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Use of dietary linoleic acid for secondary prevention
of coronary heart disease and death: evaluation of
recovered data from the Sydney Diet Heart Study and
updated meta-analysis
OPEN ACCESS
Christopher E Ramsden clinical investigator 1 2, Daisy Zamora epidemiologist 2, Boonseng
Leelarthaepin retired, original study investigator 3, Sharon F Majchrzak-Hong research chemist 1,
Keturah R Faurot epidemiology doctoral candidate2, Chirayath M Suchindran senior biostatistician4,
Amit Ringel guest researcher 1, John M Davis professor 5, Joseph R Hibbeln senior clinical
investigator 1
1Laboratory of Membrane Biophysics and Biochemistry, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda,
MD 20892, USA; 2Department of Physical Medicine and Rehabilitation, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill,
NC, USA; 3University of New South Wales, Sydney, Australia; 4Department of Biostatistics, School of Public Health, University of North Carolina at
Chapel Hill, Chapel Hill, USA; 5Department of Psychiatry, University of Illinois at Chicago, Chicago, IL, USA
Abstract
Objective To evaluate the effectiveness of replacing dietary saturated
fat with omega 6 linoleic acid, for the secondary prevention of coronary
heart disease and death.
Design Evaluation of recovered data from the Sydney Diet Heart Study,
a single blinded, parallel group, randomized controlled trial conducted
in 1966-73; and an updated meta-analysis including these previously
missing data.
Setting Ambulatory, coronary care clinic in Sydney, Australia.
Participants 458 men aged 30-59 years with a recent coronary event.
Interventions Replacement of dietary saturated fats (from animal fats,
common margarines, and shortenings) with omega 6 linoleic acid (from
safflower oil and safflower oil polyunsaturated margarine). Controls
received no specific dietary instruction or study foods. All non-dietary
aspects were designed to be equivalent in both groups.
Outcome measures All cause mortality (primary outcome),
cardiovascular mortality, and mortality from coronary heart disease
(secondary outcomes). We used an intention to treat, survival analysis
approach to compare mortality outcomes by group.
Results The intervention group (n=221) had higher rates of death than
controls (n=237) (all cause 17.6% v11.8%, hazard ratio 1.62 (95%
confidence interval 1.00 to 2.64), P=0.05; cardiovascular disease 17.2%
v11.0%, 1.70 (1.03 to 2.80), P=0.04; coronary heart disease 16.3% v
10.1%, 1.74 (1.04 to 2.92), P=0.04). Inclusion of these recovered data
in an updated meta-analysis of linoleic acid intervention trials showed
non-significant trends toward increased risks of death from coronary
heart disease (hazard ratio 1.33 (0.99 to 1.79); P=0.06) and
cardiovascular disease (1.27 (0.98 to 1.65); P=0.07).
Conclusions Advice to substitute polyunsaturated fats for saturated
fats is a key component of worldwide dietary guidelines for coronary
heart disease risk reduction. However, clinical benefits of the most
abundant polyunsaturated fatty acid, omega 6 linoleic acid, have not
been established. In this cohort, substituting dietary linoleic acid in place
of saturated fats increased the rates of death from all causes, coronary
heart disease, and cardiovascular disease. An updated meta-analysis
of linoleic acid intervention trials showed no evidence of cardiovascular
benefit. These findings could have important implications for worldwide
dietary advice to substitute omega 6 linoleic acid, or polyunsaturated
fats in general, for saturated fats.
Trial registration Clinical trials NCT01621087.
Introduction
Advice to substitute vegetable oils rich in polyunsaturated fatty
acids (PUFAs) for animal fats rich in saturated fatty acids
(SFAs) has been a cornerstone of worldwide dietary guidelines
Correspondence to: C E Ramsden Chris.Ramsden@nih.gov
Extra material supplied by the author (see http://www.bmj.com/content/346/bmj.e8707?tab=related#webextra)
Web appendix: Supplementary material
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Research
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for the past half century.1When this advice originated in the
1960s, PUFAs were regarded as a uniform molecular category
with one relevant biological mechanism—the reduction in blood
cholesterol.1 2 Omega 6 (n-6) linoleic acid (LA) was the best
known dietary PUFA at the time. Therefore, the terms “PUFA”
and “LA” were often used interchangeably when interpreting
clinical trial results and delivering dietary advice.
Since that time, there has been increased recognition that the
general category of PUFAs comprises multiple species of omega
3 (n-3) and n-6 PUFAs, each with unique biochemical properties
and perhaps divergent clinical cardiovascular effects. Favorable
biological actions of n-3 eicosapentaenoic acid and
docosahexaenoic acid (and to a lesser extent, n-3 α linolenic
acid) have been extensively described.3Clinical cardiovascular
benefits of eicosapentaenoic acid and docosahexaenoic acid
have also been reported in several4 5 but not all6 7 randomized
controlled trials.3 8
However, there is currently no clinical trial evidence indicating
that replacing SFAs with n-6 LA, without a concurrent increase
in n-3 PUFAs, lowers the risk of cardiovascular disease or death.
Thus, benefits attributed to PUFAs as a general category might
be due to n-3 PUFAs specifically, particularly eicosapentaenoic
acid and docosahexaenoic acid. Such benefits are not necessarily
generalizable to n-6 LA or other PUFA species. Since n-6 LA
is the most abundant dietary PUFA, and edible oil sources with
markedly different contents of fatty acids are commercially
available (table 1⇓),9it is important to ascertain the benefits and
risks specific to n-6 LA.
The Sydney Diet Heart Study (SDHS),10-24 a randomized
controlled trial conducted from 1966 to 1973, provides a unique
opportunity to evaluate the cardiovascular effects of replacing
SFA with n-6 LA from safflower oil. Safflower oil is a
concentrated source of LA (about 75 g LA per 100 g serving of
oil9; table 1) containing no other reported PUFAs. Increased all
cause mortality in the safflower oil group was reported in 1978,10
although deaths due to cardiovascular disease and coronary
heart disease were not reported by group. Clinical outcomes for
cardiovascular disease and coronary heart disease have been
considered to be more relevant than all cause mortality when
evaluating the evidence base25 and formulating dietary
guidelines.26 Therefore, previous meta-analyses of PUFA
intervention trials and risk of cardiovascular disease25 27 28 have
been incomplete because they were not able to include these
missing data from the SDHS.
