European Journal of Clinical Nutrition (2000) 54, 865±871 ß 2000 Macmillan Publishers Ltd All rights reserved 0954±3007/00 $15.00 www.nature.com/ejcn

Associations of -linolenic and with risk factors for coronary heart disease

WJE Bemelmans1*, FAJ Muskiet2, EJM Feskens3, JHM de Vries4, J Broer5, JF May6 and B Meyboom-de Jong1

1University of Groningen, Department of Family Medicine, Groningen, The Netherlands; 2Groningen University Hospital, Department of Pathology and Laboratory Medicine, Groningen, The Netherlands; 3National Institute of Public Health and the Environment, Department of Chronic Diseases Epidemiology, Bilthoven, The Netherlands; 4Wageningen Agricultural University, Department of and Epidemiology, Wageningen, The Netherlands; 5Public Health Department, City of Groningen, The Netherlands; and 6Groningen University Hospital, Department of Cardiology, Groningen, The Netherlands

Background: Prevention of coronary heart disease (CHD) in high-risk subjects. Objective: To investigate the associations of dietary intake of alpha- (ALA) and linoleic acid (LA) as assessed by frequency questionnaire and in the plasma cholesteryl (CE), with CHD risk factors. Design: Baseline data of a double-blind, randomized placebo-controlled trial. Subjects have hypercholester- olemia (6.0 ± 8.0 mmol=l) and at least two other CHD risk factors (n ˆ 266). Results: The reported dietary ALA and LA intakes and the LA=ALA ratio were associated with the contents in the CE (r ˆ 0.37, r ˆ 0.21, and r ˆ 0.42, respectively; P < 0.01). In multivariate analysis, CE ALA was inversely associated with diastolic pressure (r ˆ7 0.13; P < 0.05) and positively with serum triacylglycerol (r ˆ 0.13; P < 0.05), and CE LA was inversely associated with serum triacylglycerol (r ˆ7 0.32; P < 0.01). The CE LA=ALA ratio was strongly inversely associated with CE ALA (r ˆ7 0.95; P < 0.01). In the lowest quintile of CE ALA, mean dietary intake was 0.4 energy % ALA (1.2 g=day), 8.4 energy % LA and an LA=ALA ratio of 21, and in the highest quintile 0.6 energy % ALA (1.7 g=day), 6.8 energy % LA and 12 (ratio). In the lowest quintile of CE ALA the diastolic blood pressure was 4 mmHg lower (P trend < 0.05), and the serum triacylglycerol 0.3 mmol=l higher (P trend NS) when compared with the top quintile. Conclusions: In a CHD high-risk population with LA-rich background diet, these cross-sectional data suggest that replacing LA in the diet by ALA may decrease diastolic blood pressure, and may increase serum triacylglycerol concentration. Sponsorship: Dutch Prevent fund grant no. 28-2757; (). Descriptors: alpha-linolenic acid; linoleic acid; plasma cholesteryl ester; CHD risk factors; blood pressure; serum triacylglycerol European Journal of Clinical Nutrition (2000) 54, 865±871

