Alpha-Linolenic acid is hypothesized to reduce the risk of

Adipose Tissue -Linolenic Acid and Nonfatal Acute Myocardial Infarction in Costa Rica Ana Baylin, MD, DrPH; Edmond K. Kabagambe, BVM, PhD; Alberto Ascherio, MD, DrPH; Donna Spiegelman, DSc; Hannia Campos, PhD Background -Linolenic acid may protect against cardiovascular disease. We examined the association between adipose tissue -linolenic acid and nonfatal acute myocardial infarction (MI) in a population-based case-control study in Costa Rica. Methods and Results The 482 case patients with a first nonfatal acute MI and 482 population control subjects were matched by age, sex, and area of residence. Fatty acids were assessed by gas-liquid chromatography in adipose tissue samples collected from all subjects. ORs and 95% CIs were calculated from multivariate conditional logistic regression models. Subjects in the top quintiles of adipose tissue -linolenic acid had a lower risk of MI than those in the lowest quintile: OR (95% CI), 1.00; 0.80 (0.52 to 1.24); 0.53 (0.34 to 0.82); 0.44 (0.28 to 0.67); and 0.37 (0.24 to 0.59); test for trend, P 0.0001. This association was strengthened after adjustment for established MI risk factors, including smoking, physical activity, income, and adipose tissue linoleic acid and trans fatty acids (OR for the top versus lowest quintile, 0.23; 95% CI, 0.10 to 0.50; test for trend, P 0.0001). Further adjustment for the intake of saturated fat, fiber, alcohol, and vitamin E did not change this association (OR for the top versus lowest quintile, 0.23; 95% CI, 0.10 to 0.55; test for trend, P 0.0001). Conclusions The inverse association observed between -linolenic acid and nonfatal acute MI suggests that consumption of vegetable oils rich in -linolenic acid confers important protection against cardiovascular disease. (Circulation. 2003; 107:1586-1591.) Key Words: coronary disease diet fatty acids epidemiology risk factors Alpha-Linolenic acid is hypothesized to reduce the risk of coronary heart disease (CHD) 1 3 and was found to be beneficial in secondary prevention trials. 4 6 Its actual mechanism of action is not known, but its potential biological effects include the inhibition of platelet aggregation and thrombosis, reduction of blood pressure, decrease in serum lipids, and prevention of cardiac arrhythmias. 1,3 These effects can be achieved directly by -linolenic acid 3,7 or through conversion to long-chain n-3 fatty acids, including eicosapentaenoic acid and docosahexaenoic acid. The largest food sources of -linolenic acid are vegetable oils, such as flax seed oil, soybean oil, and canola oil. Other food sources of -linolenic acid include walnuts, dairy products, beans, broccoli, and green leafy vegetables. 1,8 However, these foods generally do not contribute much -linolenic acid to the diet. Nonhydrogenated soybean oil contains 7% to 10% -linolenic acid. 9 However, soybean oil is usually partially hydrogenated to produce margarines and vegetable shortenings, with lower -linolenic acid concentrations and a high content of trans fatty acids, which are positively associated with CHD. 10 Randomized clinical trials suggest that -linolenic acid is protective against the risk of myocardial infarction (MI). 4 6 However, none of the clinical trials were specifically designed to assess exclusively the effect of -linolenic acid on CHD. Results from population-based, observational studies are inconsistent: some 2,11 but not all 12,13 showed a protective effect for -linolenic acid. These inconsistencies could be partially explained by the fact that dietary sources of -linolenic and trans fatty acids usually overlap, but these nutrients have opposite effects on MI. It is also possible that the protective effects of -linolenic acid are manifested more strongly in populations with a low intake of n-3 fatty acids from fish, because most studies showing no association were carried out in populations with a higher fish intake. 