Mitochondrial dysfunction-related lipid changes occur in nonalcoholic fatty liver disease progression

Nonalcoholic fatty liver disease (NAFLD) comprises fat-accumulating conditions within hepatocytes that can cause severe liver damage and metabolic comorbidities. Studies suggest that mitochondrial dysfunction contributes to its development and progression and that the hepatic lipidome changes extensively in obesity and in NAFLD. To gain insight into the relationship between lipid metabolism and disease progression through different stages of NAFLD, we performed lipidomic analysis of plasma and liver biopsy samples from obese patients with nonalcoholic fatty liver (NAFL) or nonalcoholic steatohepatitis (NASH) and from those without NAFLD. Congruent with earlier studies, hepatic lipid levels overall increased with NAFLD. Lipid species that differed with NAFLD severity were related to mitochondrial dysfunction; specifically, hepatic cardiolipin and ubiquinone accumulated in NAFL, and levels of acylcarnitine increased with NASH. We propose that increased levels of cardiolipin and ubiquinone may help to preserve mitochondrial function in early NAFLD, but that mitochondrial function eventually fails with progression to NASH, leading to increased acylcarnitine. We also found a negative association between hepatic odd-chain phosphatidylcholine and NAFLD, which may result from mitochondrial dysfunction-related impairment of branched-chain amino acid catabolism. Overall, these data suggest a close link between accumulation of specific hepatic lipid species, mitochondrial dysfunction, and the progression of NAFLD. Nonalcoholic fatty liver disease (NAFLD) comprises fat-accumulating conditions within hepatocytes that can cause severe liver damage and metabolic comorbidities. Studies suggest that mitochondrial dysfunction contributes to its development and progression and that the hepatic lipidome changes extensively in obesity and in NAFLD. To gain insight into the relationship between lipid metabolism and disease progression through different stages of NAFLD, we performed lipidomic analysis of plasma and liver biopsy samples from obese patients with nonalcoholic fatty liver (NAFL) or nonalcoholic steatohepatitis (NASH) and from those without NAFLD. Congruent with earlier studies, hepatic lipid levels overall increased with NAFLD. Lipid species that differed with NAFLD severity were related to mitochondrial dysfunction; specifically, hepatic cardiolipin and ubiquinone accumulated in NAFL, and levels of acylcarnitine increased with NASH. We propose that increased levels of cardiolipin and ubiquinone may help to preserve mitochondrial function in early NAFLD, but that mitochondrial function eventually fails with progression to NASH, leading to increased acylcarnitine. We also found a negative association between hepatic odd-chain phosphatidylcholine and NAFLD, which may result from mitochondrial dysfunction-related impairment of branched-chain amino acid catabolism. Overall, these data suggest a close link between accumulation of specific hepatic lipid species, mitochondrial dysfunction, and the progression of NAFLD. Nonalcoholic fatty liver disease (NAFLD) is an umbrella term that is used to describe a variety of conditions characterized by a build-up of fat within liver cells. NAFLD can range from mild hepatic steatosis [nonalcoholic fatty liver (NAFL)] to the more severe nonalcoholic steatohepatitis (NASH), and can progress to fibrosis, cirrhosis, and hepatocellular carcinoma. NAFLD is the predominant cause of chronic liver disease in many parts of the world, and is present in 75–90% of adults who are morbidly obese (1.Silverman J.F. O'Brien K.F. Long S. Leggett N. Khazanie P.G. Pories W.J. Norris H.T. Caro J.F. Liver pathology in morbidly obese patients with and without diabetes.Am. J. Gastroenterol. 