance, CCQM) of K80 (CCQM-K80). These new CRMs of creatinine in human serum pool are totally native without additional creatinine spiked for enrichment. These new CRMs are capable of validating routine clinical methods for ensuring accuracy, reliability and comparability of analytical results from different clinical laboratories. They can also be used for instrument validation, development of secondary reference materials, and evaluating the accuracy of high order clinical methods for the determination of creatinine in human serum. Copyright © 2011 Elsevier B.V. All rights reserved.

Identification of a sulfate metabolite of PCB 11 in human serum

PubMed Central

Grimm, Fabian A.; Lehmler, Hans-Joachim; Koh, Wen Xin; DeWall, Jeanne; Teesch, Lynn M.; Hornbuckle, Keri C.; Thorne, Peter S.; Robertson, Larry W.; Duffel, Michael W.

2016-01-01

Despite increasing evidence for a major role for sulfation in the metabolism of lower-chlorinated polychlorinated biphenyls in vitro and in vivo, and initial evidence for potential bioactivities of the resulting sulfate ester metabolites, the formation of PCB sulfates in PCB exposed human populations had not been explored. The primary goal of this study was to determine if PCB sulfates, and potentially other conjugated PCB derivatives, are relevant classes of PCB metabolites in the serum of humans with known exposures to PCBs. In order to detect and quantify dichlorinated PCB sulfates in serum samples of 46 PCB-exposed individuals from either rural or urban communities, we developed a high-resolution mass spectrometry-based protocol using 4-PCB 11 sulfate as a model compound. The method also allowed the preliminary analysis of these 46 human serum extracts for the presence of other metabolites, such as glucuronic acid conjugates and hydroxylated PCBs. Sulfate ester metabolites derived from dichlorinated PCBs were detectable and quantifiable in more than 20 % of analyzed serum samples. Moreover, we were able to utilize this method to detect PCB glucuronides and hydroxylated PCBs, albeit at lower frequencies than PCB sulfates. Altogether, our results provide initial evidence for the presence of PCB sulfates in human serum. Considering the inability of previously employed analytical protocols for PCBs to extract these sulfate ester metabolites and the concentrations of these metabolites observed in our current study, our data support the hypothesis that total serum levels of PCB metabolites in exposed individuals may have been underestimated in the past. PMID:27816204

Competition-Mediated Pyrene-Switching Aptasensor: Probing Lysozyme in Human Serum with a Monomer-Excimer Fluorescence Switch

PubMed Central

Huang, Jin; Zhu, Zhi; Bamrungsap, Suwussa; Zhu, Guizhi; You, Mingxu; He, Xiaoxiao; Wang, Kemin; Tan, Weihong

2010-01-01

Lysozyme (Lys) plays crucial roles in the innate immune system, and the detection of Lys in urine and serum has considerable clinical importance. Traditionally, the presence of Lys has been detected by immunoassays; however, these assays are limited by the availability of commercial antibodies and tedious protein modification, and prior sample purification. To address these limitations, we report here the design, synthesis and application of a competition-mediated pyrene-switching aptasensor for selective detection of Lys in buffer and human serum. The detection strategy is based on the attachment of pyrene molecules to both ends of a hairpin DNA strand, which becomes the partially complementary competitor to an anti-Lys aptamer. In the presence of target Lys, the aptamer hybridizes with part of the competitor, which opens the hairpin such that both pyrene molecules are spatially separated. In the presence of target Lys, however, the competitor is displaced from the aptamer by the target, subsequently forming an initial hairpin structure. This brings the two pyrene moieties into close proximity to generate an excimer, which, in turn, results in a shift of fluorescence emission from ca. 400 nm (pyrene monomer) to 495 nm (pyrene excimer). The proposed method for Lys detection showed sensitivity as low as 200 pM and high selectivity in buffer. When measured by steady-state fluorescence spectrum, the detection of Lys in human serum showed a strong fluorescent background, which obscured detection of the excimer signal. However, time-resolved emission measurement (TREM) supported the potential of the method in complex environments with background fluorescence by demonstrating the temporal separation of probe fluorescence emission decay from the intense background signal. We have also demonstrated that the same strategy can be applied to the detection of small biomolecules such as adenosine triphosphate (ATP), sowing the generality of our approach. Therefore, the

Human platelet lysate: Replacing fetal bovine serum as a gold standard for human cell propagation?

PubMed

Burnouf, Thierry; Strunk, Dirk; Koh, Mickey B C; Schallmoser, Katharina

2016-01-01

The essential physiological role of platelets in wound healing and tissue repair builds the rationale for the use of human platelet derivatives in regenerative medicine. Abundant growth factors and cytokines stored in platelet granules can be naturally released by thrombin activation and clotting or artificially by freeze/thaw-mediated platelet lysis, sonication or chemical treatment. Human platelet lysate prepared by the various release strategies has been established as a suitable alternative to fetal bovine serum as culture medium supplement, enabling efficient propagation of human cells under animal serum-free conditions for a multiplicity of applications in advanced somatic cell therapy and tissue engineering. The rapidly increasing number of studies using platelet derived products for inducing human cell proliferation and differentiation has also uncovered a considerable variability of human platelet lysate preparations which limits comparability of results. The main variations discussed herein encompass aspects of donor selection, preparation of the starting material, the possibility for pooling in plasma or additive solution, the implementation of pathogen inactivation and consideration of ABO blood groups, all of which can influence applicability. This review outlines the current knowledge about human platelet lysate as a powerful additive for human cell propagation and highlights its role as a prevailing supplement for human cell culture capable to replace animal serum in a growing spectrum of applications. Copyright © 2015 Elsevier Ltd. All rights reserved.

Effects of Macromolecular Crowding on Human Adipose Stem Cell Culture in Fetal Bovine Serum, Human Serum, and Defined Xeno-Free/Serum-Free Conditions

PubMed Central

Lee, Michelle Hui Ching; Mäkinen, Laura; Ang, Xiu Min; Mannerström, Bettina; Raghunath, Michael; Miettinen, Susanna

2017-01-01

Microenvironment plays an important role for stem cell proliferation and differentiation. Macromolecular crowding (MMC) was recently shown to assist stem cells in forming their own matrix microenvironment in vitro. The ability of MMC to support adipose stem cell (ASC) proliferation, metabolism, and multilineage differentiation was studied under different conditions: fetal bovine serum- (FBS-) and human serum- (HS-) based media and xeno- and serum-free (XF/SF) media. Furthermore, the immunophenotype of ASCs under MMC was evaluated. The proliferative capacity of ASCs under MMC was attenuated in each condition. However, osteogenic differentiation was enhanced under MMC, shown by increased deposition of mineralized matrix in FBS and HS cultures. Likewise, significantly greater lipid droplet accumulation and increased collagen IV deposition indicated enhanced adipogenesis under MMC in FBS and HS cultures. In contrast, chondrogenic differentiation was attenuated in ASCs expanded under MMC. The ASC immunophenotype was maintained under MMC with significantly higher expression of CD54. However, MMC impaired metabolic activity and differentiation capacity of ASCs in XF/SF conditions. Both the supportive and inhibitory effects of MMC on ASC are culture condition dependent. In the presence of serum, MMC maintains ASC immunophenotype and enhances adipogenic and osteogenic differentiation at the cost of reduced proliferation. PMID:28465691

Effects of Macromolecular Crowding on Human Adipose Stem Cell Culture in Fetal Bovine Serum, Human Serum, and Defined Xeno-Free/Serum-Free Conditions.

PubMed

Patrikoski, Mimmi; Lee, Michelle Hui Ching; Mäkinen, Laura; Ang, Xiu Min; Mannerström, Bettina; Raghunath, Michael; Miettinen, Susanna

2017-01-01

Microenvironment plays an important role for stem cell proliferation and differentiation. Macromolecular crowding (MMC) was recently shown to assist stem cells in forming their own matrix microenvironment in vitro. The ability of MMC to support adipose stem cell (ASC) proliferation, metabolism, and multilineage differentiation was studied under different conditions: fetal bovine serum- (FBS-) and human serum- (HS-) based media and xeno- and serum-free (XF/SF) media. Furthermore, the immunophenotype of ASCs under MMC was evaluated. The proliferative capacity of ASCs under MMC was attenuated in each condition. However, osteogenic differentiation was enhanced under MMC, shown by increased deposition of mineralized matrix in FBS and HS cultures. Likewise, significantly greater lipid droplet accumulation and increased collagen IV deposition indicated enhanced adipogenesis under MMC in FBS and HS cultures. In contrast, chondrogenic differentiation was attenuated in ASCs expanded under MMC. The ASC immunophenotype was maintained under MMC with significantly higher expression of CD54. However, MMC impaired metabolic activity and differentiation capacity of ASCs in XF/SF conditions. Both the supportive and inhibitory effects of MMC on ASC are culture condition dependent. In the presence of serum, MMC maintains ASC immunophenotype and enhances adipogenic and osteogenic differentiation at the cost of reduced proliferation.

Human umbilical cord blood serum promotes growth, proliferation, as well as differentiation of human bone marrow-derived progenitor cells.

PubMed

Phadnis, Smruti M; Joglekar, Mugdha V; Venkateshan, Vijayalakshmi; Ghaskadbi, Surendra M; Hardikar, Anandwardhan A; Bhonde, Ramesh R

2006-01-01

Fetal calf serum (FCS) is conventionally used for animal cell cultures due to its inherent growth-promoting activities. However animal welfare issues and stringent requirements for human transplantation studies demand a suitable alternative for FCS. With this view, we studied the effect of FCS, human AB serum (ABS), and human umbilical cord blood serum (UCBS) on murine islets of Langerhans and human bone marrow-derived mesenchymal-like cells (hBMCs). We found that there was no difference in morphology and functionality of mouse islets cultured in any of these three different serum supplements as indicated by insulin immunostaining. A comparative analysis of hBMCs maintained in each of these three different serum supplements demonstrated that UCBS supplemented media better supported proliferation of hBMCs. Moreover, a modification of adipogenic differentiation protocol using UCBS indicates that it can be used as a supplement to support differentiation of hBMCs into adipocytes. Our results demonstrate that UCBS not only is suitable for maintenance of murine pancreatic islets, but also supports attachment, propagation, and differentiation of hBMCs in vitro. We conclude that UCBS can serve as a better serum supplement for growth, maintenance, and differentiation of hBMCs, making it a more suitable supplement in cell systems that have therapeutic potential in human transplantation programs.

Pathogen inactivation of human serum facilitates its clinical use for islet cell culture and subsequent transplantation.

PubMed

Ståhle, Magnus U; Brandhorst, Daniel; Korsgren, Olle; Knutson, Folke

2011-01-01

Serum is regarded as an essential supplement to promote survival and growth of cells during culture. However, the potential risk of transmitting diseases disqualifies the use of serum for clinical cell therapy in most countries. Hence, most clinical cell therapy programs have replaced human serum with human serum albumin, which can result in inferior quality of released cell products. Photochemical treatment of different blood products utilizing Intercept® technology has been shown to inactivate a broad variety of pathogens of RNA and DNA origin. The present study assesses the feasibility of using pathogen-inactivated, blood group-compatible serum for use in human pancreatic islet culture. Isolated human islets were cultured at 37°C for 3-4 days in CMRL 1066 supplemented with 10% of either pathogen-inactivated or nontreated human serum. Islet quality assessment included glucose-stimulated insulin release (perifusion), ADP/ATP ratio, cytokine expression, and posttransplant function in diabetic nude mice. No differences were found between islets cultured in pathogen-inactivated or control serum regarding stimulated insulin release, intracellular insulin content, and ADP/ATP ratio. Whether media was supplemented with treated or nontreated serum, islet expression of IL-6, IL-8, MCP-1, or tissue factor was not affected. The final diabetes-reversal rate of mice receiving islets cultured in pathogen-inactivated or nontreated serum was 78% and 87%, respectively (NS). As reported here, pathogen-inactivated human serum does not affect viability or functional integrity of cultured human islets. The implementation of this technology for RNA- and DNA-based pathogen inactivation should enable reintroduction of human serum for clinical cell therapy.

Affinity precipitation of human serum albumin using a thermo-response polymer with an L-thyroxin ligand

PubMed Central

2013-01-01

Background Affinity precipitation has been reported as a potential technology for the purification of proteins at the early stage of downstream processing. The technology could be achieved using reversible soluble-insoluble polymers coupled with an affinity ligand to purify proteins from large volumes of dilute solution material such as fermentation broths or plasma. In this study, a thermo-response polymer was synthesized using N-methylol acrylamide, N-isopropyl acrylamide and butyl acrylate as monomers. The molecular weight of the polymer measured by the viscosity method was 3.06 × 104 Da and the lower critical solution temperature (LCST) was 28.0°C.The recovery of the polymer above the LCST was over 95.0%. Human serum albumin (HSA) is the most abundant protein in the human serum system, and it has important functions in the human body. High purity HSA is required in pharmaceuticals. Safe and efficient purification is a crucial process during HSA production. Results A thermo-response polymer was synthesized and L-thyroxin immobilized on the polymer as an affinity ligand to enable affinity precipitation of HSA. The LCST of the affinity polymer was 31.0°C and the recovery was 99.6% of its original amount after recycling three times. The optimal adsorption condition was 0.02 M Tris–HCl buffer (pH 7.0) and the HSA adsorption capacity was 14.9 mg/g polymer during affinity precipitation. Circular dichroism spectra and a ForteBio Octet system were used to analyze the interactions between the affinity polymer and HSA during adsorption and desorption. The recovery of total HSA by elution with 1.0 mol/L NaSCN was 93.6%. When the affinity polymer was applied to purification of HSA from human serum, HSA could be purified to single-band purity according to SDS-PAGE. Conclusion A thermo-response polymer was synthesized and L-thyroxin was attached to the polymer. Affinity precipitation was used to purify HSA from human serum. PMID:24341315

Strain Background Modifies Phenotypes in the ATP8B1-Deficient Mouse

PubMed Central

Vargas, Julie C.; Xu, Hongmei; Groen, Annamiek; Paulusma, Coen C.; Grenert, James P.; Pawlikowska, Ludmila; Sen, Saunak; Elferink, Ronald P. J. Oude; Bull, Laura N.

2010-01-01

Background Mutations in ATP8B1 (FIC1) underlie cases of cholestatic disease, ranging from chronic and progressive (progressive familial intrahepatic cholestasis) to intermittent (benign recurrent intrahepatic cholestasis). The ATP8B1-deficient mouse serves as an animal model of human ATP8B1 deficiency. Methodology/Principal Findings We investigated the effect of genetic background on phenotypes of ATP8B1-deficient and wild-type mice, using C57Bl/6 (B6), 129, and (B6-129) F1 strain backgrounds. B6 background resulted in greater abnormalities in ATP8B1-deficient mice than did 129 and/or F1 background. ATP8B1-deficient pups of B6 background gained less weight. In adult ATP8B1-deficient mice at baseline, those of B6 background had lower serum cholesterol levels, higher serum alkaline phosphatase levels, and larger livers. After challenge with cholate-supplemented diet, these mice exhibited higher serum alkaline phosphatase and bilirubin levels, greater weight loss and larger livers. ATP8B1-deficient phenotypes in mice of F1 and 129 backgrounds are usually similar, suggesting that susceptibility to manifestations of ATP8B1 deficiency may be recessive. We also detected differences in hepatobiliary phenotypes between wild-type mice of differing strains. Conclusions/Significance Our results indicate that the ATP8B1-deficient mouse in a B6 background may be a better model of human ATP8B1 deficiency and highlight the importance of informed background strain selection for mouse models of liver disease. PMID:20126555

Quantitative bioanalysis of strontium in human serum by inductively coupled plasma-mass spectrometry

PubMed Central

Somarouthu, Srikanth; Ohh, Jayoung; Shaked, Jonathan; Cunico, Robert L; Yakatan, Gerald; Corritori, Suzana; Tami, Joe; Foehr, Erik D

2015-01-01

Aim: A bioanalytical method using inductively-coupled plasma-mass spectrometry to measure endogenous levels of strontium in human serum was developed and validated. Results & methodology: This article details the experimental procedures used for the method development and validation thus demonstrating the application of the inductively-coupled plasma-mass spectrometry method for quantification of strontium in human serum samples. The assay was validated for specificity, linearity, accuracy, precision, recovery and stability. Significant endogenous levels of strontium are present in human serum samples ranging from 19 to 96 ng/ml with a mean of 34.6 ± 15.2 ng/ml (SD). Discussion & conclusion: Calibration procedures and sample pretreatment were simplified for high throughput analysis. The validation demonstrates that the method was sensitive, selective for quantification of strontium (88Sr) and is suitable for routine clinical testing of strontium in human serum samples. PMID:28031925

Identification of a sulfate metabolite of PCB 11 in human serum.

PubMed

Grimm, Fabian A; Lehmler, Hans-Joachim; Koh, Wen Xin; DeWall, Jeanne; Teesch, Lynn M; Hornbuckle, Keri C; Thorne, Peter S; Robertson, Larry W; Duffel, Michael W

2017-01-01

Despite increasing evidence for a major role for sulfation in the metabolism of lower-chlorinated polychlorinated biphenyls in vitro and in vivo, and initial evidence for potential bioactivities of the resulting sulfate ester metabolites, the formation of PCB sulfates in PCB exposed human populations had not been explored. The primary goal of this study was to determine if PCB sulfates, and potentially other conjugated PCB derivatives, are relevant classes of PCB metabolites in the serum of humans with known exposures to PCBs. In order to detect and quantify dichlorinated PCB sulfates in serum samples of 46 PCB-exposed individuals from either rural or urban communities, we developed a high-resolution mass spectrometry-based protocol using 4-PCB 11 sulfate as a model compound. The method also allowed the preliminary analysis of these 46 human serum extracts for the presence of other metabolites, such as glucuronic acid conjugates and hydroxylated PCBs. Sulfate ester metabolites derived from dichlorinated PCBs were detectable and quantifiable in more than 20% of analyzed serum samples. Moreover, we were able to utilize this method to detect PCB glucuronides and hydroxylated PCBs, albeit at lower frequencies than PCB sulfates. Altogether, our results provide initial evidence for the presence of PCB sulfates in human serum. Considering the inability of previously employed analytical protocols for PCBs to extract these sulfate ester metabolites and the concentrations of these metabolites observed in our current study, our data support the hypothesis that total serum levels of PCB metabolites in exposed individuals may have been underestimated in the past. Copyright © 2016 Elsevier Ltd. All rights reserved.

[Lethal anaphylactic shock model induced by human mixed serum in guinea pigs].

PubMed

Ren, Guang-Mu; Bai, Ji-Wei; Gao, Cai-Rong

2005-08-01

To establish an anaphylactic shock model induced by human mixed serum in guinea pigs. Eighteen guinea pigs were divided into two groups: sensitized and control, The sensitized group were immunized intracutaneously with human mixed serum and then induced by endocardiac injection after 3 weeks. Symptoms of anaphylactic shock appeared in the sensitized group. The level of serum IgE were increased in the sensitized group significantly. An animal model of anaphylactic shock wer established successfully. It provide a tool for both forensic study and anaphylactic shock therapy.

Molecularly imprinted composite cryogel for albumin depletion from human serum.

PubMed

Andaç, Müge; Baydemir, Gözde; Yavuz, Handan; Denizli, Adil

2012-11-01

A new composite protein-imprinted macroporous cryogel was prepared for depletion of albumin from human serum prior to use in proteom applications. Polyhydroxyethyl-methacylate-based molecularly imprinted polymer (MIP) composite cryogel was prepared with high gel fraction yields up to 83%, and its morphology and porosity were characterized by Fourier transform infrared, scanning electron microscopy, swelling studies, flow dynamics, and surface area measurements. Selective binding experiments were performed in the presence of competitive proteins human transferrin (HTR) and myoglobin (MYB). MIP composite cryogel exhibited a high binding capacity and selectivity for human serum albumin (HSA) in the presence of HTR and MYB. The competitive adsorption amount for HSA in MIP composite cryogel is 722.1 mg/dL in the presence of competitive proteins (HTR and MYB). MIP composite cryogel column was successfully applied in the fast protein liquid chromatography system for selective depletion of albumin in human serum. The depletion ratio was highly increased by embedding beads into cryogel (85%). Finally, MIP composite cryogel can be reused many times with no apparent decrease in HSA adsorption capacity. Copyright © 2012 John Wiley & Sons, Ltd.

The importance of selecting a proper biological milieu for protein corona analysis in vitro: Human plasma versus human serum.

PubMed

Mirshafiee, Vahid; Kim, Raehyun; Mahmoudi, Morteza; Kraft, Mary L

2016-06-01

Nanoparticle (NP) exposure to biological fluids in the body results in protein binding to the NP surface, which forms a protein coating that is called the "protein corona". To simplify studies of protein-NP interactions and protein corona formation, NPs are incubated with biological solutions, such as human serum or human plasma, and the effects of this exposure are characterized in vitro. Yet, how NP exposure to these two different biological milieus affects protein corona composition and cell response has not been investigated. Here, we explore the differences between the protein coronas that form when NPs are incubated in human serum versus human plasma. NP characterization indicated that NPs that were exposed to human plasma had higher amounts of proteins bound to their surfaces, and were slightly larger in size than those exposed to human serum. In addition, significant differences in corona composition were also detected with gel electrophoresis and liquid chromatography-mass spectrometry/mass spectrometry, where a higher fraction of coagulation proteins and complement factors were found on the plasma-exposed NPs. Flow cytometry and confocal microscopy showed that the uptake of plasma-exposed NPs was higher than that of serum-exposed NPs by RAW 264.7 macrophage immune cells, but not by NIH 3T3 fibroblast cells. This difference is likely due to the elevated amounts of opsonins, such as fibrinogen, on the surfaces of the NPs exposed to plasma, but not serum, because these components trigger NP internalization by immune cells. As the human plasma better mimics the composition of the in vivo environment, namely blood, in vitro protein corona studies should employ human plasma, and not human serum, so the biological phenomena that is observed is more similar to that occurring in vivo. Copyright © 2015 Elsevier Ltd. All rights reserved.

Human Milk and Matched Serum Demonstrate Concentration of Select miRNAs.

PubMed

Qin, Wenyi; Dasgupta, Santanu; Corradi, John; Sauter, Edward R

Pregnancy-associated breast cancers (PABCs), especially those diagnosed after childbirth, are often aggressive, with a poor prognosis. Factors influencing PABC are largely unknown. Micro(mi)RNAs are present in many human body fluids and shown to influence cancer development and/or growth. In six nursing mothers, we determined if breast cancer-associated miRNAs were (1) detectable in human breast milk and (2) if detectable, their relative expression in milk fractions compared to matched serum. We evaluated by quantitative PCR the expression of 11 cancer-associated miRNAs (10a-5p, 16, 21, 100, 140, 145, 155, 181, 199, 205, 212) in breast milk cells, fat and supernatant (skim milk), and matched serum. miRNA expression was detectable in all samples. For 10/11 miRNAs, mean relative expression compared to control (ΔCt) values was lowest in milk cells, the exception being miR205. Relative concentration was highest in the skim fraction of milk for all miRNAs. Expression was higher in skim milk than matched serum for 7/11 miRNAs and in serum for 4/11 miRNAs. miR205 expression was higher in all milk fractions than in matched serum. In conclusion, the expression of breast cancer-associated miRNAs is detectable in human breast milk and serum samples. The concentration is highest in skim milk, but is also detectable in milk fat and milk cells.

Occurrence of a multimeric high-molecular-weight glyceraldehyde-3-phosphate dehydrogenase in human serum.

PubMed

Kunjithapatham, Rani; Geschwind, Jean-Francois; Devine, Lauren; Boronina, Tatiana N; O'Meally, Robert N; Cole, Robert N; Torbenson, Michael S; Ganapathy-Kanniappan, Shanmugasundaram

2015-04-03

Cellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a phylogenetically conserved, ubiquitous enzyme that plays an indispensable role in energy metabolism. Although a wealth of information is available on cellular GAPDH, there is a clear paucity of data on its extracellular counterpart (i.e., the secreted or extracellular GAPDH). Here, we show that the extracellular GAPDH in human serum is a multimeric, high-molecular-weight, yet glycolytically active enzyme. The high-molecular-weight multimers of serum GAPDH were identified by immunodetection on one- and two-dimensional gel electrophoresis using multiple antibodies specific for various epitopes of GAPDH. Partial purification of serum GAPDH by DEAE Affigel affinity/ion exchange chromatography further established the multimeric composition of serum GAPDH. In vitro data demonstrated that human cell lines secrete a multimeric, high-molecular-weight enzyme similar to that of serum GAPDH. Furthermore, LC-MS/MS analysis of extracellular GAPDH from human cell lines confirmed the presence of unique peptides of GAPDH in the high-molecular-weight subunits. Furthermore, data from pulse-chase experiments established the presence of high-molecular-weight subunits in the secreted, extracellular GAPDH. Taken together, our findings demonstrate the presence of a high-molecular-weight, enzymatically active secretory GAPDH in human serum that may have a hitherto unknown function in humans.

Detection of α-fetoprotein in human serum using carbon nanotube transistor

NASA Astrophysics Data System (ADS)

So, Hye-Mi; Park, Dong-Won; Lee, Seong-Kyu; Kim, Beom Soo; Chang, Hyunju; Lee, Jeong-O.

2009-03-01

We have fabricated antibody-coated carbon nanotube field effect transistor (CNT-FET) sensor for the detection of α-fetoprotein (AFP), single chain glycoprotein of 70 kDa that is normally expressed in the fetal liver, in human serum. The AFP-specific antibodies were immobilized on CNT with linker molecule such as pyrenebutyric acid N-hydroxysuccinimide ester. To prevent nonspecific adsorption of antigen, we performed blocking procedure using bovine serum albumin (BSA). Antibody-antigen binding was determined by measuring electrical conductance change of FET and took an average of thereshold voltage change before and after binding. Also we checked concentration-dependent conductance change in human serum using both p-type SWNT-FETs and n-type SWNT-FETs.

Identification of the trypanocidal factor in normal human serum: high density lipoprotein.

PubMed Central

Rifkin, M R

1978-01-01

The differentiation of Trypanosoma brucei from T. rhodesiense, the causative agent of human sleeping sickness, depends on their relative sensitivities to the cytotoxic effects of normal human serum. The molecule responsible for the specific lysis of T. brucei has now been isolated. Serum lipoproteins were fractionated and purified by ultracentrifugal flotation and chromatography on Bio-Gel A-5m. Trypanocidal activity was recovered in the high density lipoprotein fraction (density, 1.063-1.216 g/ml). Contamination by other serum proteins was checked by crossed immunoelectrophoresis and sodium dodecyl sulfate/acrylamide gel electrophoresis. Only a trace of beta-lipoprotein was found. The trypanocidal activity of pure human high density lipoprotein was identical to that of unfractionated serum when the following were tested: (i) time course of in vitro lysis of T. bruceli; (ii) in vivo destruction of T. brucei; (iii) relative resistance of T. rhodesiense to lysis. Rat or rabbit high density lipoprotein had no trypanocidal activity. Identification of the trypanocidal factor as high density lipoprotein was confirmed by the finding that serum from patients with Tangier disease, an autosomal recessive disorder characterized by a severe deficiency of high density lipoprotein, had no trypanocidal activity. Images PMID:210461

Binding of human serum proteins to titanium dioxide particles in vitro.

PubMed

Zaqout, Mazen S K; Sumizawa, Tomoyuki; Igisu, Hideki; Higashi, Toshiaki; Myojo, Toshihiko

2011-01-01

To determine the capacity of human serum proteins to bind to titanium dioxide (TiO(2)) particles of different polymorphs and sizes. TiO(2) particles were mixed with diluted human serum, purified human serum albumin (HSA) or purified human serum gamma-globulin (HGG) solutions. After incubation at 37°C for 1 h, the particles were sedimented by centrifugation, and proteins in the supernatant, as well as those bound to the particles, were analyzed. The total protein concentration in the supernatant was lowered by TiO(2), whereas the albumin/globulin ratio was elevated by the particles. Incubation with TiO(2) also lowered the immunoglobulin, pre-albumin, beta2-microglobulin, ceruloplasmin and retinol-binding protein levels, but not ferritin levels, in the supernatant. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), proteins in the supernatant, especially HGG, were observed to decrease, while those released from the particles (after adding 1% SDS and heating) increased, depending on the dose of TiO(2). Purified HGG and HSA were also bound to TiO(2), although the former appeared to have a higher affinity. All the proteins tested showed the highest binding potency to the amorphous particles (<50 nm) and the lowest to the rutile particles (<5,000 nm), while binding to anatase particles was intermediate. The affinity to the larger anatase was higher than that to smaller anatase particles in most cases. Human serum proteins, including the two major components, HSA and HGG, are bound by TiO(2) particles. The polymorph of the particles seems to be important for determining the binding capacity of the particles and it may affect distribution of the particles in the body.

Human Leukocyte Antigen Class I and Class II Polymorphisms and Serum Cytokine Profiles in Cervical Cancer.

PubMed

Bahls, Larissa; Yamakawa, Roger; Zanão, Karina; Alfieri, Daniela; Flauzino, Tamires; Delongui, Francieli; de Abreu, André; Souza, Raquel; Gimenes, Fabrícia; Reiche, Edna; Borelli, Sueli; Consolaro, Marcia

2017-08-31

Only a small proportion of women who are exposed to infection with high-risk human papillomavirus (HR-HPV) progress to persistent infection and develop cervical cancer (CC). The immune response and genetic background of the host may affect the risk of progression from a HR-HPV infection to lesions and cancer. However, to our knowledge, no studies has been conducted to evaluate the relationship between variability of human leukocyte antigens ( HLA ) genes and serum cytokine expression in this pathology. In the current study, we examined the associations of HLA alleles and haplotypes including Class I ( HLA-A , -B and -C ) and II ( HLA-DRB1 , -DQA1 and -DQB1 ) with serum levels of cytokines interleukin (IL)-6, tumor necrosis factor-α (TNF-α), IL-10 and IL-17 as well as risks of HPV infections, lesions and CC among admixed Brazilian women. HLA polymorphisms were associated with an increased risk or protection from HPV, lesions and CC. Additionally, we demonstrated a potential association of a HLA class I haplotype ( HLA-B*14-C*08 ) with higher IL-10 cytokine serum levels in cervical disease, suggesting an association between HLA class I and specific cytokines in cervical carcinogenesis. However, larger studies with detailed HPV types coupled with genetic data are needed to further evaluate the effects of HLA and CC by HPV genotype.

Human Leukocyte Antigen Class I and Class II Polymorphisms and Serum Cytokine Profiles in Cervical Cancer

PubMed Central

Bahls, Larissa; Yamakawa, Roger; Zanão, Karina; Alfieri, Daniela; Flauzino, Tamires; Delongui, Francieli; de Abreu, André; Souza, Raquel; Gimenes, Fabrícia; Reiche, Edna; Borelli, Sueli; Consolaro, Marcia

2017-01-01

Only a small proportion of women who are exposed to infection with high-risk human papillomavirus (HR-HPV) progress to persistent infection and develop cervical cancer (CC). The immune response and genetic background of the host may affect the risk of progression from a HR-HPV infection to lesions and cancer. However, to our knowledge, no studies has been conducted to evaluate the relationship between variability of human leukocyte antigens (HLA) genes and serum cytokine expression in this pathology. In the current study, we examined the associations of HLA alleles and haplotypes including Class I (HLA-A, -B and -C) and II (HLA-DRB1, -DQA1 and -DQB1) with serum levels of cytokines interleukin (IL)-6, tumor necrosis factor-α (TNF-α), IL-10 and IL-17 as well as risks of HPV infections, lesions and CC among admixed Brazilian women. HLA polymorphisms were associated with an increased risk or protection from HPV, lesions and CC. Additionally, we demonstrated a potential association of a HLA class I haplotype (HLA-B*14-C*08) with higher IL-10 cytokine serum levels in cervical disease, suggesting an association between HLA class I and specific cytokines in cervical carcinogenesis. However, larger studies with detailed HPV types coupled with genetic data are needed to further evaluate the effects of HLA and CC by HPV genotype. PMID:28858203

Influence of heat inactivation of human serum on the opsonization of Streptococcus mutans.

PubMed

Moore, M A; Hakki, Z W; Gregory, R L; Gfell, L E; Kim-Park, W K; Kowolik, M J

1997-12-15

Phagocytosis of bacteria, such as Streptococcus mutans, is important to host defense. One mechanism by which phagocytosis can be enhanced is by antibody or complement-mediated opsonization of bacteria. Many studies utilize opsonization of bacteria to enhance a cellular response, but little information has been found examining methodology or validity of the opsonization process following the denaturization of the serum. Human serum was inactivated by heat in order to disrupt the classical and alternative pathways of the complement cascade. S. mutans isolated from human subjects were opsonized with heat-inactivated human serum before exposing them to viable neutrophils in vitro. Luminol-dependent chemiluminescence (CL) was used to measure neutrophil activation. Human serum used to opsonize the bacteria was denatured by incubation at 57 degrees C for intervals of 30 and 60 min to inactivate complement. The results from the opsonization data indicated that there was significantly increased CL with 60-min inactivation of the serum (34% increase in mean integration mV.min; p < or = 0.05) over the nonopsonized control. This indicated a successful opsonization of the bacteria. In addition, the data demonstrate that the inactivation of serum requires a minimum of 60 min at 57 degrees C to disrupt the complement cascade, while 30- and 15-min inactivations produced no significant increase in CL activity over the control. Standard sandwich ELISA assays, detecting complement binding to S. mutans, confirmed successful heat inactivation of serum showing a significant decrease (p < or = 0.001) in complement binding to S. mutans after 30 min, but could not explain the increased CL response after 60-min heat deactivation of the serum.

Candida albicans Shaving to Profile Human Serum Proteins on Hyphal Surface

PubMed Central

Marín, Elvira; Parra-Giraldo, Claudia M.; Hernández-Haro, Carolina; Hernáez, María L.; Nombela, César; Monteoliva, Lucía; Gil, Concha

2015-01-01

Candida albicans is a human opportunistic fungus and it is responsible for a wide variety of infections, either superficial or systemic. C. albicans is a polymorphic fungus and its ability to switch between yeast and hyphae is essential for its virulence. Once C. albicans obtains access to the human body, the host serum constitutes a complex environment of interaction with C. albicans cell surface in bloodstream. To draw a comprehensive picture of this relevant step in host-pathogen interaction during invasive candidiasis, we have optimized a gel-free shaving proteomic strategy to identify both, human serum proteins coating C. albicans cells and fungi surface proteins simultaneously. This approach was carried out with normal serum (NS) and heat inactivated serum (HIS). We identified 214 human and 372 C. albicans unique proteins. Proteins identified in C. albicans included 147 which were described as located at the cell surface and 52 that were described as immunogenic. Interestingly, among these C. albicans proteins, we identified 23 GPI-anchored proteins, Gpd2 and Pra1, which are involved in complement system evasion and 7 other proteins that are able to attach plasminogen to C. albicans surface (Adh1, Eno1, Fba1, Pgk1, Tdh3, Tef1, and Tsa1). Furthermore, 12 proteins identified at the C. albicans hyphae surface induced with 10% human serum were not detected in other hypha-induced conditions. The most abundant human proteins identified are involved in complement and coagulation pathways. Remarkably, with this strategy, all main proteins belonging to complement cascades were identified on the C. albicans surface. Moreover, we identified immunoglobulins, cytoskeletal proteins, metabolic proteins such as apolipoproteins and others. Additionally, we identified more inhibitors of complement and coagulation pathways, some of them serpin proteins (serine protease inhibitors), in HIS vs. NS. On the other hand, we detected a higher amount of C3 at the C. albicans surface in

[Comparison of acetonitrile, ethanol and chromatographic column to eliminate high-abundance proteins in human serum].

PubMed

Li, Yin; Liao, Ming; He, Xiao; Zhou, Yi; Luo, Rong; Li, Hongtao; Wang, Yun; He, Min

2012-11-01

To compare the effects of acetonitrile precipitation, ethanol precipitation and multiple affinity chromatography column Human 14 removal to eliminate high-abundance proteins in human serum. Elimination of serum high-abundance proteins performed with acetonitrile precipitation, ethanol precipitation and multiple affinity chromatography column Human 14 removal. Bis-Tris Mini Gels electrophoresis and two-dimensional gel electrophoresis to detect the effect. Grey value analysis from 1-DE figure showed that after serum processed by acetonitrile method, multiple affinity chromatography column Human 14 removal method and ethanol method, the grey value of albumin changed into 157.2, 40.8 and 8.2 respectively from the original value of 19. 2-DE analysis results indicated that using multiple affinity chromatography column Human 14 method, the protein points noticeable increased by 137 compared to the original serum. After processed by acetonitrile method and ethanol method, the protein point reduced, but the low abundance protein point emerged. The acetonitrile precipitation could eliminate the vast majority of high abundance proteins in serum and gain more proteins of low molecular weight, ethanol precipitation could eliminate part of high abundance proteins in serum, but low abundance proteins less harvested, and multiple affinity chromatography column Human 14 method could effectively removed the high abundance proteins, and keep a large number of low abundance proteins.

Effect of transdermal magnesium cream on serum and urinary magnesium levels in humans: A pilot study

PubMed Central

Tanner, Amy; Sullivan, Keith; McAuley, William; Plesset, Michael

2017-01-01

Background Oral magnesium supplementation is commonly used to support a low magnesium diet. This investigation set out to determine whether magnesium in a cream could be absorbed transdermally in humans to improve magnesium status. Methods and findings In this single blind, parallel designed pilot study, n = 25 participants (aged 34.3+/-14.8y, height 171.5+/-11cm, weight 75.9 +/-14 Kg) were randomly assigned to either a 56mg/day magnesium cream or placebo cream group for two weeks. Magnesium serum and 24hour urinary excretion were measured at baseline and at 14 days intervention. Food diaries were recorded for 8 days during this period. Mg test and placebo groups’ serum and urinary Mg did not differ at baseline. After the Mg2+ cream intervention there was a clinically relevant increase in serum magnesium (0.82 to 0.89 mmol/l,p = 0.29) that was not seen in the placebo group (0.77 to 0.79 mmol/L), but was only statistically significant (p = 0.02)) in a subgroup of non-athletes. Magnesium urinary excretion increased from baseline slightly in the Mg2+ group but with no statistical significance (p = 0.48). The Mg2+ group showed an 8.54% increase in serum Mg2+ and a 9.1% increase in urinary Mg2+ while these figures for the placebo group were smaller, i.e. +2.6% for serum Mg2+ and -32% for urinary Mg2+. In the placebo group, both serum and urine concentrations showed no statistically significant change after the application of the placebo cream. Conclusion No previous studies have looked at transdermal absorbency of Mg2+ in human subjects. In this pilot study, transdermal delivery of 56 mg Mg/day (a low dose compared with commercial transdermal Mg2+ products available) showed a larger percentage rise in both serum and urinary markers from pre to post intervention compared with subjects using the placebo cream, but statistical significance was achieved only for serum Mg2+ in a subgroup of non-athletes. Future studies should look at higher dosage of magnesium cream for

Second-trimester maternal serum marker screening: maternal serum alpha-fetoprotein, beta-human chorionic gonadotropin, estriol, and their various combinations as predictors of pregnancy outcome.

PubMed

Yaron, Y; Cherry, M; Kramer, R L; O'Brien, J E; Hallak, M; Johnson, M P; Evans, M I

1999-10-01

We evaluated the value of all 3 common biochemical serum markers, maternal serum alpha-fetoprotein, beta-human chorionic gonadotropin, and unconjugated estriol, and combinations thereof as predictors of pregnancy outcome. A total of 60,040 patients underwent maternal serum screening. All patients had maternal serum alpha-fetoprotein measurements; beta-human chorionic gonadotropin was measured in 45,565 patients, and 24,504 patients had determination of all 3 markers, including unconjugated estriol. The incidences of various pregnancy outcomes were evaluated according to the serum marker levels by using clinically applied cutoff points. In confirmation of previous observations, increased maternal serum alpha-fetoprotein levels (>2.5 multiples of the median) were found to be significantly associated with pregnancy-induced hypertension, miscarriage, preterm delivery, intrauterine growth restriction, intrauterine fetal death, oligohydramnios, and abruptio placentae. Increased beta-human chorionic gonadotropin levels (>2.5 multiples of the median [MoM]) were significantly associated with pregnancy-induced hypertension, miscarriage, preterm delivery, and intrauterine fetal death. Finally, decreased unconjugated estriol levels (<0.5 MoM) were found to be significantly associated with pregnancy-induced hypertension, miscarriage, intrauterine growth restriction, and intrauterine fetal death. As with increased second-trimester maternal serum alpha-fetoprotein levels, increased serum beta-human chorionic gonadotropin and low unconjugated estriol levels are significantly associated with adverse pregnancy outcomes. These are most likely attributed to placental dysfunction. Multiple-marker screening can be used not only for the detection of fetal anomalies and aneu-ploidy but also for detection of high-risk pregnancies.

Determination of guaifenesin in human serum by capillary gas chromatography and electron capture detection.

PubMed

Sharaf, Maged H M; Stiff, Dwight D

2004-06-29

A method for the quantitation of guaifenesin in human serum has been developed and validated. The procedure involves liquid-liquid extraction of the serum sample in the presence of mephenesin as an internal standard, followed by derivatization and analysis using capillary gas chromatography (GC) and electron capture detection (ECD). Different solvents were tested for extraction of guaifenesin from serum. n-Hexane/dichloromethane (1:1, v/v) gave the highest recovery and the lowest background and was chosen as the extraction solvent. After extraction, the residue of guaifenesin was derivatized at 60 degrees C for 30 min, with trifluoroacetic acid anhydride (TFAA) in toluene in the presence of pyridine. Excess trifluoroacetic acid anhydride was removed using dilute solution of ammonium hydroxide. The method proved to be linear over the range of 25.0-1000 ng/ml. Recovery of guaifenesin from spiked samples was consistent, averaging 75.5% at 50.0 ng/ml with a range of 72.0-80.0% (N = 8 determinations) and averaging 78% at 800 ng/ml with a range of 76.0-81.0% (N = 8 determinations). The internal standard recovery was also consistent averaging 72.8% with a range of 67.0-76.0% (N = 16 determinations). Copyright 2004 Elsevier B.V.

Indirect electrochemical detection for total bile acids in human serum.

PubMed

Zhang, Xiaoqing; Zhu, Mingsong; Xu, Biao; Cui, Yue; Tian, Gang; Shi, Zhenghu; Ding, Min

2016-11-15

Bile acids level in serum is a useful index for screening and diagnosis of hepatobiliary diseases. As bile acids concentration is closely related to the degree of hepatobiliary diseases, detecting it is a vital factor to understand the stage of the diseases. The prevalent determination for bile acids is the enzymatic cycling method which has low sensitivity while reagent-consuming. It is desirable to develop a new method with lower cost and higher sensitivity. An indirect electrochemical detection (IED) for bile acids in human serum was established using the screen printed carbon electrode (SPCE). Since bile acids do not show electrochemical signals, they were converted to 3-ketosteroids by 3-α-hydroxysteroid dehydrogenase (3α-HSD) in the presence of nicotinamide adenine dinucleotide (NAD(+)), which was reduced to NADH. NADH could then be oxidized on the surface of SPCE, generating a signal that was used to calculate the total bile acids (TBA) concentration. A good linear calibration for TBA was obtained at the concentration range from 5.00μM to 400μM in human serum. Both the precisions and recoveries were sufficient to be used in a clinical setting. The TBA concentrations in 35 human serum samples by our IED method didn't show significant difference with the result by enzymatic cycling method, using the paired t-test. Moreover, our IED method is reagent-saving, sensitive and cost-effective. Copyright © 2016. Published by Elsevier B.V.

Extending the half-life of a fab fragment through generation of a humanized anti-human serum albumin Fv domain: An investigation into the correlation between affinity and serum half-life.

PubMed

Adams, Ralph; Griffin, Laura; Compson, Joanne E; Jairaj, Mark; Baker, Terry; Ceska, Tom; West, Shauna; Zaccheo, Oliver; Davé, Emma; Lawson, Alastair Dg; Humphreys, David P; Heywood, Sam

2016-10-01

We generated an anti-albumin antibody, CA645, to link its Fv domain to an antigen-binding fragment (Fab), thereby extending the serum half-life of the Fab. CA645 was demonstrated to bind human, cynomolgus, and mouse serum albumin with similar affinity (1-7 nM), and to bind human serum albumin (HSA) when it is in complex with common known ligands. Importantly for half-life extension, CA645 binds HSA with similar affinity within the physiologically relevant range of pH 5.0 - pH 7.4, and does not have a deleterious effect on the binding of HSA to neonatal Fc receptor (FcRn). A crystal structure of humanized CA645 Fab in complex with HSA was solved and showed that CA645 Fab binds to domain II of HSA. Superimposition with the crystal structure of FcRn bound to HSA confirmed that CA645 does not block HSA binding to FcRn. In mice, the serum half-life of humanized CA645 Fab is 84.2 h. This is a significant extension in comparison with < 1 h for a non-HSA binding CA645 Fab variant. The Fab-HSA structure was used to design a series of mutants with reduced affinity to investigate the correlation between the affinity for albumin and serum half-life. Reduction in the affinity for MSA by 144-fold from 2.2 nM to 316 nM had no effect on serum half-life. Strikingly, despite a reduction in affinity to 62 µM, an extension in serum half-life of 26.4 h was still obtained. CA645 Fab and the CA645 Fab-HSA complex have been deposited in the Protein Data Bank (PDB) with accession codes, 5FUZ and 5FUO, respectively.

Multiplexed Activity-based Protein Profiling of the Human Pathogen Aspergillus fumigatus Reveals Large Functional Changes upon Exposure to Human Serum*

PubMed Central

Wiedner, Susan D.; Burnum, Kristin E.; Pederson, LeeAnna M.; Anderson, Lindsey N.; Fortuin, Suereta; Chauvigné-Hines, Lacie M.; Shukla, Anil K.; Ansong, Charles; Panisko, Ellen A.; Smith, Richard D.; Wright, Aaron T.

2012-01-01

Environmental adaptability is critical for survival of the fungal human pathogen Aspergillus fumigatus in the immunocompromised host lung. We hypothesized that exposure of the fungal pathogen to human serum would lead to significant alterations to the organism's physiology, including metabolic activity and stress response. Shifts in functional pathway and corresponding enzyme reactivity of A. fumigatus upon exposure to the human host may represent much needed prognostic indicators of fungal infection. To address this, we employed a multiplexed activity-based protein profiling (ABPP) approach coupled to quantitative mass spectrometry-based proteomics to measure broad enzyme reactivity of the fungus cultured with and without human serum. ABPP showed a shift from aerobic respiration to ethanol fermentation and utilization over time in the presence of human serum, which was not observed in serum-free culture. Our approach provides direct insight into this pathogen's ability to survive, adapt, and proliferate. Additionally, our multiplexed ABPP approach captured a broad swath of enzyme reactivity and functional pathways and provides a method for rapid assessment of the A. fumigatus response to external stimuli. PMID:22865858

Multiplexed activity-based protein profiling of the human pathogen Aspergillus fumigatus reveals large functional changes upon exposure to human serum.

PubMed

Wiedner, Susan D; Burnum, Kristin E; Pederson, LeeAnna M; Anderson, Lindsey N; Fortuin, Suereta; Chauvigné-Hines, Lacie M; Shukla, Anil K; Ansong, Charles; Panisko, Ellen A; Smith, Richard D; Wright, Aaron T

2012-09-28

Environmental adaptability is critical for survival of the fungal human pathogen Aspergillus fumigatus in the immunocompromised host lung. We hypothesized that exposure of the fungal pathogen to human serum would lead to significant alterations to the organism's physiology, including metabolic activity and stress response. Shifts in functional pathway and corresponding enzyme reactivity of A. fumigatus upon exposure to the human host may represent much needed prognostic indicators of fungal infection. To address this, we employed a multiplexed activity-based protein profiling (ABPP) approach coupled to quantitative mass spectrometry-based proteomics to measure broad enzyme reactivity of the fungus cultured with and without human serum. ABPP showed a shift from aerobic respiration to ethanol fermentation and utilization over time in the presence of human serum, which was not observed in serum-free culture. Our approach provides direct insight into this pathogen's ability to survive, adapt, and proliferate. Additionally, our multiplexed ABPP approach captured a broad swath of enzyme reactivity and functional pathways and provides a method for rapid assessment of the A. fumigatus response to external stimuli.

99M-technetium labeled macroaggregated human serum albumin pharmaceutical

DOEpatents

Winchell, Harry S.; Barak, Morton; Van Fleet, III, Parmer

1977-05-17

A reagent comprising macroaggregated human serum albumin having dispersed therein particles of stannous tin and a method for instantly making a labeled pharmaceutical therefrom, are disclosed. The labeled pharmaceutical is utilized in organ imaging.

Hexons from adenovirus serotypes 5 and 48 differentially protect adenovirus vectors from neutralization by mouse and human serum

PubMed Central

Harmon, Andrew W.; Moitra, Rituparna; Xu, Zhili

2018-01-01

Adenovirus vectors are widely used in gene therapy clinical trials, and preclinical studies with these vectors are often conducted in mice. It is therefore critical to understand whether mouse studies adequately predict the behavior of adenovirus vectors in humans. The most commonly-used adenovirus vectors are derived from adenovirus serotype 5 (Ad5). The Ad5 hexon protein can bind coagulation factor X (FX), and binding of FX has a major impact on vector interactions with other blood proteins. In mouse serum, FX protects Ad5 vectors from neutralization by natural antibodies and complement. In the current study, we similarly find that human FX inhibits neutralization of Ad5 vectors by human serum, and this finding is consistent among individual human sera. We show that human IgM and human IgG can each induce complement-mediated neutralization when Ad5 vectors are not protected by FX. Although mouse and human serum had similar effects on Ad5 vectors, we found that this was not true for a chimeric Ad5 vector that incorporated hexon regions from adenovirus serotype 48. Interestingly, this hexon-chimeric vector was neutralized by human serum, but not by mouse serum. These findings indicate that studies in mouse serum accurately predict the behavior of Ad5 vectors in human serum, but mouse serum is not an accurate model system for all adenovirus vectors. PMID:29401488

Lipidomics of human umbilical cord serum: identification of unique sterol sulfates.

PubMed

Wood, Paul L; Siljander, Heli; Knip, Mikael

2017-08-01

There are currently limited lipidomics data for human umbilical cord blood. Therefore, the lipidomes of cord sera from six newborns and sera from six nonpregnant females were compared. Sera lipidomics analyses were conducted using a high-resolution mass spectrometry analytical platform. Cord serum contained a diverse array of glycerophospholipids, albeit generally at lower concentrations than monitored in adult serum. The unexpected observations were that cord serum contained several neurosteroid sulfates and bile acid sulfates that were not detectable in adult serum. Our data are the first to demonstrate that cord serum contains bile acid sulfates that are synthesized early in the hydroxylase, neutral and acidic pathways of primary bile acid biosynthesis and support previous publications of cord blood perfluoralkyl toxins in newborns.

Polymerized soluble venom--human serum albumin

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

Patterson, R.; Suszko, I.M.; Grammer, L.C.

Extensive previous studies have demonstrated that attempts to produce polymers of Hymenoptera venoms for human immunotherapy resulted in insoluble precipitates that could be injected with safety but with very limited immunogenicity in allergic patients. We now report soluble polymers prepared by conjugating bee venom with human serum albumin with glutaraldehyde. The bee venom-albumin polymer (BVAP) preparation was fractionated on Sephacryl S-300 to have a molecular weight range higher than catalase. /sup 125/I-labeled bee venom phospholipase A was almost completely incorporated into BVAP. Rabbit antibody responses to bee venom and bee venom phospholipase A were induced by BVAP. Human antisera againstmore » bee venom were absorbed by BVAP. No new antigenic determinants on BVAP were present as evidenced by absorption of antisera against BVAP by bee venom and albumin. BVAP has potential immunotherapeutic value in patients with anaphylactic sensitivity to bee venom.« less

Square Wave Voltammetric Determination of Diclofenac in Pharmaceutical Preparations and Human Serum

PubMed Central

Ciltas, Ulvihan; Yilmaz, Bilal; Kaban, Selcuk; Akcay, Bilge Kaan; Nazik, Gulsah

2015-01-01

In this study, a simple and reliable square wave voltammetric (SWV) method was developed and validated for determination of diclofenac in pharmaceutical preparations and human serum. The proposed method was based on electrooxidation of diclofenac at platinum electrode in 0.1 M TBAClO4/acetonitrile solution. The well-defined two oxidation peaks were observed at 0.87 and 1.27 V, respectively. Calibration curves that were obtained by using current values measured for second peak were linear over the concentration range of 1.5-17.5 μg mL-1 and 2-20 μg mL-1 in supporting electrolyte and serum, respectively. Precision and accuracy were also checked in all media. Intra- and inter-day precision values for diclofenac were less than 3.64, and accuracy (relative error) was better than 2.49%. Developed method in this study is accurate, precise and can be easily applied to Diclomec, Dicloflam and Voltaren tablets as pharmaceutical preparation. Also, the proposed technique was successfully applied to spiked human serum samples. No electroactive interferences from the endogenous substances were found in human serum. PMID:26330859

Square Wave Voltammetric Determination of Diclofenac in Pharmaceutical Preparations and Human Serum.

PubMed

Ciltas, Ulvihan; Yilmaz, Bilal; Kaban, Selcuk; Akcay, Bilge Kaan; Nazik, Gulsah

2015-01-01

In this study, a simple and reliable square wave voltammetric (SWV) method was developed and validated for determination of diclofenac in pharmaceutical preparations and human serum. The proposed method was based on electrooxidation of diclofenac at platinum electrode in 0.1 M TBAClO4/acetonitrile solution. The well-defined two oxidation peaks were observed at 0.87 and 1.27 V, respectively. Calibration curves that were obtained by using current values measured for second peak were linear over the concentration range of 1.5-17.5 μg mL(-1) and 2-20 μg mL(-1) in supporting electrolyte and serum, respectively. Precision and accuracy were also checked in all media. Intra- and inter-day precision values for diclofenac were less than 3.64, and accuracy (relative error) was better than 2.49%. Developed method in this study is accurate, precise and can be easily applied to Diclomec, Dicloflam and Voltaren tablets as pharmaceutical preparation. Also, the proposed technique was successfully applied to spiked human serum samples. No electroactive interferences from the endogenous substances were found in human serum.

Mangifera indica L. (Vimang) protection against serum oxidative stress in elderly humans.

PubMed

Pardo-Andreu, Gilberto L; Philip, Sarah J; Riaño, Annia; Sánchez, Carlos; Viada, Carmen; Núñez-Sellés, Alberto J; Delgado, René

2006-01-01

We searched for the protective effect of a natural extract from stem bark of Mangifera indica L. extract (Vimang) on age-related oxidative stress. Healthy subjects were classified in two groups, elderly (>65 years) and young group (<26 years). The elderly group received a daily dose of 900 mg of extract (three coated Vimang tablets, 300 mg each, before meals) for 60 days. Serum concentration of lipid peroxides, serum peroxidation potential, extracellular superoxide dismutase activity (EC-SOD), glutathione status (GSH, GSSG, GSSG/GSH ratio)) and total antioxidant status (TAS) were determined before (both experimental groups) and 15, 30, and 60 days after treatment (only elderly group). We confirmed the existence of an age-associated oxidative stress in human serum as documented by an age-related increase in serum lipoperoxides and GSSG and a decrease in serum antioxidant capacity and EC-SOD activity. Vimang tablet supplementation increased EC-SOD activity (p <0.01) and serum TAS (p <0.01). It also decreased serum thiobarbituric reactive substances (p <0.01) and GSSG levels (p <0.05). We suggested that the antioxidant components of the extract could have been utilized by the cells (especially blood and endothelial cells), sparing the intra- and extracellular antioxidant system and increasing serum peroxil scavenging capacity, thus preventing age-associated increase in GSH oxidation and lipoperoxidation. Vimang tablets prevent age-associated oxidative stress in elderly humans, which could retard the onset of age-associated disease, improving the quality of life for elderly persons.

Methodology and Applications of Disease Biomarker Identification in Human Serum

PubMed Central

Sahab, Ziad J.; Semaan, Suzan M.; Sang, Qing-Xiang Amy

2007-01-01

Biomarkers are biomolecules that serve as indicators of biological and pathological processes, or physiological and pharmacological responses to a drug treatment. Because of the high abundance of albumin and heterogeneity of plasma lipoproteins and glycoproteins, biomarkers are difficult to identify in human serum. Due to the clinical significance the identification of disease biomarkers in serum holds great promise for personalized medicine, especially for disease diagnosis and prognosis. This review summarizes some common and emerging proteomics techniques utilized in the separation of serum samples and identification of disease signatures. The practical application of each protein separation or identification technique is analyzed using specific examples. Biomarkers of cancers of prostate, breast, ovary, and lung in human serum have been reviewed, as well as those of heart disease, arthritis, asthma, and cystic fibrosis. Despite the advancement of technology few biomarkers have been approved by the Food and Drug Administration for disease diagnosis and prognosis due to the complexity of structure and function of protein biomarkers and lack of high sensitivity, specificity, and reproducibility for those putative biomarkers. The combination of different types of technologies and statistical analysis may provide more effective methods to identify and validate new disease biomarkers in blood. PMID:19662190

Development of certified reference materials for electrolytes in human serum (GBW09124-09126).

PubMed

Feng, Liuxing; Wang, Jun; Cui, Yanjie; Shi, Naijie; Li, Haifeng; Li, Hongmei

2017-05-01

Three reference materials, at relatively low, middle, and high concentrations, were developed for analysis of the mass fractions of electrolytes (K, Ca, Na, Mg, Cl, and Li) in human serum. The reference materials were prepared by adding high purity chloride salts to normal human serum. The concentration range of the three levels is within ±20% of normal human serum. It was shown that 14 units with duplicate analysis is enough to demonstrate the homogeneity of these candidate reference materials. The statistical results also showed no significant trends in both short-term stability test for 1 week at 40 °C and long-term stability test for 14 months. The certification methods of the six elements include isotope dilution inductively coupled plasma mass spectrometry (ID-ICP-MS), inductively coupled plasma optical emission spectroscopy (ICP-OES), atomic absorption spectroscopy (AAS), ion chromatography (IC), and ion-selective electrode (ISE). The certification methods were validated by international comparisons among a number of national metrology institutes (NMIs). The combined relative standard uncertainties of the property values were estimated by considering the uncertainties of the analytical methods, homogeneity, and stability. The range of the expanded uncertainties of all the elements is from 2.2% to 3.9%. The certified reference materials (CRMs) are primarily intended for use in the calibration and validation of procedures in clinical analysis for the determination of electrolytes in human serum or plasma. Graphical Abstract Certified reference materials for K, Ca, Mg, Na, Cl and Li in human serum (GBW09124-09126).

Elevated serum level of human alkaline phosphatase in obesity.

PubMed

Khan, Abdul Rehman; Awan, Fazli Rabbi; Najam, Syeda Sadia; Islam, Mehboob; Siddique, Tehmina; Zain, Maryam

2015-11-01

To investigate a correlation between serum alkaline phosphatase level and body mass index in human subjects. The comparative cross-sectional study was carried out at the National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan, from April 2012 to June 2013. Blood serum alkaline phosphatase levels were estimated and the subjects were divided into three sub-groups on the basis of their body mass. normal weight (<25kg/m2), overweight (25-27kg/m2) and obese (>27kg/m2) subjects. The serum samples were used for the estimation of clinically important biochemical parameters, using commercial kits on clinical chemistry analyser. Of the 197 subjects, 97(49%) were obese and 100(51%) were non-obese. The serum alkaline phosphatase level increased in obese (214±6.4 IU/L) compared to the non-obese subjects (184.5±5 IU/L). Furthermore, a significant linear relationship (r=0.3;p-0.0001) was found between serum alkaline phosphatase and body mass index. Other biochemical variables were not correlated to the body mass index. Over activity and higher amounts of alkaline phosphatase were linked to the development of obesity.

The development of retrosynthetic glycan libraries to profile and classify the human serum N-linked glycome.

PubMed

Kronewitter, Scott R; An, Hyun Joo; de Leoz, Maria Lorna; Lebrilla, Carlito B; Miyamoto, Suzanne; Leiserowitz, Gary S

2009-06-01

Annotation of the human serum N-linked glycome is a formidable challenge but is necessary for disease marker discovery. A new theoretical glycan library was constructed and proposed to provide all possible glycan compositions in serum. It was developed based on established glycobiology and retrosynthetic state-transition networks. We find that at least 331 compositions are possible in the serum N-linked glycome. By pairing the theoretical glycan mass library with a high mass accuracy and high-resolution MS, human serum glycans were effectively profiled. Correct isotopic envelope deconvolution to monoisotopic masses and the high mass accuracy instruments drastically reduced the amount of false composition assignments. The high throughput capacity enabled by this library permitted the rapid glycan profiling of large control populations. With the use of the library, a human serum glycan mass profile was developed from 46 healthy individuals. This paper presents a theoretical N-linked glycan mass library that was used for accurate high-throughput human serum glycan profiling. Rapid methods for evaluating a patient's glycome are instrumental for studying glycan-based markers.

Acoustic analysis of the composition of human blood serum

NASA Astrophysics Data System (ADS)

Gurbatov, S. N.; Demin, I. Yu.; Klemina, A. V.; Klemin, V. A.

2009-10-01

New acoustic methods of determining total protein, protein fractions, and lipid components of the human blood serum are presented. Acoustic methods are based on high-precision measurements of velocity and temperature dependences and frequency and temperature dependences of ultrasound absorption. Acoustic characteristics of the blood serum were measured using the method of a fixed length interferometer in acoustic cells ˜80 mcl in volume in the temperature range from 15 to 40°C and the 4-9 MHz frequency range with the acoustic analyzer developed by BIOM company. An error in measuring ultrasound velocity in the blood serum was 3 × 10-5; that of absorption, 2 × 10-2. The developed acoustic methods were clinically tested and recommended for application at clinical diagnostic laboratories with RF treatment-and-prophylactics establishments.

Proteolysis inhibition by hibernating bear serum leads to increased protein content in human muscle cells.

PubMed

Chanon, Stéphanie; Chazarin, Blandine; Toubhans, Benoit; Durand, Christine; Chery, Isabelle; Robert, Maud; Vieille-Marchiset, Aurélie; Swenson, Jon E; Zedrosser, Andreas; Evans, Alina L; Brunberg, Sven; Arnemo, Jon M; Gauquelin-Koch, Guillemette; Storey, Kenneth B; Simon, Chantal; Blanc, Stéphane; Bertile, Fabrice; Lefai, Etienne

2018-04-03

Muscle atrophy is one of the main characteristics of human ageing and physical inactivity, with resulting adverse health outcomes. To date, there are still no efficient therapeutic strategies for its prevention and/or treatment. However, during hibernation, bears exhibit a unique ability for preserving muscle in conditions where muscle atrophy would be expected in humans. Therefore, our objective was to determine whether there are components of bear serum which can control protein balance in human muscles. In this study, we exposed cultured human differentiated muscle cells to bear serum collected during winter and summer periods, and measured the impact on cell protein content and turnover. In addition, we explored the signalling pathways that control rates of protein synthesis and degradation. We show that the protein turnover of human myotubes is reduced when incubated with winter bear serum, with a dramatic inhibition of proteolysis involving both proteasomal and lysosomal systems, and resulting in an increase in muscle cell protein content. By modulating intracellular signalling pathways and inducing a protein sparing phenotype in human muscle cells, winter bear serum therefore holds potential for developing new tools to fight human muscle atrophy and related metabolic disorders.

Degalactosylated/desialylated human serum containing GcMAF induces macrophage phagocytic activity and in vivo antitumor activity.

PubMed

Kuchiike, Daisuke; Uto, Yoshihiro; Mukai, Hirotaka; Ishiyama, Noriko; Abe, Chiaki; Tanaka, Daichi; Kawai, Tomohito; Kubo, Kentaro; Mette, Martin; Inui, Toshio; Endo, Yoshio; Hori, Hitoshi

2013-07-01

The group-specific component protein-derived macrophage-activating factor (GcMAF) has various biological activities, such as macrophage activation and antitumor activity. Clinical trials of GcMAF have been carried out for metastatic breast cancer, prostate cancer, and metastatic colorectal cancer. In this study, despite the complicated purification process of GcMAF, we used enzymatically-treated human serum containing GcMAF with a considerable macrophage-stimulating activity and antitumor activity. We detected GcMAF in degalactosylated/desialylated human serum by western blotting using an anti-human Gc globulin antibody, and Helix pomatia agglutinin lectin. We also found that GcMAF-containing human serum significantly enhanced the phagocytic activity of mouse peritoneal macrophages and extended the survival time of mice bearing Ehrlich ascites tumors. We demonstrated that GcMAF-containing human serum can be used as a potential macrophage activator for cancer immunotherapy.

Experimental Study on the Efficacy of Site-Specific PEGylated Human Serum Albumins in Resuscitation From Hemorrhagic Shock.

PubMed

Song, Xinlei; Zhang, Shu; Cheng, Yanna; Zhao, Ting; Lian, Qianqian; Lu, Lu; Wang, Fengshan

2016-11-01

To evaluate the resuscitative efficacy and the effect on reperfusion injury of two site-specific PEGylated human serum albumins modified with linear or branched PEG20kDa, compared with saline, 8% human serum albumin and 25% human serum albumin, in a hemorrhagic shock model. Laboratory. Male Wistar rats. Prospective study. Rats were bled to hemorrhagic hypovolemic shock and resuscitated with different resuscitation fluids. The mean arterial pressure and blood gas variables were measured. Hemorheology analysis was performed to evaluate the influence of resuscitation on RBCs and blood viscosity. The microvascular state was indirectly characterized in terms of monocyte chemotactic protein-1 and endothelial nitric oxide synthase that related to shear stress and vasodilation, respectively. The levels of inflammation-related factors and apoptosis-related proteins were used to evaluate the reperfusion injury in lungs. The results showed that PEGylated human serum albumin could improve the level of mean arterial pressure and blood gas variables more effectively at the end of resuscitation. poly(ethylene glycol) modification was able to increase the viscosity of human serum albumin to the level of effectively enhancing the expression of monocyte chemotactic protein-1 and endothelial nitric oxide synthase, which could promote microvascular perfusion. The hyperosmotic resuscitative agents including both 25% human serum albumin and PEGylated human serum albumins could greatly attenuate lung injury. No significant therapeutic advantages but some disadvantages were found for Y shaped poly(ethylene glycol) modification over linear poly(ethylene glycol) modification, such as causing the decrease of erythrocyte deformability. Linear high molecular weight site-specific PEGylated human serum albumin is recommended to be used as a hyperosmotic resuscitative agent.

Circulating human papillomavirus DNA detected using droplet digital PCR in the serum of patients diagnosed with early stage human papillomavirus-associated invasive carcinoma.

