GLC of Serum Phenylalanine - a Gas Liquid Chromatographic Method for the Determination of Phenylalanine Serum
Gas liquid chromatographic method for quantitative analysis of phenylalanine in serum
Close, V. A.↗
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Binding of histidine and human serum albumin to dirhodium(II) tetraacetate
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Jalilehvand, Farideh↗
Serum tryptophan level after carbohydrate ingestion - Selective decline in non-albumin-bound tryptophan coincident with reduction in serum free fatty acids.
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Lipsett, D.↗
Serum tryptophan level after carbohydrate ingestion - Selective decline in non-albumin-bound tryptophan coincident with reduction in serum free fatty acids.
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Lipsett, D.↗
Effect of Serum from Chickens Treated with Clenbuterol on Myosin Accumulation, Beta-Adrenergic Receptor Population, and Cyclic AM Synthesis in Embryonic Chicken Skeletal Muscle Cell Cultures
Broiler chickens at 35 days of age were fed 1 ppm clenbuterol for 14 days. This level of dietary clenbuterol led to 5-7% increases in weights of leg and breast muscle tissue. At the end of the 14-day period, serum was prepared from both control and clenbuterol-treated chickens and was then employed as a component of cell culture media at a final concentration of 20% (v/v). Muscle cell cultures were prepared from both the leg and breast muscle groups of twelve-day chick embryos. Treatment groups included control chicken serum to which 10 nM, 50 nM, and 1 micron clenbuterol had been added, as well as cells grown in media containing 10% horse serum. Cultures were subjected to each treatment for 3 days beginning on the seventh day in culture. Neither the percent fusion nor the number of nuclei in myotubes were significantly affected by any of the treatments. The quantity of MHC was not increased by serum from clenbuterol-treated chickens in either breast and leg muscle cultures; however, MHC quantity was 50- 100% higher in cultures grown in control chicken serum to which 10 nM and 50 nM clenbuterol had also been added. The Beta-AR population was 4,000-7,000 Beta-AR per cell in cultures grown in chicken serum, with leg muscle cultures having approximately 25-30% more receptors than breast muscle cultures. Receptor population was not significantly affected by the presence of clenbuterol or by the presence of serum from clenbuterol-treated chickens. In contrast, the Beta-AR population in leg and breast muscle cultures grown in the presence of 10% horse serum was 18,000-20,000 Beta-AR per cell. Basal concentration of cAMP was not significantly affected by any of the treatments. When cultures grown in chicken serum were stimulated for 10 min with 1 micron isoproterenol, limited increases of 12-20% in cAMP concentration above basal levels were observed. However, when cultures grown in the presence of horse serum were stimulated with 1 micron isoproterenol, increases of 600-800 % in cAMP concentration above basal levels were observed. Thus, not only did cells grown in horse serum have a higher Beta-AR population, each receptor had a higher capacity for cAMP synthesis following isoproterenol stimulation. Finally, the hypothesis was tested that clenbuterol exerts its action on muscle protein content by changes in cAMP concentration. No correlation was apparent between basal cAMP concentration and MHC content.
