An assay method for separately measuring metabolites of vitamin D3 and those presumed to be derived from vitamin D2.

Abstract

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 Eisman's 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.