PubMed

Jeannot, Emmanuelle; Becette, Véronique; Campitelli, Maura; Calméjane, Marie-Ange; Lappartient, Emmanuelle; Ruff, Evelyne; Saada, Stéphanie; Holmes, Allyson; Bellet, Dominique; Sastre-Garau, Xavier

2016-10-01

Specific human papillomavirus genotypes are associated with most ano-genital carcinomas and a large subset of oro-pharyngeal carcinomas. Human papillomavirus DNA is thus a tumour marker that can be detected in the blood of patients for clinical monitoring. However, data concerning circulating human papillomavirus DNA in cervical cancer patients has provided little clinical value, due to insufficient sensitivity of the assays used for the detection of small sized tumours. Here we took advantage of the sensitive droplet digital PCR method to identify circulating human papillomavirus DNA in patients with human papillomavirus-associated carcinomas. A series of 70 serum specimens, taken at the time of diagnosis, between 2002 and 2013, were retrospectively analyzed in patients with human papillomavirus-16 or human papillomavirus-18-associated carcinomas, composed of 47 cases from the uterine cervix, 15 from the anal canal and 8 from the oro-pharynx. As negative controls, 18 serum samples from women with human papillomavirus-16-associated high-grade cervical intraepithelial neoplasia were also analyzed. Serum samples were stored at -80°C (27 cases) or at -20°C (43 cases). DNA was isolated from 200 µl of serum or plasma and droplet digital PCR was performed using human papillomavirus-16 E7 and human papillomavirus-18 E7 specific primers. Circulating human papillomavirus DNA was detected in 61/70 (87%) serum samples from patients with carcinoma and in no serum from patients with cervical intraepithelial neoplasia. The positivity rate increased to 93% when using only serum stored at -80°C. Importantly, the two patients with microinvasive carcinomas in this series were positive. Quantitative evaluation showed that circulating viral DNA levels in cervical cancer patients were related to the clinical stage and tumour size, ranging from 55 ± 85 copies/ml (stage I) to 1774 ± 3676 copies/ml (stage IV). Circulating human papillomavirus DNA is present in patients with

Circulating human papillomavirus DNA detected using droplet digital PCR in the serum of patients diagnosed with early stage human papillomavirus‐associated invasive carcinoma

PubMed Central

Jeannot, Emmanuelle; Becette, Véronique; Campitelli, Maura; Calméjane, Marie‐Ange; Lappartient, Emmanuelle; Ruff, Evelyne; Saada, Stéphanie; Holmes, Allyson; Bellet, Dominique

2016-01-01

Abstract Specific human papillomavirus genotypes are associated with most ano‐genital carcinomas and a large subset of oro‐pharyngeal carcinomas. Human papillomavirus DNA is thus a tumour marker that can be detected in the blood of patients for clinical monitoring. However, data concerning circulating human papillomavirus DNA in cervical cancer patients has provided little clinical value, due to insufficient sensitivity of the assays used for the detection of small sized tumours. Here we took advantage of the sensitive droplet digital PCR method to identify circulating human papillomavirus DNA in patients with human papillomavirus‐associated carcinomas. A series of 70 serum specimens, taken at the time of diagnosis, between 2002 and 2013, were retrospectively analyzed in patients with human papillomavirus‐16 or human papillomavirus‐18‐associated carcinomas, composed of 47 cases from the uterine cervix, 15 from the anal canal and 8 from the oro‐pharynx. As negative controls, 18 serum samples from women with human papillomavirus‐16‐associated high‐grade cervical intraepithelial neoplasia were also analyzed. Serum samples were stored at −80°C (27 cases) or at −20°C (43 cases). DNA was isolated from 200 µl of serum or plasma and droplet digital PCR was performed using human papillomavirus‐16 E7 and human papillomavirus‐18 E7 specific primers. Circulating human papillomavirus DNA was detected in 61/70 (87%) serum samples from patients with carcinoma and in no serum from patients with cervical intraepithelial neoplasia. The positivity rate increased to 93% when using only serum stored at −80°C. Importantly, the two patients with microinvasive carcinomas in this series were positive. Quantitative evaluation showed that circulating viral DNA levels in cervical cancer patients were related to the clinical stage and tumour size, ranging from 55 ± 85 copies/ml (stage I) to 1774 ± 3676 copies/ml (stage IV). Circulating human

Serum or breast milk immunoglobulins mask the self-reactivity of human natural IgG antibodies.

PubMed

Djoumerska-Alexieva, Iglika; Manoylov, Iliyan; Dimitrov, Jordan D; Tchorbanov, Andrey

2014-04-01

B cells producing IgG antibodies specific to a variety of self- or foreign antigens are a normal constituent of the immune system of all healthy individuals. These naturally occurring IgG antibodies are found in the serum, external secretions, and pooled human immunoglobulin preparations. They bind with low affinity to antigens, which can also be targets for pathologic autoantibodies. An enhancement of naturally occurring IgG autoantibody activity was observed after treatment of human IgG molecules with protein-destabilizing agents. We have investigated the interactions of human immunoglobulins that were obtained from serum or from breast milk of healthy individuals or IVIg with human liver antigens. Proteins from an individual serum or milk were isolated by two methods, one of which included exposure to low pH and the other did not. Purified serum, mucosal IgM, IgA, and the fraction containing immunoglobulin G F(ab')2 fragments each inhibited the binding of a single donor or pooled IgG to human liver antigens. Our study presents findings regarding the role of the breast milk or serum antibodies in blocking the self-reactivity of IgG antibodies. It supports the suggestion that not IVIg only, but also the pooled human IgM and IgA might possess a potent beneficial immunomodulatory activity in autoimmune patients. © 2013 APMIS. Published by John Wiley & Sons Ltd.

Determination of element levels in human serum: Total reflection X-ray fluorescence applications

NASA Astrophysics Data System (ADS)

Majewska, U.; Łyżwa, P.; Łyżwa, K.; Banaś, D.; Kubala-Kukuś, A.; Wudarczyk-Moćko, J.; Stabrawa, I.; Braziewicz, J.; Pajek, M.; Antczak, G.; Borkowska, B.; Góźdź, S.

2016-08-01

Deficiency or excess of elements could disrupt proper functioning of the human body and could lead to several disorders. Determination of their concentrations in different biological human fluids and tissues should become a routine practice in medical treatment. Therefore the knowledge about appropriate element concentrations in human organism is required. The purpose of this study was to determine the concentration of several elements (P, S, Cl, K, Ca, Cr, Fe, Cu, Zn, Se, Br, Rb, Pb) in human serum and to define the reference values of element concentration. Samples of serum were obtained from 105 normal presumably healthy volunteers (66 women aged between 15 and 78 years old; 39 men aged between 15 and 77 years old). Analysis has been done for the whole studied population and for subgroups by sex and age. It is probably first so a wide study of elemental composition of serum performed in the case of Świętokrzyskie region. Total reflection X-ray fluorescence (TXRF) method was used to perform the elemental analysis. Spectrometer S2 Picofox (Bruker AXS Microanalysis GmbH) was used to identify and measure elemental composition of serum samples. Finally, 1st and 3rd quartiles were accepted as minimum and maximum values of concentration reference range.

Exosome enrichment of human serum using multiple cycles of centrifugation.

PubMed

Kim, Jeongkwon; Tan, Zhijing; Lubman, David M

2015-09-01

In this work, we compared the use of repeated cycles of centrifugation at conventional speeds for enrichment of exosomes from human serum compared to the use of ultracentrifugation (UC). After removal of cells and cell debris, a speed of 110 000 × g or 40 000 × g was used for the UC or centrifugation enrichment process, respectively. The enriched exosomes were analyzed using the bicinchoninic acid assay, 1D gel separation, transmission electron microscopy, Western blotting, and high-resolution LC-MS/MS analysis. It was found that a five-cycle repetition of UC or centrifugation is necessary for successful removal of nonexosomal proteins in the enrichment of exosomes from human serum. More significantly, 5× centrifugation enrichment was found to provide similar or better performance than 5× UC enrichment in terms of enriched exosome protein amount, Western blot band intensity for detection of CD-63, and numbers of identified exosome-related proteins and cluster of differentiation (CD) proteins. A total of 478 proteins were identified in the LC-MS/MS analyses of exosome proteins obtained from 5× UCs and 5× centrifugations including many important CD membrane proteins. The presence of previously reported exosome-related proteins including key exosome protein markers demonstrates the utility of this method for analysis of proteins in human serum. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Probing thyroglobulin in undiluted human serum based on pattern recognition and competitive adsorption of proteins

NASA Astrophysics Data System (ADS)

Wang, Ran; Huang, Shuai; Li, Jing; Chae, Junseok

2014-10-01

Thyroglobulin (Tg) is a sensitive indicator of persistent or recurrent differentiated thyroid cancer of follicular cell origin. Detection of Tg in human serum is challenging as bio-receptors, such as anti-Tg, used in immunoassay have relatively weak binding affinity. We engineer sensing surfaces using the competitive adsorption of proteins, termed the Vroman Effect. Coupled with Surface Plasmon Resonance, the "cross-responsive" interactions of Tg on the engineered surfaces produce uniquely distinguishable multiple signature patterns, which are discriminated using Linear Discriminant Analysis. Tg-spiked samples, down to 2 ng/ml Tg in undiluted human serum, are sensitively and selectively discriminated from the control (undiluted human serum).

CCQM-K11.2 determination of glucose in human serum and CCQM-K12.2 determination of creatinine in human serum

NASA Astrophysics Data System (ADS)

Wise, Stephen A.; Phinney, Karen W.; Duewer, David L.; Sniegoski, Lorna T.; Welch, Michael J.; Pritchett, Jeanita; Pabello, Guiomar; Avila Calderon, Marco A.; Balderas, Miryan; Qinde, Liu; Kooi, Lee Tong; Rego, Eliane; Garrido, Bruno; Allegri, Gabriella; de La Cruz, Marcia; Barrabin, Juliana; Monteiro, Tânia; Lee, Hwashim; Kim, Byungjoo; Delatour, Vincent; Peignaux, Maryline; Kawaguchi, Migaku; Bei, Xu; Can, Quan; Nammoonnoy, Jintana; Schild, Katrin; Ohlendorf, Rüdiger; Henrion, Andre; Ceyhan Gören, Ahmet; Yılmaz, Hasibe; Bilsel, Mine; Konopelko, L.; Krylov, A.; Lopushanskaya, E.

2018-01-01

Glucose and creatinine are two of the most frequently measured substances in human blood/serum for assessing the health status of individuals. Because of their clinical significance, CCQM-K11 glucose in human serum and CCQM-K12 creatinine in human serum were the fourth and fifth key comparisons (KCs) performed by the Organic Analysis Working Group (OAWG). These KCs were conducted in parallel and were completed in 2001. The initial subsequent KCs for glucose, CCQM-K11.1, and creatinine, CCQM-K12.1, were completed in 2005. Measurements for the next KCs for these two measurands, CCQM-K11.2 and CCQM-K12.2, were completed in 2013. While designed as subsequent KCs, systematic discordances between the participants' and the anchor institution's results in both comparisons lead the OAWG to request reference results from two experienced laboratories that had participated in the 2001 comparisons. Based on the totality of the available information, the OAWG converted both CCQM-K11.2 and CCQM-K12.2 to 'Track C' KCs where the key comparison reference value is estimated by consensus. These comparisons highlighted that carrying out comparisons for complex chemical measurements and expecting to be able to treat them under the approaches used for formal CIPM subsequent comparisons is not an appropriate strategy. The approach used here is a compromise to gain the best value from the comparison; it is not an approach that will be used in the future. Instead, the OAWG will focus on Track A and Track C comparisons that are treated as stand-alone entities. Participation in CCQM-K11.2 demonstrates a laboratory's capabilities to measure a polar (pKow > 2), low molecular mass (100 g/mol to 500 g/mol) metabolite in human serum at relatively high concentrations (0.1 mg/g to 10 mg/g). Participation in CCQM-K12.2 demonstrates capabilities to measure similar classes of metabolites at relatively low concentrations (1 μg/g to 30 μg/g). The capabilities required for the analysis of complex

THE INACTIVATION OF PENICILLINS F, G, K, AND X BY HUMAN AND RABBIT SERUM

PubMed Central

Eagle, Harry

1947-01-01

1. Penicillins F, G, K, and X were all inactivated by human and rabbit serum, but two qualitatively distinct mechanisms were apparently involved. 2. One was a slow inactivation of all four penicillins by a relatively thermostable serum component which was not demonstrably affected by heating for 60 minutes at 56°C. (a) In both human and rabbit serum this general inactivation of penicillin behaved like a pseudo first order reaction, with a velocity constant of 0.05–0.07 for penicillin X, and 0.09–0.11 for penicillins F and G. (b) The percentage of penicillins F, G, and X inactivated per hour was independent of their concentration over the range 0.4 to 50 micrograms per cc., averaging 9.5, 10, and 6.5 per cent, respectively, in human serum, and 9,8.5, and 5 per cent in rabbit serum. (c) The rate of inactivation varied linearly with the concentration of the serum factor. (d) Penicillin X was consistently and significantly less susceptible to inactivation than any of the other penicillins. Although minor differences were observed between F and G, these were not consistent, and are of questionable significance. 3. Superimposed on this slow inactivation of penicillins F, G, K, and X by a thermostable serum component was a much faster inactivation observed only with penicillin K. (a) In both rabbit and human serum, the serum factor responsible for this inactivation was highly thermolabile, and was almost completely destroyed within 5 minutes at 56°C., leaving only a thermostable component, not affected by further heating. (b) The inactivation of K by this thermolabile component was not a first order reaction, but varied with the concentration of both serum and penicillin. At high concentrations of K, the rate of inactivation due to the thermolabile factor was negligible, and penicillin K was destroyed no more rapidly than F, G, or X. The rate of inactivation increased as the concentration of penicillin was reduced. At penicillin K concentrations of 50, 10, 2, and 0

Effects of non-enzymatic glycation in human serum albumin. Spectroscopic analysis

NASA Astrophysics Data System (ADS)

Szkudlarek, A.; Sułkowska, A.; Maciążek-Jurczyk, M.; Chudzik, M.; Równicka-Zubik, J.

2016-01-01

Human serum albumin (HSA), transporting protein, is exposed during its life to numerous factors that cause its functions become impaired. One of the basic factors - glycation of HSA - occurs in diabetes and may affect HSA-drug binding. Accumulation of advanced glycation end-products (AGEs) leads to diseases e.g. diabetic and non-diabetic cardiovascular diseases, Alzheimer disease, renal disfunction and in normal aging. The aim of the present work was to estimate how non-enzymatic glycation of human serum albumin altered its tertiary structure using fluorescence technique. We compared glycated human serum albumin by glucose (gHSAGLC) with HSA glycated by fructose (gHSAFRC). We focused on presenting the differences between gHSAFRC and nonglycated (HSA) albumin used acrylamide (Ac), potassium iodide (KI) and 2-(p-toluidino)naphthalene-6-sulfonic acid (TNS). Changes of the microenvironment around the tryptophan residue (Trp-214) of non-glycated and glycated proteins was investigated by the red-edge excitation shift method. Effect of glycation on ligand binding was examined by the binding of phenylbutazone (PHB) and ketoprofen (KP), which a primary high affinity binding site in serum albumin is subdomain IIA and IIIA, respectively. At an excitation and an emission wavelength of λex 335 nm and λem 420 nm, respectively the increase of fluorescence intensity and the blue-shift of maximum fluorescence was observed. It indicates that the glycation products decreases the polarity microenvironment around the fluorophores. Analysis of red-edge excitation shift method showed that the red-shift for gHSAFRC is higher than for HSA. Non-enzymatic glycation also caused, that the Trp residue of gHSAFRC becomes less accessible for the negatively charged quencher (I-), KSV value is smaller for gHSAFRC than for HSA. TNS fluorescent measurement demonstrated the decrease of hydrophobicity in the glycated albumin. KSV constants for gHSA-PHB systems are higher than for the unmodified serum

Alteration of human serum albumin tertiary structure induced by glycation. Spectroscopic study

NASA Astrophysics Data System (ADS)

Szkudlarek, A.; Maciążek-Jurczyk, M.; Chudzik, M.; Równicka-Zubik, J.; Sułkowska, A.

2016-01-01

The modification of human serum albumin (HSA) structure by non-enzymatic glycation is one of the underlying factors that contribute to the development of complications of diabetes and neurodegenerative diseases. The aim of the present work was to estimate how glycation of HSA altered its tertiary structure. Changes of albumin conformation were investigated by comparison of glycated (gHSA) and non-glycated human serum albumin (HSA) absorption spectra, red edge excitation shift (REES) and synchronous spectra. Effect of glycation on human serum albumin tertiary structure was also investigated by 1H NMR spectroscopy. Formation of gHSA Advanced Glycation End-products (AGEs) caused absorption of UV-VIS light between 310 nm and 400 nm while for non-glycated HSA in this region no absorbance has been registered. Analysis of red edge excitation shift effect allowed for observation of structural changes of gHSA in the hydrophobic pocket containing the tryptophanyl residue. Moreover changes in the microenvironment of tryptophanyl and tyrosyl residues brought about AGEs on the basis of synchronous fluorescence spectroscopy have been confirmed. The influence of glycation process on serum albumin binding to 5-dimethylaminonaphthalene-1-sulfonamide (DNSA), 2-(p-toluidino) naphthalene-6-sulfonic acid (TNS), has been studied. Fluorescence analysis showed that environment of both binding site I and II is modified by galactose glycation.

Determining an Optimal Cutoff of Serum β-Human Chorionic Gonadotropin for Assisting the Diagnosis of Intracranial Germinomas

PubMed Central

Zhang, Hui; Zhang, Peng; Fan, Jun; Qiu, Binghui; Pan, Jun; Zhang, Xi’an; Fang, Luxiong; Qi, Songtao

2016-01-01

Background Beta (β)-human chorionic gonadotropin (β-HCG) is used to confirm the diagnosis and plan treatment of intracranial germinomas. However, the cutoff values of serum β-HCG in diagnosis of intracranial germinomas reported in the literature are inconsistent. To establish an appropriate cutoff value of serum β-HCG for diagnosis of intracranial germinomas, we retrospectively reviewed the records of intracranial tumor patients who received serum β-HCG and α-fetoprotein (AFP) tests for diagnostic purposes at our hospital from 2005 to 2014. Methods A total of 93 intracranial germinomas and 289 intracranial non-germ cell tumors were included in this study. Receiver operating characteristic (ROC) analysis was used to evaluate the sensitivity and specificity of 3 cutoffs (0.1, 0.4, and 0.5 mIU/mL) for diagnosing intracranial germinomas. The serum β-HCG level of intracranial germinoma patients was further analyzed to investigate the effect of metastasis status and tumor location on serum β-HCG level. Results The area under the ROC curve was 0.81 (P < .001), suggesting β-HCG is an effective marker. Of the 3 cutoff values, 0.1 mIU/mL possessed a highest sensitivity (66.67%) and good specificity (91%). Although there was no β-HCG level difference between metastatic and non-metastatic intracranial germinoma patients, the diagnostic rate of metastatic neurohypophyseal germinomas was significantly higher than that of its non-metastatic counterpart (P < .05), implying that the location of the germinoma might need to be considered when β-HCG is used as a marker to predict metastasis. Conclusions Determining an optimal cutoff of serum β-HCG is helpful for assisting the diagnosis of intracranial germinoma. PMID:26771195

Resveratrol-loaded glycyrrhizic acid-conjugated human serum albumin nanoparticles wrapping resveratrol nanoparticles: Preparation, characterization, and targeting effect on liver tumors.

PubMed

Wu, Mingfang; Lian, Bolin; Deng, Yiping; Feng, Ziqi; Zhong, Chen; Wu, Weiwei; Huang, Yannian; Wang, Lingling; Zu, Chang; Zhao, Xiuhua

2017-08-01

In this study, glycyrrhizic acid-conjugated human serum albumin nanoparticles wrapping resveratrol nanoparticles were prepared to establish a tumor targeting nano-sized drug delivery system. Glycyrrhizic acid was coupled to human serum albumin, and resveratrol was encapsulated in glycyrrhizic acid-conjugated human serum albumin by high-pressure homogenization emulsification. The average particle size of sample nanoparticles prepared under the optimal conditions was 108.1 ± 5.3 nm with a polydispersity index (PDI) of 0.001, and the amount of glycyrrhizic acid coupled with human serum albumin was 112.56 µg/mg. The drug encapsulation efficiency and drug loading efficiency were 83.6 and 11.5%, respectively. The glycyrrhizic acid-conjugated human serum albumin nanoparticles wrapping resveratrol nanoparticles were characterized through laser light scattering, scanning electron microscopy, Fourier-transform infrared spectroscopy, X-ray diffraction, differential scanning calorimetry, thermogravimetric analyses, and gas chromatography. The characterization results showed that resveratrol in glycyrrhizic acid-conjugated human serum albumin nanoparticles wrapping resveratrol nanoparticles existed in amorphous state and the residual amounts of chloroform and methanol in nanoparticles were separately less than the international conference on harmonization (ICH) limit. The in vitro drug-release study showed that the nanoparticles released the drug slowly and continuously. The inhibitory rate of glycyrrhizic acid-conjugated human serum albumin nanoparticles wrapping resveratrol nanoparticles was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2 H-tetrazolium bromide method. The IC50 values of glycyrrhizic acid-conjugated human serum albumin nanoparticles wrapping resveratrol nanoparticles and resveratrol were 62.5 and 95.5 µg/ml, respectively. The target ability of glycyrrhizic acid-conjugated human serum albumin nanoparticles wrapping resveratrol nanoparticles

Activation of lipoprotein lipase by lipoprotein fractions of human serum.

PubMed

Bier, D M; Havel, R J

1970-11-01

Triglycerides in fat emulsions are hydrolyzed by lipoprotein lipase only when they are "activated" by serum lipoproteins. The contribution of different lipoprotein fractions to hydrolysis of triglycerides in soybean oil emulsion was assessed by determining the quantity of lipoprotein fraction required to give half-maximal hydrolysis. Most of the activator property of whole serum from normolipidemic, postabsorptive subjects was in high density lipoproteins. Low density lipoproteins and serum from which all lipoprotein classes were removed had little or no activity. Also, little activator was present in guinea pig serum or in very low density poor serum from an individual with lecithin:cholesterol acyltransferase deficiency, both of which are deficient in high density lipoproteins. Human very low density lipoproteins are potent activators and are much more active than predicted from their content of high density lipoprotein-protein. Per unit weight of protein, very low density lipoproteins had 13 times the activity of high density lipoproteins. These observations suggest that one or more of the major apoproteins of very low density lipoproteins, present as a minor constituent of high density lipoproteins, may be required for the activation process.

Posaconazole in Human Serum: a Greater Pharmacodynamic Effect than Predicted by the Non-Protein-Bound Serum Concentration ▿

PubMed Central

Lignell, Anders; Löwdin, Elisabeth; Cars, Otto; Chryssanthou, Erja; Sjölin, Jan

2011-01-01

It is generally accepted that only the unbound fraction of a drug is pharmacologically active. Posaconazole is an antifungal agent with a protein binding of 98 to 99%. Taking into account the degree of protein binding, plasma levels in patients, and MIC levels of susceptible strains, it can be assumed that the free concentration of posaconazole sometimes will be too low to exert the expected antifungal effect. The aim was therefore to test the activity of posaconazole in serum in comparison with that of the calculated unbound concentrations in protein-free media. Significant differences (P < 0.05) from the serum control were found at serum concentrations of posaconazole of 1.0 and 0.10 mg/liter, with calculated free concentrations corresponding to 1× MIC and 0.1× MIC, respectively, against one Candida lusitaniae strain selected for proof of principle. In RPMI 1640, the corresponding calculated unbound concentration of 0.015 mg/liter resulted in a significant effect, whereas that of 0.0015 mg/liter did not. Also, against seven additional Candida strains tested, there was an effect of the low posaconazole concentration in serum, in contrast to the results in RPMI 1640. Fluconazole, a low-grade-protein-bound antifungal, was used for comparison at corresponding concentrations in serum and RPMI 1640. No effect was observed at the serum concentration, resulting in a calculated unbound concentration of 0.1× MIC. In summary, there was a substantially greater pharmacodynamic effect of posaconazole in human serum than could be predicted by the non-protein-bound serum concentration. A flux from serum protein-bound to fungal lanosterol 14α-demethylase-bound posaconazole is suggested. PMID:21502622

Micro-RNA Profiling in Human Serum Reveals Compartment-Specific Roles of miR-571 and miR-652 in Liver Cirrhosis

PubMed Central

Roderburg, Christoph; Mollnow, Tobias; Bongaerts, Brenda; Elfimova, Natalia; Vargas Cardenas, David; Berger, Katharina; Zimmermann, Henning; Koch, Alexander; Vucur, Mihael; Luedde, Mark; Hellerbrand, Claus; Odenthal, Margarete; Trautwein, Christian; Tacke, Frank; Luedde, Tom

2012-01-01

Background and Aims Micro-RNAs (miRNAs) have recently emerged as crucial modulators of molecular processes involved in chronic liver diseases. The few miRNAs with previously proposed roles in liver cirrhosis were identified in screening approaches on liver parenchyma, mostly in rodent models. Therefore, in the present study we performed a systematic screening approach in order to identify miRNAs with altered levels in the serum of patients with chronic liver disease and liver cirrhosis. Methods We performed a systematic, array-based miRNA expression analysis on serum samples from patients with liver cirrhosis. In functional experiments we evaluated the relationship between alterations of miRNA serum levels and their role in distinct cellular compartments involved in hepatic cirrhosis. Results The array analysis and the subsequent confirmation by qPCR in a larger patient cohort identified significant alterations in serum levels of miR-513-3p, miR-571 and miR-652, three previously uncharacterized miRNAs, in patients with alcoholic or hepatitis C induced liver cirrhosis. Of these, miR-571 serum levels closely correlated with disease stages, thus revealing potential as a novel biomarker for hepatic cirrhosis. Further analysis revealed that up-regulation of miR-571 in serum reflected a concordant regulation in cirrhotic liver tissue. In isolated primary human liver cells, miR-571 was up-regulated in human hepatocytes and hepatic stellate cells in response to the pro-fibrogenic cytokine TGF-β. In contrast, alterations in serum levels of miR-652 were stage-independent, reflecting a concordant down-regulation of this miRNA in circulating monocytes of patients with liver cirrhosis, which was inducible by proinflammatory stimuli like bacterial lipopolysaccharide. Conclusion Alterations of miR571 and miR-652 serum levels in patients with chronic liver disease reflect their putative roles in the mediation of fibrogenic and inflammatory processes in distinct cellular

The influence of fatty acids on theophylline binding to human serum albumin. Comparative fluorescence study

NASA Astrophysics Data System (ADS)

Maciążek-Jurczyk, M.; Sułkowska, A.; Bojko, B.; Równicka-Zubik, J.; Szkudlarek-Haśnik, A.; Zubik-Skupień, I.; Góra, A.; Dubas, M.; Korzonek-Szlacheta, I.; Wielkoszyński, T.; Żurawiński, W.; Sosada, K.

2012-04-01

Theophylline, popular diuretic, is used to treat asthma and bronchospasm. In blood it forms complexes with albumin, which is also the main transporter of fatty acids. The aim of the present study was to describe the influence of fatty acids (FA) on binding of theophylline (Th) to human serum albumin (HSA) in the high affinity binding sites. Binding parameters have been obtained on the basis of the fluorescence analysis. The data obtained for the complex of Th and natural human serum albumin (nHSA) obtained from blood of obese patients qualified for surgical removal of stomach was compared with our previous studies on the influence of FA on the complex of Th and commercially available defatted human serum albumin (dHSA).

Human CRISP-3 binds serum alpha(1)B-glycoprotein across species.

PubMed

Udby, Lene; Johnsen, Anders H; Borregaard, Niels

2010-04-01

CRISP-3 was previously shown to be bound to alpha(1)B-glycoprotein (A1BG) in human serum/plasma. All mammalian sera are supposed to contain A1BG, although its presence in rodent sera is not well-documented. Since animal sera are often used to supplement buffers in experiments, in particular such that involve cell cultures, binding proteins present in sera might interfere in the experiments. We examined sera from five different animal species for CRISP-3 binding proteins using gel filtration and ligand blotting. We developed a rapid method for isolation of proteins that bind to human CRISP-3 and identified the isolated proteins by mass spectrometry and N-terminal sequencing. We identified A1BG as a CRISP-3 binding protein in sera from cow, horse and rabbit. CRISP-3 bound kininogen 1 in mouse serum, whereas rat serum showed no CRISP-3 binding activity. In equine serum, we furthermore detected a possible CRISP, already bound to A1BG. It seems to be a common mechanism that A1BGs bind CRISPs, also across species. Apart from the possible physiological implications hereof, complex binding of CRISPs by A1BG (and other proteins) may interfere with the detection and function of CRISPs, when these are studied in the presence of animal sera. Copyright 2009 Elsevier B.V. All rights reserved.

Simultaneous quantitation of multiple contraceptive hormones in human serum by LC-MS/MS.

PubMed

Blue, Steven W; Winchell, Andrea J; Kaucher, Amy V; Lieberman, Rachel A; Gilles, Christopher T; Pyra, Maria N; Heffron, Renee; Hou, Xuanlin; Coombs, Robert W; Nanda, Kavita; Davis, Nicole L; Kourtis, Athena P; Herbeck, Joshua T; Baeten, Jared M; Lingappa, Jairam R; Erikson, David W

2018-04-01

The objective was to develop a method to simultaneously quantify five commonly used hormonal contraceptives (HCs) and two endogenous sex steroids by liquid chromatography-tandem triple quadrupole mass spectrometry (LC-MS/MS) and apply this method to human serum samples. We developed a method to simultaneously analyze ethinyl estradiol (EE2), etonogestrel (ENG), levonorgestrel (LNG), medroxyprogesterone acetate (MPA) and norethisterone (NET), along with estradiol (E2) and progesterone (P4), in human serum for a Shimadzu Nexera-LCMS-8050 LC-MS/MS platform. We analyzed serum collected from women self-reporting use of oral contraceptives, contraceptive implants or injectable contraceptives (n=14) and normally cycling women using no HC (n=15) as well as pooled samples from women administered various HCs (ENG, n=6; LNG, n=14; MPA, n=7; NET, n=5). Limits of quantitation were 0.010ng/mL for E2, EE2 and P4; 0.020ng/mL for ENG, LNG and MPA; and 0.040ng/mL for NET. Precisions for all assays, as indicated by coefficient of variation, were less than or equal to 12.1%. Accuracies for all assays were in the range of 95%-108%. Endogenous hormone values obtained from analysis of human serum samples are in agreement with levels previously reported in the literature for normally cycling women as well as for women taking the appropriate HC. We have developed a robust, accurate and sensitive method for simultaneously analyzing commonly used contraceptive steroids and endogenous sex steroids in human serum. This analytical method can be used for quantitating contraceptive steroid levels in women for monitoring systemic exposure to determine drug interactions, nonadherence, misreporting and proper dosing. Copyright © 2018 Elsevier Inc. All rights reserved.

Complement Interaction with Trypanosomatid Promastigotes in Normal Human Serum

PubMed Central

Domínguez, Mercedes; Moreno, Inmaculada; López-Trascasa, Margarita; Toraño, Alfredo

2002-01-01

In normal human serum (NHS), axenic promastigotes of Crithidia, Phytomonas, and Leishmania trigger complement activation, and from 1.2 to 1.8 × 105 C3 molecules are deposited per promastigote within 2.5 min. In Leishmania, promastigote C3 binding capacity remains constant during in vitro metacyclogenesis. C3 deposition on promastigotes activated through the classical complement pathway reaches a 50% maximum after ∼50 s, and represents >85% of total C3 bound. In C1q- and C2-deficient human sera, promastigotes cannot activate the classical pathway (CP) unless purified C1q or C2 factors, respectively, are supplemented, demonstrating a requirement for CP factor in promastigote C3 opsonization. NHS depleted of natural anti-Leishmania antibodies cannot trigger promastigote CP activation, but IgM addition restores C3 binding. Furthermore, Leishmania binds natural antibodies in ethylenediaminetetracetic acid (EDTA)-treated NHS; after EDTA removal, promastigote-bound IgM triggers C3 deposition in natural antibody-depleted NHS. Serum collectins and pentraxins thus do not participate significantly in NHS promastigote C3 opsonization. Real-time kinetic analysis of promastigote CP-mediated lysis indicates that between 85–95% of parasites are killed within 2.5 min of serum contact. These data indicate that successful Leishmania infection in man must immediately follow promastigote transmission, and that Leishmania evasion strategies are shaped by the selective pressure exerted by complement. PMID:11854358

Radioimmunoassay for glyburide in human serum. [Sulfonylurea; /sup 14/C and /sup 125/I

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

Royer, M.W.; Ko, H.; Evans, J.S.

1976-01-01

A radioimmunoassay (RIA) has been developed to measure nanogram amounts of drug-related materials in human serum, after oral administration of 1.25, 2.5, 3.75 or 5.0 mg glyburide, G, Micronase, a sulfonylurea. Of the compounds tested, only the known hydroxy metabolites of G cross-reacted significantly. Recoveries were quantitative (100.6 percent). In normal human volunteers, peak serum drug concentrations were observed at 4.3 +- 1.4 hrs (S.D., M = 32).

Hydrolysis of pyrethroids by human and rat tissues: Examination of intestinal, liver and serum carboxylesterases

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

Crow, J. Allen; Borazjani, Abdolsamad; Potter, Philip M.

2007-05-15

Hydrolytic metabolism of pyrethroid insecticides in humans is one of the major catabolic pathways that clear these compounds from the body. Rodent models are often used to determine the disposition and clearance rates of these esterified compounds. In this study the distribution and activities of esterases that catalyze pyrethroid metabolism have been investigated in vitro using several human and rat tissues, including small intestine, liver and serum. The major esterase in human intestine is carboxylesterase 2 (hCE2). We found that the pyrethroid trans-permethrin is effectively hydrolyzed by a sample of pooled human intestinal microsomes (5 individuals), while deltamethrin and bioresmethrinmore » are not. This result correlates well with the substrate specificity of recombinant hCE2 enzyme. In contrast, a sample of pooled rat intestinal microsomes (5 animals) hydrolyze trans-permethrin 4.5-fold slower than the sample of human intestinal microsomes. Furthermore, it is demonstrated that pooled samples of cytosol from human or rat liver are {approx} 2-fold less hydrolytically active (normalized per mg protein) than the corresponding microsomal fraction toward pyrethroid substrates; however, the cytosolic fractions do have significant amounts ({approx} 40%) of the total esteratic activity. Moreover, a 6-fold interindividual variation in carboxylesterase 1 protein expression in human hepatic cytosols was observed. Human serum was shown to lack pyrethroid hydrolytic activity, but rat serum has hydrolytic activity that is attributed to a single CE isozyme. We purified the serum CE enzyme to homogeneity to determine its contribution to pyrethroid metabolism in the rat. Both trans-permethrin and bioresmethrin were effectively cleaved by this serum CE, but deltamethrin, esfenvalerate, alpha-cypermethrin and cis-permethrin were slowly hydrolyzed. Lastly, two model lipase enzymes were examined for their ability to hydrolyze pyrethroids. However, no hydrolysis products could

Binding affinity of aluminium to human serum transferrin and effects of carbohydrate chain modification as studied by HPLC/high-resolution ICP-MS--speciation of aluminium in human serum.

PubMed

Nagaoka, Megumi Hamano; Maitani, Tamio

2005-09-01

Aluminium (Al) in the blood is bound to transferrin (Tf), a glycoprotein of about 80kDa that is characterized by its need for a synergistic anion. In this focused review, the binding affinity of Al to Tf is surveyed in the context of our recent studies using on-line high-performance liquid chromatography/high-resolution inductively coupled plasma mass spectrometry (HPLC/HR-ICP-MS). Al in human serum without any in vitro Al-spikes was present in a form bound to the N-lobe site of Tf. The influences of sialic acid in the carbohydrate chain of human serum Tf (hTf) were studied using asialo-hTf, obtained by treatment with sialidase. The binding affinity of Fe was similar between asialo-hTf and native-hTf, while that of Al for asialo-hTf was larger than that for native-hTf, especially in the presence of oxalate, a synergistic anion. The above findings are discussed in relation to diseases in which the serum concentrations of carbohydrate-deficient Tf and oxalate are augmented.

ELISA measurement of stachylysin in serum to quantify human exposures to the indoor mold Stachybotrys chartarum.

PubMed

Van Emon, Jeanette M; Reed, Allan W; Yike, Iwona; Vesper, Stephen J

2003-06-01

The goal of this research was to develop a measurable indicator of human exposure to Stachyborys chartarum. Antibodies were produced against the hemolytic agent stachylysin obtained from the mold S. chartarum. These antibodies were used to develop two enzyme-linked immunosorbent assay methods for the analysis of stachylysin in human and rat sera and environmental samples. Stachylysin was measured in rat pups that received nasal instillations of S. chartarum conidia but not in control rat serum. Stachylysin in the serum of five human adults exposed to S. chartarum in water-damaged environments was 371 ng/mL but none was detected in the control serum. Stachylysin was also quantified in spore, wallboard, mycelial, and dust samples. The measurement of stachylysin may be a useful indicator in assessing human exposure to S. chartarum and in determining the presence of this indoor mold.

Targeted quantification of low ng/mL level proteins in human serum without immunoaffinity depletion

PubMed Central

Shi, Tujin; Sun, Xuefei; Gao, Yuqian; Fillmore, Thomas L.; Schepmoes, Athena A.; Zhao, Rui; He, Jintang; Moore, Ronald J.; Kagan, Jacob; Rodland, Karin D.; Liu, Tao; Liu, Alvin Y.; Smith, Richard D.; Tang, Keqi; Camp, David G.; Qian, Wei-Jun

2013-01-01

We recently reported an antibody-free targeted protein quantification strategy, termed high-pressure, high-resolution separations with intelligent selection and multiplexing (PRISM) for achieving significantly enhanced sensitivity using selected reaction monitoring (SRM) mass spectrometry. Integrating PRISM with front-end IgY14 immunoaffinity depletion, sensitive detection of targeted proteins at 50–100 pg/mL levels in human blood plasma/serum was demonstrated. However, immunoaffinity depletion is often associated with undesired losses of target proteins of interest. Herein we report further evaluation of PRISM-SRM quantification of low-abundance serum proteins without immunoaffinity depletion. Limits of quantification (LOQ) at low ng/mL levels with a median coefficient of variation (CV) of ~12% were achieved for proteins spiked into human female serum. PRISM-SRM provided >100-fold improvement in the LOQ when compared to conventional LC-SRM measurements. PRISM-SRM was then applied to measure several low-abundance endogenous serum proteins, including prostate-specific antigen (PSA), in clinical prostate cancer patient sera. PRISM-SRM enabled confident detection of all target endogenous serum proteins except the low pg/mL-level cardiac troponin T. A correlation coefficient >0.99 was observed for PSA between the results from PRISM-SRM and immunoassays. Our results demonstrate that PRISM-SRM can successful quantify low ng/mL proteins in human plasma or serum without depletion. We anticipate broad applications for PRISM-SRM quantification of low-abundance proteins in candidate biomarker verification and systems biology studies. PMID:23763644

Analysis of anhydrous glucose and human serum assisted by Raman spectroscopy

NASA Astrophysics Data System (ADS)

González, Naara; Cerecedo-Núñez, H. H.; Padilla-Sosa, P.; Flores, Aaron; May, Manuel; Basurto, Miguel; Alcántara, Gabriela; Muñiz, Omar

2017-02-01

Raman spectroscopy has been considered like a potentially important clinical tool for real-time diagnosis of disease and evaluation of living tissue, whit the proposal to development noninvasive glucose measurements in a near future, with lower power than other reported studies, in this work are reported experimental tests made with a excitation source of semiconductor laser of 785 nm and 35 mW power. Measurements were made to different glucose concentrations, with variation from 50 mg/dL to 6000 mg/dL. For this, three intervals with different ranges of concentration were analyzed, these tests were put into plastic sampling cells, making incise the beam vertically on sample. In the same way measurements to serum human are reported, for healthy volunteers had 12 hours fasting and non-fasting conditions, with it's corresponding values of glucose taken through a conventional glucometer. Freeze-dried human serum was poured on object-holder, in the case of human serum reconstitute, it was used container in which were previously kept samples. Nine spectra per test were obtained and subsequently average was calculated, the spectra were studied in a range of 500 to 1700 cm-1. This work explores the intensity variation of the bands of glucose in 1065 cm-1 and 1127 cm-1 as a function of glucose concentration. In the obtained results, there observes a behavior with positive slope in both substances, interrelation being observed between the measurements, being promissory for non-invasive measurement.

Cytochrome P450 inactivation by serum from humans with a viral infection and serum from rabbits with a turpentine-induced inflammation: the role of cytokines.

PubMed

Bleau, A M; Levitchi, M C; Maurice, H; du Souich, P

2000-08-01

Serum from humans with an acute upper respiratory viral infection and from rabbits with turpentine-induced inflammation reduce the catalytic activity of hepatic cytochrome P450 (P450). The aim of this study was to identify the serum mediators responsible for the decrease in P450 activity. Rabbit and human sera were fractionated by size exclusion chromatography and the fractions tested for their ability to reduce the activity and amount of P450 after 4 h of incubation with hepatocytes from turpentine-treated rabbits (H(INF)). Rabbit and human sera decreased P450 activity by around 40% without any change in the amount of CYP1A1 and 1A2 apoproteins. In rabbit serum, the fraction containing proteins of M(r) 23-15 kDa decreased P450 content by 41%, but did not alter the amount of the apoproteins. Anti-IL-6 antibody added to the M(r) 23-15 kDa fraction restored P450 content to 97% of control values, while anti-IL-1beta, TNF-alpha and IFN-gamma antibodies had no effect. Supporting the role of IL-6, incubation of H(INF) in the presence of IL-6 for 4 h reduced P450 content by 40%. In human serum, the fraction containing proteins of M(r) >95 kDa lowered P450 content by 43% without modifying the amounts of CYP1A1/2. Neutralization experiments showed that IFN-gamma, IL-6, and IL-1beta contributed to the decrease in P450 content. In conclusion, the present results demonstrate that IL-6, and IFN-gamma, IL-6 and IL-1beta are the serum mediators released in vivo by a turpentine-induced inflammatory reaction in the rabbit and an upper respiratory viral infection in humans, respectively, inactivating hepatic P450.

Cytochrome P450 inactivation by serum from humans with a viral infection and serum from rabbits with a turpentine-induced inflammation: the role of cytokines

PubMed Central

Bleau, Anne-Marie; Levitchi, Mihaela C; Maurice, Hélène; du Souich, Patrick

2000-01-01

Serum from humans with an acute upper respiratory viral infection and from rabbits with turpentine-induced inflammation reduce the catalytic activity of hepatic cytochrome P450 (P450). The aim of this study was to identify the serum mediators responsible for the decrease in P450 activity.Rabbit and human sera were fractionated by size exclusion chromatography and the fractions tested for their ability to reduce the activity and amount of P450 after 4 h of incubation with hepatocytes from turpentine-treated rabbits (HINF). Rabbit and human sera decreased P450 activity by around 40% without any change in the amount of CYP1A1 and 1A2 apoproteins.In rabbit serum, the fraction containing proteins of Mr 23–15 kDa decreased P450 content by 41%, but did not alter the amount of the apoproteins. Anti-IL-6 antibody added to the Mr 23–15 kDa fraction restored P450 content to 97% of control values, while anti-IL-1β, TNF-α and IFN-γ antibodies had no effect. Supporting the role of IL-6, incubation of HINF in the presence of IL-6 for 4 h reduced P450 content by 40%.In human serum, the fraction containing proteins of Mr >95 kDa lowered P450 content by 43% without modifying the amounts of CYP1A1/2. Neutralization experiments showed that IFN-γ, IL-6, and IL-1β contributed to the decrease in P450 content.In conclusion, the present results demonstrate that IL-6, and IFN-γ, IL-6 and IL-1β are the serum mediators released in vivo by a turpentine-induced inflammatory reaction in the rabbit and an upper respiratory viral infection in humans, respectively, inactivating hepatic P450. PMID:10952665

Self-Assembly of Human Serum Albumin: A Simplex Phenomenon

PubMed Central

Thakur, Garima; Prashanthi, Kovur; Jiang, Keren; Thundat, Thomas

2017-01-01

Spontaneous self-assemblies of biomolecules can generate geometrical patterns. Our findings provide an insight into the mechanism of self-assembled ring pattern generation by human serum albumin (HSA). The self-assembly is a process guided by kinetic and thermodynamic parameters. The generated protein ring patterns display a behavior which is geometrically related to a n-simplex model and is explained through thermodynamics and chemical kinetics. PMID:28930179

Possibilities of vaccine manufacture in human diploid cell strains with a serum replacement factor.

PubMed

Candal, F J; George, V G; Ades, E W

1991-07-01

Cell lines MDCK (canine kidney), BGM (Buffalo green monkey kidney) and human embryonic lung fibroblast will support viral growth efficiently in medium without serum. Both MRC-5 and WI-38 cell strains have been approved by the Food and Drug Administration for manufacturing viral vaccines against cytomegalovirus and varicella-zoster virus. In this study we examine these two cell lines and viruses for their ability to grow in medium containing a serum replacement. The serum substitute used is LPSR-1 (low protein serum replacement). Using LPSR-1 in defined medium, we demonstrate multipassage cell growth and viral cultivation and replication equivalent to those obtained in medium containing fetal bovine serum (FBS). Viral growth after complete elimination of FBS varies and depends on cell line and virus. Serum substitutes can eliminate FBS in the viral growth phase of vaccine production and reduce costs.

Multiplexed Activity-based Protein Profiling of the Human Pathogen Aspergillus fumigatus Reveals Large Functional Changes upon Exposure to Human Serum

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

Wiedner, Susan D.; Burnum, Kristin E.; Pederson, Leeanna M.

2012-08-03

Environmental and metabolic adaptability is critical for survival of the fungal human pathogen Aspergillus fumigatus in the immunocompromised lung. We employed an activity-based protein profiling (ABPP) approach utilizing a new aryl vinyl sulfonate probe and a serine hydrolase probe combined with quantitative LC-MS based accurate mass and time (AMT) tag proteomics for the identification of functional pathway adaptation of A. fumigatus to environmental variability relevant to pulmonary Invasive Aspergillosis. When the fungal pathogen was grown with human serum, metabolism and energy processes were markedly decreased compared to no serum culture. Additionally, functional pathways associated with amino acid and protein biosynthesismore » were limited as the fungus scavenged from the serum to obtain essential nutrients. Our approach revealed significant metabolic adaptation by A. fumigatus, and provides direct insight into this pathogen’s ability to survive and proliferate.« less

Analysis of human serum proteins by liquid phase isoelectric focusing and matrix-assisted laser desorption/ionization-mass spectrometry.

PubMed

Wang, Michael Z; Howard, Brandon; Campa, Michael J; Patz, Edward F; Fitzgerald, Michael C

2003-09-01

Direct matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of human serum yielded ion signals from only a fraction of the total number of peptides and proteins expected to be in the sample. We increased the number of peptide and protein ion signals observed in the MALDI-TOF mass spectra analysis of human serum by using a prefractionation protocol based on liquid phase isoelectric focusing electrophoresis. This pre-fractionation technique facilitated the MALDI-TOF MS detection of as many as 262 different peptide and protein ion signals from human serum. The results obtained from three replicate fractionation experiments on the same serum sample indicated that 148 different peptide and protein ion signals were reproducibly detected using our isoelectric focusing and MALDI-TOF MS protocol.

Surface imprinted beads for the recognition of human serum albumin.

PubMed

Bonini, Francesca; Piletsky, Sergey; Turner, Anthony P F; Speghini, Adolfo; Bossi, Alessandra

2007-04-15

The synthesis of poly-aminophenylboronic acid (ABPA) imprinted beads for the recognition of the protein human serum albumin (HSA) is reported. In order to create homogeneous recognition sites, covalent immobilisation of the template HSA was exploited. The resulting imprinted beads were selective for HSA. The indirect imprinting factor (IF) calculated from supernatant was 1.6 and the direct IF, evaluated from the protein recovered from the beads, was 1.9. The binding capacity was 1.4 mg/g, which is comparable to commercially available affinity materials. The specificity of the HSA recognition was evaluated with competitive experiments, indicating a molar ratio 4.5/1 of competitor was necessary to displace half of the bound HSA. The recognition and binding of the imprinted beads was also tested with a complex sample, human serum and targeted removal of HSA without a loss of the other protein components was demonstrated. The easy preparation protocol of derivatised beads and a good protein recognition properties make the approach an attractive solution to analytical and bio-analytical problems in the field of biotechnology.

The cholesterol-lowering effect of coconut flakes in humans with moderately raised serum cholesterol.

PubMed

Trinidad, Trinidad P; Loyola, Anacleta S; Mallillin, Aida C; Valdez, Divinagracia H; Askali, Faridah C; Castillo, Joan C; Resaba, Rosario L; Masa, Dina B

2004-01-01

This study investigated the effect of coconut flakes on serum cholesterol levels of humans with moderately raised serum cholesterol in 21 subjects. The serum total cholesterol of subjects differed and ranged from 259 to 283 mg/dL. The study was conducted in a double-blind randomized crossover design on a 14-week period, consisting of four 2-week experimental periods, with each experimental period separated by a 2-week washout period. The test foods were as follows: corn flakes as the control food, oat bran flakes as the reference food, and corn flakes with 15% and 25% dietary fiber from coconut flakes (made from coconut flour production). Results showed a significant percent reduction in serum total and low-density lipoprotein (LDL) cholesterol (in mg/dL) for all test foods, except for corn flakes, as follows: oat bran flakes, 8.4 +/- 1.4 and 8.8 +/- 6.0, respectively; 15% coconut flakes, 6.9 +/- 1.1 and 11.0 +/- 4.0, respectively; and 25% coconut flakes, 10.8 +/- 1.3 and 9.2 +/- 5.4, respectively. Serum triglycerides were significantly reduced for all test foods: corn flakes, 14.5 +/- 6.3%; oat bran flakes, 22.7 +/- 2.9%; 15% coconut flakes, 19.3 +/- 5.7%; and 25% coconut flakes, 21.8 +/- 6.0%. Only 60% of the subjects were considered for serum triglycerides reduction (serum triglycerides >170 mg/dL). In conclusion, both 15% and 25% coconut flakes reduced serum total and LDL cholesterol and serum triglycerides of humans with moderately raised serum cholesterol levels. Coconut flour is a good source of both soluble and insoluble dietary fiber, and both types of fiber may have significant role in the reduction of the above lipid biomarker. To our knowledge, this is the first study conducted to show a relationship between dietary fiber from a coconut by-product and a lipid biomarker. Results from this study serves as a good basis in the development of coconut flakes/flour as a functional food, justifying the increased production of coconut and coconut by-products.

Massive glutamine cyclization to pyroglutamic acid in human serum discovered using NMR spectroscopy.

PubMed

Nagana Gowda, G A; Gowda, Yashas N; Raftery, Daniel

2015-04-07

Glutamine is one of the most abundant metabolites in blood and is a precursor as well as end product central to numerous important metabolic pathways. A number of surprising and unexpected roles for glutamine, including cancer cell glutamine addiction discovered recently, stress the importance of accurate analysis of glutamine concentrations for understanding its role in health and numerous diseases. Utilizing a recently developed NMR approach that offers access to an unprecedented number of quantifiable blood metabolites, we have identified a surprising glutamine cyclization to pyroglutamic acid that occurs during protein removal. Intact, ultrafiltered and protein precipitated samples from the same pool of human serum were comprehensively investigated using (1)H NMR spectroscopy at 800 MHz to detect and quantitatively evaluate the phenomenon. Interestingly, although glutamine cyclization occurs in both ultrafiltered and protein precipitated serum, the cyclization was not detected in intact serum. Strikingly, due to cyclization, the apparent serum glutamine level drops by up to 75% and, concomitantly, the pyroglutamic acid level increases proportionately. Further, virtually under identical conditions, the magnitude of cyclization is vastly different for different portions of samples from the same pool of human serum. However, the sum of glutamine and pyroglutamic acid concentrations in each sample remains the same for all portions. These unexpected findings indicate the importance of considering the sum of apparent glutamine and pyroglutamic acid levels, obtained from the contemporary analytical methods, as the actual blood glutamine level for biomarker discovery and biological interpretations.

Determination of perfluorinated alkyl acid concentrations in human serum and milk standard reference materials.

PubMed

Keller, Jennifer M; Calafat, Antonia M; Kato, Kayoko; Ellefson, Mark E; Reagen, William K; Strynar, Mark; O'Connell, Steven; Butt, Craig M; Mabury, Scott A; Small, Jeff; Muir, Derek C G; Leigh, Stefan D; Schantz, Michele M

2010-05-01

Standard Reference Materials (SRMs) are certified reference materials produced by the National Institute of Standards and Technology that are homogeneous materials well characterized with values for specified properties, such as environmental contaminant concentrations. They can be used to validate measurement methods and are critical in improving data quality. Disagreements in perfluorinated alkyl acid (PFAA) concentrations measured in environmental matrices during past interlaboratory comparisons emphasized the need for SRMs with values assigned for PFAAs. We performed a new interlaboratory comparison among six laboratories and provided, for the first time, value assignment of PFAAs in SRMs. Concentrations for perfluorooctane sulfonate (PFOS), perfluorooctanoate (PFOA), and other PFAAs in two human serum and two human milk SRMs are reported. PFAA concentration measurements agreed for serum SRM 1957 using different analytical methods in six laboratories and for milk SRM 1954 in three laboratories. The interlaboratory relative standard deviation for PFOS in SRM 1957 was 7%, which is an improvement over past interlaboratory studies. Matrix interferences are discussed, as well as temporal trends and the percentage of branched vs. linear isomers. The concentrations in these SRMs are similar to the present-day average concentrations measured in human serum and milk, resulting in representative and useful control materials for PFAA human monitoring studies.

Measuring and Validating the Levels of Brain-Derived Neurotrophic Factor in Human Serum

PubMed Central

Naegelin, Yvonne; Dingsdale, Hayley; Säuberli, Katharina; Schädelin, Sabine; Kappos, Ludwig

2018-01-01

Brain-derived neurotrophic factor (BDNF) secreted by neurons is a significant component of synaptic plasticity. In humans, it is also present in blood platelets where it accumulates following its biosynthesis in megakaryocytes. BDNF levels are thus readily detectable in human serum and it has been abundantly speculated that they may somehow serve as an indicator of brain function. However, there is a great deal of uncertainty with regard to the range of BDNF levels that can be considered normal, how stable these values are over time and even whether BDNF levels can be reliably measured in serum. Using monoclonal antibodies and a sandwich ELISA, this study reports on BDNF levels in the serum of 259 volunteers with a mean value of 32.69 ± 8.33 ng/ml (SD). The mean value for the same cohort after 12 months was not significantly different (N = 226, 32.97 ± 8.36 ng/ml SD, p = 0.19). Power analysis of these values indicates that relatively large cohorts are necessary to identify significant differences, requiring a group size of 60 to detect a 20% change. The levels determined by ELISA could be validated by Western blot analyses using a BDNF monoclonal antibody. While no association was observed with gender, a weak, positive correlation was found with age. The overall conclusions are that BDNF levels can be reliably measured in human serum, that these levels are quite stable over one year, and that comparisons between two populations may only be meaningful if cohorts of sufficient sizes are assembled. PMID:29662942

Centrifugal ultrafiltration of human serum for improving immunoglobulin A quantification using attenuated total reflectance infrared spectroscopy.

PubMed

Elsohaby, Ibrahim; McClure, J Trenton; Riley, Christopher B; Bryanton, Janet; Bigsby, Kathryn; Shaw, R Anthony

2018-02-20

Attenuated total reflectance infrared (ATR-IR) spectroscopy is a simple, rapid and cost-effective method for the analysis of serum. However, the complex nature of serum remains a limiting factor to the reliability of this method. We investigated the benefits of coupling the centrifugal ultrafiltration with ATR-IR spectroscopy for quantification of human serum IgA concentration. Human serum samples (n = 196) were analyzed for IgA using an immunoturbidimetric assay. ATR-IR spectra were acquired for whole serum samples and for the retentate (residue) reconstituted with saline following 300 kDa centrifugal ultrafiltration. IR-based analytical methods were developed for each of the two spectroscopic datasets, and the accuracy of each of the two methods compared. Analytical methods were based upon partial least squares regression (PLSR) calibration models - one with 5-PLS factors (for whole serum) and the second with 9-PLS factors (for the reconstituted retentate). Comparison of the two sets of IR-based analytical results to reference IgA values revealed improvements in the Pearson correlation coefficient (from 0.66 to 0.76), and the root mean squared error of prediction in IR-based IgA concentrations (from 102 to 79 mg/dL) for the ultrafiltration retentate-based method as compared to the method built upon whole serum spectra. Depleting human serum low molecular weight proteins using a 300 kDa centrifugal filter thus enhances the accuracy IgA quantification by ATR-IR spectroscopy. Further evaluation and optimization of this general approach may ultimately lead to routine analysis of a range of high molecular-weight analytical targets that are otherwise unsuitable for IR-based analysis. Copyright © 2017 Elsevier B.V. All rights reserved.

Alteration of human serum albumin binding properties induced by modifications: A review

NASA Astrophysics Data System (ADS)

Maciążek-Jurczyk, Małgorzata; Szkudlarek, Agnieszka; Chudzik, Mariola; Pożycka, Jadwiga; Sułkowska, Anna

2018-01-01

Albumin, a major transporting protein in the blood, is the main target of modification that affects the binding of drugs to Sudlow's site I and II. These modification of serum protein moderates its physiological function, and works as a biomarker of some diseases. The main goal of the paper was to explain the possible alteration of human serum albumin binding properties induced by modifications such as glycation, oxidation and ageing, their origin, methods of evaluation and positive and negative meaning described by significant researchers.

Headspace Solid Phase Micro Extraction Gas Chromatographic Determination of Fenthion in Human Serum

PubMed Central

Kasiotis, Konstantinos M.; Souki, Helen; Tsakirakis, Angelos N.; Carageorgiou, Haris; Theotokatos, Spiridon A.; Haroutounian, Serkos A.; Machera, Kyriaki

2008-01-01

A simple and effective analytical procedure was developed for the determination of fenthion residues in human serum samples. The sample treatment was performed using the headspace solid-phase micro extraction with polyacrylate fiber, which has the advantage to require low amount of serum (1 mL) without tedious pre-treatment. The quantification of fenthion was carried out by gas chromatography-mass spectrometry and the recoveries ranged from 79 to 104% at two spiking levels for 6 replicates. Detection and quantification limits were calculated as 1.51 and 4.54 ng/mL of serum respectively. Two fenthion metabolites fenoxon and fenthion–sulfoxide were also identified. PMID:19325792

Use of mep HyperCel for polishing of human serum albumin.

PubMed

McCann, Karl B; Vucica, Yvonne; Wu, John; Bertolini, Joseph

2014-10-15

The manufacture of human serum albumin by chromatographic procedures involves gel filtration chromatography as a final polishing step. Despite this step being essential to remove high molecular weight impurity proteins and thus ensure a stable and safe final product, it is relatively inefficient. This paper explores the use of hydrophobic charge induction chromatographic media, MEP HyperCel as an alternative to Sephacryl S200HR gel filtration for the polishing of human serum albumin derived by ion exchange chromatographic purification of Cohn Supernatant I. The use of MEP HyperCel results in a product with a higher purity than achieved with gel filtration and in a less time consuming manner and with potential resource savings. MEP HyperCel appears to have great potential for incorporation into downstream processes in the plasma fractionation industry as an efficient means of achieving polishing of intermediates or capture of proteins of interest. Copyright © 2014 Elsevier B.V. All rights reserved.

Novel fluorimetric assay of trace analysis of epinephrine in human serum

NASA Astrophysics Data System (ADS)

Adeniyi, William K.; Wright, Ashleigh R.

2009-12-01

A simple, rapid, and sensitive spectrofluorimetric technique for the microdetermination of epinephrine in human serum is described. The investigation shows that trace amounts of epinephrine, antidepressant of clinical importance, can be determined without the conventional derivatization or use of fluorophores by diazotization. The method is based on the optimization of experimental parameters, such as pH, temperature, careful selection of excitation and emission wavelengths and on the use of anionic surfactant, sodium dodecyl sulfate (SDS), to enhance sensitivity. The measurement was carried out at 360 nm with excitation at 286 nm. Under the optimum conditions, a linear relationship was obtained between the fluorescence intensity and epinephrine concentration in the range of 0.10 and 1.0 μg/mL; the correlation coefficient and detection limit are 0.9953 and 0.05 μg/mL, respectively. Recovery tests indicated an efficiency of 95.5-98.7% by using known amounts of epinephrine spiked with human serum.

Efficiency of the human observer detecting random signals in random backgrounds

PubMed Central

Park, Subok; Clarkson, Eric; Kupinski, Matthew A.; Barrett, Harrison H.

2008-01-01

The efficiencies of the human observer and the channelized-Hotelling observer relative to the ideal observer for signal-detection tasks are discussed. Both signal-known-exactly (SKE) tasks and signal-known-statistically (SKS) tasks are considered. Signal location is uncertain for the SKS tasks, and lumpy backgrounds are used for background uncertainty in both cases. Markov chain Monte Carlo methods are employed to determine ideal-observer performance on the detection tasks. Psychophysical studies are conducted to compute human-observer performance on the same tasks. Efficiency is computed as the squared ratio of the detectabilities of the observer of interest to the ideal observer. Human efficiencies are approximately 2.1% and 24%, respectively, for the SKE and SKS tasks. The results imply that human observers are not affected as much as the ideal observer by signal-location uncertainty even though the ideal observer outperforms the human observer for both tasks. Three different simplified pinhole imaging systems are simulated, and the humans and the model observers rank the systems in the same order for both the SKE and the SKS tasks. PMID:15669610

Protein Turnover Measurements in Human Serum by Serial Immunoaffinity LC-MS/MS.

PubMed

Farrokhi, Vahid; Chen, Xiaoying; Neubert, Hendrik

2018-02-01

The half-life of target proteins is frequently an important parameter in mechanistic pharmacokinetic and pharmacodynamic (PK/PD) modeling of biotherapeutics. Clinical studies for accurate measurement of physiologically relevant protein turnover can reduce the uncertainty in PK/PD model-based predictions, for example, of the therapeutic dose and dosing regimen in first-in-human clinical trials. We used a targeted mass spectrometry work flow based on serial immunoaffinity enrichment ofmultiple human serum proteins from a [5,5,5- 2 H 3 ]-L-leucine tracer pulse-chase study in healthy volunteers. To confirm the reproducibility of turnover measurements from serial immunoaffinity enrichment, multiple aliquots from the same sample set were subjected to protein turnover analysis in varying order. Tracer incorporation was measured by multiple-reaction-monitoring mass spectrometry and target turnover was calculated using a four-compartment pharmacokinetic model. Five proteins of clinical or therapeutic relevance including soluble tumor necrosis factor receptor superfamily member 12A, tissue factor pathway inhibitor, soluble interleukin 1 receptor like 1, soluble mucosal addressin cell adhesion molecule 1, and muscle-specific creatine kinase were sequentially subjected to turnover analysis from the same human serum sample. Calculated half-lives ranged from 5-15 h; however, no tracer incorporation was observed for mucosal addressin cell adhesion molecule 1. The utility of clinical pulse-chase studies to investigate protein turnover can be extended by serial immunoaffinity enrichment of target proteins. Turnover analysis from serum and subsequently from remaining supernatants provided analytical sensitivity and reproducibility for multiple human target proteins in the same sample set, irrespective of the order of analysis. © 2017 American Association for Clinical Chemistry.

Biomonitoring of selenoprotein P in human serum by fast affinity chromatography coupled to ICP-MS.

PubMed

Heitland, Peter; Köster, Helmut D

2018-04-01

Most of the Se in human serum is bound to selenoprotein P (SEPP1) in which Se is present in form of selenocysteine. The SEPP1 is a new possible biomarker for the Se status and for this reason we developed a fast, simple and reliable method for the quantitative determination of SEPP1 in serum by affinity chromatography coupled to ICP-MS. It is possible to separate SEPP1 from other selenoproteins in serum in only 5 min, which allows high sample throughput in clinical laboratories. Measured and certified concentrations of total Se and Se(SEPP1) are in good agreement for the reference material SRM 1950. The SEPP1 concentration was stable in serum samples of 3 persons for a minimum of 2 weeks. Further results of method validation were described including internal and external quality assurance. The analytical method was applied for a biomonitoring study of the SEPP1 and total Se concentration in human serum of 50 occupationally non-exposed persons living in northern Germany. Concentration ranges and mean concentrations for Se(SEPP1) are 31.1-59.7 and 46.2 μg/L, respectively. The corresponding values for total Se are 62-120 and 83.5 μg/L. The mean percentage of total Se in serum present as SEPP1 is 58%. Copyright © 2018 Elsevier GmbH. All rights reserved.

Milk and serum standard reference materials for monitoring organic contaminants in human samples.

PubMed

Schantz, Michele M; Eppe, Gauthier; Focant, Jean-François; Hamilton, Coreen; Heckert, N Alan; Heltsley, Rebecca M; Hoover, Dale; Keller, Jennifer M; Leigh, Stefan D; Patterson, Donald G; Pintar, Adam L; Sharpless, Katherine E; Sjödin, Andreas; Turner, Wayman E; Vander Pol, Stacy S; Wise, Stephen A

2013-02-01

Four new Standard Reference Materials (SRMs) have been developed to assist in the quality assurance of chemical contaminant measurements required for human biomonitoring studies, SRM 1953 Organic Contaminants in Non-Fortified Human Milk, SRM 1954 Organic Contaminants in Fortified Human Milk, SRM 1957 Organic Contaminants in Non-Fortified Human Serum, and SRM 1958 Organic Contaminants in Fortified Human Serum. These materials were developed as part of a collaboration between the National Institute of Standards and Technology (NIST) and the Centers for Disease Control and Prevention (CDC) with both agencies contributing data used in the certification of mass fraction values for a wide range of organic contaminants including polychlorinated biphenyl (PCB) congeners, chlorinated pesticides, polybrominated diphenyl ether (PBDE) congeners, and polychlorinated dibenzo-p-dioxin (PCDD) and dibenzofuran (PCDF) congeners. The certified mass fractions of the organic contaminants in unfortified samples, SRM 1953 and SRM 1957, ranged from 12 ng/kg to 2200 ng/kg with the exception of 4,4'-DDE in SRM 1953 at 7400 ng/kg with expanded uncertainties generally <14 %. This agreement suggests that there were no significant biases existing among the multiple methods used for analysis.

Milk and serum standard reference materials for monitoring organic contaminants in human samples

PubMed Central

Eppe, Gauthier; Focant, Jean-François; Hamilton, Coreen; Heckert, N. Alan; Heltsley, Rebecca M.; Hoover, Dale; Keller, Jennifer M.; Leigh, Stefan D.; Patterson, Donald G.; Pintar, Adam L.; Sharpless, Katherine E.; Sjödin, Andreas; Turner, Wayman E.; Vander Pol, Stacy S.; Wise, Stephen A.

2016-01-01

Four new Standard Reference Materials (SRMs) have been developed to assist in the quality assurance of chemical contaminant measurements required for human biomonitoring studies, SRM 1953 Organic Contaminants in Non-Fortified Human Milk, SRM 1954 Organic Contaminants in Fortified Human Milk, SRM 1957 Organic Contaminants in Non-Fortified Human Serum, and SRM 1958 Organic Contaminants in Fortified Human Serum. These materials were developed as part of a collaboration between the National Institute of Standards and Technology (NIST) and the Centers for Disease Control and Prevention (CDC) with both agencies contributing data used in the certification of mass fraction values for a wide range of organic contaminants including polychlorinated biphenyl (PCB) congeners, chlorinated pesticides, polybrominated diphenyl ether (PBDE) congeners, and polychlorinated dibenzo-p-dioxin (PCDD) and dibenzofuran (PCDF) congeners. The certified mass fractions of the organic contaminants in unfortified samples, SRM 1953 and SRM 1957, ranged from 12 ng/kg to 2200 ng/kg with the exception of 4,4′-DDE in SRM 1953 at 7400 ng/kg with expanded uncertainties generally <14 %. This agreement suggests that there were no significant biases existing among the multiple methods used for analysis. PMID:23132544

Analysis of Monoclonal Antibodies in Human Serum as a Model for Clinical Monoclonal Gammopathy by Use of 21 Tesla FT-ICR Top-Down and Middle-Down MS/MS

NASA Astrophysics Data System (ADS)

He, Lidong; Anderson, Lissa C.; Barnidge, David R.; Murray, David L.; Hendrickson, Christopher L.; Marshall, Alan G.

