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.
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 microL) is mixed with 0.5 mL of reagent containing 0.5 mumol/L 25-hydroxyvitamin D [25(OH)D3] plus 3000 counts/min [3H]25(OH)D3. After 0.5 h at 37 degrees 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 approximately 10 mumol/L. 25D-binding capacity in reference-group serum samples was 4.33 (0.58 SD) mumol/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 x 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.
Vieth
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Department of Clinical Biochemistry, University of Toronto, Canada.
D000328:Adult; D001665:Binding Sites; D002112:Calcifediol / Q000097:blood; D002117:Calcitriol / Q000097:blood*; D002498:Centrifugation; D002606:Charcoal; D003911:Dextrans; D003956:Dialysis; D005260:Female; D006801:Humans; D007202:Indicators and Reagents; D008297:Male; D008875:Middle Aged; D012016:Reference Values; D012709:Serum Albumin / Q000378:metabolism; D014316:Tritium; D014462:Ultrafiltration; D014809:Vitamin D-Binding Protein / Q000378:metabolism*
D003911:Dextrans; D007202:Indicators and Reagents; D012709:Serum Albumin; D014809:Vitamin D-Binding Protein; D014316:Tritium; D002606:Charcoal; D002117:Calcitriol; D002112:Calcifediol