AN ASSAY METHOD FOR SEPARATELY MEASURING METABOLITES OF VITAMIN D3 AND THOSE PRESUMED TO BE DERIVED FROM VITAMIN D2

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An assay method for separately measuring the metabolites of vitamin D2 and D3 is investigated. Seven-week-old Wistar male rats were orally administered 200-20, 000IU of either vitamin D2 or D3 on alternate days for 12 days. The blood plasma was extracted with either methanol-chloroform for 25-OH-D and 24, 25-(OH)2-D, or dichloro-methane for 1α, 25-(OH)2-D. Each extract was applied separately to Sephadex LH-20 columns and then to high pressure liquid chromatog-raphy (HPLC). On HPLC each D2 metabolite appeared to be eluted in the region just prior to the respective D3 metabolite. 25-OH-D3, 24, 25-(OH)2-D3, and metabolites presumed to be 25-OH-D2 and 24, 25-(OH)2-D2 were measured separately by a competitive protein binding assay using rachitic rat plasma. 1α, 25-(OH)2-D3 and a metabolite suspected to be 1α, 25-(OH)2-D2 were determined by Eismans radioreceptor assay. When the rats were given graded amounts of D3 for 12 days, plasma levels of 25-OH-D3 and 24, 25-(OH)2-D3 increased in proportion to the dose levels of D3, while those of 1α, 25-(OH)2-D3 remained unchanged. The plasma levels of 24, 25-(OH)2-D3 correlated closely with those of 25-OH-D3 (γ=0.978). Hypercalcemia induced by high doses of D3 appeared to be due to the increase of 25-OH-D3 rather than to 1α, 25-(OH)2-D3.
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