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Improvement in non-alcoholic fatty liver disease severity is associated with a reduction in carotid intima-media thickness progression

  • Lokpal Bhatia
    Correspondence
    Corresponding author. Cardiologist & Clinical Research Fellow, Human Development and Health Academic Unit, University of Southampton, IDS Building, MP 887 Southampton General Hospital, Tremona Road, Southampton SO16 6YD, United Kingdom.
    Affiliations
    Human Development and Health Academic Unit, Faculty of Medicine, University of Southampton, Southampton, UK

    National Institute for Health Research Southampton Biomedical Research Centre, University of Southampton and University Hospital Southampton NHS Foundation Trust, Southampton, UK

    Wessex Cardiac Centre, University Hospital Southampton NHS Foundation Trust, Southampton, UK
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  • Eleonora Scorletti
    Affiliations
    Human Development and Health Academic Unit, Faculty of Medicine, University of Southampton, Southampton, UK

    National Institute for Health Research Southampton Biomedical Research Centre, University of Southampton and University Hospital Southampton NHS Foundation Trust, Southampton, UK
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  • Nicholas Curzen
    Affiliations
    National Institute for Health Research Southampton Biomedical Research Centre, University of Southampton and University Hospital Southampton NHS Foundation Trust, Southampton, UK

    Wessex Cardiac Centre, University Hospital Southampton NHS Foundation Trust, Southampton, UK
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  • Geraldine F. Clough
    Affiliations
    Human Development and Health Academic Unit, Faculty of Medicine, University of Southampton, Southampton, UK

    National Institute for Health Research Southampton Biomedical Research Centre, University of Southampton and University Hospital Southampton NHS Foundation Trust, Southampton, UK
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  • Philip C. Calder
    Affiliations
    Human Development and Health Academic Unit, Faculty of Medicine, University of Southampton, Southampton, UK

    National Institute for Health Research Southampton Biomedical Research Centre, University of Southampton and University Hospital Southampton NHS Foundation Trust, Southampton, UK
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  • Christopher D. Byrne
    Affiliations
    Human Development and Health Academic Unit, Faculty of Medicine, University of Southampton, Southampton, UK

    National Institute for Health Research Southampton Biomedical Research Centre, University of Southampton and University Hospital Southampton NHS Foundation Trust, Southampton, UK
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      Highlights

      • We studied the effects of high-dose n-3 PUFA vs placebo on CIMT progression over 18 months.
      • We also analysed the association between biomarkers of NAFLD severity and CIMT progression.
      • No significant effect of n-3 PUFA vs placebo on altering CIMT progression.
      • Reduced CIMT progression was independently associated with decreased liver fat and reduced CK-18 levels over 18 months.

      Abstract

      Background and aims

      n-3 polyunsaturated fatty acid (PUFA) treatment may decrease liver fat in non-alcoholic fatty liver disease (NAFLD), but uncertainty exists whether this treatment also decreases cardiovascular disease (CVD) risk in NAFLD. We tested whether 15–18 months n-3 PUFA [docosahexaenoic acid (DHA) and eicosapentaenoic acid] (Omacor/Lovaza, 4 g/day) vs placebo decreased carotid intima-media thickness (CIMT) progression, a surrogate marker of CVD risk. We also evaluated if improvement in markers of NAFLD severity was associated with decreased CIMT progression over time.

      Methods

      In a pre-specified sub-study of the WELCOME (Wessex Evaluation of fatty Liver and Cardiovascular markers in NAFLD with OMacor thErapy) trial (NCT00760513), CIMT was measured using B-mode ultrasound while NAFLD severity was assessed by measuring liver fat percentage (magnetic resonance spectroscopy) and hepatic necro-inflammation (serum cytokeratin-18 (CK-18) concentration), at baseline and end of study.

      Results

      92 patients (age 51.5 ± 10.7 years, 57.6% men) completed the study. In the treatment group (n = 45), CIMT progressed by 0.012 mm (IQR 0.005–0.020 mm) compared to 0.015 mm (IQR 0.007–0.025 mm) in the placebo group (n = 47) (p = 0.17). Reduced CIMT progression in the entire cohort was independently associated with decreased liver fat (standardized β-coefficient 0.32, p = 0.005), reduced CK-18 levels (standardized β-coefficient 0.22, p = 0.04) and antihypertensive usage (standardized β-coefficient −0.31, p = 0.009) in multivariable regression analysis after adjusting for all potential confounders. Decreased weight (standardized β-coefficient 0.30, p < 0.001) and increased DHA tissue enrichment during the 18-month study (standardized β-coefficient −0.19, p = 0.027) were both independently associated with decreased liver fat, but not with CK-18.

      Conclusion

      Improvement in two markers of NAFLD severity is independently associated with reduced CIMT progression.

