Chapter  94:  Effects of Omega-3 Fatty Acids on Cardiovascular Disease

Metabolism and Biological Effects of Essential Fatty Acids

Dietary fat is an important source of energy for biological activities in human beings. Dietary fat encompasses saturated fatty acids, which are usually solid at room temperature, and unsaturated fatty acids, which are liquid at room temperature. Unsaturated fatty acids can be further divided into monounsaturated and polyunsaturated fatty acids. Polyunsaturated fatty acids (PUFAs) can be classified on the basis of their chemical structure into two groups: omega-3 (n-3) fatty acids and omega-6 (n-6) fatty acids. The omega-3 or n-3 notation means that the first double bond from the methyl end of the molecule is in the third. The same principle applies to the omega-6 or n-6 notation. Despite their differences in structure, all fats contain the same amount of energy (9 kcal/g or 37 kJ/g).

Of all fats found in food, 2—alpha-linolenic acid (chemical abbreviation: ALA, 18:3 n-3) and linoleic acid (LA, 18:2 n-6)—cannot be synthesized in the human body, yet are necessary for proper physiological functioning. These 2 fats are called essential fatty acids. The essential fatty acids can be converted in the liver to long-chain polyunsaturated fatty acids (LC PUFAs), which have a higher number of carbon atoms and double bonds. These LC PUFAs retain the omega type (n-3 or n-6) of the parent essential fatty acids.

ALA and LA comprise the bulk of the total PUFAs consumed in a typical North American diet. Typically, LA comprises 89% of the total PUFAs consumed, while ALA comprises 9%. Smaller amounts of other PUFAs make up the remainder ¹. Both ALA and LA are present in a variety of foods. For example, LA is present in high concentrations in many commonly used oils, including safflower, sunflower, soy, and corn oil. ALA, which is consumed in smaller quantities, is present in leafy green vegetables and in some commonly used oils, including canola and soybean oil. Some novelty oils, such as flaxseed oil, contain relatively high concentrations of ALA, but these oils are not commonly found in the food supply.

The Institute of Medicine suggests that, for adults 19 and older, an adequate intake (AI) of ALA is 1.1–1.6 g/day, while an adequate daily intake of LA is 11–17 g/day ². Recommendations regarding AI differ by age and gender groups, and for special conditions such as pregnancy and lactation.

As shown in Figure 1.1, EPA and DHA can act as competitors for the same metabolic pathways as AA. In human studies, the analyses of fatty-acid compositions in both blood phospholipids and adipose tissue showed similar competitive relationship between omega-3 LC PUFAs and AA. General scientific agreement supports an increased consumption of omega-3 fatty acids and reduced intake of omega-6 fatty acids to promote good health. However, for omega-3 fatty acid intakes, the specific quantitative recommendations vary widely among countries not only in terms of different units - ratio, gram, total energy intake - but also in quantity ³. Furthermore, there remain numerous questions relating to the inherent complexities about omega-3 and omega-6 fatty acid metabolism, in particular regarding the inter-relationships between the 2 fatty acids. For example, it remains unclear to what extend ALA is converted to EPA and DHA in humans, and to what extend high intake of omega-6 fatty acids compromises any benefits of omega-3 fatty acid consumption. Without resolution of these 2 foundational questions, it remains difficult to study the importance of omega-6 to omega-3 fatty acid ratio.

Metabolic Pathways of Omega-3 and Omega-6 Fatty Acids

Omega-3 and omega-6 fatty acids share the same pools of enzymes and go through the same oxidation pathways while being metabolized (Figure 1.1). Once ingested, ALA and LA can be elongated and desaturated into LC PUFAs. LA is converted into gamma-linolenic acid (GLA, 18:3 n-6), an omega-6 fatty acid that is a positional isomer of ALA. GLA, in turn, can be converted to the long-chain omega-6 fatty acid, arachidonic acid (AA, 20:4 n-6). ALA can be converted, to a lesser extent, to the long-chain omega-3 fatty acids, eicosapentaenoic acid (EPA; 20:5 n-3) and docosahexaenoic acid (DHA; 22:6 n-3). However, the conversion from parent fatty acids into LC PUFAs occurs slowly in humans, and conversion rates are not well understood. Because of the slow rate of conversion and the importance of LC PUFAs to many physiological processes, humans must augment their level of LC PUFAs by consuming foods that are rich in these important compounds. Meat is the primary food source of AA, while fish is the primary food source of EPA.

The specific biological functions of fatty acids depend on the number and position of double bonds and the length of the acyl chain. Both EPA and AA are 20-carbon fatty acids and are precursors for the formation of prostaglandins, thromboxane, and leukotrienes—hormone-like agents that are members of a larger family of substances called eicosanoids. Eicosanoids are localized tissue hormones that seem to be 1 of the fundamental regulatory classes of molecules in most higher forms of life. They do not travel in the blood, but are created in the cells to regulate a large number of processes, including the movement of calcium and other substances into and out of cells, dilation and contraction of muscles, inhibition and promotion of clotting, regulation of secretions including digestive juices and hormones, and control of fertility, cell division, and growth ⁴.

As shown in Figure 1.1, the long-chain omega-6 fatty acid, AA, is the precursor of a group of eicosanoids including series-2 prostaglandins and series-4 leukotrienes. The omega-3 fatty acid, EPA, is the precursor to a group of eicosanoids including series-3 prostaglandins and series-5 leukotrienes. The series-2 prostaglandins and series-4 leukotrienes derived from AA are involved in intense actions (such as accelerating platelet aggregation and enhancing vasoconstriction and the synthesis of inflammatory mediators) in response to physiological stressors. The series-3 prostaglandins and series-5 leukotrienes that are derived from EPA are less physiologically potent than those derived from AA. More specifically, the series-3 prostaglandins are formed at a slower rate and work to attenuate excessive series-2 prostaglandins. Thus, adequate production of the series-3 prostaglandins, which are derived from the omega-3 fatty acid, EPA, may protect against heart attack and stroke as well as certain inflammatory diseases like arthritis, lupus, and asthma ⁴. In addition, animal studies, have demonstrated that omega-3 LC PUFAs, such as EPA and DHA, engage in multiple cytoprotective activities that may contribute to antiarrhythmic mechanisms⁵. Arrhythmias are thought to be the cause of “sudden death” in heart disease.

In addition to affecting eicosanoid production as described above, EPA also affects lipoprotein metabolism and decreases the production of other compounds—including cytokines, interleukin 1ß (IL-1ß), and tumor necrosis factor a (TNF-a)—that have pro-inflammatory effects. These compounds exert pro-inflammatory cellular actions that include stimulating the production of collagenases and increasing the expression of adhesion molecules necessary for leukocyte extravasation ⁶. The mechanism responsible for the suppression of cytokine production by omega-3 LC PUFAs remains unknown, although suppression of eicosanoid production by omega-3 fatty acids may be involved. EPA can also be converted into the longer chain omega-3 form of docosapentaenoic acid (DPA, 22:5 n-3), and then further elongated and oxygenated into DHA. EPA and DHA are frequently referred to as very long chain omega-3 fatty acids. DHA, which is thought to be important for brain development and functioning, is present in significant amounts in a variety of food products, including fish, fish liver oils, fish eggs, and organ meats. Similarly, AA can convert into an omega-6 form of DPA. Studies have reported that omega-3 fatty acids decrease triglycerides (Tg) and very low density lipoprotein (VLDL) in hypertriglyceridemic subjects, with a concomitant increase in high density lipoprotein (HDL). However, they appear to increase or have no effect on low density lipoprotein (LDL). Omega-3 fatty acids apparently lower Tg by inhibiting VLDL and apolipoprotein B-100 synthesis and decreasing post-prandial lipemia ⁷. Omega-3 fatty acids, in conjunction with transcription factors (small proteins that bind to the regulatory domains of genes), target the genes governing cellular Tg production and those activating oxidation of excess fatty acids in the liver. Inhibition of fatty acid synthesis and increased fatty acid catabolism reduce the amount of substrate available for Tg production ⁸.

