Association of dietary, circulating, and supplement fatty acids with coronary risk

Rajiv Chowdhury1, MD, PhD; Samantha Warnakula1‡, MPhil; Setor Kunutsor1‡, MD, MSt; Francesca
Crowe2, PhD; Heather A Ward3, PhD; Laura Johnson4, PhD; Oscar H Franco5, MD, PhD; Adam
Butterworth1, PhD; Nita G Forouhi6, PhD; Simon G Thompson1, FMedSci; Kay-Tee Khaw1, FMedSci;
Dariush Mozaffarian7, MD, DrPH; John Danesh1*, FRCP; Emanuele Di Angelantonio1*, MD, MSc, PhD.

1
Department of Public Health and Primary Care, University of Cambridge, Cambridge, England;
2
Cancer Epidemiology Unit, Nuffield Department of Population Health, University of Oxford, Oxford
England;
3
Department of Epidemiology and Biostatistics, School of Public Health, Imperial College London,
London, England;
4
Centre for Exercise, Nutrition and Health Sciences, University of Bristol, Bristol, England;
5
Department of Epidemiology, Erasmus MC, University Medical Center, Rotterdam, The Netherlands;
6
UK Medical Research Council Epidemiology Unit, Cambridge, England;
7
Department of Epidemiology, Harvard School of Public Health, Boston, Massachusetts, United
States.

‡Denotes equal contribution.

*Denotes equal contribution.

Text word count: 4129 (excluding abstract)

3 main figures, 1 main table

26 supplementary figures and tables, and 6 appendices

Correspondence: Dr Rajiv Chowdhury

Department of Public Health & Primary Care
University of Cambridge
Strangeways Research Laboratory
Cambridge, CB1 8RN, England
Email:rc436@medschl.cam.ac.uk
Tel: +44 1223748675; Fax: +44 1223 741339

1
ABSTRACT

Background Guidelines advocate changes in fatty acid consumption to promote cardiovascular

health.

Purpose To summarize evidence about associations between fatty acids and coronary disease.

Data Sources MEDLINE, Science Citation Index, and Cochrane Central Register of Controlled Trials
through July 2013.

Study Selection Prospective, observational studies and randomized, controlled trials.

Data Extraction Investigators extracted data about study characteristics and assessed study
biases.

Data synthesis There were 32 observational studies (512420 participants) of fatty acids from
dietary intake; 17 observational studies (25721 participants) of fatty acid biomarkers; and 27
randomized, controlled trials (105085 participants) of fatty acid supplementation. In observational
studies, relative risks for coronary disease were 1.03 (95%CI, 0.98 to 1.07) for saturated, 1.00 (CI,
0.91 to 1.10) for monounsaturated, 0.87 (CI, 0.78 to 0.97) for long-chain omega-3
polyunsaturated, 0.98 (CI, 0.90 to 1.06) for omega-6 polyunsaturated, and 1.16 (CI, 1.06 to 1.27)
for trans fatty acids when the top and bottom thirds of baseline dietary fatty acid intake were
compared. Corresponding estimates for circulating fatty acids were 1.06 (CI, 0.86 to 1.30), 1.06
(CI, 0.97 to 1.17), 0.84 (CI, 0.63 to 1.11), 0.94 (CI, 0.84 to 1.06), and 1.05 (CI, 0.76 to 1.44),
respectively. There was heterogeneity of the associations among individual circulating fatty acids
and coronary disease. In randomized, controlled trials, relative risks for coronary disease were 0.97
(CI, 0.69 to 1.36) for alpha-linolenic, 0.94 (CI, 0.86 to 1.03) for long-chain omega-3
polyunsaturated, and 0.86 (CI, 0.69 to 1.07) for omega-6 polyunsaturated fatty acid
supplementations

Limitations Potential biases from preferential publication and selective reporting.

Conclusion Current evidence does not clearly support cardiovascular guidelines that encourage high
consumption of polyunsaturated fatty acids and low consumption of total saturated fats.

Primary funding British Heart Foundation, UK Medical Research Council, Cambridge NIHR
Biomedical Research Centre, and Gates Cambridge.