2017-05-01

With the rapid growth of therapeutic monoclonal antibodies (mAbs), stringent quality control is needed to ensure clinical safety and efficacy. Monoclonal antibody primary sequence and post-translational modifications (PTM) are conventionally analyzed with labor-intensive, bottom-up tandem mass spectrometry (MS/MS), which is limited by incomplete peptide sequence coverage and introduction of artifacts during the lengthy analysis procedure. Here, we describe top-down and middle-down approaches with the advantages of fast sample preparation with minimal artifacts, ultrahigh mass accuracy, and extensive residue cleavages by use of 21 tesla FT-ICR MS/MS. The ultrahigh mass accuracy yields an RMS error of 0.2-0.4 ppm for antibody light chain, heavy chain, heavy chain Fc/2, and Fd subunits. The corresponding sequence coverages are 81%, 38%, 72%, and 65% with MS/MS RMS error 4 ppm. Extension to a monoclonal antibody in human serum as a monoclonal gammopathy model yielded 53% sequence coverage from two nano-LC MS/MS runs. A blind analysis of five therapeutic monoclonal antibodies at clinically relevant concentrations in human serum resulted in correct identification of all five antibodies. Nano-LC 21 T FT-ICR MS/MS provides nonpareil mass resolution, mass accuracy, and sequence coverage for mAbs, and sets a benchmark for MS/MS analysis of multiple mAbs in serum. This is the first time that extensive cleavages for both variable and constant regions have been achieved for mAbs in a human serum background.

Three-dimensional structure of human serum albumin

NASA Technical Reports Server (NTRS)

Carter, Daniel C.; He, Xiao-Min; Twigg, Pamela D.; Casale, Elena

1991-01-01

The binding locations to human serum albumin (HSA) of several drug molecules were determined at low resolution using crystallographic methods. The principal binding sites are located within subdomains IIA and IIIA. Preliminary studies suggest that an approach to increasing the in vivo efficacy of drugs which are rendered less effective or ineffective by virtue of their interaction with HSA, would be the use of competitive displacement in drug therapies and/or the development of a general inhibitor to the site within subdomain IIIA. These findings also suggest that the facilitated transfer of various ligands across organ/circulatory interfaces such as liver, kidney, and brain may be associated with binding to the IIIA subdomain.

Radioimmunoassay of Human Serum Thyrotrophin

PubMed Central

Hall, Reginald; Amos, Jacqueline; Ormston, Brian J.

1971-01-01

The double antibody radioimmunoassay of serum thyroid-stimulating hormone (TSH) allows measurement of circulating levels of the hormone in most normal subjects. The serum TSH level in normal subjects is 1·6 ± 0·8μU/ml. Patients with non-toxic goitre and acromegaly have normal TSH levels. Values are always raised in hypothyroid patients (with primary thyroid disease) and are significantly lowered in those with hyperthyroidism. Of the many stimuli used in an attempt to raise TSH levels in normal adult subjects only three—synthetic thyrotrophin-releasing hormone, ethinyloestradiol, and carbimazole plus iodides—have been effective. The major clinical application of the TSH immunoassay lies in the diagnosis of minor degrees of hypothyroidism. An impaired response of serum TSH to synthetic thyrotrophin-releasing hormone should also help in the diagnosis of hypopituitarism affecting TSH production. PMID:5548300

Reversible covalent binding of neratinib to human serum albumin in vitro.

PubMed

Chandrasekaran, Appavu; Shen, Li; Lockhead, Susan; Oganesian, Aram; Wang, Jianyao; Scatina, JoAnn

2010-12-01

Neratinib (HKI-272), an irreversible inhibitor of Her 2 tyrosine kinase, is currently in development as an alternative for first and second line therapy in metastatic breast cancer patients who overexpress Her 2. Following incubation of [(14)C]neratinib in control human plasma at 37°C for 6 hours, about 60% to 70% of the radioactivity was not extractable, due to covalent binding to albumin. In this study, factors that could potentially affect the covalent binding of neratinib to plasma proteins, specifically to albumin were investigated. When [(14)C]neratinib was incubated at 10 μg/mL in human serum albumin (HSA) or control human plasma, the percent binding increased with time; the highest percentages of binding (46 and 67%, respectively) were observed at 6 hours, the longest duration of incubation examined. Binding increased with increasing temperature; the highest percentages of binding to HSA or human plasma (59 and 78%) were observed at 45°C, the highest temperature tested. The binding also increased with increasing pH of incubation; the highest percentages of binding (56 and 65%) were observed at pH 8.5, the highest pH value tested. The percentages of binding were similar (53% to 57%) when a wide range of concentrations of [(14)C]neratinib (50 ng/mL to 10 μg/mL) were incubated with human plasma at 37°C for 6 hours, indicating that the binding was independent of the substrate concentration, especially in the therapeutic range (50 to 200 ng/mL). When human plasma proteins containing covalently bound [(14)C]neratinb were suspended in a 10 fold volume of phosphate buffer at pH 4.0, 6.0, 7.4, and 8.5, and further incubated at 37°C for ~ 16 hours, about 45%, 44%, 32%, and 12% of the total radioactivity, respectively, was released as unchanged [(14)C]neratinib, indicating that the binding is reversible in nature, with more released at pH 7.4 and below. In conclusion, the covalent binding of neratinib to serum albumin is pH, time and temperature dependent, but

Doxorubicin-loaded glycyrrhetinic acid modified recombinant human serum albumin nanoparticles for targeting liver tumor chemotherapy.

PubMed

Qi, Wen-Wen; Yu, Hai-Yan; Guo, Hui; Lou, Jun; Wang, Zhi-Ming; Liu, Peng; Sapin-Minet, Anne; Maincent, Philippe; Hong, Xue-Chuan; Hu, Xian-Ming; Xiao, Yu-Ling

2015-03-02

Due to overexpression of glycyrrhetinic acid (GA) receptor in liver cancer cells, glycyrrhetinic acid modified recombinant human serum albumin (rHSA) nanoparticles for targeting liver tumor cells may result in increased therapeutic efficacy and decreased adverse effects of cancer therapy. In this study, doxorubicin (DOX) loaded and glycyrrhetinic acid modified recombinant human serum albumin nanoparticles (DOX/GA-rHSA NPs) were prepared for targeting therapy for liver cancer. GA was covalently coupled to recombinant human serum albumin nanoparticles, which could efficiently deliver DOX into liver cancer cells. The resultant GA-rHSA NPs exhibited uniform spherical shape and high stability in plasma with fixed negative charge (∼-25 mV) and a size about 170 nm. DOX was loaded into GA-rHSA NPs with a maximal encapsulation efficiency of 75.8%. Moreover, the targeted NPs (DOX/GA-rHSA NPs) showed increased cytotoxic activity in liver tumor cells compared to the nontargeted NPs (DOX/rHSA NPs, DOX loaded recombinant human serum albumin nanoparticles without GA conjugating). The targeted NPs exhibited higher cellular uptake in a GA receptor-positive liver cancer cell line than nontargeted NPs as measured by both flow cytometry and confocal laser scanning microscopy. Biodistribution experiments showed that DOX/GA-rHSA NPs exhibited a much higher level of tumor accumulation than nontargeted NPs at 1 h after injection in hepatoma-bearing Balb/c mice. Therefore, the DOX/GA-rHSA NPs could be considered as an efficient nanoplatform for targeting drug delivery system for liver cancer.

Estimation of polyclonal IgG4 hybrids in normal human serum.

PubMed

Young, Elizabeth; Lock, Emma; Ward, Douglas G; Cook, Alexander; Harding, Stephen; Wallis, Gregg L F

2014-07-01

The in vivo or in vitro formation of IgG4 hybrid molecules, wherein the immunoglobulins have exchanged half molecules, has previously been reported under experimental conditions. Here we estimate the incidence of polyclonal IgG4 hybrids in normal human serum and comment on the existence of IgG4 molecules with different immunoglobulin light chains. Polyclonal IgG4 was purified from pooled or individual donor human sera and sequentially fractionated using light-chain affinity and size exclusion chromatography. Fractions were analysed by SDS-PAGE, immunoblotting, ELISA, immunodiffusion and matrix-assisted laser-desorption mass spectrometry. Polyclonal IgG4 purified from normal serum contained IgG4κ, IgG4λ and IgG4κ/λ molecules. Size exclusion chromatography showed that IgG4 was principally present in monomeric form (150 000 MW). SDS-PAGE, immunoblotting and ELISA showed the purity of the three IgG4 samples. Immunodiffusion, light-chain sandwich ELISA and mass spectrometry demonstrated that both κ and λ light chains were present on only the IgG4κ/λ molecules. The amounts of IgG4κ/λ hybrid molecules ranged from 21 to 33% from the five sera analysed. Based on the molecular weight these molecules were formed of two IgG4 heavy chains plus one κ and one λ light chain. Polyclonal IgG (IgG4-depleted) was similarly fractionated according to light-chain specificity. No evidence of hybrid IgG κ/λ antibodies was observed. These results indicate that hybrid IgG4κ/λ antibodies compose a substantial portion of IgG4 from normal human serum. © 2014 John Wiley & Sons Ltd.

Micro-bead injection spectroscopy for label-free automated determination of immunoglobulin G in human serum.

PubMed

Ramos, Inês I; Magalhães, Luís M; Barreiros, Luisa; Reis, Salette; Lima, José L F C; Segundo, Marcela A

2018-01-01

Immunoglobulin G (IgG) represents the major fraction of antibodies in healthy adult human serum, and deviations from physiological levels are a generic marker of disease corresponding to different pathologies. Therefore, screening methods for IgG evaluation are a valuable aid to diagnostics. The present work proposes a rapid, automatic, and miniaturized method based on UV-vis micro-bead injection spectroscopy (μ-BIS) for the real-time determination of human serum IgG with label-free detection. Relying on attachment of IgG in rec-protein G immobilized in Sepharose 4B, a bioaffinity column is automatically assembled, where IgG is selectively retained and determined by on-column optical density measurement. A "dilution-and-shoot" approach (50 to 200 times) was implemented without further sample treatment because interferences were flushed out of the column upon sample loading, with minimization of carryover and cross-contamination by automatically discarding the sorbent (0.2 mg) after each determination. No interference from human serum albumin at 60 mg mL -1 in undiluted sample was found. The method allowed IgG determination in the range 100-300 μg mL -1 (corresponding to 5.0-60 mg mL -1 in undiluted samples), with a detection limit of 33 μg mL -1 (1.7 mg mL -1 for samples, dilution factor of 50). RSD values were < 9.4 and < 11.7%, for intra and inter-assay precision, respectively, while recovery values for human serum spiked with IgG at high pathological levels were 97.8-101.4%. Comparison to commercial ELISA kit showed no significant difference for tested samples (n = 8). Moreover, time-to-result decreased from several hours to < 5 min and analysis cost decreased 10 times, showing the potential of the proposed approach as a point-of-care method. Graphical abstract Micro-Bead Injection Spectroscopy method for real time, automated and label-free determination of total serum human Immunoglobulin G (IgG). The method was designed for Lab

Parathyroid hormone related protein concentration in human serum and CSF correlates with age.

PubMed

Kushnir, Mark M; Peterson, Lisa K; Strathmann, Frederick G

2018-02-01

Parathyroid Hormone-Related Protein (PTHrP) is involved in intracellular calcium (Ca) regulation, and has been demonstrated to participate in regulation of Ca in brain cells, activation of neurons, and modulation of pain. However, there are conflicting reports regarding the presence of PTHrP in CSF. PTHrP and Ca were quantified in paired CSF and serum samples using mass spectrometry-based methods. Associations between PTHrP and Ca concentrations with age, sex and concentrations of nine CSF diagnostic markers in a set of 140 paired serum and CSF patient samples were evaluated. The observed median PTHrP concentration in CSF was 51 times higher than in serum; the median concentration of Ca in CSF was 1.8 times lower than in serum. We observed positive correlation between concentrations of PTHrP in CSF and serum (p=0.013). Distribution of PTHrP concentrations in serum was associated with age (p=0.0068) and the concentrations were higher in women. In samples with serum calcium concentrations within the reference intervals (n=118), central 95% distribution of concentrations for Ca-CSF, PTHrP-serum and PTHrP-CSF were 5.4 (4.5-6.1) mg/dL, 1.2 (0.5-2.5) pmol/L, 62 (22-125) pmol/L, respectively. Our data demonstrate that PTHrP is a normal constituent of human CSF with median concentrations 51 fold higher than in serum. Elevated serum PTHrP concentrations were positively correlated with age and significantly higher in women. Our data suggest that CSF could be a significant source of circulating PTHrP. Copyright © 2017 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Spectral Fluorescence Properties of an Anionic Oxacarbocyanine Dye in Complexes with Human Serum Albumin

NASA Astrophysics Data System (ADS)

Pronkin, P. G.; Tatikolov, A. S.

2015-07-01

The spectral fluorescence properties of the anionic oxacarbocyanine dye 3,3'-di-(γ-sulfopropyl)-5,5'-diphenyl-9-ethyloxacarbocyanine betaine (OCC) were studied in solutions and in complexes with human serum albumin (HSA). Interaction with HSA leads to a significant increase in the fluorescence of the dye. We studied quenching of the fluorescence of OCC in a complex with HSA by ibuprofen and warfarin. Data on quenching of fluorescence by ibuprofen indicate binding of the dye to binding site II of subdomain IIIA in the HSA molecule. Synchronous fluorescence spectra of human serum albumin in the presence of OCC showed that complexation with OCC does not lead to appreciable rearrangement of the protein molecule at the binding site.

The Human Serum Metabolome of Vitamin B-12 Deficiency and Repletion, and Associations with Neurological Function in Elderly Adults.

PubMed

Brito, Alex; Grapov, Dmitry; Fahrmann, Johannes; Harvey, Danielle; Green, Ralph; Miller, Joshua W; Fedosov, Sergey N; Shahab-Ferdows, Setareh; Hampel, Daniela; Pedersen, Theresa L; Fiehn, Oliver; Newman, John W; Uauy, Ricardo; Allen, Lindsay H

2017-08-09

Background: The specific metabolomic perturbations that occur in vitamin B-12 deficiency, and their associations with neurological function, are not well characterized. Objective: We sought to characterize the human serum metabolome in subclinical vitamin B-12 deficiency and repletion. Methods: A before-and-after treatment study provided 1 injection of 10 mg vitamin B-12 (with 100 mg pyridoxine and 100 mg thiamin) to 27 community-dwelling elderly Chileans (∼74 y old) with vitamin B-12 deficiency, as evaluated with serum vitamin B-12, total plasma homocysteine (tHcy), methylmalonic acid (MMA), and holotranscobalamin. The combined indicator of vitamin B-12 status (cB-12) was computed. Targeted metabolites [166 acylcarnitines, amino acids, sugars, glycerophospholipids, and sphingolipids (liquid chromatography-tandem mass spectrometry)], and untargeted metabolites [247 chemical entities (gas chromatography time-of-flight mass spectrometry)] were measured at baseline and 4 mo after treatment. A peripheral nerve score was developed. Differences before and after treatment were examined. For targeted metabolomics, the data from 18 individuals with adequate vitamin B-12 status (selected from the same population) were added to the before-and-after treatment data set. Network visualizations and metabolic pathways are illustrated. Results: The injection increased serum vitamin B-12, holotranscobalamin, and cB-12 ( P < 0.001), and reduced tHcy and serum MMA ( P < 0.001). Metabolomic changes from before to after treatment included increases ( P < 0.001) in acylcarnitines, plasmalogens, and other phospholipids, whereas proline and other intermediaries of one-carbon metabolism-that is, methionine and cysteine-were reduced ( P < 0.001). Direct significant correlations ( P < 0.05 after the false discovery rate procedure) were identified between acylcarnitines, plasmalogens, phospholipids, lyso-phospholipids, and sphingomyelins compared with vitamin B-12 status and nerve function

Maternal biochemical serum screening for Down syndrome in pregnancy with human immunodeficiency virus infection.

PubMed

Le Meaux, Jean-Patrick; Tsatsaris, Vassilis; Schmitz, Thomas; Fulla, Yvonne; Launay, Odile; Goffinet, François; Azria, Elie

2008-08-01

To estimate the influence of human immunodeficiency virus (HIV) infection and antiretroviral therapy on maternal serum markers levels and the false-positive rate with biochemical maternal serum screening for Down syndrome. We performed a 1:1 matched case-control study comparing 132 HIV-infected women with single pregnancy to controls selected among non-HIV-infected women matched on geographical origin and fetal sex. Of HIV-infected women, 47.7% were receiving antiretroviral therapy. Groups did not differ in multiples of the median (MoM) levels of total human chorionic gonadotrophin. The MoM alpha fetoprotein level did not differ between total HIV-infected women and control women but was significantly lower for untreated HIV-positive women compared with control women (0.91 compared with 1.03 MoM, P<.01) and compared with treated HIV-positive women (0.91 compared with 1.18 MoM, P<.01). The false-positive rate of biochemical screening did not differ between groups. Untreated HIV infection is associated with lower maternal serum alpha fetoprotein levels. Nevertheless, the false-positive rate of double-marker second-trimester Down syndrome serum screening did not appear to be affected in our sample of HIV-infected women, whether women were receiving antiretroviral therapy at the time of the test or not.

Serum-deprived human multipotent mesenchymal stromal cells (MSCs) are highly angiogenic

PubMed Central

Oskowitz, Adam; McFerrin, Harris; Gutschow, Miriam; Carter, Mary Leita; Pochampally, Radhika

2016-01-01

Recent reports have indicated that mesenchymal stromal cells (MSCs) from bone marrow have a potential in vascular remodeling and angiogenesis. Here, we report a unique phenomenon that under serum-deprived conditions MSCs survive and replicate. Secretome analysis of MSCs grown under serum-deprived conditions (SD-MSCs) identified a significant upregulation of prosurvival and angiogenic factors including VEGF-A, ANGPTs, IGF-1, and HGF. An ex vivo rat aortic assay demonstrated longer neovascular sprouts generated from rat aortic rings cultured in SD-MSC-conditioned media compared to neovascular sprouts from aortas grown in MSC-conditioned media. With prolonged serum deprivation, a subpopulation of SD-MSCs began to exhibit an endothelial phenotype. This population expressed endothelial-specific proteins including VEGFR2, Tie2/TEK, PECAM/CD31, and eNOS and also demonstrated the ability to uptake acetylated LDL. SD-MSCs also exhibited enhanced microtubule formation in an in vitro angiogenesis assay. Modified chick chorioallantoic membrane (CAM) angiogenesis assays showed significantly higher angiogenic potential for SD-MSCs compared to MSCs. Analysis of CAMs grown with SD-MSCs identified human-specific CD31-positive cells in vascular structures. We conclude that under the stress of serum deprivation MSCs are highly angiogenic and a population of these cells has the potential to differentiate into endothelial-like cells. PMID:21421339

The human serum metabolome of vitamin B-12 deficiency and repletion, and associations with neurological function

USDA-ARS?s Scientific Manuscript database

We characterize the human serum metabolome in sub-clinical vitamin B-12 (B-12) deficiency and repletion. A pre-post treatment study provided one injection of 10 mg B-12 to 27 community-dwelling elderly Chileans with B-12 deficiency evaluated with serum B-12, plasma homocysteine, methylmalonic acid a...

Human Ozone (O3) Exposure Alters Serum Profile of Lipid Metabolites

EPA Science Inventory

HUMAN OZONE (O3) EXPOSURE ALTERS SERUM PROFILE OF LIPID METABOLITES Miller, D B.1; Kodavanti, U P.2 Karoly, E D.3; Cascio W.E2, Ghio, A J. 21. UNC-Chapel Hill, Chapel Hill, N.C., United States. 2. NHEERL, U.S. EPA, RTP, N.C., United States. 3. METABOLON INC., Durham, N.C., United...

A spectroscopic and molecular docking approach on the binding of tinzaparin sodium with human serum albumin

NASA Astrophysics Data System (ADS)

Abdullah, Saleh M. S.; Fatma, Sana; Rabbani, Gulam; Ashraf, Jalaluddin M.

2017-01-01

Protein bound toxins are poorly removed by conventional extracorporeal therapies. Venous thromboembolism (VTE) is a major cause of morbidity and mortality in patients with cancer. The interaction between tinzaparin, an inhibitor of angiotensin converting enzyme and human serum albumin, a principal plasma protein in the liver has been investigated in vitro under a simulated physiological condition by UV-vis spectrophotometry and fluorescence spectrometry. The intrinsic fluorescence intensity of human serum albumin was strongly quenched by tinzaparin (TP). The binding constants and binding stoichiometry can be calculated from the data obtained from fluorescence quenching experiments. The negative value of ΔG° reveals that the binding process is a spontaneous process. Thermodynamic analysis shows that the HSA-TP complex formation occurs via hydrogen bonds, hydrophobic interactions and undergoes slight structural changes as evident by far-UV CD. It indicated that the hydrophobic interactions play a main role in the binding of TP to human serum albumin. In addition, the distance between TP (acceptor) and tryptophan residues of human serum albumin (donor) was estimated to be 2.21 nm according to the Förster's resonance energy transfer theory. For the deeper understanding of the interaction, thermodynamic, and molecular docking studies were performed as well. Our docking results suggest that TP forms stable complex with HSA (Kb ∼ 104) and its primary binding site is located in subdomain IIA (Sudlow Site I). The results obtained herein will be of biological significance in pharmacology and clinical medicine.

Correlation between model observers in uniform background and human observers in patient liver background for a low-contrast detection task in CT

NASA Astrophysics Data System (ADS)

Gong, Hao; Yu, Lifeng; Leng, Shuai; Dilger, Samantha; Zhou, Wei; Ren, Liqiang; McCollough, Cynthia H.

2018-03-01

Channelized Hotelling observer (CHO) has demonstrated strong correlation with human observer (HO) in both single-slice viewing mode and multi-slice viewing mode in low-contrast detection tasks with uniform background. However, it remains unknown if the simplest single-slice CHO in uniform background can be used to predict human observer performance in more realistic tasks that involve patient anatomical background and multi-slice viewing mode. In this study, we aim to investigate the correlation between CHO in a uniform water background and human observer performance at a multi-slice viewing mode on patient liver background for a low-contrast lesion detection task. The human observer study was performed on CT images from 7 abdominal CT exams. A noise insertion tool was employed to synthesize CT scans at two additional dose levels. A validated lesion insertion tool was used to numerically insert metastatic liver lesions of various sizes and contrasts into both phantom and patient images. We selected 12 conditions out of 72 possible experimental conditions to evaluate the correlation at various radiation doses, lesion sizes, lesion contrasts and reconstruction algorithms. CHO with both single and multi-slice viewing modes were strongly correlated with HO. The corresponding Pearson's correlation coefficient was 0.982 (with 95% confidence interval (CI) [0.936, 0.995]) and 0.989 (with 95% CI of [0.960, 0.997]) in multi-slice and single-slice viewing modes, respectively. Therefore, this study demonstrated the potential to use the simplest single-slice CHO to assess image quality for more realistic clinically relevant CT detection tasks.

Determination of miloxacin and metabolites in human serum and urine by high-pressure liquid chromatography.

PubMed Central

Yoshitake, A; Kawahara, K; Shono, F; Umeda, I; Izawa, A; Komatsu, T

1980-01-01

A sensitive and reliable high-pressure liquid chromatography (HPLC) assay for miloxacin and its two principal metabolites, 5,8-dihydro-8-oxo-2H-1,3-dioxolo[4,5-g]quinoline-7-carboxylic acid (M-1) and 1,4-dihydro-1,6-dimethoxy-7-hydroxy-4-oxoquinoline-3-carboxylic acid (M-2), in human serum and urine was developed. A strong anion-exchange Zipax SAX column using a mobile phase of 0.01 M citric acid solution containing 0.03 M sodium nitrate with pH 5.0 was used to achieve separation of the three compounds. The retention times of miloxacin, M-1, and M-2 were 3.8, 9.3, and 5.9 min, respectively. Serum and urine concentrations of these compounds as low as 10 ng/ml were measured. When results from the HPLC assay were compared with those from the microbiological assay of serum and urine samples from human subjects receiving miloxacin orally, the correlation coefficients were 0.94 for the serum and 0.99 for the urine. The HPLC assay method presents an alternative to the microbiological assay and permits future pharmacokinetic investigations of miloxacin. PMID:7416751

Surface modification of PLGA nanoparticles via human serum albumin conjugation for controlled delivery of docetaxel

PubMed Central

2013-01-01

Background Poly lactic-co-glycolic acid (PLGA) based nanoparticles are considered to be a promising drug carrier in tumor targeting but suffer from the high level of opsonization by reticuloendothelial system due to their hydrophobic structure. As a result surface modification of these nanoparticles has been widely studied as an essential step in their development. Among various surface modifications, human serum albumin (HSA) possesses advantages including small size, hydrophilic surface and accumulation in leaky vasculature of tumors through passive targeting and a probable active transport into tumor tissues. Methods PLGA nanoparticles of docetaxel were prepared by emulsification evaporation method and were surface conjugated with human serum albumin. Fourier transform infrared spectrum was used to confirm the conjugation reaction where nuclear magnetic resonance was utilized for conjugation ratio determination. In addition, transmission electron microscopy showed two different contrast media in conjugated nanoparticles. Furthermore, cytotoxicity of free docetaxel, unconjugated and conjugated PLGA nanoparticles was studied in HepG2 cells. Results Size, zeta potential and drug loading of PLGA nanoparticles were about 199 nm, −11.07 mV, and 4%, respectively where size, zeta potential and drug loading of conjugated nanoparticles were found to be 204 nm, −5.6 mV and 3.6% respectively. Conjugated nanoparticles represented a three-phasic release pattern with a 20% burst effect for docetaxel on the first day. Cytotoxicity experiment showed that the IC50 of HSA conjugated PLGA nanoparticles (5.4 μg) was significantly lower than both free docetaxel (20.2 μg) and unconjugated PLGA nanoparticles (6.2 μg). Conclusion In conclusion surface modification of PLGA nanoparticles through HSA conjugation results in more cytotoxicity against tumor cell lines compared with free docetaxel and unconjugated PLGA nanoparticles. Albumin conjugated PLGA nanoparticles may

Comprehensive identification of Vibrio vulnificus genes required for growth in human serum.

PubMed

Carda-Diéguez, M; Silva-Hernández, F X; Hubbard, T P; Chao, M C; Waldor, M K; Amaro, C

2018-12-31

Vibrio vulnificus can be a highly invasive pathogen capable of spreading from an infection site to the bloodstream, causing sepsis and death. To survive and proliferate in blood, the pathogen requires mechanisms to overcome the innate immune defenses and metabolic limitations of this host niche. We created a high-density transposon mutant library in YJ016, a strain representative of the most virulent V. vulnificus lineage (or phylogroup) and used transposon insertion sequencing (TIS) screens to identify loci that enable the pathogen to survive and proliferate in human serum. Initially, genes underrepresented for insertions were used to estimate the V. vulnificus essential gene set; comparisons of these genes with similar TIS-based classification of underrepresented genes in other vibrios enabled the compilation of a common Vibrio essential gene set. Analysis of the relative abundance of insertion mutants in the library after exposure to serum suggested that genes involved in capsule biogenesis are critical for YJ016 complement resistance. Notably, homologues of two genes required for YJ016 serum-resistance and capsule biogenesis were not previously linked to capsule biogenesis and are largely absent from other V. vulnificus strains. The relative abundance of mutants after exposure to heat inactivated serum was compared with the findings from the serum screen. These comparisons suggest that in both conditions the pathogen relies on its Na + transporting NADH-ubiquinone reductase (NQR) complex and type II secretion system to survive/proliferate within the metabolic constraints of serum. Collectively, our findings reveal the potency of comparative TIS screens to provide knowledge of how a pathogen overcomes the diverse limitations to growth imposed by serum.

24-hour human urine and serum profiles of bisphenol A: Evidence against sublingual absorption following ingestion in soup

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

Teeguarden, Justin G., E-mail: jt@pnl.gov; Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, OR 93771; Twaddle, Nathan C., E-mail: nathan.twaddle@fda.hhs.gov

Extensive first-pass metabolism of ingested bisphenol A (BPA) in the gastro-intestinal tract and liver restricts blood concentrations of bioactive BPA to < 1% of total BPA in humans and non-human primates. Absorption of ingested BPA through non-metabolizing tissues of the oral cavity, recently demonstrated in dogs, could lead to the higher serum BPA concentrations reported in some human biomonitoring studies. We hypothesized that the extensive interaction with the oral mucosa by a liquid matrix, like soup, relative to solid food or capsules, might enhance absorption through non-metabolizing oral cavity tissues in humans, producing higher bioavailability and higher serum BPA concentrations.more » Concurrent serum and urine concentrations of d6-BPA, and its glucuronide and sulfate conjugates, were measured over a 24 hour period in 10 adult male volunteers following ingestion of 30 μg d6-BPA/kg body weight in soup. Absorption of d6-BPA was rapid (t{sub 1/2} = 0.45 h) and elimination of the administered dose was complete 24 h post-ingestion, evidence against any tissue depot for BPA. The maximum serum d6-BPA concentration was 0.43 nM at 1.6 h after administration and represented < 0.3% of total d6-BPA. Pharmacokinetic parameters, pharmacokinetic model simulations, and the significantly faster appearance half-life of d6-BPA-glucuronide compared to d6-BPA (0.29 h vs 0.45 h) were evidence against meaningful absorption of BPA in humans through any non-metabolizing tissue (< 1%). This study confirms that typical exposure to BPA in food produces picomolar to subpicomolar serum BPA concentrations in humans, not nM concentrations reported in some biomonitoring studies.« less

24-hour human urine and serum profiles of bisphenol A: Evidence against sublingual absorption following ingestion in soup.

PubMed

Teeguarden, Justin G; Twaddle, Nathan C; Churchwell, Mona I; Yang, Xiaoxia; Fisher, Jeffrey W; Seryak, Liesel M; Doerge, Daniel R

2015-10-15

Extensive first-pass metabolism of ingested bisphenol A (BPA) in the gastro-intestinal tract and liver restricts blood concentrations of bioactive BPA to <1% of total BPA in humans and non-human primates. Absorption of ingested BPA through non-metabolizing tissues of the oral cavity, recently demonstrated in dogs, could lead to the higher serum BPA concentrations reported in some human biomonitoring studies. We hypothesized that the extensive interaction with the oral mucosa by a liquid matrix, like soup, relative to solid food or capsules, might enhance absorption through non-metabolizing oral cavity tissues in humans, producing higher bioavailability and higher serum BPA concentrations. Concurrent serum and urine concentrations of d6-BPA, and its glucuronide and sulfate conjugates, were measured over a 24hour period in 10 adult male volunteers following ingestion of 30μg d6-BPA/kg body weight in soup. Absorption of d6-BPA was rapid (t1/2=0.45h) and elimination of the administered dose was complete 24h post-ingestion, evidence against any tissue depot for BPA. The maximum serum d6-BPA concentration was 0.43nM at 1.6h after administration and represented <0.3% of total d6-BPA. Pharmacokinetic parameters, pharmacokinetic model simulations, and the significantly faster appearance half-life of d6-BPA-glucuronide compared to d6-BPA (0.29h vs 0.45h) were evidence against meaningful absorption of BPA in humans through any non-metabolizing tissue (<1%). This study confirms that typical exposure to BPA in food produces picomolar to subpicomolar serum BPA concentrations in humans, not nM concentrations reported in some biomonitoring studies. Published by Elsevier Inc.

Functionalizable and ultra stable nanoparticles coated with zwitterionic poly(carboxybetaine) in undiluted blood serum.

PubMed

Yang, Wei; Zhang, Lei; Wang, Shanlin; White, Andrew D; Jiang, Shaoyi

2009-10-01

A new surface chemistry presenting an abundance of functional groups for ligand immobilization in an ultra-low fouling background all in one material for nanoparticles was introduced. This surface platform, as demonstrated by zwitterionic poly(carboxybetaine acrylamide) (polyCBAA) coated nanoparticles, is not only ultra stable in undiluted human blood serum, but also can be conjugated to biomolecules conveniently and effectively. Thus, this surface chemistry is ideal to create multi-functional nanoparticles for targeted delivery and diagnostics. In addition, this work clearly shows that 10% blood serum commonly used to evaluate the stability of nanoparticles is insufficient and a new evaluation criterion with undiluted blood serum is recommended.

Effect of storage duration on cytokine stability in human serum and plasma.

PubMed

Vincent, Fabien B; Nim, Hieu T; Lee, Jacinta P W; Morand, Eric F; Harris, James

2018-06-14

Quantification of analytes such as cytokines in serum samples is intrinsic to translational research in immune diseases. Optimising pre-analytical conditions is critical for ensuring study quality, including evaluation of cytokine stability. We aimed to evaluate the effect on cytokine stability of storage duration prior to freezing of serum, and compare to plasma samples obtained from patients with systemic lupus erythematosus (SLE). Protein stability was analysed by simultaneously quantifying 18 analytes using a custom multi-analyte profile in SLE patient serum and plasma samples that had been prospectively stored at 4 °C for pre-determined periods between 0 and 30 days, prior to freezing. Six analytes were excluded from analysis, because most tested samples were above or below the limit of detection. Amongst the 12 analysed proteins, 11 did not show significant signal degradation. Significant signal degradation was observed from the fourth day of storage for a single analyte, CCL19. Proteins levels were more stable in unseparated serum compared to plasma for most analytes, with the exception of IL-37 which appears slightly more stable in plasma. Based on this, a maximum 3 days of storage at 4 °C for unseparated serum samples is recommended for biobanked samples intended for cytokine analysis in studies of human immune disease. Copyright © 2018 Elsevier Ltd. All rights reserved.

Effect of resistant and digestible rice starches on human cytokine and lactate metabolic networks in serum.

PubMed

Wang, Huisong; Pang, Guangchang

2017-05-01

Resistant starch generated after treating ordinary starch is of great significance to human health in the countries with overnutrition. However, its functional evaluation in the human body has been rarely reported. By determining the lactate metabolic flux, 12 serum enzymes expression level and 38 serum cytokines in healthy volunteers, the variation in cytokine network and lactate metabolic network in serum were investigated to compare the mechanism of the physiological effects between the two starches. The results indicated that compared with digestible starch, resistant starch had anti-inflammatory effects, increased anabolism, and decreased catabolism. Further, the intercellular communication networks including cytokine and lactate metabolic networks were mapped out. The relationship suggested that resistant starch might affect and control the secretion of cytokines to regulate lactate metabolic network in the body, promoting the development of immunometabolism. Copyright © 2017 Elsevier Ltd. All rights reserved.

Human Growth Factor Cream and Hyaluronic Acid Serum in Conjunction with Micro Laser Peel

PubMed Central

Katz, Bruce E.; Cohen, Joel L.; Biron, Julie

2010-01-01

The present study investigated the use of a novel hyaluronic acid serum in combination with a cream comprising a mixture of human growth factors in conjunction with the micro laser peel procedure for skin rejuvenation. After preconditioning the face with the hyaluronic acid serum followed by the cream twice daily for one month, 15 female volunteers between 35 to 65 years of age with demonstrable facial wrinkling received a micro laser peel on the entire face using an erbium-doped yttrium aluminium garnet laser. Immediately following the laser procedure, the subjects applied the test products twice daily until the second laser peel one month later. Immediately following the second procedure, the subjects reapplied the test products for another month. In the large majority of subjects, erythema or edema, crusts or erosions, and transitory stinging or burning sensations after the micro laser peel were minimal or mild when the skin was treated with the serum followed by the cream. The micro laser peel in conjunction with the test products helped to significantly improve hyperpigmentation, wrinkles, and texture as compared to before treatment. This study with the micro laser peel device demonstrated that a novel hyaluronic acid serum combined with the human growth factor cream can be successfully used for skin rejuvenation in conjunction with light-to-medium invasive laser skin treatments. PMID:21203354

Spectroscopic and molecular modeling studies of the interaction between cytidine and human serum albumin and its analytical application.

PubMed

Cui, Fengling; Wang, Junli; Yao, Xiaojun; Wang, Li; Zhang, Qiangzhai; Qu, Guirong

In this study, the interaction between cytidine and human serum albumin (HSA) was investigated for the first time by fluorescence spectroscopy in combination with UV absorption spectrum and molecular modeling under simulative physiological conditions. Experimental results indicated that cytidine had a strong ability to quench the intrinsic fluorescence of human serum albumin. The binding constants (K) at different temperatures, thermodynamic parameter enthalpy changes (DeltaH) and entropy changes (DeltaS) of HSA-cytidine had been calculated according to the relevant fluorescence data, which indicated that the hydrophobic and electrostatic interactions played a major role, which was in agreement with the results of molecular modeling study. In addition, the effects of other ions on the binding constants were also studied. Furthermore, synchronous fluorescence technology was successfully applied to the determination of human serum albumin added into the cytidine solution.

Serum Stabilities of Short Tryptophan- and Arginine-Rich Antimicrobial Peptide Analogs

PubMed Central

Nguyen, Leonard T.; Chau, Johnny K.; Perry, Nicole A.; de Boer, Leonie; Zaat, Sebastian A. J.; Vogel, Hans J.

2010-01-01

Background Several short antimicrobial peptides that are rich in tryptophan and arginine residues were designed with a series of simple modifications such as end capping and cyclization. The two sets of hexapeptides are based on the Trp- and Arg-rich primary sequences from the “antimicrobial centre” of bovine lactoferricin as well as an antimicrobial sequence obtained through the screening of a hexapeptide combinatorial library. Methodology/Principal Findings HPLC, mass spectrometry and antimicrobial assays were carried out to explore the consequences of the modifications on the serum stability and microbicidal activity of the peptides. The results show that C-terminal amidation increases the antimicrobial activity but that it makes little difference to its proteolytic degradation in human serum. On the other hand, N-terminal acetylation decreases the peptide activities but significantly increases their protease resistance. Peptide cyclization of the hexameric peptides was found to be highly effective for both serum stability and antimicrobial activity. However the two cyclization strategies employed have different effects, with disulfide cyclization resulting in more active peptides while backbone cyclization results in more proteolytically stable peptides. However, the benefit of backbone cyclization did not extend to longer 11-mer peptides derived from the same region of lactoferricin. Mass spectrometry data support the serum stability assay results and allowed us to determine preferred proteolysis sites in the peptides. Furthermore, isothermal titration calorimetry experiments showed that the peptides all had weak interactions with albumin, the most abundant protein in human serum. Conclusions/Significance Taken together, the results provide insight into the behavior of the peptides in human serum and will therefore aid in advancing antimicrobial peptide design towards systemic applications. PMID:20844765

Reduced Neurite Density in Neuronal Cell Cultures Exposed to Serum of Patients with Bipolar Disorder

PubMed Central

Wollenhaupt-Aguiar, Bianca; Pfaffenseller, Bianca; Chagas, Vinicius de Saraiva; Castro, Mauro A A; Passos, Ives Cavalcante; Kauer-Sant’Anna, Márcia; Kapczinski, Flavio

2016-01-01

Background: Increased inflammatory markers and oxidative stress have been reported in serum among patients with bipolar disorder (BD). The aim of this study is to assess whether biochemical changes in the serum of patients induces neurotoxicity in neuronal cell cultures. Methods: We challenged the retinoic acid-differentiated human neuroblastoma SH-SY5Y cells with the serum of BD patients at early and late stages of illness and assessed neurite density and cell viability as neurotoxic endpoints. Results: Decreased neurite density was found in neurons treated with the serum of patients, mostly patients at late stages of illness. Also, neurons challenged with the serum of late-stage patients showed a significant decrease in cell viability. Conclusions: Our findings showed that the serum of patients with bipolar disorder induced a decrease in neurite density and cell viability in neuronal cultures. PMID:27207915

Serum osteopontin concentration is decreased by exercise-induced fat loss but is not correlated with body fat percentage in obese humans.

PubMed

You, Jeong Soon; Ji, Hye-In; Chang, Kyung Ja; Yoo, Myung Chul; Yang, Hyung-In; Jeong, In-Kyung; Kim, Kyoung Soo

2013-08-01

To evaluate the extent to which fat mass contributes to serum osteopontin (OPN) concentration, we investigated whether serum OPN levels are decreased by exercise-induced fat mass loss and whether they are associated with body fat percentage in obese humans. Twenty‑three female college students were recruited to participate in an 8‑week body weight control program. Body composition [body weight, soft lean mass, body fat mass, body fat percentage, waist-hip ratio and body mass index (BMI)] were assessed prior to and following the program. Serum lipid profiles and serum adiponectin, leptin and osteopontin levels were measured from serum collected prior to and following the program. To understand the effect of fat mass loss on the serum levels of adipokine, which is mainly produced in adipose tissue, the leptin and adiponectin levels were also measured prior to and following the program. Serum leptin levels (mean ± standard error of the mean) decreased significantly following the program (from 9.82±0.98 to 7.23±0.67 ng/ml) and were closely correlated with body fat percentage. In addition, serum adiponectin levels were negatively correlated with body fat percentage, while serum adiponectin levels were not significantly altered. By contrast, serum OPN levels decreased significantly following the program (from 16.03±2.34 to 10.65±1.22 ng/ml). However, serum OPN levels were not correlated with body fat percentage, suggesting that serum OPN levels are controlled by several other factors in humans. In conclusion, a high expression of OPN in adipose tissues may not be correlated with serum OPN levels in obese humans. Thus, tissues or physiological factors other than fat mass may have a greater contribution to the serum OPN levels.

Screening the low molecular weight fraction of human serum using ATR-IR spectroscopy.

PubMed

Bonnier, Franck; Brachet, Guillaume; Duong, Romain; Sojinrin, Tobiloba; Respaud, Renaud; Aubrey, Nicolas; Baker, Matthew J; Byrne, Hugh J; Chourpa, Igor

2016-10-01

Vibrational spectroscopic techniques can detect small variations in molecular content, linked with disease, showing promise for screening and early diagnosis. Biological fluids, particularly blood serum, are potentially valuable for diagnosis purposes. The so-called Low Molecular Weight Fraction (LMWF) contains the associated peptidome and metabolome and has been identified as potentially the most relevant molecular population for disease-associated biomarker research. Although vibrational spectroscopy can deliver a specific chemical fingerprint of the samples, the High Molecular Weight Fraction (HMWF), composed of the most abundant serum proteins, strongly dominates the response and ultimately makes the detection of minor spectral variations a challenging task. Spectroscopic detection of potential serum biomarkers present at relatively low concentrations can be improved using pre-analytical depletion of the HMWF. In the present study, human serum fractionation by centrifugal filtration was used prior to analysis by Attenuated Total Reflection infrared spectroscopy. Using a model sample based on glycine spiked serum, it is demonstrated that the screening of the LMWF can be applied to quantify blinded concentrations up to 50 times lower. Moreover, the approach is easily transferable to different bodily fluids which would support the development of more efficient and suitable clinical protocols exploring vibrational spectroscopy based ex-vivo diagnostic tools. Revealing serum LMWF for spectral serological diagnostic applications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Direct determination of trace refractory elements in human serum by ETV-ICP-MS with in-situ matrix removal.

PubMed

Li, Shengqing; Hu, Bin; Jiang, Zucheng; Chen, Rui

2004-08-01

A method for in-situ removal of matrix is proposed for direct determination of trace refractory elements in human serum by ETV-ICP-MS with the use of poly(tetrafluoroethylene) (PTFE) as fluorinating reagent. Attention has been paid to investigating the vaporization behavior both of refractory elements of interest and of matrix elements (Na, K, Ca, Mg, Cl, S, and P) in a graphite furnace with the PTFE modifier present or not. It was shown that potential interferences from the organic and inorganic matrices in the serum sample could be eliminated or reduced to a negligible level by appropriate dilution of the serum and deliberate optimization of the ETV temperature program. The proposed method has been applied to the direct simultaneous determination of V, Cr, Mo, Ba, La, Ce, and W in human serum. The limits of detection for fivefold diluted serum were 0.18 (V), 0.229 (Cr), 0.050 (Mo), 0.328 (Ba), 0.031 (La), 0.038 (Ce), and 0.019 ng mL(-1) (W), respectively, and the relative standard deviations of the method were in the range 4-15% (2 ng mL(-1) in serum, n=3).

Rapid killing of Capnocytophaga canimorsus and Capnocytophaga cynodegmi by human whole blood and serum is mediated via the complement system.

PubMed

Zangenah, Salah; Bergman, Peter

2015-01-01

Capnocytophaga canimorsus (Cani) and Capnocytophaga cynodegmi (Cyno) are found in the oral cavities of dogs and cats. They can be transmitted to humans via licks or bites and cause wound infections as well as severe systemic infections. Cani is considered to be more pathogenic than Cyno, but the pathophysiological mechanisms are not elucidated. Cani has been suggested to be resistant to serum bactericidal effects. Thus, we hypothesized that the more invasive Cani would exhibit a higher degree of serum-resistance than the less pathogenic Cyno. Whole blood and serum bactericidal assays were performed against Cani- (n = 8) and Cyno-strains (n = 15) isolated from blood and wound-specimens, respectively. Analysis of complement-function was performed by heat-inactivation, EGTA-treatment and by using C1q-depleted serum. Serum and whole blood were collected from healthy individuals and from patients (n = 3) with a history of sepsis caused by Cani. Both Cani and Cyno were equally susceptible to human whole blood and serum. Cani was preferentially killed by the classical pathway of the complement-system whereas Cyno was killed by a partly different mechanism. Serum from 2/3 Cani-infected patients were deficient in MBL-activity but still exhibited the same killing effect as control sera. Both Cani and Cyno were readily killed by human whole blood and serum in a complement-dependent way. Thus, it is not likely that serum bactericidal capacity is the key determinant for the clinical outcome in Cani or Cyno-infections.

Assessment of serum pharmacokinetics and urinary excretion of albendazole and its metabolites in human volunteers.

PubMed

Ceballos, Laura; Krolewiecki, Alejandro; Juárez, Marisa; Moreno, Laura; Schaer, Fabian; Alvarez, Luis I; Cimino, Rubén; Walson, Judd; Lanusse, Carlos E

2018-01-01

Soil Transmitted Helminth (STH) infections negatively impact physical and mental development in human populations. Current WHO guidelines recommend morbidity control of these infections through mass drug administration (MDA) using albendazole (ABZ) or mebendazole. Despite major reductions in STH associated morbidity globally, not all programs have demonstrated the expected impact on prevalence of parasite infections. These therapeutic failures may be related to poor programmatic coverage, suboptimal adherence or the exposure of parasites to sub-therapeutic drug concentrations. As part of the DeWorm3 project, we sought to characterize the serum disposition kinetics and pattern of urinary excretion of ABZ and its main metabolites ABZ sulphoxide (ABZSO) and ABZ sulphone (ABZSO2) in humans, and the assessment of the duration and optimal time point where ABZ and/or its metabolites can be measured in urine as an indirect assessment of an individual's adherence to treatment. Consecutive venous blood and urine samples were collected from eight (8) human volunteers up to 72 h post-ABZ oral administration. ABZ/metabolites were quantified by HPLC. The ABZSO metabolite was the main analyte recovered both in serum and urine. ABZSO Cmax in serum was 1.20 ± 0.44 μg/mL, reached at 4.75 h post-treatment. In urine, ABZSO Cmax was 3.24 ± 1.51 μg/mL reached at 6.50 h post-ABZ administration. Pharmacokinetic data obtained for ABZ metabolites in serum and urine, including the recovery of the ABZ sulphoxide derivative up to 72 h in both matrixes and the recovery of the amino-ABZ sulphone metabolite in urine samples, are suggesting the possibility of developing a urine based method to assess compliance to ABZ treatment. Such an assay may be useful to optimize ABZ use in human patients. ClinicalTrials.gov NCT03192449.

In vitro biotransformation of tris(2-butoxyethyl) phosphate (TBOEP) in human liver and serum

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

Van den Eede, Nele, E-mail: nele.vandeneede@uantwerpen.be; Erratico, Claudio; Exarchou, Vassiliki

Tris(2-butoxyethyl) phosphate (TBOEP) is a plasticizer present in indoor dust, reaching levels of several micrograms per gram. Such levels could lead to significant daily exposure of adults and children. Currently, no toxicokinetic data are available to estimate TBOEP clearance in humans after uptake and therefore, one objective of this study was to investigate intrinsic clearance of TBOEP by human liver microsome (HLM) and serum enzymes. Another objective was to generate information to identify and prioritize several metabolites of TBOEP for investigation of human exposure by biomonitoring. 1D and 2D-NMR methodologies were successfully applied on a mixture of the metabolites tomore » confirm the structure of 3-HO-TBOEP (bis(2-butoxyethyl) 3-hydroxyl-2-butoxyethyl phosphate) and to tentatively assign structures to 1-HO-TBOEP and 2-HO-TBOEP. HO-TBOEP isomers and bis(2-butoxyethyl) phosphate (BBOEP), bis(2-butoxyethyl) hydroxyethyl phosphate (BBOEHEP) were further monitored by liquid chromatography–tandem mass spectrometry. Rates of formation of BBOEHEP and HO-TBOEP metabolites by liver enzymes were best described by the Michaelis–Menten model. Apparent K{sub m} values for BBOEHEP, 3-HO-TBOEP, and sum of 1- and 2-HO-TBOEP isomer formation were 152, 197 and 148 μM, respectively. Apparent V{sub max} values for the formation of BBOEHEP, 3-HO-TBOEP, and the sum of 1- and 2-HO-TBOEP isomers were 2560, 643, and 254 pmol/min/mg protein, respectively. No detectable formation of BBOEP occurred with liver or serum enzymes. Our findings indicate that intrinsic clearance of TBOEP is mainly catalyzed by oxidative enzymes in the liver and that its major in vitro metabolite is BBOEHEP. These findings can be applied in human biomonitoring studies and risk assessment. - Highlights: • First steps in the elucidation of TBOEP toxicokinetics • Quantification of TBOEP metabolites in human serum and liver microsomes • No detectable formation of BBOEP occurred with liver or

Ex Vivo Expansion of Human Mesenchymal Stem Cells in Defined Serum-Free Media

PubMed Central

Jung, Sunghoon; Panchalingam, Krishna M.; Rosenberg, Lawrence; Behie, Leo A.

2012-01-01

Human mesenchymal stem cells (hMSCs) are presently being evaluated for their therapeutic potential in clinical studies to treat various diseases, disorders, and injuries. To date, early-phase studies have indicated that the use of both autologous and allogeneic hMSCs appear to be safe; however, efficacy has not been demonstrated in recent late-stage clinical trials. Optimized cell bioprocessing protocols may enhance the efficacy as well as safety of hMSC therapeutics. Classical media used for generating hMSCs are typically supplemented with ill-defined supplements such as fetal bovine serum (FBS) or human-sourced alternatives. Ideally, culture media are desired to have well-defined serum-free formulations that support the efficient production of hMSCs while maintaining their therapeutic and differentiation capacity. Towards this objective, we review here current cell culture media for hMSCs and discuss medium development strategies. PMID:22645619

Displacement of Drugs from Human Serum Albumin: From Molecular Interactions to Clinical Significance.

PubMed

Rimac, Hrvoje; Debeljak, Željko; Bojić, Mirza; Miller, Larisa

2017-01-01

Human serum albumin (HSA) is the most abundant protein in human serum. It has numerous functions, one of which is transport of small hydrophobic molecules, including drugs, toxins, nutrients, hormones and metabolites. HSA has the ability to interact with a wide variety of structurally different compounds. This promiscuous, nonspecific affinity can lead to sudden changes in concentrations caused by displacement, when two or more compounds compete for binding to the same molecular site. It is important to consider drug combinations and their binding to HSA when defining dosing regimens, as this can directly influence drug's free, active concentration in blood. In present paper we review drug interactions with potential for displacement from HSA, situations in which they are likely to occur and their clinical significance. We also offer guidelines in designing drugs with decreased binding to HSA. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Human Blood Typing: A Forensic Science Approach. Part I: Background.

ERIC Educational Resources Information Center

Kobilinsky, Lawrence; Sheehan, Francis X.

1988-01-01

In this article, part I of a series, the forensic methods used in "typing" human blood, which as physical evidence is often found in the dried state, are outlined. Background information about individualization, antibody typing, fresh blood, dried blood, and additional systems is provided. (CW)

Rapidly Escalating Hepcidin and Associated Serum Iron Starvation Are Features of the Acute Response to Typhoid Infection in Humans

PubMed Central

Darton, Thomas C.; Blohmke, Christoph J.; Giannoulatou, Eleni; Waddington, Claire S.; Jones, Claire; Sturges, Pamela; Webster, Craig; Drakesmith, Hal; Pollard, Andrew J.; Armitage, Andrew E.

2015-01-01

Background Iron is a key pathogenic determinant of many infectious diseases. Hepcidin, the hormone responsible for governing systemic iron homeostasis, is widely hypothesized to represent a key component of nutritional immunity through regulating the accessibility of iron to invading microorganisms during infection. However, the deployment of hepcidin in human bacterial infections remains poorly characterized. Typhoid fever is a globally significant, human-restricted bacterial infection, but understanding of its pathogenesis, especially during the critical early phases, likewise is poorly understood. Here, we investigate alterations in hepcidin and iron/inflammatory indices following experimental human typhoid challenge. Methodology/Principal Findings Fifty study participants were challenged with Salmonella enterica serovar Typhi and monitored for evidence of typhoid fever. Serum hepcidin, ferritin, serum iron parameters, C-reactive protein (CRP), and plasma IL-6 and TNF-alpha concentrations were measured during the 14 days following challenge. We found that hepcidin concentrations were markedly higher during acute typhoid infection than at baseline. Hepcidin elevations mirrored the kinetics of fever, and were accompanied by profound hypoferremia, increased CRP and ferritin, despite only modest elevations in IL-6 and TNF-alpha in some individuals. During inflammation, the extent of hepcidin upregulation associated with the degree of hypoferremia. Conclusions/Significance We demonstrate that strong hepcidin upregulation and hypoferremia, coincident with fever and systemic inflammation, are hallmarks of the early innate response to acute typhoid infection. We hypothesize that hepcidin-mediated iron redistribution into macrophages may contribute to S. Typhi pathogenesis by increasing iron availability for macrophage-tropic bacteria, and that targeting macrophage iron retention may represent a strategy for limiting infections with macrophage-tropic pathogens such as S

Association between Nitrogen Stable Isotope Ratios in Human Hair and Serum Levels of Leptin.

PubMed

Ahn, Song Vogue; Koh, Sang-Baek; Lee, Kwang-Sik; Bong, Yeon-Sik; Park, Jong-Ku

2017-10-01

Stable isotope ratios have been reported to be potential biomarkers of dietary intake and nutritional status. High serum levels of leptin, a hormone which regulates energy metabolism and food intake, are associated with insulin resistance and metabolic syndrome. However, little is known about the association between stable isotope ratios and the metabolic risk in humans. We investigated whether the carbon and nitrogen stable isotope ratios in hair are associated with serum leptin levels. Hair samples were collected from 399 healthy adults (233 men and 166 women) aged 40 to 70 years of a community-based cohort in Korea and the bulk stable isotope ratios of carbon (δ 13 C) and nitrogen (δ 15 N) were measured for all hair samples. Serum leptin levels were analyzed by radioimmunoassay. δ 15 N showed positive correlations with serum leptin levels. In multivariate models, increasing δ 15 N were associated with elevated serum leptin levels (defined as ≥ the median values), whereas δ 13 C were not significantly associated with serum leptin levels. The odds ratio (95% confidence interval) per 1‰ increase in δ 15 N for an elevated serum leptin level was 1.58 (1.11-2.26). In participants with high body mass index, δ 15 N showed positive associations with serum leptin levels, whereas these associations were not seen in participants with low body mass index. The nitrogen stable isotopic ratio in hair is positively associated with serum leptin levels. The hair δ 15 N could be used as a clinical marker to estimate metabolic risk.

Evaluation of human platelet lysate versus fetal bovine serum for culture of mesenchymal stromal cells.

PubMed

Hemeda, Hatim; Giebel, Bernd; Wagner, Wolfgang

2014-02-01

Culture media for therapeutic cell preparations-such as mesenchymal stromal cells (MSCs)-usually comprise serum additives. Traditionally, fetal bovine serum is supplemented in basic research and in most clinical trials. Within the past years, many laboratories adapted their culture conditions to human platelet lysate (hPL), which further stimulates proliferation and expansion of MSCs. Particularly with regard to clinical application, human alternatives for fetal bovine serum are clearly to be preferred. hPL is generated from human platelet units by disruption of the platelet membrane, which is commonly performed by repeated freeze and thaw cycles. Such culture supplements are notoriously ill-defined, and many parameters contribute to batch-to-batch variation in hPL such as different amounts of plasma, a broad range of growth factors and donor-specific effects. The plasma components of hPL necessitate addition of anticoagulants such as heparins to prevent gelatinization of hPL medium, and their concentration must be standardized. Labels for description of hPL-such as "xenogen-free," "animal-free" and "serum free"-are not used consistently in the literature and may be misleading if not critically assessed. Further analysis of the precise composition of relevant growth factors, attachment factors, microRNAs and exosomes will pave the way for optimized and defined culture conditions. The use of hPL has several advantages and disadvantages: they must be taken into account because the choice of cell culture additive has major impact on cell preparations. Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Absolute quantification of DcR3 and GDF15 from human serum by LC-ESI MS

PubMed Central

Lancrajan, Ioana; Schneider-Stock, Regine; Naschberger, Elisabeth; Schellerer, Vera S; Stürzl, Michael; Enz, Ralf

2015-01-01

Biomarkers are widely used in clinical diagnosis, prognosis and therapy monitoring. Here, we developed a protocol for the efficient and selective enrichment of small and low concentrated biomarkers from human serum, involving a 95% effective depletion of high-abundant serum proteins by partial denaturation and enrichment of low-abundant biomarkers by size exclusion chromatography. The recovery of low-abundance biomarkers was above 97%. Using this protocol, we quantified the tumour markers DcR3 and growth/differentiation factor (GDF)15 from 100 μl human serum by isotope dilution mass spectrometry, using 15N metabolically labelled and concatamerized fingerprint peptides for the both proteins. Analysis of three different fingerprint peptides for each protein by liquid chromatography electrospray ionization mass spectrometry resulted in comparable concentrations in three healthy human serum samples (DcR3: 27.23 ± 2.49 fmol/ml; GDF15: 98.11 ± 0.49 fmol/ml). In contrast, serum levels were significantly elevated in tumour patients for DcR3 (116.94 ± 57.37 fmol/ml) and GDF15 (164.44 ± 79.31 fmol/ml). Obtained data were in good agreement with ELISA and qPCR measurements, as well as with literature data. In summary, our protocol allows the reliable quantification of biomarkers, shows a higher resolution at low biomarker concentrations than antibody-based strategies, and offers the possibility of multiplexing. Our proof-of-principle studies in patient sera encourage the future analysis of the prognostic value of DcR3 and GDF15 for colon cancer patients in larger patient cohorts. PMID:25823874

Investigation of the interaction of deltamethrin (DM) with human serum albumin by multi-spectroscopic method

NASA Astrophysics Data System (ADS)

Wang, Jiaman; Ma, Liang; Zhang, Yuhao; Jiang, Tao

2017-02-01

The interaction of Deltamethrin (DM) with human serum albumin (HSA) under the condition of simulating human blood pH environment (pH = 7.4) was investigated by fluorescence, UV-Vis absorbance and circular dichroism (CD) spectroscopy. It was shown that DM was a static quencher of HSA. The binding constants (Ka) are 3.598 × 104 L mol-1 (25 °C); the thermodynamic parameters (ΔH = -3.269 × 104 kJ mol-1, ΔS = -22.81 kJ mol-1 k-1, ΔG = -25889.8 kJ mol-1) obtained with the thermodynamic equation. The hydrogen bond and Vander Waals were the main driving force. The effect of DM on the conformation of HSA was observed by three-dimensional (3D) fluorescence and circular dichroism spectra, indicating that the interaction between DM and HSA was achieved through the binding of DM with the tryptophan and tyrosine residues of HSA. The study on the interaction of DM and Bovine Serum Albumin (BSA) was researched and compared. Difference exists in the interactions of with each of the serum albumins. We will verify and supplement that DM residue in animals and human metabolism, toxicology and other mechanisms are different.

Single chain variable fragment displaying M13 phage library functionalized magnetic microsphere-based protein equalizer for human serum protein analysis.

PubMed

Zhu, Guijie; Zhao, Peng; Deng, Nan; Tao, Dingyin; Sun, Liangliang; Liang, Zhen; Zhang, Lihua; Zhang, Yukui

2012-09-18

Single chain variable fragment (scFv) displaying the M13 phage library was covalently immobilized on magnetic microspheres and used as a protein equalizer for the treatment of human serum. First, scFv displaying M13 phage library functionalized magnetic microspheres (scFv@M13@MM) was incubated with a human serum sample. Second, captured proteins on scFv@M13@MM were eluted with 2 M NaCl, 50 mM glycine-hydrochloric acid (Gly-HCl), and 20% (v/v) acetonitrile with 0.5% (v/v) trifluoroacetic acid in sequence. Finally, the tightly bonded proteins were released by the treatment with thrombin. The eluates were first analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining. Results indicated that the difference of protein concentration was reduced obviously in NaCl and Gly-HCl fractions compared with untreated human serum sample. The eluates were also digested with trypsin, followed by online 2D-strong cation exchange (SCX)-RPLC-ESI-MS/MS analysis. Results demonstrated that the number of proteins identified from an scFv@M13@MM treated human serum sample was improved 100% compared with that from the untreated sample. In addition, the spectral count of 10 high abundance proteins (serum albumin, serotransferrin, α-2-macroglobulin, α-1-antitrypsin, apolipoprotein B-100, Ig γ-2 chain C region, haptoglobin, hemopexin, α-1-acid glycoprotein 1, and α-2-HS-glycoprotein) decreased evidently after scFv@M13@MM treatment. All these results demonstrate that scFv@M13@MM could efficiently remove high-abundance proteins, reduce the protein concentration difference of human serum, and result in more protein identification.

Rabbit antithymocyte globulin–induced serum sickness disease and human kidney graft survival

PubMed Central

Couvrat-Desvergnes, Grégoire; Salama, Apolline; Le Berre, Ludmilla; Evanno, Gwénaëlle; Viklicky, Ondrej; Hruba, Petra; Vesely, Pavel; Guerif, Pierrick; Dejoie, Thomas; Rousse, Juliette; Nicot, Arnaud; Bach, Jean-Marie; Ang, Evelyn; Foucher, Yohann; Brouard, Sophie; Castagnet, Stéphanie; Giral, Magali; Harb, Jean; Perreault, Hélène; Charreau, Béatrice; Lorent, Marine; Soulillou, Jean-Paul

2015-01-01

BACKGROUND. Rabbit-generated antithymocyte globulins (ATGs), which target human T cells, are widely used as immunosuppressive agents during treatment of kidney allograft recipients. However, ATGs can induce immune complex diseases, including serum sickness disease (SSD). Rabbit and human IgGs have various antigenic differences, including expression of the sialic acid Neu5Gc and α-1-3-Gal (Gal), which are not synthesized by human beings. Moreover, anti-Neu5Gc antibodies have been shown to preexist and be elicited by immunization in human subjects. This study aimed to assess the effect of SSD on long-term kidney allograft outcome and to compare the immunization status of grafted patients presenting with SSD following ATG induction treatment. METHODS. We analyzed data from a cohort of 889 first kidney graft recipients with ATG induction (86 with SSD [SSD+] and 803 without SSD [SSD–]) from the Données Informatisées et Validées en Transplantation data bank. Two subgroups of SSD+ and SSD– patients that had received ATG induction treatment were then assessed for total anti-ATG, anti-Neu5Gc, and anti-Gal antibodies using ELISA assays on sera before and after transplantation. RESULTS. SSD was significantly associated with long-term graft loss (>10 years, P = 0.02). Moreover, SSD+ patients exhibited significantly elevated titers of anti-ATG (P = 0.043) and anti-Neu5Gc (P = 0.007) IgGs in late post-graft samples compared with SSD– recipients. CONCLUSION. In conclusion, our data indicate that SSD is a major contributing factor of late graft loss following ATG induction and that anti-Neu5Gc antibodies increase over time in SSD+ patients. FUNDING. This study was funded by Société d’Accélération du Transfert de Technologies Ouest Valorisation, the European FP7 “Translink” research program, the French National Agency of Research, Labex Transplantex, the Natural Science and Engineering Research Council of Canada, and the Canadian Foundation for Innovation. PMID

Evaluation of the binding effect of human serum albumin on the properties of granules.

PubMed

Kristó, Katalin; Bajdik, János; Eros, István; Pintye-Hódi, Klára

2008-11-01

The main objective of this study was the application of a solution of human serum albumin as a granulating fluid. The properties of the granules formed were evaluated and compared with those when a conventional binder was applied in the same concentration. The powder mixture contained a soluble (mannitol) and an insoluble component (different types of cellulose). The protein solution applied exerted an appropriate aggregating effect if the system contained microcrystalline celluloses. Powdered cellulose was not suitable for the granulation with human serum albumin solution. As compared with the same concentration of the conventionally applied cellulose ethers as binder, the prepared granules exhibited a larger particle size, a significantly better compressibility, a higher breaking hardness and a favourable deformation process. These findings mainly reflect the good adhesive properties of the protein. The best compressibility and mechanical behaviour were attained on the application of the microcrystalline cellulose Vivapur type 105. This favourable behaviour may be connected with the wettability of cellulose. These results suggest that the formulation of tablets may be easier from an active agent in the serum that binds to albumin (e.g. interferon) since the amount of additives (binder) can be reduced.

Human serum miR-34a as an indicator of exposure to ionizing radiation.

PubMed

Halimi, Mohammad; Shahabi, Ahmad; Moslemi, Dariush; Parsian, Hadi; Asghari, S Mohsen; Sariri, Reyhaneh; Yeganeh, Farshid; Zabihi, Ebrahim

2016-11-01

Radiation exposure in industrial accidents or nuclear device attacks is a major public health concern. There is an urgent need for markers that rapidly identify people exposed to ionizing radiation (IR). Finding a blood-based marker is advantageous because of the ease of sample collection. This study was designed to test the hypothesis that serum miR-34a could serve as an indicator of exposure to IR. Therefore, 44 women with breast cancer, where radiotherapy was part of their therapeutic protocol, were investigated in this study. After demonstrating the appropriateness of our microRNA (miRNA) extraction efficiency and miRNA assay in human serum, we analyzed the miR-34a level in paired serum samples before and after radiotherapy. Fifty Gy X-ray irradiation in daily dose fractions of 2 Gy, 5 days per week, was used in this study. We demonstrated that IR significantly increased serum level of miR-34a. By measuring miR-34a in serum, we could distinguish irradiated patients with sensitivity of 65 % and specificity of 75 %. According to this study, serum miR-34a has the potential to be used as an indicator of radiation exposure.

Allosteric effects of gold nanoparticles on human serum albumin.

PubMed

Shao, Qing; Hall, Carol K

2017-01-07

The ability of nanoparticles to alter protein structure and dynamics plays an important role in their medical and biological applications. We investigate allosteric effects of gold nanoparticles on human serum albumin protein using molecular simulations. The extent to which bound nanoparticles influence the structure and dynamics of residues distant from the binding site is analyzed. The root mean square deviation, root mean square fluctuation and variation in the secondary structure of individual residues on a human serum albumin protein are calculated for four protein-gold nanoparticle binding complexes. The complexes are identified in a brute-force search process using an implicit-solvent coarse-grained model for proteins and nanoparticles. They are then converted to atomic resolution and their structural and dynamic properties are investigated using explicit-solvent atomistic molecular dynamics simulations. The results show that even though the albumin protein remains in a folded structure, the presence of a gold nanoparticle can cause more than 50% of the residues to decrease their flexibility significantly, and approximately 10% of the residues to change their secondary structure. These affected residues are distributed on the whole protein, even on regions that are distant from the nanoparticle. We analyze the changes in structure and flexibility of amino acid residues on a variety of binding sites on albumin and confirm that nanoparticles could allosterically affect the ability of albumin to bind fatty acids, thyroxin and metals. Our simulations suggest that allosteric effects must be considered when designing and deploying nanoparticles in medical and biological applications that depend on protein-nanoparticle interactions.