Introduction preventive effect of dietary ALA on CHD events (Ascherio et al, 1996; Hu et al, 1999). The MARGARIN study is a prevention project of coronary ALA is the vegetary precursor of the marine long chain heart disease (CHD) through a high-risk approach. The n-3 fatty (EPA; 20 : 5n-3) study group is composed of 266 hypercholesterolemic and (DHA; 22 : 6n-3). ALA com- subjects with at least two other CHD risk factors. Our petes with its n-6 counterpart linoleic acid (18 : 2n-6) for project was inspired by the Lyon Diet Heart Study. In this desaturation and chain-elongation. Linoleic acid (LA) secondary prevention trial among patients who had a is in this pathway converted to (AA; myocardial infarction, a 70% reduction in CHD events 20 : 4n-6). was observed in the experimental group on an alpha- In recent years, hypercholesterolemic subjects have been linolenic acid (ALA; 18 : 3n-3) rich Mediterranean diet. stimulated to decrease saturated intake and to consume In the ®nal report, plasma ALA content was associated with LA-enriched food products. It is accepted that LA lowers improved prognosis (de Lorgeril et al, 1994,1999). Also, serum levels (Schaefer, 1997). However, a steadily several large observational studies have reported on the increasing dietary LA=ALA ratio may have adverse health consequences (Renaud et al, 1995; Okuyama et al, 1997). An excessive amount of LA in the diet may lead to inappropriate production of n-6 (Lands, 1989). One study observed a positive association between *Correspondence: WJE Bemelmans, nutritionist, University of Groningen, Department of Family Medicine, Antonius Deusinglaan 4, 9713 AW the dietary n-6=n-3 ratio and cardiovascular mortality Groningen, The Netherlands. (Dolecek & Grandits, 1991). E-mail: [email protected] Accurate epidemiologic data of usual LA and ALA Guarantor: B Meyboom-de Jong. intake in populations are limited, mainly due to incomplete Contributors: All authors have contributed to the execution, interpretation and reporting of the study. databases. Hence, in the context of the primary Received 10 January 2000; revised 14 August 2000; accepted 20 August prevention of CHD the in¯uence of increased intake of 2000 ALA on CHD risk factors is relatively unknown (Schaefer, Alpha-linolenic acid and risk factors WJE Bemelmans et al 866 1997). Therefore, at the start of our prevention project we Samples and analysis investigated cross-sectionally the relation between dietary Serum total , HDL-cholesterol and triacylgly- ALA and LA intake and ALA and LA content of the cerol (without blanking) were determined by enzymatic plasma cholesteryl ester (CE) on the one hand and blood methods on a Vitros 950 (Ortho-Clinical Diagnostics, pressure, serum total and HDL cholesterol, and serum Rochester NY, USA). HDL-cholesterol was isolated by triacylglycerol on the other hand. precipitation of LDL and VLDL with dextran sulfate and magnesium chloride. The CE composition was determined with our method described previously (Hoving Subjects and methods et al, 1988). Brie¯y, total were isolated from plasma by organic solvent extraction. The CE were isolated by Study design solid phase extraction using an aminopropyl-silica column The MARGARIN study, abbreviated from Mediterranean and converted into their methyl by transmethylation. alpha linolenic enriched Groningen dietary intervention The methyl esters were isolated by organic solvent extrac- study, investigates two preventive strategies: ®rst, intensive tion and injected into a gas chromatograph with ¯ame group education about Mediterranean dietary habits com- ionization detection. Peak areas were recorded and pared to `care as usual' for hypercholesterolemic persons converted into mol=100 mol fatty acids. (single blind); and second, supplementation of an ALA- enriched margarine compared to an LA-rich margarine Dietary assessment (double blind). The randomized placebo-controlled study Dietaryintakewasassessedbyaself-administeredsemi-quan- design will be described in detail elsewhere (Bemelmans titative food frequency questionnaire, called the FFQ-ALA. et al, in press). The present study involves the baseline The FFQ-ALA is based on two validated questionnaires, data, collected in November 1997. The medical ethical measuring total fat and fatty acids intake (Feunekes et al, committee of the Groningen University Hospital approved 1993), or antioxidant and macronutrient intake (Ocke et al, the study design. 1997). We changed the composition of several pro- duct items to achieve the speci®c objectives of the Study group and selection MARGARIN study. The FFQ-ALA aims to accurately Inclusion criteria were a mean of two serum cholesterol assess ALA and LA intake. Further, the FFQ-ALA deter- measurements between 6 and 8 mmol=l and at least two mines the intake of total energy, fatty acids, alcohol and of the following CHD risk factors: high blood pressure dietary ®bre. The intakes of energy, the macronutrients and (diastolic  95 mmHg and=or systolic  160 mmHg) or dietary ®bre were validated with a 3 day 24 h recall method use of anti-hypertensive medication; body mass index in a pilot study (n ˆ 43). Pearson correlation coef®cients (BMI)  27 kg=m2; smoking; and diagnosis of cardiovas- (P < 0.01) were as follows: energy intake (r ˆ 0.76); total cular disease, or a ®rst-degree relative with a history of fat as percentage of energy (r ˆ 0.66); saturated fatty acids cardiovascular disease prior to the age of 60 y. Exclusion (r ˆ 0.51); monounsaturated fatty acids (r ˆ 0.52); polyun- criteria were age < 30 or > 70 y, diabetes mellitus, saturated fatty acids (r ˆ 0.70); alcohol (r ˆ 0.72); and ®bre hypothyroidism, and use of acetylsalicylic acid, anti- in grams per day (r ˆ 0.74) (data not yet published). coagulant or cholesterol-lowering drugs. The FFQ-ALA contains 165 food items, plus 12 ques- Potential participants with at least two CHD risk factors tions about the amount of added fat, and 10 questions to were invited to have their serum cholesterol measured and determine portion sizes more accurately. The consumption they were recruited in three ways. First, all inhabitants over frequency of each item is reported per day, week or month. 30 y of two municipalities received a written invitation to Average portion sizes were assigned. The product items participate in a blood pressure screening program. Second, consist of 650 and the weighed item composition is registration systems of three pharmacies and one family based on national food consumption data (Hulshof et al, physician in these two counties were screened for patients 1998). Questionnaires were checked on completeness and on anti-hypertensive drugs. Third, a local radio program unrealistic answers by the research nutritionist. Nutrient discussed the project and invited potential participants to composition of the foods was obtained from the Dutch contact the researchers. NEVO 1996-1 table (Netherlands Bureau for Nutrition Written informed consent was obtained from 282 per- Education, 1996). Data on ALA contents were provided sons. Fourteen persons dropped out prior to the baseline by a report from the National Institute of Public Health and examinations. Two persons were further excluded because the Environment (RIVM) (Voskuil & Feskens, 1994), by of diabetes mellitus, accidently discovered at baseline Dutch Nevo Foundation, and by analyses performed by the examination. department of Human Nutrition and Epidemiology of Wageningen Agricultural University (Hulshof et al, Baseline examinations 1996). If the ALA or LA values were not available for a Within 2 weeks, all subjects were investigated at the food, the contents were estimated on the basis of compar- regional hospital in the early morning after an overnight able foods. The questionnaires were processed by the fast. We determined serum total cholesterol, HDL choles- nutritional software package KOMEET1, developed by terol, triacylglycerol and the plasma CE fatty acid compo- the Department of Human Nutrition and Epidemiology of sition. Blood pressure was measured in sitting position and Wageningen Agricultural University. Data entry was bodyweight was determined without shoes and heavy double checked. clothing. Height was measured during the study in March 1998, and was assumed to be equal at baseline. A standar- Data evaluation and statistics dized questionnaire was used to provide information on With the statistical package SPSS 6.0 (NorusÏis, 1993) smoking. CVD history and use of medication. A food means and standard deviations were computed and non- frequency questionnaire was distributed 4 weeks before Gaussian distributed variables are also presented as med- baseline (see below). ians. Differences between men and women were analyzed