1990; 85: 1349-1355PubMed Google Scholar, 2.Schwenger K.J.P. Fischer S.E. Jackson T.D. Okrainec A. Allard J.P. Non-alcoholic fatty liver disease in morbidly obese individuals undergoing bariatric surgery: prevalence and effect of the pre-bariatric very low calorie diet.Obes. Surg. 2018; 28: 1109-1116Crossref PubMed Scopus (26) Google Scholar). Given that the liver is a central regulator of whole-body energy homeostasis, it is not surprising that many individuals with NAFLD are also characterized by metabolic complications, including insulin resistance, muscle atrophy, and cardiovascular disease, resulting in increased morbidity and mortality (3.Fotbolcu H. Zorlu E. Nonalcoholic fatty liver disease as a multi-systemic disease.World J. Gastroenterol. 2016; 22: 4079-4090Crossref PubMed Scopus (80) Google Scholar). NAFL is diagnosed when liver fat exceeds 5% of the total liver weight. Lipid accumulation in the liver is regulated by the interplay between the delivery of lipids to the liver and their hepatic uptake, synthesis, oxidation, and secretion of lipids. Alterations in the equilibrium of one or more of these processes can promote hepatic steatosis (4.Meex R.C.R. Watt M.J. Hepatokines: linking nonalcoholic fatty liver disease and insulin resistance.Nat. Rev. Endocrinol. 2017; 13: 509-520Crossref PubMed Scopus (323) Google Scholar). NASH is characterized by hepatic steatosis, hepatocyte ballooning, and lobular inflammation. The mechanisms that trigger hepatic inflammation are poorly understood, and to date, a liver biopsy is the only reliable method to distinguish between the different stages of NAFLD. Several studies have tried to identify lipid biomarkers of NAFL and NASH, but this has proven to be very difficult because the correlations between blood and liver lipid species drastically decrease after the transition from NAFL to NASH (5.Sa R. Zhang W. Ge J. Wei X. Zhou Y. Landzberg D.R. Wang Z. Han X. Chen L. Yin H. Discovering a critical transition state from nonalcoholic hepatosteatosis to nonalcoholic steatohepatitis by lipidomics and dynamical network biomarkers.J. Mol. Cell Biol. 2016; 8: 195-206Crossref PubMed Scopus (25) Google Scholar). We previously used lipidomics to compare serum lipid profiles from excessive chronic drinkers without liver disease to those with advanced alcoholic cirrhosis, and showed that circulating lipids associate with alcoholic liver cirrhosis and represent potential biomarkers for risk assessment (6.Meikle P.J. Mundra P.A. Wong G. Rahman K. Huynh K. Barlow C.K. Duly A.M. Haber P.S. Whitfield J.B. Seth D. Circulating lipids are associated with alcoholic liver cirrhosis and represent potential biomarkers for risk assessment.PLoS One. 2015; 10: e0130346Crossref PubMed Scopus (24) Google Scholar). The aim of the current study was to gain a more comprehensive understanding of the hepatic and plasma lipidomic changes in morbidly obese subjects with NAFL or NASH compared with morbidly obese subjects without NAFLD, and to gain insight into lipid species that are linked to disease progression through the different stages of NAFLD. We performed lipidomic analysis in liver biopsies and plasma samples from 58 subjects classified on the basis of liver histology as either normal (n = 16), steatotic (n = 10), or NASH (n = 32). Our study cohort consisted of 58 subjects with severe obesity (BMI >35) who underwent bariatric surgery. The institutional medical ethical committee of Maastricht University approved the study, and all subjects gave written informed consent before participation in the study. Just before surgery, a fasting venous blood sample was obtained from each subject. During surgery, a fasted liver biopsy was obtained. Part of the liver biopsies were fixed in formalin and embedded in paraffin, and histopathology was performed by H&E staining and Masson's trichrome staining. Subjects were classified as control (CTRL), NAFL, or NASH according to the criteria of both Brunt et al. (7.Brunt E.M. Janney C.G. Di Bisceglie A.M. Neuschwander-Tetri B.A. Bacon B.R. Nonalcoholic steatohepatitis: a proposal for grading and staging the histological lesions.Am. J. Gastroenterol. 