As noted earlier, omega-6 fatty acids are consumed in larger quantities (>10 times) than omega-3 fatty acids. Maintaining a sufficient intake of omega-3 fatty acids is particularly important since many of the body's physiologic properties depend upon their availability and metabolism.

Population Intake of Omega-3 Fatty Acids in the United States

The major source of omega-3 fatty acids is dietary intake of fish, fish oil, vegetable oils (principally canola and soybean), some nuts including walnuts, and dietary supplements. Two population-based surveys, the Continuing Food Survey of Intakes by Individuals 1994-98 (CSFII) and the third National Health and Nutrition Examination (NHANES III) 1988-94 surveys, are the main source of dietary intake data for the U.S. population. NHANES III collected information on the U.S. population aged =2 months. Mexican Americans and non-Hispanic African-Americans, children =5 years old, and adults = 60 years old were over-sampled to produce more precise estimates for these population groups. There were no imputations for missing 24-hour dietary recall data. A total of 29,105 participants had complete and reliable dietary recall. Complete descriptions of the methods used and fuller analyses are later described in this report, under “Methods: Method to Assess the Dietary Intake of Omega-3 Fatty Acids in the US population” and “Results: Population Intake of Omega-3 Fatty Acids in the United States”. CSFII 1994-96, popularly known as the What We Eat in America survey, addressed the requirements of the National Nutrition Monitoring and Related Research Act of 1990 (Public Law 101–445) for continuous monitoring of the dietary status of the American population. In CSFII 1994-96, an improved data-collection method known as the multiple-pass approach for the 24-hour recall was used. Given the large variation in intake from day-to-day, multiple 24-hours recalls are considered to be the best suited for most nutrition monitoring and will produce stable estimates of mean nutrient intakes from groups of individuals ⁹. In 1998, the Supplemental Children's Survey, a survey of food and nutrient intake by children under age of 10, was conducted as the supplement to the CSFII 1994-96. The CSFII 1994-96, 1998 surveyed 20,607 people of all ages with over-sampling of low-income population (<130% of the poverty threshold). Dietary intake data by individuals of all ages were collected over 2 nonconsecutive days by use of two 1-day dietary recalls.

Table 1.1 reports the NHANES III survey mean intake ± the standard error of the mean (SEM), as well as, the median and range for each omega-3 fatty acid. Distributions of EPA, DPA, and DHA were very skewed; therefore, the means and standard errors of the means should be used and interpreted with caution. Table 1.2 reports the CSFII survey mean and median intakes for each omega-3 fatty acid, along with SEMs, as reported in Dietary Reference Intakes by the Institute of Medicine ².

Dietary Sources of Omega-3 Fatty Acids

Omega-3 fatty acids can be found in many different sources of food, including fish, shellfish, some nuts, and various plant oils. Table 1.3 lists the amount of omega-3 fatty acids in some commonly consumed fish, shellfish, nuts, and edible oils, selected from the USDA website (accessed November 3, 2003) http://www.nal.usda.gov/fnic/foodcomp (Finfish and Shellfish Products, sr16fg15.pdf; Fats and Oils, sr16fg04.pdf; and Nut and Seed Products, sr16fg12.pdf) ¹⁰.

General Question

What are the mean and median intakes of eicosapentaenoic acid (EPA, 20:5 n-3), docosahexaenoic acid (DHA, 22:6 n-3), alpha linolenic acid (ALA, 18:3 n-3), fish, fish oil, and omega-6 fatty acids, and what is the mean and median omega-6 to omega-3 fatty acid ratio, in the US population?

• Do consumption levels differ among subpopulations?

CVD Questions

What is the efficacy or association of omega-3 fatty acids (DHA, EPA or ALA supplements, and fish consumption) in reducing CVD events (including all-cause mortality, CVD mortality, non-fatal CVD events, and new diagnosis of CVD)?

• What is the efficacy or association of omega-3 fatty acids in preventing incident CVD outcomes in people without known CVD (primary prevention) and with known CVD (secondary prevention)?

• How does the efficacy or association of omega-3 fatty acids in preventing incident CVD outcomes differ in sub-populations, including men, pre-menopausal women, post-menopausal women, and different age groups?

• What are the effects of potential confounders — such as lipid levels, body mass index (BMI), blood pressure, diabetes, aspirin use, hormone replacement therapy, and cardiovascular drugs — on associations found in prospective cohort studies?

• What is the relative efficacy of omega-3 fatty acids on different CVD outcomes? Can the CVD outcomes be ordered by strength of treatment effect of omega-3 fatty acids?

Omega-3 fatty acid variables and modifiers:

• What is the efficacy or association of specific omega-3 fatty acids (DHA, EPA, ALA), and different ratios of omega-3 fatty acid components in dietary supplements, on CVD outcomes?

• Does the ratio of omega-6 to omega-3 fatty acid intake affect the efficacy or association of omega-3 fatty acid intake on CVD outcomes?

• How does the efficacy or association of omega-3 fatty acids on CVD outcomes differ by source (e.g., dietary fish, dietary oils, dietary plants, fish oil supplement, flax seed supplement)?

• How does the efficacy or association of omega-3 fatty acids on CVD outcomes differ by different ratios of DHA, EPA, and ALA?

• Is there a threshold or dose-response relationship between omega-3 fatty acids and CVD outcomes?

• How does the duration of intervention or exposure affect the treatment effect of omega-3 fatty acids on CVD outcomes?

• Are treatment effects or the association of omega-3 fatty acids on CVD events sustained after the intervention or exposure stops?

• What is the effect or association of baseline dietary intake of omega-3 fatty acids on the efficacy of omega-3 fatty acid supplements on CVD outcomes?

• Does the use of medications for CVD and/or CVD risk factors (including lipid lowering agents and diabetes medications) affect the efficacy or association of omega-3 fatty acids?

Adverse events and drug interactions:

• What adverse events related to omega-3 fatty acid dietary supplements are reported in studies of CVD outcomes and markers?

• What adverse events related to omega-3 fatty acid dietary supplements are reported specifically among diabetics and people with CVD in studies of CVD outcomes and markers?

• What interactions between omega-3 fatty acid dietary supplements and medications are reported in studies of CVD outcomes and markers?

• What interactions between omega-3 fatty acid dietary supplements and medications are reported specifically among diabetics and people with CVD in studies of CVD outcomes and markers?

The 3^rd National Health and Nutrition Survey (NHANES III) Database

The NHANES III, 1988-94 database was used to examine the population intake of omega-3 fatty acids in the US (General Question). NHANES III was designed to collect information on the US population aged = 2 months. Mexican Americans and non-Hispanic African Americans, children = 5 years old, and adults = 60 years old were over-sampled to produce more precise estimates for these population groups. There were no imputations for missing 24-hour dietary recall data. A total of 29,105 participants had complete and reliable dietary recall.

