脂肪肝1

A Lipidomic Analysis of Nonalcoholic Fatty Liver Disease Puneet Puri,1 Rebecca A. Baillie,3 Michelle M. Wiest,3 Faridoddin Mirshahi,1 Jayanta Choudhury,1 Onpan Cheung,1 Carol Sargeant,1 Melissa J. Contos,2 and Arun J. Sanyal1 The spectrum of nonalcoholic fatty liver disease (NAFLD) includes a nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH). The specific types and amounts of lipids that accumulate in NAFLD are not fully defined. The free fatty acid (FFA), diacylglycerol (DAG), triacylglycerol (TAG), free cholesterol (FC), cholesterol ester, and phospholipid contents in normal livers were quantified and compared to those of NAFL and NASH, and the distribution of fatty acids within these classes was compared across these groups. Hepatic lipids were quantified by capillary gas chromatography. The mean (nmol/g of tissue) DAG (normal/NAFL/NASH: 1922 versus 4947 versus 3304) and TAG (13,609 versus 128,585 versus 104,036) increased significantly inNAFLD, but FFA remained unaltered (5533 versus 5929 versus 6115). There was a stepwise increase in the mean TAG/DAG ratio from normal livers to NAFL toNASH (7 versus 26 versus 31,P< 0.001). There was also a similar stepwise increment in hepatic FC (7539 versus 10,383 versus 12,863, P< 0.05 for NASH). The total phosphatidylcholine (PC) decreased in both NAFL and NASH. The FC/PC ratio increased progressively (0.34 versus 0.69 versus 0.71, P < 0.008 for both). Although the levels for linoleic acid (18:2n-6) and �-linolenic acid (18:3n-3) remained unaltered, there was a decrease in arachidonic acid (20:4n-6) in FFA, TAG, and PC (P < 0.05 for all) in NASH. Eicosapentanoic acid (20:5n-3) and docosahexanoic acid (22:6n-3) were decreased in TAG in NASH. The n-6:n-3 FFA ratio increased in NASH (P < 0.05). Conclusions: NAFLD is associated with numerous changes in the lipid composition of the liver. The potential implications are discussed. (HEPATOLOGY 2007;46:1081-1090.) Nonalcoholic fatty liver disease (NAFLD) is acommon cause of chronic liver disease in NorthAmerica.1 NAFLD is associated with insulin re- sistance and the metabolic syndrome.2,3 The clinical-his- tologic spectrum of NAFLD extends from a nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH).4 Although NASH is distinguished from NAFL by the presence of cytologic ballooning and inflamma- tion, both conditions are characterized by a fatty liver.4,5 Thus, hepatic fat accumulation is the hallmark of NAFLD. The compositions of the lipids that accumulate in the livers of subjects with NAFLD are not well characterized. Most of the published literature has focused on triglycer- ide accumulation as the key defect in NAFLD.6,7 How- ever, it is not knownwhether there are substantial changes in other lipid classes, such as cholesterol and specific phos- pholipids (PLs). Although an increase in the n-6:n-3 fatty acid ratio in total lipids in NAFLD has been described recently,8,9 the distribution of these fatty acids within spe- cific lipid classes has not been extensively characterized. Given the important biological activities of many lipids, such information could provide potential insights into the pathophysiology of NAFLD and the metabolic syn- drome. A lipidomic approach was taken to quantify the major lipid classes and the distribution of fatty acids within these classes in the liver. The specific aims of the study were to (1) quantify the absolute and relative amounts of free fatty acids (FFAs), diacylglycerol (DAG), triacylglycerol Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; CE, cholesterol ester; CL, cardiolipin; DAG, diacylglycerol; DGAT, diacylglycerol acyl transferase; EDTA, ethylene diamine tetraacetic acid; EFA, essential fatty acid; FC, free cholesterol; FFA, free fatty acid; HDL, high-density lipid; LCPUFA, long-chain polyunsaturated fatty acid; LDL, low-density lipid; LYPC, lyso- phosphatidylcholine (lysolecithin); MUFA, monounsaturated fatty acid; NAFL, nonal- coholic