Covalent Binding of a Halothane Metabolite and Neoantigen Production in Guinea Pig Liver Slices
Halothane hepatitis is a rare and potentially fatal consequence to the use of this anesthetic. The physiological basis of the disease appears to be an immune response to neoantigens formed by the covalent binding of halothane metabolites to liver protein. Liver slices were used to study the condition for halothane associated neoantigen formation in vitro. Liver slices, (1 cm diameter, 300 μm thick) from male Hartley guinea pigs (600 g) were exposed to either 1.0 or 1.7 mM halothane (media concentration) in 95% O2/5% CO2 for 12 hr. Covalent binding was determined using 14C-halothane. Neoantigens were detected by western immunoblot assay using rabbit anti-trifluoroacetylated albumin antiserum. Covalent binding was detected by 1 hr of incubation and increased linearly through 12 hr. Covalent binding preceeded and correlated with the appearance of neoantigen. By 12 hr of incubation, 5 neoantigens were seen with molecular weights ranging from 51–97 kD. These neoantigens have molecular weights similar to those seen in vivo. This in vitro model system can be used to examine the mechanism for covalent binding and neoantigen production in the hepatocyte.
KeywordsCovalent Binding Liver Slice Liver Protein Western Immunoblot Analysis Halothane Hepatitis
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