Assessment of EN-RAGE, sRAGE, and its isoforms: cRAGE, esRAGE in obese patients treated by moderate caloric restriction combined with physical activity conducted in hospital condition.

BACKGROUND: AGEs, their receptor (RAGE), and the extracellular newly identified receptor for AGEs product-binding protein (EN-RAGE) are implicated in the pathogenesis of inflammation. AIM: We analyzed serum EN-RAGE, soluble RAGE (sRAGE), and their isoforms: endogenous secretory - esRAGE and cleaved - cRAGE concentrations in lean controls (n = 74) and in patients with obesity (n = 71) treated for three weeks with moderate calorie restriction (CR) combined with physical activity in a hospital condition. METHODS: Using the ELISA method, serum sRAGE, esRAGE, and EN-RAGE were measured before and after CR. RESULTS: The serum level of sRAGE and esRAGE in patients with obesity was lower than that in non-obese individuals, contrary to cRAGE. EN-RAGE concentration was about three times higher in obese patients. Gradually, a rise in BMI resulted in sRAGE, esRAGE reduction, and EN-RAGE increase. The sRAGE concentration was sex-dependent, indicating a higher value in lean men. A moderate negative correlation was observed between BMI and all RAGE isoforms, whereas EN-RAGE displays a positive correlation. CR resulted in an expected decrease in anthropometric, metabolic, and proinflammatory parameters and EN-RAGE, but no RAGE isoforms. The ratio EN-RAGE/sRAGE was higher in obese humans than in control and was not modified by CR. CONCLUSION: Obesity decreases sRAGE and esRAGE and increases EN-RAGE concentration. Moderate CR and physical activity by decreasing inflammation reduces EN-RAGE but is insufficient to increase sRAGE and esRAGE to the extent observed in lean patients. EN-RAGE instead of sRAGE could be helpful to indicate a better outcome of moderate dietary intervention in obese subjects.

Assessment of muscle fiber adaptation in footballers using a new ELISA assay of myosin isoforms.

BACKGROUND: To measure the impact of training models on injury incidence, data of health and performance were integrated to study fiber adaptation during a competitive season. We studied football players over a season, analyzing hours of exposure to sport by serum changes in fast and slow myosin, creatine kinase and lactate dehydrogenase. METHODS: A new assay was developed to measure the myosin isoforms in 49 non-sporting volunteers and in 27 professional football players. RESULTS: Myosin isoforms in volunteers with mean ages of 30±8 were 1553 µg/L fast and 1284 µg/L slow; in the group with of 56±7 were 1426 µg/L fast and 1046 µg/L slow. Slow myosin was significantly lower in older subjects (-18%). Samples from the players in preseason had lower mean scores for fast myosin (1123 µg/L) and higher for slow myosin (2072 µg/L) than reference volunteers. During the season, myosins reached the maximum with the maximum load (1537 µg/L fast, 2195 µg/L slow but decreased and adapted to the high level of demand (425 µg/L fast, 1342 µg/L slow). CK and LDH were maximal at the pre-season (227 U/L, 333 U/L) while myosin levels were maximal at the beginning of season (1537 µg/L, 2195 µg/L). CONCLUSIONS: Measuring serum myosin isoforms we identify the type and amount of damage caused by training and matches, making it a new control tool capable of advising training towards a minimum of blood slow myosin but controlling the fast fiber participating and be able to improve the performance of the players.

Assessment of the Validity of Nuclear-Localized Androgen Receptor Splice Variant 7 in Circulating Tumor Cells as a Predictive Biomarker for Castration-Resistant Prostate Cancer.

