[The effect of purification procedures on the determination of serum level of 1,25-dihydroxyvitamin D].

The advance in knowledge of clinical disorders involving calcium and bone diseases has resulted in part from the development of assay procedures for the major vitamin D metabolites, but the values of the same samples measured in different laboratories vary considerably. They suggest that the chromatographic purification involved in these assays seems to be a critical step. In this study, we compared the results of 1,25-dihydroxyvitamin D (1,25(OH)2D) using different chromatographic purification methods: Sephadex LH-20 column chromatographic purification (non-HPLC method) described by Mallon et al. and High Performance Liquid Chromatographic purification (HPLC method) modified by Yamaoka et al. Serum 1,25(OH)2D levels of healthy volunteers before and after oral load of 4 micrograms of 1 alpha-hydroxyvitamin D3 (1 alpha OHD3) were measured by the two different methods mentioned above. A good correlation (r = 0.951, p less than 0.001) was noted in the samples before loading. The mean values of serum 1,25(OH)2D before loading did not differ significantly between HPLC method and non-HPLC method. After loading, however, the mean value of serum 1,25(OH)2D was significantly higher in non-HPLC method (p less than 0.01). The values of 1,25(OH)2D were determined in the serum added 1,25-dihydroxy-24-oxo-vitamin D3 (24-oxo-D3) (0 pg/ml, 200 pg/ml or 2000 pg/ml). In the samples added 2000 pg/ml of 24-oxo-D3, the mean value of 1,25(OH)2D was significantly higher in non-HPLC method (p less than 0.001). The affinity of 24-oxo-D3 for 1,25(OH)2D3 cytosolic receptor was evaluated in chick embryo intestinal mucosa and rachitic chick intestinal mucosa.(ABSTRACT TRUNCATED AT 250 WORDS)