2
INTRODUCTION
Dietary fats mainly comprise triacylglycerols consisting of three individual fatty acids, each linked by
an ester bond to a glycerol backbone (1-2). Based on the number of double bonds they contain,
fatty acids are classified as saturated, monounsaturated, and polyunsaturated. Specific fatty acids
within these categories tend to have different biological effects and physical properties (3). To
promote cardiovascular health (4-5), nutritional guidelines have generally encouraged low
consumption of saturated fats, high consumption of omega-3 polyunsaturated fatty acids from fish
or plant sources, and avoidance of trans fats, particularly those from partially-hydrogenated fat.
There is, however, considerable variation in international guidelines concerning optimum amounts
and types of fatty acids consumption (6-11). This variation reflects, at least in part, uncertainties in
the available evidence. For example, prospective observational studies have questioned whether
there really are associations between saturated fat consumption and cardiovascular disease (12).
However, interpretation has been complicated by potential misclassification in the self-report
questionnaires used to assess fatty acid consumption (13-15), which also lack the ability to compute
intake of specific fatty acids (16). By contrast, fatty acid biomarkers may provide more accurate
assessment of consumption, such as for polyunsaturated fatty acids (17), and/or metabolism, such
as for saturated and monounsaturated fatty acids (17-20). However, earlier analyses have generally
not assessed the consistency between findings from dietary self-report and biomarker measures of
fatty acids in coronary disease. With respect to randomised trials of fatty acid supplements for
preventing coronary disease, interpretation of results has been complicated by the differences in
dietary habits of various trial populations, the absence or presence (and type) of pre-existing
vascular disease at entry, the composition of supplementation regimens, trial duration and power,
and apparent differences in reported efficacy for coronary prevention. Furthermore, previous meta-
analyses of randomised trials were only focused on omega-3 and omega-6 supplementations(21-
22)and did not include more recent and larger trials.

To help clarify the evidence, we have conducted a systematic review and meta-analysis of data from
long-term prospective observational studies of a broad range of both dietary and biomarker fatty
acid measures in coronary disease. To put the observational evidence into context, we have
examined associations with coronary outcomes in the randomised trials of fatty acids
supplementation.

3
METHODS
Data Sources and Searches
This review was conducted using a predefined protocol and in accordance to the MOOSE and PRISMA
guidelines (Appendix 1 and 2). Studies published before 1 July 2013 were identified, without any
language restriction, through electronic searches using MEDLINE, Science Citation Index databases,
Cochrane Central Register of Controlled Trials and supplemented by scanning reference lists of
articles identified for all relevant studies and review articles (including meta-analyses), by hand
searching of relevant journals, and by correspondence with authors of included studies. The
computer-based searches combined search terms related to the exposure (eg, fatty acids,
unsaturated fatty acids etc) and coronary disease (eg, myocardial infarction, atherosclerosis,
coronary heart disease, coronary stenosis) without language restriction (Appendix 3).

Study Selection
Observational and intervention studies were included if they had reported on associations of dietary
fatty acids intake, fatty acids biomarkers (measured in whole blood, serum, plasma, erythrocyte
fraction [ie, circulating fatty acids], or adipose tissue), or fatty acids intervention (dietary or
supplements) with risk of coronary disease (defined as fatal or non-fatal myocardial infarction,
coronary heart disease, coronary insufficiency, coronary death, angina, angiographic coronary
stenosis [where possible sudden cardiac death was not included in the outcome definition]:
Appendix 4 provides study-specific outcomes definition). Observational studies were eligible if they
were prospective in design with at least one year of follow-up and involved participants from
approximately general populations (ie, participants not selected on the basis of pre-existing disease
at baseline) or with stable cardiovascular disease at study entry (defined as a diagnosis made at
least 30 days prior to baseline sampling). Intervention studies were eligible for inclusion if they were
randomised and recorded coronary outcomes as an endpoint of interest.

Data Extraction and Quality Assessment

Data on the following characteristics were extracted independently by two investigators who used
standardised protocols: sample size; study design; sampling population; location (defined as
Europe, North America, and the Asia-Pacific region); year of baseline survey; age range of
participants at baseline; gender; duration of follow-up; numbers of disease outcomes of interest and
reported effect estimates with coronary disease with each marker, degree of statistical adjustment
used, cross-sectional correlation coefficients of dietary fatty acids intake and circulating fatty acids
(where available). Where appropriate, information on sample type (serum, plasma or adipose
tissue), storage temperature, assay methods, dietary assessment tool (diet questionnaire, defined
as food frequency or diet history questionnaires; diet records, defined as all open-ended instruments
such as 24-hour recall and food diaries), type and formulation of intervention, year of
randomisation, allocation concealment, blinding of carers and participants, daily dose of
supplementations, and composition of placebo were abstracted. Discrepancies were resolved by
discussion and by adjudication of a third reviewer. We used the most up-to-date or comprehensive
information in cases of multiple publications. The Newcastle-Ottawa Scale (23) was used for

