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trans-4-Hydroxy-2-nonenal (HNE) is a cytotoxic alpha,beta-unsaturated aldehyde implicated in the pathology of multiple diseases involving oxidative damage. Oxidation of HNE by aldehyde dehydrogenases (ALDHs) to trans-4-hydroxy-2-nonenoic acid (HNEA) is a major route of metabolism in many organisms. HNE exists as two enantiomers, (R)-HNE and (S)-HNE, and in intact rat brain mitochondria, (R)-HNE is enantioselectively oxidized to HNEA. In this work, we further elucidated the basis of the enantioselective oxidation of HNE by brain mitochondria. Our results showed that (R)-HNE is oxidized enantioselectively by brain mitochondrial lysates with retention of stereoconfiguration of the C4 hydroxyl group. Purified rat ALDH5A enantioselectively oxidized (R)-HNE, whereas rat ALDH2 was not enantioselective. Kinetic data using (R)-HNE, (S)-HNE, and trans-2-nonenal in combination with computer-based modeling of ALDH5A suggest that the selectivity of (R)-HNE oxidation by ALDH5A is the result of the carbonyl carbon of (R)-HNE forming a more favorable Bürgi-Duntiz angle with the active site cysteine 293. The presence of Mg2+ ions altered the enantioselectivity of ALDH5A and ALDH2. Mg2+ ions suppressed (R)-HNE oxidation by ALDH5A to a greater extent than that of (S)-HNE. However, Mg2+ ions stimulated the enantioselective oxidation of (R)-HNE by ALDH2 while suppressing (S)-HNE oxidation. These results demonstrate that enantioselective utilization of substrates, including HNE, by ALDHs is dependent upon the ALDH isozyme and the presence of Mg 2+ ions.

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Abstracttrans-4-Hydroxy-2-nonenoic acid (HNEA) is a marker of lipid peroxidation resulting from the metabolism of trans-4-hydroxy-2-nonenal (HNE). Direct and indirect RP-HPLC methods for the separation of HNEA enantiomers were developed and compared. The indirect method involved precolumn derivatization with a chiral amino agent, (1S,2S)-(+)-2-amino-1-(4-nitrophenyl)-1,3-propanediol, and subsequent separation of diastereomers on a Spherisorb ODS2 column. The direct separation of HNEA enantiomers was performed using the chiral stationary phase, Chiralpak AD-RH. Validation parameters including limit of quantification, linear range, accuracy and precision were determined. The indirect separation method was successfully applied for the determination of enantiomeric ratio of HNEA in rat brain mitochondrial lysate, and showed that HNEA was formed (R)-enantioselectively from HNE.

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