ApoE Polymorphism and Fish Oil Supplementation in Subjects With an Atherogenic Lipoprotein Phenotype

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2000;20;1990-1997 Arterioscler Thromb Vasc BiolW. Wright, Margaret C. Murphy, Bruce A. Griffin and Christine M. Williams

Anne M. Minihane, Syrah Khan, Elizabeth C. Leigh-Firbank, Philippa Talmud, John Atherogenic Lipoprotein Phenotype

ApoE Polymorphism and Fish Oil Supplementation in Subjects With an

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ApoE Polymorphism and Fish Oil Supplementation inSubjects With an Atherogenic Lipoprotein Phenotype

Anne M. Minihane, Syrah Khan, Elizabeth C. Leigh-Firbank, Philippa Talmud, John W. Wright,Margaret C. Murphy, Bruce A. Griffin, Christine M. Williams

Abstract—The study assessed the efficacy of fish oil supplementation in counteracting the classic dyslipidemia of theatherogenic lipoprotein phenotype (ALP). In addition, the impact of the common apolipoprotein E (apoE) polymorphismon the fasting and postprandial lipid profile and on responsiveness to the dietary intervention was established. Fifty-fiveALP males (aged 34 to 69 years, body mass index 22 to 35 kg/m2, triglyceride [TG] levels 1.5 to 4.0 mmol/L, highdensity lipoprotein cholesterol [HDL-C] !1.1 mmol/l, and percent low density lipoprotein [LDL]-3 "40% total LDL)completed a randomized placebo-controlled crossover trial of fish oil (3.0 g eicosapentaenoic acid/docosahexaenoic acidper day) and placebo (olive oil) capsules with the 6-week treatment arms separated by a 12-week washout period. Inaddition to fasting blood samples, at the end of each intervention arm, a postprandial assessment of lipid metabolismwas carried out. Fish oil supplementation resulted in a reduction in fasting TG level of 35% (P!0.001), in postprandialTG response of 26% (TG area under the curve, P!0.001), and in percent LDL-3 of 26% (P!0.05). However, no changein HDL-C levels was evident (P#0.752). ANCOVA showed that baseline HDL-C levels were significantly lower inapoE4 carriers (P#0.035). The apoE genotype also had a striking impact on lipid responses to fish oil intervention.Individuals with an apoE2 allele displayed a marked reduction in postprandial incremental TG response (TG incrementalarea under the curve, P#0.023) and a trend toward an increase in lipoprotein lipase activity relative to non-E2 carriers.In apoE4 individuals, a significant increase in total cholesterol and a trend toward a reduction in HDL-C relative to thecommon homozygous E3/E3 profile was evident. Our data demonstrate the efficacy of fish oil fatty acids incounteracting the proatherogenic lipid profile of the ALP but also that the apoE genotype influences responsiveness tothis dietary treatment. (Arterioscler Thromb Vasc Biol. 2000;20:1990-1997.)

Key Words: atherogenic lipoprotein phenotype ! apoE genotype ! fish oils ! plasma lipids

The importance of hypertriglyceridemia as a risk factor forcoronary heart disease (CHD) is now well established.1–5

In addition to the ability of triglyceride-rich lipoproteins todirectly infiltrate and sequester cholesterol in the atheroma,6raised circulating triglycerides (TG) have been implicated asa major metabolic component of an “atherogenic lipoproteinphenotype” (ALP),7,8 a term frequently used to describe acollection of proatherogenic lipoprotein abnormalities. TheALP lipid profile, which occurs in up to 25% of middle-agedmales,9 is associated with a 3-fold increased CHD risk.10,11 Itis characterized by a moderate fasting hypertriglyceridemia(1.5 to 4.0 mmol/L), exaggerated postprandial lipemic re-sponses, low HDL cholesterol (HDL-C) levels (!1.1 mmol/L), and a predominance of the potentially atherogenic smalldense LDL-3 particle ("40% of total LDL).8,12,13The hypotriglyceridemic effect of fish oils is well docu-

mented, with a recent meta-analysis concluding that 3 to 4 gof eicosapentaenoic acid (EPA) and docosahexaenoic acid(DHA) per day resulted in a 25% and 34% decrease in fasting

