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Abstract

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. References

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 in NAFLD, 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 to NASH (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 α-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.)

Nonalcoholic fatty liver disease (NAFLD) is a common cause of chronic liver disease in North America.1 NAFLD is associated with insulin resistance and the metabolic syndrome.2, 3 The clinical-histologic 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 inflammation, 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 triglyceride accumulation as the key defect in NAFLD.6, 7 However, it is not known whether there are substantial changes in other lipid classes, such as cholesterol and specific phospholipids (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 specific 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 syndrome.

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 (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 development and progression of NASH.

Patients and Methods

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. References

Study Cohort

Consecutive subjects with elevated aminotransferases were screened. All subjects had routine clinical, hematologic, biochemical, and serologic evaluations. The subjects 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, α1-antitrypsin deficiency, and drug-induced liver disease) were excluded. NAFLD was suspected by the presence of abnormal liver enzymes without evidence of other liver diseases or radiologic evidence of a fatty liver.

Subjects with suspected NAFLD who were undergoing 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 Commonwealth 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 obtained from E. Merck (Darmstadt, Germany). Ethylene diamine tetraacetic acid (EDTA) and butylated hydroxytoluene were obtained from Sigma Chemical Co. (St. Louis, MO). Fatty acid methyl ester and internal PL standards 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 standards 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 development.15 Sample extracts were dried under nitrogen and spotted onto EDTA-impregnated TLC plates. Two TLC standard lanes consisting of authentic phosphatidylcholine (PC), phosphatidylethanolamine (PE), cardiolipin (CL), and FFA were spotted on the outside lanes of the TLC plate as reference samples. The chloroform/methanol/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/acetic acid (80:20:1 by volume) solvent system was employed.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 chromatographed in the reference lanes. Lipid fractions were transesterified 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 hexane containing 0.05% butylated hydroxytoluene and prepared 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 chromatograph (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 percent (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 distributed data were being compared. A P value of 0.05 or less was considered significant.

Results

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. References

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, glycosylated hemoglobin, and hepatic synthetic functions. Subjects with NASH had higher aspartate aminotransferase and alanine aminotransferase levels that approached but did not reach significance. Although the total cholesterol 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-density lipid cholesterol. Subjects with NAFL had isolated steatosis (mean grade ± SEM: 2.1 ± 0.6) or steatosis with mild inflammation (mean NAFLD activity score ± SEM: 3.2 ± 0.3), whereas those with NASH had a mean steatosis score ± SEM of 1.8 ± 0.4 and an NAFLD activity score ± SEM of 5.1 ± 0.4 (P < 0.03 versus NAFL).

Table 1. Baseline Characteristics of the Study Population
ParameterControl (n = 9)NAFL (n = 9)NASH (n = 9)
  • The data are expressed as the mean ± SEM. ALT indicates alanine aminotransferase; 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).

Gender F/M7/26/36/3
Caucasian/African American7/28/19/0
Mean age (years)46.6 ± 3.848.5 ± 4.847 ± 3.2
BMI (kg/m2)34.5 ± 4.337.5 ± 5.234 ± 1.8
Fasting glucose (mg/dl)87.5 ± 4.9119.2 ± 39.6123.3 ± 29.3
Glycosylated hemoglobin (%)5.2 ± 0.45.8 ± 0.15.8 ± 0.1
AST (normal range: 0-65 U/l)42 ± 16.636.8 ± 577.4 ± 14.2
ALT (normal range: 0-65 U/l)55.7 ± 27.449 ± 10.2119.4 ± 25.8
Alkaline phosphatase (U/l)82.4 ± 9.1113.4 ± 22.8111 ± 13.9
Total bilirubin (mg/dl)0.6 ± 0.10.5 ± 0.10.5 ± 0.1
Albumin (g/dl)4.1 ± 0.14 ± 0.34 ± 0.1
Platelets (1000/mm3)255.5 ± 27.9281.3 ± 40.5233 ± 23.3
Total cholesterol (mg/dl)166.5 ± 10.5230.6 ± 13.8*234 ± 11.2*
HDL cholesterol (mg/dl)57.5 ± 8.540.2 ± 6.350.5 ± 2.1
LDL cholesterol (mg/dl)87 ± 4127 ± 22.699 ± 3.8
Triglycerides (mg/dl)220 ± 22397.8 ± 135.2274 ± 19.4
Steatosis grade02.1 ± 0.61.8 ± 0.4
NAFLD activity score03.2 ± 0.35.1 ± 0.4
Fibrosis stage01.3 ± 0.31.8 ± 0.4