We recovered the original SDHS dataset and used modern
statistical methods to compare rates of all cause, cardiovascular,
and coronary heart disease mortality by group; and to examine
whether longitudinal dietary changes in PUFAs (that is, n-6 LA
from safflower oil) or SFAs were associated with mortality
outcomes. SDHS data recovery also allowed us to update our
previously incomplete meta-analysis published in 2010,28
permitting a comprehensive risk-benefit assessment for n-6 LA
including datasets from all known randomized controlled trials
evaluating dietary PUFAs for cardiovascular risk reduction.
Methods
SDHS data recovery and validation
We obtained permission from an original study investigator (B
Leelarthaepin, coauthor) and approval from the Office of Human
Research Protection to recover, analyze, and interpret
de-identified SDHS data stored on a 9 track magnetic tape. Part
1 of the web appendix describes the methods used to recover
the original SDHS dataset; convert these data into a useable
format; and identify, confirm, and verify the recovered data.
Only variables that exactly matched published data were
included. All matching variables were further verified by an
original study investigator (B Leelarthaepin) to ensure accuracy.
Study design and participants
The SDHS was a randomized controlled dietary trial, evaluating
the effects of increasing n-6 LA from safflower oil in place of
SFA for secondary prevention of coronary heart disease. Eligible
patients were men aged 30-59 years admitted to one of four
academic teaching hospitals (Prince Henry, Prince of Wales,
Sutherland, St George)13 affiliated with the University of New
South Wales near Sydney, Australia, for an episode of acute
myocardial infarction (86%), or acute coronary insufficiency
or angina (14%). Patients were subsequently referred to the
Prince Henry Hospital Coronary Clinic for inclusion in the trial.
Participants entered the study at least eight weeks (mean 11
weeks) after their acute coronary event, by which time most
had resumed normal activity, and many had returned to work.13
Randomization and masking
After provision of informed consent, participants were allocated
by a table of random numbers to either the dietary intervention
group (n=221) or a control group with no specific dietary
instruction (n=237). The number sequence was generated by a
research assistant and was concealed until after the medical
evaluations and testing at baseline were completed. After the
baseline phase, the study dietitians were unmasked to assign
patients to their groups and administer the interventions. Medical
investigators were initially masked to group assignment,
although the success of blinding was not evaluated. Deaths were
assigned codes from ICD-7 (international classification of
diseases, 7th revision)29 according to death certificates or final
hospital admission records.
Procedures
During the baseline phase, a testing battery of clinical,
laboratory, and dietary assessments was administered. Levels
of serum total cholesterol and triglycerides after fasting (>12
h) were assessed at the first baseline visit and again one week
later, by a modification of the methods of Abell and Van
Handel,30 31 respectively. Participants recorded their dietary
intake in a seven day food log between these two baseline visits.
Experienced dietitians assessed daily intake via a combination
of this food log and accompanying interview at the second
baseline visit.13 Data were converted to mean daily nutrient
intake using food composition tables supplemented by laboratory
analyzed values on a previously established computer
program.10 32
Diet intervention
The intervention group received instructions to increase their
PUFA intake to about 15% of food energy, and to reduce their
intake of SFA and dietary cholesterol to less than 10% of food
energy and 300 mg per day, respectively.10 To achieve these
targets, intervention participants were provided with liquid
safflower oil and safflower oil polyunsaturated margarine
(“Miracle” brand, Marrickville Margarine). Liquid safflower
oil was substituted for animal fats, common margarines and
shortenings in cooking oils, salad dressings, baked goods, and
other products, and was also taken as a supplement. Safflower
oil polyunsaturated margarine was used in place of butter and
common margarines. Safflower oil is a concentrated source of
n-6 LA (table 1)9and contains no other reported PUFAs.
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RESEARCH
Therefore, the intervention oil selectively increased n-6 LA
without a concurrent increase in n-3 PUFAs; this LA selective
PUFA intervention will be referred to as the LA intervention.
Control
The control group received no specific dietary instruction.
However, some participants began substituting polyunsaturated
margarine for butter after their coronary event.33 Because the
research team made no effort to alter the PUFA or SFA content
of control diets, such dietary changes were allowed to continue.
All non-dietary aspects of the study were designed to be
equivalent in both groups, with all participants receiving
standard medical care available at the time for secondary
prevention of coronary heart disease and other medical
conditions. This treatment included equivalent advice for all
active smokers to quit and all overweight patients to lose weight.
For follow-up, participants returned for testing three times in
the first year after entry, and every six months thereafter (web
appendix, part 4). At each visit, a clinical assessment was
performed and fasting levels of cholesterol and triglycerides
were assessed. Between each study visit, participants recorded
their dietary intake in a seven day food log. At each follow-up
visit, mean daily nutrient intakes were assessed by a combination
of the food log and dietitian interview, similar to the baseline
phase. The full food log was repeated if doubts arose about
accuracy,32 although the specific criteria used to assess accuracy
were not recovered. Duration of diet exposure and ICD-7 codes
were recorded for all study deaths.
Statistical analysis for the SDHS
The published objective of the SDHS was to evaluate whether
increasing PUFAs in place of SFAs in men with “premature
coronary heart disease might so improve survival.”10 However,
the primary survival outcome (for example, mortality from all
causes, cardiovascular disease, or coronary heart disease) was
not explicitly defined. The original sample size calculations
were not recovered. For our analyses, all cause mortality was
selected as the primary outcome, with deaths from
cardiovascular disease and coronary heart disease as secondary
outcomes.
Statistical analyses were performed using Stata version 11 (Stata
Corporation, 2009). We computed and compared descriptive
statistics for baseline demographics, clinical characteristics, and
dietary intakes by randomization assignment. We used survival
analysis methodology to compare mortality from all causes,
cardiovascular disease, and coronary heart disease between
intervention and control groups on an intention to treat basis.
Cumulative death rates were summarized using Kaplan Meier
curves, and hazard ratios were estimated using Cox proportional
hazards models.
To examine whether longitudinal changes in the prescribed
dietary nutrients (PUFA, SFA, and PUFA:SFA ratio) were
associated with mortality, we calculated hazard ratios for the
whole sample and stratified by group for each mortality outcome
as a function of time varying change from baseline. Nutrient
values were expressed as a percentage of total energy intake.
