The transport of vitamin D and its 25-hydroxy metabolite in human plasma. Isolation and partial characterization of vitamin D and 25-hydroxyvitamin D binding protein

This study reports the isolation and partial characterization of vitamin D and 25-hydroxyvitamin D binding protein (DBP), the specific transport protein for vitamin D and its 25-hydroxy metabolite in human plasma. DBP was labeled by the addition of a tracer amount of 3H-labeled 25-OH-D3 to the original plasma used for protein fractionation. Previous experiments have shown that such 25-OH-D3 added in vitro binds to the same protein normally responsible for the transport of endogenous 25-OH-D and of vitamin D. The isolation of human DBP was achieved by an extensive sequence of procedures which resulted in a final yield of only approximately 4 mg of purified DBP from a starting volume of 34 liters of plasma. Purified DBP was homogeneous in the analytical ultracentrifuge and showed a single band of protein on analytical polyacrylamide gel electrophoresis. DBP had a sedimentation constant of 3.49s and a mol wt of approximately 52,000. The molecular weight was assessed by sedimentation equilibrium analysis and also by sodium dodecyl sulfate-disc-gel electrophoresis and by gel filtration on a standardized column of Sephadex G-150. The amino acid composition of DBP was determined and was generally consistent with the estimated extinction coefficient (E1cm1% at 280 nm) of about 9.1. The isoelectric point of DBP was estimated as 4.8 from isoelectric focusing experiments. Direct study of the binding capacity of the purified DBP for added 25-OH-D3 showed that the isolated DBP had a high affinity for 25-OH-D3, with an apparent maximum binding capacity of one molecule of 25-OH-D3 per molecule of protein.     

Seasonal variations in vitamin D status and calcium absorption do not influence bone turnover in young women

OBJECTIVE: To evaluate the effect of seasonal variations in UV B-exposure on calcium absorption and bone turnover in young women with the overall goal to assess the potential benefit of a vitamin D supplementation during wintertime. DESIGN: Cross-sectional study. SETTING: Area of Bonn, Germany (51 degrees N). SUBJECTS: Thirty-eight women (24.5+/-0.5 y) studied in winter and 38 females of the same age (24.7+/-0.4 y) studied in summer. RESULTS: As estimated by a 4 d food record, both groups had similar dietary calcium and phosphorus intakes (> 1200 mg/d, respectively) covering actual recommendations. Significant reductions in serum concentrations of 25-hydroxyvitamin D (25OHD) and calcitriol, fractional calcium absorption (Fc220, measured by means of a stable strontium test), 24h urinary calcium and 24h urinary phosphorus excretion were observed during wintertime. 25OHD but not calcitriol was correlated with Fc220 values and with 24h urinary phosphorus excretion. Moreover, Fc220 was related to 24 h urinary calcium excretion. Fasting 2 h-urinary deoxypyridinoline concentrations (biomarker of bone resorption) and serum levels of carboxyterminal propeptide of type I procollagen (biomarker of bone formation) showed no differences between summer and winter. CONCLUSIONS: Our data indicate a decrease in intestinal calcium and phosphorus absorption during wintertime, most likely because of a reduction in serum 25OHD levels. Since bone turnover was not affected by the seasonal differences in mineral metabolism, there is no objective for young women with high calcium intake to supplement vitamin D during wintertime.     

Association between intestinal vitamin D receptor, calcium absorption, and serum 1,25 dihydroxyvitamin D in normal young and elderly women

This paper systematically reviews the results from epidemiologic studies investigating the hypothesis that breast cancer risk in postmenopausal women increases with increasing concentrations of estradiol in blood and with increasing urinary estrogen excretion rates. Data from 29 epidemiologic studies of endogenous hormones and postmenopausal breast cancer were used. The ratio of the average estrogen concentration in the women with breast cancer to that in the women without breast cancer (and its 95 percent confidence interval [CI]) was calculated for each study, and the results were summarized by calculating weighted averages of the log ratios. In six prospective studies of serum estradiol concentration, 329 women who subsequently developed breast cancer had, overall, a 15 percent (CI = 6-24 percent, P = 0.0003) higher mean concentration of estradiol in their blood than the 1,105 women who remained free of cancer. The results of these prospective studies did not differ significantly from each other (chi2 for heterogeneity = 8.7; degrees of freedom = 5; P > 0.1). Similar differences in mean estrogen levels were seen in the case-control studies which reported either estradiol concentrations in the blood or urinary estrogen excretion. However, the case-control studies showed significant heterogeneity among their results. The data from the prospective studies strongly suggest that breast cancer risk in postmenopausal women is associated with relatively high concentrations of endogenous estradiol.     

