Chromatographic separation of vitamin D3 sulfate and vitamin D3.

This paper describes a simple chromatographic technique on Sephadex LH20 for the separation of vitamin D3 sulfate from free vitamin D3 and its metabolites. This technique has been used in the study of vitamin D3 sulfate metabolism in rats. Seven hours after injection of vitamin D3 sulfate (35S or 35S and 3H) only the peak of vitamin D sulfoconjugate was found in chromatographic elution of serum extracts.     

Differences in the metabolism of vitamin D2 and vitamin D3 by subcellular fractions from rat liver

The 25-hydroxylation of vitamin D2 and vitamin D3 was studied in the mitochondrial fraction from rat liver and in a reconstituted system containing cytochrome P-450 from rat liver microsomes. The mitochondrial fraction catalyzed the 25-hydroxylation of vitamin D3 at least two times more effectively than the 25-hydroxylation of vitamin D2. Microsomal cytochrome P-450 catalyzed an efficient 25-hydroxylation of vitamin D3, but no 25-hydroxylation of vitamin D2 could be detected. The present results show a difference in the 25-hydroxylation of vitamin D2 and vitamin D3 in rat liver in vitro.     

The isolation and identification of vitamin D2 and vitamin D3 from Medicago sativa (alfalfa plant)

Vitamin D₂ and vitamin D₃ were isolated from Medicago sativa (alfalfa) grown under field and laboratory conditions and then irradiated with ultraviolet light. The vitamins were identified by ultraviolet absorption, mass spectroscopy, and comparison with synthetic standards on several chromatographic systems. Sun-cured, field-grown alfalfa contained vitamin D₂ at a concentration of 48 ng/g (1920 IU/kg) and vitamin D₃ at 0.63 ng/g (25 IU/kg). Laboratory-grown alfalfa, artificially irradiated, contained vitamin D₂ at a concentration of 80 ng/g and vitamin D₃ at 1.0 ng/g. Therefore, the presence of vitamin D₂, as well as vitamin D₃, has unequivocally been demonstrated in alfalfa plant tissue.     

Studies on vitamin D3 metabolism. Discrete liver cytosolic binding proteins for vitamin D3 and 25-hydroxyvitamin D3

Two separate liver cytosolic proteins have been partially purified and identified by their selectivity for binding either [1α,2α(n)-³H]vitamin D₃ or 25-hydroxy [26(27)-methyl-³H]vitamin D₃. The chromatographic properties of the two proteins were not distinguishable by ion-exchange nor were they dependent upon the vitamin D₃ nutritional status of the birds. However, in molecular exclusion chromatography, the binding proteins can be successfully resolved into two discrete entities. Their binding properties suggest that they are not identical with plasma vitamin D₃ binding protein.     

Hepatic accumulation of vitamin D3 and 25-hydroxyvitamin D3.

Concomitant intravenous administration of 25-hydroxycholecalciferol and [3H] vitamin D3 to vitamin D-depleted rats did not affect the conversion of [3H] vitamin D3 to 25-OH-[3H] vitamin D3 as indicated by a serum 25-OH-[3H] vitamin D3 to content at 3 and 24 h identical to those observed in animals receiving [3H] vitamin D3 alone. Similarly, pre-dosing with 25-OH vitamin D3 24 h earlier did not affect the conversion. Co-administration to vitamin D depleted rats of vitamin D2 or D3, at 200-fold higher doses than a control group receiving tracer [3H] vitamin D3 alone, resulted in serum 25-OH vitamin D levels that were 15-20 fold higher than the control, indicating a similar metabolic fate for synthetic and natural vitamin D in rats and the ability of increased substrate to overwhelm hepatic constraints on 25-OH vitamin D production. Following intravenous administration of 25-OH-[3H] vitamin D3 to vitamin D depleted rats, hepatic 3H content decreased in parallel with serum radioactivity. Hepatic accumulation of intravenously administered vitamin D3 ([14C] vitamin D3) alone or with 25-OH-[3H] vitamin D3, by vitamin D-depleted rats revealed a marked preference for vitamin D3; the hepatic accumulation of [14C] vitamin D3 increased to 35% of the dose by 45 min, at which time 25-OH-[3H] vitamin D3 hepatic content was 7-fold less, and decreasing. Chromatography of extracts of hepatic subcellular fractions revealed more [14C] vitamin D3 than 25-OH-[3H] vitamin D3 in the microsomes, the reported site of calciferol 25-hydroxylase. Circulating 25-OH vitamin D, therefore, has comparatively minimal potential for hepatic accumulation. Product inhibition of the calciferol 25-hydroxylase must, therefore, result from recently synthesized hepatic 25-OH vitamin D, and is not affected by exogenous 25-OH vitamin D3.     