4
assessing the quality of observational studies. This scale uses a “star” system (with maximum of
nine stars) to assess the quality of a study in three domains: selection of participants; comparability
of study groups; and the ascertainment of outcomes of interest. Studies that received a score of
nine stars were judged to be of high quality; studies that scored seven or eight stars were
considered of medium quality. The Cochrane Collaboration’s tool for assessing risk of bias was used
for assessing the validity of randomised trials (24). This tool assesses seven possible sources of
bias: random sequence generation, allocation concealment, blinding of participants and personnel,
blinding of outcome assessment, incomplete outcome data, selective reporting and other bias. For
each individual domain, studies were classified into low, unclear and high risk of bias.

Data Synthesis and Analysis

Analyses involved only within-study comparisons (ie, cases and controls were only directly compared
within each cohort) to limit potential biases. To enable a consistent approach to meta-analysis and
interpretation of findings in this review, relative risk estimates for association of fatty acids and
coronary disease that were often differently reported by each study (e.g. per unit change, or per 1-
SD change, or comparing quintiles, quartiles, thirds, and other groupings) were transformed to
consistently correspond to comparison of the top vs. bottom third of fatty acid distribution in each
study, using methods previously described (25). Briefly, log risk estimates were transformed
assuming a normal distribution, with the comparison between top and bottom thirds being
equivalent to 2.18 times the log risk ratio (RR) for a 1 standard deviation increase (or equivalently,
as 2.18/2.54 times the log RR for a comparison of extreme quarters). Standard errors of the log RRs
were calculated using published confidence limits and were transformed in the same way (Appendix
5 provides details of the statistical methods used). Where studies reported RRs with differing
degrees of adjustment for other risk factors, the multivariable adjusted estimate that did not include
adjustment for blood lipids and/or circulating fatty acids was used (as circulating lipids may act as
potential mediators for the associations between fatty acids and coronary disease (26). For
randomised intervention trials we used reported RR or calculated study-specific unadjusted RR for
the main outcomes of interest. Hazard ratios and odds ratios were assumed to approximate the
same measure of relative risk. Summary RRs were calculated by pooling the study-specific estimates
using a random-effects model that included between-study heterogeneity (parallel analyses used
fixed-effect models). Correlations of dietary fatty acids intake and circulating fatty acids were
estimated by pooling Spearman’s study-specific correlation coefficients using random effects meta-
analysis. Consistency of findings across individual studies was assessed by standard χ2 tests and the
I2 statistic (27). Heterogeneity between observational cohorts was assessed by comparing results
from studies grouped according to pre-specified study-level characteristics (such as location,
gender, year of baseline survey, duration of follow-up, numbers of outcomes recorded, outcome
definition, degree of statistical adjustment used, assay characteristics and dietary assessment) and
to the Newcastle-Ottawa Scale score using meta-regression A similar method was used to assess
heterogeneity between randomised trials by constructing groups according to pre-specified trial
characteristics (such as type and formulation of intervention, year of randomisation, allocation
concealment, blinding of carers and participants, daily dose of supplementations, and composition of

5
placebo) and to the risk of bias in each individual domain defined by the Cochrane Collaboration’s
tool for assessing risk of bias. Evidence of publication bias across studies was assessed using funnel
plots and Egger test (28). All statistical tests were two-sided and used a significance level of p
<0.05. All analyses were performed using Stata release 11 (StataCorp, College Station, Texas).

Role of the funding sources

The study was conducted and analysed independently from its funders.

6
RESULTS
Overall, seventy-two unique studies were identified (Table 1, eFigure 1, and Appendix 6 provide
study details and references). Nineteen studies were based in North America, 42 studies in Europe,
9 studies in the Asia-Pacific and 2 studies were multinational. Forty-five studies were prospective
observational cohorts and 27 were randomised trials (one trial also reported data as an
observational cohort on blood fatty acids). Forty studies involved initially healthy populations, ten
studies recruited individuals with elevated cardiovascular risk factors, and 22 recruited people with
cardiovascular disease at baseline.