Association of bone mineral density with vitamin D receptor gene polymorphism--changes in radial bone mineral density with long-term follow-up: longitudinal study

Recent studies have shown that genetic effects on bone mineral density (BMD) and bone turnover are related to vitamin D receptor (VDR) gene polymorphism. However, discordant studies have been published and it is still not clear whether VDR genotypes influence bone mass accretion and/or postmenopausal bone loss. To assess allelic influence of the VDR gene on BMD, we determined changes in 1/6-radial-BMD by several repeat measurements in the same subjects for about ten years and analyzed VDR polymorphism of BsmI restriction enzyme in 53 normal healthy Japanese women (age: 50.3 +/- 4.7 years, mean +/- SD). Twenty-seven (age: 53.2 +/- 4.7 years) of the subjects were post-menopausal (POST group). Among these 53 subjects, the distribution of bb, Bb and BB genotypes was 64.2%, 34% and 1.9%, respectively. The genotype frequencies in this study were very similar to those in previous reports concerning other Japanese women. There was no difference between the b group (women with bb genotype) and B group (women with BB or Bb genotype) in age, body weight, height, body mass index (BMI), years since menopause, serum osteocalcin and serum alkaline phosphatase values. In the POST group, BMD of the B group at menopause was lower than that of the b group (p < 0.05). About ten years after menopause, BMD did not differ significantly between these groups because the decrease in BMD in the b group was larger than that in the B group. Regarding changes in BMD in the POST group for four years after menopause, BMD of the b group was significantly decreased compared with the B group (p < 0.01). Our findings suggest that the differences in BMD by VDR genotype were larger among pre- and pri-menopausal women and seemed to decrease with years after menopause. It is suggested that there are other factors influencing BMD and postmenopausal bone loss in elderly women.     

High prevalence of vitamin D deficiency and reduced bone mass in Parkinson's disease

Despite excessive hip fractures in patients with Parkinson's disease (PD), little is known about bone changes in these patients. We measured bone mineral density (BMD; Z scores) in PD patients and analyzed its relation to serum biochemical indices and sunlight exposure. We measured BMD in 71 patients in the second metacarpals and divided the patients into two groups according to functional independence; group 1, Hoehn and Yahr stages 1 and 2; and group 2, stages 3 to 5. In four of 20 patients in group 1 (20%), the Z score was less than -1.0, indicating osteopenia. In 51 patients in group 2, 31 (61%) had a Z score less than -1.0. The group 1 patients showed a normal mean serum level of 25-hydroxyvitamin D (25-OHD; 21.7 ng/ml), while most group 2 patients were in a deficiency range (group mean 8.9 ng/ml). Many group 2 patients were sunlight deprived. Both groups had elevated serum ionized calcium levels correlating positively with Hoehn and Yahr stage and markedly depressed serum 1,25-dihydroxyvitamin D (1,25-[OH]2D) concentrations, indicating that immobilization-induced hypercalcemia had inhibited 1,25-[OH]2D production. Z scores correlated positively with 25-OHD levels and negatively with parathyroid hormone concentration and Hoehn and Yahr stage. Vitamin D deficiency due to sunlight deprivation and hypercalcemia induces compensatory hyperparathyroidism, which contributes to reduced BMD in PD patients, particularly those who are functionally dependent. Low BMD increases risk of hip fractures in patients with PD but may be improved by vitamin D supplementation.     