      Keywords

      1. Introduction

      Non-alcoholic fatty liver disease (NAFLD) is a common condition associated with obesity and type 2 diabetes, and is prevalent in up to a third of the general population [
      • Browning J.D.
      • Szczepaniak L.S.
      • Dobbins R.
      • Nuremberg P.
      • Horton J.D.
      • Cohen J.C.
      • Grundy S.M.
      • Hobbs H.H.
      Prevalence of hepatic steatosis in an urban population in the United States: impact of ethnicity.
      ]. Current evidence demonstrates a robust association between NAFLD and atherosclerotic disease such that, for example, cardiovascular (CV) mortality represents the main mode of death in NAFLD and is also linked to the severity of liver disease [
      • Bhatia L.S.
      • Curzen N.P.
      • Calder P.C.
      • Byrne C.D.
      Non-alcoholic fatty liver disease: a new and important cardiovascular risk factor?.
      ]. This progressive disease spectrum ranges from simple hepatic steatosis to varying grades of non-alcoholic steatohepatitis (NASH) and ultimately cirrhosis. Carotid intima-media thickness (IMT) is a well-established marker of subclinical atherosclerosis and is predictive of subsequent CV events in asymptomatic subjects [
      • Lorenz M.W.
      • Markus H.S.
      • Bots M.L.
      • Rosvall M.
      • Sitzer M.
      Prediction of clinical cardiovascular events with carotid intima-media thickness: a systematic review and meta-analysis.
      ]. Measurement of carotid IMT progression is commonly used in intervention trials as a surrogate end-point for adverse clinical events, as well as evaluating efficacy of specific atherosclerotic-modifying therapies [
      • Bots M.L.
      • Evans G.W.
      • Riley W.A.
      • Grobbee D.E.
      Carotid intima-media thickness measurements in intervention studies: design options, progression rates, and sample size considerations: a point of view.
      ]. Importantly, carotid IMT offers good inter-scan as well as good inter- and intra-observer reproducibility with intra-class correlation coefficients greater than 0.90 in previous trials utilising carotid IMT progression as a primary end-point [
      • Duivenvoorden R.
      • de Groot E.
      • Stroes E.S.
      • Kastelein J.J.
      Surrogate markers in clinical trials–challenges and opportunities.
      ]. Although NAFLD is described as an independent risk factor for increased carotid IMT [
      • Gastaldelli A.
      • Kozakova M.
      • Hojlund K.
      • Flyvbjerg A.
      • Favuzzi A.
      • Mitrakou A.
      • Balkau B.
      Fatty liver is associated with insulin resistance, risk of coronary heart disease, and early atherosclerosis in a large European population.
      ], no studies to date have investigated whether a reduction in the degree of fatty liver disease is associated with modification of carotid IMT progression. Our group recently described a significant reduction in liver fat percentage after 18 months treatment with high-dose n-3 polyunsaturated fatty acids (PUFAs) i.e. docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) (Omacor/Lovaza 4 g/day) versus placebo in subjects with a high level of erythrocyte DHA enrichment [
      • Scorletti E.
      • Bhatia L.
      • McCormick K.G.
      • Clough G.F.
      • Nash K.
      • Hodson L.
      • Moyses H.E.
      • Calder P.C.
      • Byrne C.D.
      • Study W.
      Effects of purified eicosapentaenoic and docosahexaenoic acids in nonalcoholic fatty liver disease: results from the Welcome study.
      ]. Furthermore, studies from other groups have shown a positive association between high dietary intake of n-3 PUFAs and less carotid atherosclerotic plaque burden [
      • Sekikawa A.
      • Curb J.D.
      • Ueshima H.
      • et al.
      Marine-derived n-3 fatty acids and atherosclerosis in Japanese, Japanese-American, and white men: a cross-sectional study.
      ], as well as a plaque-stabilizing effect on carotid atheroma and improved arterial elasticity with n-3 PUFA treatment [
      • Cawood A.L.
      • Ding R.
      • Napper F.L.
      • Young R.H.
      • Williams J.A.
      • Ward M.J.
      • Gudmundsen O.
      • Vige R.
      • Payne S.P.
      • Ye S.
      • Shearman C.P.
      • Gallagher P.J.
      • Grimble R.F.
      • Calder P.C.
      Eicosapentaenoic acid (EPA) from highly concentrated n-3 fatty acid ethyl esters is incorporated into advanced atherosclerotic plaques and higher plaque EPA is associated with decreased plaque inflammation and increased stability.
      ,
      • Hjerkinn E.M.
      • Abdelnoor M.
      • Breivik L.
      • Bergengen L.
      • Ellingsen I.
      • Seljeflot I.
      • Aase O.
      • Ole Klemsdal T.
      • Hjermann I.
      • Arnesen H.
      Effect of diet or very long chain omega-3 fatty acids on progression of atherosclerosis, evaluated by carotid plaques, intima-media thickness and by pulse wave propagation in elderly men with hypercholesterolaemia.
      ].
      The aims of this prespecified substudy of the WELCOME (Wessex Evaluation of fatty Liver and Cardiovascular markers in NAFLD with OMacor thErapy) trial [
      • Scorletti E.
      • Bhatia L.
      • McCormick K.G.
      • Clough G.F.
      • Nash K.
      • Calder P.C.
      • Byrne C.D.
      • Investigators W.T.
      Design and rationale of the WELCOME trial: a randomised, placebo controlled study to test the efficacy of purified long chain omega-3 fatty treatment in non-alcoholic fatty liver disease.
      ] were: (i) to investigate the effects of high-dose n-3 PUFA treatment over 18 months on carotid IMT progression in NAFLD subjects and (ii) to describe associations between changes in markers of NAFLD disease severity (both in terms of simple steatosis and steatohepatitis) and carotid IMT progression over that time period.

      2. Material and methods

      2.1 Subjects & study design

      The present study included participants from the WELCOME study, which was a phase IV randomised, double-blind, placebo-controlled clinical trial undertaken in asymptomatic patients with NAFLD testing the effects of 15–18 months treatment with high-dose (3.6 g/day) purified ω-3 polyunsaturated fatty acids (PUFA) comprising DHA (46%) and EPA (38%) (Omacor 4 g/day; Pronova Biopharma ASA, Lysaker, Norway; Abbott Laboratories, Southampton, UK) on improving liver fat and/or fibrosis markers as its primary outcome (www.clinicaltrials.gov NCT00760513). The design and rationale for the WELCOME study have been reported previously [
      • Scorletti E.
      • Bhatia L.
      • McCormick K.G.
      • Clough G.F.
      • Nash K.
      • Calder P.C.
      • Byrne C.D.
      • Investigators W.T.
      Design and rationale of the WELCOME trial: a randomised, placebo controlled study to test the efficacy of purified long chain omega-3 fatty treatment in non-alcoholic fatty liver disease.
      ]. Briefly, subjects were included if they had recent imaging evidence of liver fat (ultrasound, magnetic resonance imaging or computerised tomography scan) and/or histological confirmation of NAFLD as well as exclusion of all other liver conditions causing liver fat accumulation such as excess alcohol intake or evidence of cirrhosis. The study was approved by the Southampton and South West Hampshire NHS Ethics Committee and all participants gave their written informed consent.
      103 participants were randomised (Fig. 1) to either Omacor 4 g/day (n = 51) or placebo (olive oil capsules, n = 52) in a 1:1 double-blind fashion, with treatment duration of between 15 and 18 months. Compliance with the allocated medication was monitored by recording returned capsules at fixed intervals during the study. We also assessed erythrocyte EPA and DHA enrichment (between baseline and end of study) by gas chromatography [
      • Scorletti E.
      • Bhatia L.
      • McCormick K.G.
      • Clough G.F.
      • Nash K.
      • Calder P.C.
      • Byrne C.D.
      • Investigators W.T.
      Design and rationale of the WELCOME trial: a randomised, placebo controlled study to test the efficacy of purified long chain omega-3 fatty treatment in non-alcoholic fatty liver disease.
      ] to test adherence to the intervention in the DHA + EPA group and to monitor dietary contamination with DHA and EPA in the placebo group. Dietary and lifestyle changes had already been recommended to all participants as part of their routine clinical care [
      • Ratziu V.
      • Bellentani S.
      • Cortez-Pinto H.
      • Day C.
      • Marchesini G.
      A position statement on NAFLD/NASH based on the EASL 2009 special conference.
      ] and this was continued throughout the study. There were no additional specific weight-loss programs or strict dietary restrictions placed on the participants as part of the study.
      Figure thumbnail gr1
      Fig. 1Consort diagram showing recruitment for the WELCOME sub-study and the numbers of participants available for carotid analysis to test the effects of the intervention. For the reasons for withdrawal from the study, see (end of text).

