Kuiper Heather C, Stevens Jan F
Linus Pauling Institute and the Department of Pharmaceutical Sciences, Oregon State University, Corvallis, Oregon, USA.
Curr Protoc Toxicol. 2011 Feb 1;Chapter 17:Unit17.14.2. doi: 10.1002/0471140856.tx1714s45.
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.
氧化应激诱导的脂质过氧化(LPO)会导致细胞毒性和基因毒性2-烯醛的形成。LPO产物,如4-羟基-2(E)-壬烯醛(HNE)和4-氧代-2(E)-壬烯醛(ONE),由于它们与心血管疾病、神经退行性疾病以及癌症的发生有关,已成为许多研究的主题。LPO产物在与谷胱甘肽(GSH)结合并随后代谢为硫醚氨酸(MA)结合物后随尿液排出。尿LPO-MA代谢物是稳定的终产物代谢物,作为氧化应激的非侵入性体内生物标志物受到关注。本方案描述了一种使用同位素稀释液相色谱-电喷雾串联质谱(LC-MS/MS)定量分析尿液中LPO-MA代谢物的方法。其中包括从未标记的LPO产物和氘标记的MA制备标记的LPO-MA结合物的方案。