Assessment of Vitamin D Status in Slovenian Premenopausal and Postmenopausal Women, Using Total, Free, and Bioavailable 25-Hydroxyvitamin D (25(OH)D).

The objective of our study was to evaluate vitamin D status and its predictors in Slovenian premenopausal and postmenopausal women. A cross-sectional study was carried out between 1 March 2021 and 31 May 2021. A total of 319 healthy women from the Central Slovenian region aged between 44 and 65 were recruited; 176 were included in the final analysis. The vitamin D status was determined by measuring the total 25-Hydroxycholecalciferol (25(OH)D) concentration, vitamin D binding protein (DBP), and albumin and calculating the bioavailable 25(OH)D and free 25(OH)D. For the calculation of bioavailable and free 25(OH)D, we developed a new online calculator. The Endocrine Society's thresholds for vitamin D deficiency and insufficiency were used; 29.0% of premenopausal and 24.4% of postmenopausal subjects were found to be vitamin D deficient (total 25(OH)D < 50 nmol/L); 76.8% of the premenopausal and 61.7% of postmenopausal subjects were found to have insufficient levels (total 25(OH)D < 75 nmol/L). Premenopausal women had 11.8% lower total 25(OH)D, 32.2% lower bioavailable 25(OH)D, and 25.2% higher DBP than postmenopausal women. The most important predictors of vitamin D status were vitamin D supplementation and time spent in the sun. Contrary to similar studies, the vitamin D status in Slovenian postmenopausal women was significantly better than in premenopausal women. In postmenopausal women, the measurement of free or bioavailable 25(OH)D instead of the total 25(OH)D could be advantageous.

Assessment of Vitamin D Metabolism in Patients with Cushing's Disease in Response to 150,000 IU Cholecalciferol Treatment.

In this study we aimed to assess vitamin D metabolism in patients with Cushing's disease (CD) compared to healthy individuals in the setting of bolus cholecalciferol treatment. The study group included 30 adults with active CD and the control group included 30 apparently healthy adults with similar age, sex and BMI. All participants received a single dose (150,000 IU) of cholecalciferol aqueous solution orally. Laboratory assessments including serum vitamin D metabolites (25(OH)D3, 25(OH)D2, 1,25(OH)2D3, 3-epi-25(OH)D3 and 24,25(OH)2D3), free 25(OH)D, vitamin D-binding protein (DBP) and parathyroid hormone (PTH) as well as serum and urine biochemical parameters were performed before the intake and on Days 1, 3 and 7 after the administration. All data were analyzed with non-parametric statistics. Patients with CD had similar to healthy controls 25(OH)D3 levels (p > 0.05) and higher 25(OH)D3/24,25(OH)2D3 ratios (p < 0.05) throughout the study. They also had lower baseline free 25(OH)D levels (p < 0.05) despite similar DBP levels (p > 0.05) and lower albumin levels (p < 0.05); 24-h urinary free cortisol showed significant correlation with baseline 25(OH)D3/24,25(OH)2D3 ratio (r = 0.36, p < 0.05). The increase in 25(OH)D3 after cholecalciferol intake was similar in obese and non-obese states and lacked correlation with BMI (p > 0.05) among patients with CD, as opposed to the control group. Overall, patients with CD have a consistently higher 25(OH)D3/24,25(OH)2D3 ratio, which is indicative of a decrease in 24-hydroxylase activity. This altered activity of the principal vitamin D catabolism might influence the effectiveness of cholecalciferol treatment. The observed difference in baseline free 25(OH)D levels is not entirely clear and requires further study.

Association of GC Variants with Bone Mineral Density and Serum VDBP Concentrations in Mexican Population.