Young, R. B.↗
Effect of Serum from Chickens Treated with Clenbuterol on Myosin Accumulation, Beta-Adrenergic Receptor Population, and Cyclic AMP Synthesis in Embryonic Chicken Skeletal Muscle Cell Cultures
Broiler chickens at 35 d of age were fed 1 ppm clenbuterol for 14 d. This level of dietary clenbuterol led to 5-7% increases in the weights of leg and breast muscle tissue. At the end of the 14-d period, serum was prepared from both control and clenbuterol-treated chickens, and was then employed as a component of cell culture media at a final concentration of 20% (v/v). Muscle cell cultures were prepared from both the leg and the breast muscle groups of 12-d chick embryos. Treatment groups included control chicken serum to which 10 nM, 50 nM, and 1 uM clenbuterol had been added, as well as cells grown in media containing 10% horse serum. Cultures were subjected to each treatment for 3 d, beginning on the seventh d in culture. Neither the percent fusion nor the number of nuclei in myotubes was significantly affected by any of the treatments. The quantity of myosin heavy chains (MHCs) was not increased by serum from clenbuterol-treated chickens in either breast or leg muscle cultures; however, the MHC quantity was 50-150% higher in cultures grown in control chicken serum to which 10 and 50 nM clenbuterol had also been added. The B-adrenergic receptor (betaAR) population was 4000-7000 betaARs per cell in cultures grown in chicken serum with leg muscle cultures having approximately 25-30% more receptors than breast muscle Culture. Receptor population was not significantly affected by the presence of clenbuterol or by the presence of serum from clenbuterol-treated chickens. In contrast, the betaAR Population in leg and breast muscle cultures grown in the presence of 10% horse serum was 16,000-18,000 betaARs per cell. Basal concentration of cyclic adenosine 3':5'monophosphate (cAMP) was not significantly affected by the treatments. When cultures grown in chicken serum were stimulated for 10 min with 1 uM isoproterenol, limited increases of 12-20% in cAMP Concentration above the. basal levels were observed. However, when cultures grown in the presence of horse serum were stimulated with 1 uM isoproterenol, cAMP concentration was stimulated 5- to 9-fold above the basal levels. Thus, not only did cells, grown in horse serum have a higher PAR population, but also each receptor had a higher capacity for cAMP synthesis following isoproterenol stimulation. Finally, the hypothesis that clenbuterol exerts its action on muscle protein content by changes in cAMP concentration was tested. No correlation was apparent between basal cAMP concentration and MHC content.
Young, Ronald B.↗
Quantitating Iron in Serum Ferritin by Use of ICP-MS
A laboratory method has been devised to enable measurement of the concentration of iron bound in ferritin from small samples of blood (serum). Derived partly from a prior method that depends on large samples of blood, this method involves the use of an inductively-coupled-plasma mass spectrometer (ICP-MS). Ferritin is a complex of iron with the protein apoferritin. Heretofore, measurements of the concentration of serum ferritin (as distinguished from direct measurements of the concentration of iron in serum ferritin) have been used to assess iron stores in humans. Low levels of serum ferritin could indicate the first stage of iron depletion. High levels of serum ferritin could indicate high levels of iron (for example, in connection with hereditary hemochromatosis an iron-overload illness that is characterized by progressive organ damage and can be fatal). However, the picture is complicated: A high level of serum ferritin could also indicate stress and/or inflammation instead of (or in addition to) iron overload, and low serum iron concentration could indicate inflammation rather than iron deficiency. Only when concentrations of both serum iron and serum ferritin increase and decrease together can the patient s iron status be assessed accurately. Hence, in enabling accurate measurement of the iron content of serum ferritin, the present method can improve the diagnosis of the patient s iron status. The prior method of measuring the concentration of iron involves the use of an atomic-absorption spectrophotometer with a graphite furnace. The present method incorporates a modified version of the sample- preparation process of the prior method. First, ferritin is isolated; more specifically, it is immobilized by immunoprecipitation with rabbit antihuman polyclonal antibody bound to agarose beads. The ferritin is then separated from other iron-containing proteins and free iron by a series of centrifugation and wash steps. Next, the ferritin is digested with nitric acid to extract its iron content. Finally, a micronebulizer is used to inject the sample of the product of the digestion into the ICPMS for analysis of its iron content. The sensitivity of the ICP-MS is high enough to enable it to characterize samples smaller than those required in the prior method (samples can be 0.15 to 0.60 mL).