12,13 Furthermore, dietary information from questionnaires used in many studies is often prone to misclassification. Biomarkers could provide a more accurate objective measure of long-term intake compared with dietary questionnaires because they do not rely on memory or self-reported information and are not subject to interviewer bias. 14 Subcutaneous adipose tissue is considered the best choice for the Received October 22, 2002; revision received December 30, 2002; accepted January 2, 2003. From the Departments of Nutrition (A.B., E.K.K., A.A., H.C.), Epidemiology (A.A., D.S.), and Biostatistics (D.S.), Harvard School of Public Health, Boston, Mass; and Centro Centroamericano de Población, Universidad de Costa Rica, San Pedro de Montes de Oca, Costa Rica (H.C.). Correspondence to Hannia Campos, PhD, Department of Nutrition, Room 353A, Harvard School of Public Health, 665 Huntington Ave, Boston, MA 02115. E-mail hcampos@hsph.harvard.edu 2003 American Heart Association, Inc. Circulation is available at http://www.circulationaha.org DOI: 10.1161/01.CIR.0000058165.81208.C6 1586

Baylin et al Adipose Tissue -Linolenic Acid and Myocardial Infarction 1587 study of long-term fatty acid intake, mainly because of the slow turnover 15,16 and its lack of response to acute disease. 14 We previously showed that adipose tissue is an adequate biomarker of -linolenic and trans fatty acid intake in Costa Rica. 17 Cardiovascular disease is the main cause of death in numerous developing countries, 18 many of which have little possibility of increasing fish intake. Because of the large number of people living in these countries, the potential protective effect of -linolenic acid from vegetable oils needs to be studied in these populations. We used adipose tissue -linolenic acid as a biomarker of intake to investigate whether -linolenic acid is associated with a lower risk of nonfatal acute MI in Costa Rica. Methods Study Population The catchment area for this study was the metropolitan area of San José, Costa Rica. Eligible case patients (cases) were survivors of a first acute MI as diagnosed by a cardiologist at any of the 3 recruiting hospitals in the catchment area (San Juan de Dios Hospital, Calderón Guardia Hospital, and México Hospital) between 1994 and 1998. 19 All cases were confirmed by a study cardiologist according to the World Health Organization criteria for MI, which require typical symptoms plus either elevations in cardiac enzyme levels or diagnostic changes in the ECG. 20 To achieve 100% ascertainment, field-workers carried out daily visits to the 3 hospitals for recruitment of cases. Cases were ineligible if they (1) died during hospitalization, (2) were 75 years old on the day of their first MI, (3) were physically or mentally unable to answer the questionnaire, and (4) had a previous hospital admission related to cardiovascular disease. Enrollment was carried out while cases were in the hospital s step-down unit. One free-living control subject (control) for each case, matched for age ( 5 y), sex, and area of residence (county), was randomly selected using the information available at the National Census and Statistics Bureau of Costa Rica. Because of the comprehensive social services provided in Costa Rica, all persons living in the catchment area had access to medical care without regard to income. Therefore, control subjects come from the source population that gave rise to the cases and are not likely to have had cardiovascular disease that was not diagnosed because of poor access to medical care. Control subjects were ineligible if they had ever had an MI or if they were physically or mentally unable to answer the questionnaires. All cases and controls were visited at their homes for the collection of dietary and health