A sensitive and simplified radioimmunoassay for 1,25-dihydroxyvitamin D3

Abstract

A sensitive radioimmunoassay system for 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] with an improved extraction procedure has been developed. Following one-step extraction and prepurification of 1,25(OH)2D3 by 'Extrelut-1' minicolumns final purification was achieved by high-performance liquid chromatography (HPLC) using a radial compression separation system equipped with a mu Porasil cartridge. The HPLC method applied allows the purification of 4 extracts/h. Recovery of 1,25(OH)2[3H]D3 after HPLC was 77 +/- 2.6% (mean +/- SD, n = 51). Since the recovery of 1,25(OH)2[3H]D3 was very reproducible, addition of labelled steroid to each single serum sample for monitoring recovery was omitted. The sensitivity of the assay was 0.8 pg/tube resulting in a detection limit of 3 ng/l, when 1 ml of serum was extracted. Intra-assay and inter-assay coefficients of variation were 12% and 16.8%, respectively. Serum 1,25(OH)2D3 concentration in 30 normal subjects (mean age: 25 yr) was 55 +/- 12 ng/l (mean +/- SD). In 55 elderly patients (mean age: 77 yr) the 1,25(OH)2D3 serum level was 32 +/- 12 ng/l (mean +/- SD) and in three patients with chronic renal failure on 1,25(OH)2D3 therapy 146 +/- 67 ng/l (mean +/- SD). Patients with chronic renal failure had reduced 1,25(OH)2D3 serum levels (mean 5.4 ng/l, range less than 3-11 ng/l, n = 10). In one patient with renal failure, following kidney transplantation the serum 1,25(OH)2D3 and creatinine levels were monitored from the 4th to the 12th post-surgical day: a highly significant negative correlation (r = 0.85) was found.