Influence of ultraviolet B radiation on vitamin D3 metabolism in vitamin D3-resistant New World primates

We investigated the occurrence of rickets in adolescent tamarins (Saguinus imperator) residing at the Los Angeles Zoo. Compared to tamarins in the same colony without clinical evidence of bone disease (N = 6), rachitic platyrrhines (N = 3) had a decrease in their serum calcium concentration (P < .05). The affected tamarins also had lower serum 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) levels than did nonaffected colony mates, but 2–10-fold higher concentrations than in Old World primates of a comparable developmental stage. New World primates in many different genera are known to exhibit target organ resistance to the active vitamin D₃ metabolite, 1,25-(OH)₂D₃, compensated by maintenance of high circulating concentrations of 1,25-(OH)₂D₃. The relatively low serum 1,25-(OH)₂D₃ concentration in rachitic tamarins and ultraviolet B radiation deficient environment of these primates suggested that bone disease may be linked to a deficiency in substrate for 1,25-(OH)₂D₃, 25 hydroxyvtamin D₃ (25-OHD₃). Chronic exposure of platyrrhines in three different vitamin D resistant genera to an artificial UVB source resulted in 1) a significant increase in the mean serum 25-OHD₃ (P < .001) and 1,25-(OH)₂D₃ (P < .02) level over that encountered in platyrrhines not exposed to UVB; and 2) prevention of rachitic bone disease in irradiated individuals. These data further show that the serum 25-OHD₃ and 1,25-OH₂D₃ levels are positively correlated in vitamin D-resistant platyrrhines (r = 0.64; P= .0014) and suggest that a compromise in cutaneous vitamin D₃ production by means of UVB deprivation may limit necessary 1,25-(OH)₂D₃ production. © 1992 Wiley-Liss, Inc.     

Effect of vitamin D3 administration on serum 25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D3 and osteocalcin in vitamin D-deficient elderly people

In elderly institutionalized people, confined to bedroom and receiving no vitamin D supplementation, the frequency of vitamin D deficiency is found very high. Systematic administration of vitamin D has, therefore, been proposed to correct vitamin D deficiency. Within this context, we studied 40 elderly institutionalized subjects (mean age 80.5 + 7.2 yr) with low 25(OH)D3 concentrations (4.4 + 1.8 micrograms/l). Sixteen of them (Group I) had low serum calcium concentrations (less than 2.3 mmol/l) and 24 (Group II) had normal serum calcium concentrations (from 2.3 to 2.6 mmol/l). As hypocalcemia has been shown to regulate 1,25(OH)D3 production independent of PTH in animals and in humans, we compared their respective responses to the administration of vitamin D3. Subjects received a total dose of 15 mg (600,000 IU) of vitamin D3 divided into 3 i.m. injections at one month intervals and were explored before therapy and one and 6 months after the last dose of vitamin D3. The treatment induced a similar marked rise in 25(OH)D3 levels (from 4.1 + 1.7 to 24.4 + 8.7 micrograms/l for group I and from 5.1 + 1.8 to 27.2 + 8.0 micrograms/l for group II) in both groups but increased the 1,25(OH)2D3 concentrations only in group I (from 22.9 + 6.9 to 32.6 + 11.3 ng/l). Meanwhile serum calcium concentrations rose in group I (to low normal range i.e. 2.31 + 0.07 mmol/l) and were unaffected in group II. These results suggest that hypocalcemia is a potent stimulator of renal 1-hydroxylase in elderly people. Furthermore, a transient significant (P less than 0.01) increase in serum osteocalcin (from 10.6 + 4.1 to 14.1 + 5.9 micrograms/l) could be observed in group I which demonstrates for the first time that the osteocalcin response of osteoblasts to stimulation by 1,25(OH)2D3 is retained in very old people.     

Individual quantitation of vitamin D2, vitamin D3, 25-hydroxyvitamin D2, and 25-hydroxyvitamin D3 in human milk

Extraction, lipid-reduction, and chromatographic methods suitable for the resolution and subsequent quantitation of vitamin D2, vitamin D3, 25-hydroxyvitamin D2, and 25-hydroxy-vitamin D3 from human milk are described. This procedure utilizes a methanol:methylene chloride extraction, precipitation of unwanted lipids with cold methanol and ether, backwash with alkaline buffer, silica Sep-Pak preparative chromatography, normal- and reverse-phase high-performance liquid chromatography with final quantitation of the antirachitic sterols by competitive protein binding assay. The described assay was used to determine these antirachitic sterols in milk from women receiving various supplements of vitamin D or undergoing ultraviolet phototherapy.     

A fluorinated vitamin D3 analog with biopotency greater than 1 alpha, 25-dihydroxy vitamin D3

Vitamin D metabolites and analogs induce $\text{de novo}$ synthesis of a specific calcium-binding protein in embryonic chick duodenum maintained in organ culture. Using calcium-binding protein biosynthesis as a specific and sensitive biochemical indicator of intrinsic biopotency, 24,24-difluoro-1α,25-dihydroxy vitamin D₃ was found to be approximately four times more potent on a molar basis than the most active, naturally occurring metabolite, 1α,25-dihydroxy vitamin D₃.     