Online immunocapture ICP-MS for the determination of the metalloprotein ceruloplasmin in human serum.

PubMed

Bernevic, Bogdan; El-Khatib, Ahmed H; Jakubowski, Norbert; Weller, Michael G

2018-04-02

The human copper-protein ceruloplasmin (Cp) is the major copper-containing protein in the human body. The accurate determination of Cp is mandatory for the reliable diagnosis of several diseases. However, the analysis of Cp has proven to be difficult. The aim of our work was a proof of concept for the determination of a metalloprotein-based on online immunocapture ICP-MS. The immuno-affinity step is responsible for the enrichment and isolation of the analyte from serum, whereas the compound-independent quantitation with ICP-MS delivers the sensitivity, precision, and large dynamic range. Off-line ELISA (enzyme-linked immunosorbent assay) was used in parallel to confirm the elution profile of the analyte with a structure-selective method. The total protein elution was observed with the 32 S mass trace. The ICP-MS signals were normalized on a 59 Co signal. The human copper-protein Cp could be selectively determined. This was shown with pure Cp and with a sample of human serum. The good correlation with off-line ELISA shows that Cp could be captured and eluted selectively from the anti-Cp affinity column and subsequently determined by the copper signal of ICP-MS.

Serum metabolome biomarkers associate low-level environmental perfluorinated compound exposure with oxidative /nitrosative stress in humans.

PubMed

Wang, Xiaofei; Liu, Liangpo; Zhang, Weibing; Zhang, Jie; Du, Xiaoyan; Huang, Qingyu; Tian, Meiping; Shen, Heqing

2017-10-01

Previous in vivo and in vitro studies have linked perfluorinated compound (PFC) exposure with metabolic interruption, but the inter-species difference and high treatment doses usually make the results difficult to be extrapolated to humans directly. The best strategy for identifying the metabolic interruption may be to establish the direct correlations between monitored PFCs data and metabolic data on human samples. In this study, serum metabolome data and PFC concentrations were acquired for a Chinese adult male cohort. The most abundant PFCs are PFOA and PFOS with concentration medians 7.56 and 12.78 nM, respectively; in together they count around 81.6% of the total PFCs. PFC concentration-related serum metabolic profile changes and the related metabolic biomarkers were explored by using partial least squares-discriminant analysis (PLS-DA). Respectively taking PFOS, PFOA and total PFC as the classifiers, serum metabolome can be differentiated between the lowest dose group (1st quartile PFCs) and the highest PFC dose group (4th quartile PFCs). Ten potential PFC biomarkers were identified, mainly involving in pollutant detoxification, antioxidation and nitric oxide (NO) signal pathways. These suggested that low-level environmental PFC exposure has significantly adverse impacts on glutathione (GSH) cycle, Krebs cycle, nitric oxide (NO) generation and purine oxidation in humans. To the best of our knowledge, this is the first report investigating the association of environmental PFC exposure with human serum metabolome alteration. Given the important biological functions of the identified biomarkers, we suggest that PFC could increase the metabolism syndromes risk including diabetes and cardiovascular diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

Serum-free culture of primary human hepatocytes in a miniaturized hollow-fibre membrane bioreactor for pharmacological in vitro studies.

PubMed

Lübberstedt, Marc; Müller-Vieira, Ursula; Biemel, Klaus M; Darnell, Malin; Hoffmann, Stefan A; Knöspel, Fanny; Wönne, Eva C; Knobeloch, Daniel; Nüssler, Andreas K; Gerlach, Jörg C; Andersson, Tommy B; Zeilinger, Katrin

2015-09-01

Primary human hepatocytes represent an important cell source for in vitro investigation of hepatic drug metabolism and disposition. In this study, a multi-compartment capillary membrane-based bioreactor technology for three-dimensional (3D) perfusion culture was further developed and miniaturized to a volume of less than 0.5 ml to reduce demand for cells. The miniaturized bioreactor was composed of two capillary layers, each made of alternately arranged oxygen and medium capillaries serving as a 3D culture for the cells. Metabolic activity and stability of primary human hepatocytes was studied in this bioreactor in the presence of 2.5% fetal calf serum (FCS) under serum-free conditions over a culture period of 10 days. The miniaturized bioreactor showed functions comparable to previously reported data for larger variants. Glucose and lactate metabolism, urea production, albumin synthesis and release of intracellular enzymes (AST, ALT, GLDH) showed no significant differences between serum-free and serum-supplemented bioreactors. Activities of human-relevant cytochrome P450 (CYP) isoenzymes (CYP1A2, CYP3A4/5, CYP2C9, CYP2D6, CYP2B6) analyzed by determination of product formation rates from selective probe substrates were also comparable in both groups. Gene expression analysis showed moderately higher expression in the majority of CYP enzymes, transport proteins and enzymes of Phase II metabolism in the serum-free bioreactors compared to those maintained with FCS. In conclusion, the miniaturized bioreactor maintained stable function over the investigated period and thus provides a suitable system for pharmacological studies on primary human hepatocytes under defined serum-free conditions. Copyright © 2012 John Wiley & Sons, Ltd.

Analysis of human serum lipoprotein lipid composition using MALDI-TOF mass spectrometry.

PubMed

Hidaka, Hiroya; Hanyu, Noboru; Sugano, Mitsutoshi; Kawasaki, Kenji; Yamauchi, Kazuyoshi; Katsuyama, Tsutomu

2007-01-01

This study used matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify all lipid classes in human serum lipoproteins. After the major lipoproteins classes were isolated from serum by ultracentrifugation, the lipids were extracted and mixed with 2,5-dihydroxybenzoic acid (2,5-DHB) dissolved in Folch's solution (chloroform/methanol 2:1, v/v). MALDI-TOF MS analysis of the samples identified phospholipids (PLs), lysophospholipids (lysoPLs), sphingolipids (SLs), triglycerides (TGs), cholesteryl esters (CEs), and free cholesterol; it also showed the characteristics of individual fatty acid chains in serum lipids. MALDI-TOF MS allowed analysis of strongly hydrophobic and non-polar molecules such as CEs and TGs as well as hydrophilic molecules such as phospholipids. Direct analysis of fatty acids was not possible. The concentrations of lipids were not consistent with the ion peak intensities, since the extent of polarity affected the ionization characteristics of the molecules. However, lipid molecules with similar molecular structures but various fatty acid chains, such as phosphatidylcholine (PCs), were analyzed quantitatively by MALDI-TOF MS. Quantitative measurement of cholesterol was possible with the use of an internal standard. This study shows that MALDI-TOF MS can be used for direct investigation and quantitative analysis of the phospholipid composition of serum lipoproteins.

Electrochemical Detection of Anti-Breast-Cancer Agents in Human Serum by Cytochrome P450-Coated Carbon Nanotubes

PubMed Central

Baj-Rossi, Camilla; De Micheli, Giovanni; Carrara, Sandro

2012-01-01

We report on the electrochemical detection of anti-cancer drugs in human serum with sensitivity values in the range of 8–925 nA/μM. Multi-walled carbon nanotubes were functionalized with three different cytochrome P450 isoforms (CYP1A2, CYP2B6, and CYP3A4). A model used to effectively describe the cytochrome P450 deposition onto carbon nanotubes was confirmed by Monte Carlo simulations. Voltammetric measurements were performed in phosphate buffer saline (PBS) as well as in human serum, giving well-defined current responses upon addition of increasing concentrations of anti-cancer drugs. The results assert the capability to measure concentration of drugs in the pharmacological ranges in human serum. Another important result is the possibility to detect pairs of drugs present in the same sample, which is highly required in case of therapies with high side-effects risk and in anti-cancer pharmacological treatments based on mixtures of different drugs. Our technology holds potentials for inexpensive multi-panel drug-monitoring in personalized therapy. PMID:22778656

[The improvement of mixed human serum-induced anaphylactic reaction death model in guinea pigs].

PubMed

Chen, Jiong-Yuan; Lai, Yue; Li, Dang-Ri; Yue, Xia; Wang, Hui-Jun

2012-12-01

To increase the death rate of fatal anaphylaxis in guinea pigs and the detectahie level of the tryptase of mast cell in hlood serum. Seventy-four guinea pigs were randomly divided into five groups: original model group, original model control group, improved model group, improved model control group, improved model with non-anaphylaxis group. Using mixed human serum as the allergen, the way of injection, sensitization and induction were improved. ELISA was used to detect the serum mast cell tryptase and total IgE in guinea pigs of each group. The death rate of fatal anaphylaxis in original model group was 54.2% with the different degree of hemopericardium. The severe pericardial tamponade appeared in 9 guinea pigs in original model group and original model control group. The death rate of fatal anaphylaxis in improved model group was 75% without pericardial tamponade. The concentration of the serum total IgE showed no statistically difference hetween original model group and original model control group (P > 0.05), hut the serum mast cell tryptase level was higher in the original model group than that in the original model control group (P > 0.05). The concentration of the serum total IgE and the serum mast cell tryptase level were significantly higher in improved model group than that in the improved model control group (P < 0.05). The death rate of the improved model significantly increases, which can provide effective animal model for the study of serum total IgE and mast cell tryptase.

Effect of the conditions of isolation on the physicochemical properties of human serum albumin in the norm and with pathology

NASA Astrophysics Data System (ADS)

Ivanov, A. I.; Zhbankov, R. G.; Korolenko, E. A.; Korolik, E. V.; Meleshchenko, L. A.; Sarnatskaya, V. V.; Nikolaev, V. G.; Nikolaichik, V. V.; Yushko, L. A.

1997-01-01

Differential scanning calorimetry and IR spectrosocopy were used to investigate the effect of the procedure of isolation of human serum albumin on its physicochemical characteristics. It is shown that fractionation of blood plasma with ethylene glycol followed by ion exchange chromatography can be used to obtain albumin of normal donors that is similar to the albumin in the nonfractionated plasma according to melting thermograms. Endotherms of human serum albumin samples that were obtained by affinity chromatography and preparative electrophoresis are bimodal, unlike the monophasic for albumin obtained by polyethylene glycol precipitation. These changes result from a higher content of nonetherified fatty acids in the albumin samples obtained by affinity chromatography and from modification of the secondary protein structure in the samples obtained by electrophoresis. Analysis of melting thermograms of serum albumin from patients with uremia, chronic hepatitis, and peritonitis shows that fractionation of blood with polyethylene glycol preserves the thermodynamic characteristics of the various pathological serum albumins to the greatest extent. The present results demonstrate the advantage of polyethylene glycol fractionation for isolation of native preparations of normal and “pathological” human serum albumin.

Determination of estradiol valerate in pharmaceutical preparations and human serum by flow injection chemiluminescence.

PubMed

Liu, Wenwen; Xie, Liangxiao; Liu, Hongshuang; Xu, Shichao; Hu, Bingcheng; Cao, Wei

2013-01-01

A novel method for the detection of trace estradiol valerate (EV) in pharmaceutical preparations and human serum was developed by inhibition of luminol chemiluminescence (CL) by estradiol valerate on the zinc deuteroporphyrin (ZnDP)-enhanced luminol-K3 Fe(CN)6 chemiluminescence system. Under optimized experimental conditions, CL intensity and concentration of estradiol valerate had a good linear relationship in the ranges of 8.0 × 10(-8) to 1.0 × 10(-5) g/mL. Detection limit (3σ) was estimated to be 3.5 × 10(-8) g/mL. The proposed method was applied successfully for the determination of estradiol valerate in pharmaceutical preparations and human serum and recoveries were 97.0-105.0% and 95.5-106.0%, respectively. The possible mechanism of the CL system is discussed. Copyright © 2012 John Wiley & Sons, Ltd.

Systems analysis of singly and multiply O-glycosylated peptides in the human serum glycoproteome via EThcD and HCD mass spectrometry.

PubMed

Zhang, Yong; Xie, Xinfang; Zhao, Xinyuan; Tian, Fang; Lv, Jicheng; Ying, Wantao; Qian, Xiaohong

2018-01-06

Human serum has been intensively studied to identify biomarkers via global proteomic analysis. The altered O-glycoproteome is associated with human pathological state including cancer, inflammatory and degenerative diseases and is an attractive source of disease biomarkers. Because of the microheterogeneity and macroheterogeneity of O-glycosylation, site-specific O-glycosylation analysis in human serum is still challenging. Here, we developed a systematic strategy that combined multiple enzyme digestion, multidimensional separation for sample preparation and high-resolution tandem MS with Byonic software for intact O-glycopeptide characterization. We demonstrated that multiple enzyme digestion or multidimensional separation can make sample preparation more efficient and that EThcD is not only suitable for the identification of singly O-glycosylated peptides (50.3%) but also doubly (21.2%) and triply (28.5%) O-glycosylated peptides. Totally, with the strict scoring criteria, 499 non-redundant intact O-glycopeptides, 173 O-glycosylation sites and 6 types of O-glycans originating from 49 O-glycoprotein groups were identified in human serum, including 121 novel O-glycosylation sites. Currently, this is the largest data set of site-specific native O-glycoproteome from human serum samples. We expect that the strategies developed by this study will facilitate in-depth analyses of native O-glycoproteomes in human serum and provide opportunities to understand the functional roles of protein O-glycosylation in human health and diseases. The altered O-glycoproteome is associated with human pathological state and is an attractive source of disease biomarkers. However, site-specific O-glycosylation analysis is challenging because of the microheterogeneity (different glycoforms attached to one glycosylation site) and macroheterogeneity (site occupancy) of O-glycosylation. In this work, we developed a systematic strategy for intact O-glycopeptide characterization. This study took

Carbon dots based immunosorbent assay for the determination of GFAP in human serum

NASA Astrophysics Data System (ADS)

Ma, Yunsu; Xu, Guanhong; Wei, Fangdi; Cen, Yao; Song, Yueyue; Ma, Yujie; Xu, Xiaoman; Shi, Menglan; Sohail, Muhammad; Hu, Qin

2018-04-01

Glial fibrillary acidic protein (GFAP) is expressed in the central nervous system and the level of GFAP normally rises with brain injury and astroglial tumors. So, serum GFAP is used as a marker for diagnosing various types of brain damage and astroglial tumors. In this study, a new sensor based on carbon dots (CDs) linked with antibodies to specifically detect GFAP in human serum was developed. Anti-GFAP (Ab1) linked with protein A/G agarose resin (PA/G) as a capture antibody (PA/G-Ab1) and anti-GFAP (Ab2) labeled with CDs as a detection antibody (CDs-Ab2) were prepared firstly. Then the CD-linked antibody immunosorbent assay (CLAISA) method was constructed based on the sandwich conjunction reaction among PA/G-Ab1, GFAP, and CDs-Ab2. CLAISA, using the fluorescence of PA/G-Ab1-GFAP-Ab2-CDs as the direct signal, enabled the proposed immunosensor to detect GFAP sensitively with a linear range of 0.10-8.00 ng ml-1 and a detection limit of 25 pg ml-1. This method was applied to the determination of GFAP in human serum by the standard addition method, and the results showed high accuracy and precision. Considering the easy synthetic process and excellent performance of CLAISA, this method has great potential to be used to monitor GFAP in the clinic.

Polybrominated Diphenyl Ethers in Human Milk and Serum from the U.S. EPA MAMA Study: Modeled Predictions of Infant Exposure and Considerations for Risk Assessment

PubMed Central

Marchitti, Satori A.; Fenton, Suzanne E.; Mendola, Pauline; Kenneke, John F.; Hines, Erin P.

2016-01-01

Background: Serum concentrations of polybrominated diphenyl ethers (PBDEs) in U.S. women are believed to be among the world’s highest; however, little information exists on the partitioning of PBDEs between serum and breast milk and how this may affect infant exposure. Objectives: Paired milk and serum samples were measured for PBDE concentrations in 34 women who participated in the U.S. EPA MAMA Study. Computational models for predicting milk PBDE concentrations from serum were evaluated. Methods: Samples were analyzed using gas chromatography isotope-dilution high-resolution mass spectrometry. Observed milk PBDE concentrations were compared with model predictions, and models were applied to NHANES serum data to predict milk PBDE concentrations and infant intakes for the U.S. population. Results: Serum and milk samples had detectable concentrations of most PBDEs. BDE-47 was found in the highest concentrations (median serum: 18.6; milk: 31.5 ng/g lipid) and BDE-28 had the highest milk:serum partitioning ratio (2.1 ± 0.2). No evidence of depuration was found. Models demonstrated high reliability and, as of 2007–2008, predicted U.S. milk concentrations of BDE-47, BDE-99, and BDE-100 appear to be declining but BDE-153 may be rising. Predicted infant intakes (ng/kg/day) were below threshold reference doses (RfDs) for BDE-99 and BDE-153 but above the suggested RfD for BDE-47. Conclusions: Concentrations and partitioning ratios of PBDEs in milk and serum from women in the U.S. EPA MAMA Study are presented for the first time; modeled predictions of milk PBDE concentrations using serum concentrations appear to be a valid method for estimating PBDE exposure in U.S. infants. Citation: Marchitti SA, Fenton SE, Mendola P, Kenneke JF, Hines EP. 2017. Polybrominated diphenyl ethers in human milk and serum from the U.S. EPA MAMA Study: modeled predictions of infant exposure and considerations for risk assessment. Environ Health Perspect 125:706–713; http://dx.doi.org/10

Improvement in skin wrinkles using a preparation containing human growth factors and hyaluronic acid serum.

PubMed

Lee, Do Hyun; Oh, In Young; Koo, Kyo Tan; Suk, Jang Mi; Jung, Sang Wook; Park, Jin Oh; Kim, Beom Joon; Choi, Yoo Mi

2015-02-01

Skin aging is accompanied by wrinkle formation. At some sites, such as the periorbital skin, this is a relatively early phenomenon. We evaluated the anti-wrinkle effect of a preparation containing human growth factor and hyaluronic acid serum on periorbital wrinkles (crow's feet). In total, 23 Korean women (age range: 39-59 years), who were not pregnant, nursing, or undergoing any concurrent therapy, were enrolled in this study. All the patients completed an 8-week trial of twice-daily application of human growth factor and hyaluronic acid serum on the entire face. Efficacy was based on a global photodamage score, photographs, and image analysis using replicas and visiometer analysis every 4 weeks. The standard wrinkle and roughness parameters used in assessing skin by visiometer were calculated and statistically analyzed. Periorbital wrinkles were significantly improved after treatment, with improvements noted both by physician's assessment and visiometer analysis. Topical application of human growth factor and hyaluronic acid was beneficial in reducing periorbital wrinkles.

Affinity precipitation of human serum albumin using a thermo-response polymer with an L-thyroxin ligand.

PubMed

Ding, Zhaoyang; Cao, Xuejun

2013-12-17

Affinity precipitation has been reported as a potential technology for the purification of proteins at the early stage of downstream processing. The technology could be achieved using reversible soluble-insoluble polymers coupled with an affinity ligand to purify proteins from large volumes of dilute solution material such as fermentation broths or plasma. In this study, a thermo-response polymer was synthesized using N-methylol acrylamide, N-isopropyl acrylamide and butyl acrylate as monomers. The molecular weight of the polymer measured by the viscosity method was 3.06 × 104 Da and the lower critical solution temperature (LCST) was 28.0°C.The recovery of the polymer above the LCST was over 95.0%. Human serum albumin (HSA) is the most abundant protein in the human serum system, and it has important functions in the human body. High purity HSA is required in pharmaceuticals. Safe and efficient purification is a crucial process during HSA production. A thermo-response polymer was synthesized and L-thyroxin immobilized on the polymer as an affinity ligand to enable affinity precipitation of HSA. The LCST of the affinity polymer was 31.0°C and the recovery was 99.6% of its original amount after recycling three times. The optimal adsorption condition was 0.02 M Tris-HCl buffer (pH 7.0) and the HSA adsorption capacity was 14.9 mg/g polymer during affinity precipitation. Circular dichroism spectra and a ForteBio Octet system were used to analyze the interactions between the affinity polymer and HSA during adsorption and desorption. The recovery of total HSA by elution with 1.0 mol/L NaSCN was 93.6%. When the affinity polymer was applied to purification of HSA from human serum, HSA could be purified to single-band purity according to SDS-PAGE. A thermo-response polymer was synthesized and L-thyroxin was attached to the polymer. Affinity precipitation was used to purify HSA from human serum.

Quantification of the IgG2/4 kappa Monoclonal Therapeutic Eculizumab from Serum Using Isotype Specific Affinity Purification and Microflow LC-ESI-Q-TOF Mass Spectrometry.

PubMed

Ladwig, Paula M; Barnidge, David R; Willrich, Maria A V

2017-05-01

As therapeutic monoclonal antibodies (mAbs) become more humanized, traditional tryptic peptide approaches used to measure biologics in serum become more challenging since unique clonotypic peptides used for quantifying the mAb may also be found in the normal serum polyclonal background. An alternative approach is to monitor the unique molecular mass of the intact light chain portion of the mAbs using liquid chromatography-mass spectrometry (LC-MS). Distinguishing a therapeutic mAb from a patient's normal polyclonal immunoglobulin (Ig) repertoire is the primary limiting factor when determining the limit of quantitation (LOQ) in serum. The ability to selectively extract subclass specific Igs from serum reduces the polyclonal background in a sample. We present here the development of an LC-MS method to quantify eculizumab in serum. Eculizumab is a complement component 5 (C5) binding mAb that is fully humanized and contains portions of both IgG2 and IgG4 subclasses. Our group developed a method that uses Life Technologies CaptureSelect IgG4 (Hu) affinity matrix. We show here the ability to quantitate eculizumab with a LOQ of 5 mcg/mL by removing the higher abundance IgG1, IgG2, and IgG3 from the polyclonal background, making this approach a simple and efficient procedure. Graphical Abstract ᅟ.

Quantification of the IgG2/4 kappa Monoclonal Therapeutic Eculizumab from Serum Using Isotype Specific Affinity Purification and Microflow LC-ESI-Q-TOF Mass Spectrometry

NASA Astrophysics Data System (ADS)

Ladwig, Paula M.; Barnidge, David R.; Willrich, Maria A. V.

2017-05-01

As therapeutic monoclonal antibodies (mAbs) become more humanized, traditional tryptic peptide approaches used to measure biologics in serum become more challenging since unique clonotypic peptides used for quantifying the mAb may also be found in the normal serum polyclonal background. An alternative approach is to monitor the unique molecular mass of the intact light chain portion of the mAbs using liquid chromatography-mass spectrometry (LC-MS). Distinguishing a therapeutic mAb from a patient's normal polyclonal immunoglobulin (Ig) repertoire is the primary limiting factor when determining the limit of quantitation (LOQ) in serum. The ability to selectively extract subclass specific Igs from serum reduces the polyclonal background in a sample. We present here the development of an LC-MS method to quantify eculizumab in serum. Eculizumab is a complement component 5 (C5) binding mAb that is fully humanized and contains portions of both IgG2 and IgG4 subclasses. Our group developed a method that uses Life Technologies CaptureSelect IgG4 (Hu) affinity matrix. We show here the ability to quantitate eculizumab with a LOQ of 5 mcg/mL by removing the higher abundance IgG1, IgG2, and IgG3 from the polyclonal background, making this approach a simple and efficient procedure.

Molecular Structure-Affinity Relationship of Flavonoids in Lotus Leaf (Nelumbo nucifera Gaertn.) on Binding to Human Serum Albumin and Bovine Serum Albumin by Spectroscopic Method.

PubMed

Tang, Xiaosheng; Tang, Ping; Liu, Liangliang

2017-06-23

Lotus leaf has gained growing popularity as an ingredient in herbal formulations due to its various activities. As main functional components of lotus leaf, the difference in structure of flavonoids affected their binding properties and activities. In this paper, the existence of 11 flavonoids in lotus leaf extract was confirmed by High Performance Liquid Chromatography (HPLC) analysis and 11 flavonoids showed various contents in lotus leaf. The interactions between lotus leaf extract and two kinds of serum albumins (human serum albumin (HSA) and bovine serum albumin (BSA)) were investigated by spectroscopic methods. Based on the fluorescence quenching, the interactions between these flavonoids and serum albumins were further checked in detail. The relationship between the molecular properties of flavonoids and their affinities for serum albumins were analyzed and compared. The hydroxylation on 3 and 3' position increased the affinities for serum albumins. Moreover, both of the methylation on 3' position of quercetin and the C₂=C₃ double bond of apigenin and quercetin decreased the affinities for HSA and BSA. The glycosylation lowered the affinities for HSA and BSA depending on the type of sugar moiety. It revealed that the hydrogen bond force played an important role in binding flavonoids to HSA and BSA.

Evaluation of human LOX-12 as a serum marker for breast cancer.

PubMed

Singh, Abhay Kumar; Kant, Sashi; Parshad, Rajinder; Banerjee, Nirupama; Dey, Sharmistha

2011-10-22

The high concentration of prostaglandins has been associated with chronic inflammatory diseases and several types of human cancers. This is due to the over expression of inflammatory enzymes like Cyclooxygenase (COX), Lipoxygenase (LOX) etc. The aim of this study was to quantify the LOX-12 with clinicopathological parameter of breast cancer patients and its response after chemotherapy to establish serum LOX-12 as a prognostic marker. This case-controlled study was performed on 86 biopsy proven breast cancer patients. Blood and tissue samples were collected from the patients. Serum LOX-12 of the study group was quantified by Surface Plasmon Resonance (SPR) and ELISA techniques by antibody-antigen interaction strategy. A significant increase in LOX-12 levels was observed in breast cancer patients (Mean ± SD=40.54±13.61 ng/ml) as compared to healthy controls (Mean ± SD=13.42±2.4 ng/ml) (p<0.0001). Serum LOX-12 levels were significantly higher (p<0.002) in patients with lymph node involvement. More than 75% patients had shown significant (p<0.0001) reduction of LOX-12 levels after chemotherapy. This was also confirmed by ELISA. This study for the first time had co-related the quantity of serum LOX-12 with breast cancer and also with the effect of chemotherapy. Copyright © 2011 Elsevier Inc. All rights reserved.

Affinity chromatography on monolithic supports for simultaneous and high-throughput isolation of immunoglobulins from human serum.

PubMed

Martinović, Tamara; Andjelković, Uroš; Klobučar, Marko; Černigoj, Urh; Vidič, Jana; Lučić, Marina; Pavelić, Krešimir; Josić, Djuro

2017-11-01

Posttranslational modifications of immunoglobulins have been a topic of great interest and have been repeatedly reported as a major factor in disease pathology. Cost-effective, reproducible, and high-throughput (HTP) isolation of immunoglobulins from human serum is vital for studying the changes in protein structure and the following understanding of disease development. Although there are many methods for the isolation of specific immunoglobulin classes, only a few of them are applicable for isolation of all subtypes and variants. Here, we present the development of a scheme for fast and simultaneous affinity purification of α (A), γ (G), and μ (M) immunoglobulins from human serum through affinity monolith chromatography. Affinity-based monolithic columns with immobilized protein A, G, or L were used for antibody isolation. Monolithic stationary phases have a high surface accessibility of binding sites, large flow-through channels, and can be operated at high flow rates, making them the ideal supports for HTP isolation of biopolymers. The presented method can be used for HTP screening of human serum in order to simultaneously isolate all three above-mentioned immunoglobulins and determine their concentration and changes in their glycosylation pattern as potential prognostic and diagnostic disease biomarkers. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Fluorescence lifetime measurements of native and glycated human serum albumin and bovine serum albumin

NASA Astrophysics Data System (ADS)

Joshi, Narahari V.; Joshi, Virgina O. d.; Contreras, Silvia; Gil, Herminia; Medina, Honorio; Siemiarczuk, Aleksander

1999-05-01

Nonenzymatic glycation, also known as Maillard reaction, plays an important role in the secondary complications of the diabetic pathology and aging, therefore, human serum albumin (HSA) and bovine serum albumin (BSA) were glycated by a conventional method in our laboratory using glucose as the glycating agent. Fluorescence lifetime measurements were carried out with a laser strobe fluorometer equipped with a nitrogen/dye laser and a frequency doubler as a pulsed excitation source. The samples were excited at 295 nm and the emission spectra were recorded at 345 nm. The obtained decay curves were tried for double and triple exponential functions. It has been found that the shorter lifetime increases for glycated proteins as compared with that of the native ones. For example, in the case of glycated BSA the lifetime increased from 1.36 ns to 2.30 ns. Similarly, for HSA, the lifetime increases from 1.58 ns to 2.26 ns. Meanwhile, the longer lifetime changed very slightly for both proteins (from 6.52 ns to 6.72 ns). The increase in the lifetime can be associated with the environmental effect; originated from the attachment of glucose to some lysine residues. A good example is Trp 214 which is in the cage of Lys 225, Lys 212, Lys 233, Lys 205, Lys 500, Lys 199 and Lys 195. If fluorescence lifetime technique is calibrated and properly used it could be employed for assessing glycation of proteins.

Influence of human body composition on serum peak thyrotropin (TSH) after recombinant human TSH administration in patients with differentiated thyroid carcinoma.

PubMed

Castagna, Maria Grazia; Pinchera, Aldo; Marsili, Alessandro; Giannetti, Monica; Molinaro, Eleonora; Fierabracci, Paola; Grasso, Lucia; Pacini, Furio; Santini, Ferruccio; Elisei, Rossella

2005-07-01

In this study, we evaluated the influence of height, weight, body mass index (BMI), body surface area, and body composition [total lean body mass (LBM) and fat body mass] on serum peak TSH levels obtained after recombinant human (rh)TSH. Furthermore, to verify whether the serum peak TSH influenced the efficacy of radioiodine ((131)I), we compared the rate of thyroid remnant ablation according to the patients' BMI. We studied 105 patients with differentiated thyroid carcinoma who underwent rhTSH stimulation test. Serum TSH measurements were performed before and 24, 48, and 72 h after rhTSH administration. We also compared the rate of thyroid remnant ablation among 70 differentiated thyroid carcinoma patients with different BMI. The serum peak TSH after rhTSH was significantly lower in overweight and obese subjects compared with normal-weight subjects (92.1 +/- 41.8, 82.4 +/- 24.2, and 112.7 +/- 46.3 microU/ml, respectively; P = 0.01) and in males compared with females (74.6 +/- 22.3 and 105.0 +/- 43.0 microU/ml, respectively; P = 0.0002). By univariate analysis, serum peak TSH was negatively related to weight, height, body surface area, BMI, LBM, and fat body mass, but only LBM was independently associated with serum peak TSH levels. Although it was confirmed that overweight and obese patients had a lower serum peak TSH, the rate of ablation did not differ among normal-weight, overweight, and obese patients. With this study we demonstrated that LBM is the only parameter independently associated with serum peak TSH after rhTSH administration. However, the serum peak TSH does not influence the rate of (131)I remnant ablation.

CCQM-K132: low-polarity analytes in a biological matrix: vitamin D metabolites in human serum

NASA Astrophysics Data System (ADS)

Wise, Stephen A.; Tai, Susan S.-C.; Duewer, David L.; Bedner, Mary; Camara, Johanna E.; Lippa, Katrice A.; Qinde, Liu; Kang, Dukjin; Kim, Byungjoo; Quan, Can; Shi, Lianhua; Nammoonnoy, Jintana; Vamathevan, Veronica; Ceyhan Gören, Ahmet; Bilsel, Gökhan; Yilmaz, Hasibe

2017-01-01

Vitamin D is a fat-soluble vitamin that occurs primarily in two forms, vitamin D2 and vitamin D3. Vitamin D3 is produced naturally when skin is exposed to UV radiation, is naturally-occurring in foods (generally of animal origin), and is fortified in some foods and dietary supplements. Vitamin D2 occurs in food (generally plant sources) and until recently was the form most often used in dietary supplements. Vitamin D is metabolized in the body to produce several closely related, hydroxylated species (metabolites), with 25-hydroxyvitamin D3 [25(OH)D3] and 25-hydroxyvitamin D2 [25(OH)D2] as the most common metabolites measured in human serum. Concentrations of total vitamin D in human serum, calculated as the sum of 25(OH)D2 and 25(OH)D3, are typically in the 16 ng/g to 30 ng/g (40 nmol/L to 75 nmol/L) range, with 25(OH)D3 usually accounting for more than 90 % of the total. An epimer of 25(OH)D3, 3-epi-25(OH)D3, can be present at levels up to 10 % of 25(OH)D3 concentration. Seven National Metrology Institutions participated in the Track C Key Comparison CCQM-K132 low-polarity analytes in a biological matrix: vitamin D metabolites in human serum. Participants were requested to evaluate the mass fractions, expressed in ng/g, of 25(OH)D3, 25(OH)D2, and 3-epi-25(OH)D3 in two human serum materials, termed Serum Pool I and Serum Pool II. Due to the known low levels of 3-epi-25(OH)D3 in both materials and the very low level of 25(OH)D2 in Serum Pool I, the study protocol stated that key comparison reference values (KCRVs) would be assigned only to 25(OH)D3 in both materials and 25(OH)D2 in Serum Pool II. Results for 3-epi-25(OH)D3 were requested to evaluate the separation technologies employed; 3-epi-25(OH)D3 needs to be chromatographically separated from 25(OH)D3 for proper quantification of 25(OH)D3. Results for 25(OH)D2 in Serum Pool I were requested to explore measurement performance at its low level. All participants used isotope dilution liquid chromatography with

Comparison of human anxiety based on different cultural backgrounds.

PubMed

Kalwar, Santosh Kumar

2010-08-01

This work conceptualizes human behavior on the Internet. The study was conducted with 10 university participants representing two different cultural backgrounds, Asian and Western. The participants were asked to visit any Web page on the Internet for 15 minutes, for 30 minutes, and for 1 hour. The results showed that participants displayed no signs of anxiousness during the 15-minute task and very little anxiousness during the 30-minute task. Western participants showed overall more anxiousness than Asian participants. However, all participants showed anxiousness during the 1-hour task. Data on comparative human anxiety were collected on the basis of a literature review of social fun, online belonging, and community on the Internet. Only the limited set of data of the participant is discussed in this article.

Role of the Protein Corona Derived from Human Plasma in Cellular Interactions between Nanoporous Human Serum Albumin Particles and Endothelial Cells.

PubMed

Zyuzin, Mikhail V; Yan, Yan; Hartmann, Raimo; Gause, Katelyn T; Nazarenus, Moritz; Cui, Jiwei; Caruso, Frank; Parak, Wolfgang J

2017-08-16

The presence of a protein corona on various synthetic nanomaterials has been shown to strongly influence how they interact with cells. However, it is unclear if the protein corona also exists on protein particles, and if so, its role in particle-cell interactions. In this study, pure human serum albumin (HSA) particles were fabricated via mesoporous silica particle templating. Our data reveal that various serum proteins adsorbed on the particles, when exposed to human blood plasma, forming a corona. In human umbilical vein endothelial cells (HUVECs), the corona was shown to decrease particle binding to the cell membrane, increase the residence time of particles in early endosomes, and reduce the amount of internalized particles within the first hours of exposure to particles. These findings reveal important information regarding the mechanisms used by vascular endothelial cells to internalize protein-based particulate materials exposed to blood plasma. The ability to control the cellular recognition of these organic particles is expected to aid the advancement of HSA-based materials for intravenous drug delivery.

Generation of therapeutic protein variants with the human serum albumin binding capacity via site-specific fatty acid conjugation.

PubMed

Cho, Jinhwan; Lim, Sung In; Yang, Byung Seop; Hahn, Young S; Kwon, Inchan

2017-12-21

Extension of the serum half-life is an important issue in developing new therapeutic proteins and expanding applications of existing therapeutic proteins. Conjugation of fatty acid, a natural human serum albumin ligand, to a therapeutic protein/peptide was developed as a technique to extend the serum half-life in vivo by taking advantages of unusually long serum half-life of human serum albumin (HSA). However, for broad applications of fatty acid-conjugation, several issues should be addressed, including a poor solubility of fatty acid and a substantial loss in the therapeutic activity. Therefore, herein we systematically investigate the conditions and components in conjugation of fatty acid to a therapeutic protein resulting in the HSA binding capacity without compromising therapeutic activities. By examining the crystal structure and performing dye conjugation assay, two sites (W160 and D112) of urate oxidase (Uox), a model therapeutic protein, were selected as sites for fatty acid-conjugation. Combination of site-specific incorporation of a clickable p-azido-L-phenylalanine to Uox and strain-promoted azide-alkyne cycloaddition allowed the conjugation of fatty acid (palmitic acid analog) to Uox with the HSA binding capacity and retained enzyme activity. Deoxycholic acid, a strong detergent, greatly enhanced the conjugation yield likely due to the enhanced solubility of palmitic acid analog.

Structural consistency analysis of recombinant and wild-type human serum albumin

NASA Astrophysics Data System (ADS)

Cao, Hui-Ling; Sun, Li-Hua; Liu, Li; Li, Jian; Tang, Lin; Guo, Yun-Zhu; Mei, Qi-Bing; He, Jian-Hua; Yin, Da-Chuan

2017-01-01

Recombinant human serum albumin (rHSA) is potential alternatives for human serum albumin (HSA) which may ease severe shortage of HSA worldwide. In theory, rHSA and HSA are the same. Structure decides function. Therefore, the 3D structural consistency analysis of rHSA and HSA is outmost importance, which is the base of their function consistency. In this paper, the crystal structures of rHSA at resolution limit of 2.22 Å and HSA at 2.30 Å were determined by X-ray diffraction (XRD), which were deposited in the Protein Data Bank (PDB) with accession codes 4G03 (rHSA) and 4G04 (HSA). The differences between rHSA and HSA were systematically analyzed from the crystallization behavior, diffraction data and three-dimensional (3D) structure. The superimposed contrasted analysis indicated that rHSA and HSA achieved a structural similarity of 99% with an r.m.s. deviation of 0.397 Å for the corresponding overall Cα atoms. In addition, the number of α-helices in the rHSA or HSA molecule was verified to be 30. As a result, rHSA can potentially replace HSA. The study provides a theoretical and experimental basis for the clinical and additional applications of rHSA. Meanwhile, it is also a good example for applications of genetic engineering.

Hydrolysis of platelet-activating factor by human serum paraoxonase.

PubMed Central

Rodrigo, L; Mackness, B; Durrington, P N; Hernandez, A; Mackness, M I

2001-01-01

Human serum paraoxonase (human PON1) has been shown to be important in the metabolism of phospholipid and cholesteryl ester hydroperoxides, thereby preventing the oxidation of low-density lipoprotein (LDL) and retarding atherogenesis. However, the exact substrate specificity of PON1 has not been established. In the present study we show that purified PON1 hydrolyses platelet-activating factor (PAF). We could find no evidence for contamination of our preparation with authentic platelet-activating-factor acetylhydrolase (PAFAH) by immunoblotting with a PAFAH monoclonal antibody or by sequencing the purified protein. In addition the specific PAFAH inhibitor SB-222657 did not affect the ability of PON1 to hydrolyse PAF (30.1+/-2.8 micromol/min per mg of protein with no inhibitor; 31.4+/-2.2 micromol/min per mg of protein with 100 nM inhibitor) or phenyl acetate (242.6+/-30.8 versus 240.8+/-31.5 micromol/min per mg of protein with and without inhibitor respectively). SB-222657 was also unable to inhibit PAF hydrolysis by isolated human high-density lipoprotein (HDL), but completely abolished the activity of human LDL. Ostrich (Struthio camelus) HDL, which does not contain PON1, was unable to hydrolyse PAF. These data provide evidence that PON1 may limit the action of this bioactive pro-inflammatory phospholipid. PMID:11171072

Analysis of lysergic acid amide in human serum and urine after ingestion of Argyreia nervosa seeds.

PubMed

Paulke, Alexander; Kremer, Christian; Wunder, Cora; Toennes, Stefan W

2012-08-01

The ergot alkaloid lysergic acid amide (LSA) is a secondary plant constituent in a number of plants, but it is mainly present in considerable amounts in Convolvulaceae, like Argyreia nervosa. Due to its close structural similarity to lysergic acid diethylamide, LSA is considered as psychedelic and therefore promoted as so-called "legal high" in various internet forums. During a human behavioral study with orally administered seeds of A. nervosa, blood and urine samples were obtained. The present study describes the validation of a sensitive and robust high performance liquid chromatography method with fluorescence detection, which was applied to the study samples. The limit of detection (LOD) and lower limit of quantification in human serum were 0.05 and 0.17 ng/mL, respectively, and in urine, the LOD was 0.15 ng/mL. Intra- and interday precision and accuracy were below 15 % relative standard deviation with a bias better than ±15 %. No conversion of LSA to its epimer iso-LSA was noted during analyses. The LSA concentrations in the authentic human serum samples were in the range of 0.66 to 3.15 ng/mL approximately 2 h after ingestion. In urine, LSA could be found 1-24 h after ingestion; after 48 h, no LSA could be detected. The LSA epimer iso-LSA was also detected in serum and urine in varying ratios. In conclusion, LSA serum levels in the low nanogram per milliliter range correlated with severe vegetative adverse effects (nausea, weakness, fatigue, tremor, blood pressure elevation) and a psychosis-like state, which led to study termination.

Clinical significance of serum anti-human papillomavirus 16 and 18 antibodies in cervical neoplasia.

PubMed

Chay, Doo Byung; Cho, Hanbyoul; Kim, Bo Wook; Kang, Eun Suk; Song, Eunseop; Kim, Jae-Hoon

2013-02-01

To estimate the clinical significance of serum anti-human papillomavirus (HPV) antibodies and high-risk cervical HPV DNA in cervical neoplasia. The study population comprised patients who were histopathologically diagnosed with cervical intraepithelial neoplasia (CIN) 1 (n=64), CIN 2 and 3 (n=241), cervical cancer (n=170), and normal control participants (n=975). Cervical HPV DNA tests were performed through nucleic acid hybridization assay tests, and serum anti-HPV 16 and 18 antibodies were measured by competitive immunoassay. The associations of HPV DNA and anti-HPV antibodies were evaluated with demographic characteristics and compared according to the levels of disease severity. Anti-HPV antibodies were also investigated with clinicopathologic parameters, including survival data. Among various demographic characteristics, factors involving sexual behavior had a higher tendency of HPV DNA positivity and HPV seropositivity. Human papillomavirus DNA mean titer and positivity were both increased in patients with cervical neoplasia compared with those with normal control participants, but there was no statistical difference among types of cervical neoplasia. Serum anti-HPV 16 antibodies were also able to differentiate cervical neoplasia from a normal control participant and furthermore distinguished CIN 1 from CIN 2 and 3 (odd ratio 2.87 [1.43-5.78], P=.002). In cervical cancer, HPV 16 seropositivity was associated with prolonged disease-free survival according to the univariable analysis (hazard ratio=0.12 [0.01-0.94], P=.044). Serum anti-HPV 16 antibodies can distinguish cervical neoplasia from a normal control and has the advantage of identifying high-grade CIN. Moreover, in cervical cancer, HPV 16 seropositivity may be associated with a more favorable prognosis. II.

Animal serum-free expansion and differentiation of human mesenchymal stromal cells.

PubMed

Felka, Tino; Schäfer, Richard; De Zwart, Peter; Aicher, Wilhelm K

2010-04-01

Mesenchymal stromal cells (MSC) are attracting increasing interest for possible application in cell therapies. Fetal calf serum (FCS) is widely utilized for cell culture, but its use in the context of clinical applications is associated with too many risks. Therefore we tested FCS-free media for the expansion and differentiation of MSC in compliance with the European good manufacturing practice (GMP) regulations for medicinal products. MSC expansion medium was modified by replacing FCS with human plasma and platelet extract. Cells were characterized according to the defined minimal criteria for multipotent MSC. For chondrogenic differentiation, serum-free micromass cultures were employed. For adipogenic and osteogenic differentiation, the FCS was replaced by human plasma. After 28 days of incubation in differentiation media, cells were analyzed by cytochemical and immunohistochemical staining. Furthermore, mRNA expression of chondrogenic, adipogenic and osteogenic markers was investigated by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Expansion and differentiation of MSC under FCS-free conditions yielded cells with chondrogenic, adipogenic and osteogenic phenotypes and a characteristic gene expression. Chondrocytes in micromass pellets revealed an accumulation of proteoglycans and type II collagen as well as a significantly increased mRNA expression of chondrogenic marker genes. The adipocytes displayed Oil red O staining and expressed peroxisome proliferator-activated receptor gamma(2) (ppARgamma2) and lipoprotein lipase (LPL) mRNA. The osteoblasts were positive for von Kossa staining and expressed mRNA of osteogenic marker genes. The results did not indicate any spontaneous differentiation. Human plasma is a suitable FCS replacement for the expansion and differentiation of MSC, providing a feasible alternative for tissue engineering with GMP-compatible protocols.

Association of a Human FABP1 Gene Promoter Region Polymorphism with Altered Serum Triglyceride Levels.

PubMed

Peng, Xian-E; Wu, Yun-Li; Zhu, Yi-Bing; Huang, Rong-Dong; Lu, Qing-Qing; Lin, Xu

2015-01-01

Liver fatty acid-binding protein (L-FABP), also known as fatty acid-binding protein 1 (FABP1), is a key regulator of hepatic lipid metabolism. Elevated FABP1 levels are associated with an increased risk of cardiovascular disease (CVD) and metabolic syndromes. In this study, we examine the association of FABP1 gene promoter variants with serum FABP1 and lipid levels in a Chinese population. Four promoter single-nucleotide polymorphisms (SNPs) of FABP1 gene were genotyped in a cross-sectional survey of healthy volunteers (n = 1,182) from Fuzhou city of China. Results showed that only the rs2919872 G>A variant was significantly associated with serum TG concentration(P = 0.032).Compared with the rs2919872 G allele, rs2919872 A allele contributed significantly to reduced serum TG concentration, and this allele dramatically decreased the FABP1 promoter activity(P < 0.05). The rs2919872 A allele carriers had considerably lower serum FABP1 levels than G allele carriers (P < 0.01). In the multivariable linear regression analysis, the rs2919872 A allele was negatively associated with serum FABP1 levels (β = -0.320, P = 0.003), while serum TG levels were positively associated with serum FABP1 levels (β = 0.487, P = 0.014). Our data suggest that compared with the rs2919872 G allele, the rs2919872 A allele reduces the transcriptional activity of FABP1 promoter, and thereby may link FABP1 gene variation to TG level in humans.

Application of silver films with different roughness parameter for septic human serum albumin detection by Surface Enhanced Raman Spectroscopy

NASA Astrophysics Data System (ADS)

Zyubin, A. Y.; Konstantinova, E. I.; Matveeva, K. I.; Slezhkin, V. A.; Samusev, I. G.; Demin, M. V.; Bryukhanov, V. V.

2018-01-01

In this paper, the rough silver films parameters investigation, used as media for surface enhancement Raman spectroscopy for health and septic human serum albumin (HSA) study results have been presented. The detection of small concentrations of HSA isolated from blood serum and it main vibrational groups identification has been done.

Effect of transdermal magnesium cream on serum and urinary magnesium levels in humans: A pilot study.

PubMed

Kass, Lindsy; Rosanoff, Andrea; Tanner, Amy; Sullivan, Keith; McAuley, William; Plesset, Michael

2017-01-01

Oral magnesium supplementation is commonly used to support a low magnesium diet. This investigation set out to determine whether magnesium in a cream could be absorbed transdermally in humans to improve magnesium status. In this single blind, parallel designed pilot study, n = 25 participants (aged 34.3+/-14.8y, height 171.5+/-11cm, weight 75.9 +/-14 Kg) were randomly assigned to either a 56mg/day magnesium cream or placebo cream group for two weeks. Magnesium serum and 24hour urinary excretion were measured at baseline and at 14 days intervention. Food diaries were recorded for 8 days during this period. Mg test and placebo groups' serum and urinary Mg did not differ at baseline. After the Mg2+ cream intervention there was a clinically relevant increase in serum magnesium (0.82 to 0.89 mmol/l,p = 0.29) that was not seen in the placebo group (0.77 to 0.79 mmol/L), but was only statistically significant (p = 0.02)) in a subgroup of non-athletes. Magnesium urinary excretion increased from baseline slightly in the Mg2+ group but with no statistical significance (p = 0.48). The Mg2+ group showed an 8.54% increase in serum Mg2+ and a 9.1% increase in urinary Mg2+ while these figures for the placebo group were smaller, i.e. +2.6% for serum Mg2+ and -32% for urinary Mg2+. In the placebo group, both serum and urine concentrations showed no statistically significant change after the application of the placebo cream. No previous studies have looked at transdermal absorbency of Mg2+ in human subjects. In this pilot study, transdermal delivery of 56 mg Mg/day (a low dose compared with commercial transdermal Mg2+ products available) showed a larger percentage rise in both serum and urinary markers from pre to post intervention compared with subjects using the placebo cream, but statistical significance was achieved only for serum Mg2+ in a subgroup of non-athletes. Future studies should look at higher dosage of magnesium cream for longer durations. ISRCTN registry ID No. ISRTN

Biological Exposure Indices of Pyrrole Adducts in Serum and Urine for Hazard Assessment of n-Hexane Exposure

PubMed Central

Yin, Hongyin; Zhang, Chunling; Guo, Ying; Shao, Xiaoying; Zeng, Tao; Zhao, Xiulan; Xie, Keqin

2014-01-01

Background Pyrrole adducts might be used as a biomarker for monitoring occupational exposure to n-hexane, but the Biological Exposure Indices of pyrrole adducts in serum and urine are still unknown. The current study was designed to investigate the biological exposure limit of pyrrole adducts for hazard assessment of n-hexane. Methods Male Wistar rats were given daily dose of 500, 1000, 1500, 2000, 4000 mg/kg bw n-hexane by gavage for 24 weeks. The levels of pyrrole adducts in serum and urine were determined at 8, 24 hours postdose once a week. The Biological Exposure Indices was evaluated by neurological evaluation and the levels of pyrrole adducts. The difference in pyrrole adducts formation between humans and rats were estimated by using in vitro test. Results Dose-dependent effects were observed between the doses of n-hexane and pyrrole adducts in serum and urine, and the levels of pyrrole adduct in serum and urine approached a plateau at week 4. There was a significantly negative correlation between the time to paralysis and the level of pyrrole adducts in serum and urine, while a positive correlation between gait score and levels of pyrrole adducts in serum and urine was observed. In vitro, pyrrole adducts formed in human serum was about two times more than those in rat serum at the same level of 2,5-HD. Conclusion It was concluded that the BEIs of pyrrole adducts in humans were 23.1±5.91 nmol/ml in serum 8 h postdose, 11.7±2.64 nmol/ml in serum 24 h postdose, 253.8±36.3 nmol/ml in urine 8 h postdose and 54.6±15.42 nmol/ml in urine 24 h postdose. PMID:24465904

Human serum albumin hydropersulfide is a potent reactive oxygen species scavenger in oxidative stress conditions such as chronic kidney disease

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

Shibata, Akitomo; Ishima, Yu; Ikeda, Mayumi

Recently, hydropersulfide (RSSH) was found to exist in mammalian tissues and fluids. Cysteine hydropersulfide can be found in free cysteine residues as well as in proteins, and it has potent antioxidative activity. Human serum albumin (HSA) is the most abundant protein in mammalian serum. HSA possesses a free thiol group in Cys-34 that could be a site for hydropersulfide formation. HSA hydropersulfide of high purity as a positive control was prepared by treatment of HSA with Na{sub 2}S. The presence of HSA hydropersulfide was confirmed by spectroscopy and ESI-TOFMS analysis where molecular weights of HSA hydropersulfide by increments of approximatelymore » 32 Da (Sulfur atom) were detected. The fluorescent probe results showed that Alexa Fluor 680 conjugated maleimide (Red-Mal) was a suitable assay and bromotrimethylammoniumbimane bromide appeared to be a selective reagent for hydropersulfide. The effect of oxidative stress related disease on the existence of albumin hydropersulfides was examined in rat 5/6 nephrectomy model of chronic kidney disease (CKD). Interestingly, the level of hydropersulfides in rat 5/6 nephrectomy model serum was decreased by a uremic toxin that increases oxidative stress in rat 5/6 nephrectomy model. Furthermore, we demonstrated that the levels of HSA hydropersulfide in human subjects were reduced in CKD but restored by hemodialysis using Red-Mal assay. We conclude that HSA hydropersulfide could potentially play an important role in biological anti-oxidative defense, and it is a promising diagnostic and therapeutic marker of oxidative diseases. - Highlights: • Hydropersulfide can behave as potent antioxidants. • We firstly detected human serum albumin hydropersulfide in healthy subjects. • Human serum albumin hydropersulfide in human subjects were reduced in chronic kidney disease but restored by hemodialysis.« less

Development, validation and application of an ICP-MS/MS method to quantify minerals and (ultra-)trace elements in human serum.

PubMed

Meyer, Sören; Markova, Mariya; Pohl, Gabriele; Marschall, Talke A; Pivovarova, Olga; Pfeiffer, Andreas F H; Schwerdtle, Tanja

2018-09-01

Multi-element determination in human samples is very challenging. Especially in human intervention studies sample volumes are often limited to a few microliters and due to the high number of samples a high-throughput is indispensable. Here, we present a state-of-the-art ICP-MS/MS-based method for the analysis of essential (trace) elements, namely Mg, Ca, Fe, Cu, Zn, Mo, Se and I, as well as food-relevant toxic elements such as As and Cd. The developed method was validated regarding linearity of the calibration curves, method LODs and LOQs, selectivity and trueness as well as precision. The established reliable method was applied to quantify the element serum concentrations of participants of a human intervention study (LeguAN). The participants received isocaloric diets, either rich in plant protein or in animal protein. While the serum concentrations of Mg and Mo increased in participants receiving the plant protein-based diet (above all legumes), the Se concentration in serum decreased. In contrast, the animal protein-based diet, rich in meat and dairy products, resulted in an increased Se concentration in serum. Copyright © 2018 Elsevier GmbH. All rights reserved.

Epidermal growth factor- and hepatocyte growth factor-receptor activity in serum-free cultures of human hepatocytes.

PubMed

Runge, D M; Runge, D; Dorko, K; Pisarov, L A; Leckel, K; Kostrubsky, V E; Thomas, D; Strom, S C; Michalopoulos, G K

1999-02-01

Serum-free primary cultures of hepatocytes are a useful tool to study factors triggering hepatocyte proliferation and regeneration. We have developed a chemically defined serum-free system that allows human hepatocyte proliferation in the presence of epidermal growth factor and hepatocyte growth factor. DNA synthesis and accumulation were determined by [3H]thymidine incorporation and fluorometry, respectively. Western blot analyses and co-immunoprecipitations were used to investigate the association of proteins involved in epidermal growth factor and hepatocyte growth factor activation and signaling: epidermal growth factor receptor, hepatocyte growth factor receptor (MET), urokinase-type plasminogen activator and its receptor, and a member of the signal transducer and activator of transcription family, STAT-3. Primary human hepatocytes proliferated under serum-free conditions in a chemically defined medium for up to 12 days. Epidermal growth factor-receptor and MET were present and functional, decreasing over time. MET, urokinase-type plasminogen activator and urokinase-type plasminogen activator receptor co-precipitated to varying degrees during the culture period. STAT-3 co-precipitated with epidermal growth factor-receptor and MET to varying degrees. Proliferation of human hepatocytes can improve by modification of a chemically defined medium originally used for rat hepatocyte cultures. In these long-term cultures of human hepatocytes, hepatocyte growth factor and epidermal growth factor can stimulate growth and differentiation by interacting with their receptors and initiating downstream signaling. This involves complex formation of the receptors with other plasma membrane components for MET (urokinase-type plasminogen activator in context of its receptor) and activation of STAT-3 for both receptors.

Polybrominated Diphenyl Ethers in Human Milk and Serum from the U.S. EPA MAMA Study: Modeled Predictions of Infant Exposure and Considerations for Risk Assessment

EPA Science Inventory

Background: Serum concentrations of polybrominated diphenyl ethers (PBDEs) in U.S. women are believed to be among the world’s highest; however, little information exists on the partitioning of PBDEs between serum and breast milk and how this may affect infant exposure. Obj...

Polybrominated Diphenyl Ethers in Human Milk and Serum from the U.S. EPA MAMA Study: Modeled Predictions of Infant Exposure and Considerations for Risk Assessment

EPA Science Inventory

Background: Serum concentrations of polybrominated diphenyl ethers (PBDEs) in U.S. women are believed to be among the world’s highest; however, little information exists on the partitioning of PBDEs between serum and breast milk and how this may affect infant exposure. Objecti...

Generation of hematopoietic stem cells from human embryonic stem cells using a defined, stepwise, serum-free, and serum replacement-free monolayer culture method.

PubMed

Kim, So-Jung; Jung, Ji-Won; Ha, Hye-Yeong; Koo, Soo Kyung; Kim, Eung-Gook; Kim, Jung-Hyun

2017-03-01

Embryonic stem cells (ESCs) can be expanded infinitely in vitro and have the potential to differentiate into hematopoietic stem cells (HSCs); thus, they are considered a useful source of cells for HSC production. Although several technical in vitro methods for engineering HSCs from pluripotent stem cells have been developed, clinical application of HSCs engineered from pluripotent stem cells is restricted because of the possibility of xenogeneic contamination resulting from the use of murine materials. Human ESCs (CHA-hES15) were cultured on growth factor-reduced Matrigel-coated dishes in the mTeSR1 serum-free medium. When the cells were 70% confluent, we initiated HSC differentiation by three methods involving (1) knockout serum replacement (KSR), cytokines, TGFb1, EPO, and FLT3L; (2) KSR, cytokines, and bFGF; or (3) cytokines and bFGF. Among the three differentiation methods, the minimal number of cytokines without KSR resulted in the greatest production of HSCs. The optimized method resulted in a higher proportion of CD34 + CD43 + hematopoietic progenitor cells (HPCs) and CD34 + CD45 + HPCs compared to the other methods. In addition, the HSCs showed the potential to differentiate into multiple lineages of hematopoietic cells in vitro . In this study, we optimized a two-step, serum-free, animal protein-free, KSR-free, feeder-free, chemically defined monolayer culture method for generation of HSCs and hematopoietic stem and progenitor cells (HSPCs) from human ESCs.

The Effects of Fetal Gender on Serum Human Chorionic Gonadotropin and Testosterone in Normotensive and Preeclamptic Pregnancies

PubMed Central

Lorzadeh, Nahid; Kazemirad, Sirous

2012-01-01

Introduction. The aim of the present study was to evaluate the effects of fetal sex on serum human chorionic gonadotropin (hCG) and testosterone in normotensive and preeclamptic pregnancies. Materials and Methods. This is a cross-sectional study and 139 women with singleton pregnancies in the third trimester were studied. Seventy-one pregnancies were uncomplicated; among those were 35 male and 36 female fetuses. Sixty-eight pregnancies were complicated by preeclampsia; among those were 35 male and 33 female fetuses. Human chorionic gonadotropin and total testosterone were measured in maternal peripheral blood. Data analyzed by SPSS software. Results. In male-bearing pregnancies, maternal hCG and testosterone serum levels were significantly higher in preeclamptic than normotensive mothers (P < 0.001 and P < 0.001, resp.) in female-bearing pregnancies testosterone levels were significantly higher in preeclamptic than normotensive mothers (P < 0.001). Total testosterone levels were significantly higher in pregnancies with either gender and significantly higher in mlae-bearing than in female-bearing pregnancies. Conclusion. According to our results, there is a correlation between maternal serum hCG and testosterone levels and preeclampsia. Therefore these tests can be used as routine during 30–38 weeks of gestation. High maternal serum concentrations of these markers can predict preeclampsia. PMID:22518314

Deep-Dive Targeted Quantification for Ultrasensitive Analysis of Proteins in Nondepleted Human Blood Plasma/Serum and Tissues

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

Nie, Song; Shi, Tujin; Fillmore, Thomas L.

Mass spectrometry-based targeted proteomics (e.g., selected reaction monitoring, SRM) is emerging as an attractive alternative to immunoassays for protein quantification. Recently we have made significant progress in SRM sensitivity for enabling quantification of low ng/mL to sub-ng/mL level proteins in nondepleted human blood plasma/serum without affinity enrichment. However, precise quantification of extremely low abundant but biologically important proteins (e.g., ≤100 pg/mL in blood plasma/serum) using targeted proteomics approaches still remains challenging. To address this need, we have developed an antibody-independent Deep-Dive SRM (DD-SRM) approach that capitalizes on multidimensional high-resolution reversed-phase liquid chromatography (LC) separation for target peptide enrichment combined withmore » precise selection of target peptide fractions of interest, significantly improving SRM sensitivity by ~5 orders of magnitude when compared to conventional LC-SRM. Application of DD-SRM to human serum and tissue has been demonstrated to enable precise quantification of endogenous proteins at ~10 pg/mL level in nondepleted serum and at <10 copies per cell level in tissue. Thus, DD-SRM holds great promise for precisely measuring extremely low abundance proteins or protein modifications, especially when high-quality antibody is not available.« less

Per- and polyfluoroalkyl substances in human serum and urine samples from a residentially exposed community.

PubMed

Worley, Rachel Rogers; Moore, Susan McAfee; Tierney, Bruce C; Ye, Xiaoyun; Calafat, Antonia M; Campbell, Sean; Woudneh, Million B; Fisher, Jeffrey

2017-09-01

Per- and polyfluoroalkyl substances (PFAS) are considered chemicals of emerging concern, in part due to their environmental and biological persistence and the potential for widespread human exposure. In 2007, a PFAS manufacturer near Decatur, Alabama notified the United States Environmental Protection Agency (EPA) it had discharged PFAS into a wastewater treatment plant, resulting in environmental contamination and potential exposures to the local community. To characterize PFAS exposure over time, the Agency for Toxic Substances and Disease Registry (ATSDR) collected blood and urine samples from local residents. Eight PFAS were measured in serum in 2010 (n=153). Eleven PFAS were measured in serum, and five PFAS were measured in urine (n=45) from some of the same residents in 2016. Serum concentrations were compared to nationally representative data and change in serum concentration over time was evaluated. Biological half-lives were estimated for perfluorooctanoic acid (PFOA), perfluorooctane sulfonic acid (PFOS), and perfluorohexane sulfonic acid (PFHxS) using a one-compartment pharmacokinetic model. In 2010 and 2016, geometric mean PFOA and PFOS serum concentrations were elevated in participants compared to the general U.S. In 2016, the geometric mean PFHxS serum concentration was elevated compared to the general U.S. Geometric mean serum concentrations of PFOA, PFOS, and perfluorononanoic acid (PFNA) were significantly (p≤0.0001) lower (49%, 53%, and 58%, respectively) in 2016 compared to 2010. Half-lives for PFOA, PFOS, and PFHxS were estimated to be 3.9, 3.3, and 15.5years, respectively. Concentrations of PFOA in serum and urine were highly correlated (r=0.75) in males. Serum concentrations of some PFAS are decreasing in this residentially exposed community, but remain elevated compared to the U.S. general population. Published by Elsevier Ltd.

Chemometric resolution of fully overlapped CE peaks: quantitation of carbamazepine in human serum in the presence of several interferences.

PubMed

Vera-Candioti, Luciana; Culzoni, María J; Olivieri, Alejandro C; Goicoechea, Héctor C

2008-11-01

Drug monitoring in serum samples was performed using second-order data generated by CE-DAD, processed with a suitable chemometric strategy. Carbamazepine could be accurately quantitated in the presence of its main metabolite (carbamazepine epoxide), other therapeutic drugs (lamotrigine, phenobarbital, phenytoin, phenylephrine, ibuprofen, acetaminophen, theophylline, caffeine, acetyl salicylic acid), and additional serum endogenous components. The analytical strategy consisted of the following steps: (i) serum sample clean-up to remove matrix interferences, (ii) data pre-processing, in order to reduce the background and to correct for electrophoretic time shifts, and (iii) resolution of fully overlapped CE peaks (corresponding to carbamazepine, its metabolite, lamotrigine and unexpected serum components) by the well-known multivariate curve resolution-alternating least squares algorithm, which extracts quantitative information that can be uniquely ascribed to the analyte of interest. The analyte concentration in serum samples ranged from 2.00 to 8.00 mg/L. Mean recoveries were 102.6% (s=7.7) for binary samples, and 94.8% (s=13.5) for spiked serum samples, while CV (%)=4.0 was computed for five replicate, indicative of the acceptable accuracy and precision of the proposed method.

Acinetobacter baumannii Biofilm Formation in Human Serum and Disruption by Gallium

PubMed Central

Runci, Federica; Bonchi, Carlo; Frangipani, Emanuela; Visaggio, Daniela

2016-01-01

ABSTRACT Biofilm-associated infections caused by Acinetobacter baumannii are extremely recalcitrant to antibiotic treatment. We report that A. baumannii develops a mature biofilm when grown in complement-free human serum (HS). We demonstrate that 16 μM gallium nitrate (GaN) drastically reduces A. baumannii growth and biofilm formation in HS, whereas 64 μM GaN causes massive disruption of preformed A. baumannii biofilm. These findings pave the way to the repurposing of GaN as an antibiofilm agent for A. baumannii. PMID:27799219

Serum S-adenosylmethionine, but not methionine, increases in response to overfeeding in humans.

PubMed

Elshorbagy, A K; Jernerén, F; Samocha-Bonet, D; Refsum, H; Heilbronn, L K

2016-01-25

Plasma concentration of the methyl donor S-adenosylmethionine (SAM) is linearly associated with body mass index (BMI) and fat mass. As SAM is a high-energy compound and a sensor of cellular nutrient status, we hypothesized that SAM would increase with overfeeding. Forty normal to overweight men and women were overfed by 1250 kcal per day for 28 days. Serum SAM increased from 106 to 130 nmol/l (P=0.006). In stratified analysis, only those with weight gain above the median (high-weight gainers; average weight gain 3.9±0.3 kg) had increased SAM (+42%, P=0.001), whereas low-weight gainers (weight gain 1.5±0.2 kg) did not (Pinteraction=0.018). Overfeeding did not alter serum concentrations of the SAM precursor, methionine or the products, S-adenosyl-homocysteine and homocysteine. The SAM/SAH (S-adenosylhomocysteine) ratio was unchanged in the total population, but increased in high-weight gainers (+52%, P=0.006, Pinteraction =0.005). Change in SAM correlated positively with change in weight (r=0.33, P=0.041) and fat mass (r=0.44, P=0.009), but not with change in protein intake or plasma methionine, glucose, insulin or low-density lipoprotein (LDL)-cholesterol. Overfeeding raised serum SAM in proportion to the fat mass gained. The increase in SAM may help stabilize methionine levels, and denotes a responsiveness of SAM to nutrient state in humans. The role of SAM in human energy metabolism deserves further attention.

