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Title: Biomarkers of dietary omega-6 fatty acids and incident cardiovascular disease and mortality: an individual-level pooled analysis of 30 cohort studiesShort title: Omega-6 fatty acids and cardiovascular diseaseAuthors: The Fatty Acids and Outcomes Research Consortium (FORCE)*Corresponding author: Matti Marklund; The George Institute for Global Health and the Faculty of Medicine, University of New South Wales, Sydney, Australia; telephone: +1 202 718 34 43; email: mmarklund@.au.Word count: 3,736 of 5,000*Individual authors are presented in the end of manuscriptABSTRACT BackgroundGlobal dietary recommendations for and cardiovascular effects of linoleic acid, the major dietary omega-6 fatty acid, and its major metabolite, arachidonic acid, remain controversial. To address this uncertainty and inform international recommendations, we evaluated how in vivo circulating and tissue levels of linoleic acid (LA) and arachidonic acid (AA) relate to incident cardiovascular disease (CVD) across multiple international studies.MethodsWe performed harmonized, de novo, individual-level analyses in a global consortium of 30 prospective observational studies from 13 countries. Multivariable-adjusted associations of circulating and adipose tissue LA and AA biomarkers with incident total CVD and subtypes (coronary heart disease (CHD), ischemic stroke, cardiovascular mortality) were investigated according to a prespecified analytical plan. Levels of LA and AA, measured as % of total fatty acids, were evaluated linearly according to their interquintile range (i.e., the range between the mid-point of the first and fifth quintiles), and categorically by quintiles. Study-specific results were pooled using inverse-variance weighted meta-analysis. Heterogeneity was explored by age, sex, race, diabetes, statin use, aspirin use, omega-3 levels, and fatty acid desaturase 1 genotype (when available). ResultsIn 30 prospective studies with medians of follow-up ranging 2.5 to 31.9 years, 15,198 incident cardiovascular events occurred among 68,659 participants. Higher levels of LA were significantly associated with lower risks of total CVD, cardiovascular mortality, and ischemic stroke, with hazard ratios per interquintile range of 0.93 (95% CI: 0.88-0.99), 0.78 (0.70-0.85), and 0.88 (0.79-0.98), respectively, and nonsignificantly with lower CHD risk (0.94; 0.88-1.00). Relationships were similar for LA evaluated across quintiles. AA levels were not associated with higher risk of cardiovascular outcomes; comparing extreme quintiles, higher levels were associated with lower risk of total CVD (0.92; 0.86-0.99). No consistent heterogeneity by population subgroups was identified in the observed relationships. ConclusionsIn pooled global analyses, higher in vivo circulating and tissue levels of LA and possibly AA were associated with lower risk of major cardiovascular events. These results support a favorable role for LA in CVD prevention. Clinical perspectiveWhat is new? We conducted the hitherto largest pooled individual-level analysis using circulating and adipose tissue levels of linoleic acid and arachidonic acid to examine the link between omega-6 fatty acids and cardiovascular outcomes in various populations.Our approach increases statistical power and generalizability compared to individual studies; lowers the risk of publication bias and heterogeneity compared to meta-analyses of existing literature; and allows evaluation of the associations in key population subgroups. Strikingly, higher level of linoleic acid was associated with lower risks of total cardiovascular disease, ischemic stroke, and cardiovascular mortality, while arachidonic acid was not associated with cardiovascular risk.What are the clinical implications?Our findings support potential benefits of the main dietary omega-6 fatty acid, i.e., linoleic acid, for cardiovascular disease prevention.Furthermore, our results do not support any theorized cardiovascular harms of omega-6 fatty acids.Our findings provide evidence to help inform currently inconsistent global dietary recommendations on omega-6 consumption.INTRODUCTIONRecommendations for dietary consumption omega-6 (n-6) polyunsaturated fatty acids (PUFA) for cardiovascular disease (CVD) prevention remain controversial and inconsistent. ADDIN EN.CITE <EndNote><Cite><Author>Harris</Author><Year>2009</Year><RecNum>1171</RecNum><DisplayText><style face="superscript">1</style></DisplayText><record><rec-number>1171</rec-number><foreign-keys><key app="EN" db-id="frdaewwftrdav5e02x3xvdrgxe0x0pxpfr2d" timestamp="1464775769">1171</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Harris, William S.</author><author>Mozaffarian, Dariush</author><author>Rimm, Eric</author><author>Kris-Etherton, Penny</author><author>Rudel, Lawrence L.</author><author>Appel, Lawrence J.</author><author>Engler, Marguerite M.</author><author>Engler, Mary B.</author><author>Sacks, Frank</author></authors></contributors><titles><title>Omega-6 Fatty Acids and Risk for Cardiovascular Disease: A Science Advisory From the American Heart Association Nutrition Subcommittee of the Council on Nutrition, Physical Activity, and Metabolism; Council on Cardiovascular Nursing; and Council on Epidemiology and Prevention</title><secondary-title>Circulation</secondary-title></titles><periodical><full-title>Circulation</full-title></periodical><pages>902-907</pages><volume>119</volume><number>6</number><dates><year>2009</year><pub-dates><date>February 17</date></pub-dates></dates><urls><related-urls><url> For example, the American Heart Association and the Academy of Nutrition and Dietetics recommend 5-10%,PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5IYXJyaXM8L0F1dGhvcj48WWVhcj4yMDA5PC9ZZWFyPjxS

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ADDIN EN.CITE.DATA 1, 2 the United Nations Food and Agriculture Organization recommends 2.5-9%, ADDIN EN.CITE <EndNote><Cite><Author>Food and Agriculture Organization of the United Nations.</Author><Year>2010</Year><RecNum>2032</RecNum><DisplayText><style face="superscript">3</style></DisplayText><record><rec-number>2032</rec-number><foreign-keys><key app="EN" db-id="frdaewwftrdav5e02x3xvdrgxe0x0pxpfr2d" timestamp="1542145975">2032</key></foreign-keys><ref-type name="Book">6</ref-type><contributors><authors><author>Food and Agriculture Organization of the United Nations.,</author></authors></contributors><titles><title>Fats and fatty acids in human nutrition : report of an expert consultation : 10-14 November 2008, Geneva</title><secondary-title>FAO food and nutrition paper,</secondary-title></titles><pages>xvii, 166 p.</pages><number>91</number><keywords><keyword>Lipids in human nutrition Congresses.</keyword><keyword>Fatty acids in human nutrition Congresses.</keyword></keywords><dates><year>2010</year></dates><pub-location>Rome</pub-location><publisher>Food and Agriculture Organization of the United Nations</publisher><isbn>9789251067338&#xD;9251067333&#xD;0254-4725 ;</isbn><accession-num>16757899</accession-num><call-num>TX553.L5 F375 2010</call-num><urls></urls></record></Cite></EndNote>3 while the French national guidelines recommend 4%.PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5MZWdyYW5kPC9BdXRob3I+PFllYXI+MjAxMTwvWWVhcj48

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ADDIN EN.CITE.DATA 4 Pooled evidence from clinical trials and cohort studies suggests a moderate benefit of consuming n-6 PUFA, predominantly linoleic acid (LA, 18:2n-6), for coronary heart disease (CHD) risk, whether replacing saturated fat or total carbohydrate.PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5Nb3phZmZhcmlhbjwvQXV0aG9yPjxZZWFyPjIwMTA8L1ll

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ADDIN EN.CITE.DATA 8 substantial drop-out, ADDIN EN.CITE <EndNote><Cite><Author>Ramsden</Author><Year>2016</Year><RecNum>1165</RecNum><DisplayText><style face="superscript">9</style></DisplayText><record><rec-number>1165</rec-number><foreign-keys><key app="EN" db-id="frdaewwftrdav5e02x3xvdrgxe0x0pxpfr2d" timestamp="1464677791">1165</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Ramsden, Christopher E</author><author>Zamora, Daisy</author><author>Majchrzak-Hong, Sharon</author><author>Faurot, Keturah R</author><author>Broste, Steven K</author><author>Frantz, Robert P</author><author>Davis, John M</author><author>Ringel, Amit</author><author>Suchindran, Chirayath M</author><author>Hibbeln, Joseph R</author></authors></contributors><titles><title>Re-evaluation of the traditional diet-heart hypothesis: analysis of recovered data from Minnesota Coronary Experiment (1968-73)</title><secondary-title>BMJ</secondary-title></titles><periodical><full-title>BMJ</full-title></periodical><volume>353</volume><dates><year>2016</year><pub-dates><date>2016-04-12 22:34:54</date></pub-dates></dates><urls><related-urls><url> and confounding by industrial trans-fats.PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5SYW1zZGVuPC9BdXRob3I+PFllYXI+MjAxMzwvWWVhcj48

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ADDIN EN.CITE.DATA 8, 9 In addition, many of the other prior trials are limited by lack of blinding or randomization, and major dietary pattern shifts; and most are decades old, creating potentially low generalizability to contemporary diets and clinical settings. Cohort studies are limited by the common reliance on self-reported dietary habits, which can be influenced by memory errors and inaccurate nutrient databases. Thus, for many scientists, clinicians, and policy makers, the role of LA in CVD risk remains uncertain.In addition, concerns have been raised that n-6 PUFA could actually increase CVD risk, due to potential pro-inflammatory effects.PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5SYW1zZGVuPC9BdXRob3I+PFllYXI+MjAxNjwvWWVhcj48

