COMPARISON OF NON-HPLC 1α, 25(OH)₂D ASSAY SYSTEM WITH THE HPLC METHOD

Abstract

Recently, a method not requiring high performance liquid chromatography (HPLC) has been developed (Amersham Inc., U. K) for the measurement of levels of 1α, 25(OH)₂D, which demonstrates the most potent physiological effect among the metabolites of vitamin D. Until recently, purification of 1α, 25(OH)₂D fraction with HPLC has been considered indispensable prior to radioreceptor assay of this metabolite. Therefore, we used the Amersham assay system to determine whether it is possible to correclty measure the level of 1α, 25(OH)₂D without the HPLC process. The procedures we used were to extract the serum with an organic solvent, followed by fractionation of vitamin D metabolites through an LH-20 column. The products with or without the use of HPLC were submitted to radioreceptor assay using chick embryo intestinal receptors. As a result, it was found that (1) Separation and yield were improved by changing the column size, (2) Serum values 1α, 25(OH)₂D in healthy subjects obtained by HPLC and non-HPLC methods were in good agreement with the theoretical values. Although the values thus obtained in patients with chronic renal insufficiency (with or without dialysis) were somewhat lower than the theoretical values, the reuslt was consistent because there was no significant difference between the tow sets of measured values, and the correlation coefficient was r=0.990. (3) Variations within the assay were CV=16.5% for HPLC group and CV=21.4% for non-HPCL group. From the above, it was determined that 1α, 25(OH)₂D level in the serum of healthy subjects can be accurately measured by the assay system, and that it is possible to reduce the time and cost of measurement by using the assay system.