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.