Effect of vitamin D receptor gene polymorphism and lifestyle on bone mineral density and bone mineral density decrement rate

The effects of genetic and environmental factors on bone mineral density (BMD) were investigated in 108 healthy Japanese women. Of the 108 subjects, BMD (from the second to forth lumbar vertebrae) was measured in 1992 in 103, in 1993 in 100, and in both years in 95 by dual energy X-ray absorptiometry. Vitamin D receptor (VDR) gene polymorphism in intron 8 was used as a genetic marker. Information on menstruation, health status, lifestyle, quantities of nutrient intake and frequencies of food intake was obtained by questionnaire. The frequency of allele B (825bp), whose polymerase chain reaction (PCR) products cannot be cut with BsmI, was 0.259 and the frequency of allele b (650bp), whose PCR products can be cut with BsmI, was 0.741. The subjects in our study obeyed the Hardy-Weinberg law. While the frequency of allele B was 0.448 in European whites as reported by Morrison et al, it was 0.259 in our Japanese subjects, suggesting a racial difference. Z score values (average value 0, standard deviation 1) increased in the order BB, Bb and bb. This result indicates that allele B is associated with the lower BMD in Japanese, as in European whites. The BMD decrement rate increased in the order bb, Bb and BB, indicating that VDR gene polymorphism may be a regulatory factor for losing BMD. Most of lifestyle variables, calcium intake and vitamin D intake were not correlated with BMD, but the food frequency score (which was defined as values weighted in each of three food categories obtained by factor analysis) was significantly correlated with BMD. Multiple regression analysis showed significant influences of years after menopause, the food frequency score and VDR genotype on BMD. VDR genotype and years after menopause influenced the BMD decrement rate significantly in multiple regression analysis. Neither a relationship between BMD and calcium intake nor between BMD and vitamin D intake by VDR genotype was found. These results suggest that the VDR gene is a genetic factor in BMD and the BMD decrement rate in Japanese.     

The effect of vitamin D supplementation on the bone mineral density of the femoral neck is associated with vitamin D receptor genotype

Recent studies suggest that variations of the vitamin D receptor (VDR) gene are related to bone mineral density (BMD). In this study, we examined the effect of vitamin D3 supplementation on BMD at the femoral neck in relation to VDR genotype. We analyzed 81 women, age 70 years and over, who participated in a placebo-controlled clinical trial on the effect of vitamin D3 supplementation (400 IU daily for at least 2 years) on BMD and fracture incidence. VDR genotype was based on the presence (b) or absence (B) of the BsmI restriction site. Mean BMD of the right and left femoral neck was measured at baseline and after 1 and 2 years. Dietary calcium, body mass index, and years since menopause were assessed at baseline while biochemical markers were measured at baseline and after 1 year. There was no difference among the BB, Bb, and bb genotype for baseline measurements of BMD at the femoral neck (mean and SD, g/cm2: 0.70 (0.10), 0.71 (0.12), and 0.69 (0.10), respectively), nor for any of the biochemical indices. The mean increase of BMD in the vitamin D group relative to the placebo group, expressed as percentage of baseline BMD, was significantly higher (p = 0.03) in the BB (delta BMD: 4.4%, p = 0.04) and Bb genotype (delta BMD: 4.2%, p = 0.007) compared with the bb genotype (delta BMD: -0.3%, p = 0.61). No significant changes were found for any of the other measured parameters. The VDR genotype-dependent effect of vitamin D supplementation in these elderly subjects suggest a functional involvement of VDR gene variants in determining BMD.     

Influence of exogenous porcine growth hormone on vitamin D metabolism and calcium and phosphorus absorption in intact pigs

The influence of growth hormone (GH) on vitamin D metabolism and calcium and phosphorus absorption in vivo is not clear. We, therefore, measured calcium and phosphorus balance, plasma 1,25-dihydroxyvitamin D (1,25(OH)2D), and intestinal vitamin D-dependent calcium-binding protein (CaBP 9k) in intact growing pigs given exogenous GH. Six 10-week-old pigs were given two daily subcutaneous injections of 50 micrograms porcine GH/kg body weight for 2 months; six control pigs were given vehicle. They were all fed a diet containing 1.1% Ca, 0.6% P, and 1000 IU vitamin D3/kg. Apparent Ca and P absorption and retention were measured in a 10-day balance trial at the end of the 2 months. The plasma levels of Ca, P, 1,25(OH)2D, IGF-I, and GH were determined, and the duodenal and jejunal mucosal CaBP 9k content was measured at slaughter. The plasma Ca and P of GH-treated pigs were unchanged, but all aspects of mineral metabolism, including the plasma 1,25(OH)2D concentration (40%), Ca absorption and retention (70%), P absorption (33%) and retention (45%), and jejunal CaBP 9k (40%), were stimulated, in addition to an increase in the circulating IGF-I concentration.     