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ADDIN EN.CITE.DATA 10, 11Yet, stable isotope studies suggest very limited conversion of LA to AA in humans, ADDIN EN.CITE <EndNote><Cite><Author>Hussein</Author><Year>2005</Year><RecNum>1166</RecNum><DisplayText><style face="superscript">12</style></DisplayText><record><rec-number>1166</rec-number><foreign-keys><key app="EN" db-id="frdaewwftrdav5e02x3xvdrgxe0x0pxpfr2d" timestamp="1464703136">1166</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Hussein, Nahed</author><author>Ah-Sing, Eric</author><author>Wilkinson, Paul</author><author>Leach, Clare</author><author>Griffin, Bruce A.</author><author>Millward, D. Joe</author></authors></contributors><titles><title>Long-chain conversion of [13C]linoleic acid and α-linolenic acid in response to marked changes in their dietary intake in men</title><secondary-title>Journal of Lipid Research</secondary-title></titles><periodical><full-title>Journal of Lipid Research</full-title><abbr-1>J Lipid Res</abbr-1></periodical><pages>269-280</pages><volume>46</volume><number>2</number><dates><year>2005</year><pub-dates><date>February 1, 2005</date></pub-dates></dates><urls><related-urls><url> and trials show limited effects of increasing dietary LA on plasma and adipose tissue AA levels.PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5TYXJra2luZW48L0F1dGhvcj48WWVhcj4xOTk0PC9ZZWFy

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ADDIN EN.CITE.DATA 12-14 These findings indicate the importance of directly evaluating AA levels instead of inferring them from LA levels or intakes in relation to CVD risk. As LA cannot be produced endogenously (making tissue levels reasonable markers of intake), biomarker (circulating and adipose tissue) levels correlate with dietary consumption.PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5Ta2VhZmY8L0F1dGhvcj48WWVhcj4yMDA2PC9ZZWFyPjxS

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ADDIN EN.CITE.DATA 15, 16 Such objective biomarkers allow evaluation of dietary exposure of LA status independent of self-reported food habits and estimated nutrient composition of different foods. Circulating and adipose biomarkers also allow direct evaluation of AA, which is highly metabolically regulated and for which dietary estimates correlate poorly with in vivo levels. Yet, the relations between in vivo levels of LA and AA and CHD risk have been evaluated in relatively few studies, with different study designs, outcomes, exposures (e.g., lipid compartment), covariates, and statistical methodology. Results from meta-analyses of published studies using circulating or adipose tissue levels of n-6 PUFA have been contradictory.PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5IYXJyaXM8L0F1dGhvcj48WWVhcj4yMDA3PC9ZZWFyPjxS

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ADDIN EN.CITE.DATA 19-23 and remain uncertain.To address these major gaps in knowledge, we conducted a pooled analysis of harmonized, de novo, individual-level data across 30 cohort studies in the Fatty Acid and Outcome Research Consortium (FORCE) to evaluate associations of LA and AA levels with incident total CVD and subtypes (CHD, ischemic stroke, CVD mortality).METHODSStudy setting and population: FORCE Consortium The study was conducted within FORCE (), a consortium of studies with circulating or adipose tissue fatty acid biomarker measurements and ascertained chronic disease events.PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5EZWwgR29iYm88L0F1dGhvcj48WWVhcj4yMDE2PC9ZZWFy

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L0VuZE5vdGU+

ADDIN EN.CITE.DATA 24 Studies were identified and invited to participate if assessing biomarker (circulating or adipose tissue) levels of LA and AA, and incident CVD (or subtypes thereof), based on previous FORCE projects,PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5EZWwgR29iYm88L0F1dGhvcj48WWVhcj4yMDE2PC9ZZWFy

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ADDIN EN.CITE.DATA 24, 25 expert contacts, and online searches. Studies with adult participants (≥18 y) free of CVD (myocardial infarction, angina, coronary revascularization, stroke) at the time of fatty acid sampling were invited. Retrospective case-control studies were included in a sensitivity analysis if fatty acids were assessed in adipose tissue, which have a long half-life of exposure. ADDIN EN.CITE <EndNote><Cite><Author>Hodson</Author><Year>2008</Year><RecNum>754</RecNum><DisplayText><style face="superscript">26</style></DisplayText><record><rec-number>754</rec-number><foreign-keys><key app="EN" db-id="frdaewwftrdav5e02x3xvdrgxe0x0pxpfr2d" timestamp="1381921146">754</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Hodson, L.</author><author>Skeaff, C. M.</author><author>Fielding, B. A.</author></authors></contributors><titles><title>Fatty acid composition of adipose tissue and blood in humans and its use as a biomarker of dietary intake</title><secondary-title>Prog Lipid Res</secondary-title></titles><periodical><full-title>Progress in lipid research</full-title><abbr-1>Prog Lipid Res</abbr-1></periodical><pages>348-380</pages><volume>47</volume><number>5</number><dates><year>2008</year><pub-dates><date>Sep</date></pub-dates></dates><isbn>0163-7827</isbn><accession-num>WOS:000258748100005</accession-num><urls><related-urls><url>&lt;Go to ISI&gt;://WOS:000258748100005</url></related-urls></urls><electronic-resource-num>10.1016/j.plipres.2008.03.003</electronic-resource-num></record></Cite></EndNote>26 To minimize potential reverse causation, the main analysis included only prospective studies. Of 38 studies invited by September 2017, 31 participated (Table 1), while 7 were ineligible, declined to participate, or failed to respond (Supplemental Table 1 in the online-only Data Supplement). The study was approved by the institutional review boards of the participating cohorts.Fatty acid measurementsStudies measured fatty acids in differing compartments, including plasma phospholipids, erythrocytes, plasma, serum, cholesterol esters, and adipose tissue. All fatty acid levels were reported as percent of total fatty acids. Detailed information regarding fatty acid measurements in each study is provided in the Supplemental Material. Outcome assessmentIn each cohort, study participants were excluded if they were children (age <18 years) or had prevalent CVD at the time of fatty acid measurement. Among the remaining participants, we evaluated incident CVD (defined as incident CHD or stroke) and its subtypes including CHD (fatal or nonfatal myocardial infarction, CHD death, or sudden cardiac death), ischemic stroke (fatal or nonfatal ischemic stroke), and CVD mortality (the subset of fatal events from these causes). Studies that did not separately assess ischemic stroke used total stroke (n=5 studies). Detailed information on outcomes in each study is provided in the Supplemental Material.CovariatesTo minimize potential confounding, prespecified and harmonized covariates were utilized included age (years), sex (male/female), race (Caucasian/non-Caucasian, or study-specific), field center if applicable (categories), body-mass index (BMI, kg/m2), education (less than high school graduate, high school graduate, some college or vocational school, college graduate), smoking (current, former, never; if history not assessed, then current/not current), physical activity (quintiles of metabolic equivalents (METs)/ week), alcohol intake (none, 1-6 drinks/week, 1-2 drinks/day, >2 drinks/day), prevalent diabetes mellitus (defined as treatment with oral antihyperglycemic agents, insulin, or fasting plasma glucose >126 mg/dL), treated hypertension (defined as hypertension drug use; or if unavailable, as diagnosed/history of hypertension), treated hypercholesterolemia (defined as LDL-lowering drug use; if unavailable, as diagnosed/history of hypercholesterolemia), regular aspirin use (defined as ≥2 times/week), levels of α-linolenic acid (ALA; 18:3n-3), eicosapentaenoic acid (EPA; 20:5n-3), sum of trans isomers of oleic acid (trans18:1), and sum of trans isomers of LA (trans-18:2) (each expressed as % total FAs). If data did not allow such categorization, study-specific categories were used. Imputation was allowed for linear covariates if previously established in each cohort; missing indicator categories were utilized for missing covariate data in categories. Statistical analysis and poolingAll participating studies followed a prespecified, harmonized analysis protocol with standardized exclusions, exposures, outcomes, covariates, and analytical methods. In each study, de novo analyses of individual data were performed according to the protocol. Cox and weighted Cox proportional hazards models were used to estimate hazard ratios in cohort and nested unmatched case-cohort studies, respectively, with follow-up from the date of blood or adipose tissue sampling to date of incident event, death, loss to follow-up, or end of follow-up. In matched nested case-control studies, conditional logistic regression was used to estimate odds-ratios for each outcome, considered to approximate To assess potential nonlinear associations, each cohort also evaluated study-specific quintiles as indicator categories, with the lowest quintile as the reference. Studies assessing fatty acids in multiple compartments conducted separate analyses in each compartment. To investigate potential heterogeneity by other factors, associations in each study were also assessed in prespecified strata by age, sex, race, ALA and EPA levels, prevalent diabetes, drug-treated hypercholesterolemia, and regular aspirin use. Potential interactions by genotype were examined in the 14 studies with available data for rs174547 (single nucleotide polymorphism in the gene for fatty acid desaturase 1, a major genetic determinant of circulating LA and AA).PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5HdWFuPC9BdXRob3I+PFllYXI+MjAxNDwvWWVhcj48UmVj