Dietary fatty acids intake and coronary risk

Thirty-two prospective cohort studies reported on self-reported dietary fatty acids intake (512,420
participants, 15,945 incident coronary outcomes, average follow-up ranging from 5 to 23 years:
eTable 1), of which 21 recorded information using diet questionnaires and 11 using diet records. All
studies reported adjustment for at least several non-blood based vascular risk factors (eg, age, sex,
smoking, history of diabetes, and blood pressure). Thirteen studies were judged to be of high quality
and 19 of medium quality, while no studies were rated as low quality (eTable 2). Of the medium
quality studies, all showed a potential bias in the participant selection and 6 further lacked objective
confirmation of self-reported dietary intake of fatty acids by structured face-to-face interview. RRs
for coronary disease, comparing participants in the top third versus those in the bottom third of
dietary fatty acids, are presented in Figure 1. In these studies the pooled RRs (95%CIs) were: 1.03
(95%CI, 0.98 to 1.07) for total saturated fatty acids, 1.00 (CI, 0.91 to 1.10) for total
monounsaturated fatty acids, and 1.16 (1.06-1.27) for total trans fatty acids (Figure 1 and eFigure
2). Corresponding RRs for total dietary polyunsaturated fatty acids intake were 0.99 (0.86-1.14) for
total alpha-linolenic acid, 0.87 (CI, 0.78 to 0.97) for total long-chain omega-3 polyunsaturated fatty
acids, and 0.98 (CI, 0.90 to 1.06) for total omega-6 polyunsaturated fatty acids (Figure 1 and
eFigure 3). In studies of dietary fatty acid intake, there was some evidence of heterogeneity
between studies according to number of events recorded (p=0.009 for saturated and p=0.006 for
monounsaturated fatty acids) and geographical location (p=0.020 for long-chain omega-3
polyunsaturated fatty acid studies; eFigure 4). There was no material difference in the combined
RRs according to gender, year of baseline survey, dietary assessment tool, duration of follow-up,
outcome definition or degrees of statistical adjustment (eFigure 4).

Fatty acids biomarkers and coronary risk

Information on fatty acids biomarkers was available from 19 prospective studies (eTables 3 and 4).
Seventeen reported on circulating fatty acids composition (25,721 participants, 5,519 incident
coronary outcomes, mean follow-up ranged from 1.3 to 30.7 years: eTable 3), and 2 on adipose
tissue fatty acid composition (6,586 participants, 1,663 incident coronary events: eTable 4). Of
those reporting on circulating fatty acids composition, 14 used liquid chromatography, 2 used
calorimetric, and one used an enzymatic method to measure fatty acids. Six studies were judged to
be of high quality, 9 of medium quality, and 2 were rated as low quality (eTable 5 ). Of the medium
quality studies, 8 showed potential bias in participant selection and one did not control for any

7
potential risk factor in their analyses. The 2 low quality studies included participants drawn from
selected populations and also did not control for potential covariates in their analyses. All studies
reported adjustment for standard non-blood based vascular risk factors (eg, age, sex, smoking,
history of diabetes, and blood pressure). Studies tended to report on a variable number of individual
fatty acid isomers (eTable 6). The mean proportion of each individual circulating fatty acid relative
to the total is presented in eFigure 5. Among studies with available data, there were moderate
positive correlations between dietary intake and circulating composition of total omega-3 and
omega-6 polyunsaturated fatty acids, and weak positive correlations for total saturated and
monounsaturated fatty acids (eTable 7). RRs for coronary outcomes, typically adjusted for non-
blood based vascular risk factors, comparing top third versus bottom third of composite and
individual circulating fatty acid composition at baseline are presented in Figures 2 and eFigures 6-
11. For the circulating total fatty acids composition, combined RRs were: 1.06 (0.86-1.30) for total
saturated fatty acids, 1.06 (0.97-1.17) for total monounsaturated fatty acids, 0.93 (0.83-1.03) for
alpha-linolenic acid, 0.84 (0.63-1.11) for total long-chain omega-3 polyunsaturated fatty acids, 0.94
(0.84-1.06) for total omega-6 polyunsaturated fatty acids, and 1.05 (0.76-1.44) for total trans fatty
acids. Among individual saturated and monounsaturated fatty acids, RRs for palmitic, stearic and
oleic acids were 1.15 (0.96-1.37), 1.23 (0.93-1.61) and 1.09 (0.97-1.23). By contrast, margaric
acid was significantly associated with lower risk (0.77 [0.63-0.93]; Figure 2 and eFigures 6-7).
Among specific polyunsaturated fatty acids, eicosapentaenoic (0.78 [0.65-0.94]), docosahexaenoic
(0.79 [0.67-0.93]), and arachidonic (0.83 [0.74-0.92]) acids were associated with lower risk; and
dihomo-gamma linolenic (1.11 [0.93-1.33]), eicosadienoic (1.18 [0.93-1.50]), and
docosatetrahexanoic (1.20 [0.99-1.45]) acids tended towards a positive, albeit nonsignificant,
association with coronary disease (Figure 2, eFigures 8-10). Only two studies with a total of <500
cases reported on individual circulating trans fatty acids composition (eFigure 11). For circulating
total saturated fatty acid, there were some evidence of heterogeneity between studies according to
outcome definition (fatal vs non-fatal) and duration of follow-up for (p=0.003 for both). For
circulating eicosapentaenoic+docosahexaenoic fatty acids composition, there was some evidence of
heterogeneity between studies according to outcome definition (fatal vs non-fatal, p=0.004),
duration of follow-up for (p<0.001), number of events recorded (p<0.001), gender (p=0.014) and
fasting or non-fasting sampling state (p=0.037; eFigure 12). There was no material difference in
the combined RRs according to year of baseline survey, population baseline risk, geographical
location, assay characteristics (such as sample type, lipids fraction used or storage temperature), or
degrees of statistical adjustment. In 2 studies that measured adipose tissue fatty acid composition,
there were generally non-significant associations across total and specific fatty acids (eFigure 13).