TG levels in normolipidemic and hyperlipidemic individuals,respectively.14 The efficacy of fish oil consumption in reduc-ing the magnitude and duration of postprandial lipemicresponses has also been demonstrated in a limited number ofstudies, although high levels of intake have often beenused.12,15–19 Despite the potential benefits of fish oil supple-mentation in counteracting the characteristic fasting andpostprandial dyslipidemia of the ALP, no study has yetinvestigated the efficacy of this dietary intervention in anALP group.Responsiveness of plasma lipids to dietary manipulations

is highly variable, with diet-gene interactions thought toexplain, in part, interindividual responses.20 In addition,polymorphisms at specific gene loci are thought to besignificant determinants of fasting lipid levels21 and may alsoexplain the highly heterogeneous nature of individuals’ post-prandial responses to a standard fat load. ApoE is a functionaland structural component of several classes of lipoproteins,including chylomicrons, VLDLs, and their remnants, and has

Received January 6, 2000; revision accepted March 20, 2000.From the Hugh Sinclair Unit of Human Nutrition (A.M.M., E.C.L.-F., C.M.W.), Department of Food Science and Technology, University of Reading,

Reading, UK; the Centre of Cardiovascular Genetics (P.T.), UCL, London, UK; and the School of Biological Sciences (S.K., J.W.W., M.C.M., B.A.G.),University of Surrey, Guildford, UK.Correspondence to Dr Anne M. Minihane, Hugh Sinclair Unit of Human Nutrition, Department of Food Science and Technology, PO Box 226,

1990 by on January 9, 2010 atvb.ahajournals.orgDownloaded from

a major influence on the metabolism and clearance of theseparticles by the liver.22 Three common isoforms of this geneloci exist, yielding apoE2, apoE3, and apoE4, with $55% to60% of the population homozygous for the E3 allele.23Population studies have shown that plasma cholesterol,

LDL cholesterol (LDL-C), and apoB levels are highest insubjects with an E4 allele, intermediate in individuals ho-mozygous for E3, and lowest in those with an E2 allele,23–25with some evidence of greater responsiveness to cholesterol-lowering diets in subjects with an E4 allele.20,26 Data on theimpact of apoE genotype on circulating TG levels are lessconsistent. In a meta-analysis published in 1992, Dallon-geville et al26 concluded that although not observed in allstudies, the apoE2 and apoE4 alleles result in moderatelyhigher fasting TG levels in the general population. Althoughthe association between the homozygous E2/E2 genotype anddelayed postabsorptive triglyceride-rich lipoprotein clearancehas been repeatedly demonstrated,27 this genotype is presentin!1% of the population, and data on the impact of the morecommon apoE2/E3, apoE3/E4, and apoE4/E4 genotypes onlipemic responses have produced conflicting findings.5,28–31Although the effects of the apoE genotype on lipoprotein

metabolism and responsiveness to dietary fat restriction havebeen extensively studied in various populations, no study hasexamined the association between this polymorphism and thelipid profile of the ALP or the responsiveness to fish oilsupplementation. This is surprising in view of the central roleof apoE in TG metabolism, the well-established defect in TGmetabolism in an ALP, and the extensively studied TG-lowering actions of fish oils in normolipidemic and hyperlip-idemic individuals.The present study aims to examine the efficacy of fish oil

supplementation in counteracting the dyslipidemia of the ALP.In addition, the impact of the apoE genotype on the baselinelipid profile and on lipoprotein responsiveness to the inter-vention treatment is investigated.

MethodsStudy SubjectsFifty-five healthy male volunteers, aged 30 to 70 years, wererecruited through a database held at the Department of ClinicalPathology, Royal Berkshire Hospital, Reading, UK. All bloodsamples taken for lipid assessment in the West Berkshire HealthDistrict were analyzed at the Royal Berkshire Hospital, and the datawere stored on computer for up to 5 years. Individuals considered tobe at risk of an ALP, with a lipid profile of TG 1.5 to 4.0 mmol/L,total cholesterol (TC) 5.0 to 8.0 mmol/L, and HDL-C !1.1 mmol/L,were sent a letter (via their general practitioners) giving brief detailsof the objectives and protocol of the study. Those interested inparticipating attended the Hugh Sinclair Unit of Human Nutritionand completed a health and lifestyle questionnaire and provided afasting screening blood sample. Exclusion criteria for participation inthe study were as follows: diagnosed diabetes or fasting glucose"6.5 mmol/L, liver or other endocrine dysfunction, evidence ofcardiovascular disease (including angina), smoking, hypolipidemictherapy or any other medication known to interfere with lipidmetabolism, consumption of fatty acid supplements, weight-reducing diets, body mass index "35 kg/m2, blood pressure "160/95 mm Hg, and hemoglobin !130 g/L. The University of Readingand the West Berkshire Health Authority Ethics Committees ap-proved the study protocol, and each individual gave written consentbefore participating. ApoE genotyping was performed on 50 indi-viduals. Therefore, the data in the present study reflect 50 of the 55individuals who completed the study.