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ADDIN EN.CITE.DATA 28 Overall heterogeneity was assessed by the I2-statistic, with values of ~ 25%, 50%, and 75%, considered to indicate low, medium, and high heterogeneity, respectively. ADDIN EN.CITE <EndNote><Cite><Author>Huedo-Medina</Author><Year>2006</Year><RecNum>1958</RecNum><DisplayText><style face="superscript">29</style></DisplayText><record><rec-number>1958</rec-number><foreign-keys><key app="EN" db-id="frdaewwftrdav5e02x3xvdrgxe0x0pxpfr2d" timestamp="1527794767">1958</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Huedo-Medina, T. B.</author><author>Sanchez-Meca, J.</author><author>Marin-Martinez, F.</author><author>Botella, J.</author></authors></contributors><auth-address>Department of Basic Psychology &amp; Methodology, University of Murcia, Murcia, Spain. hmtania@um.es</auth-address><titles><title>Assessing heterogeneity in meta-analysis: Q statistic or I2 index?</title><secondary-title>Psychol Methods</secondary-title></titles><periodical><full-title>Psychol Methods</full-title></periodical><pages>193-206</pages><volume>11</volume><number>2</number><edition>2006/06/21</edition><keywords><keyword>*Analysis of Variance</keyword><keyword>Bias</keyword><keyword>Computer Simulation</keyword><keyword>*Confidence Intervals</keyword><keyword>Controlled Clinical Trials as Topic/statistics &amp; numerical data</keyword><keyword>*Data Interpretation, Statistical</keyword><keyword>Effect Modifier, Epidemiologic</keyword><keyword>Humans</keyword><keyword>*Meta-Analysis as Topic</keyword><keyword>Monte Carlo Method</keyword></keywords><dates><year>2006</year><pub-dates><date>Jun</date></pub-dates></dates><isbn>1082-989X (Print)&#xD;1082-989X (Linking)</isbn><accession-num>16784338</accession-num><urls><related-urls><url> Heterogeneity between prespecified subgroups was explored by meta-analyzing study-specific effect estimates from each stratum, with statistical differences between subgroups tested by meta-regression. Potential interactions by desaturase genotype were examined by meta-analyzing study-specific interaction terms. For each study, associations of n-6 PUFA with CVD per genotype at rs174547 (i.e, CC, CT, or TT) were calculated from beta coefficients and the variance-covariance matrix of the main and interaction terms.PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5EZWwgR29iYm88L0F1dGhvcj48WWVhcj4yMDE2PC9ZZWFy

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ADDIN EN.CITE.DATA 24 The genotype-specific estimates were pooled using pooled using inverse-variance weighted meta-analysis. While subgroups were prespecified, all heterogeneity analyses were considered exploratory and Bonferroni-corrected for multiple comparisons (10 subgroups; corrected α=0.005). In sensitivity analyses, we evaluated compartment-specific associations using absolute percent of total fatty acids as the unit of exposure, instead of study-specific interquintile range. In other sensitivity analyses, we censored events at maximum 10 y of follow-up, to minimize bias by changes in fatty acid levels over time; used alternative blood compartments in the overall pooled analysis for studies having more than one measure; included one retrospective study; and excluded studies assessing only fatal outcomes.Meta-analyses were performed using Stata 13 (StataCorp, College Station, TX), with two-tailed α=0.05 for the primary analyses.RESULTSThe pooled analyses included 76,356 fatty acid measurements from 68,659 participants in 30 prospective studies from 13 countries (Table 1). The studies included 18 cohort and 12 nested case-control or case-cohort studies. Most studies assessed fatty acids in blood compartments (plasma phospholipids, n=11 studies; erythrocyte phospholipids, total plasma, or cholesterol esters, n=7 studies each), while adipose tissue was less commonly used (n=3 studies). One retrospective case-control study measuring adipose tissue biomarkers was included in a sensitivity analysis, but not in the primary analyses. Across studies, mean age at baseline ranged from 49 to 77 years (Table 1 and Supplemental Table 2). Overall proportions of women and men were comparable, although some studies included one sex only (Table 1). Most participants were Caucasian, but several studies included sizable numbers of African Americans, Asians, and Hispanics (Supplemental Table 3). In most studies, up to 30% of the participants smoked, and alcohol intake was generally moderate (<1 drink/d). Education level, diabetes prevalence, and medication use varied across studies. As would be expected, levels of fatty acids varied between different compartments (Figure 1 and Supplemental Tables 2 and 4).Median study follow-up durations ranged from 2.5 to 31.9 years. Among the 30 prospective studies, 10,477 total incident CVD events, 4,508 CVD deaths, 11,857 incident CHD events, and 3,705 incident ischemic strokes occurred (Supplemental Table 5). Per interquintile range, higher LA levels were associated with 7% (95%CI: 1-12%), 22% (15-30%), and 12% (2-21%) lower incidence of total CVD, CVD mortality, and ischemic stroke, respectively (Figures 2-3, Table 2). LA levels were also nonsignificantly (P=0.065) associated with lower incidence of total CHD. Overall heterogeneity was moderate (I2=28-63%). Associations of LA with total CVD, total CHD, and CVD mortality varied by compartment (P-interaction≤0.031), with generally less prominent inverse associations in studies utilizing phospholipids (Figures 2-3). Compared to the lowest quintile, participants in the highest quintile of LA levels experienced lower risk of CVD mortality (HR=0.77; 95% CI, 0.69-0.86), with nonsignificant trends toward lower risk of total CVD (0.94; 0.87-1.01), CHD (0.92; 0.85-1.00), and ischemic stroke (0.90; 0.79-1.02) (Supplemental Table 6). There was no significant evidence of non-linear associations between LA and each outcome (P-nonlinearity>0.05 each).AA levels evaluated linearly were not significantly associated with CVD events, with a hazard ratio of 0.95 (0.90-1.01) for total CVD (Table 2, Figures 4-5). When different lipid compartments were assessed, AA levels in total plasma, but not other compartments, were associated with lower risk of total CVD (HR=0.81 (0.70-0.94) (Table 2, Figure 4). Overall heterogeneity was low to moderate (I2≤54%). When AA levels were evaluated in quintiles (Supplemental Table 7), participants in the highest quintile, compared to the lowest, experienced significantly lower incidence of total CVD (0.92; 0.86-0.99). There was evidence for a borderline nonlinear association (P-nonlinearity=0.039) between total plasma AA and ischemic stroke (Supplemental Figure 1). Associations of LA and AA with CVD outcomes did not significantly differ according to subgroups defined by age, sex, race, n-3 PUFA levels, diabetes status, statin use, aspirin use, or baseline year of fatty acid measurement (Supplemental Table 8). In 14 studies with genotype data (Supplemental Table 9), a significant interaction (P-interaction=0.002) was observed between LA and rs174547 genotype in relation to risk of ischemic stroke (Supplemental Table 10), with inverse associations appearing stronger in carriers of the major T-allele. The associations of AA with cardiovascular outcomes did not significantly vary by rs174547 genotype.In sensitivity analyses, results of compartment-specific analysis that utilized units of percent of total fatty acids, rather than study-specific interquintile ranges, were not appreciably different from the main findings (Supplemental Table 11). Results were also similar across all other sensitivity analyses (Supplemental Table 12). DISCUSSIONIn this harmonized, individual-level pooled analysis across 30 prospective studies from 13 countries, higher in vivo levels of the n-6 PUFA LA were associated with lower risk of CVD events, in particular CVD mortality and stroke. AA levels were not associated with higher risk, and were associated with lower CVD risk in some analyses. To our knowledge, this is the largest pooled analysis of fatty acid levels and CVD endpoints, including almost 70,000 individuals and 10,000 total CVD events. Our findings provide evidence to help inform currently inconsistent global dietary recommendations on n-6 PUFA consumption. LA, an essential fatty acid not synthesized by humans, is the main dietary PUFA, comprising about 85-90% of the total. While circulating and adipose tissue LA levels can be influenced by metabolism,PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5CYXlsaW48L0F1dGhvcj48WWVhcj4yMDA2PC9ZZWFyPjxS

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ADDIN EN.CITE.DATA 15, 26, 30 and consistently correlate with self-reported dietary estimates in large cohort studies, ADDIN EN.CITE <EndNote><Cite><Author>Hodson</Author><Year>2008</Year><RecNum>754</RecNum><DisplayText><style face="superscript">26</style></DisplayText><record><rec-number>754</rec-number><foreign-keys><key app="EN" db-id="frdaewwftrdav5e02x3xvdrgxe0x0pxpfr2d" timestamp="1381921146">754</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Hodson, L.</author><author>Skeaff, C. M.</author><author>Fielding, B. A.</author></authors></contributors><titles><title>Fatty acid composition of adipose tissue and blood in humans and its use as a biomarker of dietary intake</title><secondary-title>Prog Lipid Res</secondary-title></titles><periodical><full-title>Progress in lipid research</full-title><abbr-1>Prog Lipid Res</abbr-1></periodical><pages>348-380</pages><volume>47</volume><number>5</number><dates><year>2008</year><pub-dates><date>Sep</date></pub-dates></dates><isbn>0163-7827</isbn><accession-num>WOS:000258748100005</accession-num><urls><related-urls><url>&lt;Go to ISI&gt;://WOS:000258748100005</url></related-urls></urls><electronic-resource-num>10.1016/j.plipres.2008.03.003</electronic-resource-num></record></Cite></EndNote>26 including a considerable number of studies participating in the current analysis (Supplemental Table 13). Several lines of evidence support mechanisms by which dietary LA may reduce CVD. In randomized controlled feeding trials, dietary PUFA (primarily LA) as a replacement for either carbohydrates or saturated fat lowers low density lipoprotein (LDL)-cholesterol, triglycerides, and ApoB levels, and raises high density lipoprotein (HDL)-cholesterol;PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5LYXRhbjwvQXV0aG9yPjxZZWFyPjE5OTQ8L1llYXI+PFJl

