Mercapturic acid conjugates of 4-hydroxy-2-nonenal and 4-oxo-2-nonenal metabolites are in vivo markers of oxidative stress

Abstract

Oxidative stress-induced lipid peroxidation leads to the formation of cytotoxic and genotoxic 2-alkenals, such as 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE). Lipid-derived reactive aldehydes are subject to phase-2 metabolism and are predominantly found as mercapturic acid (MA) conjugates in urine. This study shows evidence for the in vivo formation of ONE and its phase-1 metabolites, 4-oxo-2-nonen-1-ol (ONO) and 4-oxo-2-nonenoic acid (ONA). We have detected the MA conjugates of HNE, 1,4-dihydroxy-2-nonene (DHN), 4-hydroxy-2-nonenoic acid (HNA), the lactone of HNA, ONE, ONO, and ONA in rat urine by liquid chromatography-tandem mass spectrometry comparison with synthetic standards prepared in our laboratory. CCl(4) treatment of rats, a widely accepted animal model of acute oxidative stress, resulted in a significant increase in the urinary levels of DHN-MA, HNA-MA lactone, ONE-MA, and ONA-MA. Our data suggest that conjugates of HNE and ONE metabolites have value as markers of in vivo oxidative stress and lipid peroxidation.

FIGURE 1.

Formation of LPO-MA conjugates from HNE. Under conditions of oxidative stress, linoleic and arachidonic acids can be oxidized to form HPNE. HPNE can then be reduced to HNE and further metabolized by aldehyde dehydrogenase (ALDH), aldo-keto reductase (AKR), and GST. These enzymes cause oxidization, reduction, or GSH conjugation, respectively. Once LPO-GSH conjugates have formed, the GSH is further metabolized to MA. The LPO-MA conjugates are analyzed in this study. It is important to note that once HNE has been reduced to DHN, it will no longer form a Michael-type conjugate with GSH due to the lack of α,β-unsaturation. Also, HNA-MA is subject to intramolecular condensation, resulting in the formation of a lactone.

FIGURE 2.

Formation of LPO-MA conjugates from ONE. HPNE can eliminate a water molecule, resulting in the formation of ONE. Similarly to HNE, ONE undergoes oxidation by aldehyde dehydrogenase (ALDH), reduction by aldo-keto reductase (AKR), and GSH conjugation by GST. Unlike HNE, ONE has two possible sites of conjugate formation, at the C-2 and C-3 positions. Thus, after reduction, the ONO formed retains its α,β-unsaturation, allowing it to form GSH conjugates via this route. The MA conjugates were analyzed in this study and are shown here as the C-2 conjugates.

FIGURE 3.

SRM chromatograms for the isobaric compounds HNE-MA and ONO-MA. A, standard reaction mixture of HNE-MA; transitions shown are from top to bottom: m/z 318 → 171, m/z 318 → 189, and m/z 318 → 162. B, standard reaction mixture of ONO-MA; transitions shown are from top to bottom: m/z 318 → 171, m/z 318 → 189, and m/z 318 → 162. C, rat urine sample; transitions shown are from top to bottom: m/z 318 → 171, m/z 318 → 189, and m/z 318 → 162. The HNE-MA gives only two peaks (8.9 and 9.3 min), whereas the ONO-MA gives three (8.9, 9.3, and 9.7 min). The standards confirm that the third peak contains only ONO-MA. This third peak was used to represent ONO-MA for comparison between oxidatively stressed rats and control rats.

FIGURE 4.

Fragmentation patterns for each chromatographic peak of ONO-MA. A, proposed mass fragments of the hemiketal form of ONO-MA with m/z 189 (β-elimination), m/z 171 (β-elimination followed by H₂O loss), and m/z 162 (mercapturate formed upon RM cleavage). B, the preferred open chain form of ONO-MA produces mass fragments with the same m/z values as the cyclic form, but the mercapturate fragment with m/z 162 is expected to predominate due to RM cleavage. C, MS/MS spectrum for the first minor peak of a standard solution of ONO-MA. The fragments with m/z 189 and m/z 171 are the most prominent. D, MS/MS spectrum recorded for the second minor ONO-MA peak, showing the m/z 171 fragment as the most abundant ion. E, MS/MS spectrum for the unique third and major peak of ONO-MA. The mercapturate peak at m/z 162 represents the most abundant fragment ion, which is suggestive of RM cleavage of the open chain form.

FIGURE 5.

SRM chromatograms of the isobaric compounds ONE-MA and HNA-MA lactone. A, standard reaction mixture of HNA-MA, showing the lactone form and SRM transitions from top to bottom: m/z 316 → 143 and m/z 316 → 162. B, standard reaction mixture of ONE-MA, the predominant SRM transition for this compound is m/z 316 → 162. C, rat urine sample; transitions are shown from top to bottom: m/z 316 → 162 and m/z 316 → 143. These compounds were separated using chromatographic system 2.

FIGURE 6.

SRM chromatograms for DHN-MA, ONA-MA, and HNA-MA. A, standard reaction mixture of DHN-MA; transitions are from top to bottom: m/z 320 → 191 and m/z 320 → 143. B, rat urine sample; transitions shown are from top to bottom: m/z 320 → 191 and m/z 320 → 143. C, standard reaction mixture of ONA-MA; transitions are from top to bottom: m/z 332 → 162, m/z 332 → 169, and m/z 332 → 84. D, rat urine sample, transitions shown are from top to bottom: m/z 332 → 162, m/z 332 → 169, and m/z 332 → 84. E, standard reaction mixture of HNA-MA, transition m/z 334 → 162. F, rat urine sample, transition m/z 334 → 162.

FIGURE 7.

Comparison of HPNE metabolites in control rats and rats oxidatively stressed with CCl₄. ONE-MA, DHN-MA, ONA-MA, and HNA-MA lactone all show a significant increase in CCl₄-dosed rats with p values of 0.002, 0.006, 0.008, and 0.048, respectively. HNE-MA, ONO-MA, and HNA-MA did not increase significantly in CCl₄-dosed rats. Data are shown as the mean ± S.E.

FIGURE 8.

LPO-induced degradation of linoleic acid and conversion into the isobaric metabolites HNE-MA and ONO-MA. DHN-MA can be formed by aldehyde reduction of HNE-MA or ketone reduction of ONO-MA. The asterisk indicates the electrophilic position. CR, carbonyl reductase; AKR, aldo-keto reductase.