Study DesignThe study was a double-blind placebo-controlled crossover studywith subjects consuming 6 g of fish oil (27.9% EPA and 22.3%DHA, Pikasol, Lube A/S) or 6 g of olive oil (placebo, Lube A/S) perday for 6 weeks. After a washout period of 12 weeks, subjects wereplaced on the opposite supplementation regime. The oils wereprovided as six 1-g oil capsules, with the fish oil supplementsproviding a total of 3.0 g of the long-chain !-3 fatty acids (EPA andDHA) per day. Fasting blood samples (30 mL) were collected at 0,3, and 6 weeks for each treatment, and at the end of eachsupplementation period (6 weeks), a postprandial assessment wascarried out. Compliance was monitored by examining plateletmembrane fatty acid composition in a randomly chosen subgroup(n#22).

Postprandial ProtocolAfter a 12-hour overnight fast, each participant completed a post-prandial assessment. The previous day, no strenuous exercise oralcohol was permitted, and a standard evening meal containing !20g fat was provided to standardize short-term fat intake. Beforeconsuming the test meals, an indwelling cannula was inserted intothe antecubital vein of the forearm, and a fasting blood sample (30mL) was taken. A relatively high-fat breakfast providing 49 g fat,109 g carbohydrate, and 18 g protein (start time, 0 minutes) andlunch providing 31 g fat, 63 g carbohydrate, and 15 g protein (330minutes) were provided. Blood samples (11 mL) were collected at 0,30, 60, 90, 150, 210, 270, 330, 360, 390, 420, and 480 minutes toassess postprandial TG and nonesterified fatty acids (NEFA). At 480minutes, 100 IU/kg of heparin was administered intravenously, anda 5-mL blood sample was collected at 15 minutes into lithiumheparin tubes to determine postheparin lipoprotein lipase (LPL)activity.

Biochemical AnalysisFasting and postprandial venous blood samples were collected into10-mL potassium EDTA tubes. All blood samples, except thosecollected for platelet phospholipid fatty acid composition, werecentrifuged at 1600g for 10 minutes, and subsamples of plasma werestored at %20°C, for TG, TC, and NEFA analysis. A 10-mL plasmasample was collected for determination of LDL subclass distribution;this sample was stored at 4°C and analyzed within 24 hours. Theplasma collected for the determination of LPL activity was stored at%80°C. HDL-C was determined by measuring cholesterol in thesupernatant after precipitation of the apoB-containing lipoproteinsby use of dextran sulfate and magnesium chloride.32 LDL-C levelswere computed by using the Friedewald formula.33Plasma samples were analyzed for TG, TC, HDL-C, and NEFA by

using the Monarch Automatic Analyser (Instrumentation Laborato-ries Ltd) and enzymatic colorimetric kits (Instrumentation Labora-tories Ltd). LPL activity was determined by using the methoddescribed by Nilsson-Ehle et al.34 LDL subclasses were separated bydensity-gradient ultracentrifugation, and the relative percentage ofsmall dense LDL-3 was calculated by integrating the respective areaunder the LDL subclass profile, as previously described by Griffin etal.35 Postprandial TG responses were expressed as area under thecurve (AUC, 0 to 480 minutes) or incremental area under the curve(IAUC, 0 to 480 minutes), calculated by use of the trapezoidal rule.Because of the shape of the postprandial NEFA response, NEFAAUC (0 to 480 minutes) is difficult to interpret, with circulatingNEFA concentrations dropping below baseline levels in the earlypostprandial period; therefore, in the data analysis, the NEFApostprandial responses were represented as NEFA AUC (270 to 480minutes).Platelets were extracted from whole blood according to the

methods of Indu and Ghafoorunissa36 and stored at %80°C forplatelet phospholipid fatty acid analysis. Butylated hydroxytoluenewas added to all solvents at a concentration of 50 mg/L to minimizeauto-oxidation during analysis. One hundred microliters of phos-phatidylethanolamine diheptadecanoic acid (0.25 mg/mL in chloro-form) was added as an internal standard to all samples. Lipids wereextracted with chloroform/methanol (2:1 [vol/vol]) according to themethod of Folch et al,37 and the phospholipid fraction was isolated

