In a randomized, placebo‐controlled trial, we tested the hypothesis that 15‐18 months of treatment with the highest licensed dose (4 g/day) of DHA+EPA was effective in ameliorating the early stages of NAFLD, 19 with primary outcomes related to (1) liver fat measured by magnetic resonance spectroscopy (MRS) scan in three discrete liver zones and (2) two algorithmically derived, histologically validated liver fibrosis scores. 20 , 21 We monitored adherence to the intervention in the DHA+EPA group, and potential contamination in the placebo group, by measuring erythrocyte enrichment of DHA or EPA between baseline and end of the study.

Omega‐3 fatty acid treatment is safe and has attracted considerable interest as a potential treatment for NAFLD. 16 A dose of 2‐4 g/day of omega‐3 fatty acids (eicosapentaenoic acid [EPA] plus docosahexaenoic acid [DHA]) is approved for treatment of hypertriglyceridemia. 17 However, whether DHA+EPA are effective in NAFLD is unproven. 18

Nonalcoholic fatty liver disease (NAFLD) is associated with metabolic syndrome (MetS) and is defined by the presence of ≥5% hepatic steatosis. NAFLD increases risk of chronic liver disease, hepatocellular carcinoma, type 2 diabetes, and cardiovascular disease, 1‐6 but there is no licensed treatment. Lifestyle change may ameliorate liver fat, 7‐9 but is difficult to achieve, and although treatment with thiazolidinediones 10 , 11 and vitamin E 12 has produced encouraging results in NAFLD, there are serious safety concerns about long‐term use of these agents. 13 - 15

All statistical analyses were performed using SPSS for Windows (version 21.0; SPSS, Inc., Chicago, IL). The primary endpoints were change in liver fat percentage and change in two histologically validated, algorithmically derived liver fibrosis biomarker scores (intention‐to‐treat [ITT] analyses). 20 , 21 Data are reported as means and 95% CIs or SDs for normally distributed variables, or as median and IQR, interquartile range for non‐normally distributed variables. Multivariable linear regression was undertaken with change in liver fat percentage or change in either of the histologically validated liver fibrosis scores as the outcome variable and adjusted for baseline. The ITT analysis included all patients randomized who had complete data (i.e., having baseline and end‐of‐study measurements), regardless of whether they were later found to be ineligible, a protocol violator, given the wrong treatment allocation, or never treated). Models are also reported as adjusted for potential confounders. For potential confounding factors that may have changed during the period of the study (e.g., weight, diet, or physical activity), we calculated the difference between baseline and end‐of‐study measurement. As a secondary analysis, the above was repeated with DHA+EPA treatment replaced by erythrocyte DHA and EPA enrichment. ITT and secondary analyses included all participants with baseline and end‐of‐study measurements, regardless of whether they were later found to be noncompliant, ineligible, or protocol violators. A P value of <0.05 was considered to be statistically significant.

Initially, we estimated that a 15% decrease in liver fat % would result from DHA+EPA treatment. 19 , 27 Subsequently, a meta‐analysis of seven studies suggested the pooled effect size of omega‐3 fatty acid treatment could be greater (Hedge's g pooled effect size = −0.97; 95% confidence interval [CI]: −1.35, −0.58; P < 0.0001). 18 With an estimated sigma of 0.3, 19 this would equal an ∼30% decrease in liver fat percentage with treatment. Adopting a more conservative estimate of the effect size (i.e., a 20% decrease in liver fat with DHA+EPA treatment), assuming a sigma of 0.3 and an alpha of 0.05 with 91 participants completing our trial, we had 86% power to detect a 20% change in liver fat (two‐tailed test). At the time of the study design, the change in liver fibrosis score 20 that equates to a clinically meaningful change was uncertain, and it was difficult to undertake a satisfactory sample‐size calculation to power the study sufficiently to test this primary outcome. However, based on the available evidence, we assumed that a 0.6‐ to 1.0‐unit change in fibrosis score might be clinically significant. 20 Consequently, to detect a minimum 0.6‐unit change in score (e.g., 9.0 at baseline and 8.4 at the end of the study) with a standard deviation (SD) of 1.0, 100 participants would provide >80% power at the 5% significance level, and with a 15% dropout of participants, there would also be >80% power to detect this effect. A sample‐size calculation was not undertaken for the NAFLD fibrosis score 20 , 21 because of the paucity of available data to guide a sample‐size calculation.