1999; 94: 2467-2474Crossref PubMed Scopus (3130) Google Scholar) and Kleiner et al. (8.Kleiner D.E. Brunt E.M. Van Natta M. Behling C. Contos M.J. Cummings O.W. Ferrell L.D. Liu Y.C. Torbenson M.S. Unalp-Arida A. Nonalcoholic Steatohepatitis Clinical Research Network Design and validation of a histological scoring system for nonalcoholic fatty liver disease.Hepatology. 2005; 41: 1313-1321Crossref PubMed Scopus (7113) Google Scholar). The CTRL group was comprised of subjects showing <5% steatotic hepatocytes. The steatotic group was comprised of individuals showing >5% steatotic hepatocytes without significant inflammation, as observed after H&E staining (no intra-acinar inflammatory foci per 20 fields with a 20 ocular and no portal tract inflammation). The third group was comprised of subjects with NASH, who were identified by the presence of steatosis, lobular inflammation, and hepatocellular ballooning. Lipid extraction of the liver and plasma was carried out using an earlier described single-phase chloroform:methanol (2:1) approach, with some modifications (9.Meikle P.J. Wong G. Tsorotes D. Barlow C.K. Weir J.M. Christopher M.J. MacIntosh G.L. Goudey B. Stern L. Kowalczyk A. Plasma lipidomic analysis of stable and unstable coronary artery disease.Arterioscler. Thromb. Vasc. Biol. 2011; 31: 2723-2732Crossref PubMed Scopus (218) Google Scholar). Briefly, liver tissues (weighing ∼0.7–1.9 mg) were first homogenized with a Bullet Blender® Gold homogenizer (Next Advance, Inc., Averill Park, NY) in 250 μl Tris-NaCl containing 100 μM of butylhydroxytoluene, and then underwent further probe sonication (S-4000, Misonix Ultrasonic, amplitude 26, 30 s on ice; Misonix) to break down the tissue completely. For lipid extraction, a 10 μl liver or plasma sample was mixed with 200 μl of chloroform/methanol (2:1 v/v) and 10 μl of internal standard mix (supplemental Table S1). The samples were rotary mixed and sonicated for 30 min at room temperature, then centrifuged (16,000 g, 15 min) to remove the insoluble pellets. The supernatants were then transferred to 96-well plates and dried using the Speedy Vac system (Savant RVT5105; Thermo Fisher Scientific). Prior to lipidomic analysis, the lipid extract was dried and redissolved in 50 μl of water-saturated butanol. Then, 50 μl of methanol containing 10 mM ammonium formate were added. The samples were subsequently centrifuged (3,350 g, 5 min, room temperature), and the supernatant containing the reconstituted lipids was transferred to glass vials (with glass inserts) for LC-MS lipidomic analysis. Targeted lipidomics for the liver and plasma specimens was performed using LC coupled with an electrospray ionization Qtrap 4000 mass spectrometer (AB Sciex), as described earlier with slight modifications (10.Weir J.M. Wong G. Barlow C.K. Greeve M.A. Kowalczyk A. Almasy L. Comuzzie A.G. Mahaney M.C. Jowett J.B. Shaw J. Plasma lipid profiling in a large population-based cohort.J. Lipid Res. 2013; 54: 2898-2908Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). HPLC separation was conducted using an Agilent 1200 series HPLC system with a Poroshell 120 EC-C18 column (2.1 × 100 mm, 2.7 