Treatment of Nonalcoholic Fatty Liver Disease with Long-Chain n-3 Polyunsaturated Fatty Acids in Humans

Abstract

Background:

Nonalcoholic fatty liver disease (NAFLD) is a major risk factor for type 2 diabetes, cardiovascular disease, and liver failure. Treatment with n-3 long-chain polyunsaturated fatty acids (n-3 LCPUFAs) produced variable success in improving NAFLD. The purpose of this review is to determine if n-3 LCPUFA will decrease markers of NAFLD, compare the efficacies of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and identify factors that contribute to discrepancies in results.

Methods:

This study reviewed published clinical studies with n-3 LCPUFA and NAFLD/nonalcoholic steatohepatitis (NASH) by using PubMed and Web of Science.

Results:

Seventeen human studies ranging in DHA 250 mg/day to a mixture of EPA+DHA 50 mL/day for 8 weeks to 2 years were identified. Results obtained varied because of different dosages of n-3 LCPUFA, EPA and DHA ratios, duration, subject characteristics, diet, exercise, compliance, methods, and other factors. Despite inconsistencies in the results reported, 13 of 17 published studies reported that n-3 LCPUFA supplementation decreased liver fat, liver enzymes, or markers of inflammation; four reported decrease in ballooning and two in fibrosis. Results also indicated that DHA was more effective than EPA in the treatment of NAFLD. Caloric restriction and supplementation with n-3 LCPUFA were additive in decreasing hepatic steatosis.

Conclusions:

n-3 PUFA decreased several markers of NAFLD; however, there was a lower observed efficacy in NASH treatment. Further long-term placebo-controlled studies with adequate power and supplementation duration and standardized and sensitive detection methods are needed to determine the efficacy of EPA and DHA individually and in a mixture to treat NAFLD and NASH.

Introduction

Nonalcoholic fatty liver disease (NAFLD) is a condition in which liver triglyceride (TG) concentration exceeds 5% of wet liver weight in patients without excessive alcohol intake (women <10 grams/day, men <20 grams/day).¹ Liver can also be classified as fatty based on histological staining of the biopsy if at least 5% hepatocytes stain positive for fat after exclusion of other causes of liver disease.² NAFLD resembles alcohol-induced liver injury, but it occurs in patients with little or no history of alcohol consumption.

The spectrum of NAFLD ranges from nonalcoholic fatty liver (NAFL) or simple steatosis (SS) to steatosis with inflammation (nonalcoholic steatohepatitis [NASH]), which may or may not be associated with fibrosis. NAFL used to be considered as benign; however, recent studies have shown that 20% of the patients with NAFLD develop NASH and 20% of those with NASH progress to cirrhosis, which can lead to portal hypertension, hepatocellular carcinoma, and increased mortality.³ Rate of progression to fibrosis from NAFL and NASH was similar in one study⁴ and slower from NAFL than NASH in others.^5,6 Regardless of these differences, NAFL can no longer be considered as benign.

Liver fat accumulation results from an imbalance among fatty acid (FA) influx (adipose tissue lipolysis and dietary intake), hepatic de novo lipogenesis [DNL], and lipid disposition (FA oxidation and very low-density lipoprotein [VLDL] secretion) from the liver.^7
–9 As determined by tracer studies, 59.0%, 26.7%, and 14.9% liver FAs in subjects with NAFLD came from adipose tissue lipolysis, DNL, and diet, respectively.¹⁰ In these patients, DNL was elevated even during the fasting state and demonstrated no diurnal variation. Thus, increased hepatic DNL and adipose tissue lipolysis are the two major contributors to increased hepatic steatosis, and interventions with these pathways should be effective in reducing the accumulation of fat in liver. Enhanced hepatic FA oxidation and VLDL-TG clearance can also reduce liver fat.

The pathogenesis of NAFLD is considered to involve two hits; first hit being increased TG accumulation due to increased insulin resistance (IR), which is associated with increased hepatic DNL and decreased β oxidation, decreased glucose uptake by muscle, and increased lipolysis in adipose tissue.¹¹ Elevated lipolysis in adipose tissue results in increased concentration of free fatty acids (FFAs) in blood, which are taken up by the liver due to increased expression of CD36 and are incorporated into liver TGs. As stated above, adipose tissue FFAs are the major source of liver TGs.

The second hit involves increased oxidation stress and inflammation in the liver and adipose tissue, which leads to the development of NASH with necroinflammation, ballooning, and fibrosis. Several other lipid metabolites, including ceramides, diglycerides (DGs), cholesterol, and proinflammatory proatherogenic cytokines, IL-6 and TNF α, are also involved in the development of NASH.^12,13 Although NAFLD and IR often occur concurrently, they can exist individually. It remains unknown which comes first.¹⁴

Two classification systems are used to group NAFLD into different classes. The first system developed by the American Association for the Study of Liver Disease proposed four classes correlating with histologic features and long-term prognosis: class 1—SS; class 2—steatosis with lobular inflammation; class 3—presence of ballooned hepatocytes; and class 4—presence of either Mallory's hyaline or fibrosis.² Classes 3 and 4 are described as NASH.¹⁵ NAFLD activity score (NAS) was proposed by the NASH Clinical Research Network.¹⁶ NAS is the weighted sum of steatosis (0 to 3); lobular inflammation (0 to 3), and hepatocyte ballooning (0 to 2). The NAS ranges from 0 to 8. An NAS <3 corresponds to not NASH; 3–4 is borderline, and 5 or greater means definitive NASH. Elevated serum transaminases with alanine aminotransferase (ALT) higher than aspartate aminotransferase (AST) are often the first indications for the existence of NAFLD.¹⁷

Prevalence and Risk Factors

NAFLD is the most common liver disease worldwide and is among the top 10 leading causes of death in the United States.^18,19 Its prevalence using ultrasound (US) is estimated to be 30% among all adults, 75%–92% in the morbidly obese, and 13%–14% in pediatric patients.²⁰ NAFLD is strongly associated with components of the metabolic syndrome (MS), including obesity, hypertension, dyslipidemia, and IR, and is believed to represent the hepatic manifestations of MS.²¹ Its prevalence is rising in parallel to the global increase in obesity and type 2 diabetes.

Although, NAFLD is more common in subjects with central obesity, it can also be found in nonobese subjects. In a study conducted in West Bengal, India, 75% patients with NAFLD had body mass index (BMI) <25 kg/m² and 54% were neither overweight nor had abdominal obesity.²² This inconsistency may be due to the genetic differences between Asian Indians and Europeans and the fact that Indians had more body fat at the same BMI. Genetic differences have also been attributed to the greater incidence of NAFLD in Hispanics than Caucasians and African Americans.²³ Thus, the incidence of NAFLD varied among different groups of population.

Other risk factors for NAFLD include diabetes, cardiovascular disease, aging,^24,25 plasma thyroid hormone (free T4),²⁶ Western diets high in cholesterol, carbohydrates, saturated, and trans and n-6 FAs and low in fiber and n-3 PUFA,^27
–29 calories from soft drinks, and meat.³⁰ Overall balance between the factors that promote and prevent NAFLD determines the occurrence and severity of NAFLD.

Other than weight loss and bariatric surgery, currently there are no validated treatments available for NAFLD. Bariatric surgery decreased steatosis in 91.6%, steatohepatitis in 81.3%, and fibrosis in 65.5%.³¹ Treatment with insulin sensitizers, such as the biguanides and thiazolidinediones, improved steatosis and inflammation, but not fibrosis.³² Furthermore, those drugs have adverse effects such as weight gain, osteoporosis, and increased risk of cardiovascular disease.^21,33 Vitamin E and obeticholic acid have been reported to decrease several markers of NASH, including the fibrosis score; however, both these treatments are effective only in a part of the subjects and also have serious adverse effects. Vitamin E increased the incidences of hemorrhagic stroke, prostate cancer, and all-cause mortality, while obeticholic acid increased IR, total and LDL cholesterol, and pruritis and decreased HDL cholesterol.^1,34

n-3 long-chain polyunsaturated fatty acids (LCPUFAs) from algal and marine sources are safe and they reduced circulating TGs and liver fat and other markers of NAFLD in a number of animal models. These findings have been reviewed in an excellent recent publication³⁵ and will not be discussed here. I will refer to results from animal studies only to compare the effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The effects of n-3 LCPUFA on human liver fat and other markers of NAFLD varied.^36,37 Several new human intervention studies investigating the effects of n-3 LCPUFA on human markers of NAFLD have been published in the last couple of years.

The purpose of this review was to determine (1) if n-3 LCPUFA will decrease liver fat and other markers of NAFLD in humans; (2) compare the efficacies of EPA and DHA in the treatment of NAFLD; and (3) identify the factors that may contribute to the inconsistencies between the results obtained. Published literature was searched between 1995 and 2016 using PubMed and Web of Science databases with the terms, fatty liver, NAFLD, or NASH, in conjunction with omega-3 PUFA, n-3 PUFA, fish oils (FOs), EPA, or DHA in humans. In this study, I will review the results from published human studies dealing with effects of n-3 LCPUFA supplementation on markers of NAFLD and NASH.

