Oxidized Low-Density Lipoproteins Trigger Hepatocellular Oxidative Stress with the Formation of Cholesteryl Ester Hydroperoxide-Enriched Lipid Droplets.

Oxidized low-density lipoproteins (oxLDLs) induce oxidative stress in the liver tissue, leading to hepatic steatosis, inflammation, and fibrosis. Precise information on the role of oxLDL in this process is needed to establish strategies for the prevention and management of non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). Here, we report the effects of native LDL (nLDL) and oxLDL on lipid metabolism, lipid droplet formation, and gene expression in a human liver-derived C3A cell line. The results showed that nLDL induced lipid droplets enriched with cholesteryl ester (CE) and promoted triglyceride hydrolysis and inhibited oxidative degeneration of CE in association with the altered expression of LIPE, FASN, SCD1, ATGL, and CAT genes. In contrast, oxLDL showed a striking increase in lipid droplets enriched with CE hydroperoxides (CE-OOH) in association with the altered expression of SREBP1, FASN, and DGAT1. Phosphatidylcholine (PC)-OOH/PC was increased in oxLDL-supplemented cells as compared with other groups, suggesting that oxidative stress increased hepatocellular damage. Thus, intracellular lipid droplets enriched with CE-OOH appear to play a crucial role in NAFLD and NASH, triggered by oxLDL. We propose oxLDL as a novel therapeutic target and candidate biomarker for NAFLD and NASH.

Simple and Sensitive Method for the Quantitative Determination of Lipid Hydroperoxides by Liquid Chromatography/Mass Spectrometry.

Lipid hydroperoxides (LOOH) are the initial products of the peroxidation of unsaturated lipids and play a crucial role in lipid oxidation due to their ability to decompose into free radicals and cause adverse effects on human health. Thus, LOOHs are commonly considered biomarkers of oxidative stress-associated pathological conditions. Despite their importance, the sensitive and selective analytical method for determination is limited, due to their low abundance, poor stability, and low ionizing efficiency. To overcome these limitations, in this study, we chemically synthesized eight fatty acid hydroperoxides (FAOOH), including FA 18:1-OOH, FA 18:2-OOH, FA 18:3-OOH, FA 20:4-OOH, FA 20:5-OOH, FA 22:1-OOH, FA 22:6-OOH as analytes, and FA 19:1-OOH as internal standard. Then, they were chemically labeled with 2-methoxypropene (2-MxP) to obtain FAOOMxP by one-step derivatization (for 10 min). A selected reaction monitoring assisted targeted analytical method was developed using liquid chromatography/tandem mass spectrometry (LC-MS/MS). The MxP-labelling improved the stability and enhanced the ionization efficiency in positive mode. Application of reverse-phase chromatography allowed coelution of analytes and internal standards with a short analysis time of 6 min. The limit of detection and quantification for FAOOH ranged from 0.1-1 pmol/µL and 1-2.5 pmol/µL, respectively. The method was applied to profile total FAOOHs in chemically oxidized human serum samples (n = 5) and their fractions of low and high-density lipoproteins (n = 4). The linoleic acid hydroperoxide (FA 18:2-OOH) and oleic acid hydroperoxide (FA 18:1-OOH) were the most abundant FAOOHs in human serum and lipoproteins. Overall, our validated LC-MS/MS methodology features enhanced detection and rapid separation that enables facile quantitation of multiple FAOOHs, therefore providing a valuable tool for determining the level of lipid peroxidation with potential diagnostic applications.