Vitamin D-binding protein (VDBP) is encoded by the GC gene and is an active participant in the control of bone metabolism. However, the effect of its major variants on VDBP concentration and bone mineral density (BMD) remains unclear. Our aim was to analyze the effect of major GC variants on serum VDBP concentration and BMD. We recruited individuals from the Health Workers Cohort Study, which includes employees of the Mexican Institute of Social Security (IMSS). A total of 1853 adults were included. The single nucleotide polymorphisms (SNPs) rs7041 and rs4588 were genotyped to identify the three best characterized haplotypes of GC. Serum VBDP, 25(OH)D and BMD were also measured. Among women, the G allele of rs7041 was associated with higher VDBP and BMD compared to homozygous TT. The A allele of rs4588 was associated with lower VDBP and BMD compared to CC homozygous. In men, GC variants were only associated with VDBP levels. We did not observe an association between free/bioavailable 25(OH)D and BMD in men and women. Our results support an association of VDBP in bone health. The G and C alleles, from rs7041 and rs4588, respectively, are associated with high concentrations of VDBP and BMD in this sample of Mexican postmenopausal women.

Longitudinal assessment of salivary vitamin D binding protein during orthodontic tooth movement.

BACKGROUND: Vitamin D is critical for bone physiology. In this study, we quantified Vitamin D Binding Protein (VitDBP) levels in saliva as a measure of Vitamin D during orthodontic tooth movement. METHODS: In this longitudinal study, saliva samples were collected from 73 orthodontic patients for 4 timepoints for the first six months of orthodontic treatment, along with dental casts at the beginning and the end of the study period. The saliva was measured for VitDBP as a biological marker for bone apposition and clinical tooth movement. We used the absolute change in Little's Irregularity Index as a quantitative measure for alignment. In addition, we measured the levels of alkaline phosphatase (ALP) in saliva as a marker of bone turnover. RESULTS: Both low (< 2.75 ng/ml) and high (> 6.48 ng/ml) VitDBP levels were associated with reduced tooth movement. Significant (p < 0.05) seasonal changes in VitDBP using a two-season year model were found with lower levels observed in the summer (Apr-Sept) than in the winter (Oct-Mar). CONCLUSIONS: Clinically significant orthodontic tooth movement is associated with an optimal range of VitDBP in saliva. Normal levels of VitDBP correlated with more orthodontic tooth movement, suggesting a "normal" range of salivary content of VitDBP. Given the strong trend that is independent of the confounding factors (ex. age, race or gender), the predictive value or salivary VitDBP for tooth movement should be studied in larger cohorts in future studies.

Association of vitamin D-binding protein and vitamin D3 with insulin and homeostatic model assessment (HOMA-IR) in overweight and obese females.

OBJECTIVE: Equivocal association the contribution of 25-hydroxyvitamin D (25(OH)D) and the well-accepted role of vitamin D-binding protein (VDBP) on bioavailability of 25(OH)D or its independent roles, has led to possible association of the VDBP in glucose metabolism. This study was conducted to evaluate the relationships among 25(OH)D, VDBP, glucose/insulin metabolism and homeostatic model assessment (HOMA-IR). Blood samples were collected from 236 obese and overweight women. VDBP and 25(OH)D levels, and biochemical parameters were measured using an enzyme-linked immunosorbent assay (ELISA). An impedance fat analyzer was utilized to acquire the body composition. RESULTS: Using the multivariate linear regression, a reverse relationship was observed between VDBP and (HOMA-IR), such that women with higher VDBP displayed lower insulin resistance. The relationship was independent of age, body mass index, standardized energy intake and physical activity (p = 0.00). No significant relationship between 25(OH)D levels, FBS, body composition or insulin resistance were observed (p > 0.2). Current study observed that higher level of VDBP may be associated with lower levels of insulin and HOMA-IR, thus the evaluation of VDBP in diverse population groups seems to have significant clinical value in evaluating the prevalence of DM or early stage of glucose intolerance.

Vitamin D deficiency in children: a challenging diagnosis!