Smith, Scott M.↗
A serum factor promotes collagenase synthesis by an osteoblastic cell line
Regulation of the synthesis of collagenase was investigated in the osteoblastic cell line, UMR 106-01. The cells were stained by the avidin-biotin-complex technique for the presence of the enzyme. By this method, it was possible to identify cells producing collagenase. Synthesis, but not secretion, was found to be constitutive in these cells with the enzyme located intracellularly in cytoplasmic vesicles and the Golgi apparatus. The amount of collagenase contained within UMR cells and the number of cells synthesizing the enzyme were proportional to the concentration of fetal bovine serum in the incubating medium. When serum was withdrawn from the osteosarcoma cells, the content of collagenase decreased with time and the enzyme became undetectable by 48 h of serum depletion. The decrease in collagenase content could be completely reversed by resupplying serum to the cells. The collagenase promoting activity of serum could not be eliminated by adsorption on activated charcoal but was retained by a dialysis membrane with a 12,000 mol wt cutoff. A range of bone-seeking hormones or agents known to affect collagenase secretion was added to the medium in an attempt to mimic the effect of serum on collagenase accumulation. None of these agonists, including parathyroid hormone, could reproduce the effect of serum on these cells, although parathyroid hormone could act as a collagenase secretagogue in the presence or absence of serum. It is concluded that fetal bovine serum contains a yet unidentified factor or factors greater than 12,000 mol wt responsible for the continued synthesis of collagenase by UMR 106-01 cells.
Non-NASA Center↗
Comparative analysis of SARS-CoV-2 neutralization titers reveals consistency between human and animal model serum and across assays
The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires ongoing monitoring to judge the ability of newly arising variants to escape the immune response. A surveillance system necessitates an understanding of differences in neutralization titers measured in different assays and using human and animal serum samples. We compared 18 datasets generated using human, hamster, and mouse serum and six different neutralization assays. Datasets using animal model serum samples showed higher titer magnitudes than datasets using human serum samples in this comparison. Fold change in neutralization of variants compared to ancestral SARS-CoV-2, immunodominance patterns, and antigenic maps were similar among serum samples and assays. Most assays yielded consistent results, except for differences in fold change in cytopathic effect assays. Hamster serum samples were a consistent surrogate for human first-infection serum samples. These results inform the transition of surveillance of SARS-CoV-2 antigenic variation from dependence on human first-infection serum samples to the utilization of serum samples from animal models.
Cell Biology↗
Retrospective Study of Serum Sclerostin Measurements in Bed Rest Subjects
Animal models and human studies suggest that osteocytes regulate the skeleton s response to mechanical unloading at the cellular level in part by an increase in sclerostin, an inhibitor of the anabolic Wnt pathway. However, few studies have reported changes in serum sclerostin in humans exposed to reduced mechanical loading. Thus, we determined changes in serum sclerostin and bone turnover markers in healthy adult men who participated in a controlled bed rest study. Seven healthy adult men (31 +/- 3 yrs old) underwent 90-day six-degree head down tilt bed rest at the University of Texas Medical Branch in Galveston's Institute for Translational Sciences - Clinical Research Center (ITS-CRC). Serum sclerostin, PTH, serum markers of bone turnover (bone specific alkaline phosphatase, RANKL/OPG, and osteocalcin), urinary calcium and phosphorus excretion, and 24 hour pooled urinary markers of bone resorption (NTX, DPD, PYD) were evaluated pre-bed rest (BL), bed rest day 28 (BR-28), bed rest day 60 (BR-60), and bed rest day 90 (BR-90). In addition, bone mineral density (BMD) was assessed by dual-energy X-ray absorptiometry (DXA) at BL, BR-60, and post bed rest day 5 (BR+5). Data are reported as mean +/- standard deviation. We used repeated measures ANOVA to compare baseline values to BR-28, BR-60, and BR-90. RESULTS Consistent with prior reports, BMD declined significantly (1-2% per month) at weight-bearing skeletal sites (spine, hip, femur neck, and calcaneus). Serum sclerostin levels were elevated above BL at BR-28 (+29% +/- 20%, p = 0.003), BR-60 (+42% +/- 31%, p < 0.001), and BR-90 (22% +/- 21%, p = 0.07). Serum PTH levels were reduced at BR-28 (-17% +/- 16%, p = 0.02), BR-60 (-24% +/- 14%, p = 0.03), and returned to baseline at BR-90 (-21% +/- 21%, p = 0.14). Serum bone turnover markers did not change, however urinary bone resorption markers and calcium were significantly elevated following bed rest (p < 0.01). CONCLUSION We observed an increase of serum sclerostin associated with decreased serum PTH and elevated bone resorption markers in otherwise healthy men subjected to long-term immobilization.