Sources of n-3 LCPUFA

Alpha linolenic acid (ALA; 18:3, n-3) and linoleic acid (LA; 18:2, n-6) are the only two FAs that are considered to be essential because human beings cannot incorporate the double bonds at n-3 and n-6 positions. While humans can elongate these FAs to the corresponding LCPUFA, the conversion of ALA to LCPUFA, particularly DHA, is insignificant. Part of this is due to the high intake of LA compared with that of ALA and the same enzymes are involved in their elongation and desaturation; ALA is also a preferred FA for mitochondrial beta oxidation. Hence, DHA must be provided in the diet or as a supplement.

n-3 LCPUFAs (EPA, DPA, DHA) are found in several foods, but their richest sources are fatty fish, including salmon, tuna, herring, mackerel, and sardines. FO, krill oil, and algal oils are commonly used supplements of n-3 LC-PUFA. The consumption of n-3 LCPUFAs is quite variable among different populations. Because n-3 LCPUFAs decrease the risk for cardiovascular disease, the American Heart Association recommended intake of EPA+DHA 500 mg/day for healthy subjects and 1 gram/day for hypertriglyceridemic subjects.³⁸ There are also recommendations for DHA intake of 100–200 mg/day for pregnant and lactating women.

Lipid and FA Composition of Livers with Steatosis and Steatohepatitis

Reported changes in liver lipids, FAs, and enzymes involved in FA synthesis and metabolism in NAFLD are listed in Table 1. Hepatic steatosis is associated with increased concentrations of TGs and DGs in NAFL, both of which usually decrease with the progression of NAFL to NASH.³⁹ Liver phospholipids from patients with NAFLD contained lower amounts of arachidonic acid (AA; 20:4, n-6), EPA (20:5, n-3), and DHA (22:6, n-3) and higher amounts of n-6 PUFA than n-3 PUFA with significant increase in the ratio between n-6 and n-3 PUFAs when compared with the normal livers (NLs).^40
–42 There was a stepwise increase in the ratio between TGs and DGs, and also free cholesterol with the progression from NL to NAFL to NASH.⁴³ Liver concentrations of FFA in this study did not increase in NAFL and NASH groups compared with concentrations in the control group because those were presumably readily incorporated into TGs and DGs, both of which increased. Since the dietary intake between the three groups of patients was similar, the differences in hepatic FA profiles were attributed to increased lipid peroxidation and defects involved in the conversion of essential FAs to their active metabolites.

Table 1.

Nonalcoholic Fatty Liver Disease-Associated Changes in Liver Lipids and Enzymes Involved in Their Metabolism

Authors	Study subjects	Major findings	Comments
Araya et al.⁴⁰	All diabetics Control n = 11 SS n = 10 NASH n = 9	For total liver lipids, AA, EPA, and DHA progressively ↓ from normal to SS to NASH, and the ratio n-6/n-3 PUFA increased. Liver TGs did not differ in concentrations of AA, EPA, and DHA between SS and NASH, but both had lower LCPUFA than control. PL had lower n-3 LCPUFA and higher n-6 LCPUFA and ratio n-6 PUFA/n-3 PUFA in both SS and NASH compared with control.	PL contained much higher concentrations of LCPUFA, particularly n-3 PUFA, compared with TGs.
Elizondo et al.⁴¹	Nondiabetic Control n = 8 NAFLD n = 12	Liver PL, AA, DHA, and DPA (n-3), total n-3 LCPUFA, and total LCPUFA, NAFLD<control; serum F-2 isoprostanes NAFLD>control.	Changes in RBC FA profile were significantly associated with those in liver lipids.
Puri et al.⁴²	Nondiabetic Control n = 9 NAFL n = 9 NASH n = 9	In total liver lipids, no difference in TG/DG and n-6/n-3 ratios between NAFL and NASH, but these ratios were increased in both groups compared with control. FFA did not differ between three groups. EPA and DHA ↓ in TGs; AA ↓ in TG, FFA, and PC.	Not Hepatic FFA, but TG, DG, FC, and n-6/n-3 PUFA increased in NAFL and NASH compared with control.
Allard et al.⁴³	Nondiabetic Control n = 17 SS n = 18 NASH n = 38	No difference in n-3 and n-6 PUFA in total liver lipids between SS and NASH. Both groups had lower n-3 and n-6 PUFA, and the ratios between AA/LA and between (EPA+DHA)/ALA compared with controls.	No significant difference in FA composition of total liver lipids between SS and NASH, except for a trend for n-3 and n-6 PUFA with NASH<SS.
Petit et al.⁴⁴	All diabetics NAFLD n = 109 w/o NAFL n = 53	RBC concentrations of 16:0, 16:1n-7, and MUFA/SFA were positively associated with liver fat determined by MRS; concentrations of GLA, AA, and DHA were negatively associated with degree of steatosis.	Did not separate NAFL and NASH. Increased RBC concentrations of n-3 and n-6 LCPUFA negatively associated with liver fat.
Auguet et al.⁴⁶	Morbidly obese NL n = 13 SS n = 47 NASH n = 67	Expression of FAS, ACC1, and LXR α in liver inversely associated with the degree of steatosis as determined by liver biopsy.	Lipogenesis seems to be downregulated in advanced stages of SS in morbidly obese.
Yamada et al.⁴⁵	Control n = 18 SS n = 63 NASH n = 40	18:0/16:0 and 18:1n-9/18:0 in total liver lipids positively associated with steatosis score; 16:1n-7/16:0 associated with lobular inflammation. Hepatic expression of SCD1, ELOVL6, SREBP1c, FAS, and PPARγ increased in NASH compared with SS.	Some of NASH patients were type 2 diabetics. Differences in gene expression and FA composition between SS and NASH.
Arendt et al.⁴⁸	Diabetes unknown Control n = 24 SS n = 20 NASH n = 19	Concentrations of hepatic AA, EPA, and DHA were NASH<SS; this did not correspond to the RBC fatty acids. Twenty-two hepatic genes were differently expressed between SS and NASH, including the overexpression of FADS1 and 2.	In NASH patients, LCPUFA were lower than SS and associations with gene expression were different compared with SS.
Walle⁴⁷	Morbidly obese Control n = 39 SS n = 23 NASH n = 30	NASH associated with higher hepatic expression of FADS 1 and 2 and SCD. Estimated activities of serum D5D, D6D, and SCD1 based on product/precursor ratios correlated positively with their hepatic mRNA expression.	Serum estimates of D5D, D6D, and SCD1 reflected liver metabolism.

AA, arachidonic acid; ACC1, acetyl-CoA carboxylase 1; ALA, alpha linolenic acid; D5D, delta-5 desaturase; D6D, delata-6 desaturase; DG, diglyceride; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; FA, fatty acid; FADS1, fatty acid desaturase-1; FADS2, fatty acid desaturase-2; FAS, fatty acid synthase; FC, free cholesterol; FFA, free fatty acid; GLA, gamma-homo-linolenic acid; LA, linoleic acid; LCPUFA, long-chain polyunsaturated fatty acid; MRS, magnetic resonance spectroscopy; MUFA, monounsaturated fatty acid; NASH, nonalcoholic steatohepatitis; NL, normal liver; PL, phospholipid; PPAR, peroxisome proliferator-activated receptor; RBC, red blood cell; SCD, stearoyl-CoA desaturase; SFA, saturated fatty acid; SREBP-1c, sterol regulatory element-binding protein-1c; SS, simple steatosis; TG, triglyceride; w/o, without.

Another study in diabetic patients with NAFLD reported that liver fat content was positively associated with the concentrations of 16:0, 16:1n-7, and the ratio between monounsaturated and saturated FAs in red blood cell (RBC) lipids. It was negatively associated with concentrations of total RBC PUFA, gamma-homo-linolenic acid (GLA), DHA, and AA.⁴⁴ Quality of fat accumulated in the liver was also associated with the steatosis score in liver tissue. Ratio between 18:0 and 16:0 was decreased in NASH compared with those with SS, and ratio between 16:n-7 and 16:0 was associated with the lobular inflammation score.⁴⁵

Changes in hepatic FA profiles are also associated with altered expression of genes involved in FA metabolism. In a study with morbidly obese women, the expression of fatty acid synthase (FAS) was significantly higher in patients with NAFL and NASH compared with those with NL, and the expression of FAS and acetyl-CoA carboxylase 1 (ACC1) was inversely associated with the grade of steatosis.⁴⁶ Authors suggested that lipogenesis was downregulated in advanced stages of steatosis. Based on hepatic gene expression and their estimated activities based on serum product/precursor ratios, the activities of delta-6-desaturase and stearoyl-CoA desaturase-1 were increased and that of delta-5-desaturase decreased in the livers of NASH patients when compared with individuals with NL.⁴⁷ NASH-related changes in serum FA composition and hepatic gene expression were independent of obesity and diet in this study.

Thus, the FA profile and the expression of enzymes involved in FA metabolism are altered in both NAFL and NASH compared with NLs. While some of the changes in the expression of FAS, ACC1, and stearoyl-CoA desaturase-1 (SCD1) are similar in both NAFL and NASH, a lipodomic study revealed differences in the expression of 22 genes between the two states of fatty liver.⁴⁸ Restoring of normal hepatic lipid and FA profiles through dietary supplements and/or dietary changes may be useful in reversing NAFL and NASH.