Effects of fatty acids supplementation on coronary outcomes

Overall, 27 randomised controlled trials reported on fatty acids supplementation, including a total of
105,085 participants among whom 6,229 had an incident coronary outcome (mean follow-up ranged
from 0.1 to 8 years: eTable 8). Eighteen trials recruited individuals with cardiovascular disease at
baseline, 8 recruited individuals with elevated cardiovascular risk factors and one involved initially
healthy participants. Four studies reported on alpha-linolenic acid supplementation (dose ranging

8
from 2 to 5.5 grams/day where dietary oil was the principal form of supplementation); 17 on long-
chain omega-3 polyunsaturated fatty acids supplementation (dose ranging from 0.3 to 6 grams/day
where capsule was the principle form of supplementation), and 8 on omega-6 polyunsaturated fatty
acids supplementation (2 using linoleic acid specific and 6 with mixed polyunsaturate intervention
where dietary supplementation consisted principally of linoleic acid). No data were available on
interventions related to saturated or monounsaturated fatty acids. Risk of bias assessment in each
trial is reported in eTable 9. All trials had low risk of bias for the random sequence generation and
the incomplete outcome data domains. One trial had an unclear risk of bias for allocation
concealment and 7 for blinding of outcome assessment. Eight trials had a high risk of bias for
blinding of participants and personnel, and 3 for selective reporting. Risk of other bias was unclear in
6 trials and high in 3. RRs for coronary outcomes comparing individuals in the intervention group
versus those in the control group were: 0.97 (0.69-1.36) for alpha-linolenic acid, 0.94 (0.86-1.03)
for total long-chain omega-3 polyunsaturated fatty acids, and 0.86 (CI, 0.69 to 1.07) for omega-6
polyunsaturated fatty acids (Figure 3 and eFigure 14). There was no significant evidence of
heterogeneity according to several trial characteristics, such as baseline population risk,
geographical location, length of follow-up, outcome definition, and number of ascertained coronary
outcomes (eFigure 15). Furthermore, overall effects of the fatty acid supplementations on coronary
disease were generally similar in the trials that had appropriate allocation concealment or blinded its
participants and carers (eFigure 15). Subsidiary analyses by excluding trials that had recorded less
than 50 coronary disease outcomes did not materially alter the results (eFigure 16). However, in a
subsidiary analysis, exclusion of one omega-6 trial which used a margarine-based supplementation
also high in trans fat, the relative risk for omega-6 polyunsaturated fatty acids was 0.81 (CI, 0.68 to
0.98).

Assessment of publication bias

There was generally no evidence of publication bias among the included observational or
intervention studies (eFigure 17).