An evaluation of the biologic activity and vitamin D receptor binding affinity of the photoisomers of vitamin D3 and previtamin D3

Skin is in the site of previtamin D3 and vitamin D3 synthesis and their isomerization in response to ultraviolet irradiation. At present, little is known about the function of the photoisomers of previtamin D3 and the vitamin D3 in skin cells. In this study we investigated the antiproliferative activity of the major photoisomers and their metabolites in the cultured human keratinocytes by determining their influence on 3H-thymidine incorporation into DNA. Our results demonstrated at both 10(-8) and 10(-6) M in a dose-dependent manner. Lumisterol, tachysterol3, 5,6-trans-vitamin D3, and 25-hydroxy-5,6-trans-vitamin D3 only induced significant inhibition at 10(-6) M. 25-Hydroxytachysterol3 was approximately 10- to 100-fold more active than tachysterol3. 7-Dehydrocholesterol was not active even at 10(-6) M. The dissociation constants of vitamin D receptor (VDR) for 25-hydroxytachysterol3, 25-hydroxy-5,6-trans-vitamin D3, and 5,6-trans-vitamin D3 were 22, 58, and 560 nM, respectively. The dissociation constants for 7-dehydrocholesterol, tachysterol, and lumisterol were greater than 20 microM. In conclusion, vitamin D3, its photoisomers and the photoisomers of previtamin D3 have antiproliferative activity in cultured human keratinocytes. However, the antiproliferative activity did not correlate with their binding affinity for VDR. The results suggest that some of the photoproducts may be metabolized to their 25-hydroxylated and 1 alpha,25-dihydroxylated counterparts before acting on VDR. Alternatively, a different receptor may recognize these photoproducts or another mechanism may be involved in modulating the antiproliferative activity of the photoisomers examined.     

Isolation of identification of 24(R)-hydroxyvitamin D3 from chicks give large doses of vitamin D3

A new metabolite of vitamin D was isolated from the blood plasma of chicks given large doses of vitamin D3. The isolation involved methanol-chloroform extraction and four column chromatographic steps. The metabolite was identified by high- and low-resolution mass spectroscopy, chemical derivatization, an comigration with authentic standard as 3 beta, 24(R)-dihydroxy-9,10-seco-5,7,10(19)-cholestatriene [24(R)-hydroxyvitamin D3]. No detectable 24-(R)-hydroxyvitamin D3 was recovered from 16 L of plasma from chicks receiving physiologic levels of vitamin D3.     

A new chromatographic system for vitamin D3 and its metabolites: resoluation of a new vitamin D3 metabolite

A simple yet powerful new chromatographic procedure for vitamin D(3) and its metabolites is described. Liquid-gel partition chromatography on Sephadex LH-20 using a solvent of various percentages of CHCl(3) in Skellysolve B (petroleum ether, bp 67-69 degrees C) permits excellent resolution of vitamin D(3), 25-hydroxyvitamin D(3), and their more polar metabolites. Of special importance is the resolution of the metabolites of vitamin D(3) more polar than 25-hydroxycholecalciferol. Because of this resolution, a new metabolite of vitamin D(3) has been demonstrated in the plasma of rats and in the intestines of chicks given 100 IU of vitamin D(3)-1,2-(3)H.     

A new in vivo metabolite of vitamin D3: 1,25,26-trihydroxyvitamin D3

The enzymatic conversion of 5-methylthioribose to methionine and its deaminated derivatives, 2-keto-4-methylmercaptobutyric acid and 2-hydroxy-4-methylmercaptobutyric acid by cell-free extracts of $\text{Enterobacter}\text{aerogenes}$ has been demonstrated. ¹⁴C-Labeled methionine was isolated from incubation mixtures with 5-methylthio[U-¹⁴C]ribose. The carbohydrate part of this compound furnishes at least part, if not all, of the four carbon chain of methionine.     

Up-regulation of the intestinal 1,25-dihydroxyvitamin D receptor during hypervitaminosis D: a comparison between vitamin D2 and vitamin D3

Concentrations of intestinal 1,25-dihydroxyvitamin D receptor were measured in rats receiving pharmacological amounts (25,000 IU/rat daily for 6 days) of either vitamin D2 or vitamin D3. The data showed that both hypervitaminosis D2 and hypervitaminosis D3 resulted in significant up-regulation of intestinal 1,25-dihydroxyvitamin D receptor (fmol/mg protein) relative to controls (409 +/- 24, vitamin D2-treated; 525 +/- 41, vitamin D3-treated; and 249 +/- 19, control). The 1,25-dihydroxyvitamin D receptor enhancement also was accompanied by elevated plasma 25-hydroxyvitamin D and hypercalcemia. These data suggest that increased target-tissue 1,25-dihydroxyvitamin D receptor may play a role in enhancing target-tissue responsiveness and, thus, have a significant role in mediating the toxic effects of hypervitaminosis D.