Clinical Trial with n-3 LCPUFA for the Treatment of NAFL and NASH

Results from cross-sectional studies discussed above revealed a lesser amount of n-3 LCPUFA and also a decrease in the ratio between n-3 and n-6 PUFAs in the livers of subjects with NAFLD than those with NLs. These findings and results from animal studies suggested that n-3 PUFA may be useful in the prevention and treatment of NAFLD. According to the literature search conducted using PubMed and Web of Science, 19 published clinical trials were found that investigated the effects of marine and algal n-3 LCPUFAs on markers of human NAFLD between 1995 and 2016. Results from these studies are summarized in Table 2. Two of these studies used purified EPA ethyl esters (EPA-EE), two used algal DHA in the absence of EPA, another one used a mixture of algal EPA and DHA, and 12 used mixtures of EPA and DHA from FOs. Three studies imposed 25%–30% caloric restriction with and without FO supplementation; one supplemented flaxseed oil along with FO.

Table 2.

Effects of Long-Chain n-3 Polyunsaturated Fatty Acids on Markers of Nonalcoholic Fatty Liver Disease in Human Subjects

Authors	Study subjects	Study design	Treatment	Major findings	Comments
Tanaka et al.⁴⁹	23 NASH patients proven by liver biopsy and US (17% T2DM)	Longitudinal with a single dose of EPA-EE and w/o a control group, 12 months	EPA-EE 2.7 grams/day, repeat US in 23, and liver biopsy in 7 at 12 months	US indicated ↓ steatosis in 17/23 subjects; second biopsy indicated 34%–59% ↓ in steatosis, inflammation, fibrosis, and ballooning in 6/7 subjects. Serum AST, ALT, ferritin, and thioredoxin also ↓ significantly; NC in BW, HOMA-IR, and adiponectin.	w/o a control group, changes due to factors other than EPA cannot be ruled out.
Sanyal et al.⁵⁰	243 NASH patients proven by liver biopsy (30%–40% T2DM)	Multicenter, DB, and parallel with 2 doses of EPA-EE and undefined placebo. Repeat liver biopsy at 12 months	EPA-EE 1.8 grams/day n = 82, 2.7 grams/day n = 86, Placebo n = 75	NC in steatosis, inflammation, ballooning, fibrosis scores, serum liver enzymes, HOMA-IR, adiponectin, keratin 18, CRP, and hyaluronic acid.	Findings disagree with those of above study, which may be due to higher intake of dietary n-3 PUFA by the Japanese than the Americans.
Nobili et al.^51 –53	60 obese/overweight NAFLD children determined by elevated ALT, ultrasound, and liver biopsy (nondiabetic)	Randomized, parallel, and placebo-controlled using DHA. Tested liver fat by US and blood samples every 6 months, and repeat liver biopsy at 18 months	Algal DHA (250 and 500 mg/day). Germ oil (LA 290 mg/day) used as placebo. Each group n = 20 (0/0/8/12) with steatosis scores of 0, 1, 2, and 3	On the basis of changes in liver fat by US (−3/−2/−1/0/+1) at 6 months of treatment number of children in placebo, DHA 250 and 500 mg groups were 0/0/7/12/1, 1/8/10/1/0, and 1/7/7/4/1, respectively. Compared with baseline, ISI improved in both DHA groups 30%–35%. ALT NC in all groups. Hepatic steatosis, ballooning, NAS, and PNHS ↓70%, 70%, 30%, and 91% at 18 months of DHA 250 mg/day as determined by second liver biopsy. Compared with 6 months, there was no further ↓ in liver fat at 12, 18, and 24 months of DHA treatment as determined by US.	Longest and only human study, which tested effects of DHA on NAFLD in the absence of EPA. Both doses of DHA were equally effective in decreasing markers of NASH and NAFLD.
Pacifico et al.⁵⁶	51 children and adolescents with MRI and biopsy-proven NAFLD	Double-blind, placebo-controlled parallel study using DHA, 6 months	Algal DHA (250 mg/day for 6 months, n = 25). Germ oil used as placebo (LA 290 mg/day, n = 26)	Hepatic fat determined by MRI ↓53% in DHA group, and ↓22% in placebo, compared with baseline. DHA also ↓ ALT, insulin, TG, visceral, and epicardial adipose tissue, which did not change in the placebo group.	Presupplement markers of fibrosis did not differ between the two groups, but there was no post-treatment biopsy.
Janczyk et al.⁵⁷	66 obese/overweight NAFLD children confirmed by US (nondiabetic)	Randomized, parallel, and placebo-controlled with algal oil (EPA:DHA = 3:2) 6 months	Algal oil dose based on body weight (<40 kg, 450 mg; 40–60 kg, 900 mg; >60 kg, 1.3 grams/day). Placebo n = 34, n-3 PUFA n = 30	At the end of 6 months, there was no difference in serum ALT, HOMA-IR, or liver steatosis between the algal and placebo oil groups; serum AST and GGT were significantly lower and adiponectin was higher in the algal than in placebo oil group.	Low sensitivity of US and EPA>DHA may have led to the failure to detect a ↓ in hepatic fat by n-3 PUFA.
Capanni et al.⁵⁸	56 NAFLD patients proven by US (nondiabetic)	Parallel study; FO supplement for treatment group and no supplement for control group, 12 months	FO 1 gram/day, (EPA 375 mg, DHA 625 mg). FO n = 42, control n = 14	Doppler perfusion index increased 50% and liver transaminases ↓40%–73% in FO group at 12 months compared with baseline. There was NC in control group.	Nonrandomized study with a small n in control group. DHA>EPA may have led to ↓ in liver steatosis.
Vega et al.⁵⁹	17 NAFLD adults confirmed by MRS (nondiabetic)	Sequential placebo 4 weeks, followed by FO 8 weeks	FO 9 grams/day, (EPA 4.6 grams and DHA 2.2 grams/day). Placebo had 18:1 trans, 72%; 18:2, 10%; and 16:0, 12%. n = 17	FO ↓ plasma TG (46%), VLDL+IDL (21%), apo B (15%), but not liver fat as determined by MRS.	Lack of FO effects on liver steatosis may be due to short duration of study and EPA>DHA.
Cussons et al.⁶⁰	25 women with PCOS; 12 with and 13 w/o NAFLD per MRS (nondiabetic)	Randomized CO, DB study with 8 weeks FO or olive oil; washout period 4 weeks	FO 4 grams/day (EPA 1.1 grams, DHA 2.2 grams/day) 8 weeks. Olive oil (4 grams/day) used as placebo. n = 25	FO ↓ liver fat compared with placebo (18.2% vs. 14.8%) in 12 women with NAFLD, but not in 13 women without NAFLD (2.4 vs. 2.7%).	DHA>EPA may have led to ↓ in steatosis even within 8 weeks of treatment.
Sofi et al.⁶¹	11 patients with NAFLD determined by US	Placebo-controlled parallel	FO (EPA 0.47 grams DHA 0.24 grams/day, and olive oil 6.5 grams/day) 1 year. Placebo Olive Oil FO n = 6, placebo n = 5	FO significantly ↓liver fat, ALT, AST, and GGT, and ↑ adiponectin, which did not change in the placebo group.	Despite the small n and small dose of n-3 PUFA, results are impressive.
Li et al.⁶²	78 NASH patients confirmed by biopsy	Parallel, single-blinded placebo-controlled, 6 months	FO (50 mL/day, EPA: DHA, 1:1), 6 months. Saline served as placebo. n = 39 for each group	Results of second liver biopsy showed significant ↓ liver fat, necroinflammation, ballooning, and fibrosis stage in the FO group compared with the control group. Plasma AST/ALT, CRP, MDA, and type IV collagen were also significantly ↓ in the FO group.	Massive dose of n-3 PUFA and an unmatched placebo of saline. Only human study besides Tanaka et al., which showed decrease in fibrosis by n-3 PUFA.
Dasarathy et al.⁶³	37 NASH patients proven by liver biopsy (all type 2 diabetics)	Randomized, DB, parallel, and placebo-controlled, 48 weeks. Repeat liver biopsy in all at 48 weeks	Purified EPA (2.16 grams/day) and DHA (1.44 grams/day). Placebo was corn oil. Placebo n = 19, n-3 PUFA n = 18	NC in liver fat and NAS determined by liver biopsy in EPA/DHA group; both responses were significantly improved in the placebo group. EPA/DHA also significantly increased HOMA-IR compared with the baseline values, which did not change in the placebo group.	No information re compliance, diets, and lifestyle changes provided. EPA>DHA. Diabetic patients. Totally unexpected results.
Scorletti et al.^64,70	103 NAFLD patients confirmed by MRS, CT, US, or biopsy (9% T2DM)	Randomized, DB, parallel, and placebo-controlled, 15–18 months.	EPA-EE (1.84 grams/day) and DHA–EE (1.52 grams/day), 15–18 months. Placebo was olive oil 4 grams/day. FO n = 51 Placebo n = 52	1% enrichment (wt% of total FAs) of DHA in RBC lipids was associated with 3.3% reduction in hepatic fat, while EPA enrichment was not associated with liver fat.	DHA enrichment of RBC may indicate effectiveness of FO to ↓ liver fat. Efficacy DHA>EPA.
Nogueira et al.⁶⁵	50 NASH patients confirmed by liver biopsy	Randomized, DB, parallel, placebo-controlled	FO along with flaxseed oil provided n-3 PUFA (ALA 590 mg/day, EPA 160 mg/day, DHA 195 mg/day, 6 months). 945 mg/day Mineral oil used as placebo FO n = 23, Placebo n = 27	NAS and TG ↓ in both groups, potentially because of unmonitored consumption of n-3 PUFA by the placebo.	Study had poor compliance. The NAS in the n-3 PUFA group was inversely associated with plasma ALA concentration and with EPA and DHA concentrations in placebo group.
Hatzitolios et al.⁶⁶	73 NAFLD patients proven by US (all nondiabetic)	Randomized, parallel study, 24 weeks.	FO 15 mL/day (EPA 2.3 grams, DHA 1.6 grams/day) FO n = 23, Atorvastatin n = 28 Orlistat n = 21	US echo pattern became normal in 35%, 61%, and 86% patients 24 weeks after treatment with FO, atorvastatin, and orlistat, respectively. Liver transaminases also significantly ↓ in all three groups.	No control group.
Spadaro et al.⁶⁷	36 NAFLD patients proven by US (all nondiabetic)	Randomized using AHA diet restricting caloric intake to 25–30 kcal/kg/day with and w/o FO, 6 months	Caloric intake restricted to 25–30 kcal/kg/day with and w/o FO 2 grams/day (EPA and DHA amounts not stated).Each group n = 18	US echo pattern improved in both groups with complete, partial, and no regression of steatosis in 33%, 50%, and17% patients in FO group and 0%, 28%, and 70% in caloric restriction group. FO, but not energy restriction, significantly ↓ serum ALT, GGT, TNF α, and HOMA-IR.	Energy restriction alone ↓ liver fat with added benefit of FO.
Zhu et al.⁶⁸	134 NAFLD subjects proven by US (all nondiabetic)	Randomized and placebo-controlled with 25%–30% caloric restriction along with FO or placebo, 24 weeks	25%–30% caloric restriction with seal or placebo oil 6 grams/day Seal oil n = 66 Placebo n = 68	Seal and placebo oils completely normalized liver fat in 20% and 7% subjects, and partially in 55% and 35% subjects, respectively. Liver enzymes significantly ↓ in both groups.	Synergistic effects of n-3 PUFA and energy restriction to ↓ liver fat. EPA and DHA amounts and the name of placebo oil not reported.
Argo et al.⁶⁹	34 NASH patients confirmed by liver biopsy (32.4% T2DM)	Randomized, DB, placebo-controlled study with 500–1000 calories restriction/day and FO, 12 months	Caloric restriction with FO 3 grams/day (EPA 1.1 grams and DHA 0.8 grams/day) and soybean oil 3 grams/day. Each group n = 17	FO treatment for 12 months significantly ↓ liver fat determined by MRI. It had no effect on ALT, HOMA-IR, and NAS and decreased markers of liver injury only in subjects who lost weight.	↓ in liver fat caused by n-3 PUFA was independent of the ↓ in body weight.