information, anthropometric measurements, and biological specimens. Data collection was completed 26 10 (mean SD) days after the MI in cases and 31 15 days after recruitment in the controls. Participation was 97% for cases and 90% for controls. All subjects gave informed consent on documents approved by the Human Subjects Committee of the Harvard School of Public Health and the University of Costa Rica. Data Collection Trained personnel visited all study participants at their homes. Sociodemographic characteristics, smoking, socioeconomic status, and medical history data were collected during an interview by use of a questionnaire with closed-ended questions. To understand the characteristics of the study population and to investigate whether nutrients not measured in adipose tissue could confound the association between adipose tissue -linolenic acid and MI, we collected data on dietary intake using a food frequency questionnaire that was developed and validated specifically to assess fatty acid intake among the Costa Rican population. 17,21 Self-reported diabetes and hypertension were validated by use of the recommended definitions by the Expert Committee on the Diagnosis and Classification of Diabetes Mellitus 22 and the Third Joint National Committee on Detection, Evaluation, and Treatment of High Blood Pressure (JNCVI). 23 We computed sensitivities, specificities, and positive and negative predictive values from the questionnaire data and measurements of blood pressure and blood sugar. These parameters showed that self-reported assessments of diabetes and hypertension are reliable in this population. 19 Anthropometric measurements and biological specimens were collected in the morning at the subject s home after an overnight fast, as described in detail elsewhere. 19 A subcutaneous adipose tissue biopsy was collected from the upper buttock with a 16-gauge needle and disposable syringe following procedures previously described. 24 Samples were stored in a cooler at 4 C and transported to the fieldwork station. At the field station, samples were stored at 80 C, and within 6 months, they were transported on dry ice to the Harvard School of Public Health for long-term storage in nitrogen tanks. Fatty Acid Analysis Fatty acids from adipose tissue were quantified by gas-liquid chromatography as described previously. 17 Peak retention times and area percentages of total fatty acids were identified by injecting known standards (NuCheck Prep) and analyzed with Agilent Technologies ChemStation A.08.03 software. Twelve duplicate samples, indistinguishable from the others, were analyzed throughout the study. The coefficients of variation for -linolenic acid, trans 18:2 fatty acid, and linoleic acid were 8.5%, 6.4%, and 5.5%, respectively. Statistical Analysis All data were analyzed with the Statistical Analysis Systems software (SAS Institute Inc). Of the 1061 subjects who were recruited, 97 were excluded because of missing data on confounders (n 9) or adipose tissue fatty acids (n 61) or died before completion of data collection (n 9) or became unmatched because of missing data (n 16), leaving 482 cases and 482 matched controls for the final analysis. The significance of differences in health characteristics and potential confounders were assessed by paired t tests and McNemar s test. ORs and 95% CIs for adipose tissue -linolenic acid quintiles were estimated by conditional logistic regression. Confounders included in the final models were smoking (never, past, 10 cigarettes/d, 10 to 20 cigarettes/d, 20 cigarettes/d), physical activity (quintiles), income (quintiles), history of diabetes (yes/no), and history of hypertension (yes/no). Other potential confounders examined but not included in the final models were body mass index, history of hypercholesterolemia, multivitamin use, aspirin use, intake of vitamin C, folate, total fat, total