Assessment of the Free Fraction of 25-Hydroxyvitamin D in Serum and Its Regulation by Albumin and the Vitamin D-Binding Protein*

We measured the free fraction of 25-hydroxyvitamin D (25OHD) in human serum and determined that 25OHD bound to a component with an affinity constant of 7 × 108 M-1 and a concentrationof 4.5 × 10-6 M. This concentration was equal to that of the vitamin D-binding protein (DBP) in the same serum sample. We removed DBP from the serum using actin affinity columns and found that the affinity for 25OHD of the remaining serum components was equivalent to that of human serum albumin (6 × 105 M-1). We then measured the free fractions of 25OHD, DBP, and albumin in normal and cirrhotic subjects. Wecalculated that 88 ± 3% (±SD) and 83 ± 8% of the 25OHD were bound to DBP in the serum of normal and cirrhotic subjects, respectively. We compared previously reported data for the free fraction and the free concentration of 1,25-dihydroxyvitamin Din these subjects with the current data for the free fraction and free concentrationof 25OHD. Thetotal concentrations and free fractions of both metabolites correlated to each other...