Quantification of a Cardiac Biomarker in Human Serum Using Extraordinary Optical Transmission (EOT)

PubMed Central

Ding, Tao; Hong, Minghui; Richards, A. Mark; Wong, Ten It; Zhou, Xiaodong; Drum, Chester Lee

2015-01-01

Nanoimprinting lithography (NIL) is a manufacturing process that can produce macroscale surface areas with nanoscale features. In this paper, this technique is used to solve three fundamental issues for the application of localized surface plasmonic resonance (LSPR) in practical clinical measurements: assay sensitivity, chip-to-chip variance, and the ability to perform assays in human serum. Using NIL, arrays of 140 nm square features were fabricated on a sensing area of 1.5 mm x 1.5 mm with low cost. The high reproducibility of NIL allowed for the use of a one-chip, one-measurement approach with 12 individually manufactured surfaces with minimal chip-to-chip variations. To better approximate a real world setting, all chips were modified with a biocompatible, multi-component monolayer and inter-chip variability was assessed by measuring a bioanalyte standard (2.5−75 ng/ml) in the presence of a complex biofluid, human serum. In this setting, nanoimprinted LSPR chips were able to provide sufficient characteristics for a ‘low-tech’ approach to laboratory-based bioanalyte measurement, including: 1) sufficient size to interface with a common laboratory light source and detector without the need for a microscope, 2) high sensitivity in serum with a cardiac troponin limit of detection of 0.55 ng/ml, and 3) very low variability in chip manufacturing to produce a figure of merit (FOM) of 10.5. These findings drive LSPR closer to technical comparability with ELISA-based assays while preserving the unique particularities of a LSPR based sensor, suitability for multiplexing and miniaturization, and point-of-care detections. PMID:25774658

Serum concentrations of polychlorinated dibenzo-p-dioxins among ceramicists.

PubMed

Demond, Avery; Jiang, Xiaohui; Broadwater, Kendra; Meeker, John; Luksemburg, William; Maier, Martha; Garabrant, David; Franzblau, Alfred

2015-01-01

Polychlorinated dibenzo-p-dioxins (PCDDs) occur naturally in ball clay at elevated concentrations. Thus, persons who habitually work with clay may be at risk for exposure to PCDDs. An earlier case report provided some evidence of elevated PCDD levels in serum for long-term hobby ceramicists; however, no previous study has measured serum dioxin concentrations among ceramicists. This study measured PCDD serum levels for 27 individuals involved in ceramics making. The average residual, defined as the average of the [log measured serum lipid concentration – log background serum lipid concentration], was calculated and then tested to determine whether it was significantly different from zero. The p-values for the average residuals indicated that the serum lipid concentrations for several PCDD congeners were elevated relative to background. The number of significant residuals increased dramatically if the background concentrations were adjusted to account for the fact that they were not contemporaneous with the measurements for the ceramicists. The ratio of the 1,2,3,6,7,8-HxCDD concentration to the 1,2,3,7,8,9-HxCDD concentration was greater than 1.0, unlike in ball clay, suggesting that although long-term working with ball clay elevates the PCDD levels in serum somewhat, it is not the predominant source of the PCDD body burden for ceramicists.

Spatial distribution of bisphenol S in surface water and human serum from Yangtze River watershed, China: Implications for exposure through drinking water.

PubMed

Wan, Yanjian; Xia, Wei; Yang, Shunyi; Pan, Xinyun; He, Zhenyu; Kannan, Kurunthachalam

2018-05-01

Bisphenol S (BPS) is an emerging environmental contaminant. The occurrence of this compound in humans and the environment is not well described. In this study, 120 surface water samples and 240 human serum samples were collected along the Yangtze River in 2015 for the determination of the occurrence of BPS. Surface water and human serum samples were extracted by solid phase extraction and liquid-liquid extraction, respectively, and analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). BPS was detected in all river water samples at concentrations that ranged from 0.18 to 14.9 ng/L (median: 0.98 ng/L), with higher concentrations in spring than summer. The median estimated daily intake (EDI) of BPS through water ingestion by infants in spring and summer was 0.12 and 0.06 ng/kg body weight (bw)/day, respectively. BPS was detected in human serum with the highest concentrations in samples from Nanjing (median: 0.65 ng/mL, maximum: 169 ng/mL) among the four cities studied. No significant gender related difference in BPS concentrations was observed in human sera, while higher concentrations were found in younger individuals than elderly. The EDI of BPS calculated based on serum concentrations of adults in Nanjing was 22.8 ng/kg bw/day. Ingestion of water accounted for <1% of the total BPS intake by the Chinese population. This is the first report of the occurrence of BPS in water from the Yangtze River and human serum from several cities located along this river in China. Copyright © 2018 Elsevier Ltd. All rights reserved.

English Proficiency and Competency Background of Social Science and Humanities Students

ERIC Educational Resources Information Center

Pelayo, Jose Maria G., III; Kutschera, P. C.; Capili, Claire Ann P.

2014-01-01

The study focuses on the background of Social Science and Humanities students (specifically in the course General Psychology) on their English education and competence. This research aims to identify the common factors of these students in terms of their English Proficiency. The students will answer survey questions that will give us information…

Human dental pulp stem cells cultured in serum-free supplemented medium

PubMed Central

Bonnamain, Virginie; Thinard, Reynald; Sergent-Tanguy, Solène; Huet, Pascal; Bienvenu, Géraldine; Naveilhan, Philippe; Farges, Jean-Christophe; Alliot-Licht, Brigitte

2013-01-01

Growing evidence show that human dental pulp stem cells (DPSCs) could provide a source of adult stem cells for the treatment of neurodegenerative pathologies. In this study, DPSCs were expanded and cultured with a protocol generally used for the culture of neural stem/progenitor cells. Methodology: DPSC cultures were established from third molars. The pulp tissue was enzymatically digested and cultured in serum-supplemented basal medium for 12 h. Adherent (ADH) and non-adherent (non-ADH) cell populations were separated according to their differential adhesion to plastic and then cultured in serum-free defined N2 medium with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). Both ADH and non-ADH populations were analyzed by FACS and/or PCR. Results: FACS analysis of ADH-DPSCs revealed the expression of the mesenchymal cell marker CD90, the neuronal marker CD56, the transferrin receptor CD71, and the chemokine receptor CXCR3, whereas hematopoietic stem cells markers CD45, CD133, and CD34 were not expressed. ADH-DPSCs expressed transcripts coding for the Nestin gene, whereas expression levels of genes coding for the neuronal markers β-III tubulin and NF-M, and the oligodendrocyte marker PLP-1 were donor dependent. ADH-DPSCs did not express the transcripts for GFAP, an astrocyte marker. Cells of the non-ADH population that grew as spheroids expressed Nestin, β-III tubulin, NF-M and PLP-1 transcripts. DPSCs that migrated out of the spheroids exhibited an odontoblast-like morphology and expressed a higher level of DSPP and osteocalcin transcripts than ADH-DPSCs. Conclusion: Collectively, these data indicate that human DPSCs can be expanded and cultured in serum-free supplemented medium with EGF and bFGF. ADH-DPSCs and non-ADH populations contained neuronal and/or oligodendrocyte progenitors at different stages of commitment and, interestingly, cells from spheroid structures seem to be more engaged into the odontoblastic lineage than the ADH

Glycoblotting method allows for rapid and efficient glycome profiling of human Alzheimer's disease brain, serum and cerebrospinal fluid towards potential biomarker discovery.

PubMed

Gizaw, Solomon T; Ohashi, Tetsu; Tanaka, Masakazu; Hinou, Hiroshi; Nishimura, Shin-Ichiro

2016-08-01

Understanding of the significance of posttranslational glycosylation in Alzheimer's disease (AD) is of growing importance for the investigation of the pathogenesis of AD as well as discovery research of the disease-specific serum biomarkers. We designed a standard protocol for the glycoblotting combined with MALDI-TOFMS to perform rapid and quantitative profiling of the glycan parts of glycoproteins (N-glycans) and glycosphingolipids (GSLs) using human AD's post-mortem samples such as brain tissues (dissected cerebral cortices such as frontal, parietal, occipital, and temporal domains), serum and cerebrospinal fluid (CSF). The structural profiles of the major N-glycans released from glycoproteins and the total expression levels of the glycans were found to be mostly similar between the brain tissues of the AD patients and those of the normal control group. In contrast, the expression levels of the serum and CSF protein N-glycans such as bisect-type and multiply branched glycoforms were increased significantly in AD patient group. In addition, the levels of some gangliosides such as GM1, GM2 and GM3 appeared to alter in the AD patient brain and serum samples when compared with the normal control groups. Alteration of the expression levels of major N- and GSL-glycans in human brain tissues, serum and CSF of AD patients can be monitored quantitatively by means of the glycoblotting-based standard protocols. The changes in the expression levels of the glycans derived from the human post-mortem samples uncovered by the standardized glycoblotting method provides potential serum biomarkers in central nervous system disorders and can contribute to the insight into the molecular mechanisms in the pathogenesis of neurodegenerative diseases and future drug discovery. Most importantly, the present preliminary trials using human post-mortem samples of AD patients suggest that large-scale serum glycomics cohort by means of various-types of human AD patients as well as the normal

Silver vanadate nanoribbons: A label-free bioindicator in the conversion between human serum transferrin and apotransferrin via surface-enhanced Raman scattering

NASA Astrophysics Data System (ADS)

Zhou, Qing; Shao, Mingwang; Que, Ronghui; Cheng, Liang; Zhuo, Shujuan; Tong, Yanhua; Lee, Shuit-Tong

2011-05-01

Silver vanadate nanoribbons were synthesized via a hydrothermal process, which exhibited surface-enhanced Raman scattering effect. This surface-enhanced substrate was stable and reproducible for identifying human serum transferrin and human serum apotransferrin in the concentration of 1×10-5 M, which further exhibited significant sensitivity in monitoring the conversion of these two proteins in turn. This result showed that the silver vanadate nanoribbon might be employed as biomonitor in such systems.

Association of a Human FABP1 Gene Promoter Region Polymorphism with Altered Serum Triglyceride Levels

PubMed Central

Zhu, Yi-bing; Huang, Rong-dong; Lu, Qing-Qing; Lin, Xu

2015-01-01

Liver fatty acid-binding protein (L-FABP), also known as fatty acid-binding protein 1 (FABP1), is a key regulator of hepatic lipid metabolism. Elevated FABP1 levels are associated with an increased risk of cardiovascular disease (CVD) and metabolic syndromes. In this study, we examine the association of FABP1 gene promoter variants with serum FABP1 and lipid levels in a Chinese population. Four promoter single-nucleotide polymorphisms (SNPs) of FABP1 gene were genotyped in a cross-sectional survey of healthy volunteers (n = 1,182) from Fuzhou city of China. Results showed that only the rs2919872 G>A variant was significantly associated with serum TG concentration(P = 0.032).Compared with the rs2919872 G allele, rs2919872 A allele contributed significantly to reduced serum TG concentration, and this allele dramatically decreased the FABP1 promoter activity(P < 0.05). The rs2919872 A allele carriers had considerably lower serum FABP1 levels than G allele carriers (P < 0.01). In the multivariable linear regression analysis, the rs2919872 A allele was negatively associated with serum FABP1 levels (β = —0.320, P = 0.003), while serum TG levels were positively associated with serum FABP1 levels (β = 0.487, P = 0.014). Our data suggest that compared with the rs2919872 G allele, the rs2919872 A allele reduces the transcriptional activity of FABP1 promoter, and thereby may link FABP1 gene variation to TG level in humans. PMID:26439934

[Construction and expression of recombinant human serum albumin-EPO fusion protein].

PubMed

Huang, Ying-Chun; Gou, Xing-Hua; Han, Lei; Li, De-Hua; Zhao, Lan-Ying; Wu, Qia-Qing

2011-05-01

OBJECTIVE To construct the recombinant plasmid pCI-HLE encoding human serum album-EPO (HSA-EPO) fusion protein and to express it in CHO cell. The cDNA encoding human serum album and EPO were amplified by PCR, and then spliced with the synsitic DNA fragment encoding GS (GGGGS), by overlap PCR extension to form LEPO. After BamH I digestion, the HSA and LEPO was ligated to generate the fusion HSA-EPO gene and was then cloned into the expression vector pCI-neo to generate the recombinant plasmid pCI-HLE. The plasmid pCI-HLE was transfected into CHO cell by liposome protocol. Then, the recombinant cells were screened by G418 and identified by PCR and Western blot. Expression of fusion protein was evaluated by Enzyme Linked Immunosorbent Assay (ELISA). Restrictive enzymes digestion and DNA sequencing revealed that HSA-EPO fusion gene was cloned into expression vector pCI-neo successfully. PCR and Western blot analysis confirmed that the fusion gene was integrated in the genome of CHO cells and expressed successfully. The HSA-EPO production varied from 86 Iu/(mL x 10(6) x 72 h) to 637 IU/(mLx 10(6) x 72 h). The results confirmed that HSA-EPO fusion gene can be expressed in the CHO cells, with EPO immunogenicity, which could serve as foundation for the development of long-lasting recombinant HSA-EPO protein.

Simple and rapid LC-MS/MS method for the absolute determination of cetuximab in human serum using an immobilized trypsin.

PubMed

Shibata, Kaito; Naito, Takafumi; Okamura, Jun; Hosokawa, Seiji; Mineta, Hiroyuki; Kawakami, Junichi

2017-11-30

Proteomic approaches using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) without an immunopurification technique have not been applied to the determination of serum cetuximab. This study developed a simple and rapid LC-MS/MS method for the absolute determination of cetuximab in human serum and applied it to clinical settings. Surrogate peptides derived from cetuximab digests were selected using a Fourier transform mass spectrometer. Reduced-alkylated serum cetuximab without immunopurification was digested for 20minutes using immobilized trypsin, and the digestion products were purified by solid-phase extraction. The LC-MS/MS was run in positive ion multiple reaction monitoring mode. This method was applied to the determination of serum samples in head and neck cancer patients treated with cetuximab. The chromatographic run time was 10minutes and no peaks interfering with surrogate peptides in serum digestion products were observed. The calibration curve of absolute cetuximab in serum was linear over the concentration range of 4-200μg/mL. The lower limit of quantification of cetuximab in human serum was 4μg/mL. The intra-assay and inter-assay precision and accuracy were less than 13.2% and 88.0-100.7%, respectively. The serum concentration range of cetuximab was 19-140μg/mL in patients. The serum cetuximab concentrations in LC-MS/MS were correlated with those in ELISA (r=0.899, P <0.01) and the mean bias was 1.5% in cancer patients. In conclusion, the present simple and rapid method with acceptable analytical performance can be helpful for evaluating the absolute concentration of serum cetuximab in clinical settings. Copyright © 2017 Elsevier B.V. All rights reserved.

Acinetobacter baumannii Biofilm Formation in Human Serum and Disruption by Gallium.

PubMed

Runci, Federica; Bonchi, Carlo; Frangipani, Emanuela; Visaggio, Daniela; Visca, Paolo

2017-01-01

Biofilm-associated infections caused by Acinetobacter baumannii are extremely recalcitrant to antibiotic treatment. We report that A. baumannii develops a mature biofilm when grown in complement-free human serum (HS). We demonstrate that 16 μM gallium nitrate (GaN) drastically reduces A. baumannii growth and biofilm formation in HS, whereas 64 μM GaN causes massive disruption of preformed A. baumannii biofilm. These findings pave the way to the repurposing of GaN as an antibiofilm agent for A. baumannii. Copyright © 2016 American Society for Microbiology.

Forster resonance energy transfer in the system of human serum albumin-xanthene dyes

NASA Astrophysics Data System (ADS)

Kochubey, V. I.; Pravdin, A. B.; Melnikov, A. G.; Konstantinova, I.; Alonova, I. V.

2016-04-01

The processes of interaction of fluorescent probes: eosin and erythrosine with human serum albumin (HSA) were studied by the methods of absorption and fluorescence spectroscopy. Extinction coefficients of probes were determined. Critical transfer radius and the energy transfer efficiency were defined by fluorescence quenching of HSA. Analysis of the excitation spectra of HSA revealed that the energy transfer process is carried out mainly between tryptophanyl and probes.

Photometric flow analysis system for biomedical investigations of iron/transferrin speciation in human serum.

PubMed

Strzelak, Kamil; Rybkowska, Natalia; Wiśniewska, Agnieszka; Koncki, Robert

2017-12-01

The Multicommutated Flow Analysis (MCFA) system for the estimation of clinical iron parameters: Serum Iron (SI), Unsaturated Iron Binding Capacity (UIBC) and Total Iron Binding Capacity (TIBC) has been proposed. The developed MCFA system based on simple photometric detection of iron with chromogenic agent (ferrozine) enables a speciation of transferrin (determination of free and Fe-bound protein) in human serum. The construction of manifold was adapted to the requirements of measurements under changing conditions. In the course of studies, a different effect of proteins on SI and UIBC determination has been proven. That was in turn the reason to perform two kinds of calibration methods. For measurements in acidic medium for SI/holotransferrin determination, the calibration curve method was applied, characterized by limit of determination and limit of quantitation on the level of 3.4 μmol L -1 and 9.1 μmol L -1 , respectively. The determination method for UIBC parameter (related to apotransferrin level) in physiological medium of pH 7.4 forced the use of standard addition method due to the strong influence of proteins on obtaining analytical signals. These two different methodologies, performed in the presented system, enabled the estimation of all three clinical iron/transferrin parameters in human serum samples. TIBC corresponding to total transferrin level was calculated as a sum of SI and UIBC. Copyright © 2017 Elsevier B.V. All rights reserved.

Determination of fluvoxamine maleate in human urine and human serum using alkaline KMnO4 -rhodamine B chemiluminescence.

PubMed

Yang, Dongqin; He, Yanyan; Chen, Funan

2017-09-01

The flow-injection chemiluminescence (FI-CL) behavior of a gold nanocluster (Au NC)-enhanced rhodamine B-KMnO 4 system was studied under alkaline conditions for the first time. In the present study, the as-prepared bovine serum albumin-stabilized Au NCs showed excellent stability and reproducibility. The addition of trace levels of fluvoxamine maleate (Flu) led to an obvious decline in CL intensity in the rhodamine B-KMnO 4 -Au NCs system, which could be used for quantitative detection of Flu. Under optimized conditions, the proposed CL system exhibited a favorable analytical performance for Flu determination in the range 2 to 100 μg ml -1 . The detection limit for Flu measurement was 0.021 μg ml -1 . Moreover, this newly developed system revealed outstanding selectivity for Flu detection when compared with a multitude of other species, such as the usual ions, uric acid and a section of hydroxy compounds. Additionally, CL spectra, UV-visible spectroscopes and fluorescence spectra were measured in order to determine the possible reaction mechanism. This approach could be used to detect Flu in human urine and human serum samples with the desired recoveries and could have promising application under physiological conditions. Copyright © 2017 John Wiley & Sons, Ltd.

Serum sialyltransferase and liver catalase activity in cachectic nude mice bearing a human malignant melanoma.

PubMed

Kondo, Y; Sato, K; Ueyama, Y; Ohsawa, N

1981-07-01

Cachexia is rare in nude mice bearing human malignant tumors even when the transplanted tumors become as large as the body size of the host. In our series on heterotransplantation of a variety of human malignant tumors into nude mice, a malignant melanoma (SEKI) was found to induce severe body weight loss in the host at the early stage of transplantation. There was no electrolyte disturbance, hyper- or hypoadrenocorticism, hyperthyroidism, or destruction of cells of vital organs to account for the weight loss. Moreover, no evidence was obtained for concomitant infection with bacteria, Mycoplasma or fungi. These cachectic mice revealed remarkably increased levels of serum sialyltransferase and decreased liver catalase activity. The removal of tumor tissues from these mice resulted in prompt recovery of body weight, serum sialyltransferase, and liver catalase activity within 1 to 2 weeks. On the basis of the results obtained, the SEKI melanoma was thought to have produced a pathophysiological state in host nude mice which was very similar to that of cachexia in cancer patients. Nude mice bearing transplants of SEKI melanoma may provide a useful system for the study of cancer cachexia in humans.

Serum 25-hydroxyvitamin d levels and C-reactive protein in persons with human immunodeficiency virus infection.

PubMed

Poudel-Tandukar, Kalpana; Poudel, Krishna C; Jimba, Masamine; Kobayashi, Jun; Johnson, C Anderson; Palmer, Paula H

2013-03-01

Human immunodeficiency virus (HIV) infection has frequently been associated with vitamin D deficiency as well as chronic inflammatory response. We tested the hypothesis of an independent relationship between serum concentrations of 25-hydroxyvitamin D [25(OH)D] and high-sensitivity C-reactive protein (CRP) in a cohort of HIV-positive people. A cross-sectional survey was conducted among 316 HIV-positive people (181 men and 135 women) aged 16 to 60 years residing in the Kathmandu Valley, Nepal. Serum high-sensitivity CRP concentrations and serum 25(OH)D levels were measured by the latex agglutination nephelometry method and the competitive protein-binding assay, respectively. The relationship between serum CRP concentrations and 25(OH)D serum level was assessed using multiple logistic regression analysis with adjustment of potential cardiovascular and HIV-related factors. The proportions of participants with 25(OH)D serum levels <20 ng/ml, 20-30 ng/ml, and ≥30 ng/ml were 83.2%, 15.5%, and 1.3%, respectively. The mean 25(OH)D serum levels in men and women were 15.3 ng/ml and 14.4 ng/ml, respectively. Participants with a 25(OH)D serum level of <20 ng/ml had a 3.2-fold higher odds of high CRP (>3 mg/liter) compared to those with a 25(OH)D serum level of ≥20 ng/ml (p=0.005). Men and women with a 25(OH)D serum level of <20 ng/ml had 3.2- and 2.7-fold higher odds of high CRP (>3 mg/liter), respectively, compared to those with a 25(OH)D serum level of ≥20 ng/ml. The relationships remained significant only in men (p =0.02) but not in women (p=0.28). The risk of having a high level of inflammation (CRP>3 mg/liter) may be high among HIV-positive men and women with a 25(OH)D serum level of <20 ng/ml.

Evaluation of an enzyme immunoassay for detection of dengue virus NS1 antigen in human serum.

PubMed

Dussart, Philippe; Labeau, Bhety; Lagathu, Gisèle; Louis, Philippe; Nunes, Marcio R T; Rodrigues, Sueli G; Storck-Herrmann, Cécile; Cesaire, Raymond; Morvan, Jacques; Flamand, Marie; Baril, Laurence

2006-11-01

We evaluated a one-step sandwich-format microplate enzyme immunoassay for detecting dengue virus NS1 antigen (Ag) in human serum by use of Platelia Dengue NS1 Ag kits (Bio-Rad Laboratories, Marnes La Coquette, France). We collected 299 serum samples from patients with dengue disease and 50 serum samples from patients not infected with dengue virus. For the 239 serum samples from patients with acute infections testing positive by reverse transcription-PCR and/or virus isolation for one of the four dengue virus serotypes, the sensitivity of the Platelia Dengue NS1 Ag kit was 88.7% (95% confidence interval, 84.0% to 92.4%). None of the serum samples from patients not infected with dengue virus tested positive with the Platelia Dengue NS1 Ag kit. A diagnostic strategy combining the Platelia Dengue NS1 Ag test for acute-phase sera and immunoglobulin M capture enzyme-linked immunosorbent assay for early-convalescent-phase sera increased sensitivity only from 88.7% to 91.9%. Thus, NS1 antigen detection with the Platelia Dengue NS1 Ag kit could be used for first-line testing for acute dengue virus infection in clinical diagnostic laboratories.

Radiation therapy induces circulating serum Hsp72 in patients with prostate cancer

PubMed Central

Hurwitz, Mark D.; Kaur, Punit; Nagaraja, Ganachari M.; Bausero, Maria A.; Manola, Judith; Asea, Alexzander

2010-01-01

Background and purpose Hsp72 found in the extracellular milieu has been shown to play an important role in immune regulation. The impact of common cancer therapies on extracellular release of Hsp72 however, has been to date undefined. Materials and methods Serum from 13 patients undergoing radiation therapy (XRT) for prostate cancer with or without hormonal therapy (ADT) was measured for levels of circulating serum Hsp72 and pro-inflammatory cytokines (IL-6 and TNF-α) using the classical sandwich ELISA technique and the relative expression of CD8+ T lymphocytes and natural killer (NK) cells was measured using flow cytometry. Mouse orthotopic xenograft of human prostate cancer tumors (DU145 and PC3) were used to validate and further characterize the response noted in the clinical setting. The biological significance of tumor released Hsp72 was studied in human dendritic cells (DC) in vitro. Results Circulating serum Hsp72 levels increased an average of 3.5-fold (median per patient 4.8-fold) with XRT but not with ADT (p = 0.0002). Increases in IL-6 (3.3-fold), TNF-α (1.8-fold), CD8+ CTL (2.1-fold) and NK cells (3.2-fold) also occurred. Using PC3 and DU145 human prostate cancer xenograft models in mice, we confirmed that XRT induces Hsp72 release primarily from implanted tumors. In vitro studies using supernatant recovered from irradiated human prostate cancer cells point to exosomes containing Hsp72 as a possible stimulator of pro-inflammatory cytokine production and costimulatory molecules expression in human DC. Conclusions The current study confirms for the first time in an actual clinical setting elevation of circulating serum Hsp72 with XRT. The accompanying studies in mice and in vitro identify the released exosomes containing Hsp72 as playing a pivotal role in stimulating pro-inflammatory immune responses. These findings, if validated, may lead to new treatment paradigms for common human malignancies. PMID:20430459

Multiple free-radical scavenging (MULTIS) capacity in cattle serum.

PubMed

Sueishi, Yoshimi; Kamogawa, Erisa; Kimura, Anna; Kitahara, Go; Satoh, Hiroyuki; Asanuma, Taketoshi; Oowada, Shigeru

2017-01-01

Multiple free-radical scavenging (MULTIS) activity in cattle and human sera was evaluated with electron spin resonance spectroscopy. Scavenging rates against six active species, namely hydroxyl radical, superoxide anion, alkoxyl radical, alkylperoxyl radical, methyl radical, and singlet oxygen were quantified. The difference in the electron spin resonance signal intensity in the presence and absence of the serum was converted into the scavenging rates. Comparative MULTIS measurements were made in sera from eight beef cattle, three fetal calves and fifteen healthy human volunteers. Further, we determined the MULTIS value of albumin, the most abundant component in serum. MULTIS values in cattle sera indicated higher scavenging activity against most free radical species tested than human sera. In particular, cattle serum scavenging activities against superoxide and methyl radical were higher than human serum by 2.6 and 3.7 fold, respectively. In cattle serum, albumin appears to play a dominant role in MULTIS activity, but in human serum that is not the case. Previous data indicated that the abundance of uric acid in bovine blood is nearly 80% less than humans; however, this difference does not explain the deviation in MULTIS profile.

Human serum albumin crystals and method of preparation

NASA Technical Reports Server (NTRS)

Carter, Daniel C. (Inventor)

1989-01-01

Human serum albumin (HSA) crystals are provided in the form of tetragonal plates having the space groups P42(sub 1)2, the crystals being grown to sizes in excess of 0.5 mm in two dimensions and a thickness of 0.1 mm. Growth of the crystals is carried out by a hanging drop method wherein a precipitant solution containing polyethylene glycol (PEG) and a phosphate buffer is mixed with an HSA solution, and a droplet of mixed solution is suspended over a well of precipitant solution. Crystals grow to the desired size in 3 to 7 days. Concentration of reagents, pH and other parameters are controlled within prescribed limits. The resulting crystals exhibit a size and quality such as to allow performance of x ray diffraction studies and enable the conduct of drug binding studies as well as genetic engineering studies.

Isolation and recovery of selected polybrominated diphenyl ethers from human serum and sheep serum: coupling reversed-phase solid-phase disk extraction and liquid-liquid extraction techniques with a capillary gas chromatographic electron capture negative ion mass spectrometric determinative technique.

PubMed

Loconto, Paul R; Isenga, David; O'Keefe, Michael; Knottnerus, Mark

2008-01-01

Polybrominated diphenyl ethers (PBDEs) are isolated and recovered with acceptable percent recoveries from human serum via liquid-liquid extraction and column chromatographic cleanup and fractionation with quantitation using capillary gas chromatography-mass spectrometry with electron capture negative ion and selected ion monitoring. PBDEs are found in unspiked serum. An alternative sample preparation approach is developed using sheep serum that utilizes a formic acid pre-treatment followed by reversed-phase solid-phase disk extraction and normal-phase solid-phase cleanup using acidified silica gel that yields>50% recoveries. When these percent recoveries are combined with a minimized phase ratio for human serum and very low instrument detection limits, method detection limits below 500 parts-per-trillion are realized.

Thermometric enzyme linked immunosorbent assay in continuous flow system: optimization and evaluation using human serum albumin as a model system.

PubMed

Borrebaeck, C; Börjeson, J; Mattiasson, B

1978-06-15

Thermometric enzyme-linked immunosorbent assay (TELISA) is described. After the procedure of optimization, human serum albumin was assayed using anti-human serum albumin bound to Sepharose CL 4-B in the enzyme thermistor unit and catalase as label on the free antigen. The model system was used for assays down to 10(-13)M and the preparation of immobilized antibodies was used repeatedly up to 100 times. Comparative studies of the TELISA technique with bromocresol green, immunoturbidimetric and rocket immunoelectrophoretic methods were carried out and showed that TELISA could be used as an alternative method.

Eimeria Species and Genetic Background Influence the Serum Protein Profile of Broilers with Coccidiosis

PubMed Central

Gilbert, Elizabeth R.; Cox, Chasity M.; Williams, Patricia M.; McElroy, Audrey P.; Dalloul, Rami A.; Ray, W. Keith; Barri, Adriana; Emmerson, Derek A.; Wong, Eric A.; Webb, Kenneth E.

2011-01-01

Background Coccidiosis is an intestinal disease caused by protozoal parasites of the genus Eimeria. Despite the advent of anti-coccidial drugs and vaccines, the disease continues to result in substantial annual economic losses to the poultry industry. There is still much unknown about the host response to infection and to date there are no reports of protein profiles in the blood of Eimeria-infected animals. The objective of this study was to evaluate the serum proteome of two genetic lines of broiler chickens after infection with one of three species of Eimeria. Methodology/Principal Findings Birds from lines A and B were either not infected or inoculated with sporulated oocysts from one of the three Eimeria strains at 15 d post-hatch. At 21 d (6 d post-infection), whole blood was collected and lesion scoring was performed. Serum was harvested and used for 2-dimensional gel electrophoresis. A total of 1,266 spots were quantitatively assessed by densitometry. Protein spots showing a significant effect of coccidia strain and/or broiler genetic line on density at P<0.05−0.01 (250 spots), P<0.01−0.001 (248 spots), and P<0.001 (314 spots) were excised and analyzed by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry. Proteins were identified in 172 spots. A total of 46 different proteins were identified. Of the spots with a corresponding protein identification, 57 showed a main effect of coccidia infection and/or 2-way interaction of coccidia infection×broiler genetic line at P<0.001. Conclusions/Significance Several of the metabolic enzymes identified in this study are potential candidates for early diagnostic markers of E. acervulina infection including malate dehydrogenase 2, NADH dehydrogenase 1 alpha subcomplex 9, and an ATP synthase. These proteins were detected only in Line A birds that were inoculated with E. acervulina. Results from this study provide a basic framework for future research aimed at uncovering the complex

Targeted quantification of low ng/mL level proteins in human serum without immunoaffinity depletion

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

Shi, Tujin; Sun, Xuefei; Gao, Yuqian

2013-07-05

We recently reported an antibody-free targeted protein quantification strategy, termed high-pressure, high-resolution separations with intelligent selection and multiplexing (PRISM) for achieving significantly enhanced sensitivity using selected reaction monitoring (SRM) mass spectrometry. Integrating PRISM with front-end IgY14 immunoaffinity depletion, sensitive detection of targeted proteins at 50-100 pg/mL levels in human blood plasma/serum was demonstrated. However, immunoaffinity depletion is often associated with undesired losses of target proteins of interest. Herein we report further evaluation of PRISM-SRM quantification of low-abundance serum proteins without immunoaffinity depletion and the multiplexing potential of this technique. Limits of quantification (LOQs) at low ng/mL levels with a medianmore » CV of ~12% were achieved for proteins spiked into human female serum using as little as 2 µL serum. PRISM-SRM provided up to ~1000-fold improvement in the LOQ when compared to conventional SRM measurements. Multiplexing capability of PRISM-SRM was also evaluated by two sets of serum samples with 6 and 21 target peptides spiked at the low attomole/µL levels. The results from SRM measurements for pooled or post-concatenated samples were comparable to those obtained from individual peptide fractions in terms of signal-to-noise ratios and SRM peak area ratios of light to heavy peptides. PRISM-SRM was applied to measure several ng/mL-level endogenous plasma proteins, including prostate-specific antigen, in clinical patient sera where correlation coefficients > 0.99 were observed between the results from PRISM-SRM and ELISA assays. Our results demonstrate that PRISM-SRM can be successfully used for quantification of low-abundance endogenous proteins in highly complex samples. Moderate throughput (50 samples/week) can be achieved by applying the post-concatenation or fraction multiplexing strategies. We anticipate broad applications for targeted PRISM

Tocopherol isomer pattern in serum and stool of human following consumption of black currant seed press residue administered in whole grain bread.

PubMed

Helbig, Dorit; Wagner, Andreas; Schubert, Rainer; Jahreis, Gerhard

2009-12-01

Serum gamma-tocopherol is thought to be associated with human health. The dietary influence of tocopherol and fibre-rich black currant seed press residue on serum and stool tocopherol concentration was investigated in a controlled human intervention study. Thirty-six women consumed bread enriched with black currant press residue (4 weeks). The resultant faecal and serum tocopherol concentrations were compared with those after a period consuming control bread without press residue and a normal baseline diet. Fibre intake and excretion, antioxidant capacity (TEAC), and vitamin C concentrations in serum and urine were also determined. Samples were obtained with a 5-day standardised diet at the end of each period. The press residue bread lead to significantly increased beta-, gamma-, delta- and total tocopherol intake, serum alpha-, beta-, gamma- and total tocopherol concentration (with and without lipid adjustment), fibre intake and urinary vitamin C concentration compared to control bread (P<0.05). Faecal excretion of total tocopherols and fibre increased compared to baseline (P<0.05). Fibre intake and excretion influence total tocopherol concentration in lipid-adjusted serum and in stool. The outstandingly high increase of serum gamma-tocopherol concentration through seed press residue consumption could be due to a presumed interruption of the enzymatic tocopherol degradation mechanism by bread constituents.

Measurements of vitamin B12 in human blood serum using resonance Raman spectroscopy

NASA Astrophysics Data System (ADS)

Tsiminis, G.; Schartner, E. P.; Brooks, J. L.; Hutchinson, M. R.

2016-12-01

Vitamin B12 (cobalamin and its derivatives) deficiency has been identified as a potential modifiable risk factor for dementia and Alzheimer's disease. Chronic deficiency of vitamin B12 has been significantly associated with an increased risk of cognitive decline. An effective and efficient method for measuring vitamin B12 concentration in human blood would enable ongoing tracking and assessment of this potential modifiable risk factor. In this work we present an optical sensor based on resonance Raman spectroscopy for rapid measurements of vitamin B12 in human blood serum. The measurement takes less than a minute and requires minimum preparation (centrifuging) of the collected blood samples.

Webinar Presentation: Suspect Screening of Environmental Organic Acids in Human Serum Using High-resolution Mass Spectrometry (HRMS)

EPA Pesticide Factsheets

This presentation, Suspect Screening of Environmental Organic Acids in Human Serum Using High-resolution Mass Spectrometry (HRMS), was given at the NIEHS/EPA Children's Centers 2016 Webinar Series: Exposome held on May 11, 2016.

Quantification of disease marker in undiluted serum using an actuating layer-embedded microcantilever

NASA Astrophysics Data System (ADS)

Hwang, Kyo Seon; Jeon, Hye Kyung; Lee, Sang-Myung; Kim, Sang Kyung; Kim, Tae Song

2009-05-01

In this study, we describe the application feasibility of a dynamic microcantilever with regard to the detection of a specific protein in human serum or real blood using an end-point analysis. The mechanical response (i.e., resonant frequency) of a functionalized dynamic microcantilever was shown to be altered by molecular interactions, which allowed for the detection of biomolecules present in small quantities without any additional signal enhancements, such as labeling. For the application of the microcantilever sensors to bioassays of serum samples, the mechanical response from the nonspecific adsorption of abundant proteins must be reduced, because it significantly influences the output signal deviation of the microcantilever sensor. We implemented a label-free prostate specific antigen (PSA) detection protocol in standard serum via our established process, which was designed to minimize nonspecific protein adsorption. PSA is a tumor marker for prostate cancer, with a threshold concentration of 2-4 ng/ml (7.2-14.4 pM) for the distinction between patients and normal individuals. The dynamic range of our dynamic microcantilever-based PSA assay on the background of standard serum ranged between 0.1 and 100 ng/ml (3.6 and 3600 pM). It was suggested that the dynamic microcantilever might allow for the sensitive label-free detection of disease markers in an actual human sample.

Bile acids induce arrhythmias in human atrial myocardium--implications for altered serum bile acid composition in patients with atrial fibrillation.

PubMed

Rainer, Peter P; Primessnig, Uwe; Harenkamp, Sandra; Doleschal, Bernhard; Wallner, Markus; Fauler, Guenter; Stojakovic, Tatjana; Wachter, Rolf; Yates, Ameli; Groschner, Klaus; Trauner, Michael; Pieske, Burkert M; von Lewinski, Dirk

2013-11-01

High bile acid serum concentrations have been implicated in cardiac disease, particularly in arrhythmias. Most data originate from in vitro studies and animal models. We tested the hypotheses that (1) high bile acid concentrations are arrhythmogenic in adult human myocardium, (2) serum bile acid concentrations and composition are altered in patients with atrial fibrillation (AF) and (3) the therapeutically used ursodeoxycholic acid has different effects than other potentially toxic bile acids. Multicellular human atrial preparations ('trabeculae') were exposed to primary bile acids and the incidence of arrhythmic events was assessed. Bile acid concentrations were measured in serum samples from 250 patients and their association with AF and ECG parameters analysed. Additionally, we conducted electrophysiological studies in murine myocytes. Taurocholic acid (TCA) concentration-dependently induced arrhythmias in atrial trabeculae (14/28 at 300 µM TCA, p<0.01) while ursodeoxycholic acid did not. Patients with AF had significantly decreased serum levels of ursodeoxycholic acid conjugates and increased levels of non-ursodeoxycholic bile acids. In isolated myocytes, TCA depolarised the resting membrane potential, enhanced Na(+)/Ca(2+) exchanger (NCX) tail current density and induced afterdepolarisations. Inhibition of NCX prevented arrhythmias in atrial trabeculae. High TCA concentrations induce arrhythmias in adult human atria while ursodeoxycholic acid does not. AF is associated with higher serum levels of non-ursodeoxycholic bile acid conjugates and low levels of ursodeoxycholic acid conjugates. These data suggest that higher levels of toxic (arrhythmogenic) and low levels of protective bile acids create a milieu with a decreased arrhythmic threshold and thus may facilitate arrhythmic events.

Preliminary crystallographic studies of four crystal forms of serum albumin

NASA Technical Reports Server (NTRS)

Carter, D. C.; Chang, B.; Ho, J. X.; Keeling, K.; Krishnasami, Z.

1994-01-01

Several crystal forms of serum albumin suitable for three-dimensional structure determination have been grown. These forms include crystals of recombinant and wild-type human serum albumin, baboon serum albumin, and canine serum albumin. The intrinsic limits of X-ray diffraction for these crystals are in the range 0.28-0.22 nm. Two of the crystal forms produced from human and canine albumin include incorporated long-chain fatty acids. Molecular replacement experiments have been successfully conducted on each crystal form using the previously determined atomic coordinates of human serum albumin illustrating the conserved tertiary structure.

Dichloro-diphenyl-trichloroethanes (DDTs) in human hair and serum in rural and urban areas in South China.

PubMed

He, Chun-Tao; Yan, Xiao; Wang, Mei-Huan; Zheng, Xiao-Bo; Chen, Ke-Hui; Guo, Mi-Na; Zheng, Jing; Chen, She-Jun

2017-05-01

Human hair has been employed as a biomarker for exposure to persistent organic pollutants (POPs), but information on the source of dichloro-diphenyl-trichloroethane (DDT) and its metabolites in hair is limited. The present study investigated the contamination of DDTs in human hair from a rural area and an urban area of South China and compared with those in human serum and indoor dust. The concentrations of ∑DDTs ranged from 2.30 to 489ng/g, with a median of 21.8ng/g in human hair. The ∑DDT concentrations (median=40.8ng/g) in female hair were significantly higher than those in male hair (median=20.6ng/g). There were significantly positive correlations between the concentrations of DDTs and ages in both the female and male hair, but the age-dependence for DDTs in serum was less significant. The profile of DDT analogues in female hair, differing from that in the male hair, was more similar to that in the indoor dust, suggesting a more important role of exogenous exposure in female hair. We estimated that exogenous source is responsible for approximately 11% and 20% of the burden of DDTs in the male and female hair, respectively. Adjusted multiple linear regression model showed significantly positive association between the p,p'-DDE concentrations in the paired hair and serum samples, indicating that endogenous origins are the primary sources of DDTs in the hair of the residents in the study areas. Our findings demonstrated that human hair is a reliable biomarker for body burden of DDTs and can be used in epidemiology research and retrospective assessment of DDT exposure. Copyright © 2017 Elsevier Inc. All rights reserved.

New Tween-80 microbiological assay of serum folate levels in humans and animals.

PubMed

Zhou, Zhenghua; Yang, Yuan; Li, Ming; Kou, Chong; Xiao, Ping; Jiang, Yan; Hong, Junrong; Huang, Chengyu

2012-01-01

The objective of this study was to develop a new Tween-80 microbiological assay (Tween-80 MBA) to determine human or animal serum folate levels and to verify its reliability. The effects of the Lactobacillius casei subspecies rhamnosus (L. casei, ATCC No. 7469) inoculum concentration, incubation time, and Tween-80 on L. casei growth were studied, and the serum folate levels were investigated. Serum samples were collected from patients with esophageal cancer (EC) and healthy control subjects in Yanting, healthy adult subjects in Chengdu, Sichuan, and in male Sprague-Dawley rats. Optimal conditions for the new MBA were as follows: 1.28 x 10(7) CFU/mL working inoculum, vitamin folic acid assay broth with 0.24% (w/w) Tween-80, and anaerobic incubation with L. casei at 37 degrees C for 22 h. Under the optimal conditions, the working curve was in simple linear rather than logarithmic equation; the linear working curve of the folic acid standard working solution concentration versus the turbidity (adsorption value) of medium with L. casei ranged from 0.05 to 1.00 microg/L; the linear correlation coefficient was 0.9989 (SD 0.0007); the recovery rate of folate was 105.4-112.7%; and the minimum concentration for detecting folate was 0.03 microg/L. The RSD within-day and between-day precisions were 5.6 and 3.3%, respectively. The serum folate level of 100 EC patients was 6.4 (SEM 0.4) microg/L which was significantly lower than that of healthy control subjects [8.0 (SEM 0.6) microg/L, n = 100, P=0.020]. The new Tween-80 MBA is considered to be a reliable method for measuring serum folate level.

CCQM K6.2 determination of total cholesterol in human serum

NASA Astrophysics Data System (ADS)

Wise, Stephen A.; Phinney, Karen W.; Duewer, David L.; Sniegoski, Lorna T.; Welch, Michael J.; Pabello, Guiomar; Avila Caldero, Marco A.; Qinde, Liu; Kooi, Lee Tong; Rego, Eliane; Garrido, Bruno; Allegri, Gabriella; de La Cruz, Marcia; Barrabin, Juliana; Puglisi, Celia; Lopez, Eduardo; Lee, Hwashim; Kim, Byungjoo; Delatour, Vincent; Heuillet, Maud; Nammoonnoy, Jintana; Ceyhan Gören, Ahmet; Bilsel, Gokhan; Konopelko, L.; Krylov, A.; Lopushanskaya, E.

2018-01-01

Cholesterol is one of the most frequently measured substances in human blood/serum to assist in assessing the health status of individuals. Because of its clinical significance, CCQM-K6 determination of cholesterol in serum was completed in 2000 as one of the first key comparison (KC) studies performed within the Organic Analysis Working Group (OAWG). The first subsequent KC for cholesterol, CCQM-K6.1, was completed in 2001. Measurements for this second subsequent, CCQM-K6.2, were completed in 2012. These subsequent comparisons were conducted to enable CCQM members that had not participated in earlier studies to demonstrate their capabilities to measure a nonpolar (pKow < ‑2), low molecular mass (100 g/mol to 500 g/mol) metabolite in human serum at relatively high concentrations (1 mg/g to 3 mg/g) found in normal populations. Successful participation in CCQM-K6.2 demonstrated capabilities in analysis of complex biological matrices including sample preparation (extraction, derivatization), LC or GC separation, and quantification using an isotope dilution mass spectrometry approach. Normally in a subsequent KC, no key comparison reference value (KCRV) would be established and assessment of performance would be via the deviation of participants' results to the anchor institute's results, adjusted to account for the anchor's performance in the original comparison versus its KCRV. Due to the very long-time period since the original key comparison, the OAWG decided that this did not represent the best approach to assess performance in what is a relatively complex measurement. Given the excellent agreement between the anchor institute's results and robust consensus summary of the participants' values, the reference value for this study was taken as the anchor institute's result and treated as a 'KCRV'. Seven of the nine participants demonstrated agreement with the reference value. Main text To reach the main text of this paper, click on Final Report. Note that this text

The establishment of a WHO Reference Reagent for anti-malaria (Plasmodium falciparum) human serum.

PubMed

Bryan, Donna; Silva, Nilupa; Rigsby, Peter; Dougall, Thomas; Corran, Patrick; Bowyer, Paul W; Ho, Mei Mei

2017-08-05

At a World Health Organization (WHO) sponsored meeting it was concluded that there is an urgent need for a reference preparation that contains antibodies against malaria antigens in order to support serology studies and vaccine development. It was proposed that this reference would take the form of a lyophilized serum or plasma pool from a malaria-endemic area. In response, an immunoassay standard, comprising defibrinated human plasma has been prepared and evaluated in a collaborative study. A pool of human plasma from a malaria endemic region was collected from 140 single plasma donations selected for reactivity to Plasmodium falciparum apical membrane antigen-1 (AMA-1) and merozoite surface proteins (MSP-1 19 , MSP-1 42 , MSP-2 and MSP-3). This pool was defibrinated, filled and freeze dried into a single batch of ampoules to yield a stable source of naturally occurring antibodies to P. falciparum. The preparation was evaluated by an enzyme-linked immunosorbent assay (ELISA) in a collaborative study with sixteen participants from twelve different countries. This anti-malaria human serum preparation (NIBSC Code: 10/198) was adopted by the WHO Expert Committee on Biological Standardization (ECBS) in October 2014, as the first WHO reference reagent for anti-malaria (Plasmodium falciparum) human serum with an assigned arbitrary unitage of 100 units (U) per ampoule. Analysis of the reference reagent in a collaborative study has demonstrated the benefit of this preparation for the reduction in inter- and intra-laboratory variability in ELISA. Whilst locally sourced pools are regularly use for harmonization both within and between a few laboratories, the presence of a WHO-endorsed reference reagent should enable optimal harmonization of malaria serological assays either by direct use of the reference reagent or calibration of local standards against this WHO reference. The intended uses of this reference reagent, a multivalent preparation, are (1) to allow cross

Human Platelet Lysate as a Xeno Free Alternative of Fetal Bovine Serum for the In Vitro Expansion of Human Mesenchymal Stromal Cells

PubMed Central

Mohammadi, Saeed; Nikbakht, Mohsen; Malek Mohammadi, Ashraf; Zahed Panah, Mahdi; Ostadali, Mohammad Reza; Nasiri, Hajar; Ghavamzadeh, Ardeshir

2016-01-01

Background: Mesenchymal stromal cells (MSCs) are employed in various different clinical settings in order to modulate immune response. Human autologous and allogeneic supplements including platelet derivatives such as platelet lysate (PL), platelet-released factors (PRF) and serum are assessed in clinical studies to replace fetal bovine serum (FBS). The immunosuppressive activity and multi-potential characteristic of MSCs appear to be maintained when the cells are expanded in platelet derivatives. Materials and Methods: Platelet-rich plasma was collected from umbrical cord blood (UCB). Platelet-derived growth factors obtained by freeze and thaw methods. CD62P expression was determined by flow cytometry. The concentration of PDGF-BB and PDGF-AB was detemined by ELISA. We tested the ability of a different concentration of PL-supplemented medium to support the ex vivo expansion of Wharton's jelly derived MSCs. We also investigated the biological/functional properties of expanded MSCs in presence of different concentration of PL. The conventional karyotyping was performed in order to study the chromosomal stability. The gene expression of Collagen I and II aggrecan and SOX-9 in the presence of different concentrations of PL was evaluated by Real-time PCR. Results: We observed 5% and 10% PL, causing greater effects on proliferation of MSCs .These cells exhibited typical morphology, immunophenotype and differentiation capacity. The genetic stability of these derivative cells from Wharton's jelly was demonstrated by a normal karyotype. Furthermore, the results of Real-time PCR analysis showed that the expression of chondrocyte specific genes was higher in MSCs in the presence of 5% and 10% PL, compared with FBS supplement. Conclusions: We demonstrated that PL could be used as an alternative safe source of growth factors for expansion of MSCs and also maintained similar growing potential and phenotype without any effect on chromosomal stability. PMID:27489592

Commercial radioimmunoassay for beta subunit of human chorionic gonadotropin: falsely positive determinations due to elevated serum luteinizing hormone

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

Fowler, J.E. Jr.; Platoff, G.E.; Kubrock, C.A.

1982-01-01

Among 17 men who had received seemingly curative treatment for unilateral non-seminomatous germ cell tumors for the testis and who had consistently normal serum human chorionic gonadotropin (HCG) levels at a reference laboratory, 7 (41%) had at least one falsely positive commercial serum HCG determination. To investigate the cause of these falsely positive determinations the authors measured the cross reactivity of luteinizing hormone (LH) and follicle stimulating hormone (FSH) standards in the commercial HCG assay, and studied the relationships between commercial HCG levels and serum LH levels, serum FSH levels and gonadal status in men with and without normal gonadalmore » function. The falsely positive HCG determinations appeared to be due to elevated serum LH levels and cross reactivity of LH in the commercial HCG assay because: 1) there was substantial cross reactivity of the LH standards in the commercial assay, 2) the serum LH was elevated in four of six men with solitary testes, 3) there was a striking correlation between elevated serum LH levels and falsely elevated commercial HCG levels in ten men with solitary or absent testes, and 4) there were no falsely positive HCG determinations in 13 normal men but there were falsely positive HCG determinations in seven of ten anorchid men.« less

Serum Ferritin: Past, Present and Future

PubMed Central

Wang, Wei; Knovich, Mary Ann; Coffman, Lan G.; Torti, Frank M.; Torti, Suzy V.

2010-01-01

Background Serum ferritin was discovered in the 1930’s, and was developed as a clinical test in the 1970’s. Many diseases are associated with iron overload or iron deficiency. Serum ferritin is widely used in diagnosing and monitoring these diseases. Scope of Review In this chapter, we discuss the role of serum ferritin in physiological and pathological processes and its use as a clinical tool. Major Conclusions Although many aspects of the fundamental biology of serum ferritin remain surprisingly unclear, a growing number of roles have been attributed to extracellular ferritin, including newly described roles in iron delivery, angiogenesis, inflammation, immunity, signaling and cancer. General Significance Serum ferritin remains a clinically useful tool. Further studies on the biology of this protein may provide new biological insights. PMID:20304033

Serum Metabolomics Investigation of Humanized Mouse Model of Dengue Virus Infection.

PubMed

Cui, Liang; Hou, Jue; Fang, Jinling; Lee, Yie Hou; Costa, Vivian Vasconcelos; Wong, Lan Hiong; Chen, Qingfeng; Ooi, Eng Eong; Tannenbaum, Steven R; Chen, Jianzhu; Ong, Choon Nam

2017-07-15

Dengue is an acute febrile illness caused by dengue virus (DENV) and a major cause of morbidity and mortality in tropical and subtropical regions of the world. The lack of an appropriate small-animal model of dengue infection has greatly hindered the study of dengue pathogenesis and the development of therapeutics. In this study, we conducted mass spectrometry-based serum metabolic profiling from a model using humanized mice (humice) with DENV serotype 2 infection at 0, 3, 7, 14, and 28 days postinfection (dpi). Forty-eight differential metabolites were identified, including fatty acids, purines and pyrimidines, acylcarnitines, acylglycines, phospholipids, sphingolipids, amino acids and derivatives, free fatty acids, and bile acid. These metabolites showed a reversible-change trend-most were significantly perturbed at 3 or 7 dpi and returned to control levels at 14 or 28 dpi, indicating that the metabolites might serve as prognostic markers of the disease in humice. The major perturbed metabolic pathways included purine and pyrimidine metabolism, fatty acid β-oxidation, phospholipid catabolism, arachidonic acid and linoleic acid metabolism, sphingolipid metabolism, tryptophan metabolism, phenylalanine metabolism, lysine biosynthesis and degradation, and bile acid biosynthesis. Most of these disturbed pathways are similar to our previous metabolomics findings in a longitudinal cohort of adult human dengue patients across different infection stages. Our analyses revealed the commonalities of host responses to DENV infection between humice and humans and suggested that humice could be a useful small-animal model for the study of dengue pathogenesis and the development of dengue therapeutics. IMPORTANCE Dengue virus is the most widespread arbovirus, causing an estimated 390 million dengue infections worldwide every year. There is currently no effective treatment for the disease, and the lack of an appropriate small-animal model of dengue infection has greatly

Determination of the binding properties of p-cresyl glucuronide to human serum albumin.

PubMed

Yi, Dan; Monteiro, Elisa Bernardes; Chambert, Stéphane; Soula, Hédi A; Daleprane, Julio B; Soulage, Christophe O

2018-04-26

p-Cresyl glucuronide (p-CG) is a by-product of tyrosine metabolism that accumulates in patients with end-stage renal disease. p-CG binding to human serum albumin in physiological conditions (37°C, pH 7.40) was studied by ultrafiltration (MWCO 10 kDa) and data were analyzed assuming one binding site. The estimated value of the association constant was 2.77×10 3  M -1 and a maximal stoichiometry of 3.80 mol per mole. At a concentration relevant for end-stage renal patients, p-CG was 23% bound to albumin. Competition experiments, using fluorescent probes, demonstrated that p-CG did not bind to Sudlow's site I or site II. The p-CG did not interfere with the binding of p-cresyl-sulfate or indoxyl sulfate to serum albumin. Copyright © 2018. Published by Elsevier B.V.

Human serum albumin binding of certain antimalarials

NASA Astrophysics Data System (ADS)

Marković, Olivera S.; Cvijetić, Ilija N.; Zlatović, Mario V.; Opsenica, Igor M.; Konstantinović, Jelena M.; Terzić Jovanović, Nataša V.; Šolaja, Bogdan A.; Verbić, Tatjana Ž.

2018-03-01

Interactions between eight in-house synthesized aminoquinolines, along with well-known chloroquine, and human serum albumin (HSA) have been studied by fluorescence spectroscopy. The synthesized aminoquinolines, despite being structurally diverse, were found to be very potent antimalarials. Fluorescence measurements indicate that three compounds having additional thiophene or benzothiophene substructure bind more strongly to HSA than other studied compounds. Competitive binding experiments indicate that these three compounds bind significantly stronger to warfarin compared to diazepam binding site. Fluorescence quenching at three temperatures (20, 25, and 37 °C) was analyzed using classical Stern-Volmer equation, and a static quenching mechanism was proposed. The enthalpy and entropy changes upon sulphur-containing compound-HSA interactions were calculated using Van't Hoff equation. Positive values of enthalpy and entropy changes indicate that non-specific, hydrophobic interactions are the main contributors to HSA-compound interaction. Molecular docking and calculated lipophilicity descriptors indicate the same, pointing out that the increased lipophilicity of sulphur-containing compounds might be a reason for their better binding to HSA. Obtained results might contribute to design of novel derivatives with improved pharmacokinetic properties and drug efficacy.

Methylglyoxal induced glycation and aggregation of human serum albumin: Biochemical and biophysical approach.

PubMed

Ahmed, Azaj; Shamsi, Anas; Khan, Mohd Shahnawaz; Husain, Fohad Mabood; Bano, Bilqees

2018-07-01

Serum protein glycation and formation of advanced glycation end products (AGEs) correlates with many diseases viz. diabetes signifying the importance of studying the glycation pattern of serum proteins. In our present study, methylglyoxal was investigated for its effect on the structure of human serum albumin (HSA); exploring the formation of AGEs and aggregates of HSA. The analytical tools employed includes intrinsic and extrinsic fluorescence, UV spectroscopy, far UV circular dichroism, Thioflavin T fluorescence, congo red binding, polyacrylamide gel electrophoresis (PAGE). UV and fluorescence spectroscopy revealed the structural transition of native HSA evident by new peaks and increased absorbance in UV spectra and quenched fluorescence in the presence of MG. Far UV CD spectroscopy revealed MG induced secondary structural alteration evident by reduced α-helical content. AGEs formation was confirmed by AGEs specific fluorescence. Increased ThT fluorescence and CR absorbance of 10mM MG incubated HSA suggests that glycated HSA results in the formation of aggregates of HSA. SEM and TEM were reported to have an insight of these aggregates. Molecular docking was also utilized to see site specific interaction of MG-HSA. This study is clinically significant as HSA is a clinically relevant protein which plays a crucial role in many diseases. Copyright © 2018 Elsevier B.V. All rights reserved.

Matrix interference from Fc-Fc interactions in immunoassays for detecting human IgG4 therapeutics.

PubMed

Partridge, Michael A; Karayusuf, Elif Kabuloglu; Dhulipala, Gangadhar; Dreyer, Robert; Daly, Thomas; Sumner, Giane; Pyles, Erica; Torri, Albert

2015-01-01

An assay measuring an IgG4 biotherapeutic in human serum used a drug-specific monoclonal antibody (mAb) capture reagent and an antihuman IgG4 mAb as detection reagent. However, serum IgG4 binding to the capture mAb via Fc-interactions was detected by the anti-IgG4 mAb, causing high background. Two approaches were developed to minimize background; incorporating a mild acid sample preparation step or using the Fab of the capture antibody. Either strategy improved signal:noise dramatically, increasing assay sensitivity >20-fold. Biophysical analyses of antibody domains indicated that noncovalent Fc oligomers could inhibit the background. Matrix interference from human IgG4 binding to the capture mAb was reduced with a Fab fragment of the drug-specific capture antibody or by incorporating a mild acid sample treatment into the assay.

Use of procainamide gels in the purification of human and horse serum cholinesterases.

PubMed Central

Ralston, J S; Main, A R; Kilpatrick, B F; Chasson, A L

1983-01-01

Two large-scale methods based primarily on the use of procainamide-Sepharose gels were developed for the purification of horse and human serum non-specific cholinesterases. With method I, the procainamide-Sepharose 4B gel was used in the first step to handle large volumes of serum. With method II, the procainamide-Sepharose 4B gel was used in the final step to obtain pure enzyme. Although both methods gave electrophoretically pure cholinesterase preparations in good yields, they were significantly more efficient at purifying the horse enzyme than the human enzyme. To study this problem, the relative binding of human and horse cholinesterases to procainamide-, methylacridinium (MAC)-, m-trimethylammoniophenyl (m-PTA)- and p-trimethylammoniophenyl (p-PTA)-Sepharose 4B gels were measured, by using two approaches. In one, binding was measured by a procedure involving equilibration of pure cholinesterase in a small volume of diluted gel slurry (4%, v/v). A partially purified preparation of Electrophorus acetylcholinesterase was included. Pure human cholinesterase bound consistently more tightly to each of the gels than did horse cholinesterase, and the acetylcholinesterase appeared to bind the gels 10-100 times more tightly than did the non-specific cholinesterases. The order of binding for the cholinesterases, beginning with the tightest, was: procainamide-Sepharose 4B, MAC-Sepharose 4B, p-PTA-Sepharose 4B and m-PTA-Sepharose 4B. For the acetylcholinesterase the order was: MAC-Sepharose 4B, procainamide-Sepharose 4B, p-PTA-Sepharose 4B and m-PTA-Sepharose 4B. The second approach involved passing native sera or partially purified sera fractions through 1 ml test columns of each of the four affinity gels to determine their retention capacity for the cholinesterases. With these impure samples, the MAC-Sepharose 4B gels proved superior to the procainamide-Sepharose 4B gels at retaining human cholinesterase, but the opposite was true for the horse cholinesterase. PMID

Biophysical studies of interaction between hydrolysable tannins isolated from Oenothera gigas and Geranium sanguineum with human serum albumin.

PubMed

Sekowski, Szymon; Ionov, Maksim; Kaszuba, Mateusz; Mavlyanov, Saidmukhtar; Bryszewska, Maria; Zamaraeva, Maria

2014-11-01

Tannins, secondary plant metabolites, possess diverse biological activities and can interact with biopolymers such as lipids or proteins. Interactions between tannins and proteins depend on the structures of both and can result in changes in protein structure and activity. Because human serum albumin is the most abundant protein in plasma and responsible for interactions with important biological compounds (e.g. bilirubin) and proper blood pressure, therefore, it is very important to investigate reactions between HSA and tannins. This paper describes the interaction between human serum albumin (HSA) and two tannins: bihexahydroxydiphenoyl-trigalloylglucose (BDTG) and 1-O-galloyl-4,6-hexahydroxydiphenoyl-β-d-glucose (OGβDG), isolated from Geranium sanguineum and Oenothera gigas leafs, respectively. Optical (spectrofluorimetric) and chiral optical (circular dichroism) methods were used in this study. Fluorescence analysis demonstrated that OGβDG quenched HSA fluorescence more strongly than BDTG. Both OGβDG and BDTG formed complexes with albumin and caused a red shift of the fluorescence spectra but did not significantly change the protein secondary structure. Our studies clearly demonstrate that the tested tannins interact very strongly with human serum albumin (quenching constant K=88,277.26±407.04 M(-1) and K=55,552.67±583.07 M(-1) respectively for OGβDG and BDTG) in a manner depending on their chemical structure. Copyright © 2014 Elsevier B.V. All rights reserved.

Evaluation of the major royal jelly proteins as an alternative to fetal bovine serum in culturing human cell lines.

PubMed

Chen, Di; Xin, Xiao-Xuan; Qian, Hao-Cheng; Yu, Zhang-Yin; Shen, Li-Rong

2016-06-01

Royal jelly (RJ) is a well-known bioactive substance. It contains large amounts of major royal jelly proteins (MRJPs), which express growth-factor-like activity in several animal and human cell lines. However, the question on whether MRJPs possess growth-factor-like activity on all types of cell cultures remains. In order to determine whether MRJPs can be used as an alternative to fetal bovine serum (FBS) in different types of human cell culture, the proliferation of the complex serum with different ratios of MRJPs/FBS (M/F) was evaluated on five cell lines: 293T, HFL-I, 231, HCT116, and Changliver using MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay. The proliferation activity of the combination of the complex M/F serum with cytokines on the test cell lines was also measured. The results demonstrated that the complex serum with M/F 6/4 possessed the highest proliferation activity similar to or in excess of FBS. However, no activity of complex medium with M/F 6/4 was observed in 231 cells, indicating a selectivity of MRJPs on cell types. Compared with the complex medium with M/F 6/4, the complex medium with M/F 6/4 together with two cytokines, epidermal growth factor (EGF) and insulin-transferrin-selenium (ITS), promoted proliferations of Changliver, 293T, HCT116, and HFL-I by 18.73%‒56.19% (P<0.01). Our findings demonstrate that MRJPs could partially replace FBS in culturing many human cell lines.

The effect of intravenous lactated Ringer's solution versus 0.9% sodium chloride solution on serum osmolality in human volunteers.

PubMed

Williams, E L; Hildebrand, K L; McCormick, S A; Bedel, M J

1999-05-01

Animal studies have shown that large volumes of IV lactated Ringer's solution (LR) decrease serum osmolality, thereby increasing cerebral water. These studies have led to recommendations to limit LR to avoid cerebral edema in neurosurgical patients. Eighteen healthy human volunteers aged 20-48 yr received 50 mL/kg LR over 1 h on one occasion and 0.9% sodium chloride (NS) on another. Venous samples were taken at baseline (T1), at infusion end (T2), and 1 h after T2 (T3). Time until first urination was noted. With LR, serum osmolality decreased by 4+/-3 mOsm/kg from T1 to T2 and increased insignificantly with NS. At T3, osmolality returned almost to baseline in the LR group. Blood pH increased from T1 to T2 with LR by 0.04+/-0.04 and decreased with NS by 0.04+/-0.04. These pH changes persisted at T3. Subjective mental changes occurred only with NS. Abdominal discomfort was more common with NS. Time until first urination was longer with NS (106+/-11 min) than with LR (75+/-10 min) (P < 0.001). In healthy humans, an infusion of large volumes of LR, but not NS, transiently decreased serum osmolality, whereas acidosis associated with NS persisted and urinary output was slower with NS. Large volumes of lactated Ringer's solution administered to healthy humans produced small transient changes in serum osmolality. Large volumes of sodium chloride did not change osmolality but resulted in lower pH.

Is DTPA a good competing chelating agent for Th(IV) in human serum and suitable in targeted alpha therapy?

PubMed

Le Du, Alicia; Sabatié-Gogova, Andrea; Morgenstern, Alfred; Montavon, Gilles

2012-04-01

The interaction between thorium and human serum components was studied using difference ultraviolet spectroscopy (DUS), ultrafiltration and high-pressure-anion exchange chromatography (HPAEC) with external inductively conducted plasma mass spectrometry (ICP-MS) analysis. Experimental data are compared with modelling results based on the law of mass action. Human serum transferrin (HSTF) interacts strongly with Th(IV), forming a ternary complex including two synergistic carbonate anions. This complex governs Th(IV) speciation under blood serum conditions. Considering the generally used Langmuir-type model, values of 10(33.5) and 10(32.5) were obtained for strong and weak sites, respectively. We showed that trace amounts of diethylene triamine pentaacetic acid (DTPA) cannot complex Th(IV) in the blood serum at equilibrium. Unexpectedly this effect is not related to the competition with HSTF but is due to the strong competition with major divalent metal ions for DTPA. However, Th-DTPA complex was shown to be stable for a few hours when it is formed before addition in the biological medium; this is related to the high kinetic stability of the complex. This makes DTPA a potential chelating agent for synthesis of (226)Th-labelled biomolecules for application in targeted alpha therapy. Copyright © 2011 Elsevier Inc. All rights reserved.

Effect of human serum albumin upon the permeabilizing activity of sticholysin II, a pore forming toxin from Stichodactyla heliantus.

PubMed

Celedón, Gloria; González, Gustavo; Gulppi, Felipe; Pazos, Fabiola; Lanio, María E; Alvarez, Carlos; Calderón, Cristian; Montecinos, Rodrigo; Lissi, Eduardo

2013-12-01

Sticholysin II (St II) is a haemolytic toxin isolated from the sea anemone Stichodactyla helianthus. The high haemolytic activity of this toxin is strongly dependent on the red cell status and the macromolecule conformation. In the present communication we evaluate the effect of human serum albumin on St II haemolytic activity and its capacity to form pores in the bilayer of synthetic liposomes. St II retains its pore forming capacity in the presence of large concentrations (up to 500 μM) of human serum albumin. This effect is observed both in its capacity to produce red blood cells haemolysis and to generate functional pores in liposomes. In particular, the capacity of the toxin to lyse red blood cells increases in the presence of human serum albumin (HSA). Regarding the rate of the pore forming process, it is moderately decreased in liposomes and in red blood cells, in spite of an almost total coverage of the interface by albumin. All the data obtained in red cells and model membranes show that St II remains lytically active even in the presence of high HSA concentrations. This stubbornness can explain why the toxin is able to exert its haemolytic activity on membranes immersed in complex plasma matrixes such as those present in living organisms.