microns; Agilent) with the thermostat set at 50°C. The solvent system consisted of solvent A [60% water, 20% methanol, and 20% THF (v/v/v) with 10 mM ammonium formate] and solvent B [75% THF, 20% methanol, and 5% water (v/v/v) with 10 mM ammonium formate]. Our gradient was as follows: starting conditions at 90% A 10% B, increasing to 20% B over 0.1 min then increasing to 35% B at 3 min, 50% B at 3.5 min, 62% B at 11 min, followed by a sharp adjustment to 89% B at 11.1 min with a shallow gradient to 92% B at 14.9 min then adjusting to 100% B at 15 min and holding until 16.5 min. The gradient was then adjusted back to starting conditions at 17 min and held for 3 min for a final run time of 20 min. This method enabled all lipid species, including diacylglycerol (DG) and triacylglycerol (TG) species, to be monitored in a single chromatographic run. As in the previous method, the quantification of the TG species was based on the neutral loss of a FA (+NH4) and the remaining FAs were assigned based on comprehensive neutral loss scans of each FA type using the pooled plasma quality control sample. Thus, we were able to differentiate the TG species containing 17:0 acyl chains (e.g., TG(16:0_17:0_18:1) from the internal standard [TG(17:0/17:0/17:0)] based on precursor mass (m/z 864.8 and m/z 866.8), the neutral loss [299.3 Da (18:1) and 287.2 Da (17:0)], and the retention time (15.65 min and 15.94 min). A total of 474 lipid species across 29 lipid classes and subclasses were detected in this study (supplemental Table S1). AB Sciex Multiquant software version 2.1.1 (AB Sciex) was used for quantification of peaks and data integration. Under normal analysis conditions, we were unable to obtain phospholipid acyl composition. However, our current chromatography allowed separation of isomers by their retention time. To facilitate identification, the FA composition of phospholipids was characterized by either collision-induced dissociation in negative ionization mode, for neutral or negatively charged species, or by collision-induced dissociation of lithium adducts in positive ionization mode for positively charged species, and subsequently used to infer structural composition of phospholipid isomers based on retention time for species that were well separated. Characterization of the phospholipid FAs was performed on whole lipid extracts of pooled plasma samples. For the analysis of lithium adducts of phosphatidylcholine (PC), alkylphosphatidylcholine, alkenylphosphatidylcholine, and SM, the 10 mM of ammonium formate in the extraction buffer and running solvents were substituted with 200 μM of lithium acetate, which provided the ability to monitor for lithium adducts for additional structural information (11.Hsu F.F. Turk J. Electrospray ionization/tandem quadrupole mass spectrometric studies on phosphatidylcholines: the fragmentation processes.J. Am. Soc. Mass Spectrom. 2003; 14: 352-363Crossref PubMed Scopus (261) Google Scholar, 12.Hsu F.F. Turk J. Thukkani A.K. Messner M.C. Wildsmith K.R. Ford D.A. Characterization of alkylacyl, alk-1-enylacyl and lyso subclasses of glycerophosphocholine by tandem quadrupole mass spectrometry with electrospray ionization.J. Mass Spectrom. 