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

• Published in: Journal of Nutritional Science and Vitaminology 1979, Vol.25(5), pp.351-360
• Main Authors: KANO, Kenichi, YOSHIDA, Hisashi, YATA, Junichi, ABE, Etsuko, TANABE, Reiko, SUDA, Tatsuo
• Format: Article
• Language: English
• Subjects: 1α,25-dihydroxy-vitamin D3; 1α,25-dihydroxyvitamin D2; 24,25-dihydroxyvitamin D2; 24,25-dihydroxyvitamin D3; 25-hydroxy-vitamin D2; 25-hydroxyvitamin D3; Animals; Binding, Competitive; Cholecalciferol - analogs & derivatives; Cholecalciferol - blood; Cholecalciferol - pharmacology; Chromatography, High Pressure Liquid; competitive protein binding assay; Dihydroxycholecalciferols - blood; Dose-Response Relationship, Drug; Ergocalciferols - blood; high pressure liquid chromatography; Hydroxycholecalciferols - blood; Hypercalcemia - chemically induced; hypervitaminosis D; Male; Protein Binding; Radioligand Assay; Rats; vitamin D2; vitamin D3
• ISSN: 0301-4800; 1881-7742
• DOI: 10.3177/jnsv.25.351

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.