      2.2 Laboratory and anthropometry measurements

      Blood samples were drawn after an overnight fast (>12 h) and serum was separated within 1 h to undergo routine biochemical assay by conventional enzymatic methods (lipids, glucose, liver transaminases). Plasma and serum samples were also frozen at −70 °C for further analysis in batches (insulin and cytokeratin-18 (CK-18) subfraction M65 measured by M65 EpiDeath enzyme-linked immunosorbent assay kit (PEVIVA, Bromma, Sweden). CK-18 is a major intermediate filament protein in hepatocytes, which is released into the extracellular compartment following epithelial cell death. M65 levels not only reflect hepatocellular apoptosis and necrosis consistent with changes seen in NASH, but they have also been shown to correlate with fibrosis progression [
      • Shen J.
      • Chan H.L.
      • Wong G.L.
      • Chan A.W.
      • Choi P.C.
      • Chan H.Y.
      • Chim A.M.
      • Yeung D.K.
      • Yu J.
      • Chu W.C.
      • Wong V.W.
      Assessment of non-alcoholic fatty liver disease using serum total cell death and apoptosis markers.
      ].
      Blood pressure was measured using a Marquette Dash 3000 monitor (GE Healthcare, Bucks, UK) on the non-dominant arm in the supine position after a minimum of 60 min rest and a mean of 3 measurements 5 min apart was taken. Body mass index (BMI) was calculated as weight (kg) divided by the square of height (m).

      2.3 Liver fat assessment

      Magnetic resonance spectroscopy (MRS) is a quick and safe, non-invasive tool to accurately quantify liver fat in NAFLD and correlates well with liver biopsy results. Due to its high sensitivity, it can detect small percentage differences in liver fat accumulation in NAFLD [
      • Mehta S.R.
      • Thomas E.L.
      • Bell J.D.
      • Johnston D.G.
      • Taylor-Robinson S.D.
      Non-invasive means of measuring hepatic fat content.
      ]. The full methodology of our MRS technique has already been described [
      • Scorletti E.
      • Bhatia L.
      • McCormick K.G.
      • Clough G.F.
      • Nash K.
      • Calder P.C.
      • Byrne C.D.
      • Investigators W.T.
      Design and rationale of the WELCOME trial: a randomised, placebo controlled study to test the efficacy of purified long chain omega-3 fatty treatment in non-alcoholic fatty liver disease.
      ]. Briefly, subjects had liver MRS scans at the start and end of the study. Three 20 × 20 × 20 mm3 spectroscopic volumes of interest (VOI) were positioned in three standard areas of the liver and the average of each VOI's lipid spectroscopic peak was used to calculate the percentage liver fat. VOIs remained constant for end of study measurements.

      2.4 Carotid ultrasonography

      The carotid arteries were studied with a duplex scanner using a 7.5 MHz linear array transducer (Philips IE33, Koninklijke Philips N.V., Netherlands) with ECG monitoring. Ultrasound parameters (dynamic range, depth range, power output and greyscale) for B-Mode carotid imaging were adjusted during image acquisition to optimize image quality. All scans were carried out according to a standardized protocol [
      • Stein J.H.
      • Korcarz C.E.
      • Hurst R.T.
      • Lonn E.
      • Kendall C.B.
      • Mohler E.R.
      • Najjar S.S.
      • Rembold C.M.
      • Post W.S.
      American Society of Echocardiography Carotid Intima-Media Thickness Task F
      Use of carotid ultrasound to identify subclinical vascular disease and evaluate cardiovascular disease risk: a consensus statement from the American Society of Echocardiography Carotid Intima-Media thickness task Force. Endorsed by the Society for vascular Medicine.
      ]. Briefly, subjects lay supine with the neck slightly rotated and a transverse scan was first performed as a screening measure and also to identify the carotid bifurcation. Longitudinal images of the near and far walls of the common, proximal portion of the internal and external carotid arteries and the carotid bifurcation were examined and multiple images of 4 cine–loop cycles of the carotid artery were recorded and stored digitally for subsequent off-line analysis using Philips Q-Lab version 8 software (Koninklijke Philips N.V., Netherlands). For each subject, a 10 mm plaque-free segment of IMT at the far wall of the common carotid artery immediately proximal to the carotid bulb was measured at ECG-gated end-diastole using QLAB automated software. An average of three different cardiac cycle measurements of IMT from each of the left and right common carotid arteries was calculated. The presence of carotid plaque at the distal common carotid, carotid bulb and proximal internal carotid arteries was also recorded [
      • Stein J.H.
      • Korcarz C.E.
      • Hurst R.T.
      • Lonn E.
      • Kendall C.B.
      • Mohler E.R.
      • Najjar S.S.
      • Rembold C.M.
      • Post W.S.
      American Society of Echocardiography Carotid Intima-Media Thickness Task F
      Use of carotid ultrasound to identify subclinical vascular disease and evaluate cardiovascular disease risk: a consensus statement from the American Society of Echocardiography Carotid Intima-Media thickness task Force. Endorsed by the Society for vascular Medicine.
      ]. All measurements were performed and analysed off-line by a single trained operator blinded to subject treatment allocation. A random sample of 36 scans (baseline and end-of-study) underwent repeat analysis at a later date to test intraobserver reproducibility and the coefficient of variation was 2.8%, which is within the limit of 6% recommended by the American Society of Echocardiography [
      • Stein J.H.
      • Korcarz C.E.
      • Hurst R.T.
      • Lonn E.
      • Kendall C.B.
      • Mohler E.R.
      • Najjar S.S.
      • Rembold C.M.
      • Post W.S.
      American Society of Echocardiography Carotid Intima-Media Thickness Task F
      Use of carotid ultrasound to identify subclinical vascular disease and evaluate cardiovascular disease risk: a consensus statement from the American Society of Echocardiography Carotid Intima-Media thickness task Force. Endorsed by the Society for vascular Medicine.
      ].