Minihane et al ApoE and Fish Oils in ALP 1991

from the crude lipid by using a Sep-Pack C18 column38 (WatersAssociates). The phospholipids were transmethylated by using a1.5% sulfuric acid in methanol solution, and the fatty acids werequantified by gas liquid chromatography with use of a CPSil 88column (Chrompak).36

ApoE GenotypingDNA was extracted from 10 mL of blood collected after an overnightfast into a potassium-EDTA tube. ApoE genotyping was performedby using the method of Bolla et al.39

Statistical AnalysisGroup results are expressed as mean&SEM. The data were checkedfor normality, and skewed parameters were logarithmically trans-formed before statistical analysis. The significance of any impact ofthe apoE genotype on the fasting biochemical profile of the ALPparticipants was established by 1-way ANCOVA, with age as acovariate. Post hoc comparisons of the lipoprotein variables betweenthe phenotype groups were carried out by the Tukey honestlysignificant difference post hoc test. The impact of fish oil supple-mentation on fasting lipid levels in the overall group (n#50) wasdetermined by 2-way repeated-measures ANOVA, with time (0, 3,and 6 weeks) and oil (fish oil and olive oil) as the independentvariables. Posttreatment postprandial AUCs and IAUCs, any differ-ences between TG levels, and the fatty acid composition of plateletphospholipids at the beginning of each arm of the study wereanalyzed by paired Student t tests. The percentage change of lipidoutcome measures after supplementation was determined as thepercent change after 0 to 6 weeks of fish oil minus the percentchange after 0 to 6 weeks of olive oil. Any impact of the apoE alleleon percent change was determined by 1-way ANCOVA, with apoE(E2, E3, and E4) as the independent variable and age as thecovariate. A value of P!0.05 was considered significant. Allstatistical analysis was performed with the SPSS statistical package(version 6.1, SPSS).

ResultsThe mean&SEM age, body mass index, lipid, and LPLactivity profile of the group (n#50) and of the apoE sub-groups are given in Table 1. In accordance with the recruit-ment strategy, participants displayed an ALP blood lipidprofile with group mean&SEM fasting TG, HDL-C, and

percent LDL-3 of 2.48&0.12 mmol/L, 0.97&0.12 mmol/L,and 61&3%, respectively. Relative allele frequencies of 0.09E2, 0.69 E3, and 0.23 E4 were observed. No individualexpressed an E2/E2 genotype, and because the homozygousE4/E4 (n#2) genotype sample size was small, individualswere classified into the following 3 phenotypes: (1) apoE2group (n#8), carrying the E2/E3 genotype; (2) apoE3 group(n#22), carrying the most common E3/E3 phenotype; and (3)apoE4 group (n#20), carrying either the E3/E4 or E4/E4genotype. One individual with an apoE2/E4 genotype couldnot be assigned to any of the groups and was thereforeexcluded from the data analysis.Age, body mass index, TG, and percent LDL-3 were

comparable among the 3 apoE subgroups (Table 1). Therewas a trend toward higher TC, LDL-C, and TC/HDL-C ratioin E4 carriers relative to noncarriers; however, these differ-ences failed to reach significance when age was included asa covariate. Significantly lower HDL-C was evident in the E4group (P#0.035). Although not of statistical significance,lower TG AUC and IAUC and higher postheparin LPLactivity were evident in the E3 group. Fasting and postpran-dial (270 to 480 minutes) NEFA concentrations were com-parable in the 3 apoE subgroups (Table 1).In the ALP group as a whole (n#50), fish oil supplemen-

tation resulted in significant reductions of 35.3% (P#0.000),23.3% (P#0.000), and 7.9% (P#0.007) in fasting TG, TGAUC, and TG IAUC, respectively (Table 2, Figure 1). ApoEgenotype had little impact on responsiveness of the fastingTG fraction; however, the apoE2 allele was associated with agreater reduction in postprandial responses after supplemen-tation. TG IAUC, which represents the postprandial TGresponse minus the baseline TG levels, is more representativeof postprandial clearance than is TG AUC, which representsfasting and postprandial TG metabolism. A significantlygreater reduction in TG IAUC (P#0.023) was evident in the

