Determination of vitamin D and its metabolites in plasma from normal and anephric man

A multiple assay capable of reliably determining vitamins D₂ and D₃ (ergocalciferol and cholecalciferol), 25(OH)D₂ (25-hydroxyvitamin D₂) and 25(OH)D₃ (25-hydroxyvitamin D₃), 24,25(OH)₂D (24,25-dihydroxyvitamin D), 25,26(OH)₂D (25,26-dihydroxyvitamin D) and 1,25(OH)₂D (1,25-dihydroxyvitamin D) in a single 3–5ml sample of human plasma was developed. The procedure involves methanol/methylene chloride extraction of plasma lipids followed by separation of the metabolites and purification from interfering contaminants by batch elution chromatography on Sephadex LH-20 and Lipidex 5000 and by h.p.l.c. (high-pressure liquid chromatography). Vitamins D₂ and D₃ and 25(OH)D₂ and 25(OH)D₃ are quantified by h.p.l.c. by using u.v. detection, comparing their peak heights with those of standards. 24,25(OH)₂D and 25,26(OH)₂D are measured by competitive protein-binding assay with diluted plasma from vitamin D-deficient rats. 1,25(OH)₂D is measured by competitive protein-binding assay with diluted cytosol from vitamin D-deficient chick intestine. Values in normal human plasma samples taken in February are: vitamin D 3.5±2.5ng/ml; 25(OH)D 31.6±9.3ng/ml; 24,25(OH)₂D 3.5±1.4ng/ml; 25,26(OH)₂D 0.7±0.5ng/ml; 1,25(OH)₂D 31±9pg/ml (means±s.d.). Values in two normal human plasma samples taken in February after 1 week of high sun exposure are: vitamin D 27.1±7.9ng/ml; 25(OH)D 56.8±4.2ng/ml; 24,25(OH)₂D 4.3±1.6ng/ml; 25,26(OH)₂D 0.5±0.2ng/ml. Values in anephric-human plasma are: vitamin D 2.7±0.8ng/ml; 25(OH)D 36.4±16.5ng/ml; 24,25(OH)₂D 1.9±1.3ng/ml; 25,26(OH)₂D 0.6±0.3ng/ml; 1,25(OH)₂D was undetectable.