fatty liver; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PL, phospho- lipid; PS, phosphatidylserine; PUFA, polyunsaturated fatty acid; SFA, saturated fatty acid; SM, sphingomyelin; TAG, triacylglycerol; TLC, thin-layer chromatography. From the 1Division of Gastroenterology, Hepatology, and Nutrition, Department of Internal Medicine, 2Department of Pathology, Virginia Commonwealth University Medical Center, Richmond, VA; and 3Lipomics Technologies, West Sacramento, CA. Received January, 2007; accepted April 6, 2007. Supported by the following grants from the National Institutes of Health to A.J.S.: K24 DK 02755-06, T32 DK 07150-31, and RO1 56331-05. This is an original work and is not under consideration for publication elsewhere. This work has been presented, in part, at the annual meeting of the European Association for Study of the Liver in Vienna, Austria, 2006. Address reprint requests to: Arun J. Sanyal, M.B.B.S., M.D., Professor of Medicine, Pharmacology, and Pathology, Virginia Commonwealth University Medical Center, MCV Box 980341, Richmond, VA 23298-0341. E-mail: ajsanyal@hsc.vcu.edu; fax: (804) 828 2037. Copyright © 2007 by the American Association for the Study of Liver Diseases. Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/hep.21763 Potential conflict of interest: Dr. Wiest owns stock in Lipomics Technology. 1081 (TAG), free cholesterol (FC), cholesterol esters (CEs), and PLs in normal livers and compare them to those of NAFL and NASH and (2) compare the distribution of fatty acids within each of these classes in these groups of subjects. The goal was to define the changes in the hepatic lipid composition in NAFLD and develop hypotheses about the potential impact of such changes on the devel- opment and progression of NASH. Patients and Methods Study Cohort Consecutive subjects with elevated aminotransferases were screened. All subjects had routine clinical, hemato- logic, biochemical, and serologic evaluations. The sub- jects with a history of excessive alcohol use (�20 g/day for males and�10 g/day for females) or other causes of liver disease (viral hepatitis B, viral hepatitis C, primary biliary cirrhosis, sclerosing cholangitis, autoimmune hepatitis, hemochromatosis, Wilson’s disease, �1-antitrypsin defi- ciency, and drug-induced liver disease) were excluded. NAFLD was suspected by the presence of abnormal liver enzymes without evidence of other liver diseases or radio- logic evidence of a fatty liver. Subjects with suspected NAFLD who were undergo- ing a liver biopsy to further evaluate their liver disease were considered for this study. Also, subjects without symptoms or signs of liver disease and normal alanine aminotransferase and sonogram undergoing abdominal surgery for unrelated reasons served as a normal control group. After an overnight fast, a core biopsy was obtained in all cases with either a 15-gauge Microvasive gun or a 16-gauge Klatskin needle. The presence of NAFLD was diagnosed according to standard clinical criteria.10,11 On the basis of the liver histology, 3 groups were studied: (1) normal histology controls, (2) NAFL, and (3) NASH. Subjects with bridging fibrosis or cirrhosis were excluded. The study was performed according to the Virginia Com- monwealth University regulations for the protection of human research subjects after the protocol was reviewed and approved by the institutional review board. Reagents All chromatography solvents were obtained from Fisher Scientific (Pittsburgh, PA). Silica Gel 60 thin-layer chromatography (TLC) plates (10 �20 cm) were ob- tained from E. Merck (Darmstadt, Germany). Ethylene diamine tetraacetic acid (EDTA) and butylated hydroxy- toluene were obtained from Sigma Chemical Co. (St. Louis, MO). Fatty acid methyl ester and internal PL stan- dards were obtained from Nu-Chek-Prep (Elysian, MN) and Avanti Polar Lipids (Alabaster, AL), respectively. Lipid Profiling The hepatic lipid profiles