ALT, alanine aminotransferase, AST, aspartate aminotransferase; CRP, C-reactive protein; CT, computed tomography; DB, double-blind; EPA-EE, eicosapentaenoic acid-ethyl ester; FO, fish oil; GGT, gamma glutamyl transpeptidase; HOMA-IR, homeostatic model of insulin resistance; IR, insulin resistance; ISI, insulin sensitivity index; MRI, magnetic resonance imaging; NC, no change; PNHS, pattern of NAFLD histological score; NAFL, nonalcoholic fatty liver; NAFLD, nonalcoholic fatty liver disease; NAS, NAFLD activity score; PCOS, polycystic ovary syndrome; US, ultrasound; T2DM, type 2 diabetes mellitus; VLDL, very low-density lipoprotein.

In a study with liver biopsy-proven NASH patients (n = 23, 17% type 2 diabetes mellitus [T2DM]), effects of EPA-EE (2.7 grams/day, 12 months) on markers of NASH were evaluated.⁴⁹ Liver steatosis using US, biochemical markers of NASH in all subjects, and repeat liver biopsy in only seven subjects were performed at the end of EPA-EE treatment. Hepatic steatosis, as determined by US, was decreased in 12/23 subjects and the mean steatosis grade decreased from 2.1 to 1.6. EPA-EE treatment also significantly decreased serum concentrations of AST and ALT, ferritin, and thioredoxin without any changes in adiponectin and homeostasis model of insulin resistance (HOMA-IR). Results of the second biopsy indicated 34%–59% reduction in hepatic steatosis, inflammation, fibrosis, and ballooning in 6/7 subjects following EPA-EE supplementation. Since the study did not have a control group, changes in response variables tested due to factors other than EPA-EE cannot be ruled out. The results of this study are at variance with those of a large, multicenter placebo-controlled study with EPA-EE in biopsy-proven 243 NASH subjects.⁵⁰ The later study comprised three groups (placebo, EPA-EE 1.8 grams/day, and EPA-EE 2.7 grams/day, 12 months) and second liver biopsy was performed in all subjects at the end of 12 months of supplementation with EPA-EE. At the end of the study, there was no difference in steatosis, inflammation, ballooning, and fibrosis scores, liver enzymes, HOMA-IR, adiponectin, Keratin 18, C-reactive protein (CRP), and hyaluronic acid between the placebo and the two treatment groups in this study. While, it is difficult to pinpoint the reasons for the inconsistency between the results from these two studies, it could be due to two major differences in the patient populations used. A study by Tanaka et al.⁴⁹ was conducted with Japanese subjects and that by Sanyal et al.⁵⁰ with Americans. It is possible that Japanese subjects may have higher basal level intake of n-3 PUFA than the Americans and thus an overall higher intake of n-3 PUFA. Furthermore, the study by Sanyal et al. contained roughly twice the percentage of diabetics compared with those in the study by Tanaka et al.; diabetics are likely to have more advanced NAFLD than nondiabetics, who are likely to be less responsive to n-3 PUFA. Further studies are needed to resolve this inconsistency and to determine the reasons for it.

Effects of algal DHA in the absence of EPA on markers of NAFL and NASH were evaluated in a randomized, parallel, and placebo-controlled trial in overweight/obese nondiabetic children with NAFLD confirmed by elevated liver enzymes, US, and liver biopsy.^51

–55 Sixty children were divided into three groups of 20 each and were served DHA 250 and 500 mg/day or placebo (germ oil, providing LA 290 mg/day) for 24 months. Liver steatosis using US and biochemical markers of NAFLD was determined at 6, 12, 18, and 24 months in all groups, and second liver biopsy was performed at 18 months in the group supplemented with DHA 250 mg/day. After 6 months of DHA supplementation, liver fat and serum TGs were significantly decreased and insulin sensitivity index was increased in both DHA groups when compared with the start of study. The effects of two doses of DHA on those markers of NAFLD were comparable. Supplementation of DHA for 12, 18, and 24 months did not result in further decrease in liver fat when compared with the values at 6 months.

Results of second liver biopsy after 18 months of DHA 250 mg supplementation revealed decreased hepatic steatosis by 70%, ballooning by 70%, NAS by 30%, and pattern of NAFLD histological score by 91%, but no change in fibrosis, when compared with the corresponding values determined by the first liver biopsy before DHA supplementation. Results from another recent double-blind, placebo-controlled parallel study with algal DHA (250 mg/day, 6 months, n = 25; placebo germ oil with 290 mg LA/day, 6 months, n = 26) in children and adolescents (age <18 years) confirmed the findings of Nobili et al.^51

–55 regarding the effects of DHA on NAFLD.⁵⁶ In the later study, DHA supplementation significantly decreased liver fat as determined by magnetic resonance imaging (MRI), HOMA-IR, and circulating ALT and TGs when compared with the baseline values. DHA also significantly decreased visceral and epicardial adipose tissue masses. None of these variables changed in the placebo group. Liver steatosis, lobular inflammation, ballooning, fibrosis, and NAS did not differ between the two groups at baseline as determined by liver biopsy. Results of these two studies are very encouraging that a low DHA dose of 250 mg/day could reverse several markers of NAFLD. Further studies with DHA in the absence of EPA in adults with NAFLD are needed.

Janczyk et al. determined the effects of an algal oil containing a mixture of EPA and DHA on hepatic steatosis and circulating liver enzymes in another study with overweight/obese nondiabetic children.⁵⁷ This was a randomized, parallel, and placebo-controlled trial with 34 children in the placebo (sunflower oil) group and 30 in the algal oil group (EPA: DHA = 3:2). There were three subgroups within the algal oil group depending upon the body weight and the dose of oil supplemented. Children weighing <40 kg received an algal oil dose of 450 mg/day, 40–60 kg received 900 mg/day, and those >60 kg received 1.3 grams/day for 6 months. At the end of 6 months of treatment, there was no difference between the two groups in liver fat determined by US, serum ALT, and HOMA-IR. Serum concentrations of AST and gamma-glutamyl transpeptidase (GGT) were significantly lower, and adiponectin was significantly higher in the algal oil group than in placebo oil group. Results of this study regarding the lack of an effect of algal oil containing a mixture of EPA and DHA on liver fat are at variance with those of the above two studies by Nobili et al. and Pacifico et al., which served algal oil containing DHA only.^52
–54,56 These differences may be due to the greater potency of DHA than EPA in decreasing liver fat. Further studies are needed to determine the efficacy of EPA and DHA individually and concomitantly and their dose based on body weight.

Capanni et al. examined the effects of supplementing FO, 1.0 gram/day (EPA 0.38 grams/day and DHA 0.62 grams/day), for 12 months in overweight/obese nondiabetic subjects with NAFLD. Of 56 study participants, 42 agreed to take the FO supplement, and the remainder 12 declined the treatment and served as control.⁵⁸ Doppler perfusion index (DPI, inversely associated with liver fat) as determined by US increased by 50% and liver transaminases decreased by 40%–73% in the FO group compared with the presupplement values; these variables remained unchanged in the subjects who declined to take FO supplements.