TABLE 1. Anthropometric and Fasting Lipid Levels of Subjects Overall (n!50) and by ApoE Phenotype

Variable All (n#50) ApoE2 (n#8) ApoE3 (n#22) ApoE4 (n#20) P *

Age, y 56&1 56&3 57&2 54&2 0.499

Body mass index, kg/m2 28.0&0.5 29.4&1.3 27.3&0.6 28.2&0.9 0.353

TC, mmol/L 6.56&0.12 6.34&0.35 6.47&0.17 6.73&0.20 0.689

LDL-C, mmol/L 4.48&0.12 4.23&0.33 4.35&0.17 4.72&0.22 0.218

HDL-C, mmol/L 0.97&0.12 1.02&0.08a 1.00&0.03a 0.91&0.03b 0.035

TC/HDL-C ratio 6.89&0.17 6.28&0.26 6.52&0.33 7.52&0.33 0.473

% LDL-3 61&3 55&10 64&5 59&5 0.418

TG, mmol/L 2.48&0.12 2.41&0.16 2.56&0.21 2.42&0.19 0.251

TG AUC, mmol/L at 480 min 1825&78 1831&98 1709&124 1946&132 0.879

TG IAUC, mmol/L at 480 min 520&35 526&113 478&52 562&50 0.276

LPL, nmol FA " mL%1 " min%1 63.7&7.8 46.0&18.8 75.5&10.7 59.2&13.7 0.624

NEFA, "mol/L 465.6&15.3 479.7&51.1 465.0&20.7 460.4&27.8 0.871

NEFA AUC (270–480 min),"mol/L at 210 min

112 015&3798 104 955&11 093 111 232&5271 115 853&7295 0.253

Values are mean&SEM. ApoE2 indicates apoE2/E3 (n#8); apoE3, apoE3/E3 (n#22); apoE4, apoE3/E4 (n#18)'apoE4/E4 (n#2);% LDL-3, percent small dense LDL; TG AUC, area under postprandial TG curve calculated by trapezoidal rule; TG IAUC, incrementalarea under postprandial TG curve calculated as TG AUC minus baseline TG level; FA, fatty acid; and LPL, 15-minute postheparin LPLactivity. Superscripts not sharing a common letter were significantly different (P!0.05).

*The intergroup differences for apoE were analyzed by 1-way ANCOVA, with age as the covariate, followed by Tukey honestlysignificant difference post hoc tests.

1992 Arterioscler Thromb Vasc Biol. August 2000

apoE2 group (27.2%) relative to either the apoE3 group(2.7%) or the apoE4 group (5.5%; Table 2, Figure 2).Fish oil supplementation resulted in a 7.1% increase in the

LDL-C fraction in the overall group (P#0.054); however,little change in TC or HDL-C levels was evident (Table 2,Figure 1). The apoE4 group demonstrated a shift in thecholesterol profile relative to non-E4 carriers, with a 3.5%increase in TC (P#0.014), a 7.4% decrease in HDL-C(P#0.126), and a 15.9% increase in LDL-C (P#0.120).A 26% reduction in the percentage of LDL in the small

dense form (percent LDL-3) was evident in the group as awhole after supplementation (Table 2, Figure 1). The 35.7%reduction evident in the apoE4 group was significantlygreater than the 17.2% reduction observed in the apoE3 group(P#0.021).A significant 7.8% decrease in fasting NEFA (P#0.012), a

7.4% decrease in NEFA AUC (270 to 480 minutes,

P#0.002), and a 14.7% increase in postheparin LPL activity(P#0.065) were also observed in the overall group (Table 2).Although there is a trend toward greater responsivenessof these outcome measures to fish oil supplementation inindividuals with an E2 allele (Figure 2), large interindi-vidual differences meant that differences failed to reachsignificance.Compliance was monitored by using platelet membrane

fatty acid composition. Fish oil supplementation resulted in ahighly significant increase in platelet membrane EPA andDHA concentration, from 0.53% to 3.16% (P#0.000) andfrom 2.50% to 3.61%, respectively (P#0.000, Table 3).Fasting TG (2.51 versus 2.55 mmol/L, P#0.329) and the