The BsmI vitamin D receptor restriction fragment length polymorphism (bb) influences the effect of calcium intake on bone mineral density

Previous studies of the vitamin D receptor (VDR) polymorphisms and bone mineral density (BMD) have suggested that there may be differences in calcium absorption among groups of women with different VDR genotypes, and that the association may be stronger in younger women. To investigate the association between the VDR polymorphisms and BMD, this study was undertaken in the Framingham Study Cohort and a group of younger volunteers. Subjects from the Framingham Study (ages 69-90 years) included those who underwent BMD testing and who had genotyping for the VDR alleles (n = 328) using polymerase chain reaction methods and restriction fragment length polymorphisms with BsmI (B absence, b presence of cut site). A group of younger volunteer subjects (ages 18-68) also underwent BMD testing and VDR genotyping (n = 94). In Framingham Cohort subjects with the bb genotype, but not the Bb or BB genotypes, there were significant associations between calcium intake and BMD at five of six skeletal sites, such that BMD was 7-12% higher in those with dietary calcium intakes greater than 800 mg/day compared with those with intakes < 500 mg/day. The data also suggested that BMD was higher in persons with the bb genotype only in the group with calcium intakes above 800 mg/day. No significant differences were found in the Framingham Cohort for age-, sex-, and weight-adjusted BMD at any skeletal site between those with the BB genotype and those with the bb genotype regardless of 25-hydroxyvitamin D levels or country of origin. In the younger volunteers, BMD of the femoral neck was 5.4% higher (p < 0.05) in the bb genotype group compared with the BB group and 11% higher (p < 0.05) in males with the bb genotype compared with the BB group. There were no significant differences at the lumbar spine. In this study, the association between calcium intake and BMD appeared to be dependent upon VDR genotype. The findings of an association between dietary calcium intake and BMD only in the bb genotype group suggests that the VDR genotype may play a role in the absorption of dietary calcium. Studies that do not consider calcium intake may not detect associations between VDR genotype and BMD. In addition, the association between VDR alleles and BMD may become less evident in older subjects.     

Effect of vitamin D deficiency on lipid composition and calcium transport in basolateral membrane vesicles from chick intestine

Vitamin D deficiency affects the lipid composition and Ca2+ uptake of intestinal basolateral membranes from chick intestine. The increased cholesterol content causes an increase in the molar ratio cholesterol/phospholipid. Phospholipid classes remain unchanged, but the percentages of arachidonic acid from the from the major phospholipid fractions are increased. After 24 hours of oral administration of 2,000 IU of cholecalciferol to vitamin D-deficient chicks, the cholesterol values do not change, but the amount of arachidonic acid returns to normal values. Ca2+ uptake driven by ATP is diminished in vesicles from intestinal basolateral membranes of vitamin D-deficient chicks. Cholecalciferol treatment returns these values to the controls which might be due mainly to the increased number of Ca2+ pump units. In conclusion, changes in lipid composition and in Ca2+ pump caused by vitamin D deficiency seems to play a role in the decrease of vesicular Ca2+ transport. A single dose of cholecalciferol restores only partially the lipid-protein changes produced by vitamin D deficiency.     