In contrast to the reduction in liver fat with FO supplementation in the above study, FO supplementation, 9 grams/day (EPA 4.6 grams/day and DHA 2.2 grams/day, 8 weeks), failed to decrease liver fat as determined by magnetic resonance spectroscopy (MRS) in 16 nondiabetic patients with NAFLD.⁵⁹ This was a sequential study, where all subjects received placebo oil (18:1, 72%; 18:2, 10%; and 16:0, 12%) for 4 weeks, followed by 8 weeks of FO treatment. Responses were monitored at the ends of placebo and FO treatments. Although, FO did not decrease liver fat, it decreased plasma TGs by 46%, (VLDL+IDL) by 21%, and apo B by 15%. The lack of decrease in liver fat with FO supplementation in this study may be due to short duration and very heterogeneous liver fat content, which ranged from 2.4% to 26.9% (mean ± SD = 10.6% ± 9.6%), and approximately one-half the concentration of DHA compared with that of EPA.

Cussons et al. investigated the effect of FO, 4 grams/day (EPA 1.1 grams/day and DHA 2.2 grams/day, 8 weeks), in a randomized, crossover double-blind study in 25 nondiabetic women with polycystic ovary syndrome (PCOS).⁶⁰ Olive oil, 4 grams/day, was used as placebo. Each supplement was served for 8 weeks with an 8-week washout period in between. FO supplementation significantly decreased mean liver fat for all 25 women as determined by MRS to 8.2% compared with 10.4% after placebo treatment. The decrease in liver fat was more noticeable in 12 women who had NAFLD (18.2% vs. 14.8%) than the 13 women who had liver fat% below the cutoff values for NAFLD (2.7 vs. 2.4%). FO supplementation also significantly reduced both systolic and diastolic blood pressure when compared with the placebo. Results of this study vary from of those of 12 women without PCOS in the study by Vega et al., which provided roughly twice the amount of FO provided in this study for the same duration and failed to find an effect of FO on liver fat.⁵⁹ While other reasons may have contributed to the differences in results between these two studies, relative abundance of EPA and DHA in the FOs used may be the more likely reason. FO used in this study contained twice the amount of DHA than that of EPA, while the reverse was the case in the study by Vega et al.⁵⁹ As mentioned above, relative efficacy of EPA and DHA in lowering liver fat needs to be determined.

Another study with FO was conducted in subjects who had elevated ALT and suggested to have NAFLD by US.⁶¹ This was a preliminary study in which five subjects took olive oil (6.5 grams/day, 1 year) and six others took FO (EPA 0.47 grams/day and DHA 0.24 grams/day, 1 year) in addition to olive oil. At the end of 1-year supplementation, DPI and plasma adiponectin significantly increased in the n-3 PUFA group (27% and 30%). US examination at the end of treatment revealed that one patient was without steatosis, three with mild, and two with severe steatosis in the n-3 PUFA group. Furthermore, AST, ALT, and GGT decreased by 40%, 35%, and 27%, respectively, in the n-3 PUFA group. Degree of steatosis and concentrations of liver enzymes did not change in the olive oil group. Even with the small dose of FO and small number of subjects, results of this study support the notion that FO improved liver functions.

A group of Chinese scientists used the maximum dose of n-3 PUFA tested for the treatment of NASH.⁶² In this study, 78 biopsy-proven subjects were divided in two groups of 39 each. n-3 PUFA group incorporated PUFA, 50 mL/day (EPA: DHA, 1:1), into their diets and the placebo group incorporated saline, 50 mL/day, for 6 months. All subjects were advised to do modest exercise (at least 150 min/week). Second liver biopsy was performed on all subjects at the end of 6 months. Results of the initial liver biopsy showed no difference in steatosis and necroinflammation grades, fibrosis stages, and ballooning scores between the two treatment groups. Results of the second liver biopsy showed significant improvement in all these four variables in the PUFA group when compared with the placebo group. At the end of 6-month intervention, the serum concentrations of liver enzymes, CRP, MDA,TG, total cholesterol, type IV collagen, and procollagen type III propeptide were also lesser in the PUFA group compared with the placebo group. This study is remarkable not only because of the dose of PUFA and unmatched placebo (saline) used, but it is also the only human study besides Tanaka et al.⁴⁹ that showed improvement in liver fibrosis following n-3 PUFA supplementation in human subjects. Authors did not disclose any adverse effects of taking such a massive dose of n-3 PUFA for 6 months. If indeed this dose of n-3 PUFA is safe and as effective as indicated by the authors, it may be something to consider for reversing NASH, but such study will have a tough time being approved by any IRB in the USA.

Dasarathy et al. compared the effects of purified EPA, 2.16 grams/day, plus DHA, 1.44 grams/day, with a corn oil placebo on liver fat and NAS in biopsy-proven NASH patients who had well-controlled type 2 diabetes.⁶³ This was a randomized, double-blind, and parallel study with a 48-week intervention with EPA/DHA (n = 18) and placebo (n = 19). Repeat liver biopsy was performed to determine the changes in markers of NASH at the end of the study in both groups. EPA/DHA treatment did not alter liver fat and NAS, which were both surprisingly improved in the placebo group. HOMA-IR did not change in the placebo group, but deteriorated in the EPA/DHA group. Several factors, including poor compliance, small n, relatively higher concentration of EPA than that of DHA, and advanced NASH in diabetic patients, may have contributed to the lack of improvement in markers of NASH by n-3 PUFA in this study.

The results of the above study differ from those of a recent randomized, double-blind, parallel, and placebo-controlled study published by Scorlletti et al.⁶⁴ In the later study, 103 NAFLD patients (9% T2DM) confirmed by liver biopsy, MRS, computed tomography, or US participated. They were divided into two groups; 52 received olive oil (4 grams/day, 15–18 months) and the remainder 51 received purified ethyl esters of EPA (1.84 grams/day) and DHA (1.52 grams/day) for the same duration as placebo. Blood markers for NAFLD and liver fat by MRS, MRI, and US were determined between the 15th and 18th month of interventions. RBC FA composition was determined to monitor compliance with the consumption of supplements. Interestingly, each 1% of DHA enrichment of RBC FAs was associated with a 3.3% decrease in liver fat as determined by MRS. Thus, a 6% DHA enrichment was associated with a 20% reduction in liver fat. In contrast to the association between liver fat and DHA enrichment, there was no association between EPA enrichment of RBC FAs and liver fat. These findings support the notion that FOs containing more DHA than EPA may be more effective in reducing liver fat. Although the n-3 PUFA supplement contained ∼55% EPA and 45% DHA, RBC enrichment of DHA was greater than that of EPA. Neither n-3 PUFA nor the placebo improved the liver or NAFLD fibrosis scores. Results from animal models of NASH that will be discussed later indicate that DHA, but not EPA, reduced markers of fibrosis.

Nogueira et al. examined the effects of a combination of ALA from flax seed oil and EPA and DHA on markers of NASH.⁶⁵ Twenty-seven biopsy-proven NASH subjects took a mixture of n-3 PUFA (ALA 590 mg+EPA 160 mg+DHA195 mg/day for 6 months), while another 23 took mineral oil as placebo for 6 months. Second liver biopsy was performed at the end of 6-month intervention. NAS improved in both groups. It was associated with the plasma concentration of ALA in the n-3 PUFA group and with the EPA and DHA concentrations in the placebo group. Authors recognize that they had problems with the compliance in the placebo group. Regardless of the poor compliance, the results of this study indicate that n-3 PUFA improved NAS.

Hatzitolios et al. compared the effects of supplementing FO, 15 mL/day (EPA 2.3 grams/day and DHA 1.6 grams/day, n = 23), with that of atorvastatin (20 mg/day, n = 28) and orlistat (3 × 120 mg/day, n = 21) for 24 weeks on markers of NAFLD in subjects with hypertriglyceridemia.⁶⁶ Circulating liver transaminases and echo pattern using US were determined before and after the supplements. Echo pattern became normal in 35%, 61%, and 86% of the subjects treated with FO, atorvastatin, and orlistat, respectively. Serum concentrations of ALT, AST, and GGT were significantly decreased post-treatment compared with pretreatment values in all three groups; however, the decreases in AST and ALT were greater in the orlistat group compared with decreases in other two groups. In addition, only the orlistat group had significant decrease in body weight following treatment. It appears that FO treatment had effects similar to those of pharmaceutical interventions.

Three different studies examined the effects of caloric restriction with and without supplementation of n-3 PUFA on markers of NAFLD. In a study by Spadaro et al., caloric intake was restricted to 25–30 kcal/kg/day for 6 months in 36 overweight/obese nondiabetic subjects with NAFLD confirmed by US.⁶⁷ One-half of the subjects also took FO, 2 grams/day (EPA and DHA contents unknown), while the other half was subjected to caloric restriction alone. Compared with pretreatment values, BMI decreased by 6.3% in the FO along with caloric restriction group and by 2.9% in the caloric restriction alone group. US echo pattern improved in both groups with complete, partial, and no regression of steatosis in 33, 50, and 17%, respectively, in patients receiving FO and caloric restriction; corresponding numbers for the caloric restriction alone group were 0%, 28%, and 70%, respectively. Circulating ALT, GGT, TG, and TNFα decreased by 30%, 29%, 25%, and 19%, respectively, in the FO group when compared with the corresponding pretreatment values; these variables did not change in the other group.