2003; 38: 752-763Crossref PubMed Scopus (83) Google Scholar). Where it was not possible to definitively characterize isomers (typically resulting from double bond position), we adopted the system of annotating with an (a) and a (b). Patient characteristics are presented as mean ± SEM (numerical data) or percent (categorical data). For 15 out of 18 characteristics shown, there was at least one missing data point (maximum seven missing for any characteristics), and they were estimated with the imputation approach. The missing data were imputed using the multiple imputation by chained equations (MICE) method, utilizing the R package “mice”;. The numerical data were analyzed with the Mann-Whitney U test, while the categorical data were analyzed with the χ2 test. A P value <0.05 was considered significant. The liver and plasma lipidomic data are presented as picomoles per micromole of PC. Mean ± SEM or percent difference to the CTRL group are used to display the lipidomic data. The Kruskal-Wallis test corrected with the Benjamini-Hochberg approach was used to determine the inter-group significance. A corrected P value <0.05 was considered significant. Mann-Whitney U tests corrected with the Dunn-Sidak approach were utilized as the post hoc test, and a P value <0.05 was considered significant. To determine the association of the lipid species or classes to the disease (i.e., NAFL and NASH), logistic regression was performed between CTRL, NAFL, and NASH. The Benjamini-Hochberg approach was applied for multiple comparisons, and a P value <0.05 was considered significant. Patients undergoing bariatric surgery were retrospectively grouped into CTRL (n = 16), NAFL (n = 10), and NASH (n = 32), as determined by histological examinations of their liver biopsies. All subjects were morbidly obese and most subjects complied with the definition of metabolic syndrome [i.e., large waist circumference (>89 cm for women and >102 cm for men, hyperglycemia (fasted glucose >5.6 mmol/l), hypertriglyceridaemia (>1.7 mmol/l), and reduced HDL cholesterol (<1.3 mmol/l in women and <1.04 mmol/l in men)]. Homeostatic model assessment of insulin resistance (HOMA-IR) (5.50 ± 0.51 vs. 3.06 ± 0.34) and blood glucose levels (6.9 ± 0.4 vs. 5.7 ± 0.3) were increased in NASH compared with CTRL, indicating a more insulin-resistant state. As expected, aspartate aminotransferase levels were higher in NASH compared with CTRL. When comparing NAFL to NASH, only glucose levels were significantly higher in individuals with NASH (Table 1).TABLE 1Subject characteristicsCharacteristicsGroupCTRLNAFLNASHNumber161032Sex (% female)81.390.065.6Age (years)42.3 ± 2.541.6 ± 3.945.5 ± 1.8BMI (kg/m2)41.0 ± 1.645.5 ± 2.448.39 ± 1.9Type II diabetes (%)6.320.034.4Glucose (mmol/l)5.7 ± 0.35.6 ± 0.36.9 ± 0.4aP < 0.05 versus CTRL.,bP < 0.05 versus NAFL.Insulin (mU/l)12.3 ± 1.320.0 ± 3.518.8 ± 1.9HOMA-IR3.06 ± 0.344.96 ± 0.765.50 ± 0.51aP < 0.05 versus CTRL.HbA1c (%)6.1 ± 0.26.5 ± 0.46.9 ± 0.2Total cholesterol (mmol/l)4.74 ± 0.225.20 ± 0.314.97 ± 0.19HDL cholesterol (mmol/l)1.13 ± 0.090.95 ± 0.081.02 ± 0.08LDL cholesterol (mmol/l)2.76 ± 0.213.30 ± 0.362.99 ± 0.17TG (mmol/l)1.87 ± 0.241.98 ± 0.322.22 ± 0.21Free FA (mmol/l)0.51 ± 0.060.59 ± 0.120.92 ± 0.19ALT (U/l)19.9 ± 1.225.3 ± 2.928.6 ± 2.5AST (U/l)15.3 ± 1.917.9 ± 2.528.2 ± 2.2cP < 0.01 versus CTRL.CRP (mg/l)9.4 ± 2.116.3 ± 5.79.6 ± 1.0Baseline characteristics of CTRL, NAFL, and NASH groups are shown as percent (for categorical data) and mean ± SEM (for nominal data). Data were analyzed using the chi-square test (categorical) or Mann-Whitney U test (nominal). HbA1c, hemoglobin A1c; AST, aspartate aminotransferase; ALT, alanine aminotransferase; CRP, C-reactive protein.a P < 0.05 versus CTRL.b P < 0.05 versus NAFL.c P < 0.01 versus CTRL. Open table in a new tab Baseline characteristics of CTRL, NAFL, and NASH groups are shown as percent (for categorical data) and mean ± SEM (for nominal data). Data were analyzed using the chi-square test (categorical) or Mann-Whitney U test (nominal). HbA1c, hemoglobin A1c; AST, aspartate aminotransferase; ALT, alanine aminotransferase; CRP, C-reactive protein. To investigate the hepatic lipidome in individuals with NAFL and NASH, the abundance of each lipid class was