ENZYME-LINKED-IMMUNOSORBENT-ASSAY FOR THE SPECIFIC DETECTION OF THE MERCAPTURIC ACID METABOLITIES OF NAPHTHALENE

The measurement of metabolites constitutes a useful tool for detection of exposure and in pharmacokinetic studies. Epoxidation with subseque nt glutathione conjugation and mercapturic acid formation is an import ant deactivation pathway for naphthalene, a toxin which presumably cau ses lung disease. The mercapturic acid conjugates of naphthalene [Naph MA (1), cetyl-S-(1,2-dihydro-1-hydroxy-2-naphthyl)cysteine (1a), and c etyl-S-(1,2-dihydro-2-hydroxy-1-naphthyl)cysteine (1b)], its most impo rtant urinary metabolites, and other structurally related derivatives, such as -(1,2,3,4-tetrahydro-2-hydroxy-1-naphthyl)cysteine (2), -(3-h ydroxy-1,2,3,4-tetrahydro-2-naphthyl)cysteine (3), and N-acetyl-S-(2-h ydroxy-1-phenyl-ethyl)cysteine (4a) and N-acetyl-S-(2-hydroxy-2-phenyl ethyl)cysteine (4b) as an isomeric mixture, were synthesized to develo p an ELISA (enzyme-linked immunosorbent assay) for the specific detect ion of NaphMA (1). Compound 1, as an isomeric mixture, was used to rai se antibodies by immunizing six rabbits with the corresponding KLH (ke yhole limpet hemocyanin) and BSA (bovine serum albumin) derivatives (1 KLH and 1BSA). The remaining compounds were covalently attached to BSA , conalbumin, and ovalbumin to be used as coating antigens. The best a ssay was obtained in a homologous system combining serum Ab2357 (1KLH) and 1BSA as coating antigen. The immunoassay has an I50 of 4-6 ng/mL and a detection limit of 1-2 ng/mL. Because of the known instability o f the mercapturic acid conjugate of naphthalene 1, leading to the full y aromatic compound 20, a system involving HPLC is described to check the stability of the NaphMA stock solutions used in the assay. Cross-r eactivity studies show high specificity toward the NaphMA. Other relat ed compounds as well as the dehydrated derivative 20 are not recognize d by the antibody in this ELISA system.