The concern about the assessment of vitamin D status is growing. Numerous publications warn about the high prevalence of vitamin D deficiency, as well as the potential role of vitamin D in non-bone health outcomes. The status of vitamin D is usually assessed by measuring serum total 25-hydroxyvitamin D (25OHD) concentration. This is the major circulating form of vitamin D and keeps an inverse correlation with serum parathyroid hormone (PTH) concentration. A value of 25OHD of 20 ng/ml is generally assumed as threshold of vitamin D sufficiency in epidemiologic studies because serum PTH tends to increase when the 25OHD concentration stands below this value. In pediatric population, very few studies have analyzed this issue and the negative relationship between serum 25OHD and serum PTH is not clear, which is the suitable circulating concentration of 25OHD and the threshold of deficiency being matters of controversy. The majority of 25OHD circulates in serum tightly bound to a globulin (DBP). According to the free hormone hypothesis, protein-bound hormones are not biologically available and it is the free form that exerts or facilitates the physiologic actions. If this is true, factors that affect DBP may alter the interpretation of total serum 25OHD measurements.

Assessment of Vitamin D-Binding Protein and Early Prediction of Nephropathy in Type 2 Saudi Diabetic Patients.

Early detection of diabetic nephropathy (DN) represents a great challenge in an attempt to reduce the burden of chronic kidney diseases in diabetic patients. This study aimed to investigate the potential early prediction role of urinary vitamin D-binding protein (uVDBP) for the diagnosis of DN and to examine the possible correlation to serum VDBP, high-sensitivity C-reactive protein (hs-CRP), and insulin resistance in these patients. Serum and urine samples were obtained from 40 healthy volunteers and 120 patients with type 2 diabetes divided into 3 groups: normoalbuminuria, microalbuminuria, and macroalbuminuria (urinary albumin excretion rate < 30, 30-300, and >300 µg/mg, resp.); n = 40/group. Serum and urinary VDBP levels were quantified by ELISA. Insulin resistance has been assessed by homeostasis model assessment index (HOMAI). Correction for urine creatinine concentration was applied for urinary quantitative measurements. uVDBP levels were significantly elevated in micro- and macroalbuminuria patient groups compared with those of the normoalbuminuria patient group and controls (820.4 ± 402.8 and 1458.1 ± 210.0 compared with 193.1 ± 141.0 and 127.7 ± 21.9 ng/mg, resp.) (P < 0.001). There was significant correlation between serum and urinary levels of VDBP in total patient group. Receiver operating characteristic analysis of uVDBP levels showed optimum cut-off value of 216.0 ng/mg corresponding to 98.8% sensitivity and 80.0% specificity and an area under the curve of 0.973 to discriminate the normoalbuminuria from the microalbuminuria groups. In multivariate analysis, ordination plot showed obvious demarcation between the study groups caused by the higher levels of uVDBP and albumin/creatinine ratio among other variables. The study findings suggested a possible clinical application of uVDPB as an early and a good marker for the detection of early renal disease in type 2 DM Saudi patients. Large-scale validation studies are warranted to confirm the results before including uVDBP with the available list of other conventional biomarkers.

Characterization of equine vitamin D-binding protein, development of an assay, and assessment of plasma concentrations of the protein in healthy horses and horses with gastrointestinal disease.