      2.5 Statistical analysis

      Statistical analysis was carried out with SPSS Version 21 (SPSS, Inc., Chicago, IL.) Mean values and standard deviations were calculated for continuous variables, or median and interquartile range values for non-normally distributed variables. Univariate comparisons of normally distributed data were performed with independent Student's t-tests. Mann–Whitney U or Wilcoxon signed rank tests were used for non-parametric data and Chi-squared tests for binary data. Pearson and Spearman correlations were used for normal and non-normally distributed data respectively. Log transformation was undertaken for non-normal variables where necessary. Exposure variables which showed a significant univariate association with the outcome variable, as well as key baseline variables that might confound the association between exposures and the outcome of interest were included in the multivariable stepwise regression model. A “difference” variable, which represented the arithmetic difference between the measurement at the end of the study minus the baseline measurement, was calculated for key exposures and potential confounders. All comparisons were two-sided and a p-value of <0.05 was considered to be statistically significant.

      3. Results

      Data from 47 participants randomized to placebo and 45 randomized to DHA + EPA treatment was available for analysis out of n = 103. Eight participants did not complete the study and a further three were excluded from the carotid analysis due to inadequate carotid data (one subject had images corrupted during transfer offline) or unsuitable images for adequate analysis (one had poor imaging quality and another had untreated familial hyperlipidaemia with significant widespread carotid plaque disease). From capsule counts at 6, 12 and 18 months, we estimated that all participants consumed >50% of their study medication and 78% consumed >75%. There were no serious adverse events attributable to study drugs. All participants consumed less than 21 units of alcohol per week at randomization apart from 1 volunteer who drank 25 units/week, with no significant difference between groups (p = 0.33). Alcohol consumption was also not associated with baseline liver fat (p = 0.45) and did not vary significantly for each subject at the end of the study (p = 0.92). The baseline characteristics of the participants according to randomization group are shown in Table 1.
      Table 1Baseline characteristics of participants in placebo and DHA + EPA groups at randomization.
      Placebo (n = 47)DHA + EPA (n = 45)p-value
      Age (years)54.2 ± 9.448.6 ± 11.20.09
      Male/Female31/1622/230.10
      Diabetes (%)34.035.60.88
      Dietary control (%)4.32.10.56
      Oral anti-diabetic (%)19.126.70.51
      Insulin use (%)10.66.70.48
      Hypertension (%)48.953.30.67
      Smoker (%)10.611.10.94
      Ex-smoker (%)36.231.10.59
      Antihypertensive use (%)36.240.00.71
      Statin use (%)46.840.00.51
      Total cholesterol (mmol/L)4.72 ± 1.105.05 ± 1.190.17
      Low density lipoprotein cholesterol (mmol/L)2.77 ± 0.832.99 ± 0.990.29
      High density lipoprotein cholesterol (mmol/L)1.11 ± 0.261.02 ± 0.270.12
      Serum triglycerides (mmol/L)1.4 (1.1, 2.0)1.8 (1.4, 2.6)0.03
      Fasting glucose (mmol/L)5.4 (5.1, 6.9)5.5 (4.9, 6.2)0.37
      Haemoglobin A1c (% total Hb)6.1 (5.7, 7.3)5.9 (5.5, 6.7)0.29
      Fasting insulin (μU/ml)11.5 (8.0, 21.1)13.9 (7.8, 19.4)0.90
      120 min oral glucose tolerance test (mmol/L)7.9 ± 2.27.5 ± 1.80.50
      Alanine transaminase (iu/L)56 (40, 73)53 (30, 72)0.38
      Aspartate aminotransferase (iu/L)41 (31, 54)37 (27, 49)0.15
      Weight (kg)93.1 ± 14.698.7 ± 17.70.10
      Body mass index (kg/m2)31.7 (28.7, 33.7)32.8 (30.6, 37.4)0.03
      Systolic blood pressure (mmHg)138 ± 16138 ± 170.86
      Diastolic blood pressure (mmHg)85 ± 885 ± 120.89
      CK-18 M65 (U/L)605 (267, 792)388 (245, 785)0.28
      MRS Liver fat (%)22.8 (13.7, 32.3)23.3 (12.7, 47.5)0.75
      Carotid IMT (mm)0.674 ± 0.0980.649 ± 0.0950.21
      Presence of any carotid plaque (%)68.155.60.22
      Erythrocyte EPA (20:5n3) (%)0.95 ± 0.390.89 ± 0.360.44
      Erythrocyte DHA (22:6n3) (%)4.21 ± 1.493.87 ± 1.290.24
      Data are means ± standard deviation (SD), or median (25th, 75th percentiles).
      Abbreviations: DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; CK-18 M65, cytokeratin-18 M65 subfraction; MRS, magnetic resonance spectroscopy; IMT, intima-media thickness.
      Baseline characteristics for the treatment and placebo groups were very similar, apart from the treatment group having higher baseline fasting serum triglyceride concentration (1.8 vs 1.4 mmol/L, p = 0.03), and a higher baseline BMI (32.8 vs 31.7, p = 0.03) compared to the control group. Liver fat, CK-18 and carotid IMT measurements were not significantly different between groups at baseline (Table 1), with mean carotid IMT of 0.674 mm in the placebo group and 0.649 mm in the treatment group (p = 0.21). There was no significant change between baseline and end-of-study statin and anti-hypertensive use between the two groups (data not shown).
      After 15–18 months of DHA + EPA treatment, carotid IMT in the treatment group progressed by 0.012 mm (0.005, 0.020 mm) or 1.7% (0.6, 3.2%), compared to 0.015 mm (0.007, 0.025 mm) or 2.4% (0.9, 3.8%) in the placebo group (p = 0.17). Furthermore, even in subjects with a >2% absolute erythrocyte DHA enrichment between baseline and end of study which has been shown to be significantly associated with liver fat reduction [
      • Scorletti E.
      • Bhatia L.
      • McCormick K.G.
      • Clough G.F.
      • Nash K.
      • Hodson L.
      • Moyses H.E.
      • Calder P.C.
      • Byrne C.D.
      • Study W.
      Effects of purified eicosapentaenoic and docosahexaenoic acids in nonalcoholic fatty liver disease: results from the Welcome study.
      ], there was no significant difference between carotid IMT percentage progression between the >2% DHA enrichment group (1.7% (0.6, 3.3%)) and the <2% DHA enrichment group (2.3% (0.9, 3.8%) (p = 0.14). Importantly, there was also no significant difference in weight change between treatment and placebo groups over the 18 months (treatment, 0.62 ± 4.68 kg vs placebo, -0.17 ± 4.53 kg, p = 0.42).
      We also examined changes (difference between end and baseline measurements) in all clinically relevant variables over the 18-month study period to evaluate univariate associations between these parameters and carotid IMT progression from baseline in the whole cohort. The correlation coefficients and p-values are presented in Table 2.
      Table 2Univariate associations between percentage carotid IMT progression and changes in relevant clinical variables between start and end of study (18 months) in the entire cohort.
      Correlation coefficientp-value
      Systolic blood pressure difference (mmHg)−0.120.26
      Diastolic blood pressure difference (mmHg)−0.040.69
      Weight difference (kg)0.300.004
      Serum TG difference (mmol/L)0.270.01
      Serum cholesterol difference (mmol/L)0.230.03
      LDL-cholesterol difference (mmol/L)0.140.26
      HDL-cholesterol difference (mmol/L)−0.060.55
      HOMA-IR % difference0.220.08
      ALT difference (iu/L)0.110.28
      AST difference (iu/L)0.100.35
      EPA % change−0.050.64
      DHA % change−0.160.14
      CK-18 M65 difference (U/L)0.270.009
      Liver fat difference (%)0.49<0.001
      Abbreviations:IMT, intima-media thickness; TG, triglycerides; LDL, low-density lipoprotein; HDL, high-density lipoprotein; HOMA-IR, homeostasis model assessment of insulin resistance; ALT, Alanine transaminase; AST, Aspartate aminotransferase; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; CK-18 M65, cytokeratin-18 M65 subfraction.
      Over the 18-month period, percentage carotid IMT progression was associated with an increase in weight among subjects (r = 0.30, p = 0.004), and inversely associated with differences in serum triglyceride (r = 0.27, p = 0.01) and total cholesterol concentrations (r = 0.23, p = 0.03). A decrease in liver fat percentage was strongly associated with reduced carotid IMT progression over 18 months (r = 0.49, p < 0.001) and change in CK-18 concentration was also positively correlated with carotid IMT progression (r = 0.27, p = 0.009) (Fig. 2).
      Figure thumbnail gr2
      Fig. 2Scatter plot of relationship between carotid IMT percentage change and liver fat change (A) and CK-18 concentration change (B) between baseline and end of study in the entire cohort.
      When we also analysed the relationship between carotid IMT progression and the same clinical variables stratified by treatment group, we found similar associations. In the DHA + EPA group, reduced carotid IMT progression was significantly associated with liver fat decrease (r = 0.52, p < 0.001), CK-18 reduction (r = 0.47, p = 0.001), weight reduction (r = 0.36, p = 0.01) and decrease in cholesterol levels (r = 0.34, p = 0.03). In the placebo group, reduced carotid IMT progression was also significantly associated with liver fat decrease (r = 0.43, p = 0.004) and decrease in triglyceride levels (r = 0.31, p = 0.04), while decrease in weight only showed a trend toward reduced carotid IMT progression in the placebo group (p = 0.06), as did reduced CK-18 levels (p = 0.13).
      In multivariable linear regression analyses of the entire cohort, after adjusting for age, sex, diabetes, smoking, BMI, triglyceride concentration, statin and antihypertensive usage at baseline, as well as changes in weight and cholesterol concentration between the start and end of study, the only independent predictors of reduced carotid IMT progression over time were decreased liver fat (standardized β-coefficient 0.32, p = 0.005), reduced CK-18 levels (standardized β-coefficient 0.22, p = 0.04), as well as use of antihypertensive drugs at baseline (standardized β-coefficient −0.31, p = 0.009). The overall model fit was R2 = 0.39 (Table 3). Weight change was not independently associated with carotid IMT progression over 18 months in the multivariate regression model (p = 0.50).
      Table 3Associations between key explanatory variables and percentage carotid IMT difference between start and end of study (18 months) in the entire cohort.
      Independent variablesβ-coefficientStandardized β-coefficient95% CI for β-coefficientp-value
      Age0.0020.102−0.003 to 0.0080.39
      Sex0.0040.009−0.097 to 0.1050.94
      Diabetes0.0680.138−0.036 to 0.1730.20
      Smoking−0.002−0.003−0.143 to 0.1390.98
      BMI at baseline<0.001−0.011−0.011 to 0.0100.92
      Statin use at baseline0.0110.022−0.091 to 0.1130.84
      Antihypertensive use at baseline−0.150−0.305−0.261 to −0.0380.009
      Weight difference (kg)0.0040.074−0.008 to 0.0160.50
      Serum total cholesterol difference (mmol/L)0.0230.095−0.027 to 0.0740.37
      Serum TG difference