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 for NASH 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 either NAFL or NASH. 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.

Table 2. Hepatic Lipid Content of the Study Groups: Total and Individual Lipid Classes
ParameterControl (n = 9)NAFL (n = 9)NASH (n = 9)
  • 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, phosphatidylcholine; PE, phosphatidylethanolamine; PUFA, polyunsaturated fatty acid; PS, phosphatidylserine; SFA, saturated fatty acid; SM, sphingomyelin; TAG, triacylglycerol.

  • *

    P < 0.05 versus the control (analysis of variance).

Total lipids15,978 ± 1,15749,991 ± 10,718*43,658 ± 6,162*
Total SFA7,189 ± 58622,889 ± 5,328*18,756 ± 3,003*
Total MUFA3,527 ± 25819,616 ± 4,93714,819 ± 2,204*
Total PUFA5,104 ± 3477,328 ± 606*9,822.62 ± 1,094*
Total n-3949 ± 153862 ± 491,336 ± 180
Total n-64,131 ± 2106,424 ± 6058,449 ± 1,012*
n-6/n-3 ratio4.8 ± 0.47.6 ± 0.8*6.9 ± 0.9*
FFA5,533 ± 1,2105,929 ± 1,4876,115 ± 584
DAG1,922 ± 4304,946 ± 1,232*3,304 ± 473*
TAG13,609 ± 2,300128,585 ± 34,201*104,035 ± 20,451*
TAG/DAG ratio726*31*
CE7,589 ± 1,5488,522 ± 1,8467,774 ± 808
FC7,538 ± 71810,382 ± 1,07912,862 ± 1,707*
CL4,447 ± 6205,023 ± 5856,063 ± 902
LYPC1,936 ± 3081,800 ± 5052,239 ± 475*
PC20,321 ± 1,09815,322 ± 1,247*16,874 ± 1,319
PE14,599 ± 81311,456 ± 494*14,828 ± 1,359
PS4,114 ± 5775,295 ± 1,7744,199 ± 706
SM4,194 ± 5873,773 ± 5905,684 ± 685

The total hepatic PL content was not significantly different 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 lysophosphatidylcholine (LYPC; i.e., lysolecithin) in subjects with NASH (P < 0.05). There was also a trend for decreased PE that was significant only for NAFL (P < 0.05); in contrast, phosphatidylserine (PS) levels remained unchanged. Although the hepatic content of sphingomyelin (SM) and CLs increased, they did not meet levels of significance.

Fatty Acid Composition of Hepatic FFAs (FFA; Table 3)

There was a trend for a progressive decrease from controls to NAFL and then NASH for n-6 and n-3 polyunsaturated 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 unaltered in both NAFL and NASH. However, there was a trend toward progressive depletion of γ-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 γ-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 unaltered in NAFL and NASH.

Table 3. Fatty Acid Composition of the FFA Lipid Class
Fatty AcidControl (n = 9)NAFL (n = 9)NASH (n = 9)
  • All values are the mean (mol %) ± SEM. FFA indicates free fatty acid; LCPUFA, long-chain polyunsaturated fatty acid (sum of 20:4n-6, 20:5n-3, and 22:6n-3); MUFA, monounsaturated fatty acid; NAFL, nonalcoholic fatty liver; NASH, nonalcoholic steatohepatitis; PUFA, polyunsaturated fatty acid; SFA, saturated fatty acid.