EPA (0.53% versus 0.66%, P#0.227) and DHA (2.55%versus 2.58%, P#0.768) composition of platelet membranephospholipids were similar at the beginning of each arm ofthe study (data not shown), indicating that the washout period

TABLE 2. Impact of ApoE Genotype on Responsiveness of Lipid and Lipoprotein Fractions toFish Oil Supplementation

Variable

% Change

P *All (n#50) ApoE2 (n#8) ApoE3 (n#22) ApoE4 (n#20)

TG %35.3&5.3† %30.7&7.6 %34.9&7.0 %37.6&10.6 0.561

TG AUC %23.3&3.0† %32.5&4.6 %18.4&4.3 %24.8&5.2 0.136

TG IAUC %7.9&5.6† %27.7&7.0a %2.7&10.0b %5.5&8.2b 0.023

TC %1.5&2.1 %1.1&3.8a %6.3&2.8a,b 3.5&3.5a,c 0.014

HDL-C %0.8&3.3 12.2&5.5 0.6&4.1 %7.4&6.3 0.806

LDL-C 7.1&3.2‡ 3.1&5.4 0.6&4.5 15.9&4.0 0.120

% LDL-3 %26.0&8.3† %31.0&40.3a %17.2&8.8a,b %35.7&13.6a,c 0.021

LPL 14.7&14.5§ 47.2&29.7 2.1&9.7 17.3&33.9 0.177

NEFA %7.8&4.8† %22.1&5.9 %5.7&5.7 %4.1&9.9 0.616

NEFA AUC %7.4&3.2† %18.3&6.7 %4.8&4.7 %5.7&6.3 0.608

Values are mean&SEM. % Change indicates percent change on fish oil minus percent change on olive oil.Superscripts not sharing a common letter were significantly different (P!0.05).

*The intergroup differences for apoE were analyzed by ANCOVA, with age as the covariate, followed by Tukeyhonestly significant difference post hoc tests.

†Statistical significance of overall group (n#50) changes in outcome parameters were calculated by repeated-measures ANOVA, with time and oil supplement (fish oil and olive oil) as the independent variables (P!0.05).

‡P#0.054; §P#0.065.

Figure 1. Responsiveness of the char-acteristic features of ALP to fish oil (FO)supplementation. OO indicates olive oil.Group means for fasting TG, percentLDL-3, and HDL-C were compared by2-way repeated-measures ANOVA, withtime and oil supplement (FO and OO)used as independent variables. Post hocTukey honestly significant differencetests were performed. Different letteringindicates that the mean values are sig-nificantly different (P!0.05). TG AUCswere compared by paired t test.

was sufficient to reverse the metabolic effects and tissueaccumulation of EPA and DHA.

DiscussionThe aim of the present investigation was to determine theefficacy of fish oil supplementation in counteracting theclassical dyslipidemia of the ALP. The impact of the commonapoE polymorphism on the fasting lipid and postprandial

profile and on the responsiveness to the fish oil interventionwas also investigated.Forty percent of the ALP subjects in the study were either

heterozygous or homozygous for the apoE4 allele, which ishigher than the 25% to 27% prevalence reported in numerouspopulation studies.5,21 Clinical studies have suggested a predis-posing role for E4 in the development of atherosclerosis andcardiovascular disease,40 which is thought to reflect an adverse

Figure 2. Impact of apoE genotype on post-prandial TG (a through c) and NEFA (d through f)responses. Incremental TG response representsthe total postprandial response minus the fastinglevels.

TABLE 3. Presupplementation and Postsupplementation Platelet MembranePUFA Composition

Olive Oil Fish Oil

P *0 wk 6 wk 0 wk 6 wk

C18:2 5.78&0.18† 5.84&0.16 5.63&0.16 5.38&0.22 0.138

C20:4 24.31&0.54 24.86&0.33 24.10&0.59 21.71&0.39 0.000

C20:5 0.65&0.13 0.86&0.17 0.53&0.14 3.16&0.16 0.000

C22:6 2.64&0.13 2.72&0.17 2.50&0.14 3.61&0.13 0.000

Values are mean&SEM. C18:2 indicates linoleic acid; C20:4, arachadonic acid; C20:5, eicosapentaenoic acid; andC22:6, docosahexaenoic acid.