We used continuous variables after testing the linearity
assumption. For these analyses, only participants with baseline
dietary measurements were included (n=429, 63 deaths). To
evaluate which of the prescribed nutrient changes could have
mediated the increased mortality observed in the LA intervention
group, we repeated the same analysis limiting the sample to the
intervention group (n=207, 35 deaths). Since safflower oil is a
concentrated source of n-6 LA containing no other reported
PUFAs (table 1), this intervention group analysis of the change
in PUFA (calculated by subtracting baseline values from values
at each subsequent visit) estimates the effects of selectively
increasing n-6 LA.
Oxidation products of n-6 LA have been implicated in the
pathogenesis of cardiovascular disease,34-46 and alcohol use and
cigarette smoking are major sources of free radical mediated
oxidation.46 39 47 48 Therefore, we hypothesized that alcohol use
or smoking at baseline modified the association between
longitudinal change in PUFA intake and mortality using
likelihood ratio tests (α=0.15). The original SDHS investigators
posited that the LA intervention would reduce serum cholesterol
as an intermediate for the prevention of cardiovascular death.
Thus, to examine whether the magnitude of postrandomization
changes in total blood cholesterol were associated with
mortality, we calculated hazard ratios for each mortality outcome
as a function of time varying change from baseline in total blood
cholesterol.
Updated meta-analysis of PUFA intervention
randomized controlled trials
To put the SDHS findings into context, we updated our prior
systematic review and meta-analysis of the effects of PUFA
interventions on risk of coronary heart disease28 49 to include the
previously missing mortality data for coronary heart disease
and cardiovascular disease from the SDHS. Web appendix 8
shows the detailed methods,28 along with descriptions of each
PUFA intervention trial, publication bias assessment,
heterogeneity analyses, pooled risk estimates for mortality
outcomes, and sensitivity analyses.
Results
Figure 1⇓shows the trial profile. The two groups were well
balanced at baseline, with no significant differences in any
demographic, laboratory, or dietary variables (tables 2⇓and
3⇓). Median follow-up was 39 months.
Table 3 summarizes nutrient intakes of the two groups at
baseline and follow-up. During follow-up, the intervention group
had a larger increase in PUFA than the control group, and larger
reductions in SFAs, dietary cholesterol, and monounsaturated
fatty acids (P<0.001). Part 5 of the web appendix shows intakes
of target nutrients at each study time point.
Table 4⇓summarizes risk factors for cardiovascular disease at
baseline and at 12 months of follow-up. Serum total cholesterol
decreased more in the LA intervention group than in the control
group (−13.3% v−5.5%; P<0.001). There were no significant
differences between groups in triglycerides, body mass index,
or systolic or diastolic blood pressure at baseline or during
follow-up.
Cumulative death rates
Compared with the control group, the intervention group had
an increased risk of all cause mortality (17.6% v11.8%; hazard
ratio 1.62 (95% confidence interval 1.00 to 2.64); P=0.051),
cardiovascular mortality (17.2% v11.0%; 1.70 (1.03 to 2.80);
P=0.037), and mortality from coronary heart disease (16.3% v
10.1%; 1.74 (1.04 to 2.92); P=0.036) (fig 2⇓).
Association of change in PUFA and saturated
fat with mortality
Table 5⇓shows results of Cox models examining the relation
between mortality risk and longitudinal changes in dietary n-6
LA, PUFA, and SFA. Among intervention patients (in whom
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RESEARCH
the PUFA increase was n-6 LA from safflower oil), an increase
of 5% of food energy from n-6 LA predicted 35% and 29%
higher risk of cardiovascular death and all cause mortality,
respectively (models adjusted for age, dietary cholesterol, body
mass index at baseline, smoking, alcohol use, and marital status).
Increases in the LA:SFA ratio in the intervention group were
also significantly associated with higher cardiovascular death
and all cause mortality; however, the reduction in SFA was not
significantly related to any mortality outcome.
Among controls (in whom PUFA changes were not specific to
n-6 LA), changes in PUFA and SFA consumption were not
significantly related to risk of death. Among the control and
intervention groups combined, an increase of 5% of food energy
from unspecified PUFA predicted about 30% higher risk of
cardiovascular death and all cause mortality. A reduction in
SFA and increase in the PUFA:SFA ratio were also associated
with increased risks of all cause and cardiovascular mortality.
Risk of death from coronary heart disease was significantly
associated with longitudinal changes in PUFA and SFA in the
adjusted (but not crude) models.
Table 6⇓shows the hazard ratios for the association between
longitudinal changes in dietary PUFA and cardiovascular
mortality, according to alcohol intake and smoking (sources of
oxidative stress) at baseline. There was evidence of effect
modification by alcohol and smoking (likelihood ratio test
P=0.04 and P=0.13, respectively) among the whole sample, but
not when restricted to the LA intervention group only. The
positive association between longitudinal increases in n-6 LA
and unspecified PUFAs and cardiovascular mortality was
significant among moderate or heavy drinkers and smokers, but
not among non-drinkers and non-smokers.
Discussion
In this evaluation of data from the SDHS, selectively increasing
the n-6 PUFA LA from safflower oil and safflower
polyunsaturated margarine increased rates of death from
cardiovascular disease, coronary heart disease, and all cause
mortality compared with a control diet rich in SFA from animal
fats and common margarines. This is the first published report
to show an increase in mortality from cardiovascular disease
and coronary heart disease, comparing this LA intervention to
the control group, and demonstrating that the magnitude of
increased n-6 LA intake was associated with higher risk of death.
Although increased all cause mortality was reported in a 1978
publication,10 cardiovascular disease and coronary heart disease
clinical outcomes, rather than all cause mortality, are the most
relevant endpoints considered when evaluating the evidence
base25 and formulating dietary guidelines for cardiovascular risk
reduction.26 Therefore, recovery of these missing data has filled
a critical gap in the published literature archive, allowing for a
more comprehensive risk-benefit assessment for n-6 LA,
including all known datasets from randomized controlled trials.