  • *

    P < 0.05 versus the control.

  • P < 0.05 versus NAFL (analysis of variance).

14:06.6 ± 1.55.4 ± 0.95.9 ± 0.7
15:01.6 ± 0.31.2 ± 0.21.6 ± 0.1
16:030 ± 0.731.5 ± 1.632.3 ± 1.2
18:019.6 ± 0.916.8 ± 2.619 ± 1.8
16:1n-71.6 ± 0.22.4 ± 0.32 ± 0.3
18:1n-71.8 ± 0.31.8 ± 0.31.8 ± 0.3
18:1n-915.8 ± 0.819.2 ± 1.917.5 ± 1.4
24:1n-91.2 ± 0.31 ± 0.31.1 ± 0.1
18:2n-67.5 ± 0.88.2 ± 1.48.3 ± 0.8
18:3n-61.1 ± 0.40.5 ± 0.20.2 ± 0.1*
20:4n-64.9 ± 0.84 ± 1.13.1 ± 0.4*
20:5n-30.4 ± 0.10.3 ± 0.10.2 ± 0.1
22:6n-31.3 ± 0.31.1 ± 0.30.8 ± 0.2
SFA59.3 ± 1.956.4 ± 2.460.4 ± 1.9
MUFA22.2 ± 1.126.2 ± 1.724.2 ± 1.8
PUFA18.4 ± 1.817.4 ± 1.715.3 ± 1.1
LCPUFA6.6 ± 1.15.4 ± 1.44.1 ± 0.6*
n-3 PUFA3.2 ± 0.52.9 ± 0.32.2 ± 0.4
n-6 PUFA15.1 ± 1.514.3 ± 1.512.9 ± 0.8
n-6/n-3 ratio5.3 ± 0.85.1 ± 0.46.7 ± 0.8*
20:4n-6/18:2n-6 ratio0.7 ± 0.10.6 ± 0.20.4 ± 0.1*
20:5n-3/18:3n-3 ratio0.6 ± 0.20.6 ± 0.20.3 ± 0.1

The α-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 docosahexanoic acid (22:6n-3), the downstream products of α-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 (MUFAs; Fig. 1B), specifically oleic acid (18:1-n9), in DAG and TAG for both NAFL and NASH.

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Figure 1. Hepatic lipid composition in subjects with metabolic syndrome and normal liver histology (controls, n = 9), nonalcoholic fatty liver (NAFL, n = 9), and nonalcoholic steatohepatitis (NASH, n = 9). (A) Distribution of lipid classes within hepatic lipids. The x axis represents free fatty acid (FFA), diacylglycerol (DAG), and triacylglycerol (TAG) lipid classes. The y axes represent lipid contents in the 3 study groups (nmol/g of tissue), with FFA and DAG displayed on the left y axis and TAG on the right y axis. NAFL and NASH had significantly increased hepatic DAG (P < 0.05) and TAG (P < 0.002). The FFA content did not change in either group. (B) There was a trend of an increase in the mole percent of saturated fatty acids (SFAs) and monounsaturated fatty acids (MUFAs) within TAG in both NAFL and NASH. The polyunsaturated fatty acid (PUFA) content of TAG, however, was significantly depleted in NAFL and NASH (P < 0.01). (C) Both n-3 (left y axis) and n-6 (right y axis) PUFAs were significantly decreased in NAFL and NASH (P < 0.02). (D) The n-6:n-3 ratio was significantly higher in NAFL and NASH (P < 0.01 for both). (E,F) Relative changes from the control (shown as 100%) are plotted. The arachidonic acid (20:4n-6), eicosapentanoic acid (20:5n-3), and docosahexanoic acid (22:6n-3) were significantly depleted in both NAFL and NASH. (B-F) The data shown are mole percent data (percentage of each fatty acid of the total fatty acids within each lipid class) within the TAG class. All values are expressed as the mean ± SEM. *P < 0.05 versus the controls. The hollow bars represent the controls, the solid bars represent NAFL, and the slashed bars represent NASH.