In a similar study, Zhu et al.⁶⁸ investigated the effects of caloric restriction along with seal or placebo oil supplementation on markers of NAFLD in overweight and hyperlipidemic subjects. One hundred thirty-four subjects with elevated ALT and AST and NAFLD confirmed by US were placed on 25%–30% caloric restriction along with seal oil, 6 grams/day (n = 66), or placebo oil (n = 68) for 24 weeks. Seal oil normalized liver fat completely in 20% and partially in 55% subjects, while the corresponding numbers for the placebo oil were 7% and 35%, respectively. Liver enzymes were significantly decreased in both groups when compared with corresponding pretreatment values.

In the third study, Argo et al. determined the effects of caloric restriction and concomitant supplementation with FO or soybean oil on metabolic and histological parameters of NASH.⁶⁹ Thirty-four patients (32.4% T2DM) with noncirrhotic NASH confirmed by liver biopsy participated in a randomized, double-blind, and placebo-controlled parallel study. Caloric intake was decreased by 500–1000 calories/day for all subjects. In addition to caloric restriction, half of the subjects were served FO, 3 grams/day (EPA 1.1 grams/day and DHA 0.8 grams/day, 12 months), and the remainder were served soybean oil, 3 grams/day. Second liver biopsy was performed at the end of 12 months of caloric restriction and FO/placebo supplementation. Liver fat determined by MRI was decreased in both groups when compared with the pretreatment values; however, the decrease was greater in the FO group than the placebo group. Decrease in liver fat in the placebo group was attributed to weight loss, but in the FO group, it was due to both weight loss and n-3 PUFA. FO supplementation did not resolve NASH or improve NAS as determined by liver biopsy. Markers for liver injury were decreased only in subjects who lost weight and took FO supplements. Furthermore, there was no decrease in HOMA-IR or ALT in both groups when compared with the pretreatment values. Results of these three studies indicate that caloric restriction alone decreased liver fat, which was further enhanced by FO.

Thus, of the 17 published studies, 13 studies (8 with FO, 2 with algal DHA in the absence of EPA, and 1 each with seal oil, FO plus flax seed oil, or EPA-EE) demonstrated improvement in markers of NAFLD following treatment with n-3 PUFA. Three studies imposed caloric restriction in addition to n-3 PUFA and the effects of weight loss and n-3 PUFA on liver fat were additive. The dose of n-3 PUFA ranged from 250 mg/day DHA to 50 mL/day of a mixture of EPA and DHA for the duration of 2 months to 2 years. Taken together, these findings support that n-3 LCPUFA improved liver enzymes, steatosis, and inflammation. Ballooning was improved in only four and fibrosis in two studies. A summary of these results is provided in Table 3.

Table 3.

Summary of Effects of n-3 Polyunsaturated Fatty Acids on Laboratory Markers of Nonalcoholic Fatty Liver Disease

Author	Steatosis	AST, ALT, & GGT	IR	TG	Inflammation	Ballooning	Fibrosis	NAS
Tanaka et al.⁴⁹	↓	↓ AST & ALT	NC	NC	↓	↓	↓	↓
Sanyal et al.⁵⁰	NC	NC	NC	↓	NC	NC	NC	NC
Nobili et al.^51 –55	↓	↓ AST, ALT, & GGT	NC	↓	↓	↓	NC	↓
Pacifico et al.⁵⁶	↓	↓ ALT	↓	↓
Janczyk et al.⁵⁷	NC	↓ AST & GGT	NC	NC
Capanni et al.⁵⁸	↓	↓ AST, ALT, & GGT		↓
Vega et al.⁵⁹	NC			↓
Cussons et al.⁶⁰	↓		NC	↓	NC
Sofi et al.⁶¹	↓	↑AST, ALT, & GGT
Dasarathy et al.⁶³	NC	NC	↑	NC	NC	NC	NC	NC
Scorletti et al.^64,70	↓			↓			NC	NC
Li et al.⁶²	↓	↓ AST & ALT		↓	↓	↓	↓
Nogueira et al.⁶⁵	NC	NC		↓	NC			↓
Hatzitolios et al.⁶⁶	↓	↓ AST & ALT		↓	↓		NC
Spadaro et al.⁶⁷	↓	↓ ALT	↓	↓	↓
Zhu et al.⁶⁸	↓	↓ AST, ALT, & GGT	↓
Argo et al.⁶⁹	↓	NC	NC	↓			NC	NC

For the empty cells in the above table, the specific response variables were not tested.

Reasons for Discrepancies

Four of 17 published human studies failed to detect decrease in liver fat following supplementation with n-3 PUFA. This may be because of short duration,⁵⁹ advanced NASH in diabetic patients,^50,63 and relatively low concentration of DHA.⁵⁷ Several other factors, including the amounts of n-6 PUFA, saturated and trans FAs, and other dietary factors impacting lipid metabolism and oxidative stress, can influence the efficacy of the n-3 PUFA in reducing liver fat. Severity and heterogeneity of NASH, sensitivity and reproducibility of the analytical methods used, compliance with the interventions, exercise, lifestyle changes, and caloric intake can all influence the outcome of the treatment with n-3 PUFA. Results of the published reports also suggest that DHA may be more effective than EPA in decreasing liver fat and inflammation of the liver. Basis for this claim will be discussed in the following section.

Is DHA More Effective Than EPA in Reducing Hepatic Steatosis and NASH?

Results of the clinical trials with n-3 PUFA suggest that the relative abundance of EPA and DHA in the FO may be a factor that determines the efficacy of the n-3 PUFA in reducing hepatic steatosis. This is supported by the facts that DHA in the absence of EPA decreased liver fat in two studies with overweight/obese children^53,56 and RBC enrichment with DHA, but not EPA, was associated with reduction in liver fat.^64,70 In a large multicenter, double-blind, and placebo-controlled study, EPA-EE failed to improve liver fat and several markers of NASH tested.⁵⁰ For studies with FO that found a decrease in liver fat, concentration of DHA in the mixture was either greater than that of EPA^58,60 or approximately same.^64,70 On the other hand, in studies that failed to detect a decrease in liver fat, the FO used either contained more EPA than DHA^57,59,63 or their concentrations were not listed.^67,68 Taken together, these findings suggest that DHA may be more effective than EPA in decreasing hepatic steatosis.

Both EPA and DHA decrease circulating TGs, inflammation, platelet aggregation, and share some other effects. However, their efficacy varies for these and other health benefits. Only DHA decreased the number of small dense LDL particles, large VLDL particles, remnant-like particles, blood pressure, and heart rate and increased the size of LDL particles, HDL cholesterol, ratio between HDL-C and LDL-C, and between HDL and TGs.^71

–76 DHA, but not EPA, decreased the circulating concentration of Apo CIII, which inhibits LPL activity and increases the conversion of VLDL into larger more buoyant LDL particles.⁷⁷ Furthermore, DHA is the major n-3 PUFA in human tissues and its concentration decreased more than that of EPA in human livers with NASH.^43,78 DHA can be retroconverted to EPA, which cannot be elongated to DHA by humans. These results from human studies again suggest a potentially greater role for DHA than EPA in treating NAFLD.

The above suggestion is also supported by results from several studies in animal models. Effects of DHA, DPA, and EPA on liver fat were compared in db/db mice fed diets supplemented with these FAs for 4 weeks. At the end of feeding experimental diets, the liver fat was DHA < DPA < EPA < Control.⁷⁹ Thus, DHA was more effective than EPA in preventing hepatic steatosis. In LDLR knockout mouse model of NAFLD, neither EPA nor DHA prevented the Western diet-induced liver steatosis; however, DHA was significantly more effective than EPA in reducing oxidative stress, inflammation, and fibrosis.⁸⁰ In other mouse models, DHA prevented the high-fat diet⁸¹ and trans FA-induced hepatic steatosis.⁸²

DHA, but not EPA, prevented the trans FA-induced increase in IR and partially prevented the decrease in adiponectin in mice.⁸² Similarly, in cultured 3T3 preadipocytes, DHA, but not EPA, increased secretion of adiponectin, which is an anti-inflammatory adipokine because it increases peroxisome proliferator-activated receptor γ (PPARγ) expression.⁸³ DHA was also more potent than EPA in inhibiting NFKB signaling and expression of TLR4 and TLR2 in monocytes and of M1 macrophages (480+/CD11+) in adipose tissue.^84
–86 DHA increased plasma superoxide dismutase activity and reduced liver fibrosis in a rat model of NASH induced by a diet deficient in choline and high in fat and oxidative stress.⁸⁷ Further studies are needed to compare the efficacy of EPA and DHA in treating NAFLD, but these results from animal studies are consistent with the notion that DHA may be more effective than EPA in preventing and treating NAFLD.

A number of nuclear transcription factors (PPARα, sterol regulatory element-binding protein-1c [SREBP-1c], LXRα, ChREBPs) that affect hepatic fat accumulation are subject to nutritional regulation. Both DHA and EPA downregulate SREBP-1 expression and increase PPARα expression. DHA was a more potent suppressor of SREBP-1 than EPA because it uniquely regulates SREBP-1 nuclear content by stimulating its proteasomal degradation.⁸⁸ DHA suppressed SREBP-1 expression in both wild-type and SREBP-1 transgenic mice, but EPA failed to suppress it in the transgenic mice.⁸⁹ SREBP-1c is the main switch controlling lipogenesis because it regulates the expression of several other transcription factors, including liver X receptor, hepatocyte nuclear factor-4α, farnesol X receptor, and PPARs. Although EPA is a more potent agonist of PPARα than DHA, its effect on hepatic steatosis was independent of PPARα activation; it was through the inhibition of SREBP-1 maturation only.⁹⁰ DHA, but not EPA, suppressed ChREBP expression, which regulates carbohydrate (glucose)-stimulated lipogenesis.⁷⁷ These findings further support that DHA may be more potent than EPA in suppressing hepatic lipogenesis.