Definitions of Key Variables

The population means and standard errors of the mean (SEM) of total polyunsaturated fatty acids (PUFAs), ALA, EPA, and DHA by sex, age, and/or income levels have been presented in a report by the National Center for Health Statistics ². However, the sub-population grouping system is different from the system that is used in Institute of Medicine (IOM) reports. In order to provide the most parsimonious interpretation of IOM reports and this evidence report, we have decided to adopt the approach used in Dietary References Intakes (DRIs) published by the IOM ². The main variables in this evidence report are defined as follows:

• Age groups: Subjects' age in months was used to form ten age groups: 2–6 months, 7–12 months, 1–3 years, 4–8 years, 9–13 years, 14–18 years, 19–30 years, 31–50 years, 51–70 years, and 71+ years. Age in months was calculated by computing the number of months between the screener questionnaire date and each subject's date of birth. Two additional age groups were created for the adult sub-population: less than 45 years old, and 45 years old and older.

• Race/ethnicity groups: Four ethnicity groups were used in this report: non-Hispanic white, non-Hispanic black, Mexican American, and others. The groups were defined by the race or ethnicity reported by respondents. Respondents were asked to identify themselves as: black; Mexican or Mexican American; white, non-Hispanic; Asian or Pacific Islander; Aleut, Eskimo, or American Indian; or other Latin American or other Spanish.

• Poverty: Two poverty income ratio (PIR) groups were created for use in analyses: PIR = 1.3 and poverty income ratio > 1.3. The numerator of the ratio was the midpoint of the respondent's family income category. The denominator was based on the poverty threshold, the respondent's age, and the calendar year of the interview.

• Urbanization: Metropolitan or non-metropolitan areas were based on the USDA's rural-urban codes that categorize counties by degree of urbanization and nearness to a metropolitan area.

• People with a history of CVD: Respondents defined in this report as having a history of CVD were those who responded “yes” to one of the following interview questions: (1) Has a doctor ever told you that you had congestive heart failure? (2) Has a doctor ever told you that you had a stroke? (3) Has a doctor ever told you that you had a heart attack? Respondents whose electrocardiography results showed a probable or possible myocardial infarction (MI), or probable or possible left-ventricular hypertrophy (LVH), by the Minnesota Code (Appendix C) were also defined as having CVD.

• Polyunsaturated fatty acids: ALA, EPA, DHA, docosapentaenoic acid (DPA, 22:5 n-3), and linoleic acid (LA, 18:2 n-6) data, estimated from a single 24-hour dietary recall, were used.

Analyses of NHANES III Data

The data were analyzed using SAS-callable SUDAAN, version 7.5.6 (Research Triangle Institute, Research Triangle Park, NC), which is a statistical analytic software program that adjusts for the complex NHANES III sample design. All analyses incorporated sampling weights that adjusted for unequal sampling probabilities. Variance estimations were made with the WR method (sampling With Replacement). Each denominator has 49 degrees of freedom. The design effect (deff4) was defined as the ratio of the properly computed actual variance of an estimated parameter to the variance based on a simple random sample of the same size.

We used simple linear regression to test the significance of the differences in daily intake of PUFAs between groups. The adjusted means for categorical covariates in the regression model were calculated with the least squares method. Statistical significance of the correlation between the dependent variables (e.g., intake of ALA) and independent variables (e.g., sex groups, age groups, CVD groups) were calculated with the Wald chi-square statistics. The details of these statistical methods are described in the SUDAAN user's manual. Since the amount of dietary PUFAs may be associated with the amount of dietary total fat, results expressed as grams per day can be misleading. Thus, all PUFAs used in the tests of significant differences between groups were measured as percent of total energy intake per day (% kcal/day).

All analyses assume a normal distribution of the nutrient intake. However, data related to EPA and DHA are very skewed. As a result, the mean and SEM estimates for these nutrients should be used and interpreted with caution. The reliability of an estimated mean or median also depends on the coefficient of variation or relative standard error (RSE), defined as the ratio between the standard error of the estimate and the estimate, multiplied by 100. Estimates with an RSE greater than 20 percent are deemed unreliable in this report.

Omega-3 Fatty Acids Search Strategy

A wide variety of search terms were used to capture the many potential sources of omega-3 fatty acids. Search terms used include the specific fatty acids, fish and other marine oils, and specific plant oils (flaxseed, linseed, rapeseed, canola, soy, walnut, mustard seed, butternut, and pumpkin seed). These terms were used in all search strategies. Because some studies evaluated the effect of nuts on CVD outcomes without specifying in the abstract the type of nuts used in the study, we performed a supplemental Medline search using the term “nut” as a text word for studies of CVD.

Cardiovascular Search Strategy

The primary search strategy was designed to address both the clinical and intermediate outcomes of CVD in humans (Appendix A). In order to identify CVD outcomes in human studies, the search was divided into 3 categories consisting of controlled trials, other studies, and reviews. These 3 categories were further divided into English and non-English subsets. To address the questions regarding stroke, the Tufts-NEMC EPC performed a separate search on the Medline database. This search yielded no additional relevant publications.

Diabetes

Because specific terms referring to diabetes had been omitted from the primary search strategy, a supplemental search strategy was conducted on March 29, 2003. The diabetes supplemental search strategy included relevant search terms for diabetes. This search strategy resulted in an additional 410 citations for screening.

Overall

The final number of citations identified by the database searches is approximate. Because the 5 main databases used in the search employ different citation formats, duplicate publications were encountered. The UO EPC eliminated most of the duplicate publications; however, because of many different permutations, it was impossible to identify all of them. We eliminated additional duplicate publications as we encountered them.

Ongoing automatic updates of Medline searches were conducted using the CVD search strategy. The last automatic update was on April 19, 2003. The UO EPC conducted a final update search of the other databases on April 10, 2003.

Abstract Screening

All abstracts identified through the literature search were screened using eligibility criteria developed in conjunction with the TEP. These criteria were designed to minimize incorrect exclusion of relevant studies. We included all English language original, experimental, or observational studies that evaluated any potential source of omega-3 fatty acids in at least 5 human subjects, regardless of the study outcomes reported in the abstract. In addition, we excluded abstracts that clearly included only subjects who had a non-CVD-related condition (e.g., cancer, schizophrenia, or organ transplant). Reports published only as letters or as abstracts in proceedings were also excluded. All abstracts were categorized to 1 or more of the key questions or as rejects.

Full Article Inclusion Criteria

Articles that passed the abstract screening process were retrieved, and the full articles were screened for eligibility. The following types of articles were rejected during this round: review articles, inappropriate human population, pediatric studies and studies conducted on subjects less than 19 years old, no mention of omega-3 fatty acid intake, dietary supplements, or fish consumption, daily dose of omega-3 fatty acid greater than 6 g, fewer than 5 subjects in omega-3 fatty acid arm(s), prospective interventional studies of less than 4 weeks duration, and no appropriate outcome of interest reported. Studies that reported only the tissue level of omega-3 fatty acid without explicitly reporting the amount of omega-3 fatty acid consumed were also excluded. However, we included studies of Mediterranean diets and studies that reported fish servings. Specific sources of omega-3 fatty acid considered acceptable included fish oils, dietary fish, canola (rapeseed) oil, soybean oil, flaxseed or linseed oil, walnuts or walnut oil, and mustard seed oil. Other sources were eligible if omega-3 fatty acid levels were reported to be greater than control. For each study that was rejected, the reason(s) for rejection was noted. For analyses of adverse events and drug interactions, all studies were included regardless of omega-3 fatty acid dose or study duration (including washout period).

Inclusion and exclusion criteria for maximal omega-3 fatty acid intake were based on discussions with the TEP, in which it was agreed that omega-3 fatty acid intake above 6 g per day is impractical and has little relevance for health care recommendations. Therefore, with the exception of studies of adverse events, the inclusion criterion for maximum daily intake was set at 6 g per day and studies of higher daily intake were excluded. The definition of omega-3 fatty acid dose varied greatly across studies. Thus, the maximal allowable dose may have applied to total daily omega-3 fatty acid, total EPA+DHA, or a total of other combinations of omega-3 fatty acids. The total did not refer to total fish oil.