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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 α-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 trend of an increase in docosahexanoic acid (22:6n-3) in NASH, this did not reach significance (Fig. 2F).

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Figure 2. Hepatic cholesterol composition in controls (n = 9), nonalcoholic fatty liver (NAFL; n = 9), and nonalcoholic steatohepatitis (NASH; n = 9). Parts B-F reflect changes within the cholesterol ester (CE) class and are from mole percent data (percentage of each fatty acid of the total fatty acids within each lipid class). (A) Distribution of CE and free cholesterol (FC) within hepatic lipids. There was a stepwise increase in the hepatic FC content from the controls to NAFL to NASH (P < 0.05, NASH versus the control). The CE content was comparable in the 3 groups. (B) The hepatic CE was significantly depleted in saturated fatty acids (SFAs; P < 0.02) and enriched with polyunsaturated fatty acids (PUFAs; P < 0.01) in both NAFL and NASH. (C) Although both n-3 (left y axis) and n-6 (right y axis) PUFAs were increased in NAFL and NASH, it was significant only for n-6 (P < 0.02). (D) The n-6:n-3 ratio was not significantly different among the 3 groups. (E,F) The relative changes from the control (shown as 100%) are plotted. The linoleic acid (18:2n-6) content was significantly higher (P < 0.01), with a trend of a decrease in arachidonic acid (20:4n-6) and a trend of an increase in docosahexanoic acid (22:6n-3) in both NAFL and NASH. All values are expressed as the mean ± SEM. *P < 0.05 versus the controls. The hollow bars represent the controls, the solid bars represent NAFL, and the slashed bars represent NASH.

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Fatty Acid Composition of Hepatic PLs

The total PC content in the liver was decreased in both NAFL and NASH, but no significant changes were seen in PS or CL (Fig. 3A,B). There was an accompanying increase in LYPC (P < 0.05 for NASH versus the control; Fig. 3A). The mole percentages of SFA, MUFA, and PUFA were unchanged in PC (Fig. 3C). There were also no significant changes in total n-6 or n-3 fatty acids in PC (Fig. 3D). Although the n-6 EFA linoleic acid (18:2n-6) content was unaltered, there was a progressive stepwise decrease in arachidonic acid (20:4n-6) that was significant for NASH (P < 0.05 versus the controls; Fig. 3E). The resultant decrease in the product/precursor ratio for the n-6 pathway (20:4n-6:18:2n-6) reached significance for NASH (P < 0.01 versus the controls).

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Figure 3. Hepatic phospholipid composition in controls (n = 9), nonalcoholic fatty liver (NAFL; n = 9), and nonalcoholic steatohepatitis (NASH; n = 9). Parts C-F show changes within phosphatidylcholine (PC) based on mole percent data (percentage of each fatty acid of the total fatty acids within each lipid class). (A) The PC content in liver was decreased in both NAFL and NASH. This was associated with a significant increase in lysophosphatidylcholine (LYPC; P < 0.05) and a trend of an increase in hepatic sphingomyelin (SM; right y axis) only in NASH. (B) No significant changes were noted in phosphatidylserine (PS) and cardiolipins (CLs), with decreased phosphatidylethanolamine (PE) in NAFL. (C) No alterations were found in the saturated fatty acid (SFA), monounsaturated fatty acid (MUFA), and polyunsaturated fatty acid (PUFA) contents of hepatic PC. (D) The n-3 (left y axis) and n-6 (right y axis) PUFAs were unchanged in NAFL and NASH. (E,F) The relative changes from controls (shown as 100%) are plotted. Arachidonic acid (20:4n-6) in hepatic PC was significantly depleted in NASH (P < 0.01). There was also a trend of a decrease in eicosapentanoic acid (20:5n-3) and docosahexanoic acid (22:6n-3) in both NAFL and NASH. All values are expressed as the mean ± SEM. *P < 0.05 versus the controls. The hollow bars represent the controls, the solid bars represent NAFL, and the slashed bars represent NASH.