All measurements (including MRS liver fat percentage) were undertaken at baseline and end of study. Fibrosis markers, including hyaluronic acid (HA), procollagen‐III N‐terminal propeptide (PIIINP), and tissue inhibitor of matrix metalloproteinase 1 (TIMP‐1), were measured along with cytokeratin 18 (CK18), which has been used to assess nonalcoholic steatohepatitis (NASH). 24 Energy expenditure was assessed by measurement of metabolic equivalent of task (MET; SenseWear Pro 3 Armband monitor; BodyMedia, Inc., Pittsburgh, PA). Any change in diet during the study was assessed by food frequency questionnaire. We also generated a “prudent diet score” as a healthy diet index, using principal component analyses. 25

Inclusion and exclusion criteria have been described previously. 19 Briefly, subjects were eligible (1) with histological confirmation of NAFLD or (2) imaging evidence of liver fat (ultrasound, magnetic resonance imaging [MRI], or computed tomography scan), features of MetS, 22 and exclusion of other liver conditions causing liver fat accumulation or cirrhosis. Subjects were also excluded if alcohol consumption was >35 units (1 unit is 7.9 g of alcohol) per week for women and >50 units per week for men, which is the threshold for harmful alcohol consumption. 23 Additional exclusion criteria were pregnancy, breastfeeding, and hypersensitivity to DHA+EPA, soya, or the excipients.

One hundred and three participants were randomized (Fig. 1 ) to either Omacor (DHA+EPA) 4 g per day (n = 51; 1 g of Omacor contains 460 mg of EPA and 380 mg of DHA as ethyl esters] or 4 g per day of placebo (olive oil; n = 52; 1 g of olive oil contains 600 mg of oleic acid plus lesser amounts of linoleic, palmitic, stearic, and α‐linolenic acids]. DHA+EPA and placebo capsules were gelatine coated and of similar appearance and taste. We assessed erythrocyte EPA and DHA enrichment (between baseline and end of study) to test adherence to the intervention in the DHA+EPA group and monitor contamination with DHA and EPA in the placebo group. Compliance with the allocated medication was also monitored by recording returned unused capsules. Adverse events (AEs) were recorded.

The design and rationale for the WELCOME study (Wessex Evaluation of fatty Liver and Cardiovascular markers in NAFLD with OMacor thErapy) have been reported previously. 19 The WELCOME study was approved by the Southampton and South West Hampshire local research ethics committee (08/H0502/165). All participants gave written informed consent. Omacor (DHA+EPA as ethyl esters), also known as Lovaza, was provided free of charge by Pronova BioPharma/Abbott (Pronova BioPharma ASA, Lysaker, Norway; Abbott Laboratories, Southampton, UK). The conduct of the trial, data analyses, and writing of the manuscript were all undertaken by the authors and were completely independent from Pronova BioPharma/Abbott. The primary endpoint was to test whether treatment with purified DHA+EPA over a maximum of 18 months and a minimum of 15 months (1) decreased liver fat percentage measured by MRS (we calculated the average liver fat percentage for three liver segments: segments 3 [inferior subsegment of the lateral segment], 5 [inferior subsegment of the anterior segment], and 8 [superior subsegment of the anterior segment]) and (2) improved two histologically validated liver fibrosis scores. 20 , 21

We further investigated the magnitude of the effect of each 1% DHA enrichment to decrease liver fat percentage adjusting for change in physical activity during the study (available in n = 82 of 91 participants). In a regression model that included liver fat percentage difference as the outcome, and age, sex, difference in body weight (kg), difference in CK18 (as a marker of apoptosis and necrosis), difference in MET, between baseline and follow‐up, and DHA percentage enrichment as the explanatory variables; each 1% DHA enrichment was associated with a 3.3% reduction in liver fat percentage (β = −3.3; 95% CI: −4.8, −1.8; P < 0.0001).

Scatter plots showing the relationships between baseline liver fat % at recruitment and the change in liver fat % between baseline and end of study for all participants stratified by erythrocyte DHA enrichment (A) ≥2% DHA enrichment (between baseline and end of study) or (B) <2% erythrocyte DHA enrichment.

Because there was clear evidence of contamination with DHA and EPA enrichment in some participants in the placebo group, and there was also poor enrichment with DHA and EPA in some participants in the treatment group (Fig. 2 ), we assessed the relationships between liver fat percentage at recruitment and the change in liver fat percentage between baseline and end of study in all participants, stratified by the prespecified threshold for erythrocyte DHA enrichment (<2% and ≥2%) and regardless of randomization group (Fig. 3 ). For those individuals achieving a ≥2% absolute increase in DHA between baseline and end of study, there was a strong inverse association between liver fat percentage at recruitment and the change in liver fat percentage between baseline and end of study.

