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Previous studies have demonstrated that patients with halothane-induced hepatitis have serum antibodies that are directed against novel liver microsomal neoantigens and have suggested that these neoantigens may play an immunopathological role in development of the patients' liver damage. These investigations have further revealed that the antibodies are directed against distinct polypeptide fractions (100 kDa, 76 kDa, 59 kDa, 57 kDa, 54 kDa) that have been covalently modified by the reactive trifluoroacetyl halide metabolite of halothane. In this paper, the trifluoroacetylated (TFA) 59-kDa neoantigen (59-kDa-TFA) recognized by the patients' antibodies was isolated from liver microsomes of halothane-treated rats by chromatography on an immunoaffinity column of anti-TFA IgG. Antibodies were raised against the 59-kDa-TFA protein and were used to purify the native protein from liver microsomes ofuntreated rats. Based upon its apparent monomeric molecular mass, NH2-terminal amino acid sequence, catalytic activity, and other physical properties, the protein has been identified as a previously characterized microsomal carboxylesterase (EC 3.1.1.1). A similar strategy may be used to purify and characterize neoantigens associated with other drug toxicities that are believed to have an immunopathological basis.It has been estimated that between 3% and 25% of all drug toxicities, which can include anaphylaxis, serum sickness, asthma, urticaria, dermatitis, fever, hemolytic anemia, thrombocytopenia, granulocytopenia, hepatitis, nephritis, vasculitis, pneumonitis, and lupus-erythematosus-like syndrome, are due to hypersensitivity (allergic) reactions (1). Although most of these drug-induced hypersensitivities have been presumed to be mediated by immunogens formed by the covalent interaction of a reactive drug metabolite with tissue carrier macromolecules (2-6), it is only in the case ofhepatitis caused by the inhalation of anesthetic halothane that this mechanism has been supported substantially by experimental evidence.Previous studies have demonstrated that the majority of halothane hepatitis patients have unique serum antibodies that react with novel neoantigens in livers of animals (1,7,8) and humans (9) treated with halothane and have suggested that these neoantigens may play an immunopathological role in development of the patients' liver damage. Characterization of these neoantigens by immunoblotting with haptenspecific anti-trifluoroacetyl (TFA) antibodies and sera from several halothane hepatitis patients has revealed that they correspond to distinct liver microsomal protein fractions (100 kDa, 76 kDa, 59 kDa, 57 kDa, 54 kDa) (10, 11) that have been covalently modified by the reactive TFA halide metabolite of halothane (11).To investigate the role of the halothane-induced neoantigens in the pathogenesis of halothane hepatitis, a general approach for their purification and characterization has been developed and utilized to identify one of them. MATERIALS AND METHODSPurification of 59-kDa-TFA Protein from Hal...

Previous investigations have shown that antibodies in sera from patients with halothane hepatitis recognize neoantigens, expressed in livers of halothane-exposed rabbits and rats, which consist of a halothane metabolite bound covalently to specific microsomal proteins. These studies have suggested that the patients' antibodies may play a role in the pathogenesis of the hepatitis. In the present investigation, human liver biopsy samples were analyzed using an immunoblotting method to seek evidence for expression of halothane-induced neoantigens in humans. Sera from four patients with halothane hepatitis, which recognized halothane-induced rabbit liver neoantigens of 100, 76 and 57 kD, reacted strongly with antigens of very similar molecular weights that were expressed in livers from two patients who had died of cardiac failure following recent anesthesia with halothane. The antigens were not expressed in normal human liver or in livers from three patients who died of cardiac failure following anesthesia with agents other than halothane. The human antigens were not recognized by antibodies present in various control sera. Recognition of the 100- and 76-kD human antigens by the patients' antibodies was greatly reduced by absorption of sera with liver microsomes from halothane-exposed rabbits, but not by absorption of sera with control rabbit microsomes. These results indicate that humans exposed to halothane express liver neoantigens which are analogous to the halothane metabolite-protein neoantigens characterized previously in halothane-exposed animals.

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