Intestinal absorption of vitamin B12 in patients with chronic pancreatic insufficiency and the effect of human duodenal juice on the intestinal uptake of vitamin B12

The mean absorption of vitamin B12 (Schilling test) was 13.1 +/- 1.0 (% +/- S.E.M.) in 21 patients with chronic pancreatic insufficiency and 17.6 +/- 1.4 in 13 control patients (p less than 0.01). There was no correlation between pancreatic bicarbonate production after secretion stimulation and vitamin B12 absorption in the patient group (r = 0.117). Human duodenal juice reduced the uptake of 57CoB12-rat intrinsic factor (IF) by perfused rat small intestinal segments in vivo (p less than 0.01) as well as the uptake of 57CoB12-human IF by purified guinea-pig intestinal brush borders in vitro (p less than 0.01). The results confirm reduced absorption of vitamin B12 in chronic pancreatic insufficiency, but the mechanism remains uncertain.     

Absence of vitamin D deficiency in young Nigerian children

OBJECTIVE: To determine the prevalence of vitamin D deficiency in young Nigerian children residing in an area where nutritional rickets is common. Study design: A randomized cluster sample of children aged 6 to 35 months in Jos, Nigeria. RESULTS: Of 218 children evaluated, no child in the study had a 25-hydroxyvitamin D (25-OHD) concentration <10 ng/mL (the generally held definition of vitamin D deficiency). Children spent an average of 8.3 hours per day outside of the home. Twenty children (9.2%) had clinical findings of rickets. Children with clinical signs of rickets were more likely to be not currently breast fed and have significantly lower serum calcium concentrations than those without signs of rickets (9.1 vs 9.4 mg/dL, respectively, P =.01). Yet, 25-OHD levels were not significantly different between those children with clinical signs of rickets and those without such clinical signs. CONCLUSION: Vitamin D deficiency was not found in this population of young children in whom clinical rickets is common. This is consistent with the hypothesis that dietary calcium insufficiency, without preexisting vitamin D deficiency, accounts for the development of clinical rickets in Nigerian children.     

Vitamin D receptor gene polymorphism is associated with urinary calcium excretion but not with bone mineral density in postmenopausal women

Polymorphism of vitamin D receptor (VDR) gene has been found to be associated with serum osteocalcin (OC) levels and bone mineral density (BMD) in Caucasian identical twins and unrelated postmenopausal women. Being ethnically different and living in a geographic area with adequate vitamin D status due to abundant sunshine exposure, it is unclear whether VDR gene polymorphism will affect bone mass in Thai population. In the present study, we investigated the association between VDR gene polymorphism and bone metabolism in Thai postmenopausal women. Subjects consisted of 84 postmenopausal women. Bsm I, Taq I and Apa I polymorphisms of VDR gene were determined by PCR-RFLP. B, T and A represent the absence of the corresponding restriction sites while b, t and a indicate the presence of the restriction sites. Data were expressed as mean +/- SE. Sixty-six subjects (78.6%) had bb genotype while 18 (21.4%) had Bb genotype. None of the subjects was found to have BB genotype. Taq I restriction site was in linkage disequilibrium to the Bsm I site. For Apa I polymorphism, 33 (39.3%), 42 (50.0%) and 9 (10.7%) of the subjects had aa, Aa and AA genotypes, respectively. There was no significant difference in serum intact OC levels and BMD at various skeletal sites among subjects with different genotypes. Despite the lack of difference in BMD and intact OC levels, subjects with bb genotype had higher 24-hour urinary calcium excretion than those with Bb genotype (bb, 6.1 +/- 0.3 mmol/day; Bb, 4.4 +/- 0.6 mmol/day; p < 0.05). The effect of Bsm I VDR genotype was still significant (p < 0.05) after dietary calcium intake was controlled using analysis of covariance. Despite the difference in urinary calcium levels, there was no significant difference in fractional excretion of calcium among subjects with different Bsm I-related genotypes, suggesting that the effect of the VDR gene polymorphism on urinary calcium excretion is more likely due to the effect on intestinal calcium absorption rather than renal tubular calcium reabsorption. We conclude that VDR genotype distributions in Thai postmenopausal women are different from those reported in Caucasians. VDR gene polymorphism does not appear to be associated with BMD or bone turnover in Thai postmenopausal women. However, Bsm I VDR polymorphism may have physiologic role in calcium homeostatasis by modulating intestinal calcium absorption.     

