Importance of the isolation of 25-hydroxyvitamin D before assay.

A protein-binding assay for determination of 25(OH)D was used to analyze the profile of chromatographed ethanol extracts of serum. There were substances in the serum of normal subjects which displaced labeled 25(OH)D3 from the binding protein and which were different from 25(OH)D or 24R,25(OH)2D. Values of 25(OH)D from serum samples without prior chromatography were higher than those obtained with chromatography (40.2 +/- 12.2 ng/ml vs. 22.3 +/- 8.2, mean +/- S.D.). This indicates a need for chromatography before assay to ensure specificity of 25(OH)D measurement.