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ADDIN EN.CITE.DATA 14, 31 and also lowers hemoglobin A1c and insulin resistance and potentially augments insulin production.PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5JbWFtdXJhPC9BdXRob3I+PFllYXI+MjAxNjwvWWVhcj48

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RW5kTm90ZT4A

ADDIN EN.CITE.DATA 32 Other potential cardiometabolic benefits of dietary LA may include favorable effects on inflammation,PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5CamVybW88L0F1dGhvcj48WWVhcj4yMDEyPC9ZZWFyPjxS

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ADDIN EN.CITE.DATA 14 blood pressure, ADDIN EN.CITE <EndNote><Cite><Author>Miura</Author><Year>2008</Year><RecNum>1174</RecNum><DisplayText><style face="superscript">33</style></DisplayText><record><rec-number>1174</rec-number><foreign-keys><key app="EN" db-id="frdaewwftrdav5e02x3xvdrgxe0x0pxpfr2d" timestamp="1464780519">1174</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Miura, Katsuyuki</author><author>Stamler, Jeremiah</author><author>Nakagawa, Hideaki</author><author>Elliott, Paul</author><author>Ueshima, Hirotsugu</author><author>Chan, Queenie</author><author>Brown, Ian J.</author><author>Tzoulaki, Ioanna</author><author>Saitoh, Shigeyuki</author><author>Dyer, Alan R.</author><author>Daviglus, Martha L.</author><author>Kesteloot, Hugo</author><author>Okayama, Akira</author><author>Curb, J. David</author><author>Rodriguez, Beatriz L.</author><author>Elmer, Patricia J.</author><author>Steffen, Lyn M.</author><author>Robertson, Claire</author><author>Zhao, Liancheng</author><author>for the International Study of Macro-Micronutrients</author><author>Blood Pressure Research Group</author></authors></contributors><titles><title>Relationship of Dietary Linoleic Acid to Blood Pressure: The International Study of Macro-Micronutrients and Blood Pressure Study</title><secondary-title>Hypertension</secondary-title></titles><periodical><full-title>Hypertension</full-title></periodical><pages>408-414</pages><volume>52</volume><number>2</number><dates><year>2008</year><pub-dates><date>August 1, 2008</date></pub-dates></dates><urls><related-urls><url> and body composition, including prevention and reduction of visceral and liver fat.PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5CamVybW88L0F1dGhvcj48WWVhcj4yMDEyPC9ZZWFyPjxS

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Tm90ZT4A

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Tm90ZT4A

ADDIN EN.CITE.DATA 6 Similarly, in meta-analyses of older, limited clinical trials, increased consumption of LA-rich vegetable oils, especially soybean oil, reduces the risk of CHD.PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5Nb3phZmZhcmlhbjwvQXV0aG9yPjxZZWFyPjIwMTA8L1ll

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ADDIN EN.CITE.DATA 5 Our findings evaluating in vivo levels of LA status across multiple global studies add strong support for cardiovascular benefits of LA. While AA has long been considered an archetypical pro-inflammatory and pro-thrombotic fatty acid, growing evidence suggests its effects may be more complex. ADDIN EN.CITE <EndNote><Cite><Author>Shearer</Author><Year>2018</Year><RecNum>2025</RecNum><DisplayText><style face="superscript">35</style></DisplayText><record><rec-number>2025</rec-number><foreign-keys><key app="EN" db-id="frdaewwftrdav5e02x3xvdrgxe0x0pxpfr2d" timestamp="1541632560">2025</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Shearer, Gregory C.</author><author>Walker, Rachel E.</author></authors></contributors><titles><title>An overview of the biologic effects of omega-6 oxylipins in humans</title><secondary-title>Prostaglandins, Leukotrienes and Essential Fatty Acids</secondary-title></titles><periodical><full-title>Prostaglandins, Leukotrienes and Essential Fatty Acids</full-title></periodical><pages>26-38</pages><volume>137</volume><dates><year>2018</year><pub-dates><date>2018/10/01/</date></pub-dates></dates><isbn>0952-3278</isbn><urls><related-urls><url> In the present investigation, AA levels were not associated with higher risk of CVD, and indeed in some analyses were associated with lower risk. These results do not provide support for adverse cardiovascular effects of AA. While AA is the precursor to potentially pro-inflammatory leukotrienes, it is also the main precursor to key anti-inflammatory metabolites, such as epoxyeicosatrienoic acids and prostaglandin E2, as well as other mediators that actively resolve inflammation, such as lipoxin A4. ADDIN EN.CITE <EndNote><Cite><Author>Shearer</Author><Year>2018</Year><RecNum>2025</RecNum><DisplayText><style face="superscript">35</style></DisplayText><record><rec-number>2025</rec-number><foreign-keys><key app="EN" db-id="frdaewwftrdav5e02x3xvdrgxe0x0pxpfr2d" timestamp="1541632560">2025</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Shearer, Gregory C.</author><author>Walker, Rachel E.</author></authors></contributors><titles><title>An overview of the biologic effects of omega-6 oxylipins in humans</title><secondary-title>Prostaglandins, Leukotrienes and Essential Fatty Acids</secondary-title></titles><periodical><full-title>Prostaglandins, Leukotrienes and Essential Fatty Acids</full-title></periodical><pages>26-38</pages><volume>137</volume><dates><year>2018</year><pub-dates><date>2018/10/01/</date></pub-dates></dates><isbn>0952-3278</isbn><urls><related-urls><url> It also gives rise to prostacyclin, a potent anti-aggregatory and vasodilatory molecule. ADDIN EN.CITE <EndNote><Cite><Author>Mitchell</Author><Year>2018</Year><RecNum>2037</RecNum><DisplayText><style face="superscript">36</style></DisplayText><record><rec-number>2037</rec-number><foreign-keys><key app="EN" db-id="frdaewwftrdav5e02x3xvdrgxe0x0pxpfr2d" timestamp="1542151123">2037</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Mitchell, J. A.</author><author>Kirkby, N. S.</author></authors></contributors><auth-address>Cardiothoracic Pharmacology, National Heart and Lung Institute, London, UK.</auth-address><titles><title>Eicosanoids, prostacyclin and cyclooxygenase in the cardiovascular system</title><secondary-title>Br J Pharmacol</secondary-title><alt-title>British journal of pharmacology</alt-title></titles><periodical><full-title>Br J Pharmacol</full-title><abbr-1>British journal of pharmacology</abbr-1></periodical><alt-periodical><full-title>Br J Pharmacol</full-title><abbr-1>British journal of pharmacology</abbr-1></alt-periodical><edition>2018/02/23</edition><dates><year>2018</year><pub-dates><date>Feb 21</date></pub-dates></dates><isbn>0007-1188</isbn><accession-num>29468666</accession-num><urls></urls><electronic-resource-num>10.1111/bph.14167</electronic-resource-num><remote-database-provider>NLM</remote-database-provider><language>eng</language></record></Cite></EndNote>36 These complex biologic effects preclude simplistic inference on health effects of AA metabolites and further support the importance of empiric assessment of relationships with clinical events, such as in our investigation.Overall, our findings provide little support for the hypothesis that LA or AA, the major n-6 PUFA, may increase CVD risk. We also identified little evidence for any interaction between n-6 and n-3 PUFA levels, consistent with prior reviews of dietary data. ADDIN EN.CITE <EndNote><Cite><Author>Harris</Author><Year>2009</Year><RecNum>1171</RecNum><DisplayText><style face="superscript">1</style></DisplayText><record><rec-number>1171</rec-number><foreign-keys><key app="EN" db-id="frdaewwftrdav5e02x3xvdrgxe0x0pxpfr2d" timestamp="1464775769">1171</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Harris, William S.</author><author>Mozaffarian, Dariush</author><author>Rimm, Eric</author><author>Kris-Etherton, Penny</author><author>Rudel, Lawrence L.</author><author>Appel, Lawrence J.</author><author>Engler, Marguerite M.</author><author>Engler, Mary B.</author><author>Sacks, Frank</author></authors></contributors><titles><title>Omega-6 Fatty Acids and Risk for Cardiovascular Disease: A Science Advisory From the American Heart Association Nutrition Subcommittee of the Council on Nutrition, Physical Activity, and Metabolism; Council on Cardiovascular Nursing; and Council on Epidemiology and Prevention</title><secondary-title>Circulation</secondary-title></titles><periodical><full-title>Circulation</full-title></periodical><pages>902-907</pages><volume>119</volume><number>6</number><dates><year>2009</year><pub-dates><date>February 17</date></pub-dates></dates><urls><related-urls><url> n-6 PUFA may also have additional metabolic benefits. For example, a recent pooled analysis from FORCE identified a strong inverse association of circulating and adipose tissue LA levels and incidence of type 2 diabetes, with no significant associations for AA.PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5XdTwvQXV0aG9yPjxZZWFyPjIwMTc8L1llYXI+PFJlY051

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ADDIN EN.CITE.DATA 25 Taken together with results of randomized controlled feeding trials of blood lipids, glucose-insulin homeostasis, and other metabolic risk factors; prospective cohort studies of self-reported consumption; and (older, methodologically limited) clinical trials of LA-rich plant oils, our novel findings do not support recommendations of somePEVuZE5vdGU+PENpdGU+PEF1dGhvcj5TaW1vcG91bG9zPC9BdXRob3I+PFllYXI+MTk5OTwvWWVh