energy, green leafy vegetables, fish, and type of oil used for cooking. In adipose tissue, we examined oleic acid, -linolenic acid, arachidonic acid, and long chain n-3 fatty acids as potential confounders. Tests for trend were performed across quintiles, using the median value for each of the quintiles modeled as a continuous variable. We tested for the statistical significance of interactions between adipose tissue -linolenic acid and total adipose tissue trans fatty acids using the likelihood ratio test after dividing both exposures in tertiles. Results Table 1 shows the characteristics of the study participants. Compared with controls, cases were more likely to be current smokers and had lower adipose tissue -linolenic acid levels (P 0.001). In this study, the average duration between MI and collection of an adipose tissue sample was 26 days (range, 10 to 93 days). In MI cases, -linolenic acid in adipose tissue was not correlated with timing of sample collection (r 0.007). The mean -linolenic acid levels by each quintile of days between MI and sample collection were 0.50%, 0.50%, 0.47%, 0.50%, and 0.51% (test for trend, P 0.05). The distribution of potential confounders by each quintile of -linolenic acid is shown in Table 2. There was a strong

1588 Circulation April 1, 2003 TABLE 1. Characteristics of MI Cases and Population-Based Controls in Costa Rica Controls (n 482) Cases (n 482) Mean SD Mean SD P Age, y 57 11 57 10 Weight, kg 69 13 69 12 0.56 Body mass index, kg/m 2 25.9 4 26 3.9 0.76 Waist-to-hip ratio 0.94 0.07 0.95 0.07 0.0001 Physical activity, METS 1.83 0.92 1.71 0.94 0.046 Income, US $/mo 570 485 462 436 0.0006 Sex, % female 26 26 Diabetes, % 12 24 0.0001 Hypertension, % 27 43 0.0001 Angina, % 4 12 0.0001 Current smokers, %* 27 43 0.0001 Dietary variables Fat, % energy Total fat 33.4 6.1 34.3 6.2 0.019 Saturated fat 11.7 2.7 12.4 2.7 0.0001 Monounsaturated fat 12.5 3.5 12.7 3.5 0.22 Polyunsaturated fat 5.6 1.5 5.5 1.6 0.14 Cholesterol, mg/1000 kcal 125 65 135 67 0.023 Carbohydrate, % energy 54.1 8.2 52.9 8.3 0.028 Protein, % energy 13.5 2.6 13.9 2.5 0.032 Vitamin E, mg/1000 kcal 17.8 35.2 15.1 26.1 0.17 Fiber, g/1000 kcal 10.7 2.9 10.3 2.9 0.027 Alcohol, g/d 15.2 20.5 17.6 26.9 0.33 Fish, g/wk 131 107 142 136 0.17 Fatty acids in adipose tissue -Linolenic acid 0.55 0.16 0.50 0.14 0.0001 Eicosapentaenoic acid 0.04 0.02 0.04 0.02 0.92 Docosapentaenoic acid 0.21 0.06 0.21 0.06 0.17 Docosahexaenoic acid 0.17 0.06 0.17 0.06 0.15 Linoleic acid 13.36 3.23 12.85 3.08 0.007 Total trans fatty acids 3.12 1.01 3.07 0.98 0.42 *Smokes 1 cigarette/d. Mean computed among alcohol users only (222 controls and 183 cases). Percent total fatty acids. positive association between -linolenic acid and trans fatty acids and linoleic acid, but not eicosapentaenoic acid, docosapentaenoic acid, or docosahexaenoic acid. An inverse relationship was observed between adipose tissue -linolenic acid and the risk of nonfatal acute MI (Table 3). Subjects in the highest quintile of -linolenic acid (0.72% of fatty acids) had a lower risk of MI than those in the lowest quintile (0.35% of fatty acids). This association was strengthened after adjustment for linoleic acid and total trans fatty acids (model 2). The CIs for this association became even tighter after adjustment for linoleic acid alone or together with trans fatty acids, suggesting an absence of collinearity (Table 3). Further adjustment for smoking, income, history of diabetes, history of hypertension, and physical activity did not change the results appreciably (model 3). Adjustment of model 3 for other adipose tissue n-3 fatty acids did not change the association. Adjustment of model 3 for dietary cholesterol (OR, 0.24; 95% CI, 0.11 to 0.54) or other dietary variables did not alter the results (model 4). We did not find any interaction between vitamin E and -linolenic acid. In