Importance: A blood test to determine whether to treat patients with metastatic castration-resistant prostate cancer (mCRPC) with an androgen receptor signaling (ARS) inhibitor or taxane is an unmet medical need. Objective: To determine whether a validated assay for the nuclear-localized androgen receptor splice variant 7 (AR-V7) protein in circulating tumor cells can determine differential overall survival among patients with mCRPC treated with taxanes vs ARS inhibitors. Design, Setting, and Participants: This blinded correlative study conducted from December 31, 2012, to September 1, 2016, included 142 patients with histologically confirmed mCRPC and who were treated at Memorial Sloan Kettering Cancer Center, The Royal Marsden, or the London Health Sciences Centre. Blood samples were obtained prior to administration of ARS inhibitors or taxanes as a second-line or greater systemic therapy for progressing mCRPC. Main Outcomes and Measures: Overall survival after treatment with an ARS inhibitor or taxane in relation to pretherapy AR-V7 status. Results: Among the 142 patients in the study (mean [SD] age, 69.5 [9.6] years), 70 were designated as high risk by conventional prognostic factors. In this high-risk group, patients positive for AR-V7 who were treated with taxanes had superior overall survival relative to those treated with ARS inhibitors (median overall survival, 14.3 vs 7.3 months; hazard ratio, 0.62; 95% CI, 0.28-1.39; P = .25). Patients negative for AR-V7 who were treated with ARS inhibitors had superior overall survival relative to those treated with taxanes (median overall survival, 19.8 vs 12.8 months; hazard ratio, 1.67; 95% CI, 1.00-2.81; P = .05). Conclusions and Relevance: This study suggests that nuclear-localized AR-V7 protein in circulating tumor cells can identify patients who may live longer with taxane chemotherapy vs ARS inhibitor treatment.

Comparison of Two ELISA Methods and Mass Spectrometry for Measurement of Vitamin D-Binding Protein: Implications for the Assessment of Bioavailable Vitamin D Concentrations Across Genotypes.

Studies using vitamin D-binding protein (DBP) concentrations to estimate free and bioavailable vitamin D have increased dramatically in recent years. Combinations of two single-nucleotide polymorphisms (SNPs) produce three major DBP isoforms (Gc1f, Gc1s, and Gc2). A recent study showed that DBP concentrations quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) did not differ by race, whereas a widely used monoclonal enzyme-linked immunosorbent assay (ELISA) quantified DBP differentially by isoform, yielding significantly lower DBP concentrations in black versus white individuals. We compared measurements of serum DBP using a monoclonal ELISA, a polyclonal ELISA, and LC-MS/MS in 125 participants in the Chronic Renal Insufficiency Cohort (CRIC). Serum free and bioavailable 25OHD were calculated based on DBP concentrations from these three assays in homozygous participants, and race differences were compared. We confirmed that the monoclonal ELISA quantifies DBP differentially by isoform and showed that the polyclonal ELISA is not subject to this bias. Whereas ≤9% of the variability in DBP concentrations quantified using either LC-MS/MS or the polyclonal ELISA was explained by genotype, 85% of the variability in the monoclonal ELISA-based measures was explained by genotype. DBP concentrations measured by the monoclonal ELISA were disproportionately lower than LC-MS/MS-based results for Gc1f homozygotes (median difference -67%; interquartile range [IQR] -71%, -64%), 95% of whom were black. In contrast, the polyclonal ELISA yielded consistently and similarly higher measurements of DBP than LC-MS/MS, irrespective of genotype, with a median percent difference of +50% (IQR +33%, +65%). Contrary to findings using the monoclonal ELISA, DBP concentrations did not differ by race, and free and bioavailable 25OHD were significantly lower in black versus white participants based on both the polyclonal ELISA and LC-MS/MS, consistent with their lower total 25OHD. Future studies of DBP and free or bioavailable vitamin D metabolites should employ DBP assays that are not biased by DBP genotype. © 2016 American Society for Bone and Mineral Research.

Assessment of naturally occurring covalent and total dimer levels in human IgG1 and IgG2.