expressed relative to the CTRL group, with CTRL values set at 100%. Levels of TG, DG, acylcarnitine, dihydroceramide, dihexosylceramide, cholesterol, cholesteryl ester (CE), cardiolipin, and ubiquinone were significantly increased in NAFL and/or NASH groups compared with CTRL. Only acylcarnitine and dihexosylceramide were significantly higher in the NASH group compared with the NAFL group (Fig. 1, supplemental Table S2). Storage of excessive energy as TG is a feature and a key diagnostic criterion of NAFLD. Many of the TG and DG species were higher in the NAFL and NASH groups compared with the CTRL group. In the NASH group, nearly all TG, but not DG, species were significantly higher compared with the NAFL group (supplemental Table S3). To better understand changes in the FA composition of TGs and DGs between groups, FA composition was calculated as a percentage of total FAs of each lipid class (Fig. 2). Individuals with NASH had significantly more saturated FAs (SFAs) (i.e., 14:0 FA, 17:0 FA, and 18:0 FA) in their TGs compared with the CTRL group (Fig. 2A), whereas the most abundant SFA (16:0) did not change. The percentage of polyunsaturated arachidonic acid (20:4 FA) in TG was progressively lower in accordance with NAFLD severity. Interestingly, the percentage of docosahexaenoic acid (22:6 FA), which is another PUFA in TG, did not differ significantly between the groups (Fig. 2B). Although total levels of DG increased to a similar extent as TG with NAFL and NASH, there were disparities between the two lipid classes in terms of FA composition. Individuals with NAFL and NASH had higher levels of MUFAs in their DGs (16:1 FA and 18:1 FA). In contrast, percentages of SFA (18:0 FA), diunsaturated (DUFA, 18:2 FA), and long-chain PUFAs (20:4, 22:5, and 22:6) declined in the DG pool of the NAFL and NASH groups (Table 2, Fig. 2C, D).TABLE 2Comparison of the FA saturation levels within the selected lipid classes between CTRL, NAFL, and NASH groupsCTRLNAFLNASHTGSFA40.3 ± 0.6641.4 ± 1.1143.1 ± 0.50aP < 0.01 versus CTRL.MUFA48.8 ± 0.5748.3 ± 0.4847.7 ± 0.42DUFA10.7 ± 0.6210.2 ± 0.749.1 ± 0.37PUFA0.20 ± 0.020.16 ± 0.020.14 ± 0.01aP < 0.01 versus CTRL.DGSFA39.4 ± 0.8936.0 ± 1.47bP < 0.05 versus CTRL.36.5 ± 0.92MUFA38.5 ± 1.7047.5 ± 1.26aP < 0.01 versus CTRL.50.1 ± 1.00cP < 0.001 versus CTRL.DUFA12.0 ± 0.6410.1 ± 0.869.10 ± 0.48aP < 0.01 versus CTRL.PUFA10.0 ± 0.846.38 ± 0.66bP < 0.05 versus CTRL.4.36 ± 0.17cP < 0.001 versus CTRL.dP < 0.05 versus NAFL.CESFA35.2 ± 0.6932.8 ± 0.8329.7 ± 0.60cP < 0.001 versus CTRL.dP < 0.05 versus NAFL.MUFA25.1 ± 0.9122.7 ± 0.9221.8 ± 0.80bP < 0.05 versus CTRL.DUFA32.3 ± 1.0835.9 ± 1.0638.9 ± 1.11aP < 0.01 versus CTRL.PUFA7.32 ± 0.498.61 ± 0.569.59 ± 0.36aP < 0.01 versus CTRL.CardiolipinSFA0.26 ± 0.050.38 ± 0.160.41 ± 0.08MUFA11.5 ± 0.4412.8 ± 1.5512.8 ± 0.36bP < 0.05 versus CTRL.DUFA87.4 ± 0.4786.0 ± 1.7086.0 ± 0.38bP < 0.05 versus CTRL.PUFA0.83 ± 0.080.76 ± 0.070.81 ± 0.06AcylcarnitineSFA62.7 ± 3.6552.3 ± 4.6957.9 ± 2.63MUFA25.0 ± 3.6832.0 ± 3.9736.0 ± 2.45bP < 0.05 versus CTRL.DUFA12.3 ± 2.5815.7 ± 4.856.19 ± 0.88PUFA−−−Data in each lipid class are shown as mean ± SEM and were analyzed with the Kruskal-Wallis test and corrected for multiple comparisons with the Benjamini-Hochberg approach. The Mann-Whitney U test corrected with the Dunn-Sidak approach was the post hoc analysis (n = 16 for CTRL; n = 10 for NAFL; n = 32 for NASH).a P < 0.01 versus CTRL.b P < 0.05 versus CTRL.c P < 0.001 versus CTRL.d P < 0.05 versus NAFL. Open table in a new tab Data in each lipid class are shown as mean ± SEM and were analyzed with the Kruskal-Wallis test and corrected for multiple comparisons with the Benjamini-Hochberg approach. The Mann-Whitney U test corrected with