European Journal of Clinical Nutrition Alpha-linolenic acid and risk factors WJE Bemelmans et al 867 for statistical signi®cance (P < 0.05) with Student's t-test. (37%) used anti-hypertensive medication (P < 0.05). More Differences in percentages of categorical variables were men (61%) than women (40%) were present smokers tested by w2. Pearson correlation coef®cients were com- (P < 0.01). puted to test for associations between variables. For this, Mean ALA and LA intake was 1.7 g=day (med- non-Gaussian distributed variables were transformed to ian ˆ 1.5 g=day) and 25.1 g=day (median ˆ 24.3 g=day), their natural logarithm (dietary and CE LA=ALA ratio). respectively, in men, and 1.2 g=day (median ˆ 1.0 g=day) In multivariate analysis, partial correlation coef®cients and 17.8 g=day (median ˆ 15.8 g=day) in women. Mean were computed to test for independent associations of dietary LA=ALA ratio was 16.7 (median ˆ 14.4) for men, nutrient intakes with CHD risk factors. and 17.0 (median ˆ 14.7) for women. As potential confounders with serum total cholesterol, Table 2 shows the mean CE fatty acid composition. No we adjusted for age, gender, BMI, saturated fatty acids and signi®cant differences between men and women were cholesterol intake, smoking and consumption of coffee. observed. The CE LA=ALA ratio was strongely inversely With HDL cholesterol we adjusted for age, gender, BMI, associated with CE ALA (r ˆ7 0.95; P < 0.01; Table 2). smoking, intake of alcohol and carbohydrates. With serum triacylglycerol, we adjusted for age, gender, BMI, smoking, ®sh consumption, intake of alcohol, carbohydrates and Association of dietary intake and content of fatty acids in ®bre. With blood pressure, we adjusted for age, gender, the cholesteryl ester BMI, smoking, alcohol intake, ®sh consumption and anti- Pearson correlation coef®cients of dietary ALA and LA hypertensive medication. intake (percentage of energy; en%) and the LA=ALA ratio To investigate which CE fatty acid predicted the depen- assessed by the FFQ-ALA, with corresponding compounds dent variables (CHD risk factors) independently from the in the CE were 0.37, 0.21 and 0.42 (P < 0.01), respectively other CE fatty acids, we used the regression equation (Table 3). In addition, the dietary LA=ALA ratio was described by Cambien et al (1988). This equation controls associated with CE AA=EPA ratio (r ˆ 0.13; P < 0.05) for structural dependencies of the CE fatty acids as per- and with the CE n-6=n-3 ratio (r ˆ 0.20; P < 0.01). Corre- centages of a composition. ANCOVA analysis provided the lation coef®cients were similar for men and women (data adjusted means of CHD risk factors for each quintile of the not shown). CE LA=ALA ratio. Associations of intake of alpha-linolenic and linoleic acid Results with confounding variables The dietary intake of ALA was inversely associated with Baseline data age (r ˆ7 0.31; P < 0.01), but the CE ALA content was In the study 266 subjects participated with at least three not associated with age (r ˆ7 0.08, NS). Dietary intake of CHD risk factors. Mean age of the female and male ALA and CE ALA were positively associated with satu- participants was 55 and 53 y, respectively (Table 1). rated fat intake and coffee consumption, and inversely with Women had higher mean BMI than men. Mean total carbohydrate intake. The dietary intake of LA was also energy intake of men and women were 11.9 MJ=day inversely associated with carbohydrate intake, and both (median ˆ 11.8 MJ=day) and 8.8 MJ=day (median ˆ dietary LA and CE LA were inversely associated with 8.4 MJ=day), respectively. More women (57%) than men alcohol intake (Table 4).