Antibody dimers, two self-associated monomers, have been detected on both recombinantly expressed and endogenous human IgG proteins. Nearly 10 years ago, Yoo et al. (2003) described low levels of IgG2 covalent dimer, in human serum, but did not quantify the levels. Here we quantify the total and covalent dimer levels of IgG2 and IgG1 in human blood, and study the origin of covalent dimer formation. Low levels (<1%) of total IgG1 and IgG2 dimers were measured in freshly prepared human plasma. Both IgG1 and IgG2 covalent dimers were also found in plasma. Whereas IgG1 covalent dimer levels were significantly reduced by steps intended to eliminate artifacts during sample preparation, IgG2 covalent dimer levels remain stable in such conditions. About 0.4% of IgG2 in plasma was in a covalent dimer form, yet very little (<0.03%) of IgG1 covalent dimer could be considered naturally occurring. IgG2 dimer also formed in vitro under conditions designed to mimic those in blood, suggesting that formation occurs in vivo during circulation. Thus, small amounts of covalent IgG2 dimer do appear to form naturally.

Assessment of adiponectin and its isoforms in Polish centenarians.

BACKGROUND: The physiological mechanisms that promote longevity remain unclear. It has been suggested that insulin sensitivity is preserved in centenarians, whereas typical aging is accompanied by increasing insulin resistance. The oldest-old individuals display raised total adiponectin levels, despite the potential correlation between enhanced adiponectin and all-cause and cardiovascular mortality. AIM: To evaluate the level of adiponectin and its isoforms in sera of centenarians and to assess associations between adiponectin and metabolic parameters. PARTICIPANTS: A group of 58 Polish centenarians (50 women and 8 men, mean age 101±1.34 years) and 68 elderly persons (55 women and 13 men, mean age 70±5.69 years) as controls. MEASUREMENTS: Serum samples were analyzed to evaluate the following parameters: adiponectin array (total adiponectin, HWM-, MMW- and LMW-adiponectin; all by ELISA methods), insulin (by IRMA methods), glucose and lipid profiles. HOMA-IR was calculated. Clinical data were collected. Statistical analyses were performed. RESULTS: The concentrations of all adiponectin isoforms were significantly higher in the oldest-old participants. In the centenarian group, total adiponectin positively correlated with age and HDL-cholesterol, and HMW-adiponectin was negatively associated with insulin and triglycerides. The long-lived participants had a lower incidence of hypertension, type 2 diabetes, overweight and obesity, with lower concentrations of serum glucose and insulin, and reduced HOMA-IR. CONCLUSION: Our findings support the thesis that centenarians possess a different adiponectin isoform pattern and have a favorable metabolic phenotype in comparison with elderly individuals. However, additional work is necessary to understand the relevance of these findings to longevity.

Assessment of the clinical significance of antigenic and functional levels of α1-proteinase inhibitor (α1-Pi) in infiltrating ductal breast carcinomas.

OBJECTIVES: To determine the clinical significance of α1-proteinase inhibitor (α1-Pi) in infiltrating ductal breast carcinoma patients. DESIGN AND METHODS: Serum levels of α1-Pi, tryptic specific inhibitory capacity and α1-Pi circulating immune complexes were determined using radial immunodiffusion, BAPNA assays and ELISA, respectively. 2-DE-MS and immunohistochemistry were performed to examine α1-Pi protein expression. RESULTS: A decreased serum level of α1-Pi was found among breast cancer patients in comparison to controls. In addition, we found a significantly decreased mean level of α1-Pi in the node metastatic group when compared to node negative patients. However, the functional activity of the inhibitor did not decrease proportionately. Through 2-DE analyses, a differential expression of α1-Pi isoforms according to tumor stage and node metastatic development was found. CONCLUSIONS: Both α1-Pi levels and specific activity could be a source of complementary clinical information and may provide useful information for a better understanding of the mechanisms of metastasis.

Assessment of ovarian reserve by using the follicle-stimulating hormone isoform distribution pattern to predict the outcome of in vitro fertilization.

Changes in FSH isoforms occur at temporally specific times during the menstrual cycle and reproductive lifespan. Changes in ratio in FSH isoforms may provide a sensitive test of ovarian reserve before IVF.