      (mmol/L)
      0.0270.096−0.035 to 0.0880.39
      Liver fat difference (%)0.0060.3210.002 to 0.0100.005
      CK-18 M65 difference (U/L)<0.0010.221<0.001 to <0.0010.04
      DHA % change−0.006−0.048−0.038 to 0.0260.70
      Model fit R2 = 0.39; Abbreviations: IMT, intima-media thickness; BMI, body mass index; TG, triglycerides; CK-18 M65, cytokeratin-18 M65 subfraction; DHA, docosahexaenoic acid.
      (“Difference” or “change” variable represents the arithmetic difference between the measurement at the end of study minus the baseline measurement for the independent variables included in the regression model).
      As reported in the WELCOME study primary outcomes paper [
      • Scorletti E.
      • Bhatia L.
      • McCormick K.G.
      • Clough G.F.
      • Nash K.
      • Hodson L.
      • Moyses H.E.
      • Calder P.C.
      • Byrne C.D.
      • Study W.
      Effects of purified eicosapentaenoic and docosahexaenoic acids in nonalcoholic fatty liver disease: results from the Welcome study.
      ], liver fat percentage decreased in both the treatment and the placebo arms of the trial, between baseline and end of study. Although there was a greater reduction in liver fat percentage in the DHA + EPA group, this result was not statistically significant (active, -8.1% ± 17.4% vs placebo, -4.5% ± 9.2% (p = 0.23). Consequently, we evaluated relevant putative univariate predictors of liver fat reduction across the entire study cohort and these are presented in Table 4 (supplementary appendix).
      Percentage liver fat change over the 18 months was significantly associated with weight change (r = 0.32, p = 0.003) as well as DHA percentage change (r = −0.26, p = 0.01), and showed a non-significant trend towards an association with change in fasting serum triglyceride concentration (r = 0.21, p = 0.06) and homeostasis model assessment of insulin resistance (HOMA-IR) percentage change (r = 0.23, p = 0.08). When we tested factors that were associated with percentage liver fat change in the entire cohort with multivariable linear regression modelling, after adjusting for age, sex, baseline percentage liver fat, change in weight and DHA percentage change, only change in weight (standardized β-coefficient 0.30, p < 0.001) and DHA percentage change (standardized β-coefficient −0.19, p = 0.027) were still independently associated with liver fat change. The overall model fit was R2 = 0.48. Baseline percentage liver fat was also independently associated with liver fat change (standardized β-coefficient −0.53, p < 0.001), which suggests that subjects with higher amounts of liver fat would have a larger absolute reduction in liver fat with increasing DHA enrichment or decrease in weight.