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The α-linolenic acid (18:3n-3) content of PC was unchanged in NAFL and NASH (Fig. 3F). There was, however, a trend for decreased downstream n-3 fatty acid products, specifically eicosapentanoic acid (20:5n-3) and docosahexanoic acid (22:6n-3). This trend was in keeping with the profile seen in TAG and DAG and in the FFA pool in the liver. Also, in keeping with the profile seen in these lipid classes, there was a progressive, but nonsignificant, trend of an increase in the total n-6:n-3 ratio in PC from 6.7 in normal controls to 7.6 in NAFL and 8.5 in NASH.

Changes in Essential n-6 and n-3 Fatty Acids

The n-6 fatty acid mole percentage was depleted in FFA, DAG, and TAG but reached significance for only TAG in both NAFL and NASH (Table 4). Arachidonic acid (20:4n-6) was relatively depleted from most lipid classes. Specifically, there were 31% and 36% decreases in the arachidonic acid (20:4n-6) content of PC (P < 0.03) and DAG (P = not significant) in NASH, respectively, the principal sources for its release for inflammatory prostaglandin synthesis.21 This decrease was noted despite no significant changes in its precursor linoleic acid (18:2n-6) in any of these classes.

Table 4. n-3 and n-6 PUFAs, n-6:n-3 Ratio, and Major n-3 and n-6 Constituents
Lipid Classn-3 PUFAn-6 PUFAn-6:n-3 Ratio
ControlNAFLNASHControlNAFLNASHControlNAFLNASH
  • All values are the mean (mol%) ± SEM. CE indicates cholesterol ester; CL, cardiolipin; DAG, diacylglycerol; FFA, free fatty acid; LYPC, lysophosphatidylcholine; NAFL, nonalcoholic fatty liver; NASH, nonalcoholic steatohepatitis; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PUFA, polyunsaturated fatty acid; PS, phosphatidylserine; SM, sphingomyelin; TAG, triacylglycerol.

  • *

    P < 0.05 versus the control.

  • P < 0.05 versus NAFL (analysis of variance).