*The statistical significance of group (n#22) changes in fatty acid composition were calculated by repeated-measures ANOVA, with time and oil supplement (fish oil and olive oil) as the independent variables.

†Percent platelet membrane phospholipid fatty acids.

effect of this polymorphism on circulating LDL particle num-bers that is due to the downregulation of LDL receptors.23,41 Therole of apoE in lipoprotein metabolism and clearance has beenextensively studied in recent years.20–27,41–45In present study, we have observed the expected trend in

TC and LDL-C, with levels in E2 carriers lower and in E4carriers higher than those observed in the homozygous E3/E3subgroup. The intergroup differences were comparable withthe 0.1- to 0.5-mmol/L differences reported in previouslarge-scale population studies.22,29,45,46 However, because ofthe relatively small sample size in the present study, the effectof apoE genotype on these outcome measures failed to reachstatistical significance.HDL-C levels were $10% lower in ALP subjects who

were E4 carriers, and these intergroup differences were foundto be significant. This finding is in agreement with the lowercirculating HDL-C concentration reported in individuals withthe E4/E3 genotype in 17 of 28 studies examined in a recentmeta-analysis.26 Removal of HDL from the circulation occursby a variety of pathways, including an apoE-dependentreceptor–mediated pathway and a hepatic lipase catabolicpathway.47 Although the precise biochemical origin of thelow HDL-C in E4 individuals remains to be established, thepreviously reported preferential association of the E4 proteinwith VLDL rather than transfer of a large portion back toHDL,48,49 lower total apoE concentrations in E4 individuals,50or altered lipase activities associated with apoE genotype51,52may be involved.In the present study, we observed no relationship between

apoE polymorphism and baseline fasting or postprandial TGlevels in subjects with an ALP profile. Divergent results arereported in the literature regarding the impact of the apoEgenotype on TG metabolism.5,26,27,29,30,48,53,54 Small samplessizes, lack of correction for the contribution of fasting TG tothe postabsorptive lipemic responses, variation in the markersof postprandial clearance used (chylomicron retinyl esters,total plasma retinyl esters, plasma TG, or non-chylomicronTG), and interaction between the apoE polymorphism andother genetic and environmental factors may be responsiblefor the discrepant results.Because of the identification of TG as the major determi-

nant of the ALP dyslipidemia, this lipid has been targeted fordietary modification as a means of correcting the character-istic lipoprotein abnormalities. Supplementation of ALP sub-jects with 6 g fish oil (3.0 g EPA/DHA) resulted in asignificant 35% (0.9-mmol/L) reduction in fasting TG levelscomparable to the mean 34% reduction observed in hypertri-glyceridemic individuals (mean TG 3.7 mmol/L) in a recentmeta-analysis.14 In light of recent estimates of risk associatedwith elevated TG levels,1 it is likely that the 0.9-mmol/Lreduction observed in the present study would have a clini-cally significant impact on CHD risk in the ALP group.A 23% reduction in postprandial lipemic responses was

also evident after the fish oil–enriched supplementationperiod, but for the group as a whole, this reflected only amodest 8% reduction in the postprandial increase in responseto the meals (TG IAUC). However, there was a significantimpact of the apoE genotype on the responsiveness of thisoutcome measure to fish oil supplementation. In the E3 andE4 subgroups, the greatest decreases in TG levels were seenin the fasted state, suggesting suppression of endogenous

VLDL secretion as the primary locus for the effect ofEPA/DHA in these groups. This conclusion is consistent witha large body of evidence supporting an inhibitory action ofn-3 polyunsaturated fatty acid (PUFA) on endogenous VLDLsecretion,55–57 with inhibition of hepatic lipogenic en-zymes,55,58 and with the stimulation of apoB degradatorypathways,57 which are thought to be responsible. Notably, n-3PUFA supplementation resulted in only small changes (3% to6%) in TG IAUC in these subgroups. However, in E2carriers, a 28% reduction in the postprandial increase incirculating TG levels (TG IAUC) was evident after fish oilsupplementation in addition to the 31% reduction in fastingTG levels. An associated 47% increase in LPL activity wasobserved in this apoE subgroup, which was higher than that inother subgroups.In the group as a whole, the fish oil intervention resulted in

