LC-MS/MS quantitation of mercapturic acid conjugates of lipid peroxidation products as markers of oxidative stress

Abstract

Oxidative stress-induced lipid peroxidation (LPO) leads to the formation of cytotoxic and genotoxic 2-alkenals. LPO products such as 4-hydroxy-2(E)-nonenal (HNE) and 4-oxo-2(E)-nonenal (ONE) have been the subject of many studies due to their association with the development of cardiovascular and neurodegenerative diseases, as well as cancer. LPO products are excreted in the urine after conjugation with glutathione (GSH) and subsequent metabolism to mercapturic acid (MA) conjugates. Urinary LPO-MA metabolites are stable end-product metabolites and have gained interest as non-invasive in vivo biomarkers of oxidative stress. This protocol describes a method for the quantitative analysis of LPO-MA metabolites in urine using isotope-dilution liquid chromatography coupled with electrospray tandem mass spectrometry (LC-MS/MS). Included are protocols for preparation of labeled LPO-MA conjugates from unlabeled LPO products and deuterium labeled MA.

Keywords: lipid peroxidation; mass spectrometry; mercapturic acid; oxidative stress.

Figure 17.14.1

Formation of LPO products from linoleic acid. Under conditions of oxidative stress, linoleic acid is oxidized to form intermediary hydroperoxy octadecadienoic acids (HPODEs) that further degrade into HNE and ONE. Both 2-alkenals are converted by phase I enzymes into HNA, DHN, ONA, and ONO.

Figure 17.14.2

HNE conjugate formation with cysteine via Michael-type addition.

Figure 17.14.3

LC-MS/MS-SRM chromatogram of LPO-MA conjugates detected in human urine. SRM transitions are: (1) HNE-MA m/z 318 → 189 and m/z 318 → 171; (2) DHN-MA m/z 320 → 191 and m/z 320 → 143; (3) HNA-MA m/z 334 → 162 and m/z 334 → 143; (4) HNAL-MA m/z 316 → 162 and m/z 316 → 143; (5) ONO-MA m/z 318 → 162; (6) ONA-MA m/z 332 → 169 and m/z 332 → 162.