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In this study we describe protein binding assays for 25-hydroxy, 24,25-dihydroxy and 1,25-dihydroxy metabolites of vitamin D in 6 ml of plasma. The procedure involves methanol/methylene chloride extraction of plasma followed by separation and purification of mono- and dihydroxylated vitamin D metabolites on two LH 20 columns with different elution solvents. The separation of dihydroxylated vitamin D metabolites is carried out by high performance liquid chromatography (HPLC). 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 plasma, as determined by this assay, was 12.2 +/- 5.9 microgram/l; of 24,25-dihydroxy vitamin D 1.5 +/- 1.0 microgram/l, and of 1,25-dihydroxy vitamin D 25.8 +/- 11.7 ng/l (means +/- S.D., n = 17, October, Düsseldorf). In April, the concentration of the vitamin D metabolites of the same subjects were: 25-hydroxy vitamin D 3.3 +/- 0.9 microgram/l; 24,25-dihydroxy vitamin D 0.17 +/- 0.04 microgram/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 one year were: 25-hydroxy vitamin D 5.7 +/- 2.9 microgram/l; 24,25-dihydroxy vitamin D 0.64 +/- 0.44 microgram/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 microgram/l; 24,25-dihydroxy vitamin D 0.27 +/- 0.15 microgram/l; 1.25-dihydroxy vitamin D 19.6 +/- 11.9 ng/l (n = 10).

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