Table 1 Baseline characteristics of 266 participants of the MARGARIN study

Men (n ˆ 118) Women (n ˆ 148) Variable Mean s.d. Mean s.d.

Age (y) 53 10 55 9* Body mass index (kg=m2) 29.0 3.2 30.5 5.2** Present smokers (%) 61 40** Number of cigarettes per day (n=day)a 12 8 14 11 Total serum cholesterol (mmol=l) 6.6 0.7 6.8 0.8 HDL cholesterol (mmol=l) 1.1 0.3 1.4 0.4** Triacylglycerol (mmol=l) 2.1 1.2 1.8 0.8* Systolic blood pressure (mmHg) 143 22 147 22 anti-hypertensive medication excluded 136 17 136 17 Diastolic blood pressure (mmHg) 88 15 85 15 anti-hypertensive medication excluded 85 15 81 16 Present use of anti-hypertensive medication (%) 37 57* Dietary nutrient intakeb Energy (MJ=day) 11.9 3.5 8.8 2.5** Total fat (percentage of energy) 38.7 6.0 37.4 7.5 P=S ratioc 0.67 0.26 0.62 0.24 Linoleic acid (g=day) 25.1 10.8 17.8 9.7** (percentage of energy) 7.9 2.4 7.4 3.0 Alpha-linolenic acid (g=day) 1.7 0.8 1.2 0.7** (percentage of energy) 0.5 0.2 0.5 0.2 Linoleic=alpha-linolenic ratio 16.7 8.6 17.0 9.1

aOnly smokers included. bWomen n ˆ 144; differences between men and women, *P < 0.05; **P < 0.01. cP=S: ratio of polyunsaturated=saturated fatty acids.

European Journal of Clinical Nutrition Alpha-linolenic acid and risk factors WJE Bemelmans et al 868 Table 2 Fatty acid composition (mol%) of the plasma cholesteryl ester and the Pearson correlation coef®cients with the content of alpha-linolenic acid, linoleic acid and the ratio in the cholesteryl ester (n ˆ 266)

Correlations with CEa

Fatty acid Mean (s.d.) ALAb LA Ratio LA=ALA

14 : 0 0.9 (0.3) 0.32** 7 0.29** 7 0.38** 16 : 0 12.5 (1.2) 7 0.17** 7 0.63** 7 0.03 16 : 1n-7 3.5 (1.5) 0.28** 7 0.72** 7 0.46** 18 : 0 1.6 (0.5) 7 0.08 7 0.22** 0 18 : 1n-7 1.1 (0.2) 7 0.13* 7 0.38** 0.02 18 : 1n-9 15.2 (2.2) 0.22** 7 0.75** 7 0.41** 18 : 2n-6 54.4 (4.6) 7 0.19** Ð 0.46** 18 : 3n-3 0.5 (0.1) Ð 7 0.19** 7 0.95** 18 : 3n-6 1.0 (0.4) 0.26** 7 0.44** 7 0.36** 20 : 3n-6 0.8 (0.2) 0.06 7 0.24** 7 0.12* 20 : 3n-9 0.08 (0.04) 0.10 7 0.48** 7 0.23** 20 : 4n-6 6.6 (1.4) 7 0.19** 7 0.26** 0.10 20 : 5n-3 1.0 (0.7) 0.17** 7 0.25** 7 0.25** 22 : 6n-3 0.6 (0.2) 0.05 7 0.07 7 0.08 Ratio LA=ALAb 113 (34) 7 0.95** 0.46** Ð

aCE ˆ cholesteryl ester. bAlpha-linolenic acid (ALA) ˆ 18 : 3n-3; linoleic acid (LA) ˆ 18 : 2n-6; LA=ALA ratio is in mol=mol. *P < 0.05; **P < 0.01.