Spatz, J. M.↗
Divergent effects of sex and calcium/vitamin D supplementation on serum magnesium and markers of bone structure and function during initial military training
Maintaining Mg status may be important for military recruits, a population that experiences high rates of stress fracture during initial military training (IMT). The objectives of this secondary analysis were to (1) compare dietary Mg intake and serum Mg in female and male recruits pre- and post-IMT, (2) determine whether serum Mg was related to parameters of bone health pre-IMT, and (3) whether Ca and vitamin D supplementation (Ca/vitamin D) during IMT modified serum Mg. Females (n 62) and males (n 51) consumed 2000 mg of Ca and 25 μg of vitamin D/d or placebo during IMT (12 weeks). Dietary Mg intakes were estimated using FFQ, serum Mg was assessed and peripheral quantitative computed tomography was performed on the tibia. Dietary Mg intakes for females and males pre-IMT were below the estimated average requirement and did not change with training. Serum Mg increased during IMT in females (0·06 ± 0·08 mmol/l) compared with males (–0·02 ± 0·10 mmol/l; P < 0·001) and in those consuming Ca/vitamin D (0·05 ± 0·09 mmol/l) compared with placebo (0·001 ± 0·11 mmol/l; P = 0·015). In females, serum Mg was associated with total bone mineral content (BMC, β = 0·367, P = 0·004) and robustness (β = 0·393, P = 0·006) at the distal 4 % site, stress–strain index of the polaris axis (β = 0·334, P = 0·009) and robustness (β = 0·420, P = 0·004) at the 14 % diaphyseal site, and BMC (β = 0·309, P = 0·009) and stress–strain index of the polaris axis (β = 0·314, P = 0·006) at the 66 % diaphyseal site pre-IMT. No significant relationships between serum Mg and bone measures were observed in males. Findings suggest that serum Mg may be modulated by Ca/vitamin D intake and may impact tibial bone health during training in female military recruits.
59 BASIC BIOLOGICAL SCIENCES↗
Temporal serum neurofilament light chain concentrations in sheep inoculated with the agent of classical scrapie
Objective: Neurofilament light chain (Nf-L) has been used to detect neuroaxonal damage in the brain caused by physical injury or disease. The purpose of this study was to determine if serum Nf-L could be used as a biomarker for pre-symptomatic detection of scrapie in sheep. Methods: Four sheep with prion protein genotype AVQQ were intranasally inoculated with the classical scrapie strain x124. Blood was collected every 4 weeks until 44 weeks post-inoculation, at which point weekly collection commenced. Serum was analyzed using single molecule array (Quanterix SR-X) to evaluate Nf-L concentrations. Results: Scrapie was confirmed in each sheep by testing homogenized brainstem at the level of the obex with a commercially available enzyme immunoassay. Increased serum Nf-L concentrations were identified above the determined cutoff during the last tenth of the respective incubation period for each sheep. Throughout the time course study, PrP Sc accumulation was not detected antemortem by immunohistochemistry in rectal tissue at any timepoint for any sheep. RT-QuIC results were inconsistently positive throughout the timepoints tested for each sheep; however, each sheep had at least one timepoint detected positive. When assessing serum Nf-L utility using receiver operator characteristic curves against different clinical parameters, such as asymptomatic and symptomatic (pruritus or neurologic signs), results showed that Nf-L was most useful at being an indicator of disease only late in disease progression when neurologic signs were present. Conclusion: Serum Nf-L concentrations in the cohort of sheep increased as disease progressed; however, serum Nf-L did not increase during the presymptomatic window. The levels increased substantially throughout the final 10% of the animals’ scrapie incubation period when other clinical signs were present. Serum Nf-L is not a reliable biomarker for pre-clinical detection of scrapie.