OBJECTIVE To purify and characterize equine vitamin D-binding protein (VDBP) from equine serum and to evaluate plasma concentrations of VDBP in healthy horses and horses with gastrointestinal injury or disease. ANIMALS 13 healthy laboratory animals (8 mice and 5 rabbits), 61 healthy horses, 12 horses with experimentally induced intestinal ischemia and reperfusion (IR), and 59 horses with acute gastrointestinal diseases. PROCEDURES VDBP was purified from serum of 2 healthy horses, and recombinant equine VDBP was obtained through a commercial service. Equine VDBP was characterized by mass spectrometry. Monoclonal and polyclonal antibodies were raised against equine VDBP, and a rocket immunoelectrophoresis assay for equine VDBP was established. Plasma samples from 61 healthy horses were used to establish working VDBP reference values for study purposes. Plasma VDBP concentrations were assessed at predetermined time points in horses with IR and in horses with naturally occurring gastrointestinal diseases. RESULTS The working reference range for plasma VDBP concentration in healthy horses was 531 to 1,382 mg/L. Plasma VDBP concentrations were significantly decreased after 1 hour of ischemia in horses with IR, compared with values prior to induction of ischemia, and were significantly lower in horses with naturally occurring gastrointestinal diseases with a colic duration of < 12 hours than in healthy horses. CONCLUSIONS AND CLINICAL RELEVANCE Plasma VDBP concentrations were significantly decreased in horses with acute gastrointestinal injury or disease. Further studies and the development of a clinically relevant assay are needed to establish the reliability of VDBP as a diagnostic and prognostic marker in horses.

Clinical Utility of Measurement of Vitamin D-Binding Protein and Calculation of Bioavailable Vitamin D in Assessment of Vitamin D Status.

BACKGROUND: The associations of vitamin D deficiency with various clinical conditions highlighted the importance of vitamin D testing. Currently, clinicians measure only the total 25-hydroxyvitamin D [25(OH)D] concentration, regardless of its bioavailability. We aimed to determine the effect of vitamin D-binding protein (VDBP) on 25(OH)D bioavailability. METHODS: Serum samples were collected from 60 healthy controls, 50 pregnant women, and 50 patients in intensive care units (ICUs). Total 25(OH)D was quantified by liquid chromatography with tandem mass spectrometry, and VDBP levels were determined by using an ELISA kit (R&D Systems, USA). The bioavailable 25(OH)D levels were calculated by using total 25(OH)D, VDBP, and albumin concentrations. RESULTS: In comparison with healthy controls, the total 25(OH)D concentration was significantly lower in ICU patients (median, 11.65 vs 18.25 ng/mL; P<0.00001), but no significant difference was noted between pregnant women (18.25 ng/mL) and healthy controls. The VDBP level was significantly lower in ICU patients (95.58 vs 167.18 µg/mL, P=0.0002) and higher in pregnant women (225.01 vs 167.18 µg/mL, P=0.008) compared with healthy controls. Nonetheless, the calculated bioavailable 25(OH)D levels of ICU patients and pregnant women were significantly lower than those of healthy controls (1.97 and 1.93 ng/mL vs 2.56 ng/mL; P=0.0073 and 0.0027). CONCLUSIONS: A single marker of the total 25(OH)D level is not sufficient to accurately evaluate vitamin D status, especially in pregnant women. In cases where VDBP concentrations may be altered, VDBP measurements and bioavailable 25(OH)D calculations may help to determine vitamin D status accurately.

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.

Vitamin D expenditure is not altered in pregnancy and lactation despite changes in vitamin D metabolite concentrations.

Pregnancy and lactation are associated with changes in vitamin D and calcium metabolism but the impact of these changes on vitamin D expenditure is unknown. We measured plasma 25(OH)D3 half-life with a stable-isotope tracer and investigated relationships with vitamin D metabolites in pregnant, lactating and 'non-pregnant, non-lactating' (NPNL) women. Vitamin D metabolites, vitamin D binding protein (DBP), PTH and 25(OH)D3 half-life were measured in third-trimester pregnant women (n22) and repeated during lactation 12 weeks post-partum (n14) and twice in NPNL women (n23 and n10, respectively) in rural Gambia where calcium intakes are low with little seasonality in UVB-exposure. 25(OH)D3 half-life was not significantly different between groups (mean(SD): 20.6(6.8), 22.6(7.7), 18.0(4.7) and 17.7(9.5) days in pregnant, lactating and NPNL women, respectively). Plasma 25(OH)D3, 1,25(OH)2D, and DBP were higher in pregnancy, and calculated free-25(OH)D3 and PTH were lower (P < 0.05). In lactation, 25(OH)D3 and 24,25(OH)2D3 were lower compared to pregnant (P < 0.001, P = 0.02) and NPNL women (P = 0.04, P = 0.07). Significant associations were observed between half-life and 25(OH)D3 (+ve) in pregnancy, and in all groups between 25(OH)D3 and free-25(OH)D3 (+ve) and PTH and 25(OH)D3 (-ve) (P < 0.0001). These data suggest that adaptive changes in pregnancy and lactation occur that prevent pronounced changes in vitamin D expenditure.

