The determination of glutathione-4-hydroxynonenal (GSHNE), E-4-hydroxynonenal (HNE), and E-1-hydroxynon-2-en-4-one (HNO) in mouse liver tissue by LC-ESI-MS

Glutathione (GSH) conjugation of 4-hydroxy-2(E)-nonenal (HNE) is an efficient means of cellular detoxification. HNE is a byproduct of lipid peroxidation which has shown toxicity but also signaling roles. E-1-hydroxynon-2-en-4-one (HNO) is another byproduct of lipid peroxidation which has the same molecular weight as HNE. This study presents the LC-MS detection of GS-HNE, HNE, and HNO in tissue samples without derivatization and with minimal sample preparation. Tissue samples were taken from wild-type mice and knock-out mice, which have been bred without the RLIP76 transfer protein. Extraction procedures were developed to determine GS-HNE and HNE levels in the mouse liver tissue. A gradient elution LC-MS method was developed for GS-HNE analysis using electrospray ionization and selected ion monitoring (SIM). The HNE/HNO method involves isocratic elution due to instability issues. Higher levels of GSHNE, HNE, and HNO were found in the knock-out animals, due to the absence of the RLIP76 transport mechanism.