Determination of vitamin D binding protein by Scatchard analysis and estimation of a free 25-hydroxy-vitamin D index

A simple method for the quantification of the vitamin D binding capacity (concentration of vitamin D binding protein, DBP) in serum is described. 25-Hydroxy-vitamin D can conveniently be measured by a binding protein assay utilizing dilute human serum as a source of DBP. The same methodology was used to determine the concentration of DBP by Scatchard analysis. In comparison to radial immunodiffusion, a correlation coefficient of 0.863 (p less than 0.001) was found with a regression line y = 0.963x + 0.08 (n = 52). Normal values were between 3 and 8 mumol/l, the coefficient of variation was less than 7% (median 4.9%). A simple routine version of this test requiring only 5 tubes per sample at a sample dilution of 1:15 000 had an interassay variation of less than 8%. The apparent affinity constants of DBP obtained by the Scatchard analysis had a very poor interassay reproducibility. Thus, the molar ratio of 25-hydroxy-vitamin D and DBP was used as a 'free 25-hydroxy-vitamin D index'. The normal range for a control group was 0.005-0.015, with samples taken in winter.