Identification of 1,25-Dihydroxycholecalciferol, a Form of Vitamin D3 Metabolically Active in the Intestine

Abstract

A biologically active vitamin Ds metabolite ("peak V metabolite") more polar than 25-hydroxycholecalciferol has been isolated from chicken intestines in pure form as a mono(trimethylsilyl)ether derivative. The molecule has been identified as 1,25-dihydroxycholecalciferol by means of mass spectrometry, ultraviolet absorption spectrophotometry, and specific chemical reactions. It has been demonstrated (1) with tritiated vitamin D3 of high specific activity that vitamin D3 is converted to metabolites more potent and rapidly acting than the parent vitamin. The most abundant of these, 25-hydroxycholecalciferol (25-HCC) (2), acts more rapidly than vitamin D3 in enhancing both intestinal calcium transport and bone mineral mobilization (3). The existence in the intestine of additional polar metabolite(s) active in promoting calcium transport is evident from work in a number of laboratories (4-8). In agreement with Haussler et al. (8) and Myrtle et al. (9), we have observed that one of these metabolite(s), the peak V metabolite, induces intestinal calcium transport in deficient chickens more rapidly than vitamin D3 or 25-HCC, although it is less effective than 25-HCC in curing rickets or inducing bone mineral mobilization.By blocking the conversion of 25-HCC to peak V metabolite by prior administration of actinomycin D, we have shown (10) that peak V, or a further metabolite of peak V, is the metabolically active form of vitamin D in the intestine. In the presence of actinomycin D, the peak V compound stimulates intestinal calcium transport, whereas 25-HCC does not. This polar metabolite has now been isolated in pure form and identified as 1,25-dihydroxycholecalciferol (1,25-DHCC).ISOLATION 1450 vitamin D-deficient chicks were each given 2.5 jug of [1,2-3H]vitamin D3 (specific activity, 3.2 X 101 dpm/,ug), orally, in vegetable oil (Wesson). 24 hr later the chicks were killed. Methanol-chloroform extracts of the small intestines were prepared, and the chloroform extract was subjected to a sequence of chromatographic separations on Sephadex LH-20 and BioRad (beads) S-X8 (BioRad Labs., Richmond, Calif.). 11 jug of highly purified peak V metabolite was obtained. 8 jug of the metabolite was then reacted with 5 jul of TBT (a combination of TMS-imidazole, bis-TMSacetamide, and trimethylchlorosilane, Pierce Chemical Co., Rockford, Ill.) and 10 jul of pyridine for 10 min. The resulting tri(trimethylsilyl)ether derivative was chromatographed on a 1 X 60 cm column of Sephadex LH-20 in 50% chloroform in petroleum ether (bp 67-690C). The tri(trimethylsilyl) derivative was partially desilylated in 100 ul of 3.6 X 10-4 M HC1 in MeOH + 50 jul pyridine at 60°C for 4 hr. This gave a mixture of di(trimethylsilyl), mono(trimethylsilyl), and unsilylated products, which were separated by chromatography on Sephadex LH-20. The metabolite and the mono-(trimethylsilylated) metabolite were separately chromatographed in methanol on Sephadex LH-20 (11) before being used for structural identification.

Complementary and alternative medicine for the treatment of multiple sclerosis

“…In vertebrates, vitamin D 3 is produced in the skin from exposure to UV radiation [126]. Vitamin D 3 is converted into 25-hydroxy-vitamin D 3 in the liver and 1,25-dihydroxyvitamin D 3 in the kidney [127]. 1,25-dihydroxy-vitamin D 3 , which is the bioactive form of vitamin D, is important for regulating the calcium and phosphorus levels in the blood by promoting their absorption from food and helping normal bone mineralization, growth and remodeling [128–130].…”

TRIENNIAL GROWTH SYMPOSIUM— Vitamin D: Bones and beyond12

“…Its structure was confirmed by synthesis (Blunt and DeLuca, 1969) and its biopotency was shown to be between 1.5 to 2 times that of vitamin D 3 itself (Blunt et al, 1968b). When it was radioactively labeled (Suda et al, 1971), 25-OH-D also disappeared before the response of intestine or bone, which ultimately led to the isolation and identification of the final active form, 1α,25-dihydroxyvitamin D 3 (1,25-(OH) 2 D 3 ; Holick et al, 1971;Suda et al, 1971;Semmler et al, 1972). This resulted in our knowledge of the activation of vitamin D in preparation for function (De-Luca, 1974), as shown in Fig.…”

The vitamin D receptor: contemporary genomic approaches reveal new basic and translational insights

“…In the early 1970s, 1,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ] was identified as both the exclusive, metabolically active form of vitamin D and a key component of what proved to be an exquisite endocrine system that regulates numerous biologic processes in higher vertebrates (1,2). This pioneering discovery ended a decades-long quest to understand the dynamic processes that govern the formation of vitamin D in the skin and its sequential activation to 25(OH) D 3 in the liver and then to 1,25(OH) 2 D 3 in the kidney.…”