Conclusions

NAFLD is the most common liver disease worldwide and among the top 10 leading causes of death in the United States. Its incidence is increasing with increase in obesity, MS, and diabetes. Other than weight loss, currently there are no validated treatments available for NAFLD. Results from human and animal studies have demonstrated that LC n-3 PUFA decreased liver fat, liver enzymes, and markers of inflammation. Only 2 of the 17 published human studies reported decrease in fibrosis and 4 reported decrease in ballooning. Several studies with animal models of NASH reported decrease in both steatosis and fibrosis following supplementation with DHA or FOs. Results from both human and animal studies also suggest that DHA may be more effective than EPA in reducing steatosis and markers of NASH and that the benefits of n-3 PUFA may be additive to those of weight loss. Long-term, randomized, double-blind, and placebo-controlled studies with standardized protocols and methods and large number of patients are needed to determine the dose, duration, and efficacy of n-3 PUFA in the treatment of NAFLD. Role of disease state, lifestyle, and dietary factors also need to be investigated.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

References

Neuschwander-Tetri

, et al. Farnesoid X nuclear receptor ligand obeticholic acid for non-cirrhotic, non-alcoholic steatohepatitis (FLINT): A multicentre, randomised, placebo-controlled trial. Lancet, 2015; 385:956–965.

Chalasani

, et al. The diagnosis and management of non-alcoholic fatty liver disease: Practice Guideline by the American Association for the Study of Liver Diseases, American College of Gastroenterology, and the American Gastroenterological Association. Hepatology, 2012; 55:2005–2023.

Henao-Mejia

, et al. Inflammasome-mediated dysbiosis regulates progression of NAFLD and obesity. Nature, 2012; 482:179–185.

McPherson

, et al. Evidence of NAFLD progression from steatosis to fibrosing-steatohepatitis using paired biopsies: Implications for prognosis and clinical management. J Hepatol, 2015; 62:1148–1155.

Pais

, et al. A systematic review of follow-up biopsies reveals disease progression in patients with non-alcoholic fatty liver. J Hepatol, 2013; 59:550–556.

Singh

, et al. Fibrosis progression in nonalcoholic fatty liver vs nonalcoholic steatohepatitis: A systematic review and meta-analysis of paired-biopsy studies. Clin Gastroenterol Hepatol, 2015; 13:643–654.e1–e9; quiz e39–e40.

Kawano

, Cohen

. Mechanisms of hepatic triglyceride accumulation in non-alcoholic fatty liver disease. J Gastroenterol, 2013; 48:434–441.

Mashek

. Hepatic fatty acid trafficking: Multiple forks in the road. Adv Nutr, 2013; 4:697–710.

Musso

, Gambino

, Cassader

. Recent insights into hepatic lipid metabolism in non-alcoholic fatty liver disease (NAFLD). Prog Lipid Res, 2009; 48:1–26.

10.

Donnelly

, et al. Sources of fatty acids stored in liver and secreted via lipoproteins in patients with nonalcoholic fatty liver disease. J Clin Invest, 2005; 115:1343–1351.

11.

Day

, James

. Steatohepatitis: A tale of two “hits”?. Gastroenterology, 1998; 114:842–845.

12.

Di Minno

, et al. Omega-3 fatty acids for the treatment of non-alcoholic fatty liver disease. World J Gastroenterol, 2012; 18:5839–5847.

13.

Masterton

, Plevris

, Hayes

. Review article: Omega-3 fatty acids—A promising novel therapy for non-alcoholic fatty liver disease. Aliment Pharmacol Ther, 2010; 31:679–692.

14.

Farese

Jr. , et al. The problem of establishing relationships between hepatic steatosis and hepatic insulin resistance. Cell Metab, 2012; 15:570–573.

15.

Neuschwander-Tetri

, Caldwell

. Nonalcoholic steatohepatitis: Summary of an AASLD Single Topic Conference. Hepatology, 2003; 37:1202–1219.

16.

Kleiner

, et al. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology, 2005; 41:1313–1321.

17.

Cave

, et al. Nonalcoholic fatty liver disease: Predisposing factors and the role of nutrition. J Nutr Biochem, 2007; 18:184–195.

18.

Adams

, Lindor

. Nonalcoholic fatty liver disease. Ann Epidemiol, 2007; 17:863–869.

19.

Zivkovic

, German

, Sanyal

. Comparative review of diets for the metabolic syndrome: Implications for nonalcoholic fatty liver disease. Am J Clin Nutr, 2007; 86:285–300.

20.

Erickson

. Nonalcoholic fatty liver disease. J Lipid Res, 2009; 50 Suppl:S412–S416.

21.

Smith

, Adams

. Non-alcoholic fatty liver disease. Crit Rev Clin Lab Sci, 2011; 48:97–113.

22.

Das

, et al. Nonobese population in a developing country has a high prevalence of nonalcoholic fatty liver and significant liver disease. Hepatology, 2010; 51:1593–1602.

23.

Browning

, et al. Prevalence of hepatic steatosis in an urban population in the United States: Impact of ethnicity. Hepatology, 2004; 40:1387–1395.

24.

Gholam

, et al. Nonalcoholic fatty liver disease in severely obese subjects. Am J Gastroenterol, 2007; 102:399–408.

25.

Portillo-Sanchez

, et al. High prevalence of nonalcoholic fatty liver disease in patients with type 2 diabetes mellitus and normal plasma aminotransferase levels. J Clin Endocrinol Metab, 2015; 100:2231–2238.

26.

Bril

, et al. Plasma thyroid hormone concentration is associated with hepatic triglyceride content in patients with type 2 diabetes. J Investig Med, 2016; 64:63–68.

27.

Cortez-Pinto

, et al. How different is the dietary pattern in non-alcoholic steatohepatitis patients?. Clin Nutr, 2006; 25:816–823.

28.

Oddy

, et al. The Western dietary pattern is prospectively associated with nonalcoholic fatty liver disease in adolescence. Am J Gastroenterol, 2013; 108:778–785.

29.

Papandreou

, Karabouta

, Rousso

. Are dietary cholesterol intake and serum cholesterol levels related to nonalcoholic Fatty liver disease in obese children?. Cholesterol, 2012; 2012:572820.

30.

Zelber-Sagi

, et al. Long term nutritional intake and the risk for non-alcoholic fatty liver disease (NAFLD): A population based study. J Hepatol, 2007; 47:711–717.

31.

Mummadi

, et al. Effect of bariatric surgery on nonalcoholic fatty liver disease: Systematic review and meta-analysis. Clin Gastroenterol Hepatol, 2008; 6:1396–1402.

32.

Stein

, Dong

, Loomba

. Insulin sensitizers in nonalcoholic fatty liver disease and steatohepatitis: Current status. Adv Ther, 2009; 26:893–907.

33.

Harrison

. Thiazolidinedione therapy for nonalcoholic steatohepatitis: Go, stop, or proceed with caution?. Hepatology, 2010; 51:366–369.

34.

Hardy

, Anstee

, Day

. Nonalcoholic fatty liver disease: New treatments. Curr Opin Gastroenterol, 2015; 31:175–183.

35.

Jump

, et al. Impact of dietary fat on the development of non-alcoholic fatty liver disease in Ldlr−/− mice. Proc Nutr Soc, 2016; 75:1–9.

36.

Parker

, et al. Omega-3 supplementation and non-alcoholic fatty liver disease: A systematic review and meta-analysis. J Hepatol, 2012; 56:944–951.

37.

Scorletti

, Byrne

. Omega-3 fatty acids, hepatic lipid metabolism, and nonalcoholic fatty liver disease. Annu Rev Nutr, 2013; 33:231–248.

38.

Aranceta

, Perez-Rodrigo

. Recommended dietary reference intakes, nutritional goals and dietary guidelines for fat and fatty acids: A systematic review. Br J Nutr, 2012; 107 Suppl 2:S8–S22.

39.

Martel

, et al. Non-alcoholic steatohepatitis: New insights from OMICS studies. Curr Pharm Biotechnol, 2012; 13:726–735.

40.

Araya

, et al. Increase in long-chain polyunsaturated fatty acid n-6/n-3 ratio in relation to hepatic steatosis in patients with non-alcoholic fatty liver disease. Clin Sci (Lond), 2004; 106:635–643.

41.

Elizondo

, et al. Polyunsaturated fatty acid pattern in liver and erythrocyte phospholipids from obese patients. Obesity (Silver Spring), 2007; 15:24–31.

42.

Puri

, et al. A lipidomic analysis of nonalcoholic fatty liver disease. Hepatology, 2007; 46:1081–1090.

43.

Allard

, et al. Nutritional assessment and hepatic fatty acid composition in non-alcoholic fatty liver disease (NAFLD): A cross-sectional study. J Hepatol, 2008; 48:300–307.

44.

Petit

, et al. Increased erythrocytes n-3 and n-6 polyunsaturated fatty acids is significantly associated with a lower prevalence of steatosis in patients with type 2 diabetes. Clin Nutr, 2012; 31:520–525.

45.