Table 3 shows baseline and end‐of‐study data for each of the primary outcomes and the results of regression models for the ITT analyses and secondary analyses. Data for change in plasma triglyceride (TG) concentration are shown for comparison (Omacor is licensed for lowering plasma TG concentrations). In the ITT analyses, in the fully adjusted model, there was a 3.64% decrease in liver fat % (β = −3.64; 95% CI: −8.0, 0.8; P = 0.1) with DHA+EPA treatment. We undertook secondary analyses (Table 3 ) to test the association between DHA or EPA enrichment and each of the primary outcomes (adjustments as per the ITT analysis). Erythrocyte DHA enrichment was independently associated with a decrease in liver fat percentage (−1.7% for each 1% DHA enrichment; β = −1.7; 95% CI: −2.9, −0.5; P = 0.007 in the fully adjusted model). In our cohort, there were only 9 patients with high NAFLD fibrosis score 21 and 14 with high liver fibrosis score. 20 There was no improvement in either liver fibrosis score with DHA+EPA or with DHA or EPA enrichment.

We hypothesized a priori that DHA+EPA intervention should produce a minimum 2% increase in erythrocyte DHA and a minimum 0.7% increase in erythrocyte EPA 26 , 28 to produce an effect. Figure 2 shows the percentage enrichment in erythrocyte DHA and EPA between baseline and end of study in participants randomized to placebo or DHA+EPA. Enrichment was highly variable in the DHA+EPA group and 5 and 6 participants in the DHA+EPA group did not reach the prespecified threshold for EPA and DHA enrichment, respectively. In the placebo group, we expected no enrichment between baseline and end of study in all participants in this group, but 3 and 4 participants reached the thresholds set for the DHA +EPA group, for EPA and DHA, respectively (Fig. 2 ). One participant in the placebo group admitted to taking cod liver oil during the study and another markedly increased consumption of fish.

From capsule counts at 6 and 12 months and at the end of study, we estimated that all participants consumed >50% of their study medication and 78% consumed >75%. No serious AEs occurred that were attributed to medication. Alcohol consumption was not associated with baseline liver fat percentage ( P = 0.93).

Ninety‐five participants completed the study (55 men and 40 women). Table 1 shows the baseline characteristics of participants according to randomization group. Table 2 shows the changes between baseline and end‐of‐trial measurements, stratified by randomization group, for the main anthropometric and biochemical variables. Inadequate MRS data were obtained for 4 of 95 participants, and end‐of‐study liver fat percentage was available on 91 participants for the primary and secondary analyses.

Discussion

Our study is the first randomized, double‐blind, placebo‐controlled trial to test the efficacy of a high‐dose DHA+EPA intervention on a quantitative measurement of liver fat in NAFLD and relate changes in erythrocyte DHA+EPA enrichment to changes in liver fat percentage. Although we were not able to prove that there was a significant effect of the intervention on the primary outcomes in the ITT analyses, our novel results show, in the secondary analyses, that erythrocyte DHA enrichment with DHA+EPA treatment is linearly associated with decreased liver fat percentage, and substantial decreases in liver fat percentage can be achieved with high‐percentage DHA enrichment. The ITT analyses showed a strong trend toward a decrease in liver fat percentage with DHA+EPA treatment, but there was strong evidence for contamination with DHA and EPA enrichment in the placebo group and poor adherence to DHA+EPA intervention in the treatment arm. (There was evidence of poor adherence to the intervention, or contamination with DHA+EPA in 10 of 95 participants completing the trial.) Poor adherence to the DHA+EPA intervention (in the treatment group), or contamination with DHA and EPA (in the placebo group), would bias the result toward the null, attenuating any effect of the DHA+EPA intervention on primary outcomes. In contrast, the secondary analysis (undertaken in all participants) circumvented this problem, because randomization to Omacor (as a dichotomous exposure variable) was replaced in the regression model by either erythrocyte DHA percentage enrichment or erythrocyte EPA percentage enrichment, as a continuous exposure variable. Thus, there was no stratification by randomization group in this analysis, and so this additional analysis allowed us to test the effect of percentage DHA enrichment (or percentage EPA enrichment), regardless of randomization group on each of the primary outcomes. The data show the effect of each 1% DHA enrichment to decrease liver fat percentage (Table 3). We have presented data on all participants in Table 3 and have not excluded protocol violators. Further regression analysis was undertaken after excluding the 2 participants who admitted to being protocol violators. With these 2 participants excluded, there was improvement in the effect of DHA+EPA treatment to decrease liver fat percentage (−4.2%; β = −4.2; 95% CI: −8.6, 0.1; P = 0.057).