9
DISCUSSION
Our findings do not clearly support cardiovascular guidelines that promote high consumption of
omega-6 polyunsaturated fatty acids and suggest reduced consumption of total saturated fatty
acids. First, we saw statistically nonsignificant associations in prospective studies of coronary
disease that involved assessment of dietary intake of omega-6 polyunsaturated fatty acids.
Conversely, dietary long-chain omega-3 polyunsaturated fatty acids was associated with lower risk
of coronary disease. We found heterogeneity of the associations between specific circulating long-
chain omega-3 and omega-6 polyunsaturated fatty acid composition and coronary disease, with
some evidence that circulating levels of eicosapentaenoic and docosahexaenoic acids (the 2 main
types of long-chain omega-3 polyunsaturated fatty acids) and arachidonic acid are each associated
with lower coronary risk. However, our meta-analysis of randomized trials of long-chain omega-3
and omega-6 polyunsaturated fatty acid supplements suggests that supplementation with these
nutrients does not statistically significantly reduce the risk for coronary outcomes. These updated
findings are in line with an earlier meta-analysis that reported limited effect of omega-3
polyunsaturated fatty acid supplements on cardiovascular disease (22). Nonetheless, further trials
are warranted because the available evidence is generally limited, especially in initially healthy
populations; hence, there is considerable interest in a large randomized trial of long-chain omega-3
polyunsaturated supplements in primary prevention currently in progress (29).

Second, we found essentially null associations between total saturated fatty acids and coronary risk
in studies using dietary intake and in those using circulating biomarkers. This apparent lack of
association in self-reported dietary studies could at least partially be explained by biases in self-
report questionnaires, especially in relation to certain foods, such as common snacks high in
saturated fats (30) (however, consumption of both saturated and monounsaturated fats is measured
reasonably well by questionnaires [31, 32]). We saw heterogeneity of effect across circulating
composition of specific saturated fatty acids. This could, at least in part, reflect biology because
circulating saturated fatty acid fractions reflect both consumption and endogenous metabolism and
synthesis (33). For example, the influence of metabolism seems particularly relevant for the de novo
synthesis of even-numbered saturated fatty acids in the body, compositions of which are largely
determined by dietary factors, including carbohydrate and alcohol consumption (33–35), and other
metabolic pathways (36, 37) rather than direct dietary intake. This is supported indirectly by the
positive yet nonsignificant associations seen for circulating blood composition of palmitic and stearic
acids (which are synthesized in the body and only weakly correlated with saturated fatty acid
consumption [32, 38]) with coronary disease. In contrast, we found a possible inverse association
between circulating margaric acid (an odd-chain saturated fatty acid that is moderately correlated
with milk and dairy fat consumption [39, 40]) and coronary disease, suggesting that odd-chain
saturated fats, which reflect milk or dairy consumption, may have less deleterious effects in risk for
coronary heart disease (41).

Third, we saw null associations of total and individual monounsaturated fatty acids with coronary
risk in studies using both dietary intake and circulating fatty acid composition. This apparent lack of

10
association is consistent with available mechanistic data, which remain contradictory about whether
monounsaturated fatty acids promote or protect against atherogenesis (42–44). In addition, total
dietary trans fatty acid intake was positively associated with coronary disease risk in our meta-
analysis, which is in line with the present guidelines that support avoidance of trans fats. However,
because only 5 published prospective cohort studies contributed to this analysis, the inclusion of
relevant data from other unpublished studies could alter the overall estimate. This association was
unclear in studies that assessed circulating trans fatty acid composition, potentially because of a
relative paucity of data on trans fatty acid biomarkers and coronary risk. Furthermore, the method
used to measure circulating fatty acids in 1 study (41) may not have been sufficient for optimal
resolution of the individual trans fatty acid isomers.

Several strengths and limitations merit careful consideration. The review provides a comprehensive
systematic synthesis of available evidence by including data from different sources of evidence and
quantifies the risk for coronary disease for a wide range of individual fatty acid isomers and several
relevant subgroups in a consistent way. Generalizability was enhanced by the involvement of
information from more than 600,000 participants in 18 countries. Most of the observational studies
were judged as reasonably high-quality. Limitations include the moderate amount of available data
on some specific circulating fatty acids and possible overestimations of associations because of
preferential publication of extreme findings or, analogously, by selective reporting of results for
particular fatty acids with striking associations. Although selective reporting seems minimal among
randomized trials, few observational studies reported on all measured circulating fatty acids.
Therefore, selective underreporting may have contributed at least in part to the observational
findings in this meta-analysis. Because most studies lacked serial assessment of fatty acids in the
same persons, relative risks in published reports may have been prone to underestimation because
of “regression dilution bias” (45). Similar considerations apply to self-reported measures of fatty
acid consumption. We assumed log-linear associations between fatty acid measures and coronary
risk because we lacked access to individual-participant data. Although we used estimates that were
unadjusted for potential mediators (such as blood lipids and circulating fatty acids), we could not
adjust consistently for potential confounding factors across all studies. In addition, although most
trials were rated as having low risk of bias, the findings from these studies should be interpreted
with caution because of the relatively small number of trials investigating _alpha-linolenic and
omega-6 polyunsaturated fatty acid interventions and the potential differences in design and
population characteristics of each trial.