Bone mineral density and bone markers in relation to vitamin D receptor gene polymorphisms in Chinese men and women

Whether vitamin D receptor gene (VDRG) polymorphism can be used as a predictor for bone turnover rate or bone mass remains controversial. Its role within various ethnic populations are also unsettled. We examined VDRG polymorphism using restrictive enzymes Bsm-I, Apa-I, and Taq-I in 155 men aged 22-88 and 113 premenopausal women aged 40-53. The bone mineral density (BMD) of the vertebrae (L2-4), proximal femur, and total body bone mineral content (tb-BMC) (women only), as well as urinary N-terminal crosslinked fragment of type I collagen (NTX), serum osteocalcin, bone isozyme of alkaline phosphatase, and caboxyterminal propeptide of type I procollagen levels were measured. Chinese men and women exhibited a low prevalence for B (absence of Bsm-I restriction site) phenotypes than white and Japanese. Within the tested samples there were 0.4% BB homozygotes, 6.7% Bb heterozygotes, and 93% bb homozygotes. The distributions of Apa-I polymorphism (9.0% AA, 42.5% Aa, and 48.5% aa) also differed from those reported for the white populations. Most of the Chinese men and women were TT homozygous (96.6%). A comparison of actual values and values adjusted for age and weight of tb-BMC and BMD at the lumbar spine, Trochanter, Ward's triangle, and femoral neck showed no significant difference among three subgroups in each of the three sets of polymorphism. Furthermore, the actual values and adjusted values (adjusted for age) of the four bone markers, respectively, showed no significant differences. We conclude that given the very low prevalence of the suspected high risk genotypes (B, A, and t), and the lack of difference among the polymorphic subgroups, VDRG polymorphism may not be an important determinant of the bone turnover rate and bone mass of Chinese men and women.     

Genetics of vitamin D 1alpha-hydroxylase deficiency in 17 families

Vitamin D-dependent rickets type I (VDDR-I), also known as pseudo-vitamin D-deficiency rickets, appears to result from deficiency of renal vitamin D 1alpha-hydroxylase activity. Prior work has shown that the affected gene lies on 12q13.3. We recently cloned the cDNA and gene for this enzyme, mitochondrial P450c1alpha, and we and others have found mutations in its gene in a few patients. To determine whether all patients with VDDR-I have mutations in P450c1alpha, we have analyzed the P450c1alpha gene in 19 individuals from 17 families representing various ethnic groups. The whole gene was PCR amplified and subjected to direct sequencing; candidate mutations were confirmed by repeat PCR of the relevant exon from genomic DNA from the patients and their parents. Microsatellite haplotyping with the markers D12S90, D12S305, and D12S104 was also done in all families. All patients had P450c1alpha mutations on both alleles. In the French Canadian population, among whom VDDR-I is common, 9 of 10 alleles bore the haplotype 4-7-1 and carried the mutation 958DeltaG. This haplotype and mutation were also seen in two other families and are easily identified because the mutation ablates a TaiI/MaeII site. Six families of widely divergent ethnic backgrounds carried a 7-bp duplication in association with four different microsatellite haplotypes, indicating a mutational hot spot. We found 14 different mutations, including 7 amino acid replacement mutations. When these missense mutations were analyzed by expressing the mutant enzyme in mouse Leydig MA-10 cells and assaying 1alpha-hydroxylase activity, none retained detectable 1alpha-hydroxylase activity. These studies show that most if not all patients with VDDR-I have severe mutations in P450c1alpha, and hence the disease should be referred to as "1alpha-hydroxylase deficiency."     

The oral strontium load test in the assessment of intestinal calcium absorption

A close correlation between calcium and strontium intestinal absorption has been described. In this study, a test using Stable Strontium has been assessed in women without abnormal calcium or bone metabolism, with no history of drugs which might affect calcium or bone metabolism. Decreasing values of Strontium intestinal absorption, according to the length of the postmenopausal period, have been observed. Besides, the Stable Strontium Test has been given in postmenopausal women with osteoporotic femur fractures. In comparison with age matched healthy women, this latter group showed a significantly lower Strontium intestinal absorption. Analogous behaviour has been reported for Calcium intestinal absorption.