Simple method for determining specific binding capacity of vitamin D-binding protein and its use to calculate the concentration of "free" 1,25-dihydroxyvitamin D

Abstract

A quantitative method to measure the specific binding capacity for 25-hydroxyvitamin D (25D-binding capacity) is described that resembles the qualitative "T3-uptake" assay. Patient's serum or standard (10 μL) is mixed with 0.5 mL of reagent containing 0.5 μmol/L 25-hydroxyvitamin D [25(OH)D3] plus 3000 counts/min [3H]25(OH)D3. After 0.5 h at 37°C, the samples are treated with dextran/ charcoal on ice for 1 h and centrifuged. The radioactivity of the bound tracer in the supernate is counted. Calibration is linear to ∼10 μmol/L. 25D-binding capacity in reference-group serum samples was 4.33 (0.58 SD) μmol/L. The relationship between the inverse of 25D-binding capacity and the free fraction of [3H]1,25-dihydroxyvitamin D3 [1,25(OH)2D3] measured by ultrafiltration isodialysis was essentially linear (r = 0.934, P <0.0001). Given this relationship, the calculated free fraction of 1,25(OH)2D3 equals 4.88 × 10-3/25D-binding capacity. The 25D-binding capacity was significantly lower in newborn babies and in adults with liver disease, and was increased during pregnancy (P <0.01 for each). This method is applicable to situations where the biologically available concentration of 1,25(OH)2D is of interest.