25-hydroxyvitamin D levels, vitamin D binding protein gene polymorphisms and incident coronary heart disease among whites and blacks: The ARIC study.

BACKGROUND: In observational studies, low 25-hydroxyvitamin D (25(OH)D) has been associated with increased risk of coronary heart disease (CHD), and this association may vary by race. Racial differences in the frequency of vitamin D binding protein (DBP) single nucleotide polymorphisms (SNPs) might account for similar bioavailable vitamin D in blacks despite lower mean 25(OH)D. We hypothesized that the associations of low 25(OH)D with CHD risk would be stronger among whites and among persons with genotypes associated with higher DBP levels. METHODS: We measured 25(OH)D by mass spectroscopy in 11,945 participants in the ARIC Study (baseline 1990-1992, mean age 57 years, 59% women, 24% black). Two DBP SNPs (rs7041; rs4588) were genotyped. We used adjusted Cox proportional hazards models to examine the association of 25(OH)D with adjudicated CHD events through December 2011. RESULTS: Over a median of 20 years, there were 1230 incident CHD events. Whites in the lowest quintile of 25(OH)D (<17 ng/ml) compared to the upper 4 quintiles had an increased risk of incident CHD (HR 1.28, 95% CI 1.05-1.56), but blacks did not (1.03, 0.82-1.28), after adjustment for demographics and behavioral/socioeconomic factors (p-interaction with race = 0.22). Results among whites were no longer significant after further adjustment for potential mediators of this association (i.e. diabetes, hypertension). There was no statistically significant interaction of 25(OH)D with the DBP SNPs rs4588 (p = 0.92) or rs7041 (p = 0.87) in relation to CHD risk. CONCLUSIONS: Low 25(OH)D was associated with incident CHD in whites, but no interactions of 25(OH)D with key DBP genotypes was found.

Safety pharmacology, toxicology and pharmacokinetic assessment of human Gc globulin (vitamin D binding protein).

Gc globulin is an important protein of the plasma actin-scavenger system. As such, it has been shown to bind free actin and prevent hypercoagulation and shock in patients with massive actin release resulting from severe tissue injuries. Treatment of such patients with Gc globulin could therefore potentially be life-saving. This article presents pre-clinical toxicology experiments conducted on purified plasma-derived human Gc globulin. The Gc globulin formulation was shown to be stable for at least 4 years with full retention of actin-binding capacity. In vitro studies did not reveal activation of the kallikrein system or the complement system and cellular studies showed no toxic effects on a variety of human cell lines. In vivo studies showed no acute toxic effects in mice, rats or guinea pigs upon intravenous infusion. A 14-day local tolerance study in rabbits showed no adverse effects, and 14-day toxicity studies in rats and horses did not show any unwanted reactions. In a 14-day toxicology study in beagle dogs, formation of antibodies was seen and in the end of the study period, three out of four dogs showed clinical immunological reactions, which could be ascribed to the formation of antibodies. The half-life, T, for human Gc globulin was 12 hr in rats, 16 hr in horses and 30 hr in dogs. The safety profile of plasma-derived Gc globulin is concluded to be consistent to that required for use in man.

Assessment of vitamin D status in male osteoporosis.