In this report, we accepted randomized controlled trials (RCTs) or prospective cohort studies with a minimum of 1-year follow-up to address CVD outcome questions. We also accepted case-control studies and cross-sectional studies that assessed the prevalence of CVD in populations with varying levels of omega-3 fatty acid consumption. In some cases, a study was reported in multiple publications (e.g., interim results might have been reported in 1 publication and various outcomes in others). For these studies, we identified and grouped articles belonging to the same overall study and used data from the latest publication, supplemented by data from earlier publications, as appropriate.

Selection of Studies for Adverse Events and Drug Interactions

Human studies that were analyzed for clinical outcomes (for this report) or for risk factors (for the accompanying report, Effects of Omega-3 Fatty Acids on Cardiovascular Disease Risk Factors) were reviewed for data on adverse events and drug interactions. The eligibility criteria for these analyses were broader than for analyses of CVD outcomes, as described above.

The Food and Drug Administration's (FDA) definition of adverse events was used [FDA]. This definition includes morbidity, mortality, and evidence of organ damage. Because fishy after-taste is almost universally reported in subjects taking fish oil supplements²⁴ it was explicitly excluded as an adverse event in this report.

Analyses of data on adverse events were limited to fish oils or omega-3 fatty acid supplements. Food-related illnesses and toxicities due to marine food sources, cooking oils, and cooking methods are beyond the purview of this report. Thus, data on mercury toxicity and carcinogenic hydrocarbons from grilling were not reviewed.

We looked for studies that evaluated potential interactions between omega-3 fatty acid supplements and commonly used drugs including, but not limited to, hormone replacement therapy, diabetes medications, aspirin, and anticoagulants. In the studies that reported serious adverse events such as clinical bleeding, we note the concurrent medications that the subjects were taking.

Quality Rating Criteria for Randomized Controlled Trials

As part of the overall omega-3 fatty acid project, the 3 collaborating EPCs agreed to use the Jadad Score and adequacy of random allocation concealment as elements to grade individual randomized controlled trials ^{26, 27}. We also agreed that individual EPCs might add other elements to this core set, as we deemed appropriate. All EPCs agreed that studies should not be graded using a single numerical quality score, as this has been found to be unreliable and arbitrary ²⁸.

The Jadad Score assesses the quality of RCTs using 3 criteria: adequacy of randomization, double blinding, and dropouts ²⁶. A study that meets all 3 criteria gets a maximum score of 5 points. Adequacy of random allocation concealment was assessed as adequate, inadequate, or unclear using criteria described by Schultz et al ²⁷.

The Jadad and Schulz scores address only some aspects of the methodological quality of RCTs. In particular, items in the core set ignore potential biases due to analytic and reporting problems in a study. To rectify this, we also assessed each RCT for the following:

• Validity of methods used to assess diet

• Errors or discrepancies in reporting results

Quality Rating Criteria for Prospective Cohort Studies

Unlike RCTs, where there is at least some empirical evidence to support the use of the core set of quality rating items, there is no empirical data to support the use of elements that should comprise a core set for non-randomized studies such as cohort and case-control studies. Because prospective cohort and case control studies do not have randomization, allocation concealment, and blinding, a core set different from that used for RCTs must be defined for these types of studies. In addition, because this report focuses on the effect of omega-3 fatty acids on CVD, the studies must estimate the amount of omega-3 fatty acid consumed by the study population as accurately as possible. We used the following criteria to assess the quality of prospective cohort studies:

• Unbiased selection of the cohort (prospective recruitment of subjects)

• Sufficiently large sample size (>1,000 subjects)

• Adequate description of the cohort

• Use of validated dietary assessment method

• Quantification of the type and amount of fish/estimates of omega-3 fatty acid intake

• Use of validated method for ascertaining clinical outcomes

• Adequate follow-up period (at least 5 years)

• Completeness of follow-up

• Analysis (multivariate adjustments) and reporting of results

Quality Rating Criteria for Case Control Studies

Criteria used to assess the quality of case control studies include:

• Valid ascertainment of cases

• Unbiased selection of cases

• Appropriateness of the control population

• Verification that the control is free of CVD

• Comparability of cases and controls with respect to potential confounders

• Validated dietary assessment method

• Appropriateness of statistical analyses

Generic Summary Quality Grade for All Studies

After evaluating each study against its design-specific quality criteria, we applied a 3 category (A, B, C) summary quality grading system that we have used in most of our previous EPC evidence reports, as well as in several evidence-based clinical practice ²⁹. This scheme defines a generic grading system for study quality that is applicable to each type of study design (i.e., RCT, cohort study, case-control study). The categories are defined as follows:

1. Least bias; results are valid. A study that mostly adheres to the commonly held concepts of high quality, including the following: a formal randomized study; clear description of the population, setting, interventions, and comparison groups; appropriate measurement of outcomes; appropriate statistical and analytic methods and reporting; no reporting errors; less than 20% dropout; clear reporting of dropouts; and no obvious bias.

2. Susceptible to some bias, but not sufficient to invalidate the results. A study that does not meet all the criteria in category A. It has some deficiencies but none likely to cause major bias. Study may be missing information making assessment of the limitations and potential problems difficult.

3. Significant bias that may invalidate the results. A study with serious errors in design, analysis, or reporting. These studies may have large amounts of missing information or discrepancies in reporting.

The summary quality grading system evaluates and grades the studies within each of the study design strata. By design, it does not attempt to assess the comparative validity of studies across different design strata. Thus, in interpreting the methodological quality of a study, one should note the study design and the quality grade that it received. For RCTs, in addition to the summary quality grade, we also indicate the Jadad score and the rating of the adequacy of allocation concealment.

While it might be desirable to rank the quality of all studies on the same scale regardless of study design, experience with this approach is limited and has never been validated. In fact, using a single rating scale for all studies creates potential problems. For example, a hierarchy of study design that places RCTs above cohort studies in terms of methodological rigor is commonly accepted. However, if an RCT is seriously flawed, the results may be more biased than a well-done cohort study.

Applicability

Applicability addresses the relevance of a given study to a population of interest. Every study applies certain eligibility criteria when selecting study subjects. Most of these criteria are explicitly stated (e.g., disease status, age, sex). Some may be implicit or due to unintentional biases, such as those related to study country, location (e.g., community vs. specialty clinic), or factors resulting in study withdrawals. The question of whether a study is applicable to a population of interest (such as Americans) is distinct from the question of the study's methodological quality. For example, due to differences in the background diets, an excellent study of Japanese men may be very applicable to people in Japan, but less applicable to Japanese American men, and even less applicable to African American men. The applicability of a study is thus dictated by the questions and populations that are of interest to those analyzing the studies.

In this report, the focus is on the US population and on specific subgroups within that population (i.e., healthy Americans, Americans with CVD, and Americans with diabetes or dyslipidemia), as specified in the scope of work for this series of evidence reports. To capture the potential applicability of studies to the different populations of interest as defined in the scope of work, we define the following target population categories:

GEN General population. Typical healthy people similar to Americans without known CVD.

CVD Cardiovascular disease population. Subjects with a history of, or currently with, 1 of the following: stroke, myocardial infarction, angina, ischemic peripheral vascular disease, or other condition as defined by the author.

We planned to include categories for diabetic and dyslipidemic populations but found no relevant studies within these categories.