In conclusion, the pattern of findings from this analysis did not yield clearly supportive evidence for
current cardiovascular guidelines that encourage high consumption of polyunsaturated fatty acids
and low consumption of saturated fats.

11
Reprint requests: Dr Rajiv Chowdhury
Department of Public Health and Primary Care
University of Cambridge
Strangeways Research Laboratory
Cambridge, CB1 8RN, England

Mailing Address of the authors:

Rajiv Chowdhury, Samantha Warnakula, Setor Kunutsor, Adam Butterworth, Kay-Tee Khaw, Simon
G Thompson, John Danesh and Emanuele Di Angelantonio: Department of Public Health and Primary
Care, University of Cambridge, Cambridge CB1 8RN, United Kingdom.

Francesca Crowe: Cancer Epidemiology Unit, Richard Doll Building, Old Road Campus, Roosevelt
Drive, Oxford, OX3 7LF, United Kingdom

Heather A Ward: Department of Epidemiology and Biostatistics, School of Public Health, Imperial
College London, South Kensington Campus, London SW7 2AZ, United Kingdom

Laura Johnson: Centre for Exercise, Nutrition and Health Sciences, University of Bristol, 8 Priory
Road, Bristol BS8 1TZ, United Kingdom

Oscar H Franco: Department of Epidemiology, Erasmus MC, University Medical Center, Office Na 29-
16 PO Box 2040, 3000 CA Rotterdam, The Netherlands

Nita G Forouhi: United Kingdom Medical Research Council Epidemiology Unit, Cambridge
Box 285, Addenbrookes Hospital, Cambridge CB2 0QQ, United Kingdom

Dariush Mozaffarian: Department of Epidemiology, Harvard School of Public Health, 655 Huntington
Avenue, Boston, Massachusetts 02115, United States

Acknowledgment
The authors cordially thank Dr Kristiina Nyyssönen; Dr Arja Erkkilä and Dr Kalevi Pyörälä for kindly
providing additional data.

12
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 MAIN TABLE AND FIGURES

Table 1. Summary of Data Included in the Current Review

Number of

Studies* Participants Coronary events

Prospective cohort studies of dietary fatty acids intake

All studies 32 512,420 15,945

Dietary questionnaire¶ 21 463,038 11,157

Diet record§ 11 49,382 4,788

Prospective cohort studies of fatty acid biomarkers

All studies 19 32,307 7,182

Circulating fatty acids composition 17 25,721 5,519

Adipose tissue fatty acids composition 2 6,586 1,663

RCTs of fatty acids supplementation 27 105,085‡ 6,229†

*5 studies reported on both circulating and diet-based exposures, and 1 study reported on both circulating fatty acids and effect of fatty
acid supplementation; ¶ includes food frequency and diet history questionnaires; § includes open-ended instruments such as 24 hour recall
and food diaries; ‡ Includes 52,588 and 52,497 total participants in intervention and control groups respectively; † Includes 3,017 and
3,212 coronary events in intervention and control groups respectively; RCT, Randomised controlled trial. Details of all individual studies
are included in the Supplementary Material.

16
 Figure 1. RRs for coronary outcomes, based in prospective cohort studies of dietary fatty acids intake.

Studies, n Participants, n Events, n RR (95% CI)*

Dietary fatty acids intake

Total saturated fatty acids 20 276 763 10 155 1.03 (0.98-1.07)

Total monounsaturated fatty acids 9 144 219 6031 1.00 (0.91-1.10)

ω-3 fatty
. acids

α-linolenic 7 157 258 7431 0.99 (0.86-1.14)

Total long-chain ω-3 16 422 786 9089 0.87 (0.78-0.97)

Total ω-6 fatty acids 8 206 376 8155 0.98 (0.90-1.06)

Total trans fatty acids 5 155 270 4662 1.16 (1.06-1.27)

0.75 1 1.25 1.50

RR (95% CI) Comparing Top vs. Bottom Thirds of
Baseline Dietary Fatty Acid Intake

Size of the data marker is proportional to the inverse of the variance of the RR. RR=relative risk.
2
*Pooled estimate based on random effects meta-analysis. Corresponding forest plots, I (95% CI) estimates and pooled RRs based on fixed-effect meta-analysis are provided in Supplement
1, available at www.annals.org.
17
 Figure 2. Relative risk for coronary outcomes, in prospective cohort studies of circulating fatty acid composition