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ADDIN EN.CITE.DATA 10 to reduce n-6 PUFA consumption or reduce the n-6:n-3 ratio (as opposed to increasing n-3 intake). Rather, the findings from the present study, together with the prior research summarized above, support independent cardioprotective benefits of LA. Our results provide important evidence that helps inform clinical and population recommendations. Dietary guidelines from several organizations, including the American Heart Association, recommend increased consumption of n-6 PUFA to prevent CVD.PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5TYWNrczwvQXV0aG9yPjxZZWFyPjIwMTc8L1llYXI+PFJl

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ADDIN EN.CITE.DATA 9, 10, 37 and other national guidelines ADDIN EN.CITE <EndNote><Cite><Author>Legrand</Author><Year>2013</Year><RecNum>2017</RecNum><DisplayText><style face="superscript">38</style></DisplayText><record><rec-number>2017</rec-number><foreign-keys><key app="EN" db-id="frdaewwftrdav5e02x3xvdrgxe0x0pxpfr2d" timestamp="1540927518">2017</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Legrand, Philippe</author></authors></contributors><titles><title>New French nutritional recommendations for fatty acids</title></titles><dates><year>2013</year></dates><urls></urls></record></Cite></EndNote>38 currently recommend avoidance of n-6 PUFA and reductions from current intake levels. Furthermore, current trends in oil production are leading to increased use of high-oleic, LA-depleted seed oils, ADDIN EN.CITE <EndNote><Cite><Author>Raatz</Author><Year>2018</Year><RecNum>2016</RecNum><DisplayText><style face="superscript">39</style></DisplayText><record><rec-number>2016</rec-number><foreign-keys><key app="EN" db-id="frdaewwftrdav5e02x3xvdrgxe0x0pxpfr2d" timestamp="1540927465">2016</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Raatz, S. K.</author><author>Conrad, Z.</author><author>Jahns, L.</author><author>Belury, M. A.</author><author>Picklo, M. J.</author></authors></contributors><auth-address>USDA-Agricultural Research Service, Grand Forks Human Nutrition Research Center, Grand Forks, ND.&#xD;Department of Food Science and Nutrition, University of Minnesota, St. Paul, MN.&#xD;Program of Human Nutrition, Ohio State University, Columbus, OH.</auth-address><titles><title>Modeled replacement of traditional soybean and canola oil with high-oleic varieties increases monounsaturated fatty acid and reduces both saturated fatty acid and polyunsaturated fatty acid intake in the US adult population</title><secondary-title>Am J Clin Nutr</secondary-title><alt-title>The American journal of clinical nutrition</alt-title></titles><periodical><full-title>The American journal of clinical nutrition</full-title><abbr-1>Am J Clin Nutr</abbr-1></periodical><alt-periodical><full-title>The American journal of clinical nutrition</full-title><abbr-1>Am J Clin Nutr</abbr-1></alt-periodical><edition>2018/08/08</edition><dates><year>2018</year><pub-dates><date>Jul 31</date></pub-dates></dates><isbn>0002-9165</isbn><accession-num>30084912</accession-num><urls></urls><electronic-resource-num>10.1093/ajcn/nqy127</electronic-resource-num><remote-database-provider>NLM</remote-database-provider><language>eng</language></record></Cite></EndNote>39 which can increase the risk of insufficient PUFA consumption in population subgroups. Our findings, combined with prior evidence from metabolic feeding trials, supports cardiovascular benefits of LA and a need to harmonize international guidelines and priorities for oilseed production and use.A unique strength of our investigation was the ability to assess associations across distinct lipid compartments across which LA (AA) levels intercorrelate to varying degrees (e.g., r=0.4-0.9),PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5Ib2Rzb248L0F1dGhvcj48WWVhcj4yMDA4PC9ZZWFyPjxS

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ADDIN EN.CITE.DATA 43 Although located in FADS1, rs174547 is also in strong linkage disequilibrium with polymorphisms in FADS2 (encoding the LA-desaturating FADS2) and has emerged as the main genetic determinant of circulating LA and AA in a recent genome-wide association study.PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5HdWFuPC9BdXRob3I+PFllYXI+MjAxNDwvWWVhcj48UmVj

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ADDIN EN.CITE.DATA 27 The T-allele has been linked to several metabolic traits including higher cholesterol (total, LDL, and HDL)PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5XaWxsZXI8L0F1dGhvcj48WWVhcj4yMDEzPC9ZZWFyPjxS

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ADDIN EN.CITE.DATA eWVhcj4yMDEzPC95ZWFyPjxwdWItZGF0ZXM+PGRhdGU+SnVuPC9kYXRlPjwvcHViLWRhdGVzPjwv

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ADDIN EN.CITE.DATA 46 The pleiotropy of the FADS cluster and the specificity for ischemic stroke rather than all CVD endpoints complicates the interpretation of the observed gene-LA interaction, which should therefore be viewed cautiously. Yet, one could also speculate that carriers of the major T-allele derive greater benefits from the established LDL-lowering effects of dietary LA and thus have accentuated health benefits –a ripe area for further investigation. Few prior meta-analyses of LA and AA levels in CVD have been performed. In one analysis of 10 published studies with 28,000 participants and 3,800 events, LA was not significantly associated with coronary events, while AA was associated with a 17% reduction in risk.PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5DaG93ZGh1cnk8L0F1dGhvcj48WWVhcj4yMDE0PC9ZZWFy

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ADDIN EN.CITE.DATA 18 In a meta-analysis of published studies acute myocardial infarction and coronary syndromes including many retrospective case-control studies, circulating and adipose tissue LA levels were inversely associated with the risk of CHD events, while overall associations for AA were null.PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5IYXJyaXM8L0F1dGhvcj48WWVhcj4yMDA3PC9ZZWFyPjxS

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ADDIN EN.CITE.DATA 17 Our investigation considerably extends these prior results by focusing on prospective studies, performing new individual-level study-specific analyses using a standardized and harmonized analysis protocol, including a much larger number of participants and events, and evaluating several major CVD outcomes. Importantly, our consortium also greatly minimizes publication bias by incorporating new (unpublished) findings from all available studies, rather than pooling only prior published results.Other strengths include use of in vivo n-6 PUFA levels, which complement self-reported dietary estimates, reduce errors from memory, and allow assessment of biologically relevant in vivo levels- especially important for AA. Outcomes in nearly all studies were defined by centralized adjudication processes or validated registries rather than from self-report alone, reducing the potential for missed or misclassified endpoints. Inclusion of cohorts from 13 countries across several continents enhances generalizability. The large numbers of participants and events allowed us to explore several potential effect modifiers and the shape of the associations. Potential limitations deserve attention. For certain compartments, such as adipose tissue, few studies were available. Most individuals were of European descent, lowering statistical power for evaluating other races/ethnicities. Despite extensive efforts to harmonize study-specific methods, some dissimilarities remained between cohorts in outcome definitions (see Expanded Methods in the Supplemental Material) and covariate categorization (Supplemental Table 3). Although such variety and unmeasured background population characteristics may increase generalizability, these may also have contributed to the moderate between-study heterogeneity observed for some exposure-outcome relationships. Fatty acids were measured once at baseline, and changes over time could lead to misclassification, which would attenuate the associations. However, reasonable temporal reproducibility has been reported for LA and AA concentrations over time.PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5Lb3Rzb3BvdWxvczwvQXV0aG9yPjxZZWFyPjIwMTA8L1ll

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ADDIN EN.CITE.DATA 47 Since few studies evaluated multiple compartments, and because cholesterol esters were only assessed by studies from Northern Europe, we were hampered in drawing any conclusions of true predictive differences between lipid fractions. Although fatty acid analytical methods were not standardized across studies, the use of a quintile-based statistical approach minimizes this concern. We did not adjust for non-fatty acid dietary factors, but pooling results across multiple cohorts with different population characteristics increases the validity of the findings. While all studies consistently adjusted for other major CVD risk factors, we cannot exclude residual confounding due to unmeasured or imprecisely measured covariates. However, the concordance of the present observed associations with other lines of evidence on cardiovascular benefits of LAPEVuZE5vdGU+PENpdGU+PEF1dGhvcj5Nb3phZmZhcmlhbjwvQXV0aG9yPjxZZWFyPjIwMTA8L1ll