59 BASIC BIOLOGICAL SCIENCES↗
Bacterial vampirism mediated through taxis to serum
Bacteria of the family Enterobacteriaceae are associated with gastrointestinal (GI) bleeding and bacteremia and are a leading cause of death, from sepsis, for individuals with inflammatory bowel diseases. The bacterial behaviors and mechanisms underlying why these bacteria are prone to bloodstream entry remain poorly understood. Herein, we report that clinical isolates of non-typhoidal Salmonella enterica serovars, Escherichia coli , and Citrobacter koseri are rapidly attracted toward sources of human serum. To simulate GI bleeding, we utilized an injection-based microfluidics device and found that femtoliter volumes of human serum are sufficient to induce bacterial attraction to the serum source. This response is orchestrated through chemotaxis and the chemoattractant L-serine, an amino acid abundant in serum that is recognized through direct binding by the chemoreceptor Tsr. We report the first crystal structures of Salmonella Typhimurium Tsr in complex with L-serine and identify a conserved amino acid recognition motif for L-serine shared among Tsr orthologues. We find Tsr to be widely conserved among Enterobacteriaceae and numerous World Health Organization priority pathogens associated with bloodstream infections. Lastly, we find that Enterobacteriaceae use human serum as a source of nutrients for growth and that chemotaxis and the chemoreceptor Tsr provide a competitive advantage for migration into enterohemorrhagic lesions. We define this bacterial behavior of taxis toward serum, colonization of hemorrhagic lesions, and the consumption of serum nutrients as ‘bacterial vampirism’, which may relate to the proclivity of Enterobacteriaceae for bloodstream infections.
59 BASIC BIOLOGICAL SCIENCES↗
Biologically active protein fragments containing specific binding regions of serum albumin or related proteins
In accordance with the present invention, biologically active protein fragments can be constructed which contain only those specific portions of the serum albumin family of proteins such as regions known as subdomains IIA and IIIA which are primarily responsible for the binding properties of the serum albumins. The artificial serums that can be prepared from these biologically active protein fragments are advantageous in that they can be produced much more easily than serums containing the whole albumin, yet still retain all or most of the original binding potential of the full albumin proteins. In addition, since the protein fragment serums of the present invention can be made from non-natural sources using conventional recombinant DNA techniques, they are far safer than serums containing natural albumin because they do not carry the potentially harmful viruses and other contaminants that will be found in the natural substances.
Carter, Daniel C.↗
Detection of Head and Neck Cancer Based on Longitudinal Changes in Serum Protein Abundance
Head and Neck Squamous Cell Carcinoma (HNSCC) is significantly represented in the military population with substantial impact on military operations. Two-thirds of new HNSCC cases are first diagnosed at advanced stage III or IV with lymph node metastases. Thus, there is an urgent need of biomarkers for early detection and risk stratification. Archived serum samples from the Department of Defense Serum Repository provide a unique resource for longitudinal studies of HNSCC in the military and the identification of early detection biomarkers. Methods: We identified 175 patients diagnosed with HNSCC with 175 matched healthy controls and retrieved a total of 978 serum samples drawn at the time of diagnosis, 2 and 4 years prior to diagnosis, and 2 years after diagnosis. Following immunoaffinity depletion, serum samples were analyzed by targeted proteomics assays for multiplexed quantification of a panel of 146 candidate protein biomarkers from the curated literature. Results: We derived a 13-protein signature that distinguishes case versus controls based on longitudinal changes in serum protein concentration. The abundances of each of the 13 proteins remain constant over time in control subjects. The area under the curve for the derived random forest classifier was 0.90. Conclusions: This 13-protein classifier is highly promising for detection of HNSCC prior to overt symptoms. Impact: Use of longitudinal samples has significant potential to identify biomarkers for detection and risk stratification.