were analyzed as previously described12 and noted in the following. Extraction and TLC. Lipids were extracted from 5-10 mg of liver tissue in the presence of internal stan- dards by Folch’s method with chloroform/methanol (2:1 vol/vol).13 Individual lipid classes from each extract were separated by preparative chromatography, as described previously.14 Briefly, TLC plates were impregnated with 1 mM EDTA (pH 5.5) and washed by ascending develop- ment.15 Sample extracts were dried under nitrogen and spotted onto EDTA-impregnated TLC plates. Two TLC standard lanes consisting of authentic phosphatidylcho- line (PC), phosphatidylethanolamine (PE), cardiolipin (CL), and FFA were spotted on the outside lanes of the TLC plate as reference samples. The chloroform/metha- nol/acetic acid/water (100:67:7:4 by volume) mobile phase employed for the separation of PL classes was a modification of the solvent system described previously.16 For the separation of lipid classes [total PL, FFA, TAG, DAG, FC, and CE], a petroleum ether/diethyl ether/ace- tic acid (80:20:1 by volume) solvent system was em- ployed.17 These methods were initially validated in tissue samples of various sizes against internal standards, and accurate data could be obtained from as little as 3-4 mg of liver. Isolation and Methylation of the Lipid Classes. Lipid classes and individual PL classes were identified by comparison with the authentic standards chromato- graphed in the reference lanes. Lipid fractions were trans- esterified in 3 N methanolic HCl under an N2 atmosphere at 100°C for 45 minutes in a sealed vial. The resulting fatty acid methyl esters were extracted with hex- ane containing 0.05% butylated hydroxytoluene and pre- pared for gas chromatography through the sealing of the hexane extracts under nitrogen. Fatty Acid Analysis. Fatty acid methyl esters were separated and quantified by capillary gas chromatography with a Hewlett-Packard (Wilmington, DE) gas chro- matograph (model 6890) equipped with a 60-m DB-23 capillary column (J&W Scientific, Folsom, CA), a flame- ionization detector, and Hewlett-Packard ChemStation software. Data Analysis Summary data for the fatty acid classes and mole per- cent (percentage of each fatty acid of the total fatty acids within each lipid class) were calculated. The results were expressed as means� SEM. A Kruskal-Wallis analysis of variance with a post hoc multiple comparison procedure was used for across-group comparisons. A Student t test was used only when 2 specific groups of normally distrib- 1082 PURI ET AL. HEPATOLOGY, October 2007 uted data were being compared. A P value of 0.05 or less was considered significant. Results Clinical, Biochemical, and Histologic Profile A total of 9 subjects in each group were studied (Table 1). The 3 groups were similar with respect to gender, ethnicity, age, body mass index, fasting blood sugar, gly- cosylated hemoglobin, and hepatic synthetic functions. Subjects with NASH had higher aspartate aminotransfer- ase and alanine aminotransferase levels that approached but did not reach significance. Although the total choles- terol was higher in subjects with NAFL and NASH than controls (P � 0.05 for both), there were no significant differences in high-density lipid cholesterol or low-den- sity lipid cholesterol. Subjects with NAFL had isolated steatosis (mean grade� SEM: 2.1� 0.6) or steatosis with mild inflammation (meanNAFLD activity score� SEM: 3.2� 0.3), whereas those with NASH had a mean steato- sis score � SEM of 1.8 � 0.4 and an NAFLD activity score� SEM of 5.1� 0.4 (P � 0.03 versus NAFL). Total Hepatic Lipid Content (Table 2) The total hepatic lipid content was markedly increased in NAFL and NASH (P� 0.001); this was driven mainly by increased TAG content (P� 0.001). Similarly, DAG was also increased significantly (P� 0.03 for both NAFL and NASH). Compared to that of normal controls, the TAG/DAG (product/precursor) ratio was significantly