FFA3.1 ± 0.52.8 ± 0.32.2 ± 0.415.1 ± 1.514.3 ± 1.512.9 ± 0.85.2 ± 0.85.1 ± 0.46.7 ± 0.8*
DAG4.0 ± 1.32.1 ± 0.42.1 ± 0.417.5 ± 1.911.8 ± 1.2*16.1 ± 0.66 ± 0.96.4 ± 1.210.3 ± 2.5
TAG1.9 ± 0.30.6 ± 0.1*1.2 ± 0.1*18.8 ± 0.710.8 ± 1.7*15.5 ± 1.1*10.2 ± 0.916.1 ± 1.4*13.2 ± 0.8*
CE1.5 ± 0.42.2 ± 0.22.7 ± 0.517.4 ± 1.928.2 ± 4.1*26.3 ± 2.8*16 ± 3.113.5 ± 2.412.8 ± 2.6
CL6.4 ± 0.75.6 ± 0.58 ± 2.132.8 ± 3.529.3 ± 3.429.9 ± 1.85.4 ± 0.75.4 ± 0.55.6 ± 1.3
LYPC2.5 ± 0.52.8 ± 0.32.8 ± 0.310.8 ± 1.212.9 ± 0.912.4 ± 0.95.5 ± 14.7 ± 0.44.85 ± 0.6
PC5.6 ± 0.84.5 ± 0.44.8 ± 0.634.5 ± 0.832.5 ± 1.733.9 ± 0.96.7 ± 0.77.6 ± 0.88.5 ± 1.6
PE12.9 ± 1.411 ± 1.112 ± 1.232.8 ± 1.333.3 ± 1.531.3 ± 1.32.8 ± 0.43.2 ± 0.32.9 ± 0.4
PS8.6 ± 1.98.7 ± 1.58.6 ± 1.118.3 ± 2.320.1 ± 2.816.6 ± 1.53.5 ± 1.22.4 ± 0.22 ± 0.1
SM3.2 ± 0.32.3 ± 0.62.8 ± 0.611.1 ± 0.810.6 ± 1.410.2 ± 1.33.5 ± 0.15.8 ± 1.34.8 ± 0.9
 Arachidonic Acid (20:4n-6)Eicosapentanoic Acid (20:5n-3)Docosahexanoic Acid (22:6n-3)
FFA4.8 ± 0.84 ± 1.13.1 ± 0.3*0.41 ± 0.10.3 ± 0.10.2 ± 0.11.3 ± 0.31.1 ± 0.30.8 ± 0.2
DAG4.8 ± 12.2 ± 0.53.1 ± 0.50.22 ± 0.10.2 ± 0.10.2 ± 0.11.1 ± 0.30.6 ± 0.20.7 ± 0.2
TAG1.3 ± 0.20.3 ± 0.1*0.3 ± 0.1*0.14 ± 0.040.02 ± 0.01*0.05 ± 0.02*0.5 ± 0.10.06 ± 0.01*0.2 ± 0.05*
CE4.2 ± 0.43.9 ± 0.73.2 ± 0.40.24 ± 0.10.3 ± 0.10.3 ± 0.10.4 ± 0.10.6 ± 0.21.0 ± 0.4
CL7.6 ± 0.57.4 ± 1.06.3 ± 0.40.17 ± 0.030.3 ± 0.10.3 ± 0.15 ± 0.74.1 ± 0.45.5 ± 1.9
LYPC4.6 ± 0.95.7 ± 0.84.5 ± 0.70.21 ± 0.10.4 ± 0.10.2 ± 0.11.3 ± 0.31.4 ± 0.31.6 ± 0.3
PC12.3 ± 1.29.5 ± 1.18.5 ± 0.6*1.0 ± 0.30.9 ± 0.20.8 ± 0.13.5 ± 0.52.5 ± 0.22.9 ± 0.5
PE22.4 ± 1.121.0 ± 1.418.8 ± 0.80.6 ± 0.20.8 ± 0.20.9 ± 0.310.8 ± 1.48.5 ± 0.99.5 ± 1.3
PS10.3 ± 2.211.2 ± 2.48.8 ± 10.4 ± 0.10.6 ± 0.10.4 ± 0.16.3 ± 1.56.2 ± 1.56.6 ± 1.0
SM3.6 ± 0.43.2 ± 0.72.7 ± 0.60.2 ± 0.10.1 ± 0.10.2 ± 0.11.5 ± 0.51.4 ± 0.41.5 ± 0.5

The mole percent of n-3 fatty acids was decreased in FFA, DAG, TAG, and PC in NASH but was significant only for TAG (Table 4). Eicosapentanoic acid (20:5n-3) and docosahexanoic acid (22:6n-3) were significantly depleted in TAG, and a trend of depletion was seen in FFA, DAG, and, to a lesser degree, PC. These changes occurred despite the unaltered content of the precursor n-3 EFA α-linolenic acid (18:3n-3). There was a trend of an increase in the n-6:n-3 ratio across several lipid classes, but it reached significance only for FFA (28% NASH versus both groups, P < 0.05) and TAG (57% NAFL and 29% NASH, versus the control, P < 0.05). Long-chain PUFAs (sum of 20:4n-6, 20:5n-3, and 22:6n-3) were significantly depleted in NASH (P < 0.05 versus the control) within FFA.

Discussion

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. References

Altered lipid homeostasis in the liver is the pathophysiologic hallmark of NAFLD.22 By using a lipidomic approach, the current study defines the amounts and types of lipids that accumulate within the liver in NAFLD and provides some novel and interesting insights into the pathophysiology of the condition.