the Dunn-Sidak approach was the post hoc analysis (n = 16 for CTRL; n = 10 for NAFL; n = 32 for NASH). Like TGs and DGs, certain CEs comprised lipid species that were more abundant in the NAFL and NASH groups compared with the CTRL group (Fig. 1; supplemental Tables S2, S3). Among the most abundant FAs in CEs, 16:1 FA and 18:2 FA were increased in subjects with NASH compared with CTRL, while levels of 16:0 FA and 18:1 FA were decreased. The 16:0 FA was also significantly lower in NAFL individuals compared with CTRL (Fig. 3A). With respect to the less abundant FAs, 16:2 FA, 17:1 FA, 18:3 FA, and 22:6 FA were increased in the NASH group as compared with the CTRL group, while 20:1 FA, 20:2 FA, and 22:4 FA were decreased. The 16:2 FA was also significantly higher in the NAFL group compared with CTRL and 20:2 FA was significantly lower in NASH compared with NAFL (Fig. 3B, C). In contrast to TGs, DGs, and CEs, there were only a few differences among the phospholipid and sphingolipid species between groups (supplemental Tables S2, S3). Notably, several phospholipid species comprising docosahexaenoic acid (22:6 FA) were significantly lower when comparing individuals with NAFL and NASH, including PC(P-16:0/22:6)(a) (NASH vs. NAFL), LPC 22:6[sn2] (NASH vs. CTRL), and PE(18:1_22:6)(a) (NASH vs. CTRL and NASH vs. NAFL). Furthermore, PC(40:7) and PC(40:8), which both contained 22:6 FA, were also lower in individuals with NASH compared with CTRL (Fig. 4A, supplemental Table S3). Although significant inter-group differences were not observed in any of the individual odd-chain PC species, logistic regression analysis indicated that total odd-chain PC was negatively associated with NAFLD. The odds ratio of total odd-chain PC, NASH versus CTRL, was 0.40 and reached statistical significance (Fig. 4A, B). In the sphingolipids class, only dihydroceramide and dihexosylceramide were significantly different between the CTRL and NAFLD groups. Interestingly, although both classes of sphingolipids were higher in individuals with NASH compared with CTRL, levels of dihydroceramide accumulated progressively with the disease severity (significant difference between NASH vs. CTRL), while levels of dihexosylceramide were only increased in the NASH group (significant NASH vs. CTRL and NASH vs. NAFL) (Fig. 1, supplemental Table S2). In line with its respective lipid class, the dihydroceramide species, Cer(d18:0/18:0), was increased in the NAFL and NASH group compared with CTRL, while the dihexosylceramide species, Hex2Cer(d18:1/18:0) and Hex2Cer(d18:1/24:1), were only increased in the NASH group. The only SM species that differed between the groups was SM(38:1), which was lower in the NASH group compared with CTRL (supplemental Table S3). Mitochondrial dysfunction has been suggested to play a pivotal role in the pathogenesis of NAFLD (13.Begriche K. Igoudjil A. Pessayre D. Fromenty B. Mitochondrial dysfunction in NASH: causes, consequences and possible means to prevent it.Mitochondrion. 2006; 6: 1-28Crossref PubMed Scopus (591) Google Scholar, 14.Koliaki C. Szendroedi J. Kaul K. Jelenik T. Nowotny P. Jankowiak F. Herder C. Carstensen M. Krausch M. Knoefel W.T. Adaptation of hepatic mitochondrial function in humans with non-alcoholic fatty liver is lost in steatohepatitis.Cell Metab. 2015; 21: 739-746Abstract Full Text Full Text PDF PubMed Scopus (541) Google Scholar). Cardiolipin, acylcarnitine, and ubiquinone are lipid classes that have been linked to mitochondrial function (15.Chicco A.J. Sparagna G.C. Role of cardiolipin alterations in mitochondrial dysfunction and disease.Am. J. Physiol. Cell Physiol. 