Table 3 Pearson correlation coef®cients of fatty acid intakes Associations of intake of alpha-linolenic and linoleic acid (percentages of energy) as assessed by food frequency questionnaire and with risk factors fatty acid contents of the plasma cholesteryl ester (n ˆ 262) In univariate analysis, the dietary intake of ALA was Food frequency questionnaire positively associated with serum triacylglycerol, but in multivariate analyses neither the intake of ALA nor of Linoleic Alpha-linolenic LA=ALA LA were associated with CHD risk factors (Table 5). In Content cholesteryl ester acid acid ratio multivariate analyses adjusted for the previous named Linoleic acid (18 : 2n-6) 0.21** 0 0.20** confounders, CE ALA was positively associated with Alpha-linolenic acid (18 : 3n-3) 7 0.18** 0.37** 7 0.39** serum triacylglycerol (r ˆ 0.13; P < 0.05), and inversely LA=ALA ratio 0.23** 7 0.34** 0.42** with diastolic blood pressure (r ˆ7 0.13; P < 0.05). The AA=EPA ratio (20 : 4n-6=20 : 5n-3) 0.15* 0 0.13* CE LA content was inversely associated with serum tria- *P < 0.05; **P < 0.01. cylglycerol (r ˆ7 0.32; P < 0.01) and inversely with HDL LA ˆ linoleic acid; ALA ˆ alpha-linolenic acid; AA ˆ arachidonic acid; cholesterol (r ˆ7 0.12; P < 0.05). However, after adjust- EPA ˆ eicosapentaenoic acid. ment for the structural interdepence of CE fatty acids with the method of Cambien et al (1988), the inverse association between CE LA and HDL cholesterol was nonsigni®cant, while the other associations remained signi®cant. The CE LA=ALA ratio was independently inversely associated with serum triacylglycerol (r ˆ7 0.21; P < 0.05), and positively with diastolic blood pressure Table 4 Pearson correlation coef®cients of alpha-linolenic acid and (r ˆ 0.13; P < 0.05). linoleic acid in the diet and in the plasma cholesteryl ester and confounding variables (n ˆ 262) Independent associations of other fatty acids in the Dietary intake Content CE cholesteryl ester with risk factors In multivariate analysis, the CE 20 : 3n-6 content was Variables (unit) ALA LA ALA LA positively associated with total serum cholesterol concen- Age (y) 7 0.31** 0.06 7 0.08 0.08 tration (r ˆ 0.16; P ˆ 0.01; n ˆ 248). None of the CE fatty Body mass index (kg=m2) 0.13* 7 0.02 7 0.06 7 0.02 acids were independently associated with the serum HDL Smoking (no. of 0.04 7 0.06 0.02 7 0.04 cholesterol concentration. Partial correlation coef®cients cigarettes=day) between serum triacylglycerol and the fatty acids were as Coffee (no. of consumptions= 0.15* 7 0.02 0.16* 7 0.02 following: CE 14 : 0 (r ˆ 0.29), CE 16 : 0 (r ˆ 0.27) and CE day) (g=day) 0.07 7 0.03 7 0.04 0 18 : 1n-7 (r ˆ 0.12) (n ˆ 252; P < 0.05). None of the fatty Nutrient intake acids were independently associated with systolic blood Saturated fatty acids 0.49** 0.06 0.30** 0.02 pressure, and CE 16 : 1n-7 was positively associated with (percentage of energy) diastolic blood pressure (r ˆ 0.13; P < 0.05; n ˆ 253). The Carbohydrates (percentage 7 0.43** 7 0.28** 7 0.20** 0.09 of energy) CE AA=EPA ratio and the CE n-6=n-3 ratio were not Alcohol (percentage 7 0.03 7 0.18** 0.07 7 0.29** signi®cantly related to blood pressure. of energy) Cholesterol (g=day) 0.29** 7 0.08 0.09 7 0.06 Dietary intake and risk factors for quintiles of fatty acid Dietary ®bre (g=day) 7 0.05 0.10 7 0.10 0.06 content in the cholesteryl ester *P < 0.05; **P < 0.01. The reported intake of ALA in the lowest quintile of CE LA ˆ Linoleic acid; ALA ˆ Alpha-linolenic acid; CE ˆ cholesteryl ester. ALA was 0.4 en% (1.2 g=day) when compared with

European Journal of Clinical Nutrition Alpha-linolenic acid and risk factors WJE Bemelmans et al 869 Table 5 Pearson correlation coef®cients (univariate) and partial correlation coef®cients (multivariate) of alpha-linolenic acid and linoleic acid in the diet and in the cholesteryl ester, and coronary heart disease risk factors (n ˆ 262)