No. of No. of No. of

Circulating blood fatty acids composition studies participants events RR (95% CI)*

Total saturated fatty acids 8 15,590 3,758 1.06 (0.86, 1.30)

14:0, Myristic 5 10,598 2,932 0.96 (0.83, 1.12)
15:0, Pentadecanoic 4 5,490 2,283 0.94 (0.67, 1.32)
16:0, Palmitic 10 25,554 4,318 1.15 (0.96, 1.37)

17:0, Margaric 4 5,490 2,283 0.77 (0.63, 0.93)

15:0, Pentadecanoic + 17:0, Margaric 4 5,490 2,283 0.81 (0.62, 1.06)

18:0, Stearic 8 22,266 3,654 1.23 (0.93, 1.61)

Total monounsaturated fatty acids 6 14,356 3,236 1.06 (0.97, 1.17)

16:1n-7, Palmitoleic 9 17,927 4,127 0.96 (0.86, 1.08)
18:1cis-9, Oleic 9 22,664 3,687 1.09 (0.97, 1.23)

Total omega-3 polyunsaturated fatty acids

18:3n-3, alpha-linolenic 8 14,945 3,426 0.93 (0.83, 1.03)

Total long-chain omega-3 4 10,558 2,753 0.84 (0.63, 1.11)

20:5n-3, Eicosapentaenoic 13 23,065 4,624 0.78 (0.65, 0.94)

22:6n-3, Docosahexaenoic 13 23,065 4,624 0.79 (0.67, 0.93)

20:5n-3, Eicosapentaenoic + 22:6n-3, Docosahexaenoic 13 20,809 4,073 0.75 (0.62, 0.89)

22:5n-3, Docosapentaenoic (clupanodonic) 4 7,155 2,565 0.64 (0.47, 0.89)

Total omega-6 polyunsaturated fatty acids 2 7,432 1,877 0.94 (0.84, 1.06)
18:2n-6, Linoleic 10 23,022 3,866 0.99 (0.77, 1.28)

18:3n-6, gamma-linolenic 4 8,285 2,259 1.03 (0.90, 1.17)

20:2n-6, Eicosadienoic 2 4,029 1,689 1.18 (0.93, 1.50)
20:3n-6, Dihomo-gamma-linolenic 6 14,189 3,214 1.11 (0.93, 1.33)
20:4n-6, Arachidonic 10 22,948 3,739 0.83 (0.74, 0.92)
22:4n-6, Docosatetraenoic 2 4,029 1,689 1.20 (0.99, 1.45)
22:5n-6, Docosapentaenoic (osbond) 2 4,029 1,689 0.97 (0.50, 1.88)

Total trans fatty acids 4 7,661 2,389 1.05 (0.76, 1.44)

18:1, trans-oleic 2 921 380 1.20 (0.39, 3.73)

18:2, trans-linoleic 2 921 380 1.36 (0.83, 2.22)

0.50 0.75 1 1.25 1.50 2.00

RR (95% CI) comparing top vs. bottom thirds

*Pooled estimate based on random effects meta-analysis. Size of the data marker is proportional to the inverse of the variance of relative risk (RR) and horizontal line
represents 95% confidence interval (CI). Corresponding forest plots, I2 (95% CI) estimates and RRs from fixed-effect meta-analysis are provided in Supplementary Material
18
 Figure 3. Effect of fatty acid supplementation on risk for coronary event,
derived from available randomized, controlled trials.

Events/Participants, n/N
Fatty Acid Supplement Studies, n
Intervention Group Control Group RR (95% CI)*

α-linolenic acid 4 199/9444 220/9422 0.97 (0.69-1.36)

Long-chain ω-3 fatty acid 17 2426/38 303 2548/38 277 0.94 (0.86-1.03)

ω-6 fatty acid† 8 459/7245 515/7231 0.86 (0.69-1.07)

0.75 1.00 1.25 1.50

Intervention Control
Better Better

RR = relative risk. Size of the data marker is proportional to the inverse of the variance of the RR.
2
*Pooled estimate based on random effects meta-analysis. Corresponding forest plots, I (95% CI) estimates and pooled RRs based on fixed-effect meta-analysis are provided in Supplement
1, available at www.annals.org.
†Includes studies with ω-6 specific intervention and mixed polyunsaturrate interventions with linoleic acid as the primary fatty acid. 19