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ADDIN EN.CITE.DATA 23 suggesting that the observed associations are not likely due to reverse causation. In summary, based on pooled individual-level analyses of prospective studies, circulating and adipose tissue biomarker concentrations of LA were inversely associated with CVD while AA was not associated with higher CVD risk. Together with prior research, these results support CVD benefits of LA. AUTHORSMatti Marklund, PhD; Jason HY Wu, PhD; Fumiaki Imamura, MS, PhD; Liana C. Del Gobbo, PhD; Amanda Fretts, PhD; Janette de Goede, PhD; Peilin Shi, PhD; Nathan Tintle, PhD; Maria Wennberg, PhD; Stella Aslibekyan, PhD; Tzu-An Chen, PhD; Marcia C. de Oliveira Otto, PhD; Yoichiro Hirakawa, MD, PhD; Helle H?jmark Eriksen, MSc; Janine Kr?ger, DrPH; Federica Laguzzi, PhD; Maria Lankinen, PhD; Rachel A. Murphy, PhD; Kiesha Prem, BSc; Cécilia Samieri, PhD; Jyrki Virtanen, PhD; Alexis C. Wood, PhD; Kerry Wong; Wei-Sin Yang; Xia Zhou, MSc; Ana Baylin, MD, DrPH; Jolanda M.A. Boer, PhD; Ingeborg A. Brouwer, PhD; Hannia Campos, PhD; Paulo H. M. Chaves, MD, PhD; Kuo-Liong Chien, MD, PHD; Ulf de Faire, MD, PhD; Luc Djoussé, MD, MPH, DSc; Gudny Eiriksdottir, MSc; Naglaa El-Abbadi, MPH; Nita G. Forouhi, FFPHM; Michael J. Gaziano, MD, MPH; Johanna M. Geleijnse, PhD; Bruna Gigante, MD, PhD; Graham Giles, PhD; Eliseo Guallar, MD, PhD; Vilmundur Gudnason, MD; Tamara Harris, MD, MS; William S. Harris, PhD; Catherine Helmer, MD, PhD; Mai-Lis Hellenius, MD, PhD; Allison Hodge, PhD; Frank B. Hu, MD,PHD; Paul F. Jacques, ScD; Jan-H?kan Jansson, MD, PhD; Anya Kalsbeek; Kay-Tee Khaw, FRCP, FFPHM; Woon-Puay Koh, PhD; Markku Laakso, MD; Karin Leander, PhD; Hung-Ju Lin, MD; Lars Lind, MD, PhD; Robert Luben, PhD; Juhua Luo, PhD; Barbara McKnight, PhD; Jaakko Mursu, PhD; Toshiharu Ninomiya, MD, PhD; Kim Overvad, PhD; Bruce M. Psaty, MD, PhD; Eric Rimm, ScD; Matthias B. Schulze, DrPH; David Siscovick, MD, MPH; Michael Skjelbo Nielsen, PhD; Albert V. Smith, PhD; Brian T. Steffen, PhD; Lyn Steffen, PhD; Qi Sun, MD, ScD; Johan Sundstr?m, MD, PhD; Michael Y. Tsai, PhD; Hugh Tunstall-Pedoe, MD; Matti I. J. Uusitupa, MD, PhD; Rob M. van Dam, PhD; Jenna Veenstra; W.M. Monique Verschuren, PhD; Nick Wareham, FRCP, PhD; Walter Willett, MD, DrPH; Mark Woodward,, PhD; Jian-Min Yuan, MD, PhD; Renata Micha, PhD; Rozenn N Lemaitre*, PhD, MPH; Dariush Mozaffarian*, MD, DrPH; Ulf Risérus*, MMed, PhD; for the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) Fatty Acids and Outcomes Research Consortium (FORCE).*These authors contributed equally to this workSOURCES OF FUNDINGFunding for the Fatty acids & Outcomes Research Consortium (FORCE): Cohort specific funding is outlined in Supplemental Table 14. Unilever provided Tufts University with a restricted grant (‘Epidemiological research on circulating polyunsaturated fatty acids in relation to cardiometabolic health within the CHARGE-consortium’) to partly support this analysis. Unilever had no role in study design, study conduct, data analysis, manuscript preparation, or decision to submit. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.DISCLOSURESDrs. Wu and Micha report research support from Unilever for this work. Dr. Mozaffarian reports research funding from the National Institutes of Health and the Gates Foundation; personal fees from GOED, DSM, Nutrition Impact, Pollock Communications, Bunge, Indigo Agriculture, Amarin, Acasti Pharma, and America’s Test Kitchen; scientific advisory board, Elysium Health (with stock options), Omada Health, and DayTwo; and chapter royalties from UpToDate; all outside the submitted work. Dr. Psaty serves on the DSMB of a clinical trial funded by the manufacturer (Zoll LifeCor) and on the Steering Committee of the Yale Open Data Access Project funded by Johnson & Johnson. No other conflicts were reported.AUTHOR AFFILIATIONS1Department of Public Health and Caring Sciences, Clinical Nutrition and Metabolism, Uppsala University, Sweden (M.M, U.R.); The George Institute for Global Health and the Faculty of Medicine, University of New South Wales, Sydney, Australia (M.M., J.HY.W., M.Wo.); 3Medical Research Council Epidemiology Unit, University of Cambridge, United Kingdom (F.I., N.G.F., N.W); Department of Medicine, Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford, California, United States (L.C.DG); Cardiovascular Health Research Unit, Department of Medicine, University of Washington, Seattle, Washington, United States (A.F., R.N.L.); Division of Human Nutrition, Wageningen University, Wageningen, Netherlands (J.dG., J.M.G); Friedman School of Nutrition Science and Policy, Tufts University, Boston, Massachusetts, United States (P.S., R.M., D.M.); Department of Mathematics and Statistics, Dordt College, Sioux Centre, Iowa, United States (N.T., A.K., J.Ve.); Department of Public Health and Clinical Medicine, Nutritional Research, Ume? University, Ume?, Sweden (M.We.); Department of Epidemiology, University of Alabama at Birmingham, Birmingham, Alabama, United States (S.A.); USDA/ARS Children’s Nutrition Research Center, Baylor College of Medicine, Houston, Texas, USA (T-A.C., A.C.W.); Division of Epidemiology, Human Genetics and Environmental Sciences, the University of Texas Health Science Center, School of Public Health; Houston, Texas, United States (M.C.dO.O); Department of Medicine and Clinical Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan (Y.H.); Unit of Epidemiology and Biostatistics, Aalborg University Hospital, Aalborg, Denmark (H.H.E.);15Department of Molecular Epidemiology, German Institute of Human Nutrition Potsdam-Rehbruecke, Nuthetal, Germany (J.K., M.B.S.); 16Unit of Cardiovascular Epidemiology, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden (F.L., U.dF.,B.G., K.L.); Institute of Public Health and Clinical Nutrition, University of Eastern Finland, Kuopio, Finland (M.Lan., J.Vi., J.M., M.I.J.U.); 18University of British Columbia, Vancouver, British Columbia, Canada (R.A.M.); Saw Swee Hock School of Public Health, National University of Singapore, Singapore (K.P., W-P.K., R.M.vD.);20Univ. Bordeaux, Inserm, Bordeaux Population Health Research Center, TUMR 1219, Bordeaux, France (C.S., C.H.); 21Centre for Epidemiology and Biostatistics, The University of Melbourne, Australia (K.W., G.G., A.H.);22Institute of Epidemiology and Preventive Medicine, College of Public Health, National Taiwan University, Taipei, Taiwan (W-S.Y., K-L.C.);23Division of Epidemiology and Community Health, School of Public Health, University of Minnesota, Minneapolis, Minnesota, United States (X.Z., L.S.);24Departments of Nutritional Sciences and Epidemiology, School of Public Health, University of Michigan, Ann Arbor, Michigan, United States (A.B.);25Centre for Nutrition, Prevention and Health Services, National Institute of Public Health and the Environment, Bilthoven, Netherlands (J.M.A.B., W.M.M.V.); 26Health Sciences, Vrije Universiteit, Amsterdam, Netherlands (I.A.B.); 27Department of Nutrition, Harvard T.H. Chan School of Public Health, Boston, Massachusetts, United States (H.C., F.B.H., E.R., Q.S., W.W.); 28Benjamin Leon for Geriatrics Research and Education, Herbert Wertheim College of Medicine, Florida International University, Miami, Florida, United States (P.H.M.C.);29Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan (H-J.L., K-L.C.); 30Brigham and Women’s Hospital, Boston Veterans Affairs Healthcare System, Massachusetts, USA (L.D); 31Icelandic Heart Association, Kópavogur, Iceland; and Faculty of Medicine, University of Iceland, Reykjavik, Iceland (A.V.S., G.E., V.G.); 32USDA Jean Mayer Human Nutrition Research Center Boston, Massachusetts, USA (N.E.A., P.F.J); 33Division of Environmental Epidemiology, Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health; Baltimore, Maryland, United States (M.J.G., E.G.); 34National Institute on Aging, Bethesda, Maryland, United States (T.H.); 35Department of Internal Medicine, Sanford School of Medicine, University of South Dakota, Sioux Fall, South Dakota, United States (W.S.H.); 36OmegaQuant Analytics, LLC, Sioux Falls, South Dakota, United States (W.S.H.); 37Department of Medicine, Cardiology Unit, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden (M.L.H.); 38Department of Epidemiology, Harvard T.H. Chan School of Public Health, Boston, Massachusetts, United States (E.R., F.B.H., W.W.); 39Division of Network Medicine, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts, United States (E.R., F.B.H., Q.S., W.W.); 40Department of Public Health and Clinical Medicine, Research Unit Skellefte?, Ume? University, Ume?, Sweden (J.H.J);41Department of Public Health and Primary Care, University of Cambridge School of Clinical Medicine, Cambridge, United Kingdom (K.T.K., R.L.); 42Duke-NUS Medical School, Singapore (W-P.K); 43Institute of Clinical Medicine, Internal medicine, University of Eastern Finland, Kuopio, Finland (M.Laa.);44Department of Medical Sciences, Uppsala University, Sweden (J.S., L.L.); 45Department of Epidemiology and Biostatistics, Indiana University, Bloomington, Indiana, United States (J.L); 46Department of Biostatistics, School of Public Health, University of Washington, Seattle, Washington, United States (B.M.K); 47Department of Epidemiology and Public Health, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan (T.N.); 48Department of Public Health, Section for Epidemiology, Aarhus University, Aarhus, Denmark (K.O.); 49Department of Cardiology, Aalborg University Hospital, Aalborg, Denmark (K.O., M.S.N); 50Cardiovascular Health Study, Departments of Medicine, Epidemiology, and Health Services, University of Washington, Seattle, Washington, United States (B.M.P); 51Kaiser Permanente Washington Health Research Institute, Seattle, Washington, United States (B.M.P); 52The New York Academy of Medicine, New York, United States (D.S.); 53Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, Minnesota, United States (B.T.S., M.Y.T.); 54Cardiovascular Epidemiology Unit, Institute of Cardiovascular Research, University of Dundee, Dundee, United Kingdom (H.T-P); 55Julius Center for Health Sciences and Primary Care, University Medical Center Utrecht, Utrecht, Netherlands (W.M.M.V.); 56Division of Cancer Control and Population Sciences, UPMC Hillman Cancer, and Department of Epidemiology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania, United States (J.M.Y.). 