Orphan Omega fatty acid hydroxylase CYP4 gene expression in Human NAFLD: Polymorphic variants of CYP4V2 gene in hepatic steatosis. Nicholas Osborne 1, Charles Leahy1, Elizabeth Olah1, Paula Rote 2, Takhar Kasmov1, Byong Song3, Yoon Kwang Lee 1, and James. P. Hardwick1 Northeast Ohio Medical University, Department of Integrative Medical Sciences, Liver Focus group, 4209 State Route 44, Rootstown, Ohio 44272, 2. Internal Medicine University of Minnesota Health care system, Minneapolis, MN 55455, 3. Laboratory of Membrane Biochemistry and Biophysics; Section of Molecular Pharmacology and Toxicology, NIAAA, 5625 Fishers Lane, Room 3N-01, MSC 9410, Bethesda, MD 20892 The Human fatty acid Omega hydroxylase gene family (CYP4) members function in the metabolism of saturated, unsaturated, and bioactive eicosanoids. Different subfamily members metabolize short-chain, medium-chain, long-chain, and very-long-chain fatty acids. Members of the CYP4A and CYP4F subfamily also metabolize the bioactive eicosanoid lipids, arachidonic acid, prostaglandins, and leukotrienes. We hypothesize that the differential regulation of CYP4 members participates in the initiation and progression of NAFLD. In this study, we analyzed the regulation, expression, and activity of orphan CYP4s that participate in fatty acid metabolism in the human liver from patients with steatosis, non-alcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma (HCC). We show that the CYP4 gene members are differentially regulated and expressed in the progression of human NAFLD. We show that lauric acid omega hydroxylase activity is increased in steatosis, NASH, and cirrhosis but decreases dramatically in hepatocellular carcinoma (HCC). This is reflected in increased acetate production in steatosis and acetyl-CoA production that reduces in NASH and cirrhosis but increases in HCC, which we attribute to increased ATP citrate lyase activity. Overall, we observed a decrease in peroxisome β-oxidation from steatosis to HCC. We analyzed the gene expression and protein levels of the orphan CYP4b1, CYP4X1, and CYP4Z1 and observed a marked increase in hepatocellular carcinoma. We also noted increased expression of CYP4F11, CYP4F12, CYP4A22, and CYP4F3 genes and protein in HCC. These results suggest increased omega-hydroxylation of fatty acid and increased production of dicarboxylic acids. Recently, Wang and others have shown that increased peroxisome dicarboxylic acid levels cause massive hepatoblastoma necrosis (Wang et al. 2020 JBC 296, 100283). Although the orphan CYP4V2 gene and protein were downregulated in HCC, we observed increased expression and protein levels in steatosis. Both microsome and lipid droplet levels of P4504V2 increased in steatosis. We also observed possible polymorphic variants of the CYP4V2 that may account for increased triglyceride accumulation in steatosis. Inactivating mutations in the Human CYP4V2 have to be associated with the retinal degenerative disease, Bietti’s crystalline dystrophy, in which cholesterol and glucosylceramides accumulate, how mutations in the CYP4V2 gene lead to increased cholesterol accumulation, which may be due to decreased peroxisome mediated formation of amino acid conjugated omega hydroxylated fatty acids. In C. elegans, the ortholog of Human CYP4V2, CYP37A1, function to produce Ascarosides (ω-hydroxylated fatty acid conjugated carbohydrates) that inhibit the conversion of cholesterol to dafachronic acids (DA). DAs are like bile acid metabolites in humans that activated FXR and LXR to control lipid and energy metabolism. It will be significant to determine if humans produce peroxisome metabolites like C. elegans Ascarosides that control bile acid synthesis.