Identification of Novel Urinary Metabolites of the Lipid Peroxidation Product 4-Hydroxy-2-nonenal in Rats Jacques Alary,* Laurent Debrauwer, Yvette Fernandez, Alain Paris, Jean-Pierre Cravedi, Laurence Dolo, Dinesh Rao, and Georges Bories Laboratoire des Xe ´ nobiotiques, INRA, BP3, 31931 Toulouse Cedex, France Received March 31, 1998 Following iv administration of 4-hydroxy-2-nonenal (HNE) and [4- 3 H]HNE to rats, 15 polar urinary metabolites accounting for about 50% of the urinary radioactivity were separated by HPLC. Among them, eight major compounds and tritiated water were quantified. The metabolites were unequivocally characterized using GC/MS and ESI/MS/MS/MS. Most of “HNE polar metabolites” originate from ω-oxidation of 4-hydroxy-2-nonenoic acid (HNA): 9-hydroxy- HNA, its mercapturic acid conjugate, and two diastereoisomers of the corresponding lactone. The oxidation of 9-hydroxy-HNA by alcohol and aldehyde dehydrogenases leads to the excretion of 9-carboxy-HNA and of the corresponding lactone mercapturic acid conjugate. 1,4-Dihydroxy- 2-nonene (DHN) originating from the reduction of HNE by alcohol dehydrogenase was to a lesser extent ω-hydroxylated, leading to 9-hydroxy-DHN which was excreted as a mercapturic acid conjugate (two diastereoisomers). Introduction Lipid peroxidation of ω-6 polyunsaturated fatty acids results in the formation of reactive aldehydes (1), of which 4-hydroxy-2-nonenal (HNE) 1 is the major and the most cytotoxic product (2). Despite its biological relevance, the in vivo metabolism of HNE still remains partially un- known. In vitro, metabolic studies carried out with rat liver subcellular fractions showed that HNE was reduced to 1,4-dihydroxy-2-nonene (DHN) by cytosolic alcohol de- hydrogenase (3) and oxidized by aldehyde dehydrogenase to 4-hydroxy-2-nonenoic acid (HNA) (4). In addition to these phase I products, the HNE-GSH conjugate was also formed after short time incubations of HNE with hepatocytes, enterocytes, and tumor cells (5). In a study carried out after iv injection of HNE into the rats, four mercapturic conjugates have been characterized in the 0-2 h urine (6), namely, 1,4-dihydroxynonene mercap- turic acid (DHN-MA), 4-hydroxynonenal mercapturic acid (HNE-MA), 4-hydroxynonenoic mercapturic acid (HNA- MA), and the corresponding lactone. The presence of these conjugates in rat urine was recently confirmed by De Zwart et al. (7). In our first study (6), the formally characterized metabolites accounted for only about one-half of the urinary radioactivity. Unidentified compounds corre- sponded to more polar metabolites remaining mostly unresolved under our chromatographic conditions. This study was designed to characterize and quantify the major “polar metabolites” of HNE to complete an overview of the pathways involved in the metabolic fate of HNE in rats. Materials and Methods Chemicals. HNE and [4- 3 H]HNE were obtained by acid hydrolysis of the corresponding synthesized diethylacetal de- rivatives (8, 9) and purified by HPLC. DHN and HNA were prepared from HNE as previously described (6). All solvents and reagents used for the preparation of buffers and HPLC eluents were of the highest commercial grade available from Merck (Nogent-sur-Marne, France) or Carlo Erba (Rueil Malmaison, France). Ultrapure water from Milli-Q system (Millipore, Saint Quentin en Yvelines, France) was used for HPLC eluent preparation. Radioactivity Determination. The samples were directly counted in a model 4330 Packard Tricarb scintillation counter (Packard Instrument Co., Downers Grove, IL) with Ultimagold (Packard) as the scintillation cocktail. Animal Treatment. Two male Wistar rats (250 g) were weakly anesthetized with diethyl ether and injected in the penis vein with 500 µL of Ringer’s solution containing a mixture of 1.2 mg of HNE and 0.6 MBq (550 ng) of [4- 3 H]HNE. The rats were housed in individual metabolism cages, and water was provided ad libitum. Twenty-four hour urine samples were collected. The urine samples were filtered through a Millex-HA 0.45 µm filter (Millipore) and stored at -20 °C until analysis. Extraction of Urinary HNE Metabolites. Filtered urine samples were diluted 1:1 with distilled water and adjusted to pH 2.5 with 1 M H3PO4. Each urine sample was loaded onto a 1 g LC-C18 Supelco cartridge (Saint Quentin Fallavier, France) preconditioned with 5 mL of methanol and 10 mL of dilute H3PO4 (pH 2.5). The cartridge was washed with 20 mL of dilute H3PO4 (pH 2.5). The aqueous phases from the column and washing were recovered and combined. The cartridge was dried * To whom correspondence should be addressed. Telephone: 33 (0)5 61 28 53 83. Fax: 33 (0)5 61 28 52 44. 1 Abbreviations: HNE, 4-hydroxy-2-nonenal; HNA, 4-hydroxy-2- nonenoic acid; 9-hydroxy-HNA, 4,9-dihydroxy-2-nonenoic acid; 8-hy- droxy-HNA, 4,8-dihydroxy-2-nonenoic acid; 9-carboxy-HNA, 4-hydroxy- 9-carboxy-2-nonenoic acid; DHN, 1,4-dihydroxy-2-nonene; 9-hydroxy- DHN, 1,4,9-trihydroxy-2-nonene; HNE-MA, 4-hydroxynonenal mercap- turic acid; DHN-MA, 1,4-dihydroxynonene mercapturic acid; HNA-MA, 4-hydroxynonenoic mercapturic acid; 9-hydroxy-HNA-MA, 4,9-dihy- droxynonenoic mercapturic acid; 3-hydroxy-HNA-MA, 3,4-dihydroxy- nonenoic mercapturic acid; 3-hydroxy-HNE-MA, 3,4-dihydroxynonenal mercapturic acid; 3-hydroxy-DHN-MA, 1,3,4-trihydroxynonene mer- capturic acid; 9-hydroxy-HNA-lactone-MA, 9-hydroxynonenoic lactone mercapturic acid; NAC, N-acetylcysteine. 1368 Chem. Res. Toxicol. 1998, 11, 1368-1376 10.1021/tx980068g CCC: $15.00 © 1998 American Chemical Society Published on Web 10/24/1998