Yamada

, et al. Characteristics of hepatic fatty acid compositions in patients with nonalcoholic steatohepatitis. Liver Int, 2015; 35:582–590.

46.

Auguet

, et al. Altered fatty acid metabolism-related gene expression in liver from morbidly obese women with non-alcoholic fatty liver disease. Int J Mol Sci, 2014; 15:22173–22187.

47.

Walle

. Fatty acid metabolism is altered in non-alcoholic steatohepatitis independent of obesity. Metabolism, 2016; 65:655–666.

48.

Arendt

, et al. Altered hepatic gene expression in nonalcoholic fatty liver disease is associated with lower hepatic n-3 and n-6 polyunsaturated fatty acids. Hepatology, 2015; 61:1565–1578.

49.

Tanaka

, et al. Highly purified eicosapentaenoic acid treatment improves nonalcoholic steatohepatitis. J Clin Gastroenterol, 2008; 42:413–418.

50.

Sanyal

, et al. No significant effects of ethyl-eicosapentanoic acid on histologic features of nonalcoholic steatohepatitis in a phase 2 trial. Gastroenterology, 2014; 147:377–384.e1.

51.

Nobili

. Non-alcoholic fatty liver disease in children and adolescents. Clin Biochem, 2014; 47:720.

52.

Nobili

, et al. Docosahexaenoic acid for the treatment of fatty liver: Randomised controlled trial in children. Nutr Metab Cardiovasc Dis, 2013; 23:1066–1070.

53.

Nobili

, et al. Docosahexaenoic acid supplementation decreases liver fat content in children with non-alcoholic fatty liver disease: Double-blind randomised controlled clinical trial. Arch Dis Child, 2011; 96:350–353.

54.

Nobili

, et al. Role of docosahexaenoic acid treatment in improving liver histology in pediatric nonalcoholic fatty liver disease. PLoS One, 2014; 9:e88005.

55.

Nobili

, et al. The use of ultrasound in clinical setting for children affected by NAFLD: Is it safe and accurate?. Ital J Pediatr, 2011; 37:36.

56.

Pacifico

, et al. A double-blind, placebo-controlled randomized trial to evaluate the efficacy of docosahexaenoic acid supplementation on hepatic fat and associated cardiovascular risk factors in overweight children with nonalcoholic fatty liver disease. Nutr Metab Cardiovasc Dis, 2015; 25:734–741.

57.

Janczyk

, et al. Omega-3 Fatty acids therapy in children with nonalcoholic Fatty liver disease: A randomized controlled trial. J Pediatr, 2015; 166:1358–1363.e1–e3.

58.

Capanni

, et al. Prolonged n-3 polyunsaturated fatty acid supplementation ameliorates hepatic steatosis in patients with non-alcoholic fatty liver disease: A pilot study. Aliment Pharmacol Ther, 2006; 23:1143–1151.

59.

Vega

, et al. Effects of N-3 fatty acids on hepatic triglyceride content in humans. J Investig Med, 2008; 56:780–785.

60.

Cussons

, et al. Omega-3 fatty acid supplementation decreases liver fat content in polycystic ovary syndrome: A randomized controlled trial employing proton magnetic resonance spectroscopy. J Clin Endocrinol Metab, 2009; 94:3842–3848.

61.

Sofi

, et al. Effects of a 1-year dietary intervention with n-3 polyunsaturated fatty acid-enriched olive oil on non-alcoholic fatty liver disease patients: A preliminary study. Int J Food Sci Nutr, 2010; 61:792–802.

62.

, et al. Efficacy of poly-unsaturated fatty acid therapy on patients with nonalcoholic steatohepatitis. World J Gastroenterol, 2015; 21:7008–7013.

63.

Dasarathy

, et al. Double-blind randomized placebo-controlled clinical trial of omega 3 fatty acids for the treatment of diabetic patients with nonalcoholic steatohepatitis. J Clin Gastroenterol, 2015; 49:137–144.

64.

Scorletti

, et al. Design and rationale of the WELCOME trial: A randomised, placebo controlled study to test the efficacy of purified long chainomega-3 fatty acid treatment in non-alcoholic fatty liver disease [corrected]. Contemp Clin Trials, 2014; 37:301–311.

65.

Nogueira

, et al. Omega-3 polyunsaturated fatty acids in treating non-alcoholic steatohepatitis: A randomized, double-blind, placebo-controlled trial. Clin Nutr, 2016; 35:578–586.

66.

Hatzitolios

, et al. Efficacy of omega-3 fatty acids, atorvastatin and orlistat in non-alcoholic fatty liver disease with dyslipidemia. Indian J Gastroenterol, 2004; 23:131–134.

67.

Spadaro

, et al. Effects of n-3 polyunsaturated fatty acids in subjects with nonalcoholic fatty liver disease. Dig Liver Dis, 2008; 40:194–199.

68.

Zhu

, et al. Effects of n-3 polyunsaturated fatty acids from seal oils on nonalcoholic fatty liver disease associated with hyperlipidemia. World J Gastroenterol, 2008; 14:6395–6400.

69.

Argo

, et al. Effects of n-3 fish oil on metabolic and histological parameters in NASH: A double-blind, randomized, placebo-controlled trial. J Hepatol, 2015; 62:190–197.

70.

Scorletti

, et al. Effects of purified eicosapentaenoic and docosahexaenoic acids in nonalcoholic fatty liver disease: Results from the Welcome study. Hepatology, 2014; 60:1211–1221.

71.

Cottin

, Sanders

, Hall

. The differential effects of EPA and DHA on cardiovascular risk factors. Proc Nutr Soc, 2011; 70:215–231.

72.

Holub

. Docosahexaenoic acid (DHA) and cardiovascular disease risk factors. Prostaglandins Leukot Essent Fatty Acids, 2009; 81:199–204.

73.

Jacobson

, et al. Effects of eicosapentaenoic acid and docosahexaenoic acid on low-density lipoprotein cholesterol and other lipids: A review. J Clin Lipidol, 2012; 6:5–18.

74.

Kelley

, Adkins

. Similarities and differences between the effects of EPA and DHA on markers of atherosclerosis in human subjects. Proc Nutr Soc, 2012; 71:322–331.

75.

Lopez-Huertas

. The effect of EPA and DHA on metabolic syndrome patients: A systematic review of randomised controlled trials. Br J Nutr, 2012; 107 Suppl 2:S185–S194.

76.

Ryan

, et al. Clinical overview of algal-docosahexaenoic acid: Effects on triglyceride levels and other cardiovascular risk factors. Am J Ther, 2009; 16:183–192.

77.

Davidson

. Omega-3 fatty acids: New insights into the pharmacology and biology of docosahexaenoic acid, docosapentaenoic acid, and eicosapentaenoic acid. Curr Opin Lipidol, 2013; 24:467–474.

78.

Shapiro

, et al. The therapeutic potential of long-chain omega-3 fatty acids in nonalcoholic fatty liver disease. Clin Nutr, 2011; 30:6–19.

79.

Gotoh

, et al. Effects of three different highly purified n-3 series highly unsaturated fatty acids on lipid metabolism in C57BL/KsJ-db/db mice. J Agric Food Chem, 2009; 57:11047–11054.

80.

Depner

, Philbrick

, Jump

. Docosahexaenoic acid attenuates hepatic inflammation, oxidative stress, and fibrosis without decreasing hepatosteatosis in a Ldlr(−/−) mouse model of western diet-induced nonalcoholic steatohepatitis. J Nutr, 2013; 143:315–323.

81.

Lin

, et al. Enhanced amelioration of high-fat diet-induced fatty liver by docosahexaenoic acid and lysine supplementations. Biomed Res Int, 2014; 2014:310981.

82.

Vemuri

, et al. Docosahexaenoic acid (DHA) but not eicosapentaenoic acid (EPA) prevents trans-10, Cis-12 conjugated linoleic acid (CLA)-induced insulin resistance in mice. Metab Syndr Relat Disord, 2007; 5:315–322.

83.

Murali

, et al. Differential effects of eicosapentaenoic acid and docosahexaenoic acid in promoting the differentiation of 3T3-L1 preadipocytes. Prostaglandins Leukot Essent Fatty Acids, 2014; 90:13–21.

84.

Lee

, et al. Differential modulation of Toll-like receptors by fatty acids: Preferential inhibition by n-3 polyunsaturated fatty acids. J Lipid Res, 2003; 44:479–486.

85.

Mullen

, Loscher

, Roche

. Anti-inflammatory effects of EPA and DHA are dependent upon time and dose-response elements associated with LPS stimulation in THP-1-derived macrophages. J Nutr Biochem, 2010; 21:444–450.

86.

Yan

, et al. Omega-3 fatty acids prevent inflammation and metabolic disorder through inhibition of NLRP3 inflammasome activation. Immunity, 2013; 38:1154–1163.

87.

Takayama

, et al. Effects of docosahexaenoic acid in an experimental rat model of nonalcoholic steatohepatitis. J Oleo Sci, 2010; 59:407–414.

88.

Jump

, Tripathy

, Depner

. Fatty acid-regulated transcription factors in the liver. Annu Rev Nutr, 2013; 33:249–269.

89.

Sekiya

, et al. Polyunsaturated fatty acids ameliorate hepatic steatosis in obese mice by SREBP-1 suppression. Hepatology, 2003; 38:1529–1539.

90.

Davidson

. Mechanisms for the hypotriglyceridemic effect of marine omega-3 fatty acids. Am J Cardiol, 2006; 98:27i–33i.