Isolation and identification of 4,25-dihydroxyvitamin D2: a novel A-ring hydroxylated metabolite of vitamin D2

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Abstract

Vitamin D2 is less toxic in rats when compared to vitamin D3. Our laboratory has been involved in research projects which were directed towards identifying the possible mechanisms responsible for the toxicity differences between vitamins D2 and D3 in rats. The present research project was designed to isolate and identify new metabolites of vitamin D2 from serum of rats which were fed toxic doses of vitamin D2. Hypervitaminosis D2 was induced in 30 rats by feeding each rat with 1000 nmol of vitamin D2/day×14 days. The rats were sacrificed on the 15th day and obtained 180 ml of serum. The lipid extract of the serum was directly analyzed by a straight phase HPLC system. The various vitamin D2 metabolites were monitored by their ultraviolet (UV) absorbance at 254 nm. One of the UV absorbing peaks did not comigrate with any of the known vitamin D2 metabolites. This unknown metabolite peak was further purified by HPLC and was then subjected to UV absorption spectrophotometry and mass spectrometry. The structure assignment of the new metabolite was established to be 4,25-dihydroxyvitamin D2 [4,25(OH)2D2] by the techniques of UV absorption spectrophotometry and mass spectrometry and by the new metabolite's susceptibility to sodium metaperiodate oxidation. At present the biological activity of this unique ‘A-ring’ hydroxylated vitamin D2 metabolite is not known. As this new metabolite is isolated from the serum of rats intoxicated with vitamin D2, we speculate that 4,25(OH)2D2 may be playing an important role in the deactivation of vitamin D2.

Introduction

Vitamin D3 (cholecalciferol) is the form of vitamin D that is synthesized by vertebrates, whereas vitamin D2 (ergocalciferol) is the major naturally occurring form of the vitamin D in plants [1]. It has been known that there are differences between both forms of the vitamins in terms of their toxicity in mammalian species [2], [3], [4], [5], [6], [7]. However, the reasons for the lesser toxicity of vitamin D2 are not fully understood. The presence of a double bond at C-22 and an extra methyl group at C-24 position, of vitamin D2 side chain (Fig. 1) is responsible for the differences from the oxidative processes known to occur on the side chain of vitamin D3 [5], [8]. As a result, several differences in the pathways of side chain metabolism of these two vitamins are noted.

The known metabolites of vitamin D2 in mammals receiving dietary vitamin D2 are depicted in Fig. 2. Vitamin D2 is metabolized in vivo to both 25-hydroxyvitamin D2 (25OHD2) and 24-hydroxyvitamin D2 (24OHD2), the major circulating metabolites of vitamin D2 in rats and humans [9], [10], [11], [12]. Both 25OHD2 and 24OHD2 are subsequently hydroxylated at C-1 position to form 1α,25-dihydroxyvitamin D2 [1α,25(OH)2D2] and 1α,24-dihydroxyvitamin D2 [1α,24(OH)2D2] respectively [13], [10]. Further 1α,25(OH)2D2 is metabolized into 1α,24(R),25-trihydroxyvitamin D2 [1α,24(R),25(OH)3D2], 1α,24(R),25,26-tetrahydroxyvitamin D2 [1α,24(R),25,26(OH)4D2] and 1α,24(S),25,28-tetrahydroxyvitamin D2 [1α,24(S),25,28(OH)4D2] respectively [8] whereas, 1α,24(OH)2D2 is converted into 1α,24(S),26-trihydroxyvitamin D2 [1α,24(S),26(OH)3D2] [14]. It is important to understand the deactivation pathways of 25OHD2 and 24OHD2 especially during hypervitaminosis D2, a condition not uncommon in clinical medicine, as vitamin D2 is used routinely as a therapeutic agent. The inactivation of 24OHD2 occurs through its conversion to 24(S),26-dihydroxyvitamin D2 [24(S),26(OH)2D2] [15], and the inactivation of 25OHD2 occurs through its further metabolism into 24(R),25-dihydroxyvitamin D2 [24(R),25(OH)2D2] [9], [16]. We demonstrated that 24(R),25(OH)2D2 is further metabolized into 24(S),25,28-trihydroxyvitamin D2 [24(S),25,28(OH)3D2] and 24(R),25,26-trihydroxyvitamin D2 [24(R),25,26(OH)3D2] in the isolated perfused rat kidney and these two metabolites circulate in vitamin D2-intoxicated rat, suggesting that the formation of the two trihydroxylated metabolites of vitamin D2 plays an important role in the deactivation of 25OHD2 [11].

During the course of a pilot study designed to identify the circulating vitamin D2 metabolites in a vitamin D2-intoxicated rat using radiolabeled vitamin D2 we found a major radiolabeled peak which did not comigrate with any of the known metabolites of vitamin D2 on a straight phase HPLC system. The metabolite peak was more polar than the standard 24(R),25(OH)2D2 but less polar than the standard 1α,25(OH)2D2. It migrated just prior to the standard 25,28-dihydroxyvitamin D2 [25,28(OH)2D2] [11]. We designed the present study to isolate and identify this unknown circulating vitamin D2 metabolite from the serum of vitamin D2-intoxicated rats. We identified this new vitamin D2 metabolite as 4,25-dihydroxyvitamin D2 [4,25(OH)2D2]. The isolation and identification of this new A-ring hydroxylated metabolite of vitamin D2 is described in this paper.

Section snippets

Vitamin D compounds

Vitamin D2 was purchased from Sigma Chemical Co. (St. Louis, MO). 25OHD2 was a gift from Dr. J.A. Campbell and Dr. J. Babcock (Upjohn, Kalamazoo, MI). 24(R),25(OH)2D2 was a gift from Dr. T. Kobayashi, Department of Hygienic Sciences, Kobe Pharmaceutical University, Kobe, Japan. All the various synthetic standards of vitamin D2 metabolites used in this study were a gift from Hoffmann-La Roche, Nutley, NJ. Authentic 24OHD2 was isolated from the serum of vitamin D2-intoxicated rats, and the

Metabolites of vitamin D2 isolated from the serum of vitamin D2-intoxicated rats

Fig. 3 shows the HPLC profile of the parent substrate and the various metabolites produced in vitamin D2-intoxicated rats. From Fig. 3 it becomes obvious that, vitamin D2 is metabolized into several metabolites as represented by the UV absorbing peaks. The identities of the known metabolites of vitamin D2 i.e. 24OHD2, 25OHD2 and 24(R),25(OH)2D2 were confirmed by their comigration with the corresponding authentic cold standards on HPLC. In addition to the aforementioned metabolites, we have

Discussion

We report the isolation and identification of 4,25(OH)2D2 a novel A-ring hydroxylated metabolite of vitamin D2 in vitamin D2-intoxicated rats. Our finding of A-ring hydroxylation of 25OHD2 is not surprising. In a previous study, Thierry-Palmer et al. [21] demonstrated for the first time the possibility of A-ring hydroxylation of 25OHD3 by rat renal microsomes in vitro. This finding was based on the mass spectrometric analysis of the metabolite and its sensitivity to NaIO4, The mass spectrum of

Acknowledgements

We gratefully acknowledge Dr. M.F. Holick and Dr. R. Ray (Boston University School of Medicine, Boston, MA) for many helpful discussions. This work was supported in part by a grant (DK-30138) from the National Institutes of Health to G.S.R.

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