Molecular Pharmacology, Vol 20, 674-680, Copyright © 1981 by the American Society for Pharmacology and Experimental Therapeutics

Application of Spin Labeling to Drug Assays

II. Determination of the Binding of [¹⁴C]-Phenytoin and Spin-Labeled Phenytoins to Albumin and Human Serum

¹ Departments of Laboratory Medicine and Pathology and Pharmacology, University of Minnesota, Minneapolis, Minnesota 55455, Research Service, Veterans Administration Medical Center, Minneapolis, Minnesota 55417, and Department of Neurology, St. Paul Ramsey Hospital, St. Paul, Minnesota 55101.

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.