Comparison with other randomized controlled
trials and updated meta-analysis
These unfavorable effects of n-6 LA shown in the SDHS are
consistent with two other randomized controlled trials, in which
experimental dietary conditions selectively increased n-6 LA
in the place of SFAs by replacing animal fats and common
margarines with corn oil.50 51 Together, these three trials provide
a rare opportunity to evaluate the specific effects of increasing
n-6 LA without confounding from concurrent increases in n-3
PUFAs. In a pooled analysis, the increased risks of death from
coronary heart disease (hazard ratio 1.33 (95% confidence
interval 0.99 to 1.79); P=0.06; fig 3⇓) and cardiovascular disease
(1.27 (0.98 to 1.65); P=0.07; fig 4⇓) approached significance.
Secondary prevention trials showed significant adverse effects
of n-6 LA on coronary heart disease mortality (1.84 (1.11 to
3.04); P=0.02; web appendix, part 8). By contrast, pooled
analysis of the four randomized controlled trials52-55 that
increased n-3 PUFAs alongside n-6 LA showed reduced
cardiovascular mortality (0.79 (0.63 to 0.99); P=0.04; fig 4).
The specific PUFA content of intervention oils was identified
as a major source of heterogeneity for all mortality outcomes
(web appendix, part 8). Therefore, benefits previously attributed
to greater intake of total PUFA might be specifically attributable
to n-3 PUFAs, and are not necessarily generalisable to n-6 LA,
or PUFAs in general. Alongside the SDHS findings, these
meta-analytic results provide supporting evidence that the
specific PUFA content of intervention oils is a critical
determinant of clinical cardiovascular outcomes, and that
selective substitution of n-6 LA for SFA is unlikely to be
beneficial, particularly in patients with established coronary
heart disease. These findings should be interpreted with some
caution, owing to the relatively small number of trials
investigating PUFA interventions and differences in design and
population characteristics of each trial. Part 8 of the web
appendix presents the complete methods, results, and limitations
of the analysis.
Among these PUFA intervention trials, the SDHS was unique
because it collected longitudinal diet data using serial seven day
food records, providing an opportunity to examine which
nutrients might have mediated the increased risk of death
observed in the study’s intervention group. Restriction of the
analysis to the intervention group—which was provided
safflower oil (a concentrated source of n-6 LA lacking n-3
PUFAs)—allowed us to estimate the specific effects of
increasing n-6 LA only. Among patients in this intervention
group, the increase in n-6 LA was associated with higher all
cause and cardiovascular mortality, providing supporting
evidence that LA itself was a key component mediating the
unfavorable effects.
This advantage of PUFA specificity from safflower oil in the
SDHS could account for the discrepancy between our findings
of increased mortality risk with selective increase in n-6 LA,
and the reported benefits of the general category of PUFAs in
pooled analyses of randomized controlled trials25 and
observational cohorts,56, which did not clearly distinguish
between n-6 LA and other PUFA species. Importantly, these
prior analyses of unspecified PUFAs have been considered
decisive evidence for the benefits of n-6 LA26 57-60 and a key
evidence base for advice to maintain or increase n-6 LA
consumption.26 57 Therefore, our finding that selectively
increasing n-6 LA from safflower oil increased the risk of death
from all causes, coronary heart disease, and cardiovascular
disease in the SDHS might have important implications for
worldwide dietary advice.
Other dietary considerations
Although our analysis focused on evaluating the effects of the
targeted reductions in SFAs and increases in n-6 LA from
safflower oil, participants in the LA intervention group also
significantly reduced their intake of monounsaturated fatty acids
(MUFA) and dietary cholesterol. This reduction in MUFA was
probably a byproduct of the reduction in foods high in SFAs
that were also rich sources of MUFA (for example, animal fats,
common margarine, shortening). Dietary cholesterol levels
decreased, owing to reductions in animal fat and egg yolk
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RESEARCH
consumption. Individual PUFA species were not recorded as
separate entities in the seven day food records. Therefore, we
cannot exclude the possibility that other PUFA species were
altered in either group. However, the safflower oil given to
intervention patients had large quantities of n-6 LA without
other PUFA species.
Another factor that could have been altered by the intervention
is dietary trans fatty acids, which are known to raise total and
low density lipoprotein cholesterol 61 and have been associated
with increased cardiovascular risk in observational studies.62
This association was not widely appreciated during the SDHS,
and the trans fatty acid content of participants’ diets was not
recorded. Restriction of common margarines and shortenings
(major sources of trans fatty acids) in the intervention group
would be expected to substantially reduce consumption of trans
fatty acids compared with the control group.
Conversely, some of this reduction in trans fatty acids in the
intervention group may have been offset by small amounts of
trans fatty acids in the safflower oil polyunsaturated margarine.
Although the precise composition of this margarine was not
specified, it was selected for the study because of its ability to
lower blood cholesterol and its high PUFA to SFA ratio,22 two
characteristics of margarines that contain comparatively low
amounts of trans fatty acids.63 Because dietary trans fatty acids
are predominantly 18-carbon MUFA isomers,64 the recorded
changes in MUFAs probably included small amounts of trans
fatty acids in both groups.
Statistical adjustment for changes in MUFAs (an imperfect
surrogate for trans fatty acids) in sensitivity analyses did not
noticeably alter the observed relation between LA and increased
risk of cardiovascular death in the intervention group (data not
shown). Collectively, these observations indicate that changes
in trans fatty acid were unlikely to play a substantial role in the
findings reported here. Nevertheless, the SDHS dataset does
not contain sufficient information to rule out the possibility that
changes in nutrients other than n-6 LA and SFAs could have
contributed to, or reduced, the observed unfavorable effects of
the LA intervention.
Reconciling results from the SDHS with the
traditional diet-heart hypothesis
Increasing dietary n-6 LA in place of SFA lowers serum total
cholesterol, primarily by reducing low density lipoprotein with
little or no effect on high density lipoprotein.61 The traditional
diet-heart hypothesis predicts that these favorable, diet induced
changes in blood lipids will diminish deposition of cholesterol
in the arterial wall, slow progression of atherosclerosis, reduce
clinical cardiovascular risk, and eventually improve survival.18 65
As expected, increasing n-6 LA from safflower oil in the SDHS
significantly reduced total cholesterol; however, these reductions
were not associated with mortality outcomes (results not shown).
Moreover, the increased risk of death in the intervention group
presented fairly rapidly and persisted throughout the trial. These
observations, combined with recent progress in the field of fatty
acid metabolism, point to a mechanism of cardiovascular disease
pathogenesis independent of our traditional understanding of
cholesterol lowering.
