Measurement of 25-hydroxyvitamin D3 in serum

We describe a method 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 (Ka = 2 X 10(10) 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 +/- 4 mug/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.