Detection of Novel Sequences Related to African Swine Fever Virus in Human Serum and Sewage▿ †

PubMed Central

Loh, Joy; Zhao, Guoyan; Presti, Rachel M.; Holtz, Lori R.; Finkbeiner, Stacy R.; Droit, Lindsay; Villasana, Zoilmar; Todd, Collin; Pipas, James M.; Calgua, Byron; Girones, Rosina; Wang, David; Virgin, Herbert W.

2009-01-01

The family Asfarviridae contains only a single virus species, African swine fever virus (ASFV). ASFV is a viral agent with significant economic impact due to its devastating effects on populations of domesticated pigs during outbreaks but has not been reported to infect humans. We report here the discovery of novel viral sequences in human serum and sewage which are clearly related to the asfarvirus family but highly divergent from ASFV. Detection of these sequences suggests that greater genetic diversity may exist among asfarviruses than previously thought and raises the possibility that human infection by asfarviruses may occur. PMID:19812170

Non-covalent binding analysis of sulfamethoxazole to human serum albumin: Fluorescence spectroscopy, UV-vis, FT-IR, voltammetric and molecular modeling.

PubMed

Naik, Praveen N; Nandibewoor, Sharanappa T; Chimatadar, Shivamurthi A

2015-06-01

This study was designed to examine the interaction of sulfamethoxazole (SMZ) with human serum albumin(HSA). Spectroscopic analysis of the emission quenching at different temperatures revealed that the quenching mechanism of human serum albumin by SMZ was static mechanism. The binding constant values for the SMZ-HSA system were obtained to be 22,500 L/mol at 288 K, 15,600 L/mol at 298 K, and 8500 L/mol at 308 K. The distance r between donor and acceptor was evaluated according to the theory of Föster energy transfer. The results of spectroscopic analysis and molecular modeling techniques showed that the conformation of human serum albumin had been changed in the presence of SMZ. The thermodynamic parameters, namely enthalpy change (∆ H 0 ) -36.0 kJ/mol, entropy change (∆ S 0 ) -41.3 J/mol K and free energy change (∆ G 0 ) -23.7 kJ/mol, were calculated by using van׳t Hoff equation. The effect of common ions on the binding of SMZ to HSA was tested.

Ultrasensitive dual-channel detection of matrix metalloproteinase-2 in human serum using gold-quantum dot core-satellite nanoprobes.

PubMed

Zheng, Tingting; Zhang, Rui; Zhang, Qingfeng; Tan, Tingting; Zhang, Kui; Zhu, Jun-Jie; Wang, Hui

2013-09-18

We have developed a robust enzymatic peptide cleavage-based assay for the ultrasensitive dual-channel detection of matrix metalloproteinase-2 (MMP-2) in human serum using gold-quantum dot (Au-QD) core-satellite nanoprobes.

The Effect of Trimethoprim on Serum Folate Levels in Humans: A Randomized, Double-Blind, Placebo-Controlled Trial.

PubMed

Meidahl Petersen, Kasper; Eplov, Kasper; Kjær Nielsen, Torben; Jimenez-Solem, Espen; Petersen, Morten; Broedbaek, Kasper; Daugaard Popik, Sara; Kallehave Hansen, Lina; Enghusen Poulsen, Henrik; Trærup Andersen, Jon

2016-01-01

Trimethoprim antagonize the actions of folate by inhibition of dihydrofolate reductase. This could diminish serum folate levels in humans and causes folate deficiency in some patients. We conducted a randomized, double-blind, placebo-controlled trial, to investigate the effect of trimethoprim on serum folate levels in healthy participants after a 7-day trial period. Thirty young, healthy males were randomly allocated to receive trimethoprim, 200 mg twice daily, and 30 were randomly allocated to placebo. Before trial initiation, participant numbers were given randomly generated treatment allocations within sealed opaque envelopes. Participants and all staff were kept blinded to treatment allocations during the trial. Serum folate was measured at baseline and at end of trial. In the 58 participants analyzed (30 in the trimethoprim group and 28 in the placebo group), 8 had folate deficiency at baseline. Within the trimethoprim group, serum folate was significantly decreased (P = 0.018) after the trial. We found a mean decrease in serum folate among trimethoprim exposed of 1.95 nmol/L, compared with a 0.21 nmol/L mean increase in the placebo group (P = 0.040). The proportion of folate-deficient participants increased significantly within the trimethoprim group (P = 0.034). No serious adverse events were observed. In conclusion, we found that a daily dose of 400 mg trimethoprim for 7 days significantly lowered serum folate levels in healthy study participants.

Serum from pregnant women with gestational diabetes mellitus increases the expression of FABP4 mRNA in primary subcutaneous human pre-adipocytes

PubMed Central

Li, Lan; Lee, Se Jin; Kook, Song Yi; Ahn, Tae Gyu; Lee, Ji Yeon

2017-01-01

Objective Gestational diabetes mellitus (GDM) is defined as glucose intolerance first detected during pregnancy. It can result in pregnancy complications such as birth injury, stillbirth. Fatty acid-binding protein 4 (FABP4), found in adipose tissue, is associated with insulin resistance, and type 2 diabetes. The aim of this study was to investigate whether FABP4 in the placenta and decidua of pregnant women with GDM is higher than that in normal pregnant women, and whether serum from pregnant women with GDM may cause adipocytes to secrete more FABP4 than does serum from a normal pregnant group. Methods We obtained placentas, deciduas, and serum from 12 pregnant women with GDM and 12 normal pregnant women and performed enzyme-linked immunosorbent assay, real time quantitative-polymerase chain reaction. We cultured human pre-adipocytes for 17 days with GDM and non-GDM serum and performed western blot, real time quantitative-polymerase chain reaction, and oil red O staining. Results Expression of FABP4 in serum, placenta and decidua of pregnant women with GDM was significantly higher than that in normal pregnant women. Serum from pregnant women with GDM increased the expression of FABP4 mRNA and decreased the expression of adiponectin mRNA in human pre-adipocytes significantly. Adipocyte cultured in GDM serum showed significantly greater lipid accumulation than those cultured in normal serum. Conclusion Our results suggest that FABP4 is higher in placenta and decidua from pregnant women with GDM. Increased circulating FABP4 in maternal serum from pregnant women with GDM may originate from adipocytes and the placenta. Circulating FABP4 can induce increased insulin resistance and decreased insulin sensitivity. PMID:28534013

Serum from pregnant women with gestational diabetes mellitus increases the expression of FABP4 mRNA in primary subcutaneous human pre-adipocytes.

PubMed

Li, Lan; Lee, Se Jin; Kook, Song Yi; Ahn, Tae Gyu; Lee, Ji Yeon; Hwang, Jong Yun

2017-05-01

Gestational diabetes mellitus (GDM) is defined as glucose intolerance first detected during pregnancy. It can result in pregnancy complications such as birth injury, stillbirth. Fatty acid-binding protein 4 (FABP4), found in adipose tissue, is associated with insulin resistance, and type 2 diabetes. The aim of this study was to investigate whether FABP4 in the placenta and decidua of pregnant women with GDM is higher than that in normal pregnant women, and whether serum from pregnant women with GDM may cause adipocytes to secrete more FABP4 than does serum from a normal pregnant group. We obtained placentas, deciduas, and serum from 12 pregnant women with GDM and 12 normal pregnant women and performed enzyme-linked immunosorbent assay, real time quantitative-polymerase chain reaction. We cultured human pre-adipocytes for 17 days with GDM and non-GDM serum and performed western blot, real time quantitative-polymerase chain reaction, and oil red O staining. Expression of FABP4 in serum, placenta and decidua of pregnant women with GDM was significantly higher than that in normal pregnant women. Serum from pregnant women with GDM increased the expression of FABP4 mRNA and decreased the expression of adiponectin mRNA in human pre-adipocytes significantly. Adipocyte cultured in GDM serum showed significantly greater lipid accumulation than those cultured in normal serum. Our results suggest that FABP4 is higher in placenta and decidua from pregnant women with GDM. Increased circulating FABP4 in maternal serum from pregnant women with GDM may originate from adipocytes and the placenta. Circulating FABP4 can induce increased insulin resistance and decreased insulin sensitivity.

Effects of illumination on human nocturnal serum melatonin levels and performance

NASA Technical Reports Server (NTRS)

Dollins, A. B.; Lynch, H. J.; Wurtman, R. J.; Deng, M. H.; Lieberman, H. R.

1993-01-01

In humans, exposure to bright light at night suppresses the normal nocturnal elevation in circulating melatonin. Oral administration of pharmacological doses of melatonin during the day, when melatonin levels are normally minimal, induces fatigue. To examine the relationship between illumination, human pineal function, and behavior, we monitored the overnight serum melatonin profiles and behavioral performance of 24 healthy male subjects. On each of three separate occasions subjects participated in 13.5 h (1630-0800 h) testing sessions. Each subject was assigned to an individually illuminated workstation that was maintained throughout the night at an illumination level of approximately 300, 1500, or 3000 lux. Melatonin levels were significantly diminished by light treatment, F(2, 36) = 12.77, p < 0.001, in a dose-dependent manner. Performance on vigilance, reaction time, and other tasks deteriorated throughout the night, consistent with known circadian variations in these parameters, but independent of ambient light intensity and circulating melatonin levels.

Facile LC-UV methods for simultaneous monitoring of ciprofloxacin and rosuvastatin in API, formulations and human serum.

PubMed

Arayne, M Saeed; Sultana, Najma; Tabassum, Arman

2015-02-01

An efficient, selective and cost-effective liquid chromatographic assay was developed and validated for the simultaneous quantification of ciprofloxacin and rosuvastatin in Active Pharmaceutical Ingredients (API), pharmaceutical formulations and in human serum. The chromatographic system consisted of mobile phase methanol-water, 90:10 v/v at pH 3.0 adjusted with o-phosphoric acid, pumped at 1.0 mL/min through a prepacked Purospher Star C18 (5 µm, 25 × 0.46 cm) column and effluent was monitored at the isosbestic point (255 nm) as well as at the λmax of individual drugs (243 and 271 nm). The method was validated over a linear concentration range of 0.25-15 µg/mL for ciprofloxacin and 0.33-20 µg/mL for rosuvastatin (r(2)  ≥ 0.999). The ranges of reliable response (limits of detection and quantitation) for ciprofloxacin were 3-15 and 9-45 ng/mL and 17-29 and 52-88 ng/mL, respectively, for rosuvastatin in all API, pharmaceutical formulations and human serum. Analytical recovery from human serum was >98% and relative standard deviation (RSD) was <2. The accuracies were 97.13-102.55 and 97.41-101.31% and precisions in RSD were 0.04-1.90 and 0.02-1.23% for ciprofloxacin and rosuvastatin, respectively. No matrix interferences, ion suppression/enhancement and carry-over were detected. The total assay run time was less than 5 min. In another study, for optimum performance the detector was programmed for multiwavelength scanning at the absorption maxima of each component. Consequently, the linearity range was improved and limit of detection and quantitation values were down to 1-4 and 4-12 ng/mL for ciprofloxacin and 3-5 and 9-15 ng/mL for rosuvastatin, respectively. The validation parameters fitted ICH guidelines through the isosbestic and individual λmax approach. The small sample volume and simplicity of preparation make this method suitable for use in human serum samples, pharmaceutical formulations, quality control, drug

Effect of recombinant human thyroid stimulating hormone on serum thyroxin and thyroid scintigraphy in euthyroid cats.

PubMed

van Hoek, Ingrid M; Peremans, Kathelijne; Vandermeulen, Eva; Duchateau, Luc; Gommeren, Kris; Daminet, Sylvie

2009-04-01

This study investigated the thyroidal response to administration of recombinant human thyroid stimulating hormone (rhTSH) by means of serum total thyroxine (TT(4)) concentration and pertechnetate uptake by the thyroid gland in six healthy euthyroid spayed female cats. A pertechnetate scan was performed on day 1 to calculate thyroid/salivary gland (T/S) uptake ratio. On day 3, 25 microg rhTSH was injected intravenously. Six hours later the thyroid scan was repeated as on day 1. Blood was drawn for serum TT(4) measurement prior to injection of rhTSH and performance of the pertechnetate scan. Statistically significant differences in mean serum TT(4) concentration, T/S uptake ratio before and 6h after rhTSH administration and T/S uptake ratio between left and right lobes were noted. We can conclude that 25 microg rhTSH increases pertechnetate uptake in the thyroid glands of cats, this should be taken into account when thyroid scintigraphy after rhTSH administration is interpreted.

Chemically defined serum-free conditions for cartilage regeneration from human embryonic stem cells.

PubMed

Yang, Dandan; Chen, Shubin; Gao, Changzhao; Liu, Xiaobo; Zhou, Yulai; Liu, Pengfei; Cai, Jinglei

2016-11-01

The aim of this study was to improve a method that induce cartilage differentiation of human embryoid stem cells (hESCs) in vitro, and test the effect of in vivo environments on the further maturation of hESCs derived cells. Embryoid bodies (EBs) formed from hESCs, with serum-free KSR-based medium and mesodermal specification related factors, CHIR, and Noggin for first 8days. Then cells were digested and cultured as micropellets in serum-free KSR-based chondrogenic medium that was supplemented with PDGF-BB, TGF β3, BMP4 in sequence for 24days. The morphology, FACS, histological staining as well as the expression of chondrogenic specific genes were detected in each stage, and further in vivo experiments, cell injections and tissue transplantations, further verified the formation of chondrocytes. We were able to obtain chondrocyte/cartilage from hESCs using serum-free KSR-based conditioned medium. qPCR analysis showed that expression of the chondroprogenitor genes and the chondrocyte/cartilage matrix genes. Morphology analysis demonstrated we got PG+COL2+COL1-particles. It indicated we obtained hyaline cartilage-like particles. 32-Day differential cells were injected subcutaneous. Staining results showed grafts developed further mature in vivo. But when transplanted in subrenal capsule, their effect was not good as in subcutaneous. Microenvironment might affect the cartilage formation. The results of this study provide an absolute serum-free and efficient approach for generation of hESC-derived chondrocytes, and cells will become further maturation in vivo. It provides evidence and technology for the hypothesis that hESCs may be a promising therapy for the treatment of cartilage disease. Copyright © 2016 Elsevier Inc. All rights reserved.

Influence of myristic acid on furosemide binding to bovine serum albumin. Comparison with furosemide-human serum albumin complex

NASA Astrophysics Data System (ADS)

Bojko, B.; Sułkowska, A.; Maciążek-Jurczyk, M.; Równicka, J.; Sułkowski, W. W.

2010-06-01

Fluorescence studies on furosemide (FUR) binding to bovine serum albumin (BSA) showed the existence of three or four binding sites in the tertiary structure of the protein. Two of them are located in subdomain IIA, while the others in subdomains IB and/or IIIA. Furosemide binding in subdomain IB is postulated on the basis of run of Stern-Volmer plot indicating the existence of two populations of tryptophans involved in the interaction with FUR. In turn, the significant participation of tyrosil residues in complex formation leads to the consideration of the subdomain IIIA as furosemide low-affinity binding site. The effect of increasing concentration of fatty acid on FUR binding in all studied binding sites was also investigated and compared with the previous results obtained for human serum albumin (HSA). For BSA the lesser impact of fatty acid on affinity between drug and albumin was observed. This is probably a result of more significant role of tyrosines in the complex formation and different polarity of microenvironment of the fluorophores when compared HSA and BSA. The most distinct differences between FUR-BSA and FUR-HSA binding parameters are observed when third fatty acid molecule is bound with the protein and rotation of domains I and II occurs. However these structural changes mostly affect FUR low affinity binding sites.

Kinetic assay of antitrypsin in human serum by a surface acoustic wave(SAW)-impedance sensor.

PubMed

Cai, Q; Wei, W; Wang, R; Nie, L; Yao, S

1996-08-01

Antitrypsin in human serum was determined by using both the SAW-impedance sensor system and spectrophotometry, indicating that the mean value for women was significantly higher than the mean value for men; the value for acute pancreasis patients is about 2-folds of the normal values, and there is no significant difference between the acute pancreasis patients and the pancreatic cancer patients.

Experimental Germ Tube Induction in Candida albicans: An Evaluation of the Effect of Sodium Bicarbonate on Morphogenesis and Comparison with Pooled Human Serum.

PubMed

Matare, Tapiwa; Nziramasanga, Pasipanodya; Gwanzura, Lovemore; Robertson, Valerie

2017-01-01

The potential of NaHCO 3 versus human serum to induce germ tube formation in Candida albicans was investigated. A total of 100 isolates were obtained from oral swabs of patients presenting with thrush. Approval for the study was granted by the Joint Research Ethics Committee (JREC/23/08). Confirmed C. albicans isolates by routine methods were tested for germ tube induction using 5 different concentrations of Tris-maleate buffered NaHCO 3 and Tris-maleate buffer control. Standard control strains included were C. albicans (ATCC 10231) and C. krusei (ATCC 6258). Microculture was done in 20  μ L inoculums on microscope slides for 3 hours at 37°C. The rate of germ tube formation at 10-minute intervals was determined on 100 isolates using the optimum 20 mM Tris-maleate buffered NaHCO 3 concentration. Parallel germ tube formation using human serum was done in test tubes. The optimum concentration of NaHCO 3 in Tris-maleate buffer for germ tube induction was 20 mM for 67% of isolates. Only 21% of isolates formed germ tubes in Tris-maleate buffer control. There was no significant difference in induction between human serum and Tris-maleate buffered NaHCO 3 . Tris-maleate buffered NaHCO 3 induced germ tube formation in C. albicans isolates at rates similar to human serum.

Garlic's ability to prevent in vitro Cu2+-induced lipoprotein oxidation in human serum is preserved in heated garlic: effect unrelated to Cu2+-chelation

PubMed Central

Pedraza-Chaverrí, José; Gil-Ortiz, Mariana; Albarrán, Gabriela; Barbachano-Esparza, Laura; Menjívar, Marta; Medina-Campos, Omar N

2004-01-01

Background It has been shown that several extracts and compounds derived from garlic are able to inhibit Cu2+-induced low density lipoprotein oxidation. In this work we explored if the ability of aqueous garlic extract to prevent in vitro Cu2+-induced lipoprotein oxidation in human serum is affected by heating (a) aqueous garlic extracts or (b) garlic cloves. In the first case, aqueous extract of raw garlic and garlic powder were studied. In the second case, aqueous extract of boiled garlic cloves, microwave-treated garlic cloves, and pickled garlic were studied. It was also studied if the above mentioned preparations were able to chelate Cu2+. Methods Cu2+-induced lipoprotein oxidation in human serum was followed by the formation of conjugated dienes at 234 nm and 37°C by 240 min in a phosphate buffer 20 mM, pH 7.4. Blood serum and CuSO4 were added to a final concentration of 0.67% and 0.0125 mM, respectively. The lag time and the area under the curve from the oxidation curves were obtained. The Cu2+-chelating properties of garlic extracts were assessed using an approach based upon restoring the activity of xanthine oxidase inhibited in the presence of 0.050 mM Cu2+. The activity of xanthine oxidase was assessed by monitoring the production of superoxide anion at 560 nm and the formation of uric acid at 295 nm. Data were compared by parametric or non-parametric analysis of variance followed by a post hoc test. Results Extracts from garlic powder and raw garlic inhibited in a dose-dependent way Cu2+-induced lipoprotein oxidation. The heating of garlic extracts or garlic cloves was unable to alter significantly the increase in lag time and the decrease in the area under the curve observed with the unheated garlic extracts or raw garlic. In addition, it was found that the garlic extracts were unable to chelate Cu2+. Conclusions (a) the heating of aqueous extracts of raw garlic or garlic powder or the heating of garlic cloves by boiling, microwave or pickling do not

Serum human epididymis secretory protein 4 as a potential biomarker of renal fibrosis in kidney transplantation recipients.

PubMed

Luo, Jinmei; Wang, Fen; Wan, Jianxin; Ye, Zhuangjian; Huang, Chumei; Cai, Yuesu; Liu, Min; Wu, Ben-Quan; Li, Laisheng

2018-05-05

Renal fibrosis remains an important cause of kidney allograft failure. The objective of this study was to evaluate the performance of serum human epididymis secretory protein 4 (HE4) as a biomarker for renal fibrosis in kidney transplant recipients. A total of 103 kidney transplantation patients were enrolled in this study, and serum HE4 concentrations were detected using the chemiluminescent microparticle immunoassay. Renal biopsy was carried out, and histological findings were assessed by immunohistochemistry. Median serum HE4 concentrations were significantly increased in kidney transplant recipients (186.2 pmol/l, interquartile range [IQR] 125.6-300.2) compared with control subjects (34.3 pmol/l, IQR 30.4-42.3, p < 0.0001). Meanwhile, serum HE4 concentrations were significantly increased along with disease severity (p < 0.0001). In addition, we found serum HE4 concentrations to be strongly correlated with the severity of fibrosis (IF/TA 0, 1, 2, and 3: 114.3, 179.0, 197.8, and 467.8 pmol/l, respectively; p < 0.0001) and serum HE4 concentrations significantly correlated with HE4 tissue expression concentrations in renal biopsy. Serum HE4 was increased in kidney transplant recipients with decreased kidney function and renal fibrosis and was correlated with the severity of the disease, suggesting that HE4 has the potential to be used as a novel clinical biomarker for evaluating kidney function and predicting renal fibrosis in kidney transplant recipients. Copyright © 2018 Elsevier B.V. All rights reserved.

Osteocalcin as a negative regulator of serum leptin concentration in humans: insight from triathlon competitions.

PubMed

Guadalupe-Grau, Amelia; Ara, Ignacio; Dorado, Cecilia; Vicente-Rodríguez, German; Perez-Gomez, Jorge; Cabrero, Javier Chavarren; Serrano-Sanchez, José A; Santana, Alfredo; Calbet, Jose A L

2010-10-01

Osteocalcin is a hormone produced by osteoblasts which acts as a negative regulator of fat mass, protecting against diet induced obesity and insulin resistance in rodents. To determine if an acute increase in osteocalcin concentration is associated with opposed changes in circulating leptin levels and insulin resistance we studied 15 middle and long distance male triathletes, (age 32.1 ± 6.9 years), before and 48 h after an Olympic (OT) or an Ironman (IT) triathlon competition. Muscle power, anaerobic capacity, body composition (dual-energy X-ray absorptiometry), and serum concentrations of testosterone, dihydrotestosterone, osteocalcin, leptin, glucose, insulin and insulin resistance (HOMA) were determined pre- and post-race. Pre- and 48 h post-race total and regional lean body mass was not altered, but fat mass was similarly increased (~250 g) 48 h after the competitions. This elicited an increase in plasma leptin of 33% after the IT while it remained unchanged after the OT, likely due to a 25% increase in plasma osteocalcin which occurred only after the OT (all p < 0.05). Post-race HOMA remained unchanged in OT and IT. Performance was normalized 48 h after the competitions, with the exception of a slightly lower jumping capacity after the IT. Serum testosterone concentration tended to decrease by 10% after the IT whilst dihydrotestosterone was reduced by 24% after the IT. In conclusion, an acute increase in serum osteocalcin concentration blunts the expected increase of serum leptin concentration that should occur with fat mass gain. This study provides evidence for osteocalcin as a negative regulator of serum leptin in humans.

Cooperative binding of drugs on human serum albumin

NASA Astrophysics Data System (ADS)

Varela, L. M.; Pérez-Rodríguez, M.; García, M.

In order to explain the adsorption isotherms of the amphiphilic penicillins nafcillin and cloxacillin onto human serum albumin (HSA), a cooperative multilayer adsorption model is introduced, combining the Brunauer-Emmet-Teller (BET) adsorption isotherm with an amphiphilic ionic adsorbate, whose chemical potential is derived from Guggenheim's theory. The non-cooperative model has been previously proved to qualitatively predict the measured adsorption maxima of these drugs [Varela, L. M., García, M., Pérez-Rodríguez, M., Taboada, P., Ruso, J. M., and Mosquera, V., 2001, J. chem. Phys., 114, 7682]. The surface interactions among adsorbed drug molecules are modelled in a mean-field fashion, so the chemical potential of the adsorbate is assumed to include a term proportional to the surface coverage, the constant of proportionality being the lateral interaction energy between bound molecules. The interaction energies obtained from the empirical binding isotherms are of the order of tenths of the thermal energy, therefore suggesting the principal role of van der Waals forces in the binding process.

Locating the Binding Sites of Pb(II) Ion with Human and Bovine Serum Albumins

PubMed Central

Belatik, Ahmed; Hotchandani, Surat; Carpentier, Robert; Tajmir-Riahi, Heidar-Ali

2012-01-01

Lead is a potent environmental toxin that has accumulated above its natural level as a result of human activity. Pb cation shows major affinity towards protein complexation and it has been used as modulator of protein-membrane interactions. We located the binding sites of Pb(II) with human serum (HSA) and bovine serum albumins (BSA) at physiological conditions, using constant protein concentration and various Pb contents. FTIR, UV-visible, CD, fluorescence and X-ray photoelectron spectroscopic (XPS) methods were used to analyse Pb binding sites, the binding constant and the effect of metal ion complexation on HSA and BSA stability and conformations. Structural analysis showed that Pb binds strongly to HSA and BSA via hydrophilic contacts with overall binding constants of KPb-HSA = 8.2 (±0.8)×104 M−1 and KPb-BSA = 7.5 (±0.7)×104 M−1. The number of bound Pb cation per protein is 0.7 per HSA and BSA complexes. XPS located the binding sites of Pb cation with protein N and O atoms. Pb complexation alters protein conformation by a major reduction of α-helix from 57% (free HSA) to 48% (metal-complex) and 63% (free BSA) to 52% (metal-complex) inducing a partial protein destabilization. PMID:22574219

The SNP at −592 of human IL-10 gene is associated with serum IL-10 levels and increased risk for human papillomavirus cervical lesion development

PubMed Central

2012-01-01

Background Women with Human Papilloma Virus (HPV) persistence are characterized by high levels of IL-10 at cervix. We have determined whether polymorphisms of IL-10 gene promoter might be associated with increased risk of squamous intraepithelial cervical lesions (SICL) and whether exist significative differences of IL-10 mRNA expression at cervix and systemic and serum IL-10 protein between SICL cases and non-Cervical Lesions (NCL). Methods Peripheral blood samples from SICL (n = 204) and NCL (n = 166) were used to detect IL-10 promoter polymorphisms at loci -592A/C (rs1800872), -819C/T (rs1800871), -1082A/G (rs1800896), -1352A/G (rs1800893), by allelic discrimination and to evaluate serum IL-10 protein. Cervical epithelial scrapings from NCL and biopsies from SICLs were used for HPV-typing and to evaluate IL-10 mRNA expression level. The systemic and local IL-10 mRNA expression levels were measured by real time-PCR. Genotypic and allelic frequencies of the selected polymorphisms were analyzed by logistic regression, adjusting by age and HPV-genotype, to determine the association with SICL. Results No significant differences were found between genotype frequencies at loci −819, -1082, and −1352. Individuals carrying at least one copy of risk allele A of polymorphism −592 had a two-fold increased risk of developing SICL [adjusted odds ratio (OR), 2.02 (95% CI, 1.26-3.25), p = 0.003], compared to NCL. The IL-10 mRNA expression and serum IL-10 protein, were significantly higher in SICL cases (p < 0.01), being higher in patients carrying the risk allele A. Conclusions The −592 polymorphism is associated with increased risk of SICL and can serve as a marker of genetic susceptibility to SICL among Mexican women. According to IL-10 levels found in SICL, IL-10 can be relevant factor for viral persistence and progression disease. PMID:23148667

Detection of teratogens in human serum using rat embryo culture: cancer and epilepsy treatments. [Detecting teratogenicity of anticonvulsant and antineoplastic drugs

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

Chatot, C. L.

1979-01-01

Growth (protein and DNA contents) of headfold stage rat embryos cultured for 48 hrs on human serum was enhanced by glucose supplementation. Embryo growth varied with the source of the serum. Sera from 3 of the 19 control subjects produced abnormal embryos. Sera from 5 subjects undergoing cancer chemotherapy and 6 subjects receiving anticonvulsants were either lethal or teratogenic to cultured rat embryos.

[Study on the interaction of doxycycline with human serum albumin].

PubMed

Hu, Tao-Ying; Chen, Lin; Liu, Ying

2014-05-01

The present study was designed to investigate the interaction of doxycycline (DC) with human serum albumin (HSA) by the inner filter effects, displacement experiments and molecular docking methods, based on classic multi-spectroscopy. With fluorescence quenching method at 298 and 310 K, the binding constants Ka, were determined to be 2. 73 X 10(5) and 0. 74X 10(5) L mol-1, respectively, and there was one binding site between DC and HSA, indicating that the binding of DC to HSA was strong, and the quenching mechanism was a static quenching. The thermodynamic parameters (enthalpy change, AH and enthropy change, delta S) were calculated to be -83. 55 kJ mol-1 and -176. 31 J mol-1 K-1 via the Vant' Hoff equation, which indicated that the interaction of DC with HSA was driven mainly by hydrogen bonding and van der Waals forces. Based on the Föster's theory of non-radiation energy transfer, the specific binding distance between Trp-214 (acceptor) and DC (donor) was 4. 98 nm, which was similar to the result confirmed by molecular docking. Through displacement experiments, sub-domain IIA of HSA was assigned to possess the high-affinity binding site of DC. Three-dimensional fluorescence spectra indicated that the binding of DC to HSA induced the conformation change of HSA and increased the disclosure of some part of hydrophobic regions that had been buried before. The results of FTIR spectroscopy showed that DC bound to HSA led to the slight unfolding of the polypeptide chain of HSA. Furthermore, the binding details between DC and HSA were further confirmed by molecular docking methods, which revealed that DC was bound at sub-domain IIA through multiple interactions, such as hydrophobic effect, polar forces and pi-pi interactions. The experimental results provide theoretical basis and reliable data for the study of the interaction between small drug molecule and human serum albumin

Surface Plasmon Resonance imaging-based sensing for anti-bovine immunoglobulins detection in human milk and serum.

PubMed

Scarano, S; Scuffi, C; Mascini, M; Minunni, M

2011-11-30

Only few papers deal with Surface Plasmon Resonance imaging (SPRi) direct detection on complex matrices, limiting the biosensor application to real analytical problems. In this work a SPRi biosensor for anti-bovine IgG detection in untreated human bodily fluids, i.e. diluted human serum and milk, was developed. Enhanced levels of cow's milk antibodies in children's serum are suspected for their possible correlation with Type 1 diabetes during childhood and their detection in real samples was up to now performed by classical immunoassays based on indirect detection. The biosensor was optimised in standard samples and then in untreated human milk for anti-bovine IgG direct detection. The key novelty of the work is the evaluation of matrix effect by applying to real samples an experimental and ex ante method previously developed for SPRi signal sampling in standard solutions, called "Data Analyzer"; it punctually visualises and analyses the behaviour of receptor spots of the array, to select only spot areas with the best specific vs. unspecific signal values. In this way, benefits provide by SPRi image analysis are exploited here to quantify and minimise drawbacks due to the matrix effect, allowing to by-pass every matrix pre-treatment except dilution. Copyright © 2011 Elsevier B.V. All rights reserved.

Pharmacokinetics and effects on serum cholinesterase activities of organophosphorus pesticides acephate and chlorpyrifos in chimeric mice transplanted with human hepatocytes.

PubMed

Suemizu, Hiroshi; Sota, Shigeto; Kuronuma, Miyuki; Shimizu, Makiko; Yamazaki, Hiroshi

2014-11-01

Organophosphorus pesticides acephate and chlorpyrifos in foods have potential to impact human health. The aim of the current study was to investigate the pharmacokinetics of acephate and chlorpyrifos orally administered at lowest-observed-adverse-effect-level doses in chimeric mice transplanted with human hepatocytes. Absorbed acephate and its metabolite methamidophos were detected in serum from wild type mice and chimeric mice orally administered 150mg/kg. Approximately 70% inhibition of cholinesterase was evident in plasma of chimeric mice with humanized liver (which have higher serum cholinesterase activities than wild type mice) 1day after oral administrations of acephate. Adjusted animal biomonitoring equivalents from chimeric mice studies were scaled to human biomonitoring equivalents using known species allometric scaling factors and in vitro metabolic clearance data with a simple physiologically based pharmacokinetic (PBPK) model. Estimated plasma concentrations of acephate and chlorpyrifos in humans were consistent with reported concentrations. Acephate cleared similarly in humans and chimeric mice but accidental/incidental overdose levels of chlorpyrifos cleared (dependent on liver metabolism) more slowly from plasma in humans than it did in mice. The data presented here illustrate how chimeric mice transplanted with human hepatocytes in combination with a simple PBPK model can assist evaluations of toxicological potential of organophosphorus pesticides. Copyright © 2014 Elsevier Inc. All rights reserved.

Comparison of proteomic profiles of serum, plasma, and modified media supplements used for cell culture and expansion

PubMed Central

Ayache, Saleh; Panelli, Monica C; Byrne, Karen M; Slezak, Stefanie; Leitman, Susan F; Marincola, Francesco M; Stroncek, David F

2006-01-01

Background The culture and expansion of human cells for clinical use requires the presence of human serum or plasma in culture media. Although these supplements have been extensively characterized in their chemical composition, only recently it has been possible to provide by high throughput protein analysis, a comprehensive profile of the soluble factors contributing to cell survival. This study analyzed and compared the presence of 100 proteins including chemokines, cytokines and soluble factors in six different types of media supplements: serum, plasma, recalcified plasma, heat inactivated serum, heat inactivated plasma and heat inactivated recalcified plasma. Methods Serum, plasma, recalcified plasma, and heat inactivated supplements were prepared from ten healthy subjects. The levels of 100 soluble factors were measured in each sample using a multiplexed ELISA assay and compared by Eisen hierarchical clustering analysis. Results A comparison of serum and plasma levels of soluble factors found that 2 were greater in plasma but 18 factors were greater in serum including 11 chemokines. The levels of only four factors differed between recalcified plasma and plasma. Heat inactivation had the greatest effect on soluble factors. Supervised Eisen hierarchical clustering indicated that the differences between heat inactivated supplements and those that were not were greater than the differences within these two groups. The levels of 36 factors differed between heat inactivated plasma and plasma. Thirty one of these factors had a lower concentration in heat inactivated plasma including 12 chemokines, 4 growth factors, 4 matrix metalloproteases, and 3 adhesion molecules. Heat inactivated decalcified plasma is often used in place of heat inactivated serum and the levels of 19 soluble factors differed between these two supplements. Conclusion Our report provides a comprehensive protein profile of serum, plasma recalcified plasma, and heat inactivated supplements. This

Radioimmunoassay determinations of p-hydroxyphenformin and of apparent phenformin in human plasma or serum

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

Alkalay, D.; Khemani, L.; Bartlett, M.F.

1978-01-01

Sensitive radioimmunoassay procedures are described for measuring p-hydroxyphenformin and apparent phenformin in human plasma or serum. The p-hydroxyphenformin procedure, which requires advance phenformin analysis, offers excellent accuracy. The determinations of apparent phenformin are influenced by the phenformin and p-hydroxyphenformin contents of the samples. Over a period of four months, the methods showed a precision associated with relative standard deviations of 12% for apparent phenformin and 13% for p-hydroxyphenformin.

A contrast-sensitive channelized-Hotelling observer to predict human performance in a detection task using lumpy backgrounds and Gaussian signals

NASA Astrophysics Data System (ADS)

Park, Subok; Badano, Aldo; Gallas, Brandon D.; Myers, Kyle J.

2007-03-01

Previously, a non-prewhitening matched filter (NPWMF) incorporating a model for the contrast sensitivity of the human visual system was introduced for modeling human performance in detection tasks with different viewing angles and white-noise backgrounds by Badano et al. But NPWMF observers do not perform well detection tasks involving complex backgrounds since they do not account for random backgrounds. A channelized-Hotelling observer (CHO) using difference-of-Gaussians (DOG) channels has been shown to track human performance well in detection tasks using lumpy backgrounds. In this work, a CHO with DOG channels, incorporating the model of the human contrast sensitivity, was developed similarly. We call this new observer a contrast-sensitive CHO (CS-CHO). The Barten model was the basis of our human contrast sensitivity model. A scalar was multiplied to the Barten model and varied to control the thresholding effect of the contrast sensitivity on luminance-valued images and hence the performance-prediction ability of the CS-CHO. The performance of the CS-CHO was compared to the average human performance from the psychophysical study by Park et al., where the task was to detect a known Gaussian signal in non-Gaussian distributed lumpy backgrounds. Six different signal-intensity values were used in this study. We chose the free parameter of our model to match the mean human performance in the detection experiment at the strongest signal intensity. Then we compared the model to the human at five different signal-intensity values in order to see if the performance of the CS-CHO matched human performance. Our results indicate that the CS-CHO with the chosen scalar for the contrast sensitivity predicts human performance closely as a function of signal intensity.

Preferential solvatation of human serum albumin in dimethylsulfoxide-H2O binary solution

NASA Astrophysics Data System (ADS)

Grigoryan, K. R.

2009-12-01

The preferential solvatation of human serum albumin (HSA) in dimethylsulfoxide (DMSO) aqueous solutions were studied using the densitometry method. It has been shown that at DMSO low concentrations HSA undergoes to preferential hydration, but at DMSO higher concentrations preferential binding of DMSO molecules to protein occurs. It has been estimated that DMSO exhibits stabilizing/destabilizing effect on HSA structure which is explained in terms of hydration/solvatation of protein, on the one hand, and the medium structure enhancement/disruption around the protein molecule, on the other hand.

Identification of drug metabolites in human plasma or serum integrating metabolite prediction, LC-HRMS and untargeted data processing.

PubMed

Jacobs, Peter L; Ridder, Lars; Ruijken, Marco; Rosing, Hilde; Jager, Nynke Gl; Beijnen, Jos H; Bas, Richard R; van Dongen, William D

2013-09-01

Comprehensive identification of human drug metabolites in first-in-man studies is crucial to avoid delays in later stages of drug development. We developed an efficient workflow for systematic identification of human metabolites in plasma or serum that combines metabolite prediction, high-resolution accurate mass LC-MS and MS vendor independent data processing. Retrospective evaluation of predictions for 14 (14)C-ADME studies published in the period 2007-January 2012 indicates that on average 90% of the major metabolites in human plasma can be identified by searching for accurate masses of predicted metabolites. Furthermore, the workflow can identify unexpected metabolites in the same processing run, by differential analysis of samples of drug-dosed subjects and (placebo-dosed, pre-dose or otherwise blank) control samples. To demonstrate the utility of the workflow we applied it to identify tamoxifen metabolites in serum of a breast cancer patient treated with tamoxifen. Previously published metabolites were confirmed in this study and additional metabolites were identified, two of which are discussed to illustrate the advantages of the workflow.

Increased EPA levels in serum phospholipids of humans after four weeks daily ingestion of one portion chicken fed linseed and rapeseed oil

PubMed Central

2012-01-01

Since the amounts of arachidonic acid (AA) and EPA in food may have implications for human health, we investigated whether a small change in chicken feed influenced the blood lipid concentration in humans ingesting the chicken. Forty-six young healthy volunteers (age 20–29) were randomly allocated into two groups in a double-blind dietary intervention trial, involving ingestion of about 160 g chicken meat per day for 4 weeks. The ingested meat was either from chickens given a feed concentrate resembling the commercial chicken feed, containing 4% soybean oil (SO), or the meat was from chickens given a feed where the soybean oil had been replaced by 2% rapeseed oil plus 2% linseed oil (RLO). Serum total cholesterol, LDL and HDL cholesterol, triacylglycerols, serum phospholipid fatty acid concentration, blood pressure, body weight and C-reactive protein were determined at baseline and post-intervention. In subjects consuming chicken meat from the RLO group there was a significantly (p < 0.001) increased concentration of EPA in serum phospholipids, and a reduced ratio between AA and EPA. The participants that had a low% of EPA + DHA in serum phospholipids (less than 4.6%), all increased their% of EPA + DHA after the four week intervention period when consuming the RLO chicken. No significant response differences in cholesterol, triacylglycerol, C-reactive protein, body weight or blood pressure were observed between the groups. This trial demonstrates that a simple change in chicken feed can have beneficial effects on amount of EPA and the AA/EPA ratio in human serum phospholipids. PMID:22913248

Increased EPA levels in serum phospholipids of humans after four weeks daily ingestion of one portion chicken fed linseed and rapeseed oil.

PubMed

Haug, Anna; Nyquist, Nicole F; Mosti, Therese J; Andersen, Malin; Høstmark, Arne T

2012-08-22

Since the amounts of arachidonic acid (AA) and EPA in food may have implications for human health, we investigated whether a small change in chicken feed influenced the blood lipid concentration in humans ingesting the chicken. Forty-six young healthy volunteers (age 20-29) were randomly allocated into two groups in a double-blind dietary intervention trial, involving ingestion of about 160 g chicken meat per day for 4 weeks. The ingested meat was either from chickens given a feed concentrate resembling the commercial chicken feed, containing 4% soybean oil (SO), or the meat was from chickens given a feed where the soybean oil had been replaced by 2% rapeseed oil plus 2% linseed oil (RLO).Serum total cholesterol, LDL and HDL cholesterol, triacylglycerols, serum phospholipid fatty acid concentration, blood pressure, body weight and C-reactive protein were determined at baseline and post-intervention. In subjects consuming chicken meat from the RLO group there was a significantly (p < 0.001) increased concentration of EPA in serum phospholipids, and a reduced ratio between AA and EPA. The participants that had a low% of EPA + DHA in serum phospholipids (less than 4.6%), all increased their% of EPA + DHA after the four week intervention period when consuming the RLO chicken. No significant response differences in cholesterol, triacylglycerol, C-reactive protein, body weight or blood pressure were observed between the groups. This trial demonstrates that a simple change in chicken feed can have beneficial effects on amount of EPA and the AA/EPA ratio in human serum phospholipids.

Direct extraction of lead (II) from untreated human blood serum using restricted access carbon nanotubes and its determination by atomic absorption spectrometry.

PubMed

Barbosa, Valéria Maria Pereira; Barbosa, Adriano Francisco; Bettini, Jefferson; Luccas, Pedro Orival; Figueiredo, Eduardo Costa

2016-01-15

Oxidized carbon nanotubes were covered with layers of bovine serum albumin to result in so-called restricted-access carbon nanotubes (RACNTs). This material can extract Pb(2+) ions directly from untreated human blood serum while excluding all the serum proteins. The RACNTs have a protein exclusion capacity of almost 100% and a maximum Pb(2+) adsorption capacity of 34.5mg g(-1). High resolution transmission electron microscopy, scanning transmission electron microscopy and energy dispersive spectroscopy were used to confirm the BSA layer and Pb(2+) adsorption sites. A mini-column filled with RACNTs was used in an on-line solid phase extraction system coupled to a thermospray flame furnace atomic absorption spectrometry. At optimized experimental conditions, the method has a detection limit as low as 2.1µg L(-1), an enrichment factor of 5.5, and inter- and intra-day precisions (expressed as relative standard deviation) of <8.1%. Recoveries of the Pb(2+) spiked samples ranged from 89.4% to 107.3% for the extraction from untreated human blood serum. Copyright © 2015 Elsevier B.V. All rights reserved.

Determination of crenolanib in human serum and cerebrospinal fluid by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS).

PubMed

Roberts, Michael S; Turner, David C; Owens, Thandranese S; Ramachandran, Abhijit; Wetmore, Cynthia; Throm, Stacy L; Stewart, Clinton F

2013-06-15

A LC-ESI-MS/MS method for the determination of crenolanib (CP-868,596) in human serum was developed and validated employing d4-CP-868,596 as an internal standard (ISTD). In addition to human serum, the method was also partially validated for crenolanib determination in human cerebrospinal fluid (CSF) samples. Sample aliquots (50μl of serum or CSF) were prepared for analysis using liquid-liquid extraction (LLE) with tert-butyl methyl ether. Chromatography was performed using a phenomenex Gemini C18 column (3μm, 100mm×4.6mm I.D.) in a column heater set at 50°C and an isocratic mobile phase (methanol/water/formic acid at a volume ratio of 25/25/0.15, v/v/v). The flow rate was 0.45mL/min, and the retention time for both analyte and ISTD was less than 3.5min. Samples were analyzed with an API-5500 LC-MS/MS system (ESI) in positive ionization mode coupled to a Shimadzu HPLC system. The ion transitions monitored were m/z 444.4→373.1 and m/z 448.2→374.2 for crenolanib and ISTD, respectively. The method was linear over the range of 5-1000ng/mL for serum and 0.5-1000ng/mL for CSF. For human serum, both intra-day and inter-day precision were <4%, while intra-day and inter-day accuracy were within 8% of nominal values. Recovery was greater than 50% for both the analyte and ISTD. For CSF samples, both intra-day and inter-day precision were <9% except at the lower limit of quantification (LLOQ) which was <17%. The intra-day and inter-day accuracy were within 11% of the nominal CSF concentrations. After validation, this method was successfully applied to the analysis of serial pharmacokinetic samples obtained from a child treated with oral crenolanib. Copyright © 2013 Elsevier B.V. All rights reserved.

Serum Antioxidant Status in Patients with Systemic Sclerosis

PubMed Central

Hassan, Iffat; Sajad, Peerzada; Majid, Sabiya; Hassan, Tehseen

2013-01-01

Background: Vascular endothelial dysfunction is a central event in pathogenesis of a variety of human diseases. Systemic sclerosis is one of such diseases. The oxidative stress and depletion of antioxidants in the serum is believed to be one of the factors in causing this dysfunction. Aims: The aim of this case control study was to compare the levels of antioxidants in the serum of patients with systemic sclerosis and the normal age and sex matched controls. Materials and Methods: Our study consisted of 16 successively admitted patients with systemic sclerosis and 16 healthy, age and sex matched controls. The age group of patient's ranged between 25 and 55 years. The duration of the disease in patients ranged from 1 to 8 years. The serum of patients and controls were assayed for the levels of antioxidants (GSH, NO, MDA, SOD and GPX) by spectrophotometry. The statistical method of analysis used was the one sample t-test. Results: The median levels of antioxidants in the control patients were: SOD-4.14 units/ml; GSH-4.76 units/ml; NO-5.58 nmol/l; MDA-0.53 nmol/l and GPX-49 μmol/l. The levels of NO, GSH and SOD were decreased in these patients with a significant P value (<0.001) whereas the levels of GPX and MDA were normal to increased with a significant P value. Conclusion: The depletion of antioxidants and oxidative stress in serum might be responsible for the vascular dysfunction and other hallmark manifestations of systemic sclerosis. Therefore micronutrient antioxidant supplements may be of therapeutic value. PMID:23723482

Systematic Evaluation of the Use of Human Plasma and Serum for Mass-Spectrometry-Based Shotgun Proteomics.

PubMed

Lan, Jiayi; Núñez Galindo, Antonio; Doecke, James; Fowler, Christopher; Martins, Ralph N; Rainey-Smith, Stephanie R; Cominetti, Ornella; Dayon, Loïc

2018-04-06

Over the last two decades, EDTA-plasma has been used as the preferred sample matrix for human blood proteomic profiling. Serum has also been employed widely. Only a few studies have assessed the difference and relevance of the proteome profiles obtained from plasma samples, such as EDTA-plasma or lithium-heparin-plasma, and serum. A more complete evaluation of the use of EDTA-plasma, heparin-plasma, and serum would greatly expand the comprehensiveness of shotgun proteomics of blood samples. In this study, we evaluated the use of heparin-plasma with respect to EDTA-plasma and serum to profile blood proteomes using a scalable automated proteomic pipeline (ASAP 2 ). The use of plasma and serum for mass-spectrometry-based shotgun proteomics was first tested with commercial pooled samples. The proteome coverage consistency and the quantitative performance were compared. Furthermore, protein measurements in EDTA-plasma and heparin-plasma samples were comparatively studied using matched sample pairs from 20 individuals from the Australian Imaging, Biomarkers and Lifestyle (AIBL) Study. We identified 442 proteins in common between EDTA-plasma and heparin-plasma samples. Overall agreement of the relative protein quantification between the sample pairs demonstrated that shotgun proteomics using workflows such as the ASAP 2 is suitable in analyzing heparin-plasma and that such sample type may be considered in large-scale clinical research studies. Moreover, the partial proteome coverage overlaps (e.g., ∼70%) showed that measures from heparin-plasma could be complementary to those obtained from EDTA-plasma.

HPLC-MS/MS method optimisation for matrix metalloproteinase 3 and matrix metalloproteinase 9 determination in human blood serum using target analysis.

PubMed

Kotnik, Petra; Krajnc, Metka Koren; Pahor, Artur; Finšgar, Matjaž; Knez, Željko

2018-02-20

A quantitative analysis of zinc endopeptidases matrix metalloproteinase 9 (MMP9) and matrix metalloproteinase 3 (MMP3) from human blood serum are presented. Both matrix metalloproteinases (MMP) are present in human blood serum and can be used as biomarkers for different diseases. The analysis was performed using LC-MS/MS with a triple quadrupole mass spectrometer, based on two specific peptides of each MMP in comparison with an enzyme-linked immunosorbent assay (ELISA). While the conditions for the LC-MS/MS analysis of MMP9 peptides were previously reported for bronchoalveolar lavage fluid, the analysis of MMP3 peptides was newly quantified for human blood serum herein for the first time. For MMP3, the linear behaviour was determined in the concentration range from 1.0-200.0ng/mL (R 2 =0.997) with an LLOD of 0.5ng/mL. For MMP9, linearity was determined in the concentration range from 6.5-65.0ng/mL (R 2 =0.995) with an LLOD of 2.0ng/mL. Copyright © 2017 Elsevier B.V. All rights reserved.

Nutrient supplemented serum-free medium increases cardiomyogenesis efficiency of human pluripotent stem cells.

PubMed

Ting, Sherwin; Lecina, Marti; Chan, Yau-Chi; Tse, Hung Fat; Reuveny, Shaul; Oh, Steve Kw

2013-07-26

To development of an improved p38 MAPK inhibitor-based serum-free medium for embryoid body cardiomyocyte differentiation of human pluripotent stem cells. Human embryonic stem cells (hESC) differentiated to cardiomyocytes (CM) using a p38 MAPK inhibitor (SB203580) based serum-free medium (SB media). Nutrient supplements known to increase cell viability were added to SB medium. The ability of these supplements to improve cardiomyogenesis was evaluated by measurements of cell viability, total cell count, and the expression of cardiac markers via flow cytometry. An improved medium containing Soy hydrolysate (HySoy) and bovine serum albumin (BSA) (SupSB media) was developed and tested on 2 additional cell lines (H1 and Siu-hiPSC). Characterization of the cardiomyocytes was done by immunohistochemistry, electrophysiology and quantitative real-time reverse transcription-polymerase chain reaction. hESC cell line, HES-3, differentiating in SB medium for 16 d resulted in a cardiomyocyte yield of 0.07 ± 0.03 CM/hESC. A new medium (SupSB media) was developed with the addition of HySoy and BSA to SB medium. This medium resulted in 2.6 fold increase in cardiomyocyte yield (0.21 ± 0.08 CM/hESC). The robustness of SupSB medium was further demonstrated using two additional pluripotent cell lines (H1, hESC and Siu1, hiPSC), showing a 15 and 9 fold increase in cardiomyocyte yield respectively. The age (passage number) of the pluripotent cells did not affect the cardiomyocyte yields. Embryoid body (EB) cardiomyocytes formed in SupSB medium expressed canonical cardiac markers (sarcomeric α-actinin, myosin heavy chain and troponin-T) and demonstrated all three major phenotypes: nodal-, atrial- and ventricular-like. Electrophysiological characteristics (maximum diastolic potentials and action potential durations) of cardiomyocytes derived from SB and SupSB media were similar. The nutrient supplementation (HySoy and BSA) leads to increase in cell viability, cell yield and cardiac

Human liver plasma membranes contain receptors for the hepatitis B virus pre-S1 region and, via polymerized human serum albumin, for the pre-S2 region.

PubMed Central

Pontisso, P; Petit, M A; Bankowski, M J; Peeples, M E

1989-01-01

Hepatitis B virus particles contain three related viral envelope proteins, the small, middle, and large S (surface) proteins. All three proteins contain the small S amino acid sequence at their carboxyl terminus. It is not clear which of these S proteins functions as the viral attachment protein, binding to a target cell receptor and initiating infection. In this report, recombinant hepatitis B surface antigen (rHBsAg) particles, which contain only virus envelope proteins, were radioactively labeled, and their attachment to human liver membranes was examined. Only the rHBsAg particles containing the large S protein were capable of directly attaching to liver plasma membranes. The attachment was saturable and could be prevented by competition with unlabeled particles or by a monoclonal antibody specific for the large S protein. In the presence of polymerized human serum albumin, both large and middle S protein-containing rHBsAg particles were capable of attaching to the liver plasma membranes. Small S protein-containing rHBsAg particles were not able to attach even in the presence of polymerized human serum albumin. These results indicate that the large S protein may be the viral attachment protein for hepatocytes, binding directly to liver plasma membranes by its unique amino-terminal (pre-S1) sequence. These results also indicate that polymerized human serum albumin or a similar molecule could act as an intermediate receptor, attaching to liver plasma membranes and to the amino acid sequence (pre-S2) shared by the middle and large S proteins but not contained in the small S protein. Images PMID:2649690

Prophylaxis with human serum butyrylcholinesterase protects guinea pigs exposed to multiple lethal doses of soman or VX.

PubMed

Saxena, Ashima; Sun, Wei; Fedorko, James M; Koplovitz, Irwin; Doctor, Bhupendra P

2011-01-01

Human serum butyrylcholinesterase (Hu BChE) is currently under advanced development as a bioscavenger for the prophylaxis of organophosphorus (OP) nerve agent toxicity in humans. It is estimated that a dose of 200mg will be required to protect a human against 2×LD(50) of soman. To provide data for initiating an investigational new drug application for the use of this enzyme as a bioscavenger in humans, we purified enzyme from Cohn fraction IV-4 paste and initiated safety and efficacy evaluations in mice, guinea pigs, and non-human primates. In mice, we demonstrated that a single dose of enzyme that is 30 times the therapeutic dose circulated in blood for at least four days and did not cause any clinical pathology in these animals. In this study, we report the results of safety and efficacy evaluations conducted in guinea pigs. Various doses of Hu BChE delivered by i.m. injections peaked at ∼24h and had a mean residence time of 78-103h. Hu BChE did not exhibit any toxicity in guinea pigs as measured by general observation, serum chemistry, hematology, and gross and histological tissue changes. Efficacy evaluations showed that Hu BChE protected guinea pigs from an exposure of 5.5×LD(50) of soman or 8×LD(50) of VX. These results provide convincing data for the development of Hu BChE as a bioscavenger that can protect humans against all OP nerve agents. Published by Elsevier Inc.

A sensitive and specific method for measurement of multiple retinoids in human serum with UHPLC-MS/MS

PubMed Central

Arnold, Samuel L. M.; Amory, John K.; Walsh, Thomas J.; Isoherranen, Nina

2012-01-01

Retinol (vitamin A) circulates at 1–4 μM concentration and is easily measured in serum. However, retinol is biologically inactive. Its metabolite, retinoic acid (RA), is believed to be responsible for biological effects of vitamin A, and hence the measurement of retinol concentrations is of limited value. A UHPLC-MS/MS method using isotope-labeled internal standards was developed and validated for quantitative analysis of endogenous RA isomers and metabolites. The method was used to measure retinoids in serum samples from 20 healthy men. In the fed state, the measured concentrations were 3.1 ± 0.2 nM for atRA, 0.1 ± 0.02 nM for 9-cisRA, 5.3 ± 1.3 nM for 13-cisRA, 0.4 ± 0.4 nM for 9,13-dicisRA, and 17.2 ± 6.8 nM for 4oxo-13-cisRA. The concentrations of the retinoids were not significantly different when measured after an overnight fast (3.0 ± 0.1 nM for atRA, 0.09 ± 0.01nM for 9-cisRA, 3.9 ± 0.2 nM for 13-cisRA, 0.3 ± 0.1 nM for 9,13-dicisRA, and 11.9 ± 1.6 nM for 4oxo-13-cisRA). 11-cisRA and 4OH-RA were not detected in human serum. The high sensitivity of the MS/MS method combined with the UHPLC separation power allowed detection of endogenous 9-cisRA and 4oxo-atRA for the first time in human serum. PMID:22192917

Anti-idiotypic antibodies to poliovirus antibodies in commercial immunoglobulin preparations, human serum, and milk.

PubMed

Hahn-Zoric, M; Carlsson, B; Jeansson, S; Ekre, H P; Osterhaus, A D; Roberton, D; Hanson, L A

1993-05-01

Our previous studies have suggested that fetal antibody production can be induced by maternal antiidiotypic antibodies transferred to the fetus via the placenta. We tested commercial Ig, sera, and milk for the presence of anti-idiotypic antibodies to poliovirus type 1, using affinity chromatography combined with ELISA systems and virus neutralization techniques. Our results indicate that commercial Ig, serum, and milk samples contain antibodies recognizing idiotypic determinants on antibodies to poliovirus. Several lines of evidence support this conclusion. Thus, in an ELISA with poliovirus as a solid phase, binding of specific antibodies could be inhibited by addition of an eluate from the Ig preparation containing anti-idiotypic antibodies against poliovirus type 1. Also, antiidiotypic antibodies from pooled human Ig, serum, and colostrum samples against poliovirus bound directly to solid-phase-attached MAb against poliovirus type 1. In addition, in a competitive inhibition ELISA, where antiidiotypic antibodies isolated from the Ig preparation competed with the poliovirus antigen for binding to monoclonal or polyclonal idiotypic antibodies on the solid phase, inhibition of antigen binding was seen at low antigen concentrations. When single-donor serum or milk was used, this inhibition was even more pronounced and could be demonstrated at almost all antigen concentrations. The finding that anti-idiotypes are present in maternal serum and milk imply, in agreement with our previous studies, that anti-idiotypes may actively induce a specific immune response in the fetus without previous exposure to the antigen by being transferred across the placenta or by being passively transferred to the newborn via mother's milk.

Selenium speciation in human serum and its implications for epidemiologic research: a cross-sectional study.

PubMed

Vinceti, Marco; Grill, Peter; Malagoli, Carlotta; Filippini, Tommaso; Storani, Simone; Malavolti, Marcella; Michalke, Bernhard

2015-01-01

Observational studies addressing the relation between selenium and human health, particularly cancer risk, yielded inconsistent results, while most recent randomized trials showed a fairly consistent pattern suggesting null or adverse effects of the metalloid. One of the most plausible explanations for such inconsistencies is inadequate exposure assessment in observational studies, commonly carried out by measuring total Se content without taking into account the specific exposure to the individual chemical forms of the metalloid, whose toxic and nutritional properties may vary greatly. Data on the distribution of these species in human blood and their correlation with overall selenium levels are very limited. The concentrations of organic and inorganic selenium species were analyzed in serum of fifty subjects sampled from the general population of the municipality of Modena, northern Italy, aged from 35 to 70 years. Samples were collected during a 30-month period, and determinations of selenium species were carried out using high pressure liquid chromatography coupled with inductively coupled plasma dynamic reaction cell mass spectrometry. The majority of selenium was found to be present as organic species, but the inorganic forms showed higher levels than expected. These species showed limited correlations with age, sex and body mass index, while the organic forms increased in subjects consuming selenium-containing dietary supplements and decreased in smokers. The length of the sample storage period strongly influenced the distribution of selenium compounds, with a clear tendency towards higher inorganic and lower organic selenium levels over time. In multivariate analysis adjusting for potential confounders, total serum selenium correlated with human serum albumin-bound selenium and, in males, with two organic species of the metalloid (selenocysteine and glutathione peroxidase-bound selenium), while little association existed with the other organic forms and the

DNA-mediated strand displacement facilitates sensitive electronic detection of antibodies in human serums.

PubMed

Dou, Baoting; Yang, Jianmei; Shi, Kai; Yuan, Ruo; Xiang, Yun

2016-09-15

We describe here the development of a sensitive and convenient electronic sensor for the detection of antibodies in human serums. The sensor is constructed by self-assembly formation of a mixed monolayer containing the small molecule epitope conjugated double stranded DNA probes on gold electrode. The target antibody binds the epitope on the dsDNA probe and lowers the melting temperature of the duplex, which facilitates the displacement of the antibody-linked strand of the duplex probe by an invading methylene blue-tagged single stranded DNA (MB-ssDNA) through the strand displacement reaction and leads to the capture of many MB-ssDNA on the sensor surface. Subsequent electrochemical oxidation of the methylene blue labels results in amplified current response for sensitive monitoring of the antibodies. The antibody assay conditions are optimized and the sensor exhibits a linear range between 1.0 and 25.0nM with a detection limit of 0.67nM for the target antibody. The sensor is also selective and can be employed to detect the target antibodies in human serum samples. With the advantages of using small molecule epitope as the antibody recognition element over traditional antigen, the versatile manipulability of the DNA probes and the unique properties of the electrochemical transduction technique, the developed sensor thus hold great potential for simple and sensitive detection of different antibodies and other proteins in real samples. Copyright © 2016 Elsevier B.V. All rights reserved.

Binding of ring-substituted indole-3-acetic acids to human serum albumin.

PubMed

Soskić, Milan; Magnus, Volker

2007-07-01

The plant hormone, indole-3-acetic acid (IAA), and its ring-substituted derivatives have recently attracted attention as promising pro-drugs in cancer therapy. Here we present relative binding constants to human serum albumin for IAA and 34 of its derivatives, as obtained using the immobilized protein bound to a support suitable for high-performance liquid chromatography. We also report their octanol-water partition coefficients (logK(ow)) computed from retention data on a C(18) coated silica gel column. A four-parameter QSPR (quantitative structure-property relationships) model, based on physico-chemical properties, is put forward, which accounts for more than 96% of the variations in the binding affinities of these compounds. The model confirms the importance of lipophilicity as a global parameter governing interaction with serum albumin, but also assigns significant roles to parameters specifically related to the molecular topology of ring-substituted IAAs. Bulky substituents at ring-position 6 increase affinity, those at position 2 obstruct binding, while no steric effects were noted at other ring-positions. Electron-withdrawing substituents at position 5 enhance binding, but have no obvious effect at other ring positions.

Nonenzymatic glycosylation of human serum albumin and its effect on antibodies profile in patients with diabetes mellitus

PubMed Central

Raghav, Alok; Alam, Khursheed

2017-01-01

Background Albumin glycation and subsequent formation of advanced glycation end products (AGEs) correlate with diabetes and associated complications. Methods Human Serum Albumin (HSA) was modified with D-glucose for a 40 day period under sterile conditions at 37°C. Modified samples along with native HSA (unmodified) were analyzed for structural modifications by UV and fluorescence, FTIR, Liquid chromatography mass spectrometry (LCMS) and X–ray crystallography. New-Zealand white female rabbits immunized with AGEs, represent auto-antibodies formation as assessed by competitive and direct binding enzyme-linked immunosorbent assay (ELISA). Neo-epitopesagainst In-vitro formed AGEs were characterized in patients with diabetes mellitus type 2 (n = 50), type 1 (n = 50), gestational diabetes (n = 50) and type 2 with chronic kidney disease (CKD) with eGFR level 60–89 mL/min (n = 50) from serum direct binding ELISA. Results Glycated-HSA showed amarked increase in hyperchromicity of 65.82%,71.98%, 73.62% and 76.63% at λ280 nm along with anincreasein fluorescence intensity of 65.82%, 71.98%, 73.62% and 76.63% in glycated-HSA compared to native. FTIR results showed theshifting of Amide I peak from 1656 cm_1 to 1659 cm_1 and Amide II peak from 1554 cm_1 to 1564 cm_1 in glycated-HSA, with anew peak appearance of carbonyl group at 1737 cm-1. LCMS chromatogram of glycated-HSA showed thepresence of carboxymethyl lysine (CML) at 279.1 m/z. Immunological analysis showed high antibody titre>1:12,800 in theserum of rabbits immunized with glycated-HSA (modified with 400 mg/dL glucose) and inhibition of 84.65% at anantigen concentration of 20μg/mL. Maximum serum auto-antibody titre was found in T2DM (0.517±0.086), T1DM (0.108±0.092), GDM (0.611±0.041) and T2DM+CKD (0.096±0.25) patients immunized with glycated-HSA (modified with 400 mg/dL glucose). Conclusions Non-enzymatic glycosylation of HSA manifests immunological complications in diabetes mellitus due to change in its

Optimizing Cationic and Neutral Lipids for Efficient Gene Delivery at High Serum Content

PubMed Central

Majzoub, Ramsey N.; Hwu, Yeu-kuang; Liang, Keng S.; Leal, Cecília; Safinya, Cyrus R.

2014-01-01

Background Cationic liposome (CL)-DNA complexes are promising gene delivery vectors with potential applications in gene therapy. A key challenge in creating CL-DNA complexes for applications is that their transfection efficiency (TE) is adversely affected by serum. In particular, little is known about the effects of high serum contents on TE even though this may provide design guidelines for applications in vivo. Methods We prepared CL-DNA complexes in which we varied the neutral lipid (DOPC, glycerol-monooleate (GMO), cholesterol), the headgroup charge and chemical structure of the cationic lipid, and the ratio of neutral to cationic lipid; we then measured the TE of these complexes as a function of serum content and assessed their cytotoxicity. We tested selected formulations in two human cancer cell lines (M21/melanoma and PC-3/prostate cancer). Results In the absence of serum, all CL-DNA complexes of custom-synthesized multivalent lipids show high TE. Certain combinations of multivalent lipids and neutral lipids, such as MVL5(5+)/GMO-DNA complexes or complexes based on the dendritic-headgroup lipid TMVLG3(8+) exhibited high TE both in the absence and presence of serum. Although their TE still dropped to a small extent in the presence of serum, it reached or surpassed that of benchmark commercial transfection reagents, in particular at high serum content. Conclusions Two-component vectors (one multivalent cationic lipid and one neutral lipid) can rival or surpass benchmark reagents at low and high serum contents (up to 50%, v/v). We suggest guidelines for optimizing the serum resistance of CL-DNA complexes based on a given cationic lipid. PMID:24753287

Multi-analyte method development for analysis of brominated flame retardants (BFRs) and PBDE metabolites in human serum.

PubMed

Lu, Dasheng; Jin, Yu'e; Feng, Chao; Wang, Dongli; Lin, Yuanjie; Qiu, Xinlei; Xu, Qian; Wen, Yimin; She, Jianwen; Wang, Guoquan; Zhou, Zhijun

2017-09-01

Commonly, analytical methods measuring brominated flame retardants (BFRs) of different chemical polarities in human serum are labor consuming and tedious. Our study used acidified diatomaceous earth as solid-phase extraction (SPE) adsorbent and defatting material to simultaneously determine the most abundant BFRs and their metabolites with different polarities in human serum samples. The analytes include three types of commercial BFRs, tetrabromobisphenol A (TBBPA), hexabromocyclododecane (HBCD) isomers, and polybrominated biphenyl ethers (PBDEs), and dominant hydroxylated BDE (OH-PBDE) and methoxylated BDE (MeO-PBDE) metabolites of PBDEs. The sample eluents were sequentially analyzed for PBDEs and MeO-BDEs on online gel permeation chromatography/gas chromatography-electron capture-negative ionization mass spectrometry (online GPC GC-ECNI-MS) and for TBBPA, HBCD, and OH-BDEs on liquid chromatography-tandem mass spectrometry (LC-MS/MS). Method recoveries were 67-134% with a relative standard deviation (RSD) of less than 20%. Method detection limits (MDLs) were 0.30-4.20 pg/mL fresh weight (f.w.) for all analytes, except for BDE-209 of 16 pg/mL f.w. The methodology was also applied in a pilot study, which analyzed ten real samples from healthy donors in China, and the majority of target analytes were detected with a detection rate of more than 80%. To our knowledge, it is the first time for us in effectively determining BFRs of most types in one aliquot of human serum samples. This new analytical method is more specific, sensitive, accurate, and time saving for routine biomonitoring of these BFRs and for integrated assessment of health risk of BFR exposure.

