Protein Binding Assays for 25-Hydroxy, 24,25-Dihydroxy and 1,25-Dihydroxy Metabolites of Vitamin D in Human Plasma

Abstract

Protein binding assays for 25-hydroxy, 24,25-dihydroxy and 1,25-dihydroxy metabolites of vitamin D in 6 ml of plasma are described. The procedure involves methanol/methylene chloride extraction of plasma followed by separation and purification of mono- and dihydroxylated vitamin D metabolites on 2 LH 20 columns with different elution solvents. The separation of dihydroxylated vitamin D metabolites is carried out by high performance liquid chromatography. 25-Hydroxy and 24,25-dihydroxy vitamin D are measured by competitive protein binding assays with diluted homogenate from vitamin D-deficient rat kidneys. 1,25-Dihydroxy vitamin D is measured by competitive protein binding assay with diluted cytosol from vitamin D-deficient chick intestine. The concentration of 25-hydroxy vitamin D in human plamsa, as determined this assay, was 12.2 .+-. 5.9 .mu.g/l; of 24,25-dihydroxy vitamin D 1.5 .+-. 1.0 .mu.g/l; and of 1,25-dihydroxy vitamin D 25.8 .+-. 11.7 ng/l (means .+-. SD, n = 17, October). In April, the concentration of the vitamin D metabolites of the same subjects were: 25-hydroxy vitamin D 3.3 .+-. 0.9 .mu.g/l; 24,25-dihydroxy vitamin D 0.17 .+-. 0.04 .mu.g/l; and 1,25-dihydroxy vitamin D 38.3 .+-. 10.2 ng/l. Mean values in nonselected human plasma samples of hospitalized patients (n = 84) taken during 1 yr were: 25-hydroxy vitamin D 5.7 .+-. 2.9 .mu.g/l; 24,25-dihydroxy vitamin D 0.64 .+-. 0.44 .mu.g/l; 1,25-dihydroxy vitamin D 53.3 .+-. 27.6 ng/l. Mean values in pancreatectomized subjects were: 25-hydroxy vitamin D 4.2 .+-. 2.6 .mu.g/l; 24,25-dihydroxy vitamin D 0.27 .+-. 0.15 .mu.g/l; 1,25-dihydroxy vitamin D 19.6 .+-. 11.9 ng/l (n = 10).