Proposed mechanistic model linking dietary
LA to cardiovascular pathogenesis
Omega-6 LA is the most abundant fatty acid in native low
density lipoprotein particles.66 Oxidized LA metabolites
(OXLAMs) are the most abundant oxidized fatty acids in
oxidized low density lipoprotein,34 67 which is potentially more
atherogenic than unmodified low density lipoprotein.46 A
potential mechanism contributing to higher cardiovascular
mortality in the LA intervention group is a diet induced increase
in the production of bioactive OXLAMs, including 9- and
13-hydroperoxy-octadecadienoic acid, and 9- and
13-hydroxy-octadecadienoic acid. These OXLAMs are enriched
in the lipid laden, macrophage foam cells; vascular endothelial
cells; and migrating vascular smooth muscle cells of
atherosclerotic lesions.35-38 OXLAMs, particularly the isomers
and enantiomers produced by free radical mediated
oxidation,35 39 68 have been mechanistically linked to
cardiovascular disease pathogenesis. Mechanisms include
inducing the formation of macrophage foam cells41 42; endothelial
cell activation43; migration, proliferation, and foam cell
formation of vascular smooth muscle cells44 69; and inhibition
of lysosomal hydrolysis of low density lipoprotein cholesteryl
esters70 (fig 5⇓).
Major sources of free radical mediated oxidative stress, such as
cigarette smoking and chronic alcohol exposure, increase the
oxidation of low density lipoprotein fatty acids46-48; smokers and
drinkers are reported to have increased concentrations of LA
oxidation products in atherosclerotic lesions.39 OXLAMs have
both proatherogenic and antiatherogenic actions, although the
isomers and enantiomers that predominate in such oxidation
are suspected to have deleterious properties, particularly in
established atherosclerotic lesions.35 39 68
Our model proposed here predicts that oxidative stress combined
with diets high in n-6 LA facilitate this oxidation, leading to
OXLAM mediated atherosclerotic progression and increased
cardiovascular mortality. Consistent with this model, the link
between the magnitude of increase in LA and mortality was
robust in smokers and drinkers in the SDHS, suggesting that
diets high in n-6 LA may be particularly detrimental in the
context of oxidative stress induced by smoking or alcohol.
Lowering dietary LA as a strategy for
cardiovascular risk reduction
If OXLAM mediated atherosclerotic progression did contribute
to the increased cardiovascular mortality seen in the SDHS,
then diet induced reductions in OXLAMs could potentially
reduce cardiovascular risk. We recently showed that lowering
n-6 LA in human diets for 12 weeks reduced OXLAMs and
their precursor LA in circulation,71 and increased erythrocyte
n-3 eicosapentaenoic acid and docosahexaenoic acid.72 Higher
levels of these n-3 fatty acids are associated with reduced risk
of coronary heart disease in some73-75 but not all76 observational
studies. These findings provide biochemical mechanisms that
are relevant to our proposed model, linking dietary LA to the
increased cardiovascular mortality seen in the SDHS, and also
suggest a dietary strategy (that is, LA lowering) that may warrant
evaluation for risk reduction of cardiovascular disease.
Limitations and strengths of the SDHS
The SDHS had several important strengths. The combination
of randomization, provision of a specific study oil (safflower)
delivering n-6 LA but not n-3 PUFAs, and detailed longitudinal
diet assessments allowed for estimation of the specific effects
of LA. Although several observational studies have reported
associations between LA and risk of coronary heart disease,77-79
the food frequency questionnaires used may have limited ability
to distinguish the respective intakes of n-6 LA and n-3 α
linolenic acid.80 81 Discrimination among PUFA species is limited
in observational studies, owing to the wide variability in n-6
LA and n-3 α linolenic acid contents of apparently similar food
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items (table 1); the lack of vegetable oil and PUFA labeling
requirements on packaged foods; and the lack of consumer
appreciation for the specific vegetable oils used when dining
away from home, which accounts for a large share of total PUFA
consumption.82 Therefore, provision of a study oil containing a
large quantity of n-6 LA without other PUFA species was a
distinct advantage of the design of the SDHS. Other SDHS
strengths included the relatively long term follow-up (median
39 months) and the use of ICD coded mortality outcomes as
objective endpoints.
The SDHS also had several important limitations. Some control
participants began substituting polyunsaturated margarine for
butter—leading to substantial, but comparatively modest, dietary
changes in the same direction as the intervention group. These
changes may have attenuated the observed differences in
mortality between the groups, leading to an underestimation of
the adverse effects of the intervention. Consistent with other
trials investigating the secondary prevention of coronary heart
disease, both groups made healthy lifestyle modifications (for
example, smoking reduction), probably from personal
reassessment that accompanies a coronary event.
Importantly, these non-dietary components of the intervention
were designed to be equivalent in both groups. Among 29
participants with missing diet data at baseline, there were a
disproportionate number of deaths in the intervention group
(four deaths from coronary heart disease) compared with the
control group (no deaths). This disproportionate loss could have
attenuated the association reported here, between increase in
dietary n-6 LA with higher mortality. Consistent with this
interpretation, a sensitivity analysis that imputed missing data
by randomization group found more precise and slightly stronger
associations between increases in LA and mortality (web
appendix, part 7).
Because the fatty acid content of blood or adipose tissue was
not measured, changes in these fatty acid pools could not be
used to validate the data obtained by serial administration of
seven day food records. However, the significant reduction in
blood cholesterol seen in the LA intervention group provides
biological data that is broadly consistent with the longitudinal
diet data.
Without access to the original study protocol, we cannot fully
appraise the accuracy of outcome ascertainment and other
quality aspects of the SDHS. Another potential limitation was
incomplete data recovery, owing to our inability to identify
some of the study variables recorded on punch cards. However,
we were able to identify, confirm, and verify each of the key
variables for the dietary, laboratory, and coded mortality
outcomes that were required to interpret the main study findings
(web appendix, part 1).
The adverse effects of increasing n-6 LA from 6% of food
energy to 15% in this cohort are not necessarily generalizable
to lower LA intakes; therefore, some caution should be used
when extrapolating results to other populations. Finally, because
the SDHS investigated secondary prevention of coronary heart
disease in men aged 30-59 years, results are not necessarily
generalizable to women, men aged younger than 30 years or
older than 59 years, or populations without established coronary
heart disease.