59 BASIC BIOLOGICAL SCIENCES↗
Longitudinal analysis of host protein serum signatures of treatment and recovery in pulmonary tuberculosis
A better understanding of treatment progression and recovery in pulmonary tuberculosis (TB) infectious disease is crucial. This study analyzed longitudinal serum samples from pulmonary TB patients undergoing interventional treatment to identify surrogate markers for TB-related outcomes. Serum that was collected at baseline and 8, 17, 26, and 52 weeks from 30 TB patients experiencing durable cure were evaluated and compared using a sensitive LC-MS/MS proteomic platform for the detection and quantification of differential host protein signatures relative to timepoint. The global proteome signature was analyzed for statistical differences across the time course and between disease severity and treatment groups. A total of 676 proteins showed differential expression in the serum over these timepoints relative to baseline. Comparisons to understand serum protein dynamics at 8 weeks, treatment endpoints at 17 and 26 weeks, and post-treatment at 52 weeks were performed. The largest protein abundance changes were observed at 8 weeks as the initial effects of antibiotic treatment strongly impacted inflammatory and immune modulated responses. However, the largest number of proteome changes was observed at the end of treatment time points 17 and 26 weeks respectively. Post-treatment 52-week results showed an abatement of differential proteome signatures from end of treatment, though interestingly those proteins uniquely significant at post-treatment were almost exclusively downregulated. Patients were additionally stratified based upon disease severity and compared across all timepoints, identifying 461 discriminating proteome signatures. These proteome signatures collapsed into discrete expression profiles with distinct pathways across immune activation and signaling, hemostasis, and metabolism annotations. Insulin-like growth factor (IGF) and Integrin signaling maintained a severity signature through 52 weeks, implying an intrinsic disease severity signature well into the post-treatment timeframe. Previous proteome studies have primarily focused on the 8-week timepoint in relation to culture conversion status. While this study confirms previous observations, it also highlights some differences. The inclusion of additional end of treatment and post-treatment time points offers a more comprehensive assessment of treatment progression within the serum proteome. Examining the expression dynamics at these later time periods will help in the investigation of relapse patients and has provided indicative markers of response and recovery.
59 BASIC BIOLOGICAL SCIENCES↗
Association of Serum Bile Acid and Unsaturated Fatty Acid Profiles with the Risk of Diabetic Retinopathy in Type 2 Diabetic Patients
Aim: We aimed to identify the ability of serum bile acids (BAs) and unsaturated fatty acids (UFAs) profiles to predict the development of diabetic retinopathy (DR) in type 2 diabetes mellitus (T2DM) patients. Methods: We first used univariate and multivariate analysis to compare 15 serum BA and 11 UFA levels in healthy control (HC) group (n = 82), T2DM patients with DR (n = 58) and T2DM patients without DR (n = 60). Forty T2DM patients were considered for validation. Then, the receiver operating characteristic curve (ROC) and decision curve analysis were used to assess the diagnostic value and clinical benefit of serum biomarkers alone, clinical variables alone or in combination, and the area under the curve (AUC), integrated discrimination improvement (IDI), and net reclassification improvement (NRI) were used to further assess whether the addition of biomarkers significantly improved the predictive ability of the model. Results: Orthogonal partial least squares-discriminant analysis (OPLS-DA) of serum BAs and UFAs separated the three cohorts including HC, T2DM patients with or without DR. The difference in serum BA and UFA profiles of T2DM patients with or without DR was mainly manifested in the three metabolites of taurolithocholic acid (TLCA), tauroursodeoxycholic acid (TUDCA) and arachidonic acid (AA). Together, they had an AUC of 0.785 (0.918 for validation cohort) for predicting DR in T2DM patients. After adjusting for numerous confounding factors, TLCA, TUDCA, and AA were independent predictors that differentiated T2DM with or without DR. The results of AUC, IDI, and NRI demonstrated that adding these three biomarkers to a model with clinical variables statistically increased their predictive value and were replicated in our independent validation cohort. Conclusion: These findings highlight the association of three metabolites, TLCA, TUDCA and AA, with DR and may indicate their potential value in the pathogenesis of DR.
60 APPLIED LIFE SCIENCES↗
Preliminary crystallographic studies of four crystal forms of serum albumin
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.
Serum Albumin/chemistry↗