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Assessment of circulating 25(OH)D and 1,25(OH)2D: emergence as clinically important diagnostic tools.

One of the major factors responsible for the explosion of knowledge related to vitamin D metabolism and its relation to clinical disease was the introduction of competitive protein-binding assays for 25(OH)D 1 and 1,25(OH)2D. 2 These assays were introduced more than three decades ago, and were based on assessing circulating 25(OH)D or 1,25(OH)2D using the vitamin D-binding protein or chick intestinal receptor, respectively, as the primary binding agents. Both of these assays used 3 H-labeled compounds as reporters. Although these assays were valid, they were also relatively cumbersome, especially the 1,25(OH)2D procedure. Through the years, assays for both of these metabolites have advanced. Some advances have been for the better, some have not. These methodologies include radioimmunoassay (RIA), enzyme-linked immunoassay (ELISA), high-performance liquid chromatography (HPLC), liquid chromatography coupled with mass spectrometry (LC-MS), and random access automated assay (RAAA) based on chemiluminescence assay technology.