BACKGROUND: Clinical assessment of vitamin D status often relies on measuring total circulating 25-hydroxyvitamin D3 (25OHD3), but much of each vitamin D metabolite is bound to plasma vitamin D-binding protein (DBP), such that the percentage of free vitamin is very low. We hypothesized that measurement of free rather than total 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and 25OHD3 may provide better assessment of vitamin D status. We therefore aimed to assess vitamin D status in men with idiopathic osteoporosis, in whom possible secondary causes of osteoporosis had been excluded, and to determine the extent of change in biologically active "free" vitamin D caused by variation in plasma DBP concentrations. METHODS: We measured 1,25(OH)2D3 and 25OHD3 in plasma samples from 56 men with idiopathic osteoporosis [mean (SD) age, 59.6 (13.6) years; range, 21-86 years] and 114 male controls [62.4 (10.4) years; range, 44-82 years]. RESULTS: Mean total plasma 25OHD3 in the 56 men with osteoporosis and the 114 controls was 44.7 (21) and 43.3 (17) nmol/L, respectively; total plasma 1,25(OH)2D3 measured in randomly selected men with osteoporosis (n = 50) and controls (n = 50) was 90 (37) and 103 (39) pmol/L, respectively. Mean plasma DBP was significantly higher (P <0.001) in men with osteoporosis [224 (62) mg/L; n = 56] than in the controls [143 (34) mg/L; n = 114], but calculated free plasma 25OHD3 and 1,25(OH)2D3 were significantly lower in the osteoporotic men than in controls [6.1 (3.1) vs 9.1 (4.4) pmol/L (P <0.00001) and 77 (37) vs 142 (58) fmol/L (P <0.00001), respectively]. CONCLUSIONS: Measurement of total vitamin D metabolites alone, although providing a crude assessment of vitamin D status, may not give an accurate indication of the free (biologically active) form of the vitamin. The ratio of total 25OHD3 and 1,25(OH)2D3 to plasma DBP, rather than total circulating vitamin D metabolites, may provide a more useful index of biological activity. Further studies are required to substantiate this hypothesis.

Genetic diversity of serum proteins in three subpopulations of the Maria Gond tribe of Madhya Pradesh, India.

The phenotype and allele frequency distribution of group specific component (GC), transferrin (TF), alpha-1-antitrypsin (PI) and apolipoprotein E (APOE) was determined by isoelectric focusing of plasma samples from three subpopulations (Bison Horn Maria of the Kuakonda and Tokapal Block, and Abuj Maria of the Abujmar Hills of the Orchha block) of the Maria Gond tribe of Madhya Pradesh, India. A considerable level of allele frequency variation was observed in these subpopulations, which highlighted social and geographical isolation among them. The average heterozygosity for these IEF subtype systems was high (29-39%) and the gene diversity among these subpopulation groups was of low to moderate range (1.4%). The overall analysis showed that these polymorphisms are useful anthropological markers for micro-evolutionary and genetic structure studies.

Development of coagglutination reagents for serological grouping of streptococci.

Coagglutination reagents for the rapid serological grouping of groups A, B, C, F and G Streptococcus have been developed. Antisera to groups A, B, C, F and G Streptococcus were raised in rabbits. After absorption with cross-reacting antigens, the specific antibodies were coated on Staphylococcus protein-A and used as group-specific coagglutination reagents. The sensitivity of the reagents for groups A, C and G Streptococcus was 100 per cent and the specificity was 100, 100, and 98.77 per cent, respectively. The sensitivity and specificity of these reagents were consistent up to 12 months, although specificity declined with longer storage. The in-house coagglutination reagents for groups A, C and G streptococcus were also tested in comparison with the commercially available Streptococcus Phadebact test and yielded almost identical results. Sensitivity of the in-house of group B Streptococcus reagent was low, while the group F reagent gave a high incidence of false positive reaction.

New rare variants and interpretation of the genetic diversity at the group-specific component (GC) locus in populations of India.