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Identification of heart rate-associated loci and their effects on cardiac conduction and rhythm disorders. Nature Genetics. 2013;45:621-+.47.Kotsopoulos J, Tworoger SS, Campos H, Chung FL, Clevenger CV, Franke AA, Mantzoros CS, Ricchiuti V, Willett WC, Hankinson SE and Eliassen AH. Reproducibility of plasma and urine biomarkers among premenopausal and postmenopausal women from the Nurses' Health Studies. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology. 2010;19:938-46.Figure 1.Figure 1. Concentration of A) linoleic acid (LA; 18:2n6) and B) arachidonic acid (AA; 20:4n6) across different biomarker compartments measured in the 31 contributing studies. Fatty acid (FA) concentrations are expressed as % of total FA, and indicated as median (circles) and interquintile range (lines; defined as the range between the midpoint of the bottom quintile [10th percentile] and the top quintile [90th percentile]), respectively. For MPCDRF and the MORGEN, values are only shown for controls.*Total number of individual fatty acids measured in the biomarker compartmentFigure 2.Figure 2. Associations of linoleic acid (LA; 18:2n6) with total CVD (A) and CVD mortality (B) in pooled analysis of 30 prospective studies. Study-specific estimates for hazard ratio (HR) per interquintile range (i.e., range between the midpoint of the bottom quintile [10th percentile] and the top quintile [90th percentile]) of biomarker linoleic acid were pooled based on the following order: 1) adipose tissue, 2) erythrocyte phospholipid, 3) plasma phospholipid 4) cholesterol ester, and 5) total plasma. Study weights are indicated (grey squares) by individual biomarker compartment and overall. Study-specific analyses were conducted using models that included the following covariates: age (linear), sex (male/female), race (binary: Caucasian/non-Caucasian, or study-specific), field or clinical center if applicable (study-specific categories), body-mass index (BMI, linear), education (less than high school graduate, high school graduate, some college or vocational school, college graduate), smoking (current, former, or never; if former not assessed, then current or not current), physical activity (quintiles of metabolic equivalents (METs) per week; or if METs unavailable, quintiles of study-specific definitions of physical or leisure activity), alcohol intake (none, 1-6 drinks/week, 1-2 drink/day, >2 drink/day [14 g alcohol=1 standard drink]), diabetes mellitus (yes or no; defined as treatment with oral hypoglycemic agents, insulin, or fasting plasma glucose >126 mg/dL), treated hypertension (yes or no; defined as hypertension drug use; or if unavailable, as diagnosed/history of hypertension according to study-specific definitions), treated hypercholesterolemia (yes or no; defined as lipid-lowering drug use; if unavailable, as diagnosed/history of hypercholesterolemia according to study-specific definitions), regular aspirin use (yes or no), biomarker concentrations of α-linolenic acid (ALA; 18:3n-3), eicosapentaenoic acid (EPA; 20:5n-3), sum of trans-18:1 fatty acids, and sum of trans-18:2 fatty acids (all linear; expressed as % total fatty acids). See Table 1 footnote for abbreviations of cohorts.Figure 3.Figure 3. Associations of linoleic acid (LA; 18:2n6) with total CHD (A) and ischemic stroke (B) in pooled analysis of 30 prospective studies. Study-specific estimates for hazard ratio (HR) per interquintile range (i.e., range between the midpoint of the bottom quintile [10th percentile] and the top quintile [90th percentile]) of biomarker linoleic acid were pooled based on the following order: 1) adipose tissue, 2) erythrocyte phospholipid, 3) plasma phospholipid 4) cholesterol ester, and 5) total plasma. Study weights are indicated (grey squares) by individual biomarker compartment and overall. Study-specific analyses were conducted using models that included the following covariates: age (linear), sex (male/female), race (binary: Caucasian/non-Caucasian, or study-specific), field or clinical center if applicable (study-specific categories), body-mass index (BMI, linear), education (less than high school graduate, high school graduate, some college or vocational school, college graduate), smoking (current, former, or never; if former not assessed, then current or not current), physical activity (quintiles of metabolic equivalents (METs) per week; or if METs unavailable, quintiles of study-specific definitions of physical or leisure activity), alcohol intake (none, 1-6 drinks/week, 1-2 drink/day, >2 drink/day [14 g alcohol=1 standard drink]), diabetes mellitus (yes or no; defined as treatment with oral hypoglycemic agents, insulin, or fasting plasma glucose >126 mg/dL), treated hypertension (yes or no; defined as hypertension drug use; or if unavailable, as diagnosed/history of hypertension according to study-specific definitions), treated hypercholesterolemia (yes or no; defined as lipid-lowering drug use; if unavailable, as diagnosed/history of hypercholesterolemia according to study-specific definitions), regular aspirin use (yes or no), biomarker concentrations of α-linolenic acid (ALA; 18:3n-3), eicosapentaenoic acid (EPA; 20:5n-3), sum of trans-18:1 fatty acids, and sum of trans-18:2 fatty acids (all linear; expressed as % total fatty acids). See Table 1 footnote for abbreviations of cohorts.Figure 4.Figure 4. Associations of arachidonic acid (AA; 20:4n6) with total CVD (A) and CVD mortality (B) in pooled analysis of 30 prospective studies. Study-specific estimates for hazard ratio (HR) per interquintile range (i.e., range between the midpoint of the bottom quintile [10th percentile] and the top quintile [90th percentile]) of biomarker linoleic acid were pooled based on the following order: 1) adipose tissue, 2) erythrocyte phospholipid, 3) plasma phospholipid 4) cholesterol ester, and 5) total plasma. Study weights are indicated (grey squares) by individual biomarker compartment and overall. Study-specific analyses were conducted using models that included the following covariates: age (linear), sex (male/female), race (binary: Caucasian/non-Caucasian, or study-specific), field or clinical center if applicable (study-specific categories), body-mass index (BMI, linear), education (less than high school graduate, high school graduate, some college or vocational school, college graduate), smoking (current, former, or never; if former not assessed, then current or not current), physical activity (quintiles of metabolic equivalents (METs) per week; or if METs unavailable, quintiles of study-specific definitions of physical or leisure activity), alcohol intake (none, 1-6 drinks/week, 1-2 drink/day, >2 drink/day [14 g alcohol=1 standard drink]), diabetes mellitus (yes or no; defined as treatment with oral hypoglycemic agents, insulin, or fasting plasma glucose >126 mg/dL), treated hypertension (yes or no; defined as hypertension drug use; or if unavailable, as diagnosed/history of hypertension according to study-specific definitions), treated hypercholesterolemia (yes or no; defined as lipid-lowering drug use; if unavailable, as diagnosed/history of hypercholesterolemia according to study-specific definitions), regular aspirin use (yes or no), biomarker concentrations of α-linolenic acid (ALA; 18:3n-3), eicosapentaenoic acid (EPA; 20:5n-3), sum of trans-18:1 fatty acids, and sum of trans-18:2 fatty acids (all linear; expressed as % total fatty acids). See Table 1 footnote for abbreviations of cohorts.Figure 5.Figure 5. Associations of arachidonic acid (AA; 20:4n6) with total CHD (A) and ischemic stroke (B) in pooled analysis of 30 prospective studies. Study-specific estimates for hazard ratio (HR) per interquintile range (i.e., range between the midpoint of the bottom quintile [10th percentile] and the top quintile [90th percentile]) of biomarker linoleic