Assessment of metallothionein and antibodies to metallothionein in normal and autistic children having exposure to vaccine-derived thimerosal.

Allergic autoimmune reaction after exposure to heavy metals such as mercury may play a causal role in autism, a developmental disorder of the central nervous system. As metallothionein (MT) is the primary metal-detoxifying protein in the body, we conducted a study of the MT protein and antibodies to metallothionein (anti-MT) in normal and autistic children whose exposure to mercury was only from thimerosal-containing vaccines. Laboratory analysis by immunoassays revealed that the serum level of MT did not significantly differ between normal and autistic children. Furthermore, autistic children harboured normal levels of anti-MT, including antibodies to isoform MT-I (anti-MT-I) and MT-II (anti-MT-II), without any significant difference between normal and autistic children. Our findings indicate that because autistic children have a normal profile of MT and anti-MT, the mercury-induced autoimmunity to MT may not be implicated in the pathogenesis of autism.

Pituitary-ovarian axis during lactational amenorrhoea. II. Longitudinal assessment of serum FSH polymorphism before and after recovery of menstrual cycles.

BACKGROUND: The association of normal serum levels of immunoassayable gonadotrophins with anovulation during lactational amenorrhoea (LA) has not been fully explained. METHODS: Serum FSH polymorphism was analysed in 10 women during LA between days 60 and 70 post-partum and again, in the mid-follicular phase (MFP), after resuming menstrual cyclicity. FSH microheterogeneity was characterized according to charge, using preparative isoelectric focusing, and according to the inner structure of carbohydrate chains, using lectin chromatography. RESULTS: A significantly higher proportion of FSH charge isoforms isolated below pH 4.10 and a lower proportion of FSH isoforms bearing highly branched oligosaccharides were observed during LA when compared to MFP. Further analysis with higher resolution showed that FSH charge isoforms, isolated in the lower pH range in LA, corresponded to FSH molecules bearing highly branched and biantennary oligosaccharides. FSH isoforms bearing hybrid-type oligosaccharides were only present during LA. The circulating FSH isoform mix was significantly less bioactive in LA than in MFP. LA is characterized by a more acidic mix of FSH isoforms, containing hormone bearing less processed oligosaccharides, with decreased biopotency in comparison with the follicular phase. CONCLUSIONS: This FSH microheterogeneity may be one of the critical factors contributing to incomplete follicular development and anovulation during LA.

Comparison of HPLC and small column (CDTect) methods for disialotransferrin.

BACKGROUND: Current methods for determination of carbohydrate-deficient transferrin (CDT) are based on separation of the CDT fraction by ion-exchange chromatography on minicolumns and quantification by immunoassay. Alternatively, the transferrin isoforms can be separated by HPLC anion-exchange chromatography and quantified by absorbance. This method has been reported to improve the validity of CDT as a marker of chronic alcohol abuse. METHODS: HPLC on either MonoQ or ResourceQ anion-exchange columns was used to separate and quantify isoforms of transferrin with detection at 460 nm. The result was expressed as the percentage of the disialo form (pI 5.7) of total transferrin (DST). The commercial CDTect assay was used as a comparison method. Serum samples from nondrinkers (n = 57), moderate drinkers (n = 77), and heavy drinkers (n = 139) were analyzed. RESULTS: In ROC analysis for differentiation between moderate and heavy drinkers, the area under the curve (AUC) for the HPLC method was 0.87 (95% confidence interval, 0.81-0.93), whereas that for CDTect was 0.72 (95% confidence interval, 0.64-0.80). At 90% specificity, the sensitivity of DST was 63% (95% confidence interval, 53-73%) compared with 33% (22-44%) for CDT. The reference interval of the HPLC method was 0.68-1.7%. CONCLUSIONS: The HPLC anion-exchange method for quantification of CDT provides substantially better separation between moderate and heavy drinkers than the CDTect method.