      4. Discussion

      This is the first study to document a novel association between an improvement in NAFLD severity, both in terms of markers of simple steatosis and steatohepatitis, and attenuation of carotid IMT progression during a randomized double-blind, placebo-controlled trial [
      • Scorletti E.
      • Bhatia L.
      • McCormick K.G.
      • Clough G.F.
      • Nash K.
      • Hodson L.
      • Moyses H.E.
      • Calder P.C.
      • Byrne C.D.
      • Study W.
      Effects of purified eicosapentaenoic and docosahexaenoic acids in nonalcoholic fatty liver disease: results from the Welcome study.
      ]. Although increased n-3 PUFA intake appears to be associated with reduced carotid atheroma burden in observational studies [
      • Saravanan P.
      • Davidson N.C.
      • Schmidt E.B.
      • Calder P.C.
      Cardiovascular effects of marine omega-3 fatty acids.
      ], our study did not show that high-dose n-3 PUFAs over 18 months had a significant independent beneficial effect on carotid IMT progression in subjects with NAFLD, even in those with significant increases in erythrocyte DHA levels compared to baseline. This lack of an effect of PUFA therapy is in contrast to the results of a recent cross sectional study of 847 subjects by Dai et al., which showed that high erythrocyte DHA (but not EPA) concentrations were associated with decreased carotid IMT and plaque burden [
      • Dai X.W.
      • Zhang B.
      • Wang P.
      • Chen C.G.
      • Chen Y.M.
      • Su Y.X.
      Erythrocyte membrane n-3 fatty acid levels and carotid atherosclerosis in Chinese men and women.
      ]. Our findings are instead, consistent with a recent randomized study by Lonn et al., which showed that a much lower daily dose of n-3 PUFAs (1 g) given to subjects with varying degrees of insulin resistance, had no favourable effect on carotid IMT progression compared to placebo [
      • Lonn E.M.
      • Bosch J.
      • Diaz R.
      • et al.
      Effect of insulin glargine and n-3FA on carotid intima-media thickness in people with dysglycemia at high risk for cardiovascular events: the glucose reduction and atherosclerosis continuing evaluation study (ORIGIN-GRACE).
      ]. Consequently, although n-3 fatty acids have been reported to have several CV benefits including antithrombotic, anti-atherosclerotic and anti-inflammatory effects, as well as improvements in blood pressure and endothelial function [
      • Wang C.
      • Harris W.S.
      • Chung M.
      • Lichtenstein A.H.
      • Balk E.M.
      • Kupelnick B.
      • Jordan H.S.
      • Lau J.
      n-3 Fatty acids from fish or fish-oil supplements, but not alpha-linolenic acid, benefit cardiovascular disease outcomes in primary- and secondary-prevention studies: a systematic review.
      ], randomised trials have so far failed to show consistent benefits of n-3 PUFAs over placebo in reducing CV outcomes [
      • Kromhout D.
      • Yasuda S.
      • Geleijnse J.M.
      • Shimokawa H.
      Fish oil and omega-3 fatty acids in cardiovascular disease: do they really work?.
      ]. One criticism of these studies has been that these trials tested too low doses of PUFA treatment. However, despite our study dose of 4 g/day being the highest licensed dose for PUFA therapy, we did not find any benefit of n-3 PUFAs on carotid IMT progression compared to placebo.
      A previous systematic review reported a significant association between NAFLD and carotid IMT, showing an estimated increase of 13% in carotid IMT for patients with liver fat, compared to controls without liver fat [
      • Sookoian S.
      • Pirola C.J.
      Non-alcoholic fatty liver disease is strongly associated with carotid atherosclerosis: a systematic review.
      ]. When we evaluated our entire cohort to investigate univariate associations between risk factors for CVD and carotid IMT progression over 18 months, we found that changes in weight, serum triglyceride and total cholesterol concentration all had significant positive correlations with increased carotid IMT progression. This result is perhaps not unexpected given the strong association between lipid levels and the metabolic syndrome with carotid disease [
      • Bhatia L.S.
      • Curzen N.P.
      • Byrne C.D.
      Nonalcoholic fatty liver disease and vascular risk.
      ]. However, and importantly, we also showed that a decrease in simple hepatic steatosis severity quantified by liver MRS as the gold-standard for non-invasive assessment of liver fat percentage, was independently associated with reduced carotid IMT progression over 18 months; even after adjusting for all measured confounding factors including standard CV risk factors, weight change and relevant medication use (i.e. statins and antihypertensives), which are known to have an impact on carotid IMT progression [
      • Chambless L.E.
      • Folsom A.R.
      • Davis V.
      • Sharrett R.
      • Heiss G.
      • Sorlie P.
      • Szklo M.
      • Howard G.
      • Evans G.W.
      Risk factors for progression of common carotid atherosclerosis: the atherosclerosis risk in communities study, 1987-1998.
      ,
      • Lakka T.A.
      • Lakka H.M.
      • Salonen R.
      • Kaplan G.A.
      • Salonen J.T.
      Abdominal obesity is associated with accelerated progression of carotid atherosclerosis in men.
      ,
      • Kang S.
      • Wu Y.
      • Li X.
      Effects of statin therapy on the progression of carotid atherosclerosis: a systematic review and meta-analysis.
      ,
      • Markus R.A.
      • Mack W.J.
      • Azen S.P.
      • Hodis H.N.
      Influence of lifestyle modification on atherosclerotic progression determined by ultrasonographic change in the common carotid intima-media thickness.
      ]. Furthermore, we found that changes in CK-18 levels from baseline to the end of study were also independently associated with carotid IMT progression after adjusting for the same confounding factors as above. As CK-18 levels have been shown to correlate well with histological features of hepatic inflammation and fibrosis, including its use for monitoring disease progression in NASH [
      • Feldstein A.E.
      • Wieckowska A.
      • Lopez A.R.
      • Liu Y.C.
      • Zein N.N.
      • McCullough A.J.
      Cytokeratin-18 fragment levels as noninvasive biomarkers for nonalcoholic steatohepatitis: a multicenter validation study.
      ,
      • Vuppalanchi R.
      • Jain A.K.
      • Deppe R.
      • Yates K.
      • Comerford M.
      • Masuoka H.C.
      • Neuschwander-Tetri B.A.
      • Loomba R.
      • Brunt E.M.
      • Kleiner D.E.
      • Molleston J.P.
      • Schwimmer J.B.
      • Lavine J.E.
      • Tonascia J.
      • Chalasani N.
      Relationship between changes in serum levels of Keratin 18 and changes in liver histology in children and adults with nonalcoholic fatty liver disease.
      ,
      • Tsutsui M.
      • Tanaka N.
      • Kawakubo M.
      • Sheena Y.
      • Horiuchi A.
      • Komatsu M.
      • Nagaya T.
      • Joshita S.
      • Umemura T.
      • Ichijo T.
      • Matsumoto A.
      • Yoshizawa K.
      • Aoyama T.
      • Tanaka E.
      • Sano K.
      Serum fragmented cytokeratin 18 levels reflect the histologic activity score of nonalcoholic fatty liver disease more accurately than serum alanine aminotransferase levels.
      ], our findings suggest that an improvement in steatohepatitis severity may also be independently associated with reduced carotid IMT progression. In a cross-sectional study, Targher et al. previously showed a progressive independent relationship between carotid IMT and increasing severity of NAFLD in 85 patients even after adjusting for classical CV risk factors and the metabolic syndrome [
      • Targher G.
      • Bertolini L.
      • Padovani R.
      • Rodella S.
      • Zoppini G.
      • Zenari L.
      • Cigolini M.
      • Falezza G.
      • Arcaro G.
      Relations between carotid artery wall thickness and liver histology in subjects with nonalcoholic fatty liver disease.
      ]. Our prospective, randomized-controlled trial confirms as well as extends that finding, showing that increasing severity of NAFLD is independently associated with carotid IMT progression over 18 months.
      Several observational and case–control studies have shown an increased incidence of adverse CV events in NAFLD subjects compared to the general population [
      • Bhatia L.S.
      • Curzen N.P.
      • Calder P.C.
      • Byrne C.D.
      Non-alcoholic fatty liver disease: a new and important cardiovascular risk factor?.
      ], with a higher rate of CV-related mortality in NASH compared to simple hepatic steatosis [
      • Ekstedt M.
      • Franzen L.E.
      • Mathiesen U.L.
      • Thorelius L.
      • Holmqvist M.
      • Bodemar G.
      • Kechagias S.
      Long-term follow-up of patients with NAFLD and elevated liver enzymes.
      ,
      • Soderberg C.