Conclusions
In this cohort, substituting dietary n-6 LA in place of SFA
increased the risks of death from all causes, coronary heart
disease, and cardiovascular disease. An updated meta-analysis
of LA intervention trials showed no evidence of cardiovascular
benefit. These findings could have important implications for
worldwide dietary advice to substitute n-6 LA, or PUFAs in
general, for SFA.
We thank the original SDHS team of researchers for their contributions:
Ralph Blacket (principal investigator), Joan Woodhill (coprincipal
investigator, lead dietitian), Jean Palmer (coinvestigator), Charles
McGilchrist (statistician), Lear Bernstein (senior dietitian), and Janet
Aitken (lipid laboratory); the physicians affiliated with the University of
New South Wales teaching hospitals for their contributions and referral
of participants; all the patients who participated in the study; John Svee
(Data Conversion Resource, Westminster, CO, USA), and Steve Morgan
and John Glauvitz (Data Recovery Systems, Morgan Hill, CA, USA) for
providing technical expertise in data recovery and conversion; Toni
Calzone for advice regarding recovery of magnetic tape data; Paul
Blacket for efforts searching for study data; and Natalie Kress, Michael
Donovan, Jie Qu, and Katherine Ness for proofreading the manuscript.
Contributors: CER designed and directed the project; located, managed,
and validated the recovered data; developed the proposed mechanistic
model; and was the main writer of the manuscript. BL was an investigator
in the original trial, provided the nine track magnetic tape, verified the
study methodology and ethical considerations, confirmed validity of
recovered data, and revised the manuscript. SFMH performed the
literature review, managed and validated recovered data, and assisted
in writing and revising the manuscript. John Svee of the Data Conversion
Resource completed the data recovery and conversion to modern
spreadsheet format, in collaboration with CER and SFMH. DZ conducted
the statistical analyses of the main trial, and KRF conducted the
meta-analyses, in collaboration with SFMH, CER, JMD, and CMS. JMD,
DZ, KRF, and CMS revised the manuscript. AR prepared the illustration
of the proposed model and revised the manuscript. JRH directed the
project and critically revised the manuscript. All authors contributed to
analyses or interpretation of results and to the intellectual content of
the manuscript. CER is the guarantor.
Funding: The Life Insurance Medical Research Fund of Australia and
New Zealand provided a grant in support of this the SDHS. The
Intramural Program of the National Institute on Alcohol Abuse and
Alcoholism, National Institutes of Health, supported data recovery and
evaluation. The funding source had no role in study design, data
collection, analysis, interpretation, or writing of the report.
Competing interests: All authors have completed the ICMJE uniform
disclosure form at www.icmje.org/coi_disclosure.pdf (available on
request from the corresponding author) and declare: support from the
Life Insurance Medical Research Fund of Australia and New Zealand
and the Intramural Program of the National Institute on Alcohol Abuse
and Alcoholism for the submitted work; no financial relationships with
any organizations that might have an interest in the submitted work in
the previous three years; no other relationships or activities that could
appear to have influenced the submitted work.
Ethical approval: The SDHS study protocol and patient consent forms
were approved by the Dean of the Faculty of Medicine, University of
New South Wales, Sydney, Australia. Medical research and clinical
practice procedures were carried out according to the June 1964 World
Medical Association Declaration of Helsinki and the Australian National
Health and Medical Research Council guidelines, which provided the
most current ethical principles for medical research involving humans.
Patients provided consent to participate without coercion, and were free
to refuse participation or withdraw at any time. The Office of Human
Research Protection for the National Institutes of Health reviewed these
conditions and determined that these de-identified data were suitable
for the current analyses (exemption no 5744).
Data sharing: The dataset is available from the corresponding author
at chris.ramsden@nih.gov. Data sharing consent was not obtained, but
the presented data are anonymised and risk of identification is low.
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BMJ 2013;346:e8707 doi: 10.1136/bmj.e8707 Page 6 of 18
RESEARCH
What is already known on this topic?
Increasing dietary omega-6 linoleic acid in the place of saturated fat lowers total cholesterol and low density lipoprotein cholesterol
Advice to substitute linoleic acid for saturated fat is one component of dietary guidelines to reduce the risk of coronary heart disease;
however, clinical benefits specific to linoleic acid have not been established
A comprehensive analysis of the effects of linoleic acid on death from coronary heart disease and cardiovascular disease was previously
not possible, owing to missing outcome data from the Sydney Diet Heart Study, a randomized controlled clinical trial
What this study adds
In this cohort, substituting omega 6 linoleic acid for saturated fat did not provide the intended benefits, but increased all cause morality,
cardiovascular death, and death from coronary heart disease
An updated meta-analysis incorporating these missing data showed no evidence of benefit, and suggested a possible increased risk of
cardiovascular disease from replacing saturated fat with omega-6 linoleic acid
These findings could have important implications for worldwide dietary advice to substitute omega-6 linoleic acid (or polyunsaturated
fatty acids in general) for saturated fatty acids
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Accepted: 14 December 2012
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RESEARCH
Tables
Table 1| Content of n-6 LA and n-3 α LNA in commercially available edible oils
α LNA (g per 100 g of cooking oil)LA (g per 100 g of cooking oil)Cooking oil
Depends on specific oilDepends on specific oilVegetable oil*
0.074.6Safflower†
0.065.7Sunflower†
0.051.5Cottonseed
0.253.5Corn
7.050.3Soybean
9.118.6Canola
0.89.8Olive
1.42.3Butter oil
0.01.8Coconut
n-6 LA=omega 6 linoleic acid; n-3 α LNA=omega 3 α linolenic acid. Fatty acid contents of oils vary to some extent by season, latitude, and other conditions. USDA
National Nutrient Database numbers: safflower 04510, sunflower 04510, cottonseed 04502, corn 04518, soybean 04669, canola 04582, olive 04053, butter 01003,
coconut 04047.9
*Food items labeled “vegetable oil” may contain one or more of the above oils.
†Varieties of safflower and sunflower oils with lower LA content are commercially available.