The omega‐3 index is the sum of DHA and EPA in erythrocyte membranes and is expressed as a percentage of total erythrocyte fatty acids.29 We anticipated an erythrocyte sum of DHA and EPA (omega‐3 index)29 of between 4 and 5 at baseline (which is associated with an intermediate level of cardiovascular risk [CVR]) and found this to be the case. Furthermore, we considered that a 0.7% increase in EPA and a 2% increase in DHA would be sufficient to improve the index to a value proposed to result in significantly decreased CVR.29

We did not specifically select participants with high liver fat % at baseline, but patients with high liver fat percentage would likely derive most benefit from achieving good DHA enrichment, because we show that, for a reasonably large (6%) enrichment in DHA, there was a (6 × 3.3%) = ∼20% decrease in liver fat percentage. Consequently, for individuals with >80% liver fat, such an absolute reduction in liver fat would be considerable. This benefit, and greater, was noted in many of the individuals treated with DHA+EPA.

We found no suggestion of benefit of DHA+EPA treatment on the two liver fibrosis scores that might have indicated an effect of treatment in NASH, but we suggest that treatment with DHA+EPA needs to be tested for a longer period than 18 months if any improvement in liver fibrosis is secondary to improvements in liver fat. Importantly, we did not observe any deterioration in either fibrosis score during the period of the study.

Very recently, Dasarathy et al. investigated the effects of omega‐3 fatty acids in 37 patients with diabetes and NAFLD.30 These investigators found no effect of omega‐3 fatty acids on liver fat, but there was no estimation of tissue enrichment of omega‐3 fatty acids in this trial. By contrast, Nobili et al. tested the effect of 18 months of treatment with DHA in children with NAFLD. Liver biopsy was undertaken before and after treatment. In this study, the investigators found that DHA improved hepatic steatosis, ballooning, and inflammation.31

Previous studies have investigated the effect of omega‐3 fatty acids in NAFLD.18,32‐34 These studies differed from ours with regard to (1) duration of the treatment, (2) dosage of omega‐3 fatty acids, (3) composition of the omega‐3 fatty acid treatment, and (4) lack of testing for both adherence to the omega‐3 intervention and for contamination with omega‐3 fatty acids obtained for other readily available sources, using DHA and EPA measurements. Notably, the limitations of these studies were the nonblinding of participants and investigators, the lack of a placebo for the control group, the semiquantitative measures for assessing changes in liver fat percentage, and the short periods of the intervention.

We selected the highest licensed dose of purified DHA+EPA, because 4 g/day of Omacor has been shown to be effective in hypertriglyceridemia.17 By using such a pharmaceutical‐grade preparation of omega‐3 fatty acids, we also avoided the fat‐soluble vitamins, A and D, contained within fish oil preparations.

The main limitation of our study is the small sample size. However, ours was a proof‐of‐concept study to test efficacy of the DHA+EPA intervention and DHA and EPA enrichment to decrease liver fat percentage and liver fibrosis scores. We tested whether the intervention changed both liver fibrosis scores, analyzing each of the fibrosis score as continuous variables, and did not attempt to treat the variables as categorical variables to define more‐severe forms of liver fibrosis.

A priori, we set an alcohol threshold to exclude harmful alcohol intake, which, we acknowledge, is higher than the usual threshold to define NAFLD. However, only 1 man and 1 woman had previously consumed alcohol above UK Governmental Guidelines (21 and 14 units of alcohol per week for men and women, respectively) preceding enrollment. Exclusion of these 2 subjects did not affect the results (data not shown), and inclusion of baseline alcohol consumption as a continuous exposure variable in the regression model shown in Table 3 did not affect the association between DHA and liver fat percentage. Furthermore, in this model, alcohol intake was not associated with liver fat percentage (β = 0.005; 95% CI: −0.304, 0.314; P = 0.98). However, we documented alcohol consumption only at baseline and at end of study; therefore, there is the possibility that, during the study, alcohol consumption may have changed. The issue of histological monitoring for NAFLD in this trial is discussed in detail in the design and rationale of the study article.19 Although many participants who were recruited had a baseline diagnostic liver biopsy, a further biopsy was not feasible in this trial at the end of the study (see previous work19). That said, MRS is currently considered the noninvasive gold‐standard technique for assessing liver fat percentage and has excellent reproducibility and sensitivity,35, 36 with a coefficient of variance of only 8%,35 and liver fat signals of only 0.2% are clearly evident above the noise level.36 Furthermore, liver biopsy is invasive, expensive, and subject to sampling variability, and many investigators currently consider it a high‐risk procedure that is unacceptable as a research test for monitoring NAFLD. Also, liver biopsy evaluates only a tiny portion (0.05 cm3) of the liver (800‐1,000 cm3), and NAFLD is often a patchy disease.

In conclusion, erythrocyte DHA enrichment with DHA+EPA treatment is linearly associated with decreased mean liver fat percentage, calculated from liver fat percentage in three discrete liver regions, in patients with NAFLD. These data suggest that substantial decreases in liver fat percentage can be achieved with high levels of erythrocyte DHA enrichment in patients with NAFLD.