A meta-analysis of the association of serum ischaemia-modified albumin levels with human hypothyroidism and hyperthyroidism.

PubMed

Reddy, Varikasuvu Seshadri; Bukke, Suman; Mahato, Khageshwar; Kumar, Vinod; Reddy, Netala Vasudeva; Munikumar, Manne; Vodelu, Bramahanapally

2017-02-28

Serum levels of ischaemia-modified albumin (IMA) have been studied as a novel and simple measure of oxidative stress (OXS) in different thyroid pathologies. However, results of available studies in the literature were not consistent. This meta-analysis was attempted to quantify the overall effect size for serum IMA levels in human hypothyroidism (HT) and hyperthyroidism (HYT) and to study its associations with the thyroid profile. Databases of PubMed/Medline, EMBASE, Google Scholar, Web of Science and Science Direct were searched for articles. Data on serum IMA levels in HT, HYT patients and euthyroid controls were extracted to compute standardized mean differences (SMD) by the random-effects model. The associations between IMA and thyroid profile were computed by the meta-analysis of correlation coefficients. IMA levels in HT patients (SMD=1.12; Z=2.76; P=0.006) and HYT patients (SMD=1.64; Z=2.57; P=0.01) were significantly higher than in euthyroid controls and the thyroid treatment showed a favourble effect on serum IMA levels. There were strong and significant correlations between IMA and hormonal status in HT and HYT groups. This meta-analysis showing increased IMA level in both HT and HYT patients and its association with thyroid profile suggests that serum IMA could be used as a simple measure of increased OXS in thyroid dysfunction. © 2017 The Author(s).

Human serum-derived protein removes the need for coating in defined human pluripotent stem cell culture

PubMed Central

Pijuan-Galitó, Sara; Tamm, Christoffer; Schuster, Jens; Sobol, Maria; Forsberg, Lars; Merry, Catherine L. R.; Annerén, Cecilia

2016-01-01

Reliable, scalable and time-efficient culture methods are required to fully realize the clinical and industrial applications of human pluripotent stem (hPS) cells. Here we present a completely defined, xeno-free medium that supports long-term propagation of hPS cells on uncoated tissue culture plastic. The medium consists of the Essential 8 (E8) formulation supplemented with inter-α-inhibitor (IαI), a human serum-derived protein, recently demonstrated to activate key pluripotency pathways in mouse PS cells. IαI efficiently induces attachment and long-term growth of both embryonic and induced hPS cell lines when added as a soluble protein to the medium at seeding. IαI supplementation efficiently supports adaptation of feeder-dependent hPS cells to xeno-free conditions, clonal growth as well as single-cell survival in the absence of Rho-associated kinase inhibitor (ROCKi). This time-efficient and simplified culture method paves the way for large-scale, high-throughput hPS cell culture, and will be valuable for both basic research and commercial applications. PMID:27405751

Human Capital Background and the Educational Attainment of Second-Generation Immigrants in France

ERIC Educational Resources Information Center

Dos Santos, Manon Domingues; Wolff, Francois-Charles

2011-01-01

In this paper, we study the impact of parental human capital background on ethnic educational gaps between second-generation immigrants using a large data set conducted in France in 2003. Estimates from censored random effect ordered Probit regressions show that the skills of immigrants explain in the most part, the ethnic educational gap between…

Efficiency of the human observer for detecting a Gaussian signal at a known location in non-Gaussian distributed lumpy backgrounds.

PubMed

Park, Subok; Gallas, Bradon D; Badano, Aldo; Petrick, Nicholas A; Myers, Kyle J

2007-04-01

A previous study [J. Opt. Soc. Am. A22, 3 (2005)] has shown that human efficiency for detecting a Gaussian signal at a known location in non-Gaussian distributed lumpy backgrounds is approximately 4%. This human efficiency is much less than the reported 40% efficiency that has been documented for Gaussian-distributed lumpy backgrounds [J. Opt. Soc. Am. A16, 694 (1999) and J. Opt. Soc. Am. A18, 473 (2001)]. We conducted a psychophysical study with a number of changes, specifically in display-device calibration and data scaling, from the design of the aforementioned study. Human efficiency relative to the ideal observer was found again to be approximately 5%. Our variance analysis indicates that neither scaling nor display made a statistically significant difference in human performance for the task. We conclude that the non-Gaussian distributed lumpy background is a major factor in our low human-efficiency results.

Activity of the de novo engineered antimicrobial peptide WLBU2 against Pseudomonas aeruginosa in human serum and whole blood: implications for systemic applications.

PubMed

Deslouches, Berthony; Islam, Kazi; Craigo, Jodi K; Paranjape, Shruti M; Montelaro, Ronald C; Mietzner, Timothy A

2005-08-01

Cationic amphipathic peptides have been extensively investigated as a potential source of new antimicrobials that can complement current antibiotic regimens in the face of emerging drug-resistant bacteria. However, the suppression of antimicrobial activity under certain biologically relevant conditions (e.g., serum and physiological salt concentrations) has hampered efforts to develop safe and effective antimicrobial peptides for clinical use. We have analyzed the activity and selectivity of the human peptide LL37 and the de novo engineered antimicrobial peptide WLBU2 in several biologically relevant conditions. The host-derived synthetic peptide LL37 displayed high activity against Pseudomonas aeruginosa but demonstrated staphylococcus-specific sensitivity to NaCl concentrations varying from 50 to 300 mM. Moreover, LL37 potency was variably suppressed in the presence of 1 to 6 mM Mg(2+) and Ca(2+) ions. In contrast, WLBU2 maintained its activity in NaCl and physiologic serum concentrations of Mg(2+) and Ca(2+). WLBU2 is able to kill P. aeruginosa (10(6) CFU/ml) in human serum, with a minimum bactericidal concentration of <9 microM. Conversely, LL37 is inactive in the presence of human serum. Bacterial killing kinetic assays in serum revealed that WLBU2 achieved complete bacterial killing in 20 min. Consistent with these results was the ability of WLBU2 (15 to 20 microM) to eradicate bacteria from ex vivo samples of whole blood. The selectivity of WLBU2 was further demonstrated by its ability to specifically eliminate P. aeruginosa in coculture with human monocytes or skin fibroblasts without detectable adverse effects to the host cells. Finally, WLBU2 displayed potent efficacy against P. aeruginosa in an intraperitoneal infection model using female Swiss Webster mice. These results establish a potential application of WLBU2 in the treatment of bacterial sepsis.

Activity of the De Novo Engineered Antimicrobial Peptide WLBU2 against Pseudomonas aeruginosa in Human Serum and Whole Blood: Implications for Systemic Applications

PubMed Central

Deslouches, Berthony; Islam, Kazi; Craigo, Jodi K.; Paranjape, Shruti M.; Montelaro, Ronald C.; Mietzner, Timothy A.

2005-01-01

Cationic amphipathic peptides have been extensively investigated as a potential source of new antimicrobials that can complement current antibiotic regimens in the face of emerging drug-resistant bacteria. However, the suppression of antimicrobial activity under certain biologically relevant conditions (e.g., serum and physiological salt concentrations) has hampered efforts to develop safe and effective antimicrobial peptides for clinical use. We have analyzed the activity and selectivity of the human peptide LL37 and the de novo engineered antimicrobial peptide WLBU2 in several biologically relevant conditions. The host-derived synthetic peptide LL37 displayed high activity against Pseudomonas aeruginosa but demonstrated staphylococcus-specific sensitivity to NaCl concentrations varying from 50 to 300 mM. Moreover, LL37 potency was variably suppressed in the presence of 1 to 6 mM Mg2+ and Ca2+ ions. In contrast, WLBU2 maintained its activity in NaCl and physiologic serum concentrations of Mg2+ and Ca2+. WLBU2 is able to kill P. aeruginosa (106 CFU/ml) in human serum, with a minimum bactericidal concentration of <9 μM. Conversely, LL37 is inactive in the presence of human serum. Bacterial killing kinetic assays in serum revealed that WLBU2 achieved complete bacterial killing in 20 min. Consistent with these results was the ability of WLBU2 (15 to 20 μM) to eradicate bacteria from ex vivo samples of whole blood. The selectivity of WLBU2 was further demonstrated by its ability to specifically eliminate P. aeruginosa in coculture with human monocytes or skin fibroblasts without detectable adverse effects to the host cells. Finally, WLBU2 displayed potent efficacy against P. aeruginosa in an intraperitoneal infection model using female Swiss Webster mice. These results establish a potential application of WLBU2 in the treatment of bacterial sepsis. PMID:16048927

Cabazitaxel-loaded human serum albumin nanoparticles as a therapeutic agent against prostate cancer

PubMed Central

Qu, Na; Lee, Robert J; Sun, Yating; Cai, Guangsheng; Wang, Junyang; Wang, Mengqiao; Lu, Jiahui; Meng, Qingfan; Teng, Lirong; Wang, Di; Teng, Lesheng

2016-01-01

Cabazitaxel-loaded human serum albumin nanoparticles (Cbz-NPs) were synthesized to overcome vehicle-related toxicity of current clinical formulation of the drug based on Tween-80 (Cbz-Tween). A salting-out method was used for NP synthesis that avoids the use of chlorinated organic solvent and is simpler compared to the methods based on emulsion-solvent evaporation. Cbz-NPs had a narrow particle size distribution, suitable drug loading content (4.9%), and superior blood biocompatibility based on in vitro hemolysis assay. Blood circulation, tumor uptake, and antitumor activity of Cbz-NPs were assessed in prostatic cancer xenograft-bearing nude mice. Cbz-NPs exhibited prolonged blood circulation and greater accumulation of Cbz in tumors along with reduced toxicity compared to Cbz-Tween. Moreover, hematoxylin and eosin histopathological staining of organs revealed consistent results. The levels of blood urea nitrogen and serum creatinine in drug-treated mice showed that Cbz-NPs were less toxic than Cbz-Tween to the kidneys. In conclusion, Cbz-NPs provide a promising therapeutic for prostate cancer. PMID:27555767

Measurement of tamsulosin in human serum by liquid chromatography–tandem mass spectrometry☆

PubMed Central

Upreti, Rita; Homer, Natalie Z.M.; Naredo, Gregorio; Cobice, Diego F.; Hughes, Katherine A.; Stewart, Laurence H.; Walker, Brian R.; Andrew, Ruth

2013-01-01

A simple, sensitive and robust method to extract tamsulosin from human serum, and quantify by liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed and validated and is applicable as a measure of compliance in clinical research. Tamsulosin was extracted from human serum (100 μL) via liquid–liquid extraction with methyl tert-butyl ether (2 mL) following dilution with 0.1 M ammonium hydroxide (100 μL), achieving 99.9% analyte recovery. Internal standard, d9-finasteride, was synthesised in-house. Analyte and internal standard were separated on an Ascentis® Express C18 (100 mm × 3 mm, 2.7 μm) column using a gradient elution with mobile phases methanol and 2 mM aqueous ammonium acetate (5:95, v/v). Total run-time was 6 min. Tamsulosin was quantified using a triple quadrupole mass spectrometer operated in multi-reaction-monitoring (MRM) mode using positive electrospray ionisation. Mass transitions monitored for quantitation were: tamsulosin m/z 409 → 228 and d9-finasteride m/z 382 → 318, with the structural formulae of ions confirmed by Fourier transform ion cyclotron resonance mass spectrometry (within 10 ppm). The limit of quantitation was 0.2 ng/mL, and the method was validated in the linear range 0.2–50 ng/mL with acceptable inter- and intra-assay precision and accuracy and stability suitable for routine laboratory practice. The method was successfully applied to samples taken from research volunteers in a clinical study of benign prostatic hyperplasia. PMID:23743242

Measurement of tamsulosin in human serum by liquid chromatography-tandem mass spectrometry.

PubMed

Upreti, Rita; Homer, Natalie Z M; Naredo, Gregorio; Cobice, Diego F; Hughes, Katherine A; Stewart, Laurence H; Walker, Brian R; Andrew, Ruth

2013-07-01

A simple, sensitive and robust method to extract tamsulosin from human serum, and quantify by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated and is applicable as a measure of compliance in clinical research. Tamsulosin was extracted from human serum (100μL) via liquid-liquid extraction with methyl tert-butyl ether (2mL) following dilution with 0.1M ammonium hydroxide (100μL), achieving 99.9% analyte recovery. Internal standard, d9-finasteride, was synthesised in-house. Analyte and internal standard were separated on an Ascentis(®) Express C18 (100mm×3mm, 2.7μm) column using a gradient elution with mobile phases methanol and 2mM aqueous ammonium acetate (5:95, v/v). Total run-time was 6min. Tamsulosin was quantified using a triple quadrupole mass spectrometer operated in multi-reaction-monitoring (MRM) mode using positive electrospray ionisation. Mass transitions monitored for quantitation were: tamsulosin m/z 409→228 and d9-finasteride m/z 382→318, with the structural formulae of ions confirmed by Fourier transform ion cyclotron resonance mass spectrometry (within 10ppm). The limit of quantitation was 0.2ng/mL, and the method was validated in the linear range 0.2-50ng/mL with acceptable inter- and intra-assay precision and accuracy and stability suitable for routine laboratory practice. The method was successfully applied to samples taken from research volunteers in a clinical study of benign prostatic hyperplasia. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

Brominated flame retardant (BFRs) and Dechlorane Plus (DP) in paired human serum and segmented hair.

PubMed

Qiao, Lin; Zheng, Xiao-Bo; Yan, Xiao; Wang, Mei-Huang; Zheng, Jing; Chen, She-Jun; Yang, Zhong-Yi; Mai, Bi-Xian

2018-01-01

Brominated flame retardants (BFRs) and Dechlorane Plus (DP) were measured in both human hair and paired serum samples from a cohort of university students in South China. Segmental analysis was conducted to explore gender difference and the relationships between the hair and serum. The concentrations of total PBDEs in the hair and serum samples were in a range of 0.28-34.1ng/g dry weight (dw) and 0.16-156ng/g lipid weight (lw), respectively. Concentrations of ∑DPs (sum of the syn-DP and anti-DP isomers) in all hair samples ranged from nd-5.45ng/g dry weight. Concentrations of most PBDEs and decabromodiphenylethane (DBDPE) in distal segments (5-10cm from the scalp) were higher than those in the proximal segments (0-5cm from the scalp) (t-test, p < 0.05), which could be due to the longer exposure time of distal segments. The proximal segments exhibited a unique congener profile, more close to that in the serum rather than the distal segments of hair. An obvious gender difference was found in the levels of ∑PBDEs using integrated hair samples, while the difference disappeared when considering alone the proximal segments of hair (0-5cm from scalp) for both genders. This paper provides supplement to the current knowledge on sources of BFRs and DPs in hair and declares the importance of segmental analysis. Copyright © 2017 Elsevier Inc. All rights reserved.

Serum From Advanced Heart Failure Patients Promotes Angiogenic Sprouting and Affects the Notch Pathway in Human Endothelial Cells

PubMed Central

Pannella, Micaela; Caliceti, Cristiana; Fortini, Francesca; Aquila, Giorgio; Sega, Francesco Vieceli Dalla; Pannuti, Antonio; Fortini, Cinzia; Morelli, Marco Bruno; Fucili, Alessandro; Francolini, Gloria; Voltan, Rebecca; Secchiero, Paola; Dinelli, GiovannI; Leoncini, Emanuela; Ferracin, Manuela; Hrelia, Silvana; Miele, Lucio; Rizzo, Paola

2017-01-01

It is unknown whether components present in heart failure (HF) patients' serum provide an angiogenic stimulus. We sought to determine whether serum from HF patients affects angiogenesis and its major modulator, the Notch pathway, in human umbilical vein endothelial cells (HUVECs). In cells treated with serum from healthy subjects or from patients at different HF stage we determined: (1) Sprouting angiogenesis, by measuring cells network (closed tubes) in collagen gel. (2) Protein levels of Notch receptors 1, 2, 4, and ligands Jagged1, Delta-like4. We found a higher number of closed tubes in HUVECs treated with advanced HF patients serum in comparison with cells treated with serum from mild HF patients or controls. Furthermore, as indicated by the reduction of the active form of Notch4 (N4IC) and of Jagged1, advanced HF patients serum inhibited Notch signalling in HUVECs in comparison with mild HF patients' serum and controls. The circulating levels of NT-proBNP (N-terminal of the pro-hormone brain natriuretic peptide), a marker for the detection and evalutation of HF, were positively correlated with the number of closed tubes (r = 0.485) and negatively with Notch4IC and Jagged1 levels in sera-treated cells (r = −0.526 and r = −0.604, respectively). In conclusion, we found that sera from advanced HF patients promote sprouting angiogenesis and dysregulate Notch signaling in HUVECs. Our study provides in vitro evidence of an angiogenic stimulus arising during HF progression and suggests a role for the Notch pathway in it. PMID:26987674

Fetal bovine serum enables cardiac differentiation of human embryonic stem cells.

PubMed

Bettiol, Esther; Sartiani, Laura; Chicha, Laurie; Krause, Karl Heinz; Cerbai, Elisabetta; Jaconi, Marisa E

2007-10-01

During development, cardiac commitment within the mesoderm requires endoderm-secreted factors. Differentiation of embryonic stem cells into the three germ layers in vitro recapitulates developmental processes and can be influenced by supplements added to culture medium. Hence, we investigated the effect of fetal bovine serum (FBS) and KnockOut serum replacement (SR) on germ layers specification and cardiac differentiation of H1 human embryonic stem cells (hESC) within embryoid bodies (EB). At the time of EB formation, FBS triggered an increased apoptosis. As assessed by quantitative PCR on 4-, 10-, and 20-day-old EB, FBS promoted a faster down-regulation of pluripotency marker Oct4 and an increased expression of endodermal (Sox17, alpha-fetoprotein, AFP) and mesodermal genes (Brachyury, CSX). While neuronal and hematopoietic differentiation occurred in both supplements, spontaneously beating cardiomyocytes were only observed in FBS. Action potential (AP) morphology of hESC-derived cardiomyocytes indicated that ventricular cells were present only after 2 months of culture. However, quantification of myosin light chain 2 ventricular (mlc2v)-positive areas revealed that mlc2v-expressing cardiomyocytes could be detected already after 2 weeks of differentiation, but not in all beating clusters. In conclusion, FBS enabled cardiac differentiation of hESC, likely in an endodermal-dependent pathway. Among cardiac cells, ventricular cardiomyocytes differentiated over time, but not as the predominant cardiac cell subtype.

Determining optimal therapy of dogs with chronic enteropathy by measurement of serum citrulline

PubMed Central

Gerou‐Ferriani, Magda; Allen, Rhiannon; Noble, Peter‐John M.; German, Alexander J.; Caldin, Marco

2018-01-01

Background Serum concentration of citrulline is a useful biomarker in human intestinal disease and indicates globally reduced enterocyte mass and absorptive function in various disease states. Objectives To determine whether serum citrulline concentration is a biomarker in chronic enteropathy (CE) in dogs, to provide useful information regarding optimal treatment or to predict outcome. Animals Seventy‐four dogs with CE and 83 breed‐ and age‐matched hospital controls with no clinical signs of intestinal disease. Methods Retrospective study. Outcome was determined and dogs were categorized by response to treatment as having food‐responsive enteropathy (FRE), antibiotic‐responsive diarrhea (ARD), or idiopathic inflammatory bowel disease (IBD). Disease severity was quantified by the CIBDAI scoring index. Results Serum citrulline concentration did not differ between dogs with CE (median, 8.4 µg/mL, 5th‐95th percentile 2.0‐19.6) and controls (median, 8.1 µg/mL, 5th‐95th percentile 2.2‐19.7, P = .91). Serum citrulline concentration was similar between dogs with FRE (median, 9.1 µg/mL, 5th‐95th percentile 2.0‐18.9), ARD (median, 13.0 µg/mL, 5th‐95th percentile 1.6‐19.2), IBD (median, 8.4 µg/mL, 5th‐95th percentile 2.1‐21.0; P = .91). Serum citrulline did not correlate to CIBDAI or to serum albumin concentration. Conclusions and Clinical Importance In our study, serum citrulline concentration was not associated with efficacy of treatment or outcome in dogs with CE. PMID:29663515

Simultaneous determination of neonicotinoid insecticides in human serum and urine using diatomaceous earth-assisted extraction and liquid chromatography-tandem mass spectrometry.

PubMed

Yamamuro, Tadashi; Ohta, Hikoto; Aoyama, Mika; Watanabe, Daisuke

2014-10-15

A rapid and sensitive analytical method was developed for simultaneous determination of eight neonicotinoid insecticides (acetamiprid, clothianidin, dinotefuran, flonicamid, imidacloprid, nitenpyram, thiacloprid and thiamethoxam) and three specific metabolites of acetamiprid (N-desmethylacetamiprid, 5-(N-acetyl-N-methylaminomethyl)-2-chloropyridine and 5-(N-acetylaminomethyl)-2-chloropyridine) in human serum and urine. A diatomaceous earth-assisted extraction using Extrelut NT3 column with chloroform/2-propanol (3:1, v/v) as eluent was selected for the single step cleanup procedure for all the target compounds. Qualitative and quantitative analyses were conducted by liquid chromatography-tandem mass spectrometry with multiple reaction monitoring mode. The limits of detection and the limits of quantification of eleven compounds were in the ranges of 0.1-0.2ng/mL and 0.5-10ng/mL for serum, 0.1-1ng/mL and 1-10ng/mL for urine, respectively. The extraction recoveries were between 80.9% and 101.8% for serum samples, 91.9% and 106% for urine samples. The intra-day RSDs and the inter-day RSDs were less than 11.5% and 13.2% for serum, less than 8.3% and 8.8% for urine. The proposed procedure will be suitable for forensic investigations of human poisoning cases with neonicotinoid insecticides. This is the first report of simultaneous determination of eight neonicotinoids in serum and urine samples. Copyright © 2014. Published by Elsevier B.V.

CNS germinoma with elevated serum human chorionic gonadotropin level: Clinical characteristics and treatment outcome

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

Ogino, Hiroyuki; Shibamoto, Yuta; Takanaka, Tsuyoshi

2005-07-01

Purpose: The prognostic significance of human chorionic gonadotropin (HCG) level in central nervous system germinoma remains controversial. The purpose of this study was to compare clinical characteristics and prognosis of germinoma patients with normal and high HCG titers in the serum. Methods and Materials: We undertook a multi-institutional retrospective analysis of 103 patients with central nervous system germinoma whose serum HCG and/or {beta}-HCG level had been measured before treatment between 1984 and 2002. All patients had been treated with radiation therapy either alone (n = 66) or in combination with chemotherapy (n = 37) with a median dose of 47.8more » Gy. Results: HCG and/or {beta}-HCG level in the serum was high in 39% of all patients. The proportion of HCG-producing tumors was higher in the lesions at the basal ganglia than in the lesions at the other sites. No correlation was found between tumor size and HCG level, but there seemed to be a weak correlation between size and {beta}-HCG. The 5- and 10-year survival rates were 96% and 94%, respectively, in both patient groups with normal and high HCG (p = 0.99). The 5- and 10-year relapse-free survival rates were 87% and 82%, respectively, in patients with normal HCG level and were both 87% in patients with high HCG (p = 0.74). Also, no other patient-, tumor-, or treatment-related factors seemed to influence the prognosis of the patients. Conclusion: Serum HCG level does not seem to influence patient prognosis when treated with sufficient doses of radiation. Relationship between tumor size and site and HCG level should be investigated further.« less

Renal targeted delivery of triptolide by conjugation to the fragment peptide of human serum albumin.

PubMed

Yuan, Zhi-xiang; Wu, Xiao-juan; Mo, Jingxin; Wang, Yan-li; Xu, Chao-qun; Lim, Lee Yong

2015-08-01

We have previously demonstrated that peptide fragments (PFs) of the human serum albumin could be developed as potential renal targeting carriers, in particular, the peptide fragment, PF-A299-585 (A299-585 representing the amino acid sequence of the human serum albumin). In this paper, we conjugated triptolide (TP), the anti-inflammatory Chinese traditional medicine, to PF-A299-585 via a succinic acid spacer to give TPS-PF-A299-585 (TP loading 2.2% w/w). Compared with the free TP, TPS-PF-A299-585 exhibited comparable anti-inflammatory activity in the lipopolysaccharide stimulated MDCK cells, but was significantly less cytotoxic than the free drug. Accumulation of TPS-PF-A299-585 in the MDCK cells in vitro and in rodent kidneys in vivo was demonstrated using FITC-labeled TPS-PF-A299-585. Renal targeting was confirmed in vivo in a membranous nephropathic (MN) rodent model, where optical imaging and analyses of biochemical markers were combined to show that TPS-PF-A299-585 was capable of alleviating the characteristic symptoms of MN. The collective data affirm PF-A299-585 to be a useful carrier for targeting TP to the kidney. Copyright © 2015 Elsevier B.V. All rights reserved.

Alteration of methotrexate binding to human serum albumin induced by oxidative stress. Spectroscopic comparative study

NASA Astrophysics Data System (ADS)

Maciążek-Jurczyk, M.; Sułkowska, A.; Równicka-Zubik, J.

2016-01-01

Changes of oxidative modified albumin conformation by comparison of non-modified (HSA) and modified (oHSA) human serum albumin absorption spectra, Red Edge Excitation Shift (REES) effect and fluorescence synchronous spectra were investigated. Studies of absorption spectra indicated that changes in the value of absorbance associated with spectral changes in the region from 200 to 250 nm involve structural alterations related to variations in peptide backbone conformation. Analysis of the REES effect allowed for the observation of changes caused by oxidation in the region of the hydrophobic pocket containing the tryptophanyl residue. Synchronous fluorescence spectroscopy confirmed changes of the position of the tryptophanyl and tyrosil residues fluorescent band. Effect of oxidative stress on binding of methotrexate (MTX) was investigated by spectrofluorescence, UV-VIS and 1HNMR spectroscopy. MTX caused the fluorescence quenching of non-modified (HSA) and modified (oHSA) human serum albumin molecule. The values of binding constants, Hill's coefficients and a number of binding sites in the protein molecule in the high affinity binding site were calculated for the binary MTX-HSA and MTX-oHSA systems. For these systems, qualitative analysis in the low affinity binding sites was performed with the use of the 1HNMR technique.

Serum HSP70

PubMed Central

Dutta, Sudhir K.; Girotra, Mohit; Singla, Montish; Dutta, Anand; Stephen, F. Otis; Nair, Padmanabhan P.; Merchant, Nipun B.

2014-01-01

Objectives Heat shock protein 70 (HSP70) is overexpressed in human pancreatic cancer cell lines. To determine if serum HSP70 levels are elevated in patients with pancreatic cancer and can function as a biomarker for early detection of pancreatic cancer. Methods Study subjects were divided into 3 groups: histologically proven pancreatic cancer (PC; n = 23), chronic pancreatitis (CP; n = 12), and matched normal control subjects (C; n = 10). Serum HSP70 levels were determined using a novel immunoelectrophoresis method developed and validated by the authors. Significance of difference between the groups was analyzed with analysis of variance (ANOVA). Receiver operating characteristic (ROC) curve analysis was performed to discriminate patients with pancreatic cancer from normal controls. Results The mean ± SE serum HSP70 levels in the PC, CP, and C groups were 1.68 ± 0.083 ng/mL, 0.40 ± 0.057 ng/mL, and 0.04 ng/mL, respectively. Serum HSP70 levels in the PC group were significantly higher compared with either the CP or C groups (P < 0.01). The sensitivity and specificity of elevated serum HSP70 in the PC group was 74% and 90%, respectively. Conclusions Serum HSP70 levels are significantly increased in patients with pancreatic cancer and may be useful as an additional biomarker for the detection of pancreatic cancer. PMID:22158074

[Determination of ferulic acid absorbed into human serum from oral decoction of rhizoma chuanxiong and pharmacokinetic study of ferulic acid].

PubMed

Sun, S; Huang, X; Zhang, L; Ren, P; Chang, Z

2001-10-01

To improve the HPLC method for determining ferulic acid(FA) in the human serum and to research FA clinical pharmacokinetics. Serum concentrations of FA were determined by HPLC using methanol-water-acetic acid (40:59.7:0.3, v/v) as the mobile phase and the column packed with ODS (150 mm x 4.6 mm, 5 microns) as a fixed phase and the flow rate was 1.0 ml/min. FA was detected at 320 nm wave length. The internal standard was coumarin. The serum samples were treated by the water-boiling method. Peak of FA in serum was recognized by photodiode-array detection. FA and internal standard were separated completely under the condition described as above. FA was linear in the range of (40.16-8032 ng/ml) (r = 0.9975). The CV was less than 10%, the average recovery was 100.3%, and the limit concentration in serum was 25.1 ng/ml. PK was in accordance with open bicameral model. This method can be applied to clinical pharmacokinetic study of FA.

A Human Serum-Based Enzyme-Free Continuous Glucose Monitoring Technique Using a Needle-Type Bio-Layer Interference Sensor

PubMed Central

Seo, Dongmin; Paek, Sung-Ho; Oh, Sangwoo; Seo, Sungkyu; Paek, Se-Hwan

2016-01-01

The incidence of diabetes is continually increasing, and by 2030, it is expected to have increased by 69% and 20% in underdeveloped and developed countries, respectively. Therefore, glucose sensors are likely to remain in high demand in medical device markets. For the current study, we developed a needle-type bio-layer interference (BLI) sensor that can continuously monitor glucose levels. Using dialysis procedures, we were able to obtain hypoglycemic samples from commercial human serum. These dialysis-derived samples, alongside samples of normal human serum were used to evaluate the utility of the sensor for the detection of the clinical interest range of glucose concentrations (70–200 mg/dL), revealing high system performance for a wide glycemic state range (45–500 mg/dL). Reversibility and reproducibility were also tested over a range of time spans. Combined with existing BLI system technology, this sensor holds great promise for use as a wearable online continuous glucose monitoring system for patients in a hospital setting. PMID:27669267

A Human Serum-Based Enzyme-Free Continuous Glucose Monitoring Technique Using a Needle-Type Bio-Layer Interference Sensor.

PubMed

Seo, Dongmin; Paek, Sung-Ho; Oh, Sangwoo; Seo, Sungkyu; Paek, Se-Hwan

2016-09-24

The incidence of diabetes is continually increasing, and by 2030, it is expected to have increased by 69% and 20% in underdeveloped and developed countries, respectively. Therefore, glucose sensors are likely to remain in high demand in medical device markets. For the current study, we developed a needle-type bio-layer interference (BLI) sensor that can continuously monitor glucose levels. Using dialysis procedures, we were able to obtain hypoglycemic samples from commercial human serum. These dialysis-derived samples, alongside samples of normal human serum were used to evaluate the utility of the sensor for the detection of the clinical interest range of glucose concentrations (70-200 mg/dL), revealing high system performance for a wide glycemic state range (45-500 mg/dL). Reversibility and reproducibility were also tested over a range of time spans. Combined with existing BLI system technology, this sensor holds great promise for use as a wearable online continuous glucose monitoring system for patients in a hospital setting.

Specific IgA and IgG antibodies in paired serum and breast milk samples in human strongyloidiasis.

PubMed

Mota-Ferreira, Daniela M L; Gonçalves-Pires, Maria do Rosário F; Júnior, Alvaro Ferreira; Sopelete, Mônica C; Abdallah, Vânia O S; Costa-Cruz, Julia M

2009-02-01

Strongyloidiasis, caused by the nematode Strongyloides stercoralis, is one of the major worldwide parasitic infections in humans. Breastfeeding may offer a potential protection against this infection. Feces, serum and milk samples were obtained from 90 lactating women from Clinical Hospital of Universidade Federal de Uberlândia, Brazil. The fecal samples were collected for parasitological diagnosis and the serum and milk samples were examined for specific S. stercoralis IgA and IgG antibodies using the indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA). Fecal examination showed that the rate of prevalence of S. stercoralis infection in the lactating women was 4.4%. IFAT manifested a 16.7% positivity rate for specific IgA antibody in serum and a 28.9% rate in milk samples; specific IgG was 41.1% in serum and 25.5% in milk samples. According to ELISA the positivity rate for specific IgA antibody was 21.1% in serum and 42.2% in milk samples; specific IgG was 40% in serum and 18.9% in milk samples. In serum samples, these immunological tests showed a concurrence of 91.1% and 94.4%, respectively, in detecting specific IgA and IgG antibodies. In milk samples, they showed a concurrence of 70% and 78.9%, respectively, in detecting specific IgA and IgG antibodies. There was a statistically significant difference between concordant and discordant results of immunological tests (P<0.0001). IFAT and ELISA highly concurred in their detection of specific S. stercoralis IgA and IgG antibodies in serum and in milk samples reconfirming prior studies that the serological method is a complement to the direct diagnosis of the parasite, and suggesting that immunological methods using milk samples can also be helpful. Furthermore, in endemic areas, infants may acquire antibodies to S. stercoralis from breast milk, possibly, contributing to the enhancement of specific mucosal immunity against this parasite.

Ternary mixed-mode silica sorbent of solid-phase extraction for determination of basic, neutral and acidic drugs in human serum.

PubMed

Jin, Shupei; Qiao, Yinghua; Xing, Jun

2018-06-01

In this study, a ternary mixed-mode silica sorbent (TMSS) with octamethylene, carboxyl, and amino groups was prepared via Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click reaction and a subsequent reduction of azide to primary amine. While used in solid-phase extraction (SPE), the retention behavior of TMSS towards a total of nine kinds of basic, neutral, and acidic drugs was investigated in detail. The results revealed that hydrophobic, ion-exchange interaction, and electrostatic repulsion between TMSS and the analytes were closely related to the retention behavior of TMSS. Besides, the log K ow value of the analyte was also a factor influencing the retention behavior of analytes on TMSS. The nine analytes could be retained by TMSS simultaneously and then, were eluted into two fractions according to the acid-base property of the analytes for further determinations. The acidic and neutral analytes were in one fraction, and the basic ones in the other fraction. When used to treat the human serum spiked with the nine drugs, TMSS offered higher recoveries than BakerBond CBA and comparable recoveries to Oasis WCX. It should be noted TMSS had better purifying capability for human serum than Oasis WCX. Under the optimized SPE conditions, a method of SPE hyphenated to high-performance liquid chromatography-ultraviolet detection (HPLC-UV) for determination of the basic, neutral, and acidic drugs spiked in human serum was established. For the nine drugs, the linear ranges were all between 5.0 and 1000 μg L -1 with correlation coefficients (R 2 ) above 0.9990, and the limits of detection (LODs) were in the range of 0.8-2.3 μg L -1 . The intra-day and inter-day relative standard deviations (RSDs) were less than 5.3 and 8.8%, respectively. Graphical abstract Treating drugs in human serum by SPE with ternary mixed-mode silica sorbent.

[Enrichment of glycoproteins in human serum using concanavalin A-functionalized magnetic nanoparticles and identification by mass spectrometry].

PubMed

Li, Feng; Kang, Jingwu

2014-04-01

Biomedical sciences, and in particular biomarker research, demand efficient glycoprotein enrichment platforms. Herein novel magnetic nanoparticles with an average size around 135 nm in diameter were prepared for the enrichment of glycoproteins in human serum. The prepared magnetic nanoparticles possessed uniform core/shell/shell structure which was composed of 8 nm magnetite internal core and double layers consisting of silica and poly glycidyl methacrylate (GMA). The latter was constructed by seed polymerization. Modified by a polyethylene hydrophilic linker, it made the surfaces of the magnetic nanoparticles highly hydrophilic so as to reduce the nonspecific adsorption of proteins. We examined affinity purification of glycoprotein in diluted human serum using our prepared magnetic nanoparticles with immobilization of concanavalin A (MNP @ ConA). The enriched proteins were reduced, alkylated and digested with trypsin. These peptides then were separated by offline two-dimensional chromatography. Protein identification was realized with nano-high performance liquid chromatography-orbitrap mass spectrometry. A total of 80 proteins were identified, among them 76 proteins were found to be glycoproteins by use of bioinformatic tools. /3-2-Glycoprotein 1 present in serum at low mass concentration around 0.000 01 g/L was also identified. This demonstrates the capability of magnetic nanoparticle for recovering minute amounts of glycoproteins from a fluid exhibiting a dynamic concentration range more than 12 orders of magnitude. Overall, MNP @ ConA has been proven to be an efficient alternative to currently available immobilization supports.

A patient with progressive myelopathy and antibodies to human T-cell leukemia virus type I and human immunodeficiency virus type 1 in serum and cerebrospinal fluid.

PubMed

Aboulafia, D M; Saxton, E H; Koga, H; Diagne, A; Rosenblatt, J D

1990-04-01

A 52-year-old human immunodeficiency virus type 1-seropositive bisexual black man was evaluated at UCLA because of the recent onset of progressive lower-extremity weakness. Initial neurologic examination showed that the patient's distal weakness was greater than his proximal weakness, with bilateral foot drop and electrophysiologic evidence of denervation in the distal lower extremities. Magnetic resonance imaging of the brain and spinal cord disclosed no abnormalities. Subsequent neurologic evaluation 8 months later showed a myelopathy, with progression of lower-extremity weakness, spasticity, and flexor spasms, and urinary incontinence, as well as the peripheral neuropathy noted previously. A second magnetic resonance imaging scan of the brain showed patchy foci of increased signal intensity in white matter and cortex, with mild generalized cerebral and cerebellar atrophy and no lesions in the spinal cord. Specimens of the patient's serum and cerebrospinal fluid contained antibodies to human immunodeficiency virus type 1. Additionally, specimens of his serum and cerebrospinal fluid were tested for antibody to human T-cell leukemia virus type I by Western blotting and radioimmunoprecipitation, and found to be positive for human T-cell leukemia virus type I gag, env, and tax antibodies. The primary cause of severe myelopathy in this patient may be infection with human T-cell leukemia virus type I rather than with human immunodeficiency virus type 1. Treatment with prednisolone resulted in improvement of the lower-extremity weakness, reduction in flexor spasms, and slower but significant improvement in urinary symptoms. Patients who are infected with human immunodeficiency virus type 1 and have unusual motor findings should be tested for concomitant human T-cell leukemia virus type I infection.

Probable levetiracetam-related serum alkaline phosphatase elevation

PubMed Central

2012-01-01

Background Levetiracetam (LEV) is an antiepileptic drug with a favorable tolerability and safety profile with little or no effect on liver function. Case presentation Here, we reported an epileptic pediatric patient who developed a significant elevation in serum alkaline phosphatase level (ALP) during LEV monotherapy. Moreover, the serum ALP level was surprisingly decreased to normal after LEV discontinuation. The Naranjo Adverse Drug Reaction Probability Scale score was 6, indicating firstly LEV was a probable cause for the increased serum ALP. Conclusions Cautious usage and concerns of the LEV-associated potential ALP elevation should be considered when levetiracetam is prescribed to epilepsy patients, especially pediatric patients. PMID:22994584

Serum profiling of healthy aging identifies phospho- and sphingolipid species as markers of human longevity.

PubMed

Montoliu, Ivan; Scherer, Max; Beguelin, Fiona; DaSilva, Laeticia; Mari, Daniela; Salvioli, Stefano; Martin, Francois-Pierre J; Capri, Miriam; Bucci, Laura; Ostan, Rita; Garagnani, Paolo; Monti, Daniela; Biagi, Elena; Brigidi, Patrizia; Kussmann, Martin; Rezzi, Serge; Franceschi, Claudio; Collino, Sebastiano

2014-01-01

As centenarians well represent the model of healthy aging, there are many important implications in revealing the underlying molecular mechanisms behind such successful aging. By combining NMR metabonomics and shot-gun lipidomics in serum we analyzed metabolome and lipidome composition of a group of centenarians with respect to elderly individuals. Specifically, NMR metabonomics profiling of serum revealed that centenarians are characterized by a metabolic phenotype distinct from that of elderly subjects, in particular regarding amino acids and lipid species. Shot- gun lipidomics approach displays unique changes in lipids biosynthesis in centenarians, with 41 differently abundant lipid species with respect to elderly subjects. These findings reveal phospho/sphingolipids as putative markers and biological modulators of healthy aging, in humans. Considering the particular actions of these metabolites, these data are suggestive of a better counteractive antioxidant capacity and a well-developed membrane lipid remodelling process in the healthy aging phenotype.

Animal models to assess the therapeutic efficacy of human serum and serum-converted platelet lysates for dry eye syndrome: Seeing is believing.

PubMed

Tseng, Ching-Li; Seghatchian, Jerard; Burnouf, Thierry

2015-08-01

There is much interest in the clinical use of serum-converted human blood or platelet concentrates in regenerative medicine, most specifically for wound healing and tissue repair of soft and hard tissues. The scientific rationale supporting the clinical efficacy of these preparations is based on the expectation that their physiological mixture of natural growth factors can orchestrate cell expansion and differentiation in vivo. However, a lack of standardization and regulatory oversight of these blood materials maintain a perception of uncertainty in the scientific and medical community on the value of these preparations for some clinical indications. More studies are needed to understand the mechanism of action underlying their expected efficacy and standardize their use, and benefit from their biological versatility. One application of serum is as eye drop for treating dry eye syndrome (DES), a multifactorial disease of the ocular surface, which has a prevalence of 15% of more in the population. DES can lead to chronic inflammation of the ocular surface, surface impairment in the cornea and conjunctiva, and, in patients with Sjogren syndrome, result in a disruption of the ocular surface epithelium. Objective experimental assessment of safety and efficacy of serum eye drops can help establish scientific rationale in optimal product composition and use. This can be achieved, first, through cell cultures with relevant cell models, before considering, then, animal studies using DES animal models. Several models have been evaluated and are reported in this concise review. The model we have developed encompasses the use of rabbits, where their eyes are treated with 0.1% benzalkonium chloride (BAC), a common preservative in ophthalmic agents, 3 times daily for 4 weeks. This relatively mild treatment results in moderate DES pathology, with a stable shortage of tear secretion throughout a 7-week study period, which we found suitable for assessing efficacy of serum eye

Spectroscopic analysis of the impact of oxidative stress on the structure of human serum albumin (HSA) in terms of its binding properties

NASA Astrophysics Data System (ADS)

Maciążek-Jurczyk, M.; Sułkowska, A.

2015-02-01

Oxygen metabolism has an important role in the pathogenesis of rheumatoid arthritis (RA). Reactive oxygen species (ROS) are produced in the course of cellular oxidative phosphorylation and by activated phagocytic cells during oxidative bursts, exceed the physiological buffering capacity and result in oxidative stress. ROS result in oxidation of serum albumin, which causes a number of structural changes in the spatial structure, may influence the binding and cause significant drug interactions, particularly in polytherapy. During the oxidation modification of amino acid residues, particularly cysteine and methionine may occur. The aim of the study was to investigate the influence of oxidative stress on human serum albumin (HSA) structure and evaluate of possible alterations in the binding of the drug to oxidized human serum albumin (oHSA). HSA was oxidized by a chloramine-T (CT). CT reacts rapidly with sulfhydryl groups and at pH 7.4 the reaction was monitored by spectroscopic techniques. Modification of free thiol group in the Cys residue in HSA was quantitatively determined by the use of Ellman's reagent. Changes of albumin conformation were examined by comparison of modified (oHSA) and nonmodified human serum albumin (HSA) absorption spectra, emission spectra, red-edge shift (REES) and synchronous spectroscopy. Studies of absorption spectra indicated that changes in the value of absorbance associated with spectral changes in the region of 200-250 nm involve structural alterations in peptide backbone conformation. Synchronous fluorescence spectroscopy technique confirmed changes of position of tryptophanyl and tyrosyl residues fluorescent band caused by CT. Moreover analysis of REES effect allowed to observe structural changes caused by CT in the region of the hydrophobic pocket containing the tryptophanyl residue. Effect of oxidative stress on binding of anti-rheumatic drugs, sulfasalazine (SSZ) and sulindac (SLD) in the high and low affinity binding sites was

Simple method for determining human serum 2,2-diphenyl-1-picryl-hydrazyl (DPPH) radical scavenging activity - possible application in clinical studies on dietary antioxidants.

PubMed

Chrzczanowicz, Jacek; Gawron, Anna; Zwolinska, Anna; de Graft-Johnson, Jeffrey; Krajewski, Wojciech; Krol, Maciej; Markowski, Jaroslaw; Kostka, Tomasz; Nowak, Dariusz

2008-01-01

2,2-Diphenyl-1-picryl-hydrazyl (DPPH) radical decomposition in alcohol solution is widely used, characterizing plant antioxidants that can rise in serum after fruit and vegetable intake. However, this test failed reproducible results with serum due to protein precipitation. We describe the application of serum deproteinization with acetonitrile relating to the DPPH test. Assay sensitivity, linearity, repeatability and storage effect were determined in serum samples deproteinized with an equal volume of acetonitrile. Associations between the DPPH test and the ferric reducing ability of serum (FRAP) method, measuring total antioxidant potential, were evaluated in sera from 78 healthy non-smoking men. The effect of a single ingestion of 1 L of cloudy apple juice on the serum DPPH radical scavenging activity in healthy volunteers was also investigated. Assay linearity was within 5-25 microL (r=0.99, p<0.01). With 25 microL-deproteinized serum, coefficient of variation was 4.2% and detection limit was 0.5% of the initial amount of decomposed DPPH radical over 30 min incubation. There was no sera activity decrease over 14 days storage at -20 degrees C. Mean values of DPPH radical scavenging activity and FRAP obtained in human serum were 11.2+/-3.3% and 382.0+/-88.1 micromol/L, respectively. A positive significant linear correlation was observed between these two methods (r=0.42, p<0.01). Serum supplementation with 50 micromol/L of catechin, gallic acid, ascorbic acid or uric acid enhanced DPPH test results. One brisk serving of 1 L of apple juice caused a significant increment of serum DPPH radical scavenging activity (1.9+/-1.9%, p<0.01) in 12 healthy subjects 1 h after juice ingestion. Applicability of the DPPH test to deproteinized serum with acetonitrile revealed numerous advantages, validating its practicability, simplicity and cost effectiveness as a tool in the estimation of antioxidant status in humans.

Menaquinones content of human serum and feces

USDA-ARS?s Scientific Manuscript database

Bacterially-synthesized menaquinones (MKn) may contribute to vitamin K (VK) nutriture. There are limited data on interindividual variability in endogenous MK synthesis and its relation to circulating forms of VK. Serum and fecal VK concentrations were assessed in 13 healthy adults (45-65 yr) consumi...

Low serum zinc is associated with elevated risk of cadmium nephrotoxicity

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

Lin, Yu-Sheng, E-mail: Lin.Yu-Sheng@epa.gov; Ho, Wen-Chao; Caffrey, James L.

Background: Despite animal evidence suggests that zinc modulates cadmium nephrotoxicity, limited human data are available. Objective: To test the hypothesis that low serum zinc concentrations may increase the risk of cadmium-mediated renal dysfunction in humans. Methods: Data from 1545 subjects aged 20 or older in the National Health and Nutrition Examination Survey (NHANES), 2011–2012 were analyzed. Renal function was defined as impaired when estimated glomerular filtration rate (eGFR) fell below 60 ml/min/1.73 m{sup 2} and/or the urinary albumin-to-creatinine ratio surpassed 2.5 in men and 3.5 mg/mmol in women. Results: Within the study cohort, 117 subjects had reduced eGFR and 214more » had elevated urinary albumin. After adjusting for potential confounders, subjects with elevated blood cadmium (>0.53 μg/L) were more likely to have a reduced eGFR (odds ratio [OR]=2.21, 95% confidence interval [CI]: 1.09–4.50) and a higher urinary albumin (OR=2.04, 95% CI: 1.13–3.69) than their low cadmium (<0.18 μg/L) peers. In addition, for any given cadmium exposure, low serum zinc is associated with elevated risk of reduced eGFR (OR=3.38, 95% CI: 1.39–8.28). A similar increase in the odds ratio was observed between declining serum zinc and albuminuria but failed to reach statistical significance. Those with lower serum zinc/blood cadmium ratios were likewise at a greater risk of renal dysfunction (p<0.01). Conclusions: This study results suggest that low serum zinc concentrations are associated with an increased risk of cadmium nephrotoxicity. Elevated cadmium exposure is global public health issue and the assessment of zinc nutritional status may be an important covariate in determining its effective renal toxicity. - Highlights: • Blood cadmium was associated with increased risk of nephrotoxicity. • Low serum zinc may exacerbate risk of cadmium-mediated renal dysfunction. • Both zinc deficiency and elevated cadmium exposure are global public health issues

Early serum human chorionic gonadotropin (hCG) trends after medication abortion.

PubMed

Pocius, Katherine D; Maurer, Rie; Fortin, Jennifer; Goldberg, Alisa B; Bartz, Deborah

2015-06-01

Despite increased reliance on human chorionic gonadotropin (hCG) for early pregnancy monitoring, there is limited information about hCG trends soon after medication abortion. The purpose of this study was to determine if there is a predictable decline in serum hCG values shortly after medication abortion. This is a retrospective study of women with early intrauterine pregnancies who underwent medication abortion with mifepristone and misoprostol and had a serum hCG level on Day 1 (day of mifepristone) and a repeat value on Day 2 to 6. The percent hCG decline was calculated from baseline to repeat measure, with repeat values from the same patient accounted for through repeated measure analysis of variance. Eighty-eight women with a mean gestational age of 5.5 weeks and median baseline hCG of 5220 IU met study criteria over a 3-year period. The mean decline (±SD) in hCG from the Day 1 baseline value was 56.9%±29.5% on Day 3, 73.5%±38.6% on Day 4, 86.1%±8.8% on Day 5, and 92.9%±3.4% on Day 6. Eighty-two women (93% of the cohort) had a complete abortion without further intervention. The least square means hCG decline among these women was 57.6% [95% confidence interval (CI): 50.3-64.9%] on Day 3, 78.9% (95% CI: 75.0-82.8%) on Day 4 and 86.2% (95% CI: 81.3-91.1%) on Day 5. There is a rapid decline in serum hCG within the first few days after early medication abortion. Further research is needed to delineate how soon after medication abortion this decline may be specific enough to confirm abortion completion. This study provides the largest cohort of patients followed with serial hCG values in the first few days after medication abortion. Our findings demonstrate the trend in hCG decline in this population, which may be predictable by Day 5. Copyright © 2015 Elsevier Inc. All rights reserved.

Quantification of a biomarker of acetaminophen protein adducts in human serum by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry: clinical and animal model applications.

PubMed

Cook, Sarah F; King, Amber D; Chang, Yan; Murray, Gordon J; Norris, Hye-Ryun K; Dart, Richard C; Green, Jody L; Curry, Steven C; Rollins, Douglas E; Wilkins, Diana G

2015-03-15

The aims of this study were to develop, validate, and apply a high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) method for quantification of protein-derived 3-(cystein-S-yl)-acetaminophen (APAP-Cys) in human serum. Formation of acetaminophen (APAP) protein adducts is thought to be a critical, early event in the development of APAP-induced hepatotoxicity, and quantification of these protein adducts in human serum represents a valuable tool for assessment of APAP exposure, metabolism, and toxicity. In the reported procedure, serum samples were first dialyzed or passed through gel filtration columns to remove APAP-Cys not covalently bound to proteins. Serum eluates were then subjected to enzymatic protease digestion to liberate protein-bound APAP-Cys. Norbuprenorphine-D3 was utilized as an internal standard (IS). APAP-Cys and IS were recovered from digested serum by protein precipitation with acetonitrile, and sample extracts were analyzed by HPLC-ESI-MS/MS. The method was validated by assessment of intra- and inter-assay accuracy and imprecision on two different analytical instrument platforms. APAP-Cys could be accurately quantified from 0.010 to 10μM, and intra- and inter-assay imprecision were <15% on both analytical instruments. APAP-Cys was stable in human serum for three freeze-thaw cycles and for 24h at ambient temperature. Extracted samples were stable when stored in refrigerated autosamplers for the typical duration of analysis or when stored at -20°C for six days. Results from process efficiency and matrix effect experiments indicated adequate recovery from human serum and insignificant ion suppression or enhancement. The utility and sensitivity of the reported procedure were illustrated by analysis of clinical samples collected from subjects taking chronic, therapeutic doses of APAP. Applicability to other biological matrices was also demonstrated by measurement of protein-derived APAP-Cys in plasma

Early maternal serum ß-human chorionic gonadotropin (ß-hCG) levels and sex-related growth difference of IVF embryos.

PubMed

Esh-Broder, Efrat; Oron, Galia; Son, Weon-Young; Holzer, Hananel; Tulandi, Togas

2015-10-01

Maternal serum ß-human chorionic gonadotropin (ß-hCG) represents the trophoblastic cell mass and is an indirect measurement of embryo development at early implantation stage. Studies in animals and human embryos detected sex-related growth differences (SRGD) in favour of male embryos during the pre-implantation period. The purpose of our study was to correlate SRGD and maternal serum ß-hCG at 16 days after embryo transfer. We retrospectively analysed all (fresh and frozen) non-donor, single embryo transfers (SET), elective and not elective, that were performed between December 2008 and December 2013. We included ß-hCG values from day 16 after oocyte collection of pregnancies resulting in live birth. Neonatal gender was retrieved from patient files. Male and female embryos were further grouped to cleavage and blastocyst stage transfers. Regression analysis for confounding variables included maternal age, maternal body mass index (BMI), use of micromanipulation (ICSI), embryo quality (grade), assisted hatching, day of transfer and fresh or frozen embryo transfer. Seven hundred eighty-six non-donor SETs resulted in live birth. After including only day 16 serum ß-hCG results, 525 SETs were analysed. Neonatal gender was available for 522 cases. Mean maternal serum ß-hCG levels were similar, 347 ± 191 IU/L in the male newborn group and 371 ± 200 IU/L in the female group. The difference between ß-hCG levels remained insignificant after adjusting for confounding variables. Early maternal ß-hCG levels after embryo transfers did not represent SRGD in our study.

Characterization of Insulin Degrading Enzyme and other Aβ Degrading Proteases in Human Serum: a Role in Alzheimer’s disease?

PubMed Central

Liu, Zhiheng; Zhu, Haihao; Fang, Guang Guang; Walsh, Kathryn; Mwamburi, Maya; Wolozin, Benjamin; Abdul-Hay, Same O.; Ikezu, Tsuneya; Lessring, Malcolm A.; Qiu, Wei Qiao

2013-01-01

Sporadic Alzheimer’s disease (AD) patients have low amyloid-β peptide (Aβ) clearance in the central nervous system (CNS). The peripheral Aβ clearance may also be important but its role in AD remains unclear. We aimed to study the Aβ degrading proteases including insulin degrading enzyme (IDE), angiotensin converting enzyme (ACE) and others in blood. Using the fluorogenic substrate V—a substrate of IDE and other metalloproteases, we showed that human serum degraded the substrate V, and the activity was inhibited by adding increasing dose of Aβ. The existence of IDE activity was demonstrated by the inhibition of insulin, amylin or EDTA, and further confirmed by immunocapture of IDE using monoclonal antibodies. The involvement of ACE was indicated by the ability of the ACE inhibitor, lisinopril, to inhibit the substrate V degradation. To test the variations of substrate V degradation in humans, we used serum samples from a homebound elderly population with cognitive diagnoses. Compared with the elderly who had normal cognition, those with probable AD and amnestic mild cognitive impairment (amnestic MCI) had lower peptidase activities. Probable AD or amnestic MCI as an outcome remained negatively associated with serum substrate V degradation activity after adjusting for the confounders. The elderly with probable AD had lower serum substrate V degradation activity compared with those who had vascular dementia. The blood proteases mediating Aβ degradation may be important for the AD pathogenesis. More studies are needed to specify each Aβ degrading protease in blood as a useful biomarker and a possible treatment target for AD. PMID:22232014

Interaction between Saikosaponin D, Paeoniflorin, and Human Serum Albumin.

PubMed

Liang, Guo-Wu; Chen, Yi-Cun; Wang, Yi; Wang, Hong-Mei; Pan, Xiang-Yu; Chen, Pei-Hong; Niu, Qing-Xia

2018-01-27

Saikosaponin D (SSD) and paeoniflorin (PF) are the major active constituents of Bupleuri Radix and Paeonia lactiflora Pall , respectively, and have been widely used in China to treat liver and other diseases for many centuries. We explored the binding of SSD/PF to human serum albumin (HSA) by using fluorospectrophotometry, circular dichroism (CD) and molecular docking. Both SSD and PF produced a conformational change in HSA. Fluorescence quenching was accompanied by a blue shift in the fluorescence spectra. Co-binding of PF and SSD also induced quenching and a conformational change in HSA. The Stern-Volmer equation showed that quenching was dominated by static quenching. The binding constant for ternary interaction was below that for binary interaction. Site-competitive experiments demonstrated that SSD/PF bound to site I (subdomain IIA) and site II (subdomain IIIA) in HSA. Analysis of thermodynamic parameters indicated that hydrogen bonding and van der Waals forces were mostly responsible for the binary association. Also, there was energy transfer upon binary interaction. Molecular docking supported the experimental findings in conformation, binding sites and binding forces.

Bilirubin photoisomers in rhesus monkey serum.

PubMed

Okada, Hitoshi; Itoh, Susumu; Nii, Kohichiroh; Sugino, Masashiro; Fuke, Noriko; Koyano, Kosuke; Yasuda, Saneyuki; Kusaka, Takashi

2018-05-23

As rhesus monkeys exhibit physiological jaundice during the neonatal period, we used rhesus monkey serum to examine changes in bilirubin photoisomers. Bilirubin-rhesus monkey serum solution was irradiated with blue light-emitting diode, and changes in the absorbance and bilirubin fraction were compared with those in bilirubin- human serum albumin (HSA) and bilirubin-rat albumin solutions. The λ max decreased with light irradiation. The mean production rate of cyclobilirubin IXα was 1.98, 199 and 0.76 × 10 -2 /min in rhesus monkey serum, HSA and rat albumin, respectively. There was no significant difference between rhesus monkey serum and HSA. The (ZE)-bilirubin IXα/(ZZ)-bilirubin IXα ratio was 0.33, 0.45, and 0.10, respectively, differing significantly among the groups. The (EZ)-bilirubin IXα/(ZZ)-bilirubin IXα ratio was 0.020, 0.010, and 0.062, respectively, with no significant difference between rhesus monkey serum and HSA. The production rate of (EZ)-cyclobilirubin XIIIα(= (ZE)-cyclobilirubin XIIIα) was 0.73, 1.60, and 0.51 × 10 -2 /min, respectively, with differing significantly among the groups. The (EZ)-bilirubin IIIα/(ZZ)-bilirubin IIIα ratio was significantly different among the groups at 0.20, 0.38, and 0.15, respectively. This is the first report demonstrating the photoisomerization of bilirubin in rhesus monkey serum and the animal with the same cyclobilirubin production rate as HSA.Rhesus monkeys may be used as an animal model for neonatal hyperbilirubinemia in humans to evaluate the efficacy of phototherapy. Copyright © 2018 Elsevier B.V. All rights reserved.

Importance of neonatal FcR in regulating the serum half-life of therapeutic proteins containing the Fc domain of human IgG1: a comparative study of the affinity of monoclonal antibodies and Fc-fusion proteins to human neonatal FcR.

PubMed

Suzuki, Takuo; Ishii-Watabe, Akiko; Tada, Minoru; Kobayashi, Tetsu; Kanayasu-Toyoda, Toshie; Kawanishi, Toru; Yamaguchi, Teruhide

2010-02-15

The neonatal FcR (FcRn) binds to the Fc domain of IgG at acidic pH in the endosome and protects IgG from degradation, thereby contributing to the long serum half-life of IgG. To date, more than 20 mAb products and 5 Fc-fusion protein products have received marketing authorization approval in the United States, the European Union, or Japan. Many of these therapeutic proteins have the Fc domain of human IgG1; however, the serum half-lives differ in each protein. To elucidate the role of FcRn in the pharmacokinetics of Fc domain-containing therapeutic proteins, we evaluated the affinity of the clinically used human, humanized, chimeric, or mouse mAbs and Fc-fusion proteins to recombinant human FcRn by surface plasmon resonance analysis. The affinities of these therapeutic proteins to FcRn were found to be closely correlated with the serum half-lives reported from clinical studies, suggesting the important role of FcRn in regulating their serum half-lives. The relatively short serum half-life of Fc-fusion proteins was thought to arise from the low affinity to FcRn. The existence of some mAbs having high affinity to FcRn and a short serum half-life, however, suggested the involvement of other critical factor(s) in determining the serum half-life of such Abs. We further investigated the reason for the relatively low affinity of Fc-fusion proteins to FcRn and suggested the possibility that the receptor domain of Fc-fusion protein influences the structural environment of the FcRn binding region but not of the FcgammaRI binding region of the Fc domain.

Chemometrics enhanced HPLC-DAD performance for rapid quantification of carbamazepine and phenobarbital in human serum samples.

PubMed

Vosough, Maryam; Ghafghazi, Shiva; Sabetkasaei, Masoumeh

2014-02-01

This paper describes development and validation of a simple and efficient bioanalytical procedure for simultaneous determination of phenobarbital and carbamazepine in human serum samples using high performance liquid chromatography with photodiode-array detection (HPLC-DAD) regarding a fast elution methodology in less than 5 min. Briefly, this method consisted of a simple deproteinization step of serum samples followed by HPLC analysis on a Bonus-RP column using an isocratic mode of elution with acetonitrile/K2HPO4 (pH=7.5) buffer solution (45:55). Due to the presence of serum endogenous components as non-calibrated components in the sample, second-order calibration based on multivariate curve resolution-alternating least squares (MCR-ALS), has been applied on a set of absorbance matrices collected as a function of retention time and wavelengths. Acceptable resolution and quantification results were achieved in the presence of matrix interferences and the second-order advantage was fully exploited. The average recoveries for carbamazepine and phenobarbital were 89.7% and 86.1% and relative standard deviation values were lower than 9%. Additionally, computed elliptical joint confidence region (EJCR) confirmed the accuracy of the proposed method and indicated the absence of both constant and proportional errors in the predicted concentrations. The developed method enabled the determination of the analytes in different serum samples in the presence of overlapped profiles, while keeping experimental time and extraction steps at minimum. Finally, the serum concentration levels of carbamazepine in three time intervals were reported for morphine-dependents who had received carbamazepine for treating their neuropathic pain. © 2013 Elsevier B.V. All rights reserved.

Enzymatic-induced upconversion photoinduced electron transfer for sensing tyrosine in human serum.

PubMed

Wu, Qiongqiong; Fang, Aijin; Li, Haitao; Zhang, Youyu; Yao, Shouzhuo

2016-03-15

This paper reports a novel nanosensor for tyrosine based on photoinduced electron-transfer (PET) between NaYF4:Yb, Tm upconversion nanoparticles (UCNPs) and melanin-like polymers. Melanin-like films were obtained from catalytic oxidation of tyrosine by tyrosinase, and deposited on the surface of UCNPs, and then quenched the fluorescence of UCNPs. Under the optimized conditions, the fluorescence quenching of UCNPs showed a good linear response to tyrosine concentration in the range of 0.8-100 μΜ with a detection limit of 1.1 μΜ. Meanwhile, it showed good sensitivity, stability and has been successfully applied to the detection of tyrosine in human serum. Copyright © 2015 Elsevier B.V. All rights reserved.

Polysialylated N-Glycans Identified in Human Serum Through Combined Developments in Sample Preparation, Separations and Electrospray ionization-mass spectrometry

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

Kronewitter, Scott R.; Marginean, Ioan; Cox, Jonathan T.

The N-glycan diversity of human serum glycoproteins, i.e. the human blood serum N-glycome, is complex due to the range of glycan structures potentially synthesizable by human glycosylation enzymes. The reported glycome, however, is limited by methods of sample preparation, available analytical platforms, e.g., based upon electrospray ionization-mass spectrometry (ESI-MS), and software tools for data analysis. In this report, several improvements have been implemented in sample preparation and analysis to extend ESI-MS glycan characterization and to provide an improved view of glycan diversity. Sample preparation improvements include acidified, microwave-accelerated, PNGase F N-glycan release, and sodium borohydride reduction were optimized to improvemore » quantitative yields and conserve the number of glycoforms detected. Two-stage desalting (during solid phase extraction and on the analytical column) increased the sensitivity by reducing analyte signal division between multiple reducing-end-forms or cation adducts. On-line separations were improved by using extended length graphitized carbon columns and adding TFA as an acid modifier to a formic acid/reversed phase gradient which provides additional resolving power and significantly improved desorption of both large and heavily sialylated glycans. To improve MS sensitivity and provide gentler ionization conditions at the source-MS interface, subambient pressure ionization with nanoelectrospray (SPIN) has been utilized. When method improvements are combined together with the Glycomics Quintavariate Informed Quantification (GlyQ-IQ) recently described1 these technologies demonstrate the ability to significantly extend glycan detection sensitivity and provide expanded glycan coverage. We demonstrate application of these advances in the context of the human serum glycome, and for which our initial observations include detection of a new class of heavily sialylated N-glycans, including polysialylated N-glycans.« less

Phase separation in solutions of monoclonal antibodies and the effect of human serum albumin

PubMed Central

Wang, Ying; Lomakin, Aleksey; Latypov, Ramil F.; Benedek, George B.

2011-01-01

We report the observation of liquid-liquid phase separation in a solution of human monoclonal antibody, IgG2, and the effects of human serum albumin, a major blood protein, on this phase separation. We find a significant reduction of phase separation temperature in the presence of albumin, and a preferential partitioning of the albumin into the antibody-rich phase. We provide a general thermodynamic analysis of the antibody-albumin mixture phase diagram and relate its features to the magnitude of the effective interprotein interactions. Our analysis suggests that additives (HSA in this report), which have moderate attraction with antibody molecules, may be used to forestall undesirable proetin condensation in antibody solutions. Our findings are relevant to understanding the stability of pharmaceutical solutions of antibodies and the mechanisms of cryoglobulinemia. PMID:21921237

Serum antibodies to huntingtin interacting protein-1: a new blood test for prostate cancer.

PubMed

Bradley, Sarah V; Oravecz-Wilson, Katherine I; Bougeard, Gaelle; Mizukami, Ikuko; Li, Lina; Munaco, Anthony J; Sreekumar, Arun; Corradetti, Michael N; Chinnaiyan, Arul M; Sanda, Martin G; Ross, Theodora S

2005-05-15

Huntingtin-interacting protein 1 (HIP1) is frequently overexpressed in prostate cancer. HIP1 is a clathrin-binding protein involved in growth factor receptor trafficking that transforms fibroblasts by prolonging the half-life of growth factor receptors. In addition to human cancers, HIP1 is also overexpressed in prostate tumors from the transgenic adenocarcinoma of the mouse prostate (TRAMP) mouse model. Here we provide evidence that HIP1 plays an important role in mouse tumor development, as tumor formation in the TRAMP mice was impaired in the Hip1null/null background. In addition, we report that autoantibodies to HIP1 developed in the sera of TRAMP mice with prostate cancer as well as in the sera from human prostate cancer patients. This led to the development of an anti-HIP1 serum test in humans that had a similar sensitivity and specificity to the anti-alpha-methylacyl CoA racemase (AMACR) and prostate-specific antigen tests for prostate cancer and when combined with the anti-AMACR test yielded a specificity of 97%. These data suggest that HIP1 plays a functional role in tumorigenesis and that a positive HIP1 autoantibody test may be an important serum marker of prostate cancer.

Preparation and value assignment of standard reference material 968e fat-soluble vitamins, carotenoids, and cholesterol in human serum.