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RESEARCH
Table 2| Baseline characteristics of 458 randomized participants
Intervention group (n=221)Control group (n=237)
48.7 (6.82)49.1 (6.55)Age (years)*
25.1 (2.39)25.4 (2.60)Body mass index*
136.6 (20.1)136.9 (21.1)Systolic blood pressure (mm Hg)*†
88.5 (12.5)88.5 (12.0)Diastolic blood pressure (mm Hg)*†
281.3 (63.4)282.0 (55.6)Total cholesterol (mg/dL; 1 mg/dL=0.03 mmol/L)*
189.0 (199.1)185.9 (132.9)Triglycerides (mg/dL; 1 mg/dL=0.01 mmol/L)*
84.0 (12.9)82.2 (11.6)Fasting glucose (mg/dL; 1 mg/dL=0.06 mmol/L)*
6.7 (1.55)6.7 (1.55)Uric acid (mg/dL; 1 mg/dL=59.48 µmol/L)*
Marital status
204 (92.3)206 (86.9)Married
17 (7.7)31 (13.1)Single/divorced/widowed
Presenting event related to coronary heart disease
192 (86.9)203 (85.7)Myocardial infarction
29 (13.1)34 (14.3)Acute angina or coronary insufficiency
Smoking status
158 (71.5)163 (68.8)Smokers at admission
Alcohol use at admission (kcal; 1 kcal=4.18 kJ)
60 (27.2)64 (27.0)Non-drinker
78 (35.3)86 (36.3)Light (<200 kcal/day)
37 (16.7)44 (18.6)Moderate (200-500 kcal/day)
46 (20.8)43 (18.1)Heavy (>200 kcal/day)
Dyspnea
133 (60.2)148 (62.4)No dyspnea
62 (28.0)69 (29.1)On severe exertion
26 (11.8)20 (8.4)On mild exertion/at rest
Glucose metabolism§
157 (71.0)171 (72.2)Normal
46 (20.8)53 (22.4)Prediabetes
18 (8.1)13 (5.5)Diabetes
Data are no (%) of participants unless stated otherwise.
*Data are mean (standard deviation).
†Blood pressure measured in 5 unit intervals.
§Glucose response classification based on results of a challenge of 50 mg oral glucose, according to 1968 criteria of Joplin and Wright.83
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Table 3| Baseline and follow-up dietary data in the SDHS, for 426 participants with baseline and at least one follow-up diet record
P
Follow-up†Baseline*
Nutrient
Change from
baseline
Intervention
(n=205)
Change from
baselineControl (n=221)
Intervention
(n=205)Control (n=221)
<0.001+9.3§15.4 (12.3-17.9)+2.28.4 (6.7-10.9)6.1 (3.0-9.2)6.2 (3.2-9.2)PUFA‡
<0.001−6.99.3 (8.2-10.9)−2.113.5 (11.4-15.6)16.2 (13.4-19.3)15.6 (13.0-18.7)SFA‡
<0.001+1.341.72 (1.31-2.08)+0.220.63 (0.45-0.92)0.38 (0.16-0.65)0.41 (0.18-0.68)PUFA:SFA ratio
<0.001−3.411.2 (10.1-12.7)−0.714.0 (12.3-15.2)14.6 (13.2-16.5)14.7 (12.8-16.9)MUFA‡
0.87−1.938.3 (36.1-46.3)−1.138.1 (34.3-41.2)40.2 (36.6-43.4)39.2 (35.0-43.5)Total fat‡
0.31+1.441.3 (36.1-46.3)+0.140.6 (35.6-44.8)39.9 (35.2-46.1)40.5 (37.0-45.2)Carbohydrate‡
0.25+0.414.8 (13.4-16.8)+1.215.3 (13.4-17.3)14.4 (12.6-16.5)14.1 (12.4-16.3)Protein‡
0.42+0.73.1 (0.7-8.9)+1.74.0 (0.9-8.7)2.4 (0.0-8.9)2.3 (0.0-8.1)Alcohol‡
0.07−1672256 (1958-2574)−1902194 (1804-2524)2423 (1972-2860)2384 (2072-2770)Energy (kcal/day;
1 kcal=4.18 kJ)
<0.001−239238 (203-283)−108331 (269-408)477 (355-621)439 (344-593)Cholesterol
(mg/day)
*Data are median (interquartile range) from a single seven day food record administered before randomization.
†Data are median summaries (interquartile range), with each participant assigned one value based on the average of their seven day food records after randomization.
Comparisons between diets were calculated with the Mann Whitney U test.
‡Data are percentage of food energy.
§From safflower oil.
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Table 4| Risk factors for cardiovascular disease
12 month follow-upBaseline
P*Intervention (n=179)Control (n=192)Intervention (n=221)Control (n=237)
<0.001243.9 (237.4 to 250.4)266.5 (259.1 to 273.8)281.3 (272.9 to 289.7)282.0 (274.8 to 289.1)Total cholesterol (mg/dL; 1 mg/dL=0.026
mmol/L)
0.06135.5 (126.0 to 145.1)151.8 (133.9 to 169.7)189.0 (162.6 to 215.4)185.9 (168.8 to 202.9)Triglycerides (mg/dL; 1 mg/dL=0.011
mmol/L)
0.2624.3 (24.0 to 24.6)24.5 (24.1 to 24.9)25.1 (24.8 to 25.3)25.4 (25.1 to 25.8)Body mass index
0.49136.4 (133.8 to 139.0)136.5 (133.4 to 139.5)136.6 (133.9 to 139.3)136.9 (134.2 to 139.6)Systolic blood pressure (mm Hg)
0.3887.5 (85.7 to 89.3)87.9 (86.0 to 89.9)88.5 (86.9 to 90.2)88.5 (86.9 to 90.0)Diastolic blood pressure (mm Hg)
Data are mean (95% confidence interval) at baseline and 12 months after randomization.
*P values=between group differences, assessed by ttest.