As part of our ongoing genetic studies, 2635 samples from 30 populations of the Indian subcontinent have been analyzed for GC subtype variation. Several rare variants were found and have been characterized using isoelectric focusing in 3 M urea and with an immobilized pH gradient and by comparison with known standards. Six new variants, 1A33, 2A22, 2A23, 2A24, 1C56, and 1C59, have been discovered in geographically and ethnically distant populations of the Indian subcontinent. Variants 1C11 and 1C21 have been reported previously in the French population and variant 1C36 has been reported in Nepalis, but all three variants were observed for the first time in the populations of India. The distribution of the rare variants in the Indian subcontinent was examined for anthropological usefulness for differentiation of Indian populations. The data for GC allele frequencies from 144 populations of India were compared for geographic and ethnic variation. A high frequency of the GC*IF allele was observed in populations of eastern India, and various ethnic groups from the eastern zone were clearly differentiated from similar ethnic populations from the rest of India. GC*2 allele frequency data were further correlated with various geographic and climatic variables, such as longitude, latitude, sunshine, solar radiation, and mean annual temperature. A significant positive correlation between GC*2 frequency and latitude was observed. The GC*2 allele frequency showed a cline increasing from south to north. Significant negative correlation was also observed between GC*2 frequency and sunshine, solar radiation, and mean temperature. A possible interpretation for selection of the GC*2 allele in areas with low levels of insolation is provided.

Genetic variation of serum proteins (GC, TF and PI subtypes) in the Baigas of Madhya Pradesh, India.

The distribution of group specific component (GC), transferrin (TF) and alpha-1-antitrypsin (PI) subtypes was determined by isoelectric focusing of plasma samples from 140 unrelated individuals belonging to the Baiga tribe of Madhya Pradesh, India. The allele frequencies observed for GC (GC*1S = 0.6742, GC*1F = 0.1326 and GC*2 = 0.1932), TF (TF*C1 = 0.7920, TF*C2 = 0.1606, TF*C3 = 0.0146 and TF*D = 0.0328) and PI (PI*M1 = 0.7179, PI*M2 = 0.1750, PI*M3 = 0.0893, PI*S = 0.0107 and PI*F = 0.0071) demonstrate appreciable heterogeneity of the Baigas compared to other populations of the region.

Simultaneous subtyping of group specific component and esterase D by isoelectric focusing on agarose gels.

A quick, sensitive and economical technique has been developed to subtype GC and ESD simultaneously on the same agarose IEF gel. This method could be a useful tool for forensic application.

Assessment of the free fraction of 25-hydroxyvitamin D in serum and its regulation by albumin and the vitamin D-binding protein.

We measured the free fraction of 25-hydroxyvitamin D (25OHD) in human serum and determined that 25OHD bound to a component with an affinity constant of 7 X 10(8) M-1 and a concentration of 4.5 X 10(-6) M. This concentration was equal to that of the vitamin D-binding protein (DBP) in the same serum sample. We removed DBP from the serum using actin affinity columns and found that the affinity for 25OHD of the remaining serum components was equivalent to that of human serum albumin (6 X 10(5) M-1). We then measured the free fractions of 25OHD, DBP, and albumin in normal and cirrhotic subjects. We calculated that 88 +/- 3% (+/- SD) and 83 +/- 8% of the 25OHD were bound to DBP in the serum of normal and cirrhotic subjects, respectively. We compared previously reported data for the free fraction and the free concentration of 1,25-dihydroxyvitamin D in these subjects with the current data for the free fraction and free concentration of 25OHD. The total concentrations and free fractions of both metabolites correlated to each other and to the DBP and albumin concentrations in these subjects, but the free concentrations of these metabolites did not. We conclude that 25OHD, like 1,25-dihydroxyvitamin D, is transported in blood bound primarily to DBP and albumin. Changes in the concentrations of DBP and albumin affected the total and free fractions of 25OHD in serum, but the actual free concentration of 25OHD was independent of such changes.