Reported dietary intake Content cholesteryl ester

ALA LA ALA LA

Parameter UV MV UV MV UV MV UV MV

Total serum cholesterola (mmol=l) 7 0.09 7 0.06 7 0.07 7 0.08 0.04 0.07 7 0.06 7 0.09 HDL cholesterolb (mmol=l) 7 0.06 0 7 0.07 7 0.11 0.07 0.02 7 0.11 7 0.12* Triacylglycerolc (mmol=l) 0.13* 0.08 7 0.05 7 0.02 0.12 0.13* 7 0.34** 7 0.32** Systolic blood pressured (mmHg) 7 0.12 7 0.05 0.10 0.06 7 0.12 7 0.09 0.04 0.04 Diastolic blood pressured (mmHg) 7 0.06 7 0.04 0.06 0.04 7 0.14* 7 0.13* 7 0.03 0

*P < 0.05; **P < 0.01; LA ˆ linoleic acid; ALA ˆ alpha-linolenic acid; CE ˆ cholesteryl ester, UV ˆ univariate; MV ˆ multivariate. aAdjusted for age, gender, BMI, intake of saturated fatty acids, dietary cholesterol intake, smoking and consumption of coffee. bAdjusted for age, gender, BMI, smoking, intake of alcohol and carbohydrates. cAdjusted for age, gender, BMI, smoking, consumption of ®sh, intake of alcohol, carbohydrate and dietary ®bre. dAdjusted for age, gender, BMI, smoking, intake of alcohol, consumption of ®sh, and anti-hypertensive medication.

Table 6 Adjusted means of dietary intake and coronary heart disease risk factors, for quintiles of alpha-linolenic acid in the cholesteryl ester

Quintiles CE ALA 1 2 3 4 5 P trend

n 58 50 50 54 50 CE ALA 0.36 0.44 0.50 0.58 0.75 ** Dietary nutrient intake Linolenic acid (en%) 0.4 0.5 0.5 0.5 0.6 ** (g=day) 1.2 1.3 1.4 1.5 1.7 ** Linoleic acid (en%) 8.4 7.6 8.1 7.3 6.8 ** (g=day) 24 21 21 21 19 * Dietary LA=ALA ratio 21 18 18 15 12 ** CHD risk factors Diastolic blood pressurea (mmHg) 89 88 88 83 85 * Anti-hypertensive medication excluded (n ˆ 138) 85 85 85 80 82 Systolic blood pressure (mmHg) 146 144 147 143 143 Anti-hypertensive medication excluded (n ˆ 138) 137 139 137 134 135 Serum triacylglycerolb (mmol=l) 1.9 1.9 1.8 2.1 2.2

*P < 0.05; **P < 0.01. LA ˆ linoleic acid: ALA ˆ alpha 7 linolenic acid; CE ˆ cholesteryl ester; FFQ ˆ food frequency questionnaire; CHD ˆ coronary heart disease. aAdjusted for age, gender, body mass index, alcohol intake, smoking, ®sh consumption, anti- hypertensive medication. bAdjusted for age, gender, body mass index, alcohol intake, smoking, ®sh consumption, carbohydrate and ®ber intake.