Even though a study may focus on a specific target population, limited study size, eligibility criteria, and the patient recruitment process may result in a narrow population sample that is of limited applicability, even to the target population. To address this issue, we categorized studies within a target population into 1 of 3 levels (I, II, III) of applicability that are defined as follows:

1. Sample is representative of the target population. It should be sufficiently large to cover both sexes, a wide age range, and other important features of the target population (e.g., diet).

2. Sample is representative of a relevant sub-group of the target population, but not the entire population. For example, while the Nurses Health Study is the largest such study and the results are highly applicable to women, it is nonetheless representative only of women. A fish oil study in Japan, where the background diet is very different from that of the US, also falls into this category.

3. Sample is representative of a narrow subgroup of subjects only, and is of limited applicability to other subgroups. For example, a study of the oldest-old men or a study of a population on a highly controlled diet.

In the summary tables, each study receives a combined applicability grade comprised of the target population (GEN or CVD) and the 3-level grade (I, II, III). For example, GEN-I represents a study of subjects representative of the general population in the US, such as a study of the NHANES population. Studies such as the Nurses Health Study and the Health Professionals Study are graded GEN-II because of each study's focus on a single sex. If several studies of complementary populations (e.g., the Nurses Health Study and the Health Professionals Study) were viewed together, they would offer highly applicable evidence for the general population and receive a grade of GEN-I.

Sample Size

The study sample size provides a quantitative measure of the weight of the evidence. In general, large studies provide more precise estimates of efficacy and associations. In addition, large studies are more likely to be generalizable; however, large size alone does not guarantee broad applicability.

Results of Randomized Clinical Trials

RCTs typically report a relative risk or the number of events for the outcome of interest. When relative risk was reported, we calculated it along with the confidence interval to verify the accuracy of the reporting. We also calculated it when only the number of events was reported. We present the adjusted relative risks when these were reported.

Results of Observational Studies

Prospective cohort studies typically categorize subjects into different quantiles (e.g., tertiles, quartiles, quintiles) of omega-3 fatty acid or fish intake and report the associated relative risk for the outcome of interest. For studies that report both unadjusted and multivariate adjusted results, we report the adjusted results in the evidence and summary tables.

Due to the heterogeneous nature of the studies (e.g., different population, background diet, dietary assessment method, and methods used to report estimates of fish or omega-3 fatty acid intake), meta-analyses were not feasible for this group of studies. To succinctly report each study's results and to help readers interpret them, we created a qualitative score or “overall effect” metric to supplement the main quantitative results in the summary tables. The overall effect metric is defined as follows:

+ + Clinically meaningful benefit demonstrated. Study reported on the clinical outcome of interest in 1 or both of the following ways:

• statistically significant trend of benefit for the quantile estimates of fish/omega-3 fatty acid intake

• at least one-half of the quantile estimates of fish/omega-3 fatty acid intake reported statistically significant beneficial effects of at least a 10% relative risk (RR) reduction (i.e., RR < 0.9), and no quantile reported a statistically significant adverse outcome

+ A clinically meaningful beneficial trend exists but is not conclusive. Study reported on the clinical outcome of interest in 1 or both of the following ways:

• a borderline significant (0.10 > P > 0.05) trend of benefit for the quantile estimates of fish/omega-3 fatty acid intake

• non-significant but potentially clinically meaningful effect (RR <0.9) in at last one-half of the quantile estimates, and no quantile reported a statistically significant adverse outcome

0 Clinically meaningful effect not demonstrated or is unlikely. Study reported clinically unimportant differences between low/no fish intake with various higher levels of fish intake. The majority of the quantiles of estimates of fish/omega-3 fatty acid intake reported less than 10% relative difference compared with the reference (i.e., 1.1>RR>0.9)

- Harmful effect demonstrated or is likely. Study reported on the clinical outcome of interest in one or both of the following ways:

• a positive association (P<.10) between quantile estimates of fish/omega-3 fatty acid intake and increased risk

• several quantile estimates reported RR >1.1

Evidence and Summary Tables

We report the evidence in 3 complementary forms:

1. Evidence tables offer a detailed description of the studies we identified that address each of the key questions. These tables provide detailed information about the study design, patient characteristics, inclusion and exclusion criteria, interventions and comparators evaluated, and outcomes. A study, regardless of how many interventions or outcomes were reported, appears once in the evidence tables. Evidence tables are grouped into RCTs and observational studies (cohorts, case-control, cross-sectional). Within each group, the studies are ordered alphabetically by the first author's last name to allow for easy searching within the tables.

2. Summary tables succinctly report on each study using summary measures of the main outcomes. These tables were developed by condensing information from the evidence tables and are designed to facilitate comparisons and synthesis across studies. Summary tables include important concise information regarding study size, intervention and control, study population (e.g., general population or CVD), outcome measures, methodological quality, and applicability. A study with multiple populations, methods of reporting estimates of omega-3 fatty acid intake, or clinical outcomes may appear multiple times in different summary tables. Because there were few RCTs and almost as many outcomes to report, we organized the RCTs into 2 groups (trials of omega-3 fatty acid supplements and trials of diet or dietary advice) to reduce the number of tables and minimize redundant information.

   Summary tables for prospective cohort and case-control studies were organized based on clinical outcomes. For each of the clinical outcomes is a table for estimates of omega-3 fatty acid consumption and a table for estimates of fish consumption. Within each table, cohort studies preceded case-control studies and studies are ordered by the number of study subjects.

3. Summary matrices provide an alternative to meta-analysis (when meta-analysis is not feasible) to facilitate the synthesis of a body of evidence. A summary matrix organizes potentially disparate studies into more homogeneous subgroups by their methodological quality and applicability grades. This allows the reader to appreciate the number of studies available and the effect size of these studies. Because there were too few RCTs and too few cohort studies of the CVD population, summary matrices were created only for prospective cohort studies for the general population in this report. Each summary matrix has applicability grades as row headings and methodological quality grades as column headings. Thus, 3 applicability grades and 3 methodological quality grades create a matrix with 9 cells. Studies assessed with a specific combination of methodological and applicability grades are displayed in their respective cells. Information displayed includes study name, study size, a measure of the effect size, and other information that may help to interpret the results.

Adverse Events Reporting

Separate adverse events evidence tables were not created. Most of these studies were included in the evidence tables of RCTs in this report or in the accompanying risk factor report. In this report, we produced summary tables on adverse events for two categories of studies: RCTs or crossover studies that compared an omega-3 fatty acid supplement with a control, and single arm cohort studies. For RCTs, we report the number and percentage of adverse events for both the omega-3 fatty acid arm and control arms for the following categories: clinical bleeding (nasal, hematuria, gastrointestinal, and other bleeding), gastrointestinal complaints, diarrhea, headaches, and withdrawals due to adverse events. We noted the dosages of omega-3 fatty acid and the control, as well as the study duration and the number of study subjects. For single arm studies, similar information was summarized. For studies that simply reported that they observed no adverse events, we created a simpler summary table listing only the information about the dosage, study size, and duration.

Average Intake Estimates of ALA, EPA, DHA, and LA in the US Population (Tables 3.2–3.9)

Analyses of intake estimates of ALA, EPA, DHA, and LA in the US population are based on the 29,000+ NHANES III respondents who had a complete and reliable 24-hour dietary recall. This sample is representative of about 200,000,000 non-institutionalized civilians in the United States. The mean intake ± SEM of ALA, EPA, DHA, and LA were 1.33±0.02, 0.04±0.003, 0.07±0.004, and 14.13±0.20 grams per day, respectively. These estimates were equivalent to 0.55±0.004, 0.02±0.001, 0.03±0.002, and 5.79±0.05 percent of total energy intake per day, respectively. The distributions of EPA and DHA intake estimates were very skewed. More than 50% of subjects had less than 0.0001 or zero grams per day of EPA or DHA intake. Therefore, the means and SEMs for EPA and DHA should be used and interpreted with caution.

