Abstract

The concentration-dependent binding of four nitroxide spin-labeled derivatives of phenytoin (Compound I) to bovine serum albumin and human sera has been investigated. The spin label moiety was attached to phenytoin either at position 3-N via a carbimidomethyl linkage (Compound II) or at a 4'-phenyl position via an amide linkage (Compounds III-V). Two of the phenyl labels (Compounds III and IV) differed only in the presence of a double bond in the pyrroline ring in Compound IV, and Compound V had the amide linkage reversed as compared with Compound III. The results of the binding studies were compared with those obtained by equilibrium dialysis of [¹⁴C]phenytoin. The spin labels were found to bind less strongly than [¹⁴C]phenytoin, with the order of binding of the spin-labeled phenytoins being IV > III > II > V. Studies with salicylic acid and phenylbutazone, known competitors of phenytoin binding, showed that the competition of spin label II paralleled that of [¹⁴C]phenytoin more than did the 4'-phenyl labels (III-V). These results suggest that the phenyl rings play a significant role in the binding of phenytoin to albumin. The binding of the 3-N spin-labeled phenytoin (II) paralleled that of [¹⁴C]phenytoin in human sera and should prove useful in the determination of free phenytoin levels in sera.