DETERMINATION OF NON-PROTEIN-BOUND PLASMA 1,25-DIHYDROXYVITAMIN-D BY SYMMETRICAL (RATE) DIALYSIS

Most of the total circulating 1,25-dihydroxyvitamin D (1,25(OH)(2)D) i s bound to plasma proteins, mainly vitamin D-binding protein (DBP) and albumin. Only a small fraction in plasma exists in the free form. It is widely assumed that the non-protein-bound free hormone reflects the biologically active fraction. We describe a dialysis method for the d etermination of plasma free 1,25(OH)(2)D which is relatively easy to p erform. In this symmetric or ''rate'' dialysis method, identical sampl es are placed at both sides of a membrane. At one side, tritiated 1,25 (OH)(2)D is added and the rate of transfer of this tritiated 1,25(OH)( 2)D through a dialysis membrane is directly related to the free fracti on of plasma 1,25(OH)(2)D. This method is much less susceptible toward tracer impurities than indirect equilibrium dialysis and centrifugal ultrafiltration. Moreover, it requires much less tracer. The intraassa y coefficient of variation for the determination of the free fraction is 1.0%; the interassay variation is 7.7%. Comparison of the free frac tion of 23 samples assessed with both centrifugal ultrafiltration and symmetric dialysis showed much higher values using the former method. No significant correlation between the two methods was found. The free fraction of 1,25(OH)(2)D in normal subjects as assessed with symmetri c dialysis ranges from 0.049 to 0.103%. (C) 1998 Academic Press.