2007; 292: C33-C44Crossref PubMed Scopus (492) Google Scholar, 16.Botham K.M. Napolitano M. Bravo E. The emerging role of disturbed CoQ metabolism in nonalcoholic fatty liver disease development and progression.Nutrients. 2015; 7: 9834-9846Crossref PubMed Scopus (9) Google Scholar). Unlike other phospholipid classes, cardiolipin levels were significantly higher in the NAFL and NASH groups, as compared with the CTRL group (Fig. 1). When the FA composition of cardiolipin was analyzed, the percentage of 18:2 FA, the most abundant FA in the cardiolipin pool, was lower in NASH (not significant under Kruskal-Wallis test; Dunn-Sidak corrected Mann-Whitney U test P value = 0.039). The percentage of MUFAs was increased in NASH, while the percentage of diunsaturated FAs (DUFAs) was decreased (supplemental Table S4). Nearly all acylcarnitine FAs were increased in individuals with NASH (supplemental Table S3). However, there were no differences in acylcarnitine FA composition between groups when expressed as a percentage of the total (supplemental Table S4). Some FAs showed a trend toward significance. Specifically, acylcarnitine 18:1 was increased in NASH compared with CTRL (not significant under Kruskal-Wallis test; Dunn-Sidak corrected Mann-Whitney U test P value = 0.015, NASH vs. CTRL), while acylcarnitine 18:2 was decreased in NASH versus CTRL (not significant under Kruskal-Wallis test; Dunn-Sidak corrected Mann-Whitney U test P value = 0.082, NASH vs. CTRL) (supplemental Table S4). Levels of ubiquinone were increased with NAFL and NASH, compared with CTRL (Fig. 1). Several earlier studies examined the potential of predicting NAFLD by measuring plasma lipids (17.Anjani K. Lhomme M. Sokolovska N. Poitou C. Aron-Wisnewsky J. Bouillot J.L. Lesnik P. Bedossa P. Kontush A. Clement K. Circulating phospholipid profiling identifies portal contribution to NASH signature in obesity.J. Hepatol. 2015; 62: 905-912Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 18.Gorden D.L. Myers D.S. Ivanova P.T. Fahy E. Maurya M.R. Gupta S. Min J. Spann N.J. McDonald J.G. Kelly S.L. Biomarkers of NAFLD progression: a lipidomics approach to an epidemic.J. Lipid Res. 2015; 56: 722-736Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). However, none of the lipid biomarkers have been widely recognized as strong or reliable predictors to assess NAFLD in clinical settings. To investigate the possibility and accuracy of detecting NAFLD by assessing plasma lipids, we performed plasma lipid profiling using plasma samples from the CTRL, NAFL, and NASH subjects. The results were analyzed using logistic regression. Interestingly, acylcarnitines were the only plasma lipids that were significantly different between groups; more specifically, it was shown that acylcarnitines were increased in NASH versus CTRL subjects (Table 3, supplemental Tables S5, S6).TABLE 3Plasma lipid species as potential predictors of NAFLDNAFL versus CTRLNASH versus CTRLNASH versus NAFLOR >1SM(41:1)Acylcarnitine 12:0Hex3Cer(d18:1/22:0)SM(42:1)Acylcarnitine 14:1SM(36:2)(a)UbiquinoneAcylcarnitine 14:2SM(38:1)Acylcarnitine 16:1SM(41:1)PE(P-16:0/22:4)SM(42:2)(b)CE(16:1)PC(20:0_20:4)CE(17:1)OR <1PC(35:2)(a)PC(18:0_22:5)(a)PC(17:0_22:6)(a)PI(40:5)(a)PC(40:7)PC(40:8)LPC 17:0[sn2]PE(O-40:6)LPE 16:0[sn2]PI(40:5)(a)Data of CTRL, NAFL, and NASH groups were analyzed pairwise with logistic regression. Only lipid species with significant (P < 0.05) uncorrected Ps are listed here. The (a) and (b) behind the lipid names of some species indicate that the lipid is one of the structural isomers separated by LC, but with an undetermined full chemical structure. Lipid species are categorized by the values of their odds ratios (OR). Open table in a new tab Data of CTRL, NAFL, and NASH groups were analyzed pairwise with logistic regression. Only lipid