An immunochemical assay was developed to detect carbonyl moieties that result from oxidative damage to proteins. Bovine serum albumin was reacted with hydroxyl radicals generated via a Fenton-like mechanism or by a radiolysis mechanism. The resulting albumin-derived carbonyls were reacted with 2,4-dinitrophenylhydrazine, giving the corresponding hydrazones, which were detected by Western blot using anti-dinitrophenyl antisera. The immunoblot demonstrated a concentration-dependent increase in carbonyl formation, as well as fragmentation of the albumin into two distinct bands with molecular masses of 51 and 45 kDa when oxidized with the Fenton-like mechanism, and 62 and 46 kDa when oxidized by radiolysis. Analysis of the immunoblot using laser densitometry indicated a linear relationship between carbonyl groups and increasing treatment from radiolysis. This immunochemical assay was approximately 3 orders of magnitude more sensitive than the spectrophotometric method and was able to determine the molecular mass of carbonyl-modified polypeptides in the detection of oxidative damage.

The mechanism of the hepatotoxicity of the analgesic acetaminophen is believed to be mediated by covalent binding to protein; however, critical targets which effect the toxicity are unknown. It has been shown that mitochondrial respiration in vivo is inhibited in mice as early as 1 h following a hepatotoxic dose of acetaminophen, and it is postulated that covalent binding to critical mitochondrial proteins may be important. A time course of mitochondrial proteins stained with anti-acetaminophen in an immunoblot detected two major adducts of 50 and 67 kDa as early as 30 min after a hepatotoxic dose of acetaminophen in mice. To further understand the role of covalent binding to mitochondrial proteins and acetaminophen hepatotoxicity, we have purified and identified a 50 kDa mitochondrial protein which becomes covalently bound to a reactive metabolite of acetaminophen. An N-terminal sequence of the 50 kDa adduct was 100% homologous with the deduced amino acid sequence of glutamate dehydrogenase. In addition, the purified protein was immunochemically reactive with rat liver anti-glutamate dehydrogenase. Enzyme activity of glutamate dehydrogenase was significantly decreased in mice 1 h following hepatotoxic treatment with acetaminophen. These data suggest that acetaminophen hepatotoxicity may in part be mediated by covalent binding to glutamate dehydrogenase.