Consumption Levels of US Subpopulations: Age, Gender, Ethnicity, Socio-economic Status, Urban vs Rural (Tables 3.10–3.14)

In general, the mean intake of ALA and that of LA were highest among adults between age 18 and age 50. The intakes were higher in non-Hispanic blacks and whites than in Mexican Americans and other races/ethnicities. Males consumed more grams per day of ALA and LA than did females. However, an inverse pattern was observed for both ALA and LA when expressed as percent of the total energy intake per day: at the same energy intake level, males consumed less ALA and LA than did females. Results from each table are summarized below.

• Adults vs Youths: Adults consumed significantly more ALA (+0.05±0.01 %kcal/day) and LA (+0.59±0.07 %kcal/day) than did youths (see Table 3.10).

• Males vs Females: Males had a significantly lower intake of ALA (-0.02±0.01 %kcal/day) and LA (-0.28±0.07 %kcal/day) than did females (see Table 3.11).

• Race/Ethnicity Groups: Compared to the reference group, non-Hispanic whites, non-Hispanic blacks, and Mexican Americans all had a significantly higher intake of both ALA and LA on average. The intakes of omega-3 fatty acids among non-Hispanic whites, non-Hispanic blacks, and Mexican Americans were similar. The mean difference ± SED (standard error of the difference) ranged from 0.04±0.01 to 0.09±0.01 (%kcal/day) for ALA, and from 0.43±0.14 to 0.61±0.15 (%kcal/day) for LA (see Table 3.12).

• Urban vs Rural Living Area: No significant differences in the average intake of ALA and LA were found when people living in metro areas were compared to those living in non-metro areas (see Table 3.13).

• Poverty Index Ratio (PIR): People who had a PIR = 1.3 consumed significantly less ALA (-0.04±0.01 %kcal/day) and LA (-0.28±0.06 %kcal/day) than people who had a PIR > 1.3 (see Table 3.14)

Average Intake Estimates of ALA, EPA, DHA, and LA in Individuals with and without Cardiovascular Disease (Tables 3.15–3.19)

A sub-population of NHANES III participants aged 18 and older was used for the analyses of the estimated mean intakes of ALA, EPA, DHA, and LA among individuals with and without a history of CVD (see definition for CVD in Chapter 2). Of the 16,683 adults in NHANES III, 12.7% (2,121) had CVD, while 87.3% (14,562) had no CVD (Table 3.15).

There was no significant difference in the mean intake of LA (%kcal/day) between people with and without CVD (Table 3.16). However, people with CVD consumed significantly less ALA than those without CVD (-0.02±0.01 %kcal/day, P = .04) (Table 3.17). The means ± SEMs of EPA and DHA for people with CVD and those without CVD are shown in Table 3.18 and Table 3.19, respectively. The distributions of EPA and DHA intake estimates were very skewed, so the means and SEMs for EPA and DHA should be used and interpreted with caution. For the same reason, no statistical tests for the differences between people with CVD and those without CVD were performed.

The crude means ± SEMs for people with CVD and those without CVD could be misleading because significant differences in the mean intake of ALA and LA were found among gender, age, and race/ethnicity groups. After adjusting for sex, age, and race/ethnicity, people with CVD still had a significantly lower intake of ALA compared to people without CVD (0.54±0.01 vs 0.57±0.01 %kcal/day, respectively, P = .02). Based on a typical total energy intake of 2,000 kilocalories per day, our results show that people with CVD consumed 0.67g per day less ALA than people without CVD. We found no significant difference in the mean intake of LA between the 2 groups after adjusting for sex, age, and race/ethnicity. In both ALA and LA models, gender and races were strong predictors of CVD. The regression and least-square results are shown in detail in Appendix D.

Estimates of Average Omega-3 Fatty Acid or Fish Intake in Countries Outside the US

We found no population-based dietary surveys based on single or multiple 24-hour dietary recalls for countries other than the US. However, reports of average fish consumption from the European Investigation into Cancer and Nutrition (EPIC) study provide good estimates for fish intake among the European population³⁰. The EPIC study was a cohort study (rather than a population-based survey) on diet and cancer that included more than 480,000 men and women from 10 European countries. The consumption (in grams/day) of total fish and fish products and at least 10 classifications of fish sub-groups was estimated for each country and different geographical areas by gender. The main results demonstrated that fish intake varies greatly throughout Europe, with the highest consumption in centers in Spain (51–120 g/d) and the lowest in centers in Germany (16–24 g/d). The mean daily intake of total fatty fish, which is usually high in omega-3 fatty acids, was the highest in centers in Spain (18–42 g/d) and the lowest in centers in the Netherlands (6–8 g/d)³¹. We found no report on the estimated amount of omega-3 fatty acids consumed by EPIC study participants.

A few other cross-cultural studies and a household budget survey in Spain estimate per capita intakes of major food groups per day. These studies observed large differences in fish consumption across the 21 countries. Japan was found to have a high per capita fish consumption of about 100 g/capita/day ³². An increased trend in per capita fish and shellfish consumption (62–88 g/capita/day) was found in Spain between 1964 and 1991 ³³.

Summary of Studies Analyzed

We screened over 7,464 abstracts that were indexed as English language articles concerning humans. Based on this initial review, we retrieved and screened 768 full text articles for potentially relevant human data. We subsequently examined 118 articles that passed a screen for studies that might have CVD clinical outcome data. We rejected 80 articles. Thirty of the rejected articles were reviews or commentaries that did not provide primary data. The reasons for rejecting the remaining 50 articles are listed in the section, Excluded Studies.

Thirty-nine unique studies fulfilled our inclusion criteria for reporting mortality or CVD clinical outcomes with a follow-up duration of 1 year or longer (interim reports or articles reporting different outcomes from the same overall study were counted as a single study). The 39 studies included: 12 randomized controlled trials (RCTs), 22 unique prospective cohort studies (including 4 studies that each contributed 2 separate articles on different analyses), 4 case-control studies, and 1 cross-sectional study. We created evidence and summary tables for these studies and included the studies in our analyses. Evidence Table 1 provides detailed information about the RCTs, and Evidence Table 2 describes prospective cohort, case-control, and cross-sectional studies. The summary tables present information about the study population, study design and duration, the frequency or amount of omega-3 fatty acid supplements or fish or fish oil consumed, dietary assessment method, main results, study quality, and study applicability. Studies are ordered by study size in each summary table.

For all practical purposes, CVD populations were studied with RCTs and the general population was studied with prospective cohort and case-control studies. Thus, in this section we first discuss results of the secondary prevention studies (i.e., studies of the CVD population), which are comprised of 11 RCTs and 1 cohort study. This is followed by a discussion of the primary prevention studies (or studies of the general population), which are comprised mostly of prospective cohort studies and 1 RCT.

For the non-randomized studies, data on each outcome are presented in 2 tables. One table presents outcomes based on estimates of omega-3 fatty acid or fish oil consumption, the other presents outcomes based on estimates of fish consumption. Because of the large amount of outcomes data reported in the prospective cohort studies, we created an “overall effect” metric to reduce this volume of information and to help interpret the results of these studies (see Chapter 2, Methods). This metric is used in the summary matrices (Tables 3.40–3.51).