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Table 5| Mortality outcomes according to longitudinal changes in dietary fatty acid intake
Mortality
Model§Diet variable
Coronary heart diseaseCardiovascular diseaseAll cause
PHazard ratio (95% CI)PHazard ratio (95% CI)PHazard ratio (95% CI)
LA intervention group only (n=207)*
0.221.20 (0.90 to 1.59)0.031.37 (1.04 to 1.79)0.051.31 (1.00 to 1.71)1PUFA (LA
specific; per 5
en% increase) 0.191.21 (0.91 to 1.61)0.031.35 (1.03 to 1.75)0.051.29 (1.00 to 1.67)2
0.150.74 (0.50 to 1.11)0.180.77 (0.52 to 1.13)0.180.77 (0.52 to 1.13)1SFA (per 5
en% increase) 0.220.78 (0.52 to 1.16)0.310.82 (0.56 to 1.21)0.290.81 (0.56 to 1.19)2
0.111.44 (0.93 to 2.23)0.031.59 (1.04 to 2.43)0.051.51 (1.00 to 2.30)1LA:SFA ratio
(per 1 unit
increase) 0.091.47 (0.94 to 2.29)0.031.62 (1.05 to 2.48)0.041.55 (1.01 to 2.36)2
Control group only (n=222)†
0.991.00 (0.62 to 1.61)0.751.07 (0.69 to 1.67)0.711.08 (0.71 to 1.66)1PUFA
(unspecified;
per 5 en%
increase)
0.931.02(0.65 to 1.60)0.681.09 (0.72 to 1.66)0.651.10 (0.73 to 1.65)2
0.930.98 (0.58 to 1.65)0.650.89 (0.54 to 1.47)0.280.77 (0.48 to 1.24)1SFA (per 5
en% increase) 0.840.95 (0.57 to 1.58)0.540.86 (0.53 to 1.39)0.250.76 (0.48 to 1.21)2
0.130.56 (0.27 to 1.18)0.530.78 (0.36 to 1.70)0.660.84 (0.39 to 1.80)1PUFA:SFA
ratio (per 1 unit
increase) 0.460.77 (0.39 to 1.52)0.991.00 (0.52 to 1.93)0.881.05 (0.56 to 1.97)2
Whole sample (n=429)‡
0.091.19 (0.97 to 1.47)<0.011.29 (1.07 to 1.57)0.021.26 (1.04 to 1.52)1PUFA
(unspecified;
per 5 en%
increase)
0.031.26 (1.02 to 1.54)<0.011.35 (1.12 to 1.63)<0.011.31 (1.09 to 1.58)2
0.080.77 (0.58 to 1.03)0.060.77 (0.58 to 1.01)0.030.74 (0.57 to 0.97)1SFA (per 5
en% increase) 0.030.72 (0.54 to 0.97)0.020.72 (0.54 to 0.95)<0.010.70 (0.53 to 0.91)2
0.141.27 (0.93 to 1.73)0.031.39 (1.03 to 1.87)0.041.35 (1.01 to 1.80)1PUFA:SFA
ratio (per 1 unit
increase) 0.031.44 (1.04 to 1.98)<0.011.58 (1.17 to 2.13)<0.011.53 (1.14 to 2.05)2
En%=percentage of food energy. Analysis includes 429 patients with diet data at baseline. Missing follow-up data were imputed for three patients with baseline
data who died within two months after randomization; median values were used for their respective group assignment.
*No of deaths: 35 (all cause), 34 (cardiovascular), 32 (coronary heart disease).
†No of deaths: 28 (all cause), 26 (cardiovascular), 24 (coronary heart disease).
‡No of deaths: 63 (all cause), 60 (cardiovascular), 56 (coronary heart disease).
§Model 1: crude model. Model 2: adjusted for age, dietary cholesterol intake, baseline body mass index, smoking, alcohol use, and marital status.
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Table 6| Risk of cardiovascular death according to longitudinal changes in dietary PUFA, stratified by sources of oxidative stress
Risk of cardiovascular death
Subgroup PHazard ratio (95% CI)n
LA intervention group only (n=207; per 5 en% increase in n-6 LA†)
Alcohol use* (kcal/day; 1 kcal=4.18 kJ)
<0.011.70 (1.18 to 2.46)79Moderate/heavy (>200 kcal/day)
0.711.09 (0.69 to 1.72)71Light (<200 kcal/day )
0.251.40 (0.79 to 2.50)57None
Smoking status*
0.011.55 (1.12 to 2.12)147Yes
0.751.08 (0.67 to 1.68)60No
Whole sample (n=429; per 5 en% increase in unspecified PUFA)
Alcohol use* (kcal/day)
<0.011.66 (1.23 to 2.25)162Moderate/heavy (>200 kcal/day)
0.051.43 (1.00 to 2.04)150Light (<200 kcal/day)
0.600.91 (0.65 to 1.28)117None
Smoking status*
<0.011.46 (1.15 to 1.86)301Yes
0.621.08 (0.79 to 1.49)128No
En%=percentage of food energy.
*Assessed at acute hospital admission.
†From safflower oil.
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Figures
Fig 1 Trial profile. An intention to treat analysis included all randomized participants. Participant exclusion data from before
randomization were not recovered. Numbers lost to follow-up were comparable in the two groups; reasons for dropout were
not recovered
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Fig 2 Kaplan-Meier estimates of five year cumulative death rates after randomization to the intervention or control group.
Results of Cox proportional hazards model include all follow-up data (≤83 months) on an intention to treat basis
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Fig 3 Updated meta-analysis of effects of LA selective interventions and mixed n-3/n-6 PUFA interventions on risk of death
from coronary heart disease. LA selective interventions selectively increased n-6 LA without a concurrent increase in n-3
PUFAs. Mixed PUFA interventions increased n-3 PUFAs and n-6 LA. PUFA interventions replaced high SFA control diets
in each trial. *Significant heterogeneity between groups. Full methods and results in part 8 of the web appendix
Fig 4 Updated meta-analysis of effects of LA selective interventions and mixed n-3/n-6 PUFA interventions on risk of
cardiovascular death. LA selective interventions selectively increased n-6 LA without a concurrent increase in n-3 PUFAs.
Mixed PUFA interventions increased n-3 PUFAs and n-6 LA. PUFA interventions replaced high SFA control diets in each
trial. *Significant heterogeneity between groups. Full methods and results in part 8 of the web appendix
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Fig 5 Proposed mechanistic model linking dietary LA to cardiovascular disease pathogenesis.34-48 Conversion of LA to
OXLAMs can proceed enzymatically, or via free radical mediated oxidative stress. Major sources of oxidative stress such
as cigarette smoking and chronic alcohol exposure facilitate LA oxidation and production of oxidized low density lipoprotein
(LDL). OXLAMs are the most abundant oxidized fatty acids in oxidized LDL and in atherosclerotic lesions. OXLAMs have
been mechanistically linked to cardiovascular pathogenesis via multiple mechanisms. LDL=low density lipoprotein;
CD36=cluster of differentiation 36 scavenger receptor; LOX 1=lectin like oxidized LDL receptor 1
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