Measurement of 25 hyrdoxyvitamin D3 in serum

Abstract

A method is described for measuring 25 hydroxyvitamin D3 in serum. Extraction with dichloromethane/methanol (2/1 by vol), followed by chromatography on a column of Sephadex LH 20, resulted in an overall analytical recovery of 82% ± 3.5% (SD). Diluted normal rat serum was used as binding protein because it contains a transport protein that has both a high affinity (K(a) = 2 x 1010 liter/mol) and a high capacity (3 x 10-6 mol/liter) for 25 hydroxyvitamin D3. There is no advantage in using more complex binding proteins derived either from rachitic animals or from cytosol preparations. Concentrations of 25 hydroxyvitamin D3 (13.4 ± 4μg/liter) in the serum of apparently normal Belgian subjects are lower than those reported for North Americans, but resemble those reported for the United Kingdom.