ISOTOPE EFFECT IN THE BINDING OF TRITIUM AND C-14-LABELED CORTISOL TOCORTICOSTEROID-BINDING GLOBULIN IN SERUM

We have determined the free cortisol concentration in serum using eith er the Amicon MPS-1 ultrafiltration-centrifugation method (I) or equil ibrium dialysis (II). If procedure I was used we found that [1,2,6,7-H -3]-, and [4-C-14]cortisol had a lower affinity than unlabelled cortis ol for corticosteroid binding globulin (CBG). The binding affinity (K- a) to three separate CBG-containing samples was 8-18 times lower for [ 1,2,6,7-H-3]cortisol and 30-90 times lower for [4-C-14]cortisol, when compared with that of unlabelled cortisol. This difference in affinity to CBG was not observed if method II was used for the free cortisol d eterminations. The observed isotope effect in method I is not caused b y unspecific binding to material such as the Amicon MPS-1 chamber or t o impurities in the tracer. We suggest that the centrifugation step du ring ultrafiltration changed the conformation of CBG, thereby reducing its affinity for labelled cortisol. It is concluded that incorrect re sults will be obtained if radiolabelled cortisol is used for determini ng the free cortisol content of plasma with the Amicon MPS-1 device. ( C) 1997 Elsevier Science Ltd.