Background: Measurement of circulating 25-hydroxyvitamin D [25(OH)D] is important in the management of metabolic bone disease. The aim of this study was to compare two widely used methods for the quantification of circulating 25(OH)D with attention to their abilities to measure 25-hydroxylated ergocalciferol (vitamin D(2)) [25(OH)D(2)] and cholecalciferol (vitamin D(3)) [25(OH)D(3)].
Methods: We used two commercially available, Food and Drug Administration-approved, radioiodine ((125)I)-based RIA kits for the detection of 25(OH)D (DiaSorin, Stillwater, MN and IDS Ltd, Tyne and Wear, United Kingdom). These methods were tested for general assay performance, including antibody specificity. Results were compared with those of an HPLC-based direct ultraviolet detection method.
Results: Within- and between-run CVs were </=10%. Both methods quantitatively recovered 25(OH)D(3) added to serum, but only the DiaSorin kit quantitatively recovered 25(OH)D(2). The primary antibody in the IDS kit had unequal reactivities with pure 25(OH)D(2) and 25(OH)D(3), whereas the DiaSorin primary antibody reacted with them equally. In 50 patient samples assayed by HPLC, the IDS method, but not the DiaSorin method, underestimated total circulating 25(OH)D when significant circulating 25(OH)D(2) was present in patient samples.
Conclusions: Some immunoassays may underestimate total 25(OH)D when 25(OH)D(2) constitutes an appreciable part of the total.