a borderline significant 15% increase in LPL activity. Alimited number of human studies have generally failed toshow an increased postheparin LPL activity with high fish oildiets,17,18,56,59 although 2 recent reports have found significantincreases.60,61 Zampelas et al60 have shown elevated posthep-arin LPL activity 9 hours after meals enriched with n-3 PUFAcompared with saturated fatty acid, and Harris et al61 ob-served a 62% increase in endogenous LPL activity in healthyvolunteers after 3 weeks of fish oil supplementation (5 g/d).Several mechanisms may be responsible for this observedincrease in LPL activity, including changes in LPL geneexpression, transport of newly synthesized LPL to the vascu-lar wall, or a change in NEFA metabolism. Circulating NEFAis a potent inhibitor of LPL,62 with a buildup of releasedNEFA at the capillary endothelium, resulting in the detach-ment of LPL from the vascular walls, thereby losing itshydrolytic activity. In the present study, there was a trendtoward a greater reduction in fasting and postprandial NEFAconcentrations in the apoE2 subgroup, indicative of moreeffective NEFA removal from the circulation, which mayhave contributed to the greater increase in LPL activity in thissubgroup.In addition to the impact on the TG profile, fish oil

intervention also had a marked influence on TC and LDL-Clevels and on the LDL density profile. Because LDL isderived from VLDL catabolism, reductions in VLDL mightbe expected to lower LDL-C. However, in agreement withprevious studies,56,63 an increase (7%) in levels was observedin the group as a whole. In addition to suppressing VLDLproduction, n-3 PUFA is also known to influence VLDLcomposition, with a shift in the distribution of VLDL sub-classes toward the smaller VLDL particles.57,63 Becausesmaller VLDL particles represent the primary precursor ofLDL, this qualitative change in VLDL may result in a greaterrate of conversion to LDL.64 The adverse effect of fish oils onLDL-C was strongest in E4 carriers, in whom a 16% increasewas observed. ApoE4 is known to selectively associate withVLDL,26,50 which may enhance its catabolism to LDL (viathe apoE-dependent catabolic pathway) and, in combinationwith the downregulated state of the LDL receptor in thissubgroup, may explain the greater increase in LDL-C levels.This potentially proatherogenic increase in LDL concen-

trations, particularly in the apoE4 carriers, may be offset bythe significant reduction in the percentage of LDL as LDL-3.Caution is required in drawing conclusions regarding the

atherogenic consequences of these 2 opposing changes,because lack of LDL compositional data means that it is notpossible to calculate the absolute concentration of cholesterolin the LDL subfractions, which may be the relevant measure.Although strong correlations between TG and LDL-3 con-centrations have been observed in a recent study of ours (datanot shown)65 and other studies,11,12 early work investigatingthe impact of n-3 PUFA on the LDL profile failed to observeany beneficial changes in LDL composition changes.55,64,66However, these studies used indirect measures of LDLparticle size and composition, because density gradient ultra-centrifugation techniques used in the present study were notavailable. More recent studies are in agreement with ourfindings,67,68 although Homma et al69 actually found de-creases in LDL particle size after n-3 PUFA supplementation,and Patti et al70 observed no change in LDL composition inpatients with non–insulin-dependent diabetes mellitus afterfish oil treatment.In conclusion, the presence of an apoE4 allele exaggerates

the HDL abnormalities of the ALP. Fish oil supplementationis effective in correcting fasting and postprandial ALP dys-lipidemia. However, apoE genotype is an important determi-nant of responsiveness to this dietary intervention. Thegreatest benefits were evident in apoE2 carriers, in whom theobserved reduction in fasting and postabsorptive TG levels, adecrease in the atherogenic LDL-3, and a trend toward higherHDL-C levels would be expected to be associated with aclinically meaningful reduction in CHD risk. In apoE4carriers, the hypotriglyceridemic benefits may be counter-acted by a potential proatherogenic shift in the cholesterolprofile.

AcknowledgmentsWe are grateful to the Biotechnology and Biological SciencesResearch Council (BBSRC) for funding this project and to Lube A/S,Hadsund, Denmark, for providing us with the oil capsules.

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