In discussing results for the CVD and general populations, evidence for the following CVD clinical outcomes is presented: all-cause mortality, CVD deaths (deaths due to strokes, cardiac and peripheral vascular diseases), cardiac deaths, sudden death, myocardial infarction (MI), stroke, and all CVD events. It should be noted that different studies reported different combinations of these outcomes, and that the definitions for some of the outcomes varied across studies. For example, coronary deaths, ischemic deaths, cardiac deaths, and fatal myocardial infarction have largely overlapping but not identical meanings, as defined by individual studies. We placed the outcome reported by a study under the most similar common definition, as judged by a clinician-methodologist member of the EPC.

Tables 3.20–3.23 and 3.25 summarize the 12 RCTs. Six of the RCTs were trials of omega-3 fatty acid supplements, and 6 were trials of diets or dietary advice. Only 1 of the 12 trials, a large study that compared linseed oil (ALA) with sunflower oil, was a primary prevention study conducted in the general population. The remaining 11 trials were secondary prevention studies conducted in patients with known CVD. This profile was reversed among the 22 prospective cohort studies (which included 26 separate papers), as all but 1 of the cohort studies were conducted in the general population.

Tables 3.24–3.39 summarize the results of the prospective cohort, case-control, and cross-sectional studies. Studies are ordered by study size in each table. Data on each outcome are presented in 2 tables: 1 table presents outcomes based on estimates of omega-3 fatty acid or fish oil consumption, the other presents outcomes based on estimates of fish consumption. Because of the large amount of data reported in the prospective cohort studies, we created an “overall effect” metric to help in interpreting the results of these studies (see Chapter 2, Methods). This metric is reported by outcome in Tables 3.40–3.51.

Information about omega-3 fatty acid consumption varied across studies. In the RCTs of omega-3 fatty acid supplements, the amount and composition of omega-3 fatty acid is known and reported, whereas in the diet/dietary advice trials, estimates of the average amount of omega-3 fatty acids consumed by subjects are reported. In the prospective cohort studies, the amount of omega-3 fatty acid was not prescribed. As a result, omega-3 fatty acid intake and the amount or frequency of fish intake were estimated and reported as different quantiles corresponding to the observed relative risk of the outcomes.

Secondary Prevention Studies (Tables 3.20–3.24)

Evidence for the effects of the consumption of omega-3 fatty acids, omega-3 fatty acid supplements, or fish on CVD outcomes in populations known to have CVD was derived from 11 RCTs and 1 prospective cohort study. The 11 RCTs include 5 trials of omega-3 fatty acid supplements and 6 diet or dietary advice trials.

Characteristics of the omega-3 fatty acids supplements trials (Table 3.20–3.21). Of the 5 RCTs of omega-3 fatty acid supplements, 4 examined EPA+DHA supplements, The methodological quality of all 4 RCTs of EPA+DHA supplements was generally good (grade A or B)^34–37. Data on women are limited. The fifth is the single RCT with both an ALA arm and an EPA+DHA arm and the methodological quality was poor (grade C)³⁸.

The study populations of these 5 trials were rated as CVD-I (highly applicable) to CVD-II (relevant subgroups). One of the trials, the GISSI-Prevenzione trial, is the largest secondary prevention study with over 11,000 patients randomized ^{35, 39}. The other 3 EPA+DHA trials, combined, contributed fewer than 1,000 patients. The study subjects in these 3 smaller trials were MI survivors, patients with other vascular diseases, or patients with significant CVD risks. Most of the omega-3 fatty acid arms used a combination of EPA+DHA, although the dosages vary from 0.27 g/d to 4.8 g/d. The types of control also varied across the studies. The GISSI study used vitamin E or no vitamin E in a factorial design. Three of the studies used an equivalent amount of non-omega-3 oil as a control. The duration of the trials ranged from 2 to 3.5 years, and most were conducted outside the US.

The ALA trial was conducted in India and had a duration of 1 year. This trial compared 2.9 g/d of ALA in the form of mustard oil in 1 treatment arm and a combination of EPA+DHA in another treatment arm with a non-oil placebo³⁸. The methodological quality of this study was poor (grade C).

Characteristics of the diet and dietary advice trials. (Tables 3.22–3.23) Evidence for the effects of diet or dietary advice on CVD outcomes in populations known to have CVD was derived from 6 RCTs. About 4,000 patients were studied in the trials, and trial duration ranged from 2 to 5 years.

Two of the trials of diet and dietary advice were conducted among males from the ^{40, 41}. The amount of omega-3 fatty acid consumption in these 2 trials can only be estimated. The methodological quality of the trials was poor (grade C) and the study populations were rated as CVD-II (relevant subgroups). Two other trials reported estimates of EPA intake. The weekly EPA consumption in the first of these trials was 0.6 g in the control group and 2.4 g in the intervention group. Weekly EPA consumption in the second trial was 0.12g in the control group and 2.7 g in the intervention group.

Four trials provided estimates of daily ALA consumption. In the control groups of these trials, estimated ALA consumption ranged from 0.67 g/d to 1 g/d. Estimated ALA intake of the intervention groups was at least double that of the control groups (range 1.8 g/d to 6.3 g/d^{42 43–45}. The methodological quality of 3 of the 4 trials was poor (Grade C). The applicability of the trials ranged from CVD-I (highly applicable) to CVD-III (limited applicability). The subjects were mostly MI survivors or those at significant CVD risk. The study by Bemelmans et al. randomized patients in a factorial design to consume a margarine rich in ALA or LA, and to receive nutritional education or not ⁴⁵. The amount of margarine prescribed was not fixed, but instead was based on the participants' usual consumption patterns. The study by ⁴⁴ was conducted among patients in India. Two-thirds of the participants were vegetarians, which limits the applicability of the study results to the US population.

There was 1 prospective cohort study⁴⁶ (Table 3.24) in a CVD population that associated estimates of daily fish consumption with CVD outcomes. The methodological quality of this study was good (grade B). The study populations were rated as CVD-II (relevant subgroups). This study lasted 5 years and included 415 subjects with known coronary artery disease. A 4-day food record was used to assess the daily fish intake. Fish intake was divided into 3 categories: no intake, below medium consumption (57 g/d), and above medium consumption.

CVD Outcomes of Secondary Prevention Studies

Results from the secondary prevention studies are summarized by outcome, below.

All-cause mortality. Ten RCTs reported all-cause mortality (Tables 3.20–3.23). Of these, 4 ^34–37 used omega-3 fatty acid supplements. The quality of the 4 studies was generally good (grade A or B).

The all-cause mortality rate for control groups in the 10 RCTs ranged from 3.6% to 9.8% over a period of 1 to 3.5 years of follow-up. The largest study ³⁵ found significant reduction of all-cause mortality with a relative risk reduction of 21% over 2 to 3.5 years. The amount of omega-3 fatty acid used in the intervention arms of this study was 0.85 g/d of EPA+DHA.

The 2 largest diet/dietary advice trials ^{41, 47} were both of poor quality (Grade C). In the first trial⁴⁷, the amount of omega-3 fatty acid in the diet in the intervention arms was 2.4g/week of EPA. This trial found a significant reduction of all-cause mortality with a relative risk of 27%⁴⁷. However, the 10 year follow-up to this trial found no long-term benefit of fish advice in the same group of patients taking a similar amount of EPA ⁴⁸.

Of the 4 diet/dietary advice trials that provided estimates of ALA consumption ^{42, 43, 44, 45}, 3 found significant or near-significant reduction of all-cause mortality with a relative risk reduction of 25% to 56% over 2 to 5 years. The quality of these studies were fair to poor (grade B or C). The amount of omega-3 fatty acid in the diet in the intervention arms data ranged from about 1 to 6.3 g/d of ALA. Because these trials were interventions based on diet, the daily variations in the amount of omega-3 fatty acids would make the interpretations of their results difficult.