increased in both NAFL and NASH (7 versus 26 versus 31, P� 0.001 for both). The n-6 fatty acid content in the total lipids increased from the controls to NAFL and NASH (mean � SEM: 4131 � 210 versus 6424 � 605 versus 8449� 1012 nmol/gm, P� 0.03 forNASH versus the controls) and was associated with a significantly higher total n-6:n-3 ratio in NAFL and NASH (P� 0.05 for both versus the controls). However, the FFA content did not increase in eitherNAFL orNASH. Also, there was a stepwise increment in FC from normal to NAFL to NASH (P � 0.05 for the control versus NASH). This was, however, not accompanied by an increase in CEs in either NAFL or NASH. The total hepatic PL content was not significantly dif- ferent across the 3 groups (mean � SEM: controls � 49,889 � 3220 nmol/g of tissue, NAFL � 42,671 � 3714 nmol/g of tissue, and NASH � 49,614 � 2599 nmol/g of tissue, P� not significant). However, despite a significant increase in the total lipids and FC, the total PC content was decreased (P � 0.03 for NAFL versus the control). This was accompanied by an increase in lyso- phosphatidylcholine (LYPC; i.e., lysolecithin) in subjects Table 1. Baseline Characteristics of the Study Population Parameter Control (n � 9) NAFL (n � 9) NASH (n � 9) Gender F/M 7/2 6/3 6/3 Caucasian/African American 7/2 8/1 9/0 Mean age (years) 46.6� 3.8 48.5� 4.8 47� 3.2 BMI (kg/m2) 34.5� 4.3 37.5� 5.2 34� 1.8 Fasting glucose (mg/dl) 87.5� 4.9 119.2� 39.6 123.3� 29.3 Glycosylated hemoglobin (%) 5.2� 0.4 5.8� 0.1 5.8� 0.1 AST (normal range: 0-65 U/l) 42� 16.6 36.8� 5 77.4� 14.2 ALT (normal range: 0-65 U/l) 55.7� 27.4 49� 10.2 119.4� 25.8 Alkaline phosphatase (U/l) 82.4� 9.1 113.4� 22.8 111� 13.9 Total bilirubin (mg/dl) 0.6� 0.1 0.5� 0.1 0.5� 0.1 Albumin (g/dl) 4.1� 0.1 4� 0.3 4� 0.1 Platelets (1000/mm3) 255.5� 27.9 281.3� 40.5 233� 23.3 Total cholesterol (mg/dl) 166.5� 10.5 230.6� 13.8* 234� 11.2* HDL cholesterol (mg/dl) 57.5� 8.5 40.2� 6.3 50.5� 2.1 LDL cholesterol (mg/dl) 87� 4 127� 22.6 99� 3.8 Triglycerides (mg/dl) 220� 22 397.8� 135.2 274� 19.4 Steatosis grade 0 2.1� 0.6 1.8� 0.4 NAFLD activity score 0 3.2� 0.3 5.1� 0.4† Fibrosis stage 0 1.3� 0.3 1.8� 0.4 The data are expressed as the mean � SEM. ALT indicates alanine amino- transferase; AST, aspartate aminotransferase; BMI, body mass index; HDL, high- density lipid; LDL, low-density lipid; NAFL, nonalcoholic fatty liver; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis. *P � 0.05 versus the control (analysis of variance). †P � 0.05 versus NAFL (t test). Table 2. Hepatic Lipid Content of the Study Groups: Total and Individual Lipid Classes Parameter Control (n � 9) NAFL (n � 9) NASH (n � 9) Total lipids 15,978� 1,157 49,991� 10,718* 43,658� 6,162* Total SFA 7,189� 586 22,889� 5,328* 18,756� 3,003* Total MUFA 3,527� 258 19,616� 4,937 14,819� 2,204* Total PUFA 5,104� 347 7,328� 606* 9,822.62� 1,094* Total n-3 949� 153 862� 49 1,336� 180 Total n-6 4,131� 210 6,424� 605 8,449� 1,012* n-6/n-3 ratio 4.8� 0.4 7.6� 0.8* 6.9� 0.9* FFA 5,533� 1,210 5,929� 1,487 6,115� 584 DAG 1,922� 430 4,946� 1,232* 3,304� 473* TAG 13,609� 2,300 128,585� 34,201* 104,035� 20,451* TAG/DAG ratio 7 26* 31* CE 7,589� 1,548 8,522� 1,846 7,774� 808 FC 7,538� 718 10,382� 1,079 12,862� 1,707* CL 4,447� 620 5,023� 585 6,063� 902 LYPC 1,936� 308 1,800� 505 2,239� 475* PC 20,321� 1,098 15,322� 1,247* 16,874� 1,319 PE 14,599� 813 11,456� 494* 14,828� 1,359 PS 4,114� 577 5,295� 1,774 4,199� 706 SM 4,194� 587 3,773� 590 5,684� 685 All values are expressed as the mean (nmol/g of tissue) � SEM. CE indicates cholesterol ester; CL, cardiolipin; DAG, diacylglycerol; FC, free cholesterol; FFA, free fatty acid; LYPC, lysophosphatidylcholine; MUFA, monounsaturated fatty acid; NAFL, nonalcoholic fatty liver; NASH, nonalcoholic steatohepatitis; PC, phosphati- dylcholine; PE, phosphatidylethanolamine; PUFA, polyunsaturated fatty acid; PS, phosphatidylserine; SFA, saturated fatty acid; SM, sphingomyelin; TAG, triacyl- glycerol. *P � 0.05 versus the control (analysis of variance). HEPATOLOGY, Vol. 46, No. 4, 2007 PURI ET AL. 1083 with NASH (P � 0.05). There was also a trend for de- creased PE that was significant only forNAFL (P� 0.05); in contrast, phosphatidylserine (PS) levels remained un- changed. Although the hepatic content of sphingomyelin (SM) and CLs increased, they did not meet levels of sig- nificance. Fatty Acid Composition of Hepatic FFAs (FFA; Table 3) There was a trend for a progressive decrease from con- trols to NAFL and then NASH for n-6 and n-3 polyun- saturated fatty acids (PUFAs) within hepatic FFA. The content of linoleic acid (18:2n-6), the starting point for processing n-6 essential fatty acids (EFAs),18 was unal- tered in both NAFL and NASH. However, there was a trend toward progressive depletion of �-linolenic acid (18:3n-6), which is immediately downstream of linoleic acid,18,19 from the control to NAFL to NASH (P� 0.01 versus NASH). This was also seen with arachidonic acid (20:4-n6), which is further downstream of �-linolenic acid, with a significant decrease in NASH (P � 0.05 versus the controls). The product/precursor ratio for the n-6 pathway (20:4n-6:18:2n-6) trended downward, reaching significance for NASH (P � 0.03 versus the controls). The levels of mead acid (20:3n-9), which are typically increased in EFA deficiency,20 remained unal- tered in NAFL and NASH. The �-linolenic acid (18:3n-3), the starting point for processing n-3 EFA,18 was unaltered. There was a trend for a progressive decrease from the controls to NAFL to NASH for eicosapentanoic acid (20:5n-3) and docosa- hexanoic acid (22:6n-3), the downstream products of �-linolenic acid (18:3n-3), which approached but did not reach significance in NASH. The product/precursor ratio for the n-3 pathway (20:5n-3:18:3n-3) also trended downward, approaching significance for NASH. Fatty Acid Composition of Hepatic DAG and TAG Both DAG and TAG were significantly increased in subjects with NAFL and NASH (Fig. 1A). There was a trend for increased saturated fatty acids (SFAs) in DAG and TAG; this was driven by increased palmitate (16:0) but offset by decreased stearic acid (18:0). There was also a trend for increased monounsaturated fatty acids (MU- FAs; Fig. 1B), specifically oleic acid (18:1-n9), in DAG and TAG for both NAFL and NASH. In contrast, there was a significant decrease in PUFA associated with DAG and TAG in NAFL and NASH (Fig. 1B). The molar percentages of n-3 and n-6 fatty acids in TAG were decreased in both NAFL and NASH (Fig. 1C); however, the decrease in n-6 was less than that in the n-3 fatty acids, resulting in a net significant increase in the n-6:n-3 fatty acid proportions in TAG (Fig. 1D). There was a significant depletion of arachidonic acid (20: 4n-6), a key n-6 fatty acid (Fig. 1E). Also, eicosapentanoic acid (20:5n-3) and docosahexanoic acid (22:6n-3), the 2 downstream products of �-linolenic acid (18:3n-3) in the n-3 pathway, were significantly depleted (Fig. 1F). These changes were qualitatively similar to the changes seen in the FFA and DAG pools. Hepatic CE Fatty Acid Composition The hepatic FC content increased progressively from controls with normal histology to NAFL to NASH (P� 0.05 for NASH versus the control; Fig. 2A). The total CE content was not, however, significantly changed in either NAFL or NASH. There was a de-enrichment of SFA and a mild, insignificant increase in MUFA, specifically oleic acid. There was a significant enrichment of the CEs with PUFA (Fig. 2B,C). Both n-6 and n-3 PUFAs increased in NAFL and NASH, but the findings were significant only for n-6 fatty acids (Fig. 2C). The overall n-6:n-3 ratio did not change significantly (Fig. 2D). Although linoleic acid (18:2n-6) was particularly enriched within the CEs in both NAFL and NASH, the arachidonic acid levels were not altered significantly (Fig. 2E). Although there was a Table 3. Fatty Acid Composition of the FFA