an analysis stratifying the population by the median intake of vitamin E, we found similar associations in low and high strata of vitamin E intake. The major source of -linolenic acid in the Costa Rican population is partially hydrogenated soybean oil used for cooking. Because of partial hydrogenation, this oil is also the main source of trans fatty acids, but other important sources of trans fatty acids include margarine and commercial baked products. Our control population consisted of 36% palm oil users, 49% soybean oil users, and 15% users of other types of oil. Because of the expected opposite effects of -linolenic

Baylin et al Adipose Tissue -Linolenic Acid and Myocardial Infarction 1589 TABLE 2. Distribution of Potential Confounders in the Population-Based Controls in Costa Rica Quintiles of -Linolenic Acid in Adipose Tissue 1 2 3 4 5 Median adipose tissue -linolenic acid* 0.36 0.46 0.53 0.61 0.75 Body mass index, kg/m 2 26.9 25.9 25.9 26.1 24.6 Waist-to-hip ratio 0.95 0.94 0.94 0.93 0.92 Income, US $/mo 437 467 593 642 657 Diabetes, % 17.7 9.5 9.4 7.4 11.2 Hypertension, % 35.6 27.6 20.0 25.0 27.6 Physical activity, METS 1.88 1.73 1.84 1.81 1.86 Current smokers, % 36.7 30.7 19.0 27.6 26.7 Alcohol, g/d 30.9 15.4 20.1 10.9 9.4 Saturated fat, % energy 12.5 11.9 11.5 11.4 10.8 Cholesterol, mg/1000 kcal 139 134 128 116 106 Vitamin E, mg/1000 kcal 13.7 15.0 14.3 22.2 25.9 Fiber, g/1000 kcal 10.7 10.7 10.8 10.0 11.1 Soybean oil users, % 24.3 42.5 56.5 56.9 70.4 Fish, g/wk 109 115 130 144 149 Adipose tissue fatty acids* Linoleic acid 10.0 11.7 13.1 14.9 17.1 Eicosapentaenoic acid 0.04 0.04 0.04 0.04 0.04 Docosapentaenoic acid 0.21 0.20 0.21 0.20 0.21 Docosahexaenoic acid 0.17 0.16 0.17 0.16 0.18 Total trans fat 2.27 2.78 3.17 3.54 3.81 *Percent of fatty acids. Mean computed among alcohol users only. acid and trans fatty acids, we carried out an analysis to show the joint effect of these variables (Figure). It is clear that the greatest protection is observed among those with high -linolenic acid and low trans fatty acids, and the interaction between these 2 variables was significant (P 0.05). Discussion We conducted a case-control study to assess the relationship between adipose tissue -linolenic acid and the risk of MI in TABLE 3. Costa Rica. We found that -linolenic acid is associated with a large and significant reduction in the risk of nonfatal acute MI. The greatest protection was found among those with high -linolenic acid and low total trans fatty acids in adipose tissue. Our data are consistent with those from clinical trials 4 6 on -linolenic acid and MI. For instance, the reduction in the risk of nonfatal MI reported by Singh et al 4 (OR, 0.47; 95% CI, 0.28 to 0.79) is comparable to that observed in our study (OR, 0.23; 95% CI, 0.10 to 0.50). However, some 5 of the ORs for the Risk of Nonfatal Acute MI by Quintile of -Linolenic Acid in Adipose Tissue in Costa Rica Quintiles of Adipose Tissue -Linolenic Acid 1 2 3 4 5 P for Trend Median adipose -linolenic acid 0.35 0.43 0.50 0.59 0.72 Ratio, cases/controls 117/75 112/81 93/100 83/110 77/116 Age-, sex-, and 1.00 0.80(0.52 1.24) 0.53(0.34 0.82) 0.44(0.28 0.67) 0.37(0.24 0.59) 0.0001 residence-adjusted Model 1 1.00 0.75(0.47 1.20) 0.41(0.24 0.70) 0.29(0.16 0.52) 0.22(0.11 0.42) 0.0001 Model 2 1.00 0.73(0.45 1.17) 0.40(0.23 0.68) 0.26(0.14 0.48) 0.18(0.09 0.37) 0.0001 Model 3 1.00 0.80(0.46 1.39) 0.52(0.28 0.96) 0.34(0.17 0.68) 0.23(0.10 0.50) 0.0001 Model 4 1.00 0.93(0.51 1.68) 0.60(0.31 1.15) 0.32(0.16 0.68) 0.23(0.10 0.55) 0.0001 Model 1: Adjusted for adipose tissue linoleic acid Model 2: Adjusted for linoleic acid and total trans fatty acids, both in adipose tissue. Model 3: Model 2 smoking, income, history of diabetes, history of hypertension, and physical activity. Model 4: Model 3 dietary variables (intake of saturated fat, fiber, alcohol, and vitamin E).

