4-Hydroxy-2-nonenal (HNE), an aldehyde end product of lipid peroxidation in biological systems, is capable of producing a range of powerful biological effects. Despite its biological relevance, the metabolic fate of this aldehyde is unknown in vivo. This study examines the urinary excretion of HNE in the rat and the nature of metabolites formed. Following iv administration of [3H]HNE, the majority of the dose appeared in urine (67.1% after 48 h). The radio-HPLC metabolic profile showed that no unchanged parent compound was detected in urine whereas at least four metabolites were present, most of them corresponding to mercapturic acid conjugates. Two major pathways were involved in the biotransformation of HNE in vivo: (i) reduction/oxidation of the aldehyde group, and (ii) conjugation to endogenous glutathione leading to mercapturic acid conjugates in urine. These end products were isolated by HPLC and identified by mass spectrometry as HNE mercapturic acid, 1,4-dihydroxynonene mercapturic acid, 4-hydroxynonenoic mercapturic acid, and the corresponding lactone.

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The formation of 4-hydroxy-2-nonenal (HNE) conjugates with glutathione (GSH) by Michael addition and subsequent cleavage to yield the related mercapturic acid (MA) conjugates are a major detoxication process. To characterize the metabolic pathways involved in the formation of urinary HNE-MA conjugates in the rat, the metabolism of HNE-thioethers (HNE-GSH, HNE-MA, and HNE-Cys) by rat liver and kidney cytosolic fractions was investigated. The experimental results showed that HNE-GSH is a good substrate for cytosolic incubations whereas HNE-MA and HNE-Cys are poorly metabolized. About 80% of the urinary MA conjugates originate from the primary and major HNE metabolite, namely, the hemiacetalized HNE-GSH. The direct reduction of HNE-GSH by a cytosolic aldo-keto reductase (NADPH) leads to 1,4-dihydroxynonene-GSH (DHN-GSH) and subsequently to DHN-MA. The direct oxidation of HNE-GSH by aldehyde dehydrogenase (NAD)(+) leads to 4-hydroxynonenoic-lactone-GSH, the partial hydrolysis of which occurs at physiological pH and accounts for the corresponding 4-hydroxynonenoic-GSH. Both the spontaneous- and the glutathione S-transferases-catalyzed retro-Michael cleavages of HNE-GSH and HNA-lactone-GSH are the source of HNE and HNA-lactone, respectively. This latter compound, with both lipophilic and electrophilic properties, is available for microsomal omega-hydroxylation by cytochrome P450 4A enzymes and conjugation with thiol groups and therefore is the most likely candidate for the formation of omega-hydroxylated HNE-mercapturic acid conjugates excreted in rat urine.

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