Radioimmunoassay of 1,25-dihydroxy vitamin D2: studies on the metabolism of vitamin D2 in man.

A sensitive radioimmunoassay for 1,25-dihydroxy vitamin D2 was developed using a sheep antiserum which preferentially reacts with 1-hydroxylated forms of vitamin D. An improved isolation procedure was also developed using acetonitrile for the initial extraction of serum followed by chromatography on cartridges of C18 silica and high pressure liquid chromatography eluted with a ternary solvent system to separate 1,25-dihydroxy vitamin D2 and 1,25-dihydroxy vitamin D3. 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3 were separated by further reverse phase high pressure liquid chromatography prior to competitive protein binding assay. The limits of detection were 4.3 pmol/1 (2.0 pg/ml) for the 1,25-dihydroxy metabolites and 1.25 nmol/1 (0.5 ng/ml) for both 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3. 25-hydroxy vitamin D2 ranged from 2.0 to 11.3 nmol/1 (0.8-4.5 ng/ml) with a mean of 4.75 nmol/1 (1.9 ng/ml) in thirteen healthy British adults and this accounted for 9.0% of the mean total 25-hydroxy vitamin D. 1,25-dihydroxy vitamin D2 was detected in the sera of only one of these subjects whereas 1,25-dihydroxy vitamin D3 was present in all ranging from 48 to 163 pmol/1 (20-65 pg/ml) with a mean of 100 pmol/1 (42 pg/ml). Both 1,25-dihydroxy vitamin D2 and 1,25-dihydroxy vitamin D3 were detected in the sera of hypoparathyroid patients treated with vitamin D2 but the relationship between 25-hydroxy vitamin D and 1,25-dihydroxy vitamin D was complex. For example, when an excess of 25-hydroxy vitamin D2 was present the serum concentration of 1,25-dihydroxy vitamin D3 was disproportionately high. Conversely, in patients who had previously been treated with vitamin D2 but were receiving only vitamin D3 at the time of study, the major 25-hydroxy metabolite was in the vitamin D3 form and there was a disproportionately high amount of 1,25-dihydroxy vitamin D2. Total 1,25-dihydroxy vitamin D ranged from 110 to 400 pmol/1 (45-165 pg/ml) and was above the upper limit of normal for 1,25-dihydroxy vitamin D3 in half of these hypoparathyroid patients treated with pharmacological doses of vitamin D.