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

**Background:** Non-alcoholic fatty liver disease (NAFLD) affects 10–35% of the global population, with 3–5% having non-alcoholic steatohepatitis (NASH). NASH can progress to cirrhosis and hepatocellular carcinoma and is projected to become the leading indication for liver transplantation in the United States. Patients with NASH have elevated plasma oxidized low-density lipoproteins (oxLDL), which contain lipid hydroperoxides. However, the direct effects of oxLDL versus native LDL (nLDL) on hepatocyte lipid droplet formation and composition had not been systematically compared.

**Methods:** LDL was isolated from healthy human serum by ultracentrifugation and gel filtration chromatography. OxLDL was prepared by copper sulfate oxidation (0.06 mM CuSO₄) for 2, 8, or 24 h at 37°C. Based on lipid hydroperoxide content, oxLDL (2 h) was selected as the mildly oxidized form for cell experiments. Human liver-derived C3A cells were exposed to nLDL or oxLDL (2 h) at 200 ng protein/10⁴ cells for 22–24 h. Lipid profiles (cholesteryl esters [CE], triacylglycerols [TG], phosphatidylcholines [PC], and their hydroperoxides) were analyzed by Orbitrap LC-MS/MS. Gene expression was measured by real-time PCR. Lipid droplets were visualized by fluorescence microscopy using SRfluor (neutral lipids), Liperfluo (lipid hydroperoxides), and Hoechst 33342 (nuclei). Cell toxicity was assessed by LDH release.

**Key Results:** Cell toxicity did not differ between groups at any concentration tested. Fluorescence imaging showed that nLDL increased non-oxidized lipid droplets, while oxLDL increased both oxidized and non-oxidized lipid droplets. LC-MS/MS revealed that nLDL significantly increased total CE levels (all five detected molecular species) compared to control and oxLDL groups. In contrast, oxLDL significantly increased CE-OOH levels (CE-OOH 18:1, CE-OOH 18:2, and CE-OOH 22:6), with the sum of CE-OOH increased only in oxLDL-treated cells. Total TG levels were significantly reduced in both nLDL and oxLDL groups versus control. TG-OOH levels showed no significant differences among groups. PC levels were significantly higher in nLDL-supplemented cells. The PC-OOH/PC ratio, used as an index of cellular oxidative stress, was significantly increased only in oxLDL-supplemented cells (Con. vs. oxLDL, p = 0.093; nLDL vs. oxLDL, p = 0.103). Gene expression analysis showed: SOAT1 (CE biosynthesis) was significantly reduced in oxLDL versus control and nLDL; LIPE (CE degradation) was markedly reduced in both nLDL and oxLDL groups; ATGL (TG degradation) was significantly increased only in nLDL; SREBP1 (fatty acid regulation) was significantly decreased in oxLDL; FASN (fatty acid synthase) was significantly reduced in both nLDL and oxLDL; SCD1 (fatty acid desaturation) was significantly reduced in oxLDL; and CAT (antioxidant enzyme) was significantly increased only in nLDL.

**Clinical Implications:** This study provides direct evidence that oxLDL promotes oxidative stress in hepatocytes by delivering lipid hydroperoxides that accumulate in lipid droplets, particularly CE-OOH, and by increasing the PC-OOH/PC ratio, a marker of oxidative cellular damage. The finding that nLDL induces CAT expression and suppresses CE oxidation suggests a protective mechanism that is lost with oxLDL exposure. These results position oxLDL as a potential novel therapeutic target and candidate biomarker for NAFLD/NASH. Limitations include the use of a single cell line (C3A), which may not fully recapitulate in vivo hepatocyte biology, and the inability to determine whether this model more closely represents NAFLD or NASH. Further studies using primary hepatocytes, other cell lines, and in vivo models are needed.