Isolation and identification of 4,25-dihydroxyvitamin D-2: a novel A-ring hydroxylated metabolite of vitamin D-2

Vitamin D-2 is less toxic in rats when compared to vitamin D-3. Our laborat ory has been involved in research projects which were directed towards iden tifying the possible mechanisms responsible for the toxicity differences be tween vitamins D-2 and D-3 in rats. The present research project was design ed to isolate and identify new metabolites of vitamin D2 from serum of rats which were fed toxic doses of vitamin D-2. Hypervitaminosis D-2 was induce d in 30 rats by feeding each rat with 1000 nmol of vitamin D-2/day x 14 day s. The rats were sacrificed on the 15th day and obtained 180 ml of serum. T he lipid extract of the serum was directly analyzed by a straight phase HPL C system, The various vitamin D-2 metabolites were monitored by their ultra violet (UV) absorbance at 254 nm. One of the UV absorbing peaks did nut com igrate with any of the known vitamin D-2 metabolites, This unknown metaboli te peak was further purified by HPLC and was then subjected to UV absorptio n spectrophotometry and mass spectrometry. The structure assignment of the new metabolite was established to be 4,25-dihydroxyvitamin D-2 [4,25(OH)(2) D-2] by the techniques of UV absorption spectrophotometry and mass spectrom etry and by the new metabolite's susceptibility to sodium metaperiodate oxi dation, At present the biological activity of this unique 'A-ring' hydroxyl ated vitamin D-2 metabolite is not known, As this new metabolite is isolate d from the serum of rats intoxicated with vitamin D-2, we speculate that 4, 25(OH)(2)D-2 may be playing an important role in the deactivation of vitami n D-2. (C) 1999 Elsevier Science Ltd, All rights reserved.