0.6 en% (1.7 g=day) in the top quintile (Table 6). In the and a positive association with serum triacylglycerol con- lowest quintile of CE ALA the adjusted mean diastolic and centration (r ˆ7 0.13; P < 0.05), while CE LA was inver- systolic blood pressures were 89 and 146 mmHg, respec- sely associated (r ˆ7 0.32; P < 0.01). In this population, tively, as compared to 85 mmHg (P for trend < 0.05) and according to the Framingham CHD risk function, a 143 mmHg (P for trend NS) in the top quintile. In the decrease in diastolic blood pressure of 4 mmHg, that is lowest vs the top quintile of CE ALA the adjusted mean the difference between the lowest and top quintile of CE serum triacylglycerol concentrations were 1.9 and ALA, implies a 5% (men) to 6% (women) relative decrease 2.2 mmol=l(P for trend NS; Table 6). In the lowest vs in risk of developing CHD, ceteris paribus (Anderson et al, the top quintile of CE LA the adjusted mean serum 1991). Serum triacylglycerol is not included in this CHD triacylglycerol concentrations were 2.4 and 1.6 mmol=l(P risk function, but an increase of 0.3 mmol=l may increase for trend < 0.01). The trend of diastolic blood pressure the CVD risk with 11% in men and 4.5% in women among the CE LA quintiles was J-shaped (P for quadratic (Austin, 1999). Therefore, the replacement of LA trend < 0.01). The mean diastolic blood pressure in the with ALA seems to have a net equal effect on risk of lowest quintile was 90 mmHg, in the second quintile developing CHD. 83 mmHg, and in the top quintile 89 mmHg (data not It should be noted that even in the highest quintile of CE shown). ALA the dietary LA=ALA ratio (12.4) was higher than recommended. A dietary LA=ALA ratio between 4.0 and Discussion 10.0 is stated as nutritional goal (Minister of National Health and Welfare, Canada, 1990; WHO and FAO Joint At baseline of our CHD prevention project, the hypercho- Consultation, 1995). Hence, we could not compare the lesterolemic participants reported a high LA intake (mean mean risk factors in a quintile with low ALA intake and ˆ 7.5Æ 2.8 en%). Dietary fatty acid intake is re¯ected into high LA=ALA ratio to a quintile with a mean LA=ALA the CE fatty acid composition (Zock et al, 1997). An ratio in the recommended range. independent inverse association was found of plasma CE Based on the correlation coef®cients with the CE fatty ALA and diastolic blood pressure (r ˆ7 0.13; P < 0.05), acid pro®le, we conclude that the FFQ-ALA reasonably