The single prospective cohort study (Table 3.24) compared subjects who consumed fish to those who did not and reported an at least 50% relative risk reduction in all-cause mortality and CVD death with any amount of fish intake ⁴⁶.

Sudden death. (Tables 3.20, 3.22, 3.25). Six RCTs reported data on sudden deaths Four studies ^{35 38, 43, 44} Singh reported a significant or near-significant large reduction of this outcome (relative risk [RR] 0.06 to 0.55). The reduction of sudden deaths in these studies was observed in both the fish oil group and the ALA oil group. However, of the 4 studies, 3 (a Mediterranean diet study ⁴³ and 2 Indian studies ^{38, 44} ) were poorly designed (grade C).

An early trial by Leren⁴² randomized 206 men 1-to-2-years post-MI to a cholesterol lowering diet and followed them for 5 years. There were no differences between subjects on the diet and those in the control group. However, a new report by Burr et al⁴¹ found that persons taking fish oil supplements have an increased risk of sudden death risk, although this study is also of poor quality (grade C).

Stroke. (Tables 3.21, 3.23, 3.26). Six trials reported data on stroke Strokes occurred in 0% to 3% of subjects in control groups. Three of the trials ^{34, 35, 37} were of fish oil supplements; the methodological quality of these trials was generally good (2 studies of grade B and 1 study of grade A) and each reported trends of increased strokes. However, the 3 diet/dietary advice trials ^{43 44, 45} (which were of poor quality 2 studies of grade C and of 1 grade B) reported trends of fewer strokes. None of the results from the 6 studies were statistically significant.

Other CVD outcomes. One study consistently reported no beneficial effect of omega-3 fatty acids on any CVD outcomes (Tables 3.20 and 3.22) ³⁶. This study randomized 300 patients to 1.7 g/d of EPA+DHA or an equivalent amount of corn oil and followed subjects for 1.5 years. Of note, 15% of the study subjects died during the study, and about 40% of the subjects had been taking fish oil before the trial.

Three of the RCTs were too small, with 59 and 120 subjects each^{34, 37}, or had too few CVD events⁴⁵ to provide meaningful results.

Reports of other outcomes, such as CVD deaths, cardiac deaths, sudden death, fatal and non-fatal MI, were inconsistently reported. The overall beneficial results were similar across studies.

Primary Prevention Studies (Tables 3.25–3.39)

Evidence for the effects of the consumption of omega-3 fatty acids, omega-3 fatty acid supplements, or fish on CVD outcomes in the general population is derived from 22 prospective cohort studies, 4 case-control studies, 1 cross-sectional study, and 1 RCT. The methodological quality of most of the studies within their study design category was good (grades A or B); 4 prospective cohort studies were graded as poor (grade C).

We found only 1 RCT that examined omega-3 fatty acid supplements in the general population. (Tables 3.25–3.26) The methodological quality of this study was poor (grade C). The study, which compares linseed oil (5.5 g/d of ALA) with sunflower seed oil (0.14 g/d ALA), was conducted in Norway more than 30 years ago⁴⁹ and lasted 1 year. It is the largest of all ALA supplement trials, with over 13,000 subjects. Presumably, subjects had high background omega-3 fatty acid levels because of characteristically large consumption of fish. There were too few all-cause mortality or CVD events in the control group, and it reported no benefit on any of the CVD outcomes. This trial does not contribute substantively to the assessment of the effect of omega-3 fatty acid supplements on CVD outcomes. The major conclusion one can draw from this study is that ALA, given at a dose of 0.14 g/d for 1 year, has no effect on CVD outcomes in the general population with a high fish consumption background diet.

The 22 prospective cohort studies were conducted in many parts of the world, including the US, China, Japan, and countries in the Mediterranean and Northern Europe. Most of the cohorts had several thousand subjects. The majority of the studies received an applicability grade of GEN-II, reflecting either relevant subgroups or differences in the background diet of the study population when compared with the US population. Several of the large population studies conducted in the US were graded as GEN-II because of single sex (male or female) cohorts. If viewed together, however, these studies would provide evidence that is highly applicable to the US population (GEN-I). Study duration in the cohort studies ranged from 4 to 30 years. The number of subjects followed in the cohorts ranged from 272 to as many as 223,170; many of the cohorts had tens of thousands of study subjects.

Most of the studies used the food frequency questionnaire to estimate the dietary fish intake. Most studies provided quantitative estimates of the amount of fish consumed (many also quantified the amount of EPA+DHA intake) and categorized them into various quantiles (e.g., tertiles, quartiles, quintiles), although some studies reported only the frequency of fish consumption or simply whether fish was consumed.

CVD Outcomes of Primary Prevention Studies

Results from the primary prevention studies are summarized by outcome, below.

All-cause mortality. Ten studies that followed a total of about 100,000 subjects for an average of over 10 years provided data on all-cause mortality.(Tables 3.27–3.28) Three of the 10 studies provided estimates of fish oil intake, and 9 provided estimates of fish consumption. The studies were conducted in the US, China, Japan, Denmark, Holland, and the UK. All 3 studies that provided estimates of fish oil intake reported a significant reduction (++ overall effect) of all-cause mortality ^50–52. The results of studies reporting estimates of fish intake were heterogeneous — 1 study ⁵³ reported a small but significant increase of all-cause mortality with increasing fish intake, and 5 studies found no benefit ^{50, 54–57}. Of the studies finding no benefit, 1 (by Nagata et al.) reported no benefit using estimates of fish consumption but observed beneficial results using estimates of fish oil. One study showed significant benefit⁵⁸, and 1 study showed a trend for benefit⁵⁹.

CVD deaths, cardiac deaths, and MI. The outcomes of CVD deaths (Tables 3.29–3.30), cardiac deaths (Tables 3.31–3.32), and MI (Tables 3.35–3.36) were similar. Most of the large cohort studies reported significant reduction of clinical events. Among the large cohort studies, only the Physicians' Health Study (PHS) failed to report a significant beneficial effect of fish consumption ⁵⁸.

Sudden death. Two prospective cohort studies reported data on sudden death. (Tables 3.33–3.34). These studies provided estimates of both fish and fish oil consumption. The Physicians' Health Study, which followed 20,551 subjects for 12 years, reported an approximately 50% overall relative risk reduction even with a small amount of fish intake (>0.3 g of fish oil per month or eating fish once a month)⁵⁸. A smaller study also found a significant reduction of arrhythmic deaths at higher levels of fish intake ⁶⁰. However, in the same study opposite results were seen with consumption of fried fish or fish sandwiches⁶⁰. Another smaller follow-up study of 30 years duration found a significant trend of reduction in sudden death ⁵⁶. A case-control study of 827 subjects in the US also reported a significant inverse association of sudden death with increasing fish intake⁶¹.

Stroke. Nine prospective cohort studies and 1 case-control study provided data on stroke. (Tables 3.37–3.38) Five of the cohort studies estimated the amount of fish oil consumed, and 8 estimated fish intake. These studies included the large US cohorts of the Nurses' Health Study (NHS)⁶², Health Professionals Study (HPS) ⁶³, and the Physicians' Health Study ⁶⁴, which followed subjects for 14, 12, and 4 years, respectively. Together, these 3 studies comprised a total of about 145,000 men and women. Only the Health Professionals Study⁶³ reported a significant reduction of ischemic strokes with any level of fish consumption above the lowest quintile. In the Nurses' Health Study ⁶², there was a non-significant trend of decreased strokes with increasing fish consumption. Other studies showed a weak benefit, no benefit, or an increased risk of strokes. The fish oil estimates and fish estimates yielded similar results.