Simultaneous measurement of 1.25-dihydroxy-vitamin D, 24.25-dihydroxy-vitamin D and 25-hydroxy-vitamin D from a single two milliliters serum specimen. Preliminary clinical application

A simple method for extraction, purification and separation of the principal vitamin D metabolites from a single serum sample is described. The method involved extraction of serum with acetonitrile followed by a first purification employing C-18 Sep-pak cartridges eluted with methanol/water and acetonitrile. Final separation before assay was carried out by high pressure liquid chromatography. 1.25-dihydroxy-vitamin D was measured with radioimmunoassay using an antiserum (S11) with high selectivity for 1 alpha-OH function of the hormone at a final dilution of 1:100,000. 24.25-dihydroxy-vitamin D and 25-hydroxy-vitamin D were measured employing a competitive binding assay with normal rat serum at a final dilution of 1:10,000 as source of binding protein. The mean (+/- SD) serum 1.25-dihydroxy-vitamin D, 24.25-dihydroxy-vitamin D and 25-hydroxy-vitamin D concentrations for a group of healthy subjects were 50.4 +/- 17.3 pg/ml, 2.3 +/- 2.6 ng/ml and 20.8 +/- 12.3 ng/ml, respectively. 1.25-dihydroxy-vitamin D concentrations were low or undetectable in patients on dialysis or with mild renal failure. High 1.25-dihydroxy-vitamin D levels were found in 2 out of 17 patients with primary hyperparathyroidism. In 4 normal subjects treated for two weeks with large doses of 25-hydroxy-vitamin D, serum 25-hydroxy-vitamin D rose from 12.5 ng/ml to 119 ng/ml and from 0.89 ng/ml to 15 ng/ml, respectively; no changes in the 1.25-dihydroxy-vitamin D assay were found.