1590 Circulation April 1, 2003 Odds ratios for combined effect of -linolenic acid and trans fatty acids in adipose tissue in Costa Rica. Subjects in first tertile of -linolenic acid and third tertile of trans fatty acids served as reference group. Test for interaction, P 0.05. trials included additional elements in the intervention diets that are considered healthy, eg, increased intake of fruits, vegetables, and nuts. Therefore, it is difficult to isolate a singular role of -linolenic acid. The Lyon Heart Study, 5 in particular, reported a 73% risk reduction in fatal plus nonfatal MI. The beneficial effect obtained from the experimental diet could be attributed to many other components of the diet. 25 However, when plasma fatty acids were measured 2 months after randomization, only -linolenic acid was significantly associated with improved prognosis. 5 Some 4,11,26 but not all 12,13,27,28 population-based studies are consistent with ours in reporting a protective effect between -linolenic acid and MI. Trans fatty acids and -linolenic acid share food sources, making it difficult to disentangle their antagonistic effects, especially when there is a great overlap in the food sources, as may occur in studies with small sample sizes. In the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study, a marginally significant trend for -linolenic acid (RR by quintiles: 1.00, 0.95, 0.94, 0.87, 0.75; test for trend, P 0.05) 27 was found only after adjustment for trans, monounsaturated, and saturated fat. Studies that found an increased risk of MI for -linolenic acid were strongly confounded by trans fatty acid intake. 13,28 Trans isomers may interfere with biological functions of essential fatty acids by competing with linoleic and -linolenic acids for the 6/5 - desaturase enzyme. 29 In our study, we observed a significant interaction between -linolenic acid and trans fatty acids in adipose tissue. Our data show a protective effect between -linolenic acid and the risk of nonfatal acute MI and are consistent with other studies 4,11 but somewhat different from the EURAMIC 12 and Nurses Health Study, 2 which reported a nonsignificant inverse association with nonfatal MI. These differences could, in part, be a result of differences in the assessment of -linolenic acid intake (food frequency questionnaire versus adipose tissue) and the potential for a threshold effect for -linolenic acid. For instance, in the EURAMIC study, centers with lower mean adipose tissue -linolenic acid levels tended to show a stronger effect than those with higher levels. 12 Another potential explanation for this finding could be the low intake of fish in Costa Rica (median intake, 14.5 g/d; mostly lean and canned fish) compared with that in European countries eg, 25 g/d in Finland. 30 Thus, -linolenic acid may play a more prominent role in MI among populations with a low intake of marine n-3 fatty acids. The main advantage of this study is the use of biomarkers as indicators of intake. Nevertheless, we cannot discard the possibility that biomarkers are also prone to misclassification because of laboratory error and may not reflect intake accurately because of differences in absorption or metabolism. Even so, tissue concentrations of fatty acids may be more relevant to disease than are dietary intakes. 31 Adipose tissue -linolenic acid was 2-fold higher in the highest than in the lowest quintile. The difference in adipose tissue linolenic acid content between the highest and lowest quintiles, 0.38%, corresponds to 0.3 g/d of intake. Approximately 0.3 g of -linolenic acid is contained in 1 teaspoon of soybean oil or canola oil, which are rich sources (7% to 10%) of -linolenic acid when they are not partially hydrogenated. Thus, the difference in adipose tissue -linolenic acid is significant to practical nutrition. In summary, we found that -linolenic acid is protective against MI in a population with low fish intake. In view of the results, we can conclude that diets rich in -linolenic acid may be beneficial in the prevention of nonfatal acute MI, and this benefit is even larger among populations with low levels of trans fatty acids. Acknowledgments This study was supported by grants HL-49086 and HL60692 from the National Institutes of Health. We are grateful to Xinia Siles for data collection and study management, the study participants, the staff of Proyecto Salud Coronaria, San José, Costa Rica, and the staff and medical personnel at the recruiting hospitals and at the Centro de Estadistica y Census in Costa Rica. References 1. Simopoulos AP. Essential fatty acids in health and chronic disease. Am J Clin Nutr. 1999;70:560S 569S. 2. Hu FB, Stampfer MJ, Manson JE, et al. Dietary intake of alpha-linolenic acid and risk of fatal ischemic heart disease among women. Am J Clin Nutr. 1999;69:890 897. 3. Lanzmann-Petithory D. Alpha-linolenic acid and cardiovascular diseases. J Nutr Health Aging. 2001;5:179 183. 4. Singh RB, Dubnov G, Niaz MA, et al. Effect of an Indo-Mediterranean diet on progression of coronary artery disease in high risk patients (Indo-Mediterranean Diet Heart Study): a randomised single-blind trial. 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