      • Stal P.
      • Askling J.
      • Glaumann H.
      • Lindberg G.
      • Marmur J.
      • Hultcrantz R.
      Decreased survival of subjects with elevated liver function tests during a 28-year follow-up.
      ]. The aetiology of increased CV risk appears to be multifactorial, with NAFLD acting as a pathological marker of ectopic fat accumulation, insulin resistance and low-grade systemic inflammation. These factors result in multiple deranged pathophysiological processes including abnormal metabolism of glucose, fatty acids and lipoproteins, worsening subclinical inflammation, increased oxidative stress, hypercoaguability, endothelial dysfunction and progression of atherosclerosis [
      • Bhatia L.S.
      • Curzen N.P.
      • Byrne C.D.
      Nonalcoholic fatty liver disease and vascular risk.
      ,
      • Byrne C.D.
      • Targher G.
      NAFLD: a multisystem disease.
      ]. These observations appear to be consistent with our finding of an association between a reduction in markers of NAFLD severity and decreased carotid IMT progression. When we also evaluated the potential causes of improvement in liver fat using multivariable linear regression modelling, we found that percentage DHA enrichment (either through dietary contamination in the placebo arm or good compliance with n-3 PUFA in the treatment arm) and weight loss during the trial in the entire cohort were independently associated with improvements in liver fat. Thus, we reason that both these factors contributed to improvements in liver fat during the study. Although weight loss in obesity has been shown to significantly attenuate carotid IMT progression [
      • Karason K.
      • Wikstrand J.
      • Sjostrom L.
      • Wendelhag I.
      Weight loss and progression of early atherosclerosis in the carotid artery: a four-year controlled study of obese subjects.
      ,
      • de las Fuentes L.
      • Waggoner A.D.
      • Mohammed B.S.
      • Stein R.I.
      • Miller 3rd, B.V.
      • Foster G.D.
      • Wyatt H.R.
      • Klein S.
      • Davila-Roman V.G.
      Effect of moderate diet-induced weight loss and weight regain on cardiovascular structure and function.
      ,
      • Cooper J.N.
      • Columbus M.L.
      • Shields K.J.
      • Asubonteng J.
      • Meyer M.L.
      • Sutton-Tyrrell K.
      • Goodpaster B.H.
      • DeLany J.P.
      • Jakicic J.M.
      • Barinas-Mitchell E.
      Effects of an intensive behavioral weight loss intervention consisting of caloric restriction with or without physical activity on common carotid artery remodeling in severely obese adults.
      ], we did not observe an independent association in our study after adjusting for relevant variables, including changes in NAFLD severity. Plausible explanations for this could be that our sample size was too small to detect a difference, or that weight changes were not large enough to observe a response (i.e. more than 5% body weight [
      • Cooper J.N.
      • Columbus M.L.
      • Shields K.J.
      • Asubonteng J.
      • Meyer M.L.
      • Sutton-Tyrrell K.
      • Goodpaster B.H.
      • DeLany J.P.
      • Jakicic J.M.
      • Barinas-Mitchell E.
      Effects of an intensive behavioral weight loss intervention consisting of caloric restriction with or without physical activity on common carotid artery remodeling in severely obese adults.
      ]).
      There are limitations to our study. As this was a pre-specified sub-study of the WELCOME trial [
      • Scorletti E.
      • Bhatia L.
      • McCormick K.G.
      • Clough G.F.
      • Nash K.
      • Calder P.C.
      • Byrne C.D.
      • Investigators W.T.
      Design and rationale of the WELCOME trial: a randomised, placebo controlled study to test the efficacy of purified long chain omega-3 fatty treatment in non-alcoholic fatty liver disease.
      ] with carotid IMT progression as a key secondary outcome of the trial, we did not undertake sample size or power calculations to determine the appropriate number of participants necessary to test the effect of the intervention with respect to carotid IMT modification. Consequently, our study may have lacked sufficient power to prove that treatment with high dose DHA + EPA caused a decrease in carotid IMT progression. Furthermore, although we observed a significant independent association between two markers of NAFLD severity (liver fat percentage and CK-18 concentration) and carotid IMT progression, our study comprised relatively small numbers of participants, and we suggest that further larger studies are necessary to confirm these findings. Thirdly, although CK-18 can be used as a non-invasive marker of steatohepatitis, the gold-standard of quantifying liver inflammation and fibrosis would have been a liver biopsy. Due to concerns of the potential, albeit small, risk associated with serial liver biopsies, we did not consider invasive assessment of NALFD severity an appropriate component of our study methodology. Fourthly, the sensitivity of carotid IMT as a discriminatory measure is likely to vary widely among studies using different methodologies. However, we sought to minimize this by adhering to the recommended guidelines on the optimal measurement and reporting of carotid IMT studies [
      • Stein J.H.
      • Korcarz C.E.
      • Hurst R.T.
      • Lonn E.
      • Kendall C.B.
      • Mohler E.R.
      • Najjar S.S.
      • Rembold C.M.
      • Post W.S.
      American Society of Echocardiography Carotid Intima-Media Thickness Task F
      Use of carotid ultrasound to identify subclinical vascular disease and evaluate cardiovascular disease risk: a consensus statement from the American Society of Echocardiography Carotid Intima-Media thickness task Force. Endorsed by the Society for vascular Medicine.
      ]. As a comparison, an analysis of the placebo groups from several large randomized placebo-controlled trials showed that the overall weighted rate of change in mean carotid IMT was 0.0147 mm/year [
      • Bots M.L.
      • Evans G.W.
      • Riley W.A.
      • Grobbee D.E.
      Carotid intima-media thickness measurements in intervention studies: design options, progression rates, and sample size considerations: a point of view.
      ], which is higher than our placebo group estimated change of 0.010 mm/year. However, our cohort represents a lower CV risk population compared to most previous trial data, of which the majority encompass secondary prevention cohorts and this could consequently explain the smaller annual progression rate in our study. Finally, it is possible that 18 months of high-dose PUFA therapy is inadequate to observe a significant biological effect on carotid IMT progression.
      In conclusion, we have shown for the first time that an improvement in NAFLD severity over 18 months is associated with a beneficial effect on carotid IMT progression, a surrogate marker for cardiovascular outcomes [
      • Lorenz M.W.
      • Markus H.S.
      • Bots M.L.
      • Rosvall M.
      • Sitzer M.
      Prediction of clinical cardiovascular events with carotid intima-media thickness: a systematic review and meta-analysis.
      ]. However, we observed no significant effect of the n-3 PUFA intervention over 18 months on carotid IMT progression. Given that there is now increasing evidence that NAFLD portends a poorer CV outcome independent of several CV risk factors including the metabolic syndrome [
      • Bhatia L.S.
      • Curzen N.P.
      • Calder P.C.
      • Byrne C.D.
      Non-alcoholic fatty liver disease: a new and important cardiovascular risk factor?.
      ], and that worsening grades of NAFLD also contribute to progressive cardiometabolic risk, we suggest that further larger prospective studies should be performed to confirm these findings including the evaluation of other biomarkers of increased CV risk or adverse CV outcomes in NAFLD patients. Although lifestyle changes such as weight loss, increased exercise and reducing dietary fat intake are the only universally recommended therapeutic strategies with proven benefit to reduce NAFLD severity [
      • Ratziu V.
      • Bellentani S.
      • Cortez-Pinto H.
      • Day C.
      • Marchesini G.
      A position statement on NAFLD/NASH based on the EASL 2009 special conference.
      ], and there are currently no established licensed pharmacological treatments for this disease, our findings of improved CIMT progression should encourage the ongoing trials of various therapeutic strategies, including active lifestyle intervention, to reduce NAFLD severity [
      • Musso G.
      • Cassader M.
      • Rosina F.
      • Gambino R.
      Impact of current treatments on liver disease, glucose metabolism and cardiovascular risk in non-alcoholic fatty liver disease (NAFLD): a systematic review and meta-analysis of randomised trials.
      ], which may then ultimately confer improved CV outcomes in the NAFLD population.