It is widely believed that elevated FFA in the liver is a major cause of cell injury and death in NASH.23, 24 This is supported by the presence of elevated circulating FFA levels in NASH and the ability of FFA to induce apoptosis in isolated cell systems.2, 25 The current study challenges this paradigm by the novel observation that, despite an increase in the total lipid content, the FFA content of the liver remains unaltered in NAFLD. It is of course possible that circulating FFA has effects independent of the intracellular FFA levels.

The current study further corroborates the existing evidence that increased TAG accumulation is the hallmark of NAFLD.6, 9 The product/precursor ratio, a functional measure of enzyme activity, revealed a marked step up from DAG to TAG in both NAFL and NASH, raising the possibility that the enzyme diacylglycerol acyl transferase (DGAT) plays an important role in the development of hepatic steatosis. Indeed, the knockdown of DGAT activity ameliorates the fatty liver seen in the ob/ob mouse.26

DAG is derived from lipogenesis and membrane PL.27 The current study does not address the origin or location of DAG in the cell; however, the decrease in PC and the increase in SM suggest that the membrane PC may contribute to the observed increase in DAG along with lipogenesis. Also, the protein kinase C–activating properties of DAG are most efficiently carried out by PUFA-enriched species of DAG.28 The potential impact of decreased PUFA in DAG and the location of DAG within hepatocytes now await investigation.

A striking and novel observation is the progressive increase in FC from NAFL to NASH. Hepatocellular FC reflects its synthesis, the hydrolysis of CEs made in the cell or taken up from circulation, on the one hand, and its utilization for CE and bile acid synthesis, biliary excretion, and efflux into circulation on the other hand.29 NAFLD is associated with several abnormalities that could potentially contribute to the observed increase in FC. These include mitochondrial abnormalities and impaired adenosine triphosphate synthesis2, 30 and the relative depletion of n-3 fatty acids (shown in this study), which increases 3-hydroxy-3-methylglutaryl coenzyme A reductase activity.31 The unchanged level of CEs, despite increasing FC, further suggests a defect in acyl coenzyme A cholesterol acyl transferase activity. It is thus likely that the increase in FC is multifactorial in origin, and the relative contributions of the specific mechanisms remain to be quantified.

FC is well known to be highly cytotoxic.32 Increasing FC levels are often associated with increased PC synthesis.33 Surprisingly, NAFLD was associated with the depletion of PC despite increasing FC content. Although a lipidomic approach allows only limited inferences to be drawn about its mechanism, the increase in SM, DAG, and LYPC in NASH suggests that increased turnover due to phospholipase A2 activity may play a role. We found a 4-fold to 6-fold induction of phospholipase A2 genes in NASH (unpublished data). The resultant FC/PC ratio is significantly elevated in NAFLD (Fig. 4), and this is likely to contribute to the genesis of cell injury and apoptosis in NASH.34 Also, the increase in lysolecithin along with the decrease in PC in NASH suggests that oxidative stress may contribute to these changes; increased lysolecithin is likely to cause membrane toxicity and promote inflammation.35, 36

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Figure 4. The free cholesterol (FC)/phosphatidylcholine (PC) ratio in the 3 study groups is shown. There was a significant alteration (P < 0.008) in the FC/PC ratio with a 2-fold increase in both nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH). All values are expressed as the mean ± SEM. *P < 0.05 versus the controls.

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Another potentially important observation is related to the changes in PUFA in NAFLD. There was a decrease in the downstream n-6 (arachidonic acid: 20:4n-6) and n-3 (eicosapentanoic acid: 20:5n-3, docosahexanoic acid: 22:6n-3) fatty acids in both NAFL and NASH. The unaltered levels of precursor n-6 (linoleic acid: 18:2n-6) and n-3 (α-linolenic acid: 18:3n-3) and the unchanged mead acid (20:3n-9) levels indicate that this is not due to an EFA deficiency. The decreased product/precursor ratios for both n-6 and n-3 pathways in FFA (Table 3) suggest that the activity of Δ6-desaturase and Δ5-desaturase, which catalyze the conversion of these precursor EFAs to downstream n-6 and n-3 fatty acids, may be reduced. NAFLD is associated with several factors, such as high saturated fats and cholesterol, that inhibit desaturase activity.37, 38 The desaturase activity and the role of these factors are subjects for future studies.