European Journal of Clinical Nutrition Alpha-linolenic acid and risk factors WJE Bemelmans et al 870 estimates dietary ALA intake and LA=ALA ratio, and less 12% while the concentration of EPA was unaltered. Vas- well estimates LA intake (r ˆ 0.21; P < 0.01). The latter cular prostacyclin synthesis increased, resulting in lower correlation is lower than described previously (James et al, blood pressure (Rupp et al, 1996). Apparently, ALA can 1993; Sarkkinen et al, 1994), and this may be due to greater be converted into EPA and subsequently alters prosta- focus on ALA by compiling the FFQ-ALA. Nevertheless, noid production, without raised EPA concentration in since the trend in LA intake among the quintiles of CE LA membranes. was signi®cant, the FFQ-ALA may reasonably indicate LA For effective CHD prevention, the impact of increased intake as percentage of energy. A limitation of the present ALA intake on CHD risk factors needs to be further study was that blood pressure was measured once, while a investigated in a prospective study design. These cross- duplo measurement may be more accurate. The absence of sectional results suggest that the overall net effect on CHD a signi®cant association of CE ALA with systolic blood risk may be equal when LA in the diet is replaced with pressure may be due to this fact. ALA. Increasing dietary ALA intake in a CHD high- The small variance in total and HDL cholesterol level risk population with an LA-rich background diet may due to the inclusion criteria may have prevented a proper decrease mean blood pressure, and may increase serum investigation of their associations with ALA and LA. Other triacylglycerol concentration. studies found either a negative association between CE LA and serum total cholesterol (Moilanen et al, 1986; Sandker et al, 1993), or no relation (De-Backer et al, 1989). The Acknowledgements ÐWe thank Ingrid Martini, Groningen University strong inverse association of CE LA with serum triacylgly- Hospital, for analyzing the fatty acid pro®les of the cholesteryl ester. cerol (r ˆ7 0.32; P < 0.01) was found previously (Moila- Thanks are also due to Claudia Oomen (MSc), National Institute of Public nen et al, 1986; Cambien et al, 1988; Rosseneu et al, 1994). Health and the Environment, for critically reading the manuscript, and also However, according to dietary intake, previous research has to Klaas Groenier (MSc), Department of Family Medicine, for statistical shown that both dietary LA and ALA lowered serum advice. triacylglycerol (Singer et al, 1990a,b) and our results also showed no independent association of dietary ALA intake with serum triacylglycerol (r ˆ 0.08; NS). An experimental References study showed that replacement of LA with ALA did not in¯uence serum lipid levels (Pang et al, 1998). Therefore, it Anderson KM, Wilson PWF, Odell PM & Kannel WB (1991): An updated appears unlikely that increasing ALA intake and thereby coronary risk pro®le. A statement for health professionals. Circulation 83, 356 ± 362. lowering the dietary LA=ALA ratio would cause an Ascherio A, Rimm EB, Giovannucci EL, Spiegelman D, Stampfer M & increase of serum triacylglycerol level. Willett WC (1996): Dietary fat and risk of coronary heart disease in According to blood pressure, CE ALA was inversely men: cohort follow up study in the United States. Br. Med. J. 13, associated with diastolic blood pressure (r ˆ7 0.13; 84 ± 90. P < 0.05), whereas CE LA was not associated. Previously Austin MA (1999): Epidemiology of hypertriglyceridemia and cardiovas- cular disease. Am. J. Cardiol. 83, 13F ± 16F. described relations between CE LA and blood pressure Bemelmans WJE, Broer J, de Vries JHM, Hulshof KFAM, May JF, were either negative (Uusitupa et al, 1994), positive Meyboom-de Jong B (2000). Impact of mediterranean diet education (Masana et al, 1996) or absent (Cambien et al, 1988; De- versus posted lea¯et on dietary habits and serum cholesterol in a high Backer et al, 1989). In a study with 399 males, an increase risk population for cardiovascular disease. Public Health Nutr. in press. of 1% in adipose ALA content was associated with a Berry EM & Hirsch J (1986): Does dietary linolenic acid in¯uence blood pressure? Am. J. Clin. Nutr. 44, 336 ± 340. decrease of 5 mmHg in systolic, diastolic and mean arterial Cambien F, Warnet JM, Vernier V, Ducimetiere P, Jacqueson A, Flament blood pressure (Berry & Hirsch, 1986). One epidemiologi- C, Orssaud G, Richard JL & Claude JR (1988): An epidemiologic cal study found an inverse relation between dietary ALA appraisal of the associations between the fatty acids esterifying serum intake and blood pressure (Salonen et al, 1988). Controlled cholesterol and some cardiovascular risk factors in middle-aged men. experiments, with small numbers of males only, proved the Am. J. Epidemiol. 127, 75 ± 86. De-Backer G, De-Craene I, Rosseneu M, Vercaemst R & Kornitzer M hypotensive effect of the marine n-3 fatty acids to be (1989): Relationship between serum cholesteryl ester composition, greater than ALA (Singer et al, 1986; Kestin et al, 1990). dietary habits and coronary risk factors in middle-aged men. Athero- Nevertheless, increasing ALA intake in this population may sclerosis 78(2 ± 3), 237 ± 243. lower blood pressure, and this hypotensive effect may be Dolecek TA & Grandits G (1991): Dietary polyunsaturated fatty acids and mortality in the Multiple Risk Factor Intervention Trial (MRFIT). mediated through in¯uencing the dietary LA=ALA ratio. World Rev. Nutr. Diet 66, 205 ± 216. Supplementation of n-3 fatty acids may lower blood pres- Feunekes GIJ, Staveren van WA, Vries de JHM, Burema J & Hautvast sure because of their competition with n-6 fatty acids for JGAJ (1993): Relative and biomarker-based validity of a food-fre- the (Knapp, 1991). Endogenous quency questionnaire estimating intake of and cholesterol. Am. J. synthesis of vasoactive eicosanoids may alter when the Clin. Nutr. 58, 489 ± 496. Gerster H (1998): Can adults adequately convert alpha-linolenic acid balance between n-3 and n-6 fatty acid intake is changed. (18 : 3n-3) to eicosapentaenoic acid (20:5n-3) and docosahexaenoic Against a high LA background diet the already low con- acid (22:6n-3)? Int. J. Vitam. Nutr. Res. 68, 159 ± 173. version of ALA to EPA is further reduced (Gerster, 1998). Hoving EB, Jansen G, Volmer M, Doormaal JJ van & Muskiet FAJ (1988): In the present study, dietary LA=ALA ratio and CE Pro®ling of plasma cholesterol ester and fatty acids as their LA=ALA ratio were associated with CE AA=EPA ratio methyl esters by capillary , preceded by a rapid aminopropyl-silica column chromatographic separation of lipid classes. (r ˆ 0.13; P < 0.05 and r ˆ 0.41; P < 0.01), but CE J. Chromatogr. Biomed. Applic. 434, 395 ± 409. AA=EPA ratio was not associated with blood pressure. Hu FB, Stampfer MJ, Manson JAE, Rimm EB, Wolk A, Colditz GA, Hence, the competition between ALA and LA for Hennekens CH & Willett WC (1999): Dietary intake of a-linolenic acid metabolic conversion and subsequent competition for the and risk of fatal ischemic heart disease among women. Am. J. Clin. enzyme cyclooxygenase may play a role, but other mechan- Nutr. 69, 890 ± 897. Hulshof KFAM, Kistemaker C & Bouman M (1998): De inname van isms could be involved (Singer, 1992). In hypertensive rats energie en voedingsstoffen door Nederlandse bevolkingsgroepen-Voed- increasing ALA intake was investigated against a high LA selconsumptiepeiling 1997 ± 1998. Report no. V98.805 TNO Nutrition background diet. The content of ALA in the aorta rose by and Food Research Institute: Zeist.

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