PubMed

Thomas, Jeanice B; Duewer, David L; Mugenya, Isaac O; Phinney, Karen W; Sander, Lane C; Sharpless, Katherine E; Sniegoski, Lorna T; Tai, Susan S; Welch, Michael J; Yen, James H

2012-01-01

Standard Reference Material 968e Fat-Soluble Vitamins, Carotenoids, and Cholesterol in Human Serum provides certified values for total retinol, γ- and α-tocopherol, total lutein, total zeaxanthin, total β-cryptoxanthin, total β-carotene, 25-hydroxyvitamin D(3), and cholesterol. Reference and information values are also reported for nine additional compounds including total α-cryptoxanthin, trans- and total lycopene, total α-carotene, trans-β-carotene, and coenzyme Q(10). The certified values for the fat-soluble vitamins and carotenoids in SRM 968e were based on the agreement of results from the means of two liquid chromatographic methods used at the National Institute of Standards and Technology (NIST) and from the median of results of an interlaboratory comparison exercise among institutions that participate in the NIST Micronutrients Measurement Quality Assurance Program. The assigned values for cholesterol and 25-hydroxyvitamin D(3) in the SRM are the means of results obtained using the NIST reference method based upon gas chromatography-isotope dilution mass spectrometry and liquid chromatography-isotope dilution tandem mass spectrometry, respectively. SRM 968e is currently one of two available health-related NIST reference materials with concentration values assigned for selected fat-soluble vitamins, carotenoids, and cholesterol in human serum matrix. This SRM is used extensively by laboratories worldwide primarily to validate methods for determining these analytes in human serum and plasma and for assigning values to in-house control materials. The value assignment of the analytes in this SRM will help support measurement accuracy and traceability for laboratories performing health-related measurements in the clinical and nutritional communities.

Disparate Proteome Responses of Pathogenic and Non-pathogenic Aspergilli to Human Serum Measured by Activity-Based Protein Profiling (ABPP)

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

Wiedner, Susan D.; Ansong, Charles; Webb-Robertson, Bobbie-Jo M.

2013-07-01

Aspergillus fumigatus is the primary pathogen causing the devastating pulmonary disease Invasive Aspergillosis in immunocompromised individuals. Genomic analysis shows high synteny between A. fumigatus and closely related rarely pathogenic Neosartorya fischeri and Aspergillus clavatus genomes. To investigate the presence of unique or highly inducible protein reactivity in the pathogen, we applied activity-based protein profiling to compare protein reactivity of all three fungi over time in minimal media growth and in response to human serum. We found 350 probe-reactive proteins exclusive to A. fumigatus, including known virulence associated proteins, and 13 proteins associated with stress response exclusive to A. fumigatus culturemore » in serum. Though the fungi are highly orthologous, A. fumigatus has significantly more activity across varied biological process. Only 50% of expected orthologs of measured A. fumigatus reactive proteins were observed in N. fischeri and A. clavatus. Human serum induced processes uniquely or significantly represented in A. fumigatus include actin organization and assembly, transport, and fatty acid, cell membrane, and cell wall synthesis. Additionally, signaling proteins regulating vegetative growth, conidiation, and cell wall integrity, required for appropriate cellular response to external stimuli, had higher reactivity over time in A. fumigatus and N. fisheri, but not in A. clavatus. Together, we show that measured proteins and physiological processes identified solely or significantly over-represented in A. fumigatus reveal a unique adaptive response to human protein not found in closely related, but rarely aspergilli. These unique protein reactivity responses may reveal how A. fumigatus initiates pulmonary invasion leading to Invasive Aspergillosis.« less

Human serum albumin (HSA) nanoparticles: reproducibility of preparation process and kinetics of enzymatic degradation.

PubMed

Langer, K; Anhorn, M G; Steinhauser, I; Dreis, S; Celebi, D; Schrickel, N; Faust, S; Vogel, V

2008-01-22

Nanoparticles prepared from human serum albumin (HSA) are versatile carrier systems for drug delivery and can be prepared by an established desolvation process. A reproducible process with a low batch-to-batch variability is required for transfer from the lab to an industrial production. In the present study the batch-to-batch variability of the starting material HSA on the preparation of nanoparticles was investigated. HSA can build dimers and higher aggregates because of a free thiol group present in the molecule. Therefore, the quality of different HSA batches was analysed by size exclusion chromatography (SEC) and analytical ultracentrifugation (AUC). The amount of dimerised HSA detected by SEC did not affect particle preparation. Higher aggregates of the protein detected in two batches by AUC disturbed nanoparticle formation at pH values below 8.0. At pH 8.0 and above monodisperse particles between 200 and 300 nm could be prepared with all batches, with higher pH values leading to smaller particles. Besides human derived albumin a particle preparation was also feasible based on recombinant human serum albumin (rHSA). Under comparable preparation conditions monodisperse nanoparticles could be achieved and the same effects of protein aggregates on particle formation were observed. For nanoparticulate drug delivery systems the enzymatic degradation is a crucial parameter for the release of an embedded drug. For this reason, besides the particle preparation process, particle degradation in the presence of different enzymes was studied. Under acidic conditions HSA as well as rHSA nanoparticles could be digested by pepsin and cathepsin B. At neutral pH trypsin, proteinase K, and protease were suitable for particle degradation. It could be shown that the kinetics of particle degradation was dependent on the degree of particle stabilisation. Therefore, the degree of particle stabilisation will influence drug release after cellular accumulation of HSA nanoparticles.

Optimal background matching camouflage.

PubMed

Michalis, Constantine; Scott-Samuel, Nicholas E; Gibson, David P; Cuthill, Innes C

2017-07-12

Background matching is the most familiar and widespread camouflage strategy: avoiding detection by having a similar colour and pattern to the background. Optimizing background matching is straightforward in a homogeneous environment, or when the habitat has very distinct sub-types and there is divergent selection leading to polymorphism. However, most backgrounds have continuous variation in colour and texture, so what is the best solution? Not all samples of the background are likely to be equally inconspicuous, and laboratory experiments on birds and humans support this view. Theory suggests that the most probable background sample (in the statistical sense), at the size of the prey, would, on average, be the most cryptic. We present an analysis, based on realistic assumptions about low-level vision, that estimates the distribution of background colours and visual textures, and predicts the best camouflage. We present data from a field experiment that tests and supports our predictions, using artificial moth-like targets under bird predation. Additionally, we present analogous data for humans, under tightly controlled viewing conditions, searching for targets on a computer screen. These data show that, in the absence of predator learning, the best single camouflage pattern for heterogeneous backgrounds is the most probable sample. © 2017 The Authors.

Quantitation of ortho-Cresyl Phosphate Adducts to Butyrylcholinesterase in Human Serum by Immunomagnetic-UHPLC-MS/MS

PubMed Central

Johnson, Darryl; Carter, Melissa D.; Crow, Brian S.; Isenberg, Samantha L.; Graham, Leigh Ann; Erol, H. Akin; Watson, Caroline M.; Pantazides, Brooke G.; van der Schans, Marcel J.; Langenberg, Jan P.; Noort, Daan; Blake, Thomas A.; Thomas, Jerry D.; Johnson, Rudolph C.

2017-01-01

Tri-ortho-cresyl phosphate (ToCP) is an anti-wear, flame retardant additive used in industrial lubricants, hydraulic fluids, and gasoline. The neurotoxic effects of ToCP arise from the liver-activated metabolite 2-(o-cresyl)-4H-1,3,2-benzodioxaphosphoran-2-one (cresyl saligenin phosphate or CBDP), which inhibits esterase enzymes including butyrylcholinesterase (BChE). Following BChE adduction, CBDP undergoes hydrolysis to form the aged adduct ortho-cresyl phosphoserine (oCP-BChE), thus providing a biomarker of CBDP exposure. Previous studies have identified ToCP in aircraft cabin and cockpit air, but assessing human exposure has been hampered by the lack of a laboratory assay to confirm exposure. This work presents the development of an immunomagnetic-UHPLC-MS/MS method for the quantitation of unadducted BChE and the long-term CBDP biomarker, oCP-BChE, in human serum. The method has a reportable range from 2.0 ng/mL to 150 ng/mL, which is consistent with the sensitivity of methods used to detect organophosphorus nerve agent protein adducts. The assay demonstrated high intraday and interday accuracy (≥ 85%) and precision (RSD ≤ 15%) across the calibration range. The method was developed for future analyses of potential human exposure to CBDP. Analysis of human serum inhibited in vitro with CBDP demonstrated that the oCP-BChE adduct was stable for at least 72 hours at 4, 22 and 37 °C. Compared to a previously reported assay, this method requires 75% less sample volume, reduces analysis time by a factor of 20, and demonstrates a 3-fold improvement in sensitivity. PMID:26149113

Detection of Human Epididymis Protein 4 (HE4) in Human Serum Samples Using a Specific Monoclonal Antibody-Based Sandwich Enzyme-Linked Immunosorbent Assay (ELISA).

PubMed

Zhou, Lijun; Lv, Zhiqiang; Shao, Jing; Xu, Ying; Luo, Xiaohong; Zhang, Yuming; Hu, Yang; Zhang, Wenji; Luo, Shuhong; Fang, Jianmin; Wang, Ying; Duan, Chaohui; Huang, Ruopan

2016-09-01

The human epididymis protein 4 (HE4) may have high specificity in the detection of malignant diseases, making the development of an immunoassay for HE4 essential. In our study, a fusion gene was constructed encoded with the HE4 protein. This protein was then produced in the bacterial cells (Escherichia coli) and used to immunize mice in order to eventually generate hybridomas specific to HE4. The hybridoma supernatants were then screened, and four positive anti-HE4 cell lines were selected. These cell lines produce monoclonal antibodies against HE4 epitopes, as demonstrated in the Western blot as well as by direct enzyme-linked immunosorbent assay (ELISA). Using the developed antibodies, we successfully identified several good antibody pairs from the hybridomas, which allowed for the development of a sandwich ELISA to measure HE4 levels. By using the HE4 ELISA, we measured HE4 levels of 60 clinical human serum samples. Compared with the Food and Drug Administration (FDA) approved kit (Roche), our results showed a strong positive correlation to those of the FDA-approved kit. In summary, highly sensitive antibody pairs were screened against HE4, and a sandwich ELISA was developed as an accurate analytical tool for the detection of HE4 in human serum, which could be especially valuable for diagnosing ovarian carcinomas. © 2015 Wiley Periodicals, Inc.

Practical methods for handling human periodontal ligament stem cells in serum-free and serum-containing culture conditions under hypoxia: implications for regenerative medicine.

PubMed

Murabayashi, Dai; Mochizuki, Mai; Tamaki, Yuichi; Nakahara, Taka

2017-07-01

Stem cell-based therapies depend on the reliable expansion of patient-derived mesenchymal stem cells (MSCs) in vitro. The supplementation of cell culture media with serum is associated with several risks; accordingly, serum-free media are commercially available for cell culture. Furthermore, hypoxia is known to accelerate the expansion of MSCs. The present study aimed to characterize the properties of periodontal ligament-derived MSCs (PDLSCs) cultivated in serum-free and serum-containing media, under hypoxic and normoxic conditions. Cell growth, gene and protein expression, cytodifferentiation potential, genomic stability, cytotoxic response, and in vivo hard tissue generation of PDLSCs were examined. Our findings indicated that cultivation in serum-free medium does not affect the MSC phenotype or chromosomal stability of PDLSCs. PDLSCs expanded in serum-free medium exhibited more active growth than in fetal bovine serum-containing medium. We found that hypoxia does not alter the cell growth of PDLSCs under serum-free conditions, but inhibits their osteogenic and adipogenic cytodifferentiation while enabling maintenance of their multidifferentiation potential regardless of the presence of serum. PDLSCs expanded in serum-free medium were found to retain common MSC characteristics, including the capacity for hard tissue formation in vivo. However, PDLSCs cultured in serum-free culture conditions were more susceptible to damage following exposure to extrinsic cytotoxic stimuli than those cultured in medium supplemented with serum, suggesting that serum-free culture conditions do not exert protective effects against cytotoxicity on PDLSC cultures. The present work provides a comparative evaluation of cell culture in serum-free and serum-containing media, under hypoxic and normoxic conditions, for applications in regenerative medicine.

Final report for CCQM-K107: total elements and selenomethionine in human serum

NASA Astrophysics Data System (ADS)

Goenaga Infante, Heidi

2016-01-01

Routine tests that measure the concentration of electrolytes in serum are needed for diagnosis and management of renal, endocrine, acid-base, water balance and other conditions such as screening D- and A-vitamin disorders, kidney insufficiency, bone diseases and leukaemia. The diagnostic concentration ranges for many such markers are narrow, requiring reference methods with small uncertainty. Serum concentration of total selenium (Se) is important in health studies but there is increasing interest in the speciation of selenium compounds in clinical samples such as serum and individual Se- Species are bio-indicators of Se status. The last CCQM IAWG key comparison for elements in the clinical area (CCQM-K14: Ca in human serum) was organized in 2003 and the previous key comparison (CCQM-K60) for Se and Se species used a wheat flour sample. Therefore, the CCQM IAWG agreed that CCQM-K107 and a parallel pilot study CCQM-P146 should be carried out. The candidate human serum sample used for both CCQM-K107 and P146 is of high complexity and contains approximately 1000-fold lower concentrations of selenium methionine (SeMet) than those encountered in the CCQM-K60 wheat flour. This significantly broadens the scope and degree of difficulty of earlier measurements in this field. A total of eleven institutes participated in CCQM-K107 (11 participants for total elements and 7 for SeMet). The performance of the majority of the K107 participants for all the measurands was very good, illustrating their ability to obtain accurate results for analytes such as electrolytes at mg kg-1 level, essential elements at µg kg-1 level and selenium species at µg kg-1 level in a complex biological fluid. The range of agreement between participants was within the interval of ± 0.1% for Ca and up to ± 1.8% for Fe. CMC claims based on total elements in this study may include other elements with similar core competencies (e.g. Se, Cu, Zn) in a wide range of biological materials (including liquids

Restricted access carbon nanotubes for direct extraction of cadmium from human serum samples followed by atomic absorption spectrometry analysis.

PubMed

Barbosa, Adriano F; Barbosa, Valéria M P; Bettini, Jefferson; Luccas, Pedro O; Figueiredo, Eduardo C

2015-01-01

In this paper, we propose a new sorbent that is able to extract metal ions directly from untreated biological fluids, simultaneously excluding all proteins from these samples. The sorbent was obtained through the modification of carbon nanotubes (CNTs) with an external bovine serum albumin (BSA) layer, resulting in restricted access carbon nanotubes (RACNTs). The BSA layer was fixed through the interconnection between the amine groups of the BSA using glutaraldehyde as cross-linker. When a protein sample is percolated through a cartridge containing RACNTs and the sample pH is higher than the isoelectric point of the proteins, both proteins from the sample and the BSA layer are negatively ionized. Thus, an electrostatic repulsion prevents the interaction between the proteins from the sample on the RACNTs surface. At the same time, metal ions are adsorbed in the CNTs (core) after their passage through the chains of proteins. The Cd(2+) ion was selected for a proof-of-principle case to test the suitability of the RACNTs due to its toxicological relevance. RACNTs were able to extract Cd(2+) and exclude almost 100% of the proteins from the human serum samples in an online solid-phase extraction system coupled with thermospray flame furnace atomic absorption spectrometry. The limits of detection and quantification were 0.24 and 0.80 μg L(-1), respectively. The sampling frequency was 8.6h(-1), and the intra- and inter-day precisions at the 0.80, 15.0, and 30.0 μg L(-1) Cd(2+) levels were all lower than 10.1% (RSD). The recoveries obtained for human blood serum samples fortified with Cd(2+) ranged from 85.0% to 112.0%. The method was successfully applied to analyze Cd(2+) directly from six human blood serum samples without any pretreatment, and the observed concentrations ranged from

Serial Sampling of Serum Protein Biomarkers for Monitoring Human Traumatic Brain Injury Dynamics: A Systematic Review.

PubMed

Thelin, Eric Peter; Zeiler, Frederick Adam; Ercole, Ari; Mondello, Stefania; Büki, András; Bellander, Bo-Michael; Helmy, Adel; Menon, David K; Nelson, David W

2017-01-01

The proteins S100B, neuron-specific enolase (NSE), glial fibrillary acidic protein (GFAP), ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), and neurofilament light (NF-L) have been serially sampled in serum of patients suffering from traumatic brain injury (TBI) in order to assess injury severity and tissue fate. We review the current literature of serum level dynamics of these proteins following TBI and used the term "effective half-life" ( t 1/2 ) in order to describe the "fall" rate in serum. Through searches on EMBASE, Medline, and Scopus, we looked for articles where these proteins had been serially sampled in serum in human TBI. We excluded animal studies, studies with only one presented sample and studies without neuroradiological examinations. Following screening (10,389 papers), n  = 122 papers were included. The proteins S100B ( n  = 66) and NSE ( n  = 27) were the two most frequent biomarkers that were serially sampled. For S100B in severe TBI, a majority of studies indicate a t 1/2 of about 24 h, even if very early sampling in these patients reveals rapid decreases (1-2 h) though possibly of non-cerebral origin. In contrast, the t 1/2 for NSE is comparably longer, ranging from 48 to 72 h in severe TBI cases. The protein GFAP ( n  = 18) appears to have t 1/2 of about 24-48 h in severe TBI. The protein UCH-L1 ( n  = 9) presents a t 1/2 around 7 h in mild TBI and about 10 h in severe. Frequent sampling of these proteins revealed different trajectories with persisting high serum levels, or secondary peaks, in patients with unfavorable outcome or in patients developing secondary detrimental events. Finally, NF-L ( n  = 2) only increased in the few studies available, suggesting a serum availability of >10 days. To date, automated assays are available for S100B and NSE making them faster and more practical to use. Serial sampling of brain-specific proteins in serum reveals different temporal trajectories that should be

Development and Validation of HPLC Method for Determination of Crocetin, a constituent of Saffron, in Human Serum Samples.

PubMed

Mohammadpour, Amir Hooshang; Ramezani, Mohammad; Tavakoli Anaraki, Nasim; Malaekeh-Nikouei, Bizhan; Amel Farzad, Sara; Hosseinzadeh, Hossein

2013-01-01

The present study reports the development and validation of a sensitive and rapid extraction method beside high performance liquid chromatographic method for the determination of crocetin in human serum. The HPLC method was carried out by using a C18 reversed-phase column and a mobile phase composed of methanol/water/acetic acid (85:14.5:0.5 v/v/v) at the flow rate of 0.8 ml/min. The UV detector was set at 423 nm and 13-cis retinoic acid was used as the internal standard. Serum samples were pretreated with solid-phase extraction using Bond Elut C18 (200mg) cartridges or with direct precipitation using acetonitrile. The calibration curves were linear over the range of 0.05-1.25 µg/ml for direct precipitation method and 0.5-5 µg/ml for solid-phase extraction. The mean recoveries of crocetin over a concentration range of 0.05-5 µg/ml serum for direct precipitation method and 0.5-5 µg/ml for solid-phase extraction were above 70 % and 60 %, respectively. The intraday coefficients of variation were 0.37- 2.6% for direct precipitation method and 0.64 - 5.43% for solid-phase extraction. The inter day coefficients of variation were 1.69 - 6.03% for direct precipitation method and 5.13-12.74% for solid-phase extraction, respectively. The lower limit of quantification for crocetin was 0.05 µg/ml for direct precipitation method and 0.5 µg/ml for solid-phase extraction. The validated direct precipitation method for HPLC satisfied all of the criteria that were necessary for a bioanalytical method and could reliably quantitate crocetin in human serum for future clinical pharmacokinetic study.

Development and Validation of HPLC Method for Determination of Crocetin, a constituent of Saffron, in Human Serum Samples

PubMed Central

Mohammadpour, Amir Hooshang; Ramezani, Mohammad; Tavakoli Anaraki, Nasim; Malaekeh-Nikouei, Bizhan; Amel Farzad, Sara; Hosseinzadeh, Hossein

2013-01-01

Objective(s): The present study reports the development and validation of a sensitive and rapid extraction method beside high performance liquid chromatographic method for the determination of crocetin in human serum. Materials and Methods: The HPLC method was carried out by using a C18 reversed-phase column and a mobile phase composed of methanol/water/acetic acid (85:14.5:0.5 v/v/v) at the flow rate of 0.8 ml/min. The UV detector was set at 423 nm and 13-cis retinoic acid was used as the internal standard. Serum samples were pretreated with solid-phase extraction using Bond Elut C18 (200mg) cartridges or with direct precipitation using acetonitrile. Results: The calibration curves were linear over the range of 0.05-1.25 µg/ml for direct precipitation method and 0.5-5 µg/ml for solid-phase extraction. The mean recoveries of crocetin over a concentration range of 0.05-5 µg/ml serum for direct precipitation method and 0.5-5 µg/ml for solid-phase extraction were above 70 % and 60 %, respectively. The intraday coefficients of variation were 0.37- 2.6% for direct precipitation method and 0.64 - 5.43% for solid-phase extraction. The inter day coefficients of variation were 1.69 – 6.03% for direct precipitation method and 5.13-12.74% for solid-phase extraction, respectively. The lower limit of quantification for crocetin was 0.05 µg/ml for direct precipitation method and 0.5 µg/ml for solid-phase extraction. Conclusion: The validated direct precipitation method for HPLC satisfied all of the criteria that were necessary for a bioanalytical method and could reliably quantitate crocetin in human serum for future clinical pharmacokinetic study. PMID:23638292

Computer simulation of the active site of human serum cholinesterase

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

Kefang Jiao; Song Li; Zhengzheng Lu

1996-12-31

The first 3D-structure of acetylchelinesterase from Torpedo California electric organ (T.AChE) was published by JL. Sussman in 1991. We have simulated 3D-structure of human serum cholinesterase (H.BuChE) and the active site of H.BuChE. It is discovered by experiment that the residue of H.BuChE is still active site after a part of H.BuChE is cut. For example, the part of 21KD + 20KD is active site of H.BuChE. The 20KD as it is. Studies on these peptides by Hemelogy indicate that two active peptides have same negative electrostatic potential maps diagram. These negative electrostatic areas attached by acetyl choline with positivemore » electrostatic potency. We predict that 147...236 peptide of AChE could be active site because it was as 20KD as with negative electrostatic potential maps. We look forward to proving from other ones.« less

[Effect of new peptide bioregulators livagen and epitalon on enkephalin-degrading enzymes in human serum].

PubMed

Kost, N V; Sokolov, O Iu; Gabaeva, M V; Zolotarev, Iu A; Malinin, V V; Khavinson, V Kh

2003-01-01

The effect of new peptide bioregulators--Livagen (Lys-Glu-Asp-Ala) and Epitalon (Ala-Glu-Asp-Gly)--on endogenous opioid system was studied, particularly, their ability to change the activity of enkephalin-degrading enzymes from serum and interact with opioid receptors of the brain membrane fraction. Enkephalinase activity was assayed in vitro by the rate of 3H-Leu-enkephalin hydrolysis in the presence of the tested peptides. Livagen and Epitalon inhibited enkephalin-degrading enzymes from human serum. Livagen proved to be more efficient also as compared to well-known peptidase inhibitors such as puromycin, leupeptin, and D-PAM. The dose-inhibitory effect curves for Livagen and Epitalon were plotted; their IC50 equaled 20 and 500 microM, respectively. The interaction between the peptides and opioid receptors was estimated using a radioreceptor method with [3H][D-Ala2, D-Leu5]-enkephalin. No interaction was observed between the tested peptides and mu- or delta-opioid receptors of the membrane fraction from the rat brain.

A retrospective analysis of 25% human serum albumin supplementation in hypoalbuminemic dogs with septic peritonitis

PubMed Central

Horowitz, Farrah B.; Read, Robyn L.; Powell, Lisa L.

2015-01-01

This study describes the influence of 25% human serum albumin (HSA) supplementation on serum albumin level, total protein (TP), colloid osmotic pressure (COP), hospital stay, and survival in dogs with septic peritonitis. Records of 39 dogs with septic peritonitis were evaluated. In the HSA group, initial and post-transfusion TP, albumin, COP, and HSA dose were recorded. In the non-supplemented group, repeated values of TP, albumin, and COP were recorded over their hospitalization. Eighteen dogs survived (53.8% mortality). Repeat albumin values were higher in survivors (mean 23.9 g/L) and elevated repeat albumin values were associated with HSA supplementation. Repeat albumin and TP were higher in the HSA supplemented group (mean 24 g/L and 51.9 g/L, respectively) and their COP increased by 5.8 mmHg. Length of hospitalization was not affected. Twenty-five percent HSA increases albumin, TP, and COP in canine patients with septic peritonitis. Higher postoperative albumin levels are associated with survival. PMID:26028681

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.

Validation of the analytical method for the simultaneous determination of selected polybrominated diphenyl ethers, polychlorinated biphenyls and organochlorine pesticides in human blood serum by gas chromatography with microelectron capture detector.

PubMed

Matuszak, Małgorzata; Minorczyk, Maria; Góralczyk, Katarzyna; Hernik, Agnieszka; Struciński, Paweł; Liszewska, Monika; Czaja, Katarzyna; Korcz, Wojciech; Łyczewska, Monika; Ludwicki, Jan K

2016-01-01

Polybrominated diphenyl ethers (PBDEs) as other persistent organic pollutants like polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs) pose a significant hazard to human health, mainly due to interference with the endocrine system and carcinogenetic effects. Humans are exposed to these substances mainly through a food of animal origin. These pollutants are globally detected in human matrices which requires to dispose reliable and simple analytical method that would enable further studies to assess the exposure of specific human populations to these compounds. The purpose of this study was to modify and validate of the analytical procedure for the simultaneous determination of selected PBDEs, PCBs and OCPs in human blood serum samples. The analytical measurement was performed by GC-µECD following preparation of serum samples (denaturation, multiple extraction, lipid removal). Identity of the compounds was confirmed by GC-MS. The method was characterised by the appropriate linearity, good repeatability (CV below 20%). The recoveries ranged from 52.9 to 125.0% depending on compound and level of fortification. The limit of quantification was set at 0.03 ng mL(-1) of serum. The modified analytical method proved to be suitable for the simultaneous determination of selected PBDEs, PCBs and OCPs in human blood serum by GC-µECD with good precision.

Human Platelet Lysate as a Replacement for Fetal Bovine Serum in Limbal Stem Cell Therapy.

PubMed

Suri, Kunal; Gong, Hwee K; Yuan, Ching; Kaufman, Stephen C

2016-10-01

To evaluate the use of human platelet lysate (HPL) as an alternative supplement for limbal explant culture. Culture media were prepared using either 10% pooled HPL (PHPL), single donor HPL, or fetal bovine serum (FBS). Limbal tissues, obtained from the Minnesota Lions Eye Bank, were cultured in each medium on plastic plates or on denuded amniotic membrane (AM). Immunofluorescence staining was performed for ABCG2, tumor protein p63α, and cytokeratin 3 (K3). Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was used to evaluate the expression of ABCG2 and p63. Limbal explants grown in each medium were labeled with bromodeoxyuridine (BrdU) to assess the proliferative capacity in each medium. Concentration of growth factors including epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β), and platelet derived growth factor (PDGF) in HPL and PHPL was compared to that in human serum (HS). Immunofluorescence staining on AM showed prominent expression of ABCG2, p63α but sparse expression of K3 in HPL and PHPL supplemented medium. Real time-PCR showed 1.7 fold higher expression of ABCG2 in PHPL supplemented medium (p = 0.03), and similar expression of p63 in HPL and PHPL supplemented medium compared to FBS medium. The proliferation assay showed that LSCs retained their proliferative potential in HPL supplemented medium. Higher concentration of growth factors were found in HPL, compared to HS. Human platelet lysate has higher concentration of grown factors and is effective in maintaining growth and stem cell phenotype of corneal limbal explant cultures.

Can profiles of poly- and Perfluoroalkyl substances (PFASs) in human serum provide information on major exposure sources?

PubMed

Hu, Xindi C; Dassuncao, Clifton; Zhang, Xianming; Grandjean, Philippe; Weihe, Pál; Webster, Glenys M; Nielsen, Flemming; Sunderland, Elsie M

2018-02-01

Humans are exposed to poly- and perfluoroalkyl substances (PFASs) from diverse sources and this has been associated with negative health impacts. Advances in analytical methods have enabled routine detection of more than 15 PFASs in human sera, allowing better profiling of PFAS exposures. The composition of PFASs in human sera reflects the complexity of exposure sources but source identification can be confounded by differences in toxicokinetics affecting uptake, distribution, and elimination. Common PFASs, such as perfluorooctanoic acid (PFOA), perfluorooctane sulfonic acid (PFOS) and their precursors are ubiquitous in multiple exposure sources. However, their composition varies among sources, which may impact associated adverse health effects. We use available PFAS concentrations from several demographic groups in a North Atlantic seafood consuming population (Faroe Islands) to explore whether chemical fingerprints in human sera provide insights into predominant exposure sources. We compare serum PFAS profiles from Faroese individuals to other North American populations to investigate commonalities in potential exposure sources. We compare individuals with similar demographic and physiological characteristics and samples from the same years to reduce confounding by toxicokinetic differences and changing environmental releases. Using principal components analysis (PCA) confirmed by hierarchical clustering, we assess variability in serum PFAS concentrations across three Faroese groups. The first principal component (PC)/cluster consists of C9-C12 perfluoroalkyl carboxylates (PFCAs) and is consistent with measured PFAS profiles in consumed seafood. The second PC/cluster includes perfluorohexanesulfonic acid (PFHxS) and the PFOS precursor N-ethyl perfluorooctane sulfonamidoacetate (N-EtFOSAA), which are directly used or metabolized from fluorochemicals in consumer products such as carpet and food packaging. We find that the same compounds are associated with the same

Serum levels of antioxidant vitamins and mineral elements of human immunodeficiency virus positive subjects in Sokoto, Nigeria.

PubMed

Bilbis, Lawal S; Idowu, Dorcas B; Saidu, Yusuf; Lawal, Mansur; Njoku, Chibueze H

2010-01-01

Undernourishment and micronutrient deficiencies exacerbate immunosuppression, oxidative stress, acceleration of human immunodeficiency virus (HIV) replication and CD4 T-cell depletion in HIV-infected individuals. The current work reports the serum levels of antioxidant vitamins (vitamins A, C and E) and minerals (Zn, Fe, Cu) in 90 HIV positive subjects attending the Usmanu Danfodiyo University Teaching Hospital (UDUTH), Sokoto, Nigeria. The serum levels of the micronutrients were correlated with the CD4 count of the subjects. The results showed that the HIV positive subjects have significantly lower (P < 0.05) levels of vitamins A, C and E. Also, serum Zn, Fe, Cu and CD4 count were also significantly (P < 0.05) lower compared with the HIV negative subjects. Micronutrient deficiencies were more pronounced in HIV positive subjects with CD4 counts less than 200 cell/μl. The results based on age and sex showed no significant (P > 0.05) difference. Vitamins A, E and C and Zn and Fe showed positive correlation with CD4 count of the HIV positive subjects. The results suggest that the HIV subjects in the study area have lowered serum levels of antioxidant micronutrients and that the levels decrease with increase in the severity of the infection. These may increase the chances of the symptomatic and asymptomatic subjects progressing into full-blown Acquired Immunodeficiency Syndrome.

Treatment of obesity hypoventilation syndrome and serum leptin.

PubMed

Yee, Brendon J; Cheung, Jane; Phipps, Paul; Banerjee, Dev; Piper, Amanda J; Grunstein, Ronald R

2006-01-01

Leptin is a protein produced by adipose tissue that circulates to the brain and interacts with receptors in the hypothalamus to inhibit eating. In obese humans, serum leptin is up to four times higher than in lean subjects, indicating that human obesity is associated with a central resistance to the weight-lowering effects of leptin. Although the leptin-deficient mouse (ob/ob) develops obesity hypoventilation syndrome (OHS), in humans with OHS, serum leptin is a better predictor of awake hypercapnia in obesity than the body mass index (BMI). This suggests that central leptin resistance may promote the development of OHS in humans. We speculated that the reversal of OHS by regular non-invasive ventilation (NIV) therapy decreases leptin levels. The aim of this study was to investigate whether ventilatory treatment of OHS would alter circulating leptin concentrations. We measured fasting serum leptin levels, BMI, spirometry and arterial blood gases in 14 obese hypercapnic subjects undergoing a diagnostic sleep study. The average age of the subjects was (mean +/- SE) 62 +/- 13 years, BMI 40.9 +/- 2.2 kg/m(2), PaCO(2) 6.7 +/- 0.2 kPa, PaO(2 )8.9 +/- 0.4 kPa and total respiratory disturbance index 44 +/- 35 events/hour. Subjects were clinically reviewed after a median of 2.3 years (range 1.6-3) with repeat investigations. Nine patients were regular NIV users and 5 were non-users. NIV users had a significant reduction in serum leptin levels (p = 0.001), without a change in BMI. In these patients, there was a trend towards an improved daytime hypercapnia and hypoxemia, while in the 5 non-users, no changes in serum leptin, BMI or arterial blood gases occurred. Regular NIV use reduces serum leptin in OHS. Leptin may be a modulator of respiratory drive in patients with OHS.

Determination of bisphenol A in human serum by high-performance liquid chromatography with multi-electrode electrochemical detection.

PubMed

Inoue, K; Kato, K; Yoshimura, Y; Makino, T; Nakazawa, H

2000-11-10

A simple and sensitive method using high-performance liquid chromatography with multi-electrode electrochemical detection (HPLC-ED) including a coulometric array of four electrochemical sensors has been developed for the determination of bisphenol A in water and human serum. For good separation and detection of bisphenol A, a CAPCELL PAK UG 120 C18 reversed-phase column and a mobile phase consisting of 0.3% phosphoric acid-acetonitrile (60:40) were used. The detection limit obtained by the HPLC-ED method was 0.01 ng/ml (0.5 pg), which was more than 3000-times higher than the detection limit obtained by the ultraviolet (UV) method, and more than 200-times higher than the detection limit obtained by the fluorescence (FL) method. Bisphenol A in water and serum samples was pretreated by solid-phase extraction (SPE) after removing possible contamination derived from a plastic SPE cartridges and water used for the pretreatment. A trace amount (ND approximately 0.013 ng/ml) of bisphenol A was detected from the parts of cartridges (filtration column, sorbent bed and frits) by extraction with methanol, and it was completely removed by washing with at least 15 ml of methanol in the operation process. The concentrations of bisphenol A in tap water and Milli-Q-purified water were found to be 0.01 and 0.02 ng/ml, respectively. For that reason, bisphenol A-free water was made to trap bisphenol A in water using an Empore disk. In every pretreatment, SPE methods using bisphenol A-free water and washing with 15 ml of methanol were done in water and serum samples. The yields obtained from the recovery tests using water to which 0.5 or 0.05 ng/ml of bisphenol A was added were 83.8 to 98.2%, and the RSDs were 3.4 to 6.1%, respectively. The yields obtained from the recovery tests by OASIS HLB using serum to which 1.0 ng/ml or 0.1 ng/ml of bisphenol A was added were 79.0% and 87.3%, and the RSDs were 5.1% and 13.5%, respectively. The limits of quantification in water and serum sample

Investigation into the interaction of losartan with human serum albumin and glycated human serum albumin by spectroscopic and molecular dynamics simulation techniques: A comparison study.

PubMed

Moeinpour, Farid; Mohseni-Shahri, Fatemeh S; Malaekeh-Nikouei, Bizhan; Nassirli, Hooriyeh

2016-09-25

The interaction between losartan and human serum albumin (HSA), as well as its glycated form (gHSA) was studied by multiple spectroscopic techniques and molecular dynamics simulation under physiological conditions. The binding information, including the binding constants, effective quenching constant and number of binding sites showed that the binding partiality of losartan to HSA was higher than to gHSA. The findings of three-dimensional fluorescence spectra demonstrated that the binding of losartan to HSA and gHSA would alter the protein conformation. The distances between Trp residue and the binding sites of the drug were evaluated on the basis of the Förster theory, and it was indicated that non-radiative energy transfer from HSA and gHSA to the losartan happened with a high possibility. According to molecular dynamics simulation, the protein secondary and tertiary structure changes were compared in HSA and gHSA for clarifying the obtained results. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Human platelet lysate is a successful alternative serum supplement for propagation of monocyte-derived dendritic cells.

PubMed

Švajger, Urban

2017-04-01

Clinical protocols for dendritic cell (DC) generation from monocytes require the use of animal serum-free supplements. Serum-free media can also require up to 1% of serum supplementation. In addition, recommendations based on the 3Rs (Refinement, Reduction, Replacement) principle also recommend the use of non-animal sera in in vitro studies. The aim of this study was to explore the potential use of platelet lysate (PL) for generation of optimally differentiated DCs from monocytes. Cells were isolated from buffy coats from healthy volunteers using immunomagnetic selection. DCs were differentiated in RPMI1640 supplemented with either 10% fetal bovine serum (FBS), 10% AB serum or 10% PL with the addition of granulocyte monocyte colony stimulating factor and interleukin-4. Generated DCs were assessed for their morphology, viability, endocytotic capacity, surface phenotype (immature, mature and tolerogenic DCs) and activation of important signaling pathways. DC function was evaluated on the basis of their allostimulatory capacity, cytokine profile and ability to induce different T-helper subsets. DCs generated with PL displayed normal viability, morphology and endocytotic capacity. Their differentiation and maturation phenotype was comparable to FBS-cultured DCs. They showed functional plasticity and up-regulated tolerogenic markers in response to their environment. PL-cultured mature DCs displayed unhindered allostimulatory potential and the capacity to induce Th1 responses. The use of PL allowed for activation of crucial signaling proteins associated with DC differentiation and maturation. This study demonstrates for the first time that human PL represents a successful alternative to FBS in differentiation of DCs from monocytes. DCs display the major phenotypic and functional characteristics compared with existing culture protocols. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Direct Evidence of Intrinsic Blue Fluorescence from Oligomeric Interfaces of Human Serum Albumin.

PubMed

Bhattacharya, Arpan; Bhowmik, Soumitra; Singh, Amit K; Kodgire, Prashant; Das, Apurba K; Mukherjee, Tushar Kanti

2017-10-10

The molecular origin behind the concentration-dependent intrinsic blue fluorescence of human serum albumin (HSA) is not known yet. This unusual blue fluorescence is believed to be a characteristic feature of amyloid-like fibrils of protein/peptide and originates due to the delocalization of peptide bond electrons through the extended hydrogen bond networks of cross-β-sheet structure. Herein, by combining the results of spectroscopy, size exclusion chromatography, native gel electrophoresis, and confocal microscopy, we have shown that the intrinsic blue fluorescence of HSA exclusively originates from oligomeric interfaces devoid of any amyloid-like fibrillar structure. Our study suggests that this low energy fluorescence band is not due to any particular residue/sequence, but rather it is a common feature of self-assembled peptide bonds. The present findings of intrinsic blue fluorescence from oligomeric interfaces pave the way for future applications of this unique visual phenomenon for early stage detection of various protein aggregation related human diseases.

Computer-assisted design and synthesis of a highly selective smart adsorbent for extraction of clonazepam from human serum.

PubMed

Aqababa, Heydar; Tabandeh, Mehrdad; Tabatabaei, Meisam; Hasheminejad, Meisam; Emadi, Masoomeh

2013-01-01

A computational approach was applied to screen functional monomers and polymerization solvents for rational design of molecular imprinted polymers (MIPs) as smart adsorbents for solid-phase extraction of clonazepam (CLO) form human serum. The comparison of the computed binding energies of the complexes formed between the template and functional monomers was conducted. The primary computational results were corrected by taking into calculation both the basis set superposition error (BSSE) and the effect of the polymerization solvent using the counterpoise (CP) correction and the polarizable continuum model, respectively. Based on the theoretical calculations, trifluoromethyl acrylic acid (TFMAA) and acrylonitrile (ACN) were found as the best and the worst functional monomers, correspondingly. To test the accuracy of the computational results, three MIPs were synthesized by different functional monomers and their Langmuir-Freundlich (LF) isotherms were studied. The experimental results obtained confirmed the computational results and indicated that the MIP synthesized using TFMAA had the highest affinity for CLO in human serum despite the presence of a vast spectrum of ions. Copyright © 2012 Elsevier B.V. All rights reserved.

Serum from calorie-restricted animals delays senescence and extends the lifespan of normal human fibroblasts in vitro.

PubMed

de Cabo, Rafael; Liu, Lijuan; Ali, Ahmed; Price, Nathan; Zhang, Jing; Wang, Mingyi; Lakatta, Edward; Irusta, Pablo M

2015-03-01

The cumulative effects of cellular senescence and cell loss over time in various tissues and organs are considered major contributing factors to the ageing process. In various organisms, caloric restriction (CR) slows ageing and increases lifespan, at least in part, by activating nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylases of the sirtuin family. Here, we use an in vitro model of CR to study the effects of this dietary regime on replicative senescence, cellular lifespan and modulation of the SIRT1 signaling pathway in normal human diploid fibroblasts. We found that serum from calorie-restricted animals was able to delay senescence and significantly increase replicative lifespan in these cells, when compared to serum from ad libitum fed animals. These effects correlated with CR-mediated increases in SIRT1 and decreases in p53 expression levels. In addition, we show that manipulation of SIRT1 levels by either over-expression or siRNA-mediated knockdown resulted in delayed and accelerated cellular senescence, respectively. Our results demonstrate that CR can delay senescence and increase replicative lifespan of normal human diploid fibroblasts in vitro and suggest that SIRT1 plays an important role in these processes.

Serum from calorie-restricted animals delays senescence and extends the lifespan of normal human fibroblasts in vitro

PubMed Central

Ali, Ahmed; Price, Nathan; Zhang, Jing; Wang, Mingyi; Lakatta, Edward; Irusta, Pablo M.

2015-01-01

The cumulative effects of cellular senescence and cell loss over time in various tissues and organs are considered major contributing factors to the ageing process. In various organisms, caloric restriction (CR) slows ageing and increases lifespan, at least in part, by activating nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylases of the sirtuin family. Here, we use an in vitro model of CR to study the effects of this dietary regime on replicative senescence, cellular lifespan and modulation of the SIRT1 signaling pathway in normal human diploid fibroblasts. We found that serum from calorie-restricted animals was able to delay senescence and significantly increase replicative lifespan in these cells, when compared to serum from ad libitum fed animals. These effects correlated with CR-mediated increases in SIRT1 and decreases in p53 expression levels. In addition, we show that manipulation of SIRT1 levels by either over-expression or siRNA-mediated knockdown resulted in delayed and accelerated cellular senescence, respectively. Our results demonstrate that CR can delay senescence and increase replicative lifespan of normal human diploid fibroblasts in vitro and suggest that SIRT1 plays an important role in these processes. (185 words). PMID:25855056

Interaction of imatinib mesylate with human serum transferrin: The comparative spectroscopic studies

NASA Astrophysics Data System (ADS)

Śliwińska-Hill, Urszula

2017-02-01

Imatinib mesylate (Imt) is a tyrosine kinase inhibitor mainly used in the treatment of Philadelphia chromosome-positive chronic myelogenous leukemia (Ph + CML). Human serum transferrin is the most abundant serum protein responsible for the transport of iron ions and many endogenous and exogenous ligands. In this study the mechanism of interactions between the imatinib mesylate and all states of transferrin (apo-Tf, Htf and holo-Tf) has been investigated by fluorescence, ultraviolet-visible (UV-vis), circular dichroism (CD) and zeta potential spectroscopic methods. Based on the experimental results it was proved that under physiological conditions the imatinib mesylate binds to the each form of transferrin with a binding constant c.a. 105 M- 1. The thermodynamic parameters indicate that hydrogen bonds and van der Waals were involved in the interaction of apo-Tf with the drug and hydrophobic and ionic strength participate in the reaction of Htf and holo-Tf with imatinib mesylate. Moreover, it was shown that common metal ions, Zn2 + and Ca2 + strongly influenced apo-Tf-Imt binding constant. The CD studies showed that there are no conformational changes in the secondary structure of the proteins. No significant changes in secondary structure of the proteins upon binding with the drug and instability of apo-Tf-Imt system are the desirable effects from pharmacological point of view.

Evidence to support a contribution of polyreactive antibodies to HLA serum reactivity

PubMed Central

Gao, Baoshan; Rong, Chunshu; Porcheray, Fabrice; Moore, Carolina; Girouard, Timothy C.; Saidman, Susan L.; Wong, Waichi; Fu, Yaowen; Zorn, Emmanuel

2015-01-01

Background Assessing the serum reactivity to HLA is essential for the evaluation of transplant candidates and the follow-up of allograft recipients. In this study, we look for evidence at the clonal level that polyreactive antibodies cross-reactive to apoptotic cells and multiple autoantigens can also react to HLA and contribute to the overall serum reactivity. Methods We immortalized B cell clones from the blood of two kidney transplant recipients and characterized their reactivity to self-antigens, apoptotic cells as well as native, denatured and cryptic HLA determinants using ELISA, immunofluorescence, flow cytometry and Luminex assays. We also assessed the reactivity of 300 pre-transplant serum specimens to HLA and apoptotic cells. Results We report here 4 distinct B cell clones cross-reactive to self and HLA class I. All 4 clones reacted to numerous HLA class I alleles but did not appear to target canonical “shared” epitopes. In parallel experiments, we observed a strong correlation between IgG reactivity to HLA and apoptotic cells in pre-transplant serum samples collected from 300 kidney transplant recipients. Further analysis revealed that samples with higher reactivity to apoptotic cells displayed significantly higher class I percent PRA compared to samples with low reactivity to apoptotic cells. Conclusions We provide here 1) proof of principle at the clonal level that human polyreactive antibodies can cross-react to HLA, multiple self-antigens and apoptotic cells and 2) supportive evidence that polyreactive antibodies contribute to overall HLA reactivity in the serum of patients awaiting kidney transplant. PMID:26285015

Development of a serum-free co-culture of human intestinal epithelium cell-lines (Caco-2/HT29-5M21)

PubMed Central

Nollevaux, Géraldine; Devillé, Christelle; El Moualij, Benaïssa; Zorzi, Willy; Deloyer, Patricia; Schneider, Yves-Jacques; Peulen, Olivier; Dandrifosse, Guy

2006-01-01

Background The absorptive and goblet cells are the main cellular types encountered in the intestine epithelium. The cell lineage Caco-2 is a model commonly used to reproduce the features of the bowel epithelium. However, there is a strong debate regarding the value of Caco-2 cell culture to mimick in vivo situation. Indeed, some authors report in Caco-2 a low paracellular permeability and an ease of access of highly diffusible small molecules to the microvilli, due to an almost complete lack of mucus. The HT29-5M21 intestinal cell lineage is a mucin-secreting cellular population. A co-culture system carried out in a serum-free medium and comprising both Caco-2 and HT29-5M21 cells was developed. The systematic use of a co-culture system requires the characterization of the monolayer under a given experimental procedure. Results In this study, we investigated the activity and localization of the alkaline phosphatase and the expression of IAP and MUC5AC genes to determine a correlation between these markers and the cellular composition of a differentiated monolayer obtained from a mixture of Caco-2 and HT29-5M21 cells. We observed that the culture conditions used (serum-free medium) did not change the phenotype of each cell type, and produced a reproducible model. The alkaline phosphatase expression characterizing Caco-2 cells was influenced by the presence of HT29-5M21 cells. Conclusion The culture formed by 75% Caco-2 and 25% HT29-5M21 produce a monolayer containing the two main cell types of human intestinal epithelium and characterized by a reduced permeability to macromolecules. PMID:16670004

Polychlorinated biphenyls in Spanish adults: Determinants of serum concentrations

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

Agudo, Antonio, E-mail: a.agudo@iconcologia.net; Goni, Fernando; CIBER Epidemiologia y Salud Publica

Background: Polychlorinated biphenyls (PCBs) are persistent compounds that may pose an environmental hazard to humans, food being the main source of exposure for the general population. Objective: To measure the serum concentrations of the main PCBs in subjects from the general population in Spain, and to assess potential determinants of such concentrations. Methods: Serum was obtained from blood samples of 953 subjects aged 35-64 years, residents in five Spanish regions (three from the North and two from the South), randomly selected from the EPIC-Spain cohort. Blood collection took place during 1992-1996 and four PCB congeners (118, 138, 153 and 180)more » were determined by means of gas chromatography with electron-capture detection (GC-ECD). Results: The concentration of total PCBs was 459 ng/g lipids (or 3.1 {mu}g/l); the corresponding figures for PCB 153 were 186 ng/g lipids and 1.25 {mu}g/l. Men had higher values than women, PCB levels increased with age, and serum concentration of PCBs was higher in northern regions. Body mass index (BMI) was inversely related to PCB concentrations, and fish intake was the dietary factor showing the greatest association with serum PCBs. The pattern described was similar for each congener separately. Conclusions: We found concentrations similar to those reported in European countries where blood collection was carried during the same period. Regional differences within Spain are not fully explained by anthropometric or dietary factors. The inverse association with BMI suggests that in the mid-1990s there was still ongoing or recent exposure to PCBs in Spain.« less

Isomer-Specific Binding Affinity of Perfluorooctanesulfonate (PFOS) and Perfluorooctanoate (PFOA) to Serum Proteins.

PubMed

Beesoon, Sanjay; Martin, Jonathan W

2015-05-05

Perfluorooctanesulfonate (PFOS) and perfluorooctanoate (PFOA) are among the most prominent contaminants in human serum, and these were historically manufactured as technical mixtures of linear and branched isomers. The isomers display unique pharmacokinetics in humans and in animal models, but molecular mechanisms underlying isomer-specific PFOS and PFOA disposition have not previously been studied. Here, ultrafiltration devices were used to examine (i) the dissociation constants (Kd) of individual PFOS and PFOA isomers with human serum albumin (HSA) and (ii) relative binding affinity of isomers in technical mixtures spiked to whole calf serum and human serum. Measurement of HSA Kd's demonstrated that linear PFOS (Kd=8(±4)×10(-8) M) was much more tightly bound than branched PFOS isomers (Kd range from 8(±1)×10(-5) M to 4(±2)×10(-4) M). Similarly, linear PFOA (Kd=1(±0.9)×10(-4) M) was more strongly bound to HSA compared to branched PFOA isomers (Kd range from 4(±2)×10(-4) M to 3(±2)×10(-4) M). The higher binding affinities of linear PFOS and PFOA to total serum protein were confirmed when both calf serum and human serum were spiked with technical mixtures. Overall, these data provide a mechanistic explanation for the longer biological half-life of PFOS in humans, compared to PFOA, and for the higher transplacental transfer efficiencies and renal clearance of branched PFOS and PFOA isomers, compared to the respective linear isomer.

[Effects of n-hexane exposure on human serum myelin basic proteins].

PubMed

Zhou, Wei; Yi, Juan; Huang, Hui-Ping; Xiang, Ying-Ping; He, Jia-Xi; Liu, Qing-Jun; Huang, Xian-Qing

2011-06-01

To explore the effects of n-hexane on expression of serum myelin proteins (MBP) in workers occupationally exposed to n-hexane. In this study, 269 workers exposed to n-hexane for more than one year and 104 subjects not exposed to n-hexane served as the exposure group and the control group, respectively. The urinary 2,5-hexanedione levels in all subjects were detected. On the basis of urinary 2,5-hexanedione levels, the exposure group was divided into the high exposure sub-group and low exposure sub-group. The serum myelin basic protein (MBP) levels were measured by ELISA kit. The mean concentration of urinary 2,5-hexanedione in the exposed group was (3.10 +/- 1.35) mg/L. The concentration of urinary 2,5-hexanedione in the control group was undetectable. The levels of serum MBP in the high exposure sub-group and low exposure sub-group were (2.43 +/- 0.24) and (1.62 +/- 0.23) microg/L, respectively, which were significantly higher than that (0.78 +/- 0.12) microg/L in the controls (P < 0.01). Pearson correlation analysis showed the positive correlation between serum MBP levels and urinary 2,5-hexanedione levels (r = 0.781, P < 0.01). The results of present study showed that the serum MBP levels of workers occupationally exposed to n-hexane significantly elevated, and the serum MBP can serve as the effective biomarker of n-hexane exposure.

Liver lipase and high-density lipoprotein. Lipoprotein changes after incubation of human serum with rat liver lipase.

PubMed

Groot, P H; Scheek, L M; Jansen, H

1983-05-16

Human sera were incubated with rat liver lipase after inactivation of lecithin:cholesterol acyltransferase, and the changes in serum lipoprotein composition were measured. In the presence of liver lipase serum triacylglycerol and phosphatidylcholine were hydrolyzed. The main changes in the concentrations of these lipids were found in the high-density lipoprotein fraction. Subfractionation of high-density lipoprotein by rate-zonal ultracentrifugation showed a prominent decrease in all constituents of high-density lipoprotein2, a smaller decrease in the 'light' high-density lipoprotein3 and an increase in the 'heavy' high-density lipoprotein3. These data support a concept in which liver lipase is involved in high-density lipoprotein2 phospholipid and triacylglycerol catabolism and suggest that as a result of this action high-density lipoprotein2 is converted into high-density lipoprotein3.

Increased production of 4 kDa amyloid beta peptide in serum deprived human primary neuron cultures: possible involvement of apoptosis.

PubMed

LeBlanc, A

1995-12-01

The etiology of the amyloid beta peptide in sporadic Alzheimer's disease (AD) is not known. Amyloid beta peptide (A beta), a proteolytic product of the amyloid precursor protein (APP), is deposited in the senile plaques and cerebrovascular tissues of individuals with either sporadic or familial AD (FAD). Increased A beta production from mutant APPs in FAD fosters the hypothesis that overexpression of A beta plays a primary role in the pathogenesis of AD. The absence of APP mutations in sporadic AD which displays identical pathological features than FAD such as synapse and neuronal loss, senile plaques and neurofibrillary tangles, suggests other causes for overexpression and/or deposition of A beta. To investigate the effect of neuronal death on APP metabolism and A beta secretion, human primary neuron cultures were induced to undergo apoptosis by serum deprivation. Serum deprived neurons display shrunken and rounded morphology, contain condensed chromatine and fragmented DNA, which are characteristic of apoptosis. In serum deprived neurons, metabolism of APP through the nonamyloidogenic secretory pathway is decreased to 20% from 40% in control cultures whereas 4kDa A beta is increased three- to fourfold. The results suggest that human neurons undergoing apoptosis generate excess A beta and indicates a possible mechanism for increased A beta in the absence of APP mutations.

Determination of phosphonoformate (foscarnet) in calf and human serum by automated solid-phase extraction and high-performance liquid chromatography with amperometric detection.

PubMed

Ba, B B; Corniot, A G; Ducint, D; Breilh, D; Grellet, J; Saux, M C

1999-03-05

An isocratic high-performance liquid chromatographic method with automated solid-phase extraction has been developed to determine foscarnet in calf and human serums. Extraction was performed with an anion exchanger, SAX, from which the analyte was eluted with a 50 mM potassium pyrophosphate buffer, pH 8.4. The mobile phase consisted of methanol-40 mM disodium hydrogenphosphate, pH 7.6 containing 0.25 mM tetrahexylammonium hydrogensulphate (25:75, v/v). The analyte was separated on a polyether ether ketone (PEEK) column 150x4.6 mm I.D. packed with Kromasil 100 C18, 5 microm. Amperometric detection allowed a quantification limit of 15 microM. The assay was linear from 15 to 240 microM. The recovery of foscarnet from calf serum ranged from 60.65+/-1.89% for 15 microM to 67.45+/-1.24% for 200 microM. The coefficient of variation was < or = 3.73% for intra-assay precision and < or =7.24% for inter-assay precision for calf serum concentrations ranged from 15 to 800 microM. For the same samples, the deviation from the nominal value ranged from -8.97% to +5.40% for same day accuracy and from -4.50% to +2.77% for day-to-day accuracy. Selectivity was satisfactory towards potential co-medications. Replacement of human serum by calf serum for calibration standards and quality control samples was validated. Automation brought more protection against biohazards and increase in productivity for routine monitoring and pharmacokinetic studies.

Mayonnaise contributes to increasing postprandial serum β-carotene concentration through the emulsifying property of egg yolk in rats and humans.

PubMed

Takeda, Sayaka; Kimura, Mamoru; Marushima, Ranko; Takeuchi, Ayako; Takizawa, Kazue; Ogino, Yuji; Masuda, Yasunobu; Kunou, Masaaki; Hasegawa, Mineo; Maruyama, Chizuko

2011-01-01

We performed in vitro, animal, and human studies to clarify the effect of mayonnaise on β-carotene intake and its mechanism. In an artificial gastric juice model, we examined the transfer of β-carotene from grated carrot to mayonnaise or vegetable oil. Mayonnaise was more easily dispersed in artificial gastric juice than vegetable oil. The β-carotene concentration was greater in mayonnaise than vegetable oil. In rats, the postprandial serum β-carotene concentration in the mayonnaise group (β-carotene with mayonnaise) was higher than that in the control (β-carotene only) and vegetable oil (β-carotene with vegetable oil) groups. Continuous feeding of dietary β-carotene (14 d), employing mayonnaise or egg yolk, resulted in an increased accumulation of β-carotene in the liver. In a human study, diets were provided in the form of (1) carrot as a control (CON), (2) carrot juice (JU), (3) carrot with oil (OIL) and (4) carrot with mayonnaise (MS). Following collection of fasting blood samples, nine adult males consumed one of the four diets in random order. Fasting and postprandial changes in serum β-carotene were assessed at 2, 3, 4, 6 and 8 h following ingestion of each diet. The incremental areas under the curves of serum β-carotene concentration were higher following MS than following both CON and JU. In conclusion, we suggest that mayonnaise contributes to raising the serum β-carotene concentration when consumed with carrots rich in β-carotene, and that its mechanism is related to the emulsifying property of the egg yolk contained in mayonnaise.

Extending the Serum Half-Life of G-CSF via Fusion with the Domain III of Human Serum Albumin

PubMed Central

Zhao, Shuqiang; Zhang, Yu; Tian, Hong; Chen, Xiaofei; Cai, Di; Yao, Wenbing; Gao, Xiangdong

2013-01-01

Protein fusion technology is one of the most commonly used methods to extend the half-life of therapeutic proteins. In this study, in order to prolong the half-life of Granulocyte colony stimulating factor (G-CSF), the domain III of human serum albumin (3DHSA) was genetically fused to the N-terminal of G-CSF. The 3DHSA-G-CSF fusion gene was cloned into pPICZαA along with the open reading frame of the α-factor signal under the control of the AOX1 promoter. The recombinant expression vector was transformed into Pichia pastoris GS115, and the recombinant strains were screened by SDS-PAGE. As expected, the 3DHSA-G-CSF showed high binding affinity with HSA antibody and G-CSF antibody, and the natural N-terminal of 3DHSA was detected by N-terminal sequencing. The bioactivity and pharmacokinetic studies of 3DHSA-G-CSF were respectively determined using neutropenia model mice and human G-CSF ELISA kit. The results demonstrated that 3DHSA-G-CSF has the ability to increase the peripheral white blood cell (WBC) counts of neutropenia model mice, and the half-life of 3DHSA-G-CSF is longer than that of native G-CSF. In conclusion, 3DHSA can be used to extend the half-life of G-CSF. PMID:24151579

Quantitation of ortho-cresyl phosphate adducts to butyrylcholinesterase in human serum by immunomagnetic-UHPLC-MS/MS.

PubMed

Johnson, Darryl; Carter, Melissa D; Crow, Brian S; Isenberg, Samantha L; Graham, Leigh Ann; Erol, H Akin; Watson, Caroline M; Pantazides, Brooke G; van der Schans, Marcel J; Langenberg, Jan P; Noort, Daan; Blake, Thomas A; Thomas, Jerry D; Johnson, Rudolph C

2015-04-01

Tri-ortho-cresyl phosphate (ToCP) is an anti-wear, flame retardant additive used in industrial lubricants, hydraulic fluids and gasoline. The neurotoxic effects of ToCP arise from the liver-activated metabolite 2-(o-cresyl)-4H-1,3,2-benzodioxaphosphoran-2-one (cresyl saligenin phosphate or CBDP), which inhibits esterase enzymes including butyrylcholinesterase (BChE). Following BChE adduction, CBDP undergoes hydrolysis to form the aged adduct ortho-cresyl phosphoserine (oCP-BChE), thus providing a biomarker of CBDP exposure. Previous studies have identified ToCP in aircraft cabin and cockpit air, but assessing human exposure has been hampered by the lack of a laboratory assay to confirm exposure. This work presents the development of an immunomagnetic-UHPLC-MS/MS method for the quantitation of unadducted BChE and the long-term CBDP biomarker, oCP-BChE, in human serum. The method has a reportable range from 2.0 ng/ml to 150 ng/ml, which is consistent with the sensitivity of methods used to detect organophosphorus nerve agent protein adducts. The assay demonstrated high intraday and interday accuracy (≥85%) and precision (RSD ≤ 15%) across the calibration range. The method was developed for future analyses of potential human exposure to CBDP. Analysis of human serum inhibited in vitro with CBDP demonstrated that the oCP-BChE adduct was stable for at least 72 h at 4, 22 and 37 °C. Compared to a previously reported assay, this method requires 75% less sample volume, reduces analysis time by a factor of 20 and demonstrates a threefold improvement in sensitivity. Published 2015. This article is a U.S. Government work and is in the public domain in the USA. Published 2015. This article is a U.S. Government work and is in the public domain in the USA.

The cytotoxic effect of spiroflavanone derivatives, their binding ability to human serum albumin (HSA) and a DFT study on the mechanism of their synthesis

NASA Astrophysics Data System (ADS)

Budzisz, Elzbieta; Paneth, Piotr; Geromino, Inacrist; Muzioł, Tadeusz; Rozalski, Marek; Krajewska, Urszula; Pipiak, Paulina; Ponczek, Michał B.; Małecka, Magdalena; Kupcewicz, Bogumiła

2017-06-01

This paper examines the cytotoxic effect of nine compounds with spiropyrazoline structures, and determines the reaction mechanism between diazomethane and selected benzylideneflavanones, their lipophilicity, and their binding ability to human serum albumin. The cytotoxic effect was determined on two human leukaemia cell lines (HL-60 and NALM-6) and melanoma WM-115 cells, as well as on normal human umbilical vein endothelial cells (HUVEC). The highest cytotoxicity was exhibited by compound B7: it was found to have an IC50 of less than 10 μM for all three cancer cell lines, with five to 12-fold lower sensitivity against normal cells (HUVEC). All the compounds exhibit comparable affinity energy in human serum albumin binding (from -8.1 to -8.6 kcal mol-1) but vary in their binding sites depending on the substituent. X-ray crystallography of two derivatives confirmed their synthetic pathway, and their structures were carefully examined.

Influenza Virus Hemagglutinin Stalk-Specific Antibodies in Human Serum are a Surrogate Marker for In Vivo Protection in a Serum Transfer Mouse Challenge Model.

PubMed

Jacobsen, Henning; Rajendran, Madhusudan; Choi, Angela; Sjursen, Haakon; Brokstad, Karl A; Cox, Rebecca J; Palese, Peter; Krammer, Florian; Nachbagauer, Raffael

2017-09-19

The immunogenicity of current influenza virus vaccines is assessed by measuring an increase of influenza virus-specific antibodies in a hemagglutination inhibition assay. This method exclusively measures antibodies against the hemagglutinin head domain. While this domain is immunodominant, it has been shown that hemagglutination inhibition titers do not always accurately predict protection from disease. In addition, several novel influenza virus vaccines that are currently under development do not target the hemagglutinin head domain, but rather more conserved sites, including the hemagglutinin stalk. Importantly, antibodies against the hemagglutinin stalk do not show activity in hemagglutination inhibition assays and will require different methods for quantification. In this study, we tested human serum samples from a seasonal influenza virus vaccination trial and an avian H5N1 virus vaccination trial for antibody activities in multiple types of assays, including binding assays and also functional assays. We then performed serum transfer experiments in mice which then received an H1N1 virus challenge to assess the in vivo protective effects of the antibodies. We found that hemagglutinin-specific antibody levels measured in an enzyme-linked immunosorbent assay (ELISA) correlated well with protection from weight loss in mice. In addition, we found that weight loss was also inversely correlated with the level of serum antibody-dependent cellular cytotoxicity (ADCC) as measured in a reporter assay. These findings indicate that protection is in part conferred by Fc-dependent mechanisms. In conclusion, ELISAs can be used to measure hemagglutinin-specific antibody levels that could serve as a surrogate marker of protection for universal influenza virus vaccines. IMPORTANCE Influenza viruses are a serious concern for public health and cause a large number of deaths worldwide every year. Current influenza virus vaccines can confer protection from disease, but they often

Serum Soluble (Pro)Renin Receptor Levels in Maintenance Hemodialysis Patients

PubMed Central

Amari, Yoshifumi; Morimoto, Satoshi; Nakajima, Fumitaka; Ando, Takashi; Ichihara, Atsuhiro

2016-01-01

The (pro)renin receptor [(P)RR] is cleaved by furin to generate soluble (P)RR [s(P)RR], which reflects the status of the tissue renin-angiotensin system. Hemodialysis patients have advanced atherosclerosis. The aim of this study was to investigate the relationships between serum s(P)RR levels and background factors, including indices of atherosclerosis, in hemodialysis patients. Serum s(P)RR levels were measured in hemodialysis patients and clearance of s(P)RR through the membrane of the dialyzer was examined. Furthermore, relationships between serum s(P)RR levels and background factors were assessed. Serum s(P)RR levels were significantly higher in hemodialysis patients (30.4 ± 6.1 ng/ml, n = 258) than those in subjects with normal renal function (21.4 ± 6.2 ng/ml, n = 39, P < 0.0001). Clearance of s(P)RR and creatinine were 56.9 ± 33.5 and 147.6 ± 9.50 ml/min, respectively. Serum s(P)RR levels were significantly higher in those with ankle-brachial index (ABI) of < 0.9, an indicator of severe atherosclerosis, than those with ABI of ≥ 0.9 (32.2 ± 5.9 and 30.1 ± 6.2 ng/ml, respectively, P < 0.05). An association between low ABI and high serum s(P)RR levels was observed even after correction for age, history of smoking, HbA1c, and LDL-C. Serum s(P)RR levels were significantly higher in hemodialysis patients when compared with subjects with normal renal function, although s(P)RR is dialyzed to some extent, but to a lesser extent than creatinine. High serum s(P)RR levels may be associated with atherosclerosis independent of other risk factors, suggesting that serum s(P)RR could be used as a marker for atherosclerotic conditions in hemodialysis patients. PMID:27367528

Serum-free Erythroid Differentiation for Efficient Genetic Modification and High-Level Adult Hemoglobin Production.

PubMed

Uchida, Naoya; Demirci, Selami; Haro-Mora, Juan J; Fujita, Atsushi; Raines, Lydia N; Hsieh, Matthew M; Tisdale, John F

2018-06-15

In vitro erythroid differentiation from primary human cells is valuable to develop genetic strategies for hemoglobin disorders. However, current erythroid differentiation methods are encumbered by modest transduction rates and high baseline fetal hemoglobin production. In this study, we sought to improve both genetic modification and hemoglobin production among human erythroid cells in vitro . To model therapeutic strategies, we transduced human CD34 + cells and peripheral blood mononuclear cells (PBMCs) with lentiviral vectors and compared erythropoietin-based erythroid differentiation using fetal-bovine-serum-containing media and serum-free media. We observed more efficient transduction (85%-93%) in serum-free media than serum-containing media (20%-69%), whereas the addition of knockout serum replacement (KSR) was required for serum-free media to promote efficient erythroid differentiation (96%). High-level adult hemoglobin production detectable by electrophoresis was achieved using serum-free media similar to serum-containing media. Importantly, low fetal hemoglobin production was observed in the optimized serum-free media. Using KSR-containing, serum-free erythroid differentiation media, therapeutic adult hemoglobin production was detected at protein levels with β-globin lentiviral transduction in both CD34 + cells and PBMCs from sickle cell disease subjects. Our in vitro erythroid differentiation system provides a practical evaluation platform for adult hemoglobin production among human erythroid cells following genetic manipulation.