Arachidonic acid (20:4n-6) is released from membrane PL by phospholipase A2 and from phosphatidylinositol bisphosphate through DAG by phospholipase C and is rapidly converted into proinflammatory prostaglandins, thromboxanes, and leukotrienes by cycloxygenase.39 It is possible that increased utilization of arachidonic acid (20:4n-6) may also contribute to the observed decrease in its levels in NAFLD. If true, the modulation of phospholipases and cyclooxygenase may provide another mechanism to control the inflammatory pathways in NASH.

There was a trend of decreased n-3 fatty acids, eicosapentanoic (20:5n-3) and docosahexanoic (22:6n-3) acids, across multiple lipid classes, reaching significance for TAG. Although this did not reach statistical significance in several lipid classes, probably because of the low sample size, the direction of the changes was consistent and qualitatively similar across these classes, suggesting that they are real. These 2 n-3 fatty acids have profound anti-inflammatory, antiproliferative, immunomodulatory, and metabolic effects.40–43 A priori, the observed trend for the depletion of these key n-3 fatty acids could promote steatosis, inflammation, dyslipidemia, cell injury, and risk of carcinogenesis in NASH. This risk is further likely to be accentuated if the observed decrease in arachidonic acid (20:4n-6) is also partly due to increased utilization for inflammatory prostaglandin and leukotriene synthesis. These possibilities must be confirmed by direct methods. If true, they will provide a rationale for the use of eicosapentanoic (20:5n-3) and docosahexanoic (22:6n-3) acid supplementation for the treatment of NASH.

It must be pointed out that the biological implications of the changes in the lipid composition are likely to be complex and difficult to predict simply on the basis of lipidomic data. The biological effects of lipids depend on their location (membrane versus cytosolic versus nuclear) and amount,44, 45 They may function as key signaling molecules, such as DAG, as transcriptional regulators, such as PUFAs on steroyl regulatory element binding protein-1c, or as regulators of enzyme activity, such as PUFAs on lipid oxidation.46–48 They may further affect membrane fluidity and can be subjected to lipid peroxidation. Moreover, different classes of lipids (for example, n-3 versus n-6) may have opposing net effects on specific processes, such as inflammation, and their relative proportions determine the net biological effect.40 Therefore, the current study mainly allows the generation of hypotheses that now need to be confirmed in more focused studies.

The potential limitations of this study are the obese nature of the control group and the small size of the study population. It is possible that the lipid composition of the liver in the control group may be different from that of lean normal individuals and that the changes seen in NAFL and NASH may be even more accentuated with respect to lean normals. Despite the small study population size, several findings reached statistical significance. Before this study, there were no data to generate sample size estimates. However, post hoc analyses suggest that the power of this study to demonstrate the observed changes in TAG, DAG, and FC was between 80%-90% for NASH. The power of the other findings, such as decreased PC and the depletion of arachidonic acid, was, however, lower.

In summary, the current study provides several observations about the alterations in lipid homeostasis in NAFLD that may be of significance in the pathophysiology of this condition. It disproves the paradigm that hepatic FFAs are increased in NAFLD. It also demonstrates the presence of increased DAG, the altered fatty acid composition of DAG, increased FC, decreased PC, decreased levels of arachidonic acid, and also key n-3 fatty acids and an increase in the n-6:n-3 fatty acid ratio. All of these observations could play a role in the pathogenesis of NASH. It also provides a rationale for the use of n-3 fatty acid supplementation as a treatment of NASH. These exciting possibilities now await further exploration.

References

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. References