acid were pooled based on the following order: 1) adipose tissue, 2) erythrocyte phospholipid, 3) plasma phospholipid 4) cholesterol ester, and 5) total plasma. Study weights are indicated (grey squares) by individual biomarker compartment and overall. Study-specific analyses were conducted using models that included the following covariates: age (linear), sex (male/female), race (binary: Caucasian/non-Caucasian, or study-specific), field or clinical center if applicable (study-specific categories), body-mass index (BMI, linear), education (less than high school graduate, high school graduate, some college or vocational school, college graduate), smoking (current, former, or never; if former not assessed, then current or not current), physical activity (quintiles of metabolic equivalents (METs) per week; or if METs unavailable, quintiles of study-specific definitions of physical or leisure activity), alcohol intake (none, 1-6 drinks/week, 1-2 drink/day, >2 drink/day [14 g alcohol=1 standard drink]), diabetes mellitus (yes or no; defined as treatment with oral hypoglycemic agents, insulin, or fasting plasma glucose >126 mg/dL), treated hypertension (yes or no; defined as hypertension drug use; or if unavailable, as diagnosed/history of hypertension according to study-specific definitions), treated hypercholesterolemia (yes or no; defined as lipid-lowering drug use; if unavailable, as diagnosed/history of hypercholesterolemia according to study-specific definitions), regular aspirin use (yes or no), biomarker concentrations of α-linolenic acid (ALA; 18:3n-3), eicosapentaenoic acid (EPA; 20:5n-3), sum of trans-18:1 fatty acids, and sum of trans-18:2 fatty acids (all linear; expressed as % total fatty acids). See Table 1 footnote for abbreviations of cohorts.Table 1. Characteristics of 31 studies and baseline characteristics of individual study participants with linoleic acid (LA; 18:2n6) and arachidonic acid (AA; 20:4n6) biomarker measures and follow-up for cardiovascular disease incidence or mortality.Study*CountryStudy design?Age, y (mean)Sex (% male)BMI, kg/m2 (mean)Biomarker compartment?Year of biomarker samplingOutcome assessed§AGES-ReykjavikIcelandPC773927.1PP2002-2006All||ARICUSAPC545227.0PP1987-1989AllCCCCTaiwanPC615523.3TP1992-2000AllCHSUSAPC733626.7PP1992-1993AllCRSCosta RicaRCC587326.2AT1994-2004Non-fatal MIDCHDenmarkPNC576126.6AT#1993-1997Total CHDEPIC-NorfolkUKPCC634926.5PP1993-1997AllEPIC-PotsdamGermanyPC503726.0RBC1994-1998Total CVDFHSUSAPC664328.2RBC2005-2008AllHPFSUSAPCC6510025.8RBC, TP1993-1995Total CVD, CHD, & strokeHSJapanPC614223.1TP2002-2003AllKIHDFinlandPC5210026.7TP1984-1989AllMCCSAustraliaPC564627.2PP1990-1994Fatal CVD, CHD, & ischemic strokeMESAUSAPC624728.3PP2000-2002AllMETSIMFinlandPC5510026.5CE, PP, RBC2006-2010Total CVDMORGEN (CHD)NetherlandsPCC527926.2CE1993-1997Fatal CHDMORGEN (Stroke)NetherlandsPCC505325.9CE1993-1997Ischemic strokeMPCDRFNetherlandsPCC517025.9CE1987-1991Fatal CHDNHSUSAPCC60025.6RBC, TP1989-1990Total CVD, CHD & strokeNSHDS ISwedenPCC547926.2PP1987-1994Total CHDNSHDS IISwedenPCC547626.4PP1987-1999Total CHDNSHDS IIISwedenPCC556126.7PP1987-1995Ischemic strokePHSUSAPCC6910025.7RBC1995-2001Total CHDPIVUSSwedenPC704726.9CE, PP2001-2004AllSCHSSingaporePCC666523.0TP1994-2005Total CHDSHHECUKPC495225.6AT1985-1986All60YOSwedenPC604826.8CE1997-1998All3C StudyFrancePC753926.0TP1999-2000AllULSAM-50**SwedenPC5010025.0CE1970-1973AllULSAM-70**SwedenPC7110026.4AT1991-1995AllWHIMSUSAPC70028.2RBC1996All*AGES-Reykjavik: Age, gene/environment susceptibility – Reykjavik Study; ARIC: Atherosclerosis Risk in Communities; CCCC: Chin-Shan Community Cardiovascular Cohort Study; CHS: Cardiovascular Health Study; CRS: Costa Rica study on adults; DCH: Diet, Cancer, and Health study; EPIC: European Prospective Investigation into Cancer; FHS: Framingham Heart Study; HPFS: Health Professionals Follow-up Study; HS: The Hisayama Study; KIHD: Kuopio Ischaemic Heart Disease Risk Factor Study; MCCS: Melbourne Collaborative Cohort Study; MESA: Multi-Ethnic Study of Atherosclerosis; METSIM: Metabolic syndrome in men study; MORGEN: Monitoring Project on Risk Factors for Chronic Diseases; MPCDRF: Monitoring Project on Cardiovascular Disease Risk Factors; NHS I: Nurses’ Health Study I; NSHDS I-III: Northern Sweden Health and Disease Study; PHS: Physicians’ Health Study; PIVUS: Prospective Investigation of the Vasculature in Uppsala Seniors; SCHS, Singapore Chinese Health Study; SHHEC, Scottish Heart Health Extended Cohort; 60YO, 60-year-old Swedish men and women; 3C Study: Three City Study; ULSAM-50 &-70: Uppsala Longitudinal Study of Adult Men investigations at ages 50 y and 70 y, respectively. ?PC, prospective cohort; PCC, prospective nested case-control; PNC, prospective nested case-cohort; RCC, retrospective case-control. ?AT, adipose tissue; CE, cholesterol ester; PP, plasma phospholipid; RBC, erythrocyte phospholipid; TP, total plasma. §CVD, cardiovascular disease; CHD, coronary heart disease; MI, myocardial infarction. ||All specified outcomes (total CVD, CVD mortality, total CHD, and ischemic stroke) were assessed. #In DCH, the association of adipose tissue arachidonic acid, but not linoleic acid, with total CHD was evaluated. **Fatty acids were measured in cholesterol ester and adipose tissue at the first and third ULSAM investigation, respectively. Table 2. Risk of incident CVD according to objective biomarker levels of linoleic acid (18:2n6) and arachidonic acid (20:4n6) in 30 pooled prospective cohort studies.Multivariable-adjusted hazard ratio (95% CI)per interquintile range*OutcomeBiomarkerStudies (n)Cases (n)Linoleic acidArachidonic acidTotal CVDPhospholipid146 8531.00 (0.92-1.09)0.95 (0.87-1.03)Total plasma62 7420.90 (0.78-1.03)0.81 (0.70-0.94)Cholesterol esters41 3000.74 (0.63-0.88)1.03 (0.88-1.20)Adipose tissue21 4120.87 (0.75-1.01)0.98 (0.87-1.10)Overall?2110 4770.93 (0.88-0.99)0.95 (0.90-1.01)CVD mortalityPhospholipid93 0570.89 (0.79-1.00)0.93 (0.83-1.05)Total plasma46790.66 (0.50-0.86)0.85 (0.66-1.09)Cholesterol esters34730.56 (0.43-0.73)0.99 (0.76-1.29)Adipose tissue24180.60 (0.44-0.82)1.02 (0.84-1.23)Overall?174 5080.78 (0.70-0.85)0.94 (0.86-1.02)Total CHDPhospholipid146 0751.01 (0.93-1.10)0.96 (0.90-1.03)Total plasma72 4300.86 (0.74-1.00)0.86 (0.74-1.01)Cholesterol esters51 1780.78 (0.65-0.94)1.02 (0.85-1.23)Adipose tissue3?3 2550.88 (0.74-1.03)1.10 (0.98-1.23)Overall? 26?11 8570.94 (0.88-1.00)0.99 (0.94-1.04)Ischemic strokePhospholipid122 3270.95 (0.82-1.10)0.98 (0.85-1.13)Total plasma61 1050.84 (0.66-1.06)0.93 (0.73-1.18)Cholesterol esters45980.67 (0.51-0.88)1.13 (0.89-1.43)Adipose tissue24050.87 (0.65-1.15)0.91 (0.74-1.11)Overall?213 7050.88 (0.79-0.98)0.99 (0.90-1.10)*Based on harmonized, de novo individual-elvel analyses in each cohort, pooled using inverse-variance weighted meta-analysis. Risk was assessed according to the interquintile range (i.e., range between the midpoint of the bottom quintile [10th percentile] and the top quintile [90th percentile]) of each fatty acid, corresponding to the difference between the midpoint of the first and fifth quintiless. Study-specific analyses were adjusted for age (years), sex (male/female), race (Caucasian/non-Caucasian, or study-specific), field or clinical center if applicable (study-specific categories), body-mass index (BMI, kg/m2), education (less than high school graduate, high school graduate, some college or vocational school, college graduate), smoking (current, former, or never; if former not assessed, then current or not current), physical activity (quintiles of metabolic equivalents (METs) per week; or if METs unavailable, quintiles of study-specific definitions of physical or leisure activity), alcohol intake (none, 1-6 drinks/week, 1-2 drink/day, >2 drink/day [14 g alcohol=1 standard drink]), diabetes mellitus (yes/no; defined as treatment with oral hypoglycemic agents, insulin, or fasting plasma glucose >126 mg/dL), treated hypertension (yes/no; defined as hypertension drug use; or if unavailable, as diagnosed/history of hypertension according to study-specific definitions), treated hypercholesterolemia (yes or no; defined as lipid-lowering drug use; if unavailable, as diagnosed/history of hypercholesterolemia according to study-specific definitions), regular aspirin use (yes/no), biomarker concentrations of α-linolenic acid (ALA; 18:3n-3), eicosapentaenoic acid (EPA; 20:5n-3), sum of trans-18:1 fatty acids, and sum of trans-18:2 fatty acids (each expressed as % total fatty acids). ?For studies that assessed LA and AA levels in more than one biomarker compartment, the primary compartment for that study was pre-selected for pooled analyses based on the following order: 1) adipose tissue, 2) erythrocyte phospholipid, 3) plasma phospholipid 4) cholesterol ester, and 5) total plasma.?Because the Diet, Cancer and Health study assessed associations of AA, but not LA, with total CHD (n cases=2138), a total of, 2 studies (n cases= 1117) evaluated adipose tissue LA and 25 studies (n cases=9719) assessed any biomarker level of LA in relation to total CHD. ................
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