25-hydroxyvitamin D2, 25-hydroxyvitamin D3 and total 24,25-dihydroxyvitamin D in human serum

An assay procedure capable of determining 25(OH)D2 (25-hydroxyvitamin D2), 25(OH)D3 (25-hydroxyvitamin D3) and total 24,25(OH)2D (24,25-dihydroxyvitamin D) in a single 2-5 ml sample of human serum is described. The assay involves methanol/chloroform extraction of serum lipids followed by separation of the mono- and dihydroxyvitamin D metabolites and purification from interfering contaminants by chromatography on Sephadex LH-20 and by high-performance liquid chromatography (HPLC). 25(OH)D2 and 25(OH)D3 are quantified by HPLC using UV detection. Total 24,25(OH)2D is routinely measured by a competitive protein-binding (CPB) assay with human serum as a source of the binding protein. Mean values (+/- SD) in 16 normal human serum samples taken in October-November were: 25(OH)D2 5.1 +/- 2.2 ng/ml (12.8 +/- 5.5 nmol/l); 25(OH)D3 16.6 +/- 4.4 ng/ml (41.5 +/- 11.0 nmol/l); total 25(OH)D 21.7 +/- 4.9 ng/ml (54.3 +/- 12.3 nmol/l); total 24,25(OH)2D 1.6 +/- 0.6 ng/ml (3.8 +/- 1.4 nmol/l) and the ratio 24,25(OH)2D/25(OH)D 0.066 +/- 0.024. The ratio was 0.048 +/- 0.009 in women and 0.081 +/- 0.022 in men (p less than 0.002). Values in three serum samples taken from patients receiving vitamin D3 therapy were: 25(OH)D2 4.1 +/- 2.7 ng/ml (10.3 +/- 6.8 nmol/l); 25(OH)D3 46.6 +/- 20.9 ng/ml (116.5 +/- 52.3 nmol/l); total 24,25(OH)2D 3.4 +/- 1.9 ng/ml (8.2 +/- 4.6 nmol/l); 24,25(OH)2D/25(OH)D 0.075 +/- 0.022.