      Acknowledgements

      Omacor/Lovaza and placebo were provided by Pronova Biopharma through Abbott Laboratories. The authors wish to thank Annette West, who performed the fatty acid analysis, Debbie Smith, Christine Glenn and Keith McCormick for sample preparation and biochemical analyses, and research nurses Gillian Wise, Bridget Clancy, Sanchia Triggs and Norma Diaper, without whose help the study would not have been possible, Lucinda England for invaluable research governance administration, Yueqing Cheng and Karen Long for data managing, and Helen Moyses for statistical advice.

      Appendix A. Supplementary data

      Sources of funding

      The study was supported by NIHR through the NIHR Biomedical Research Unit in Nutrition and Lifestyle and by Diabetes UK.

      Disclosures

      Philip Calder serves on the Scientific Advisory Boards of Pronova BioPharma, Aker Biomarine, Dutch State Mines (DSM), Solutex, Sancilio and the Danone Research Centre for Specialised Nutrition; acts as a consultant to Smartfish, Mead Johnson Nutritionals, Vifor Pharma, Amarin Corporation and Enzymotec; and has recently received speaking honoraria from Pronova BioPharma, Smartfish, Dutch State Mines (DSM), Fresenius Kabi, B. Braun and Vifor Pharma.
      Nicholas Curzen has received unrestricted research grants from Boston Scientific, Haemonetics, Heartflow, Medtronic and St Jude Medical; has received honoraria for speaking or consultancy from Haemonetics, Heartflow, St Jude Medical; and has also received unrestricted educational grant from Volcano.

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