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Markers of Endogenous Desaturase Activity and Risk of
Coronary Heart Disease in the CAREMA Cohort Study
Yingchang Lu
1,2
*, Anika Vaarhorst
3
, Audrey H. H. Merry
4
, Martijn E. T. Dolle
´
2
, Robert Hovenier
1
,
Sandra Imholz
2
, Leo J. Schouten
5
, Bastiaan T. Heijmans
3
, Michael Mu
¨ller
1
, P. Eline Slagboom
3
, Piet A. van
den Brandt
4,5
, Anton P. M. Gorgels
6
, Jolanda M. A. Boer
2
, Edith J. M. Feskens
1
*
1Division of Human Nutrition, Wageningen University and Research Center, Wageningen, The Netherlands, 2National Institute for Public Health and the Environment
(RIVM), Bilthoven, The Netherlands, 3Department of Molecular Epidemiology, Leiden University Medical Center, Leiden, The Netherlands, 4Department of Epidemiology,
CAPHRI School for Public Health and Primary Care, Maastricht University, Maastricht, The Netherlands, 5Department of Epidemiology, GROW School of Oncology and
Developmental Biology, Maastricht University, Maastricht, The Netherlands, 6Department of Cardiology, University Hospital Maastricht, Maastricht, The Netherlands
Abstract
Background:
Intakes of n-3 polyunsaturated fatty acids (PUFAs), especially EPA (C20:5n-3) and DHA (C22:6n-3), are known to
prevent fatal coronary heart disease (CHD). The effects of n-6 PUFAs including arachidonic acid (C20:4n-6), however, remain
unclear. d-5 and d-6 desaturases are rate-limiting enzymes for synthesizing long-chain n-3 and n-6 PUFAs. C20:4n-6 to
C20:3n-6 and C18:3n-6 to C18:2n-6 ratios are markers of endogenous d-5 and d-6 desaturase activities, but have never been
studied in relation to incident CHD. Therefore, the aim of this study was to investigate the relation between these ratios as
well as genotypes of FADS1 rs174547 and CHD incidence.
Methods:
We applied a case-cohort design within the CAREMA cohort, a large prospective study among the general Dutch
population followed up for a median of 12.1 years. Fatty acid profile in plasma cholesteryl esters and FADS1 genotype at
baseline were measured in a random subcohort (n = 1323) and incident CHD cases (n = 537). Main outcome measures were
hazard ratios (HRs) of incident CHD adjusted for major CHD risk factors.
Results:
The AA genotype of rs174547 was associated with increased plasma levels of C204n-6, C20:5n-3 and C22:6n-3 and
increased d-5 and d-6 desaturase activities, but not with CHD risk. In multivariable adjusted models, high baseline d-5
desaturase activity was associated with reduced CHD risk (Pfor trend = 0.02), especially among those carrying the high
desaturase activity genotype (AA): HR (95% CI) =0.35 (0.15–0.81) for comparing the extreme quintiles. High plasma DHA
levels were also associated with reduced CHD risk.
Conclusion:
In this prospective cohort study, we observed a reduced CHD risk with an increased C20:4n-6 to C20:3n-6 ratio,
suggesting that d-5 desaturase activity plays a role in CHD etiology. This should be investigated further in other
independent studies.
Citation: Lu Y, Vaarhorst A, Merry AHH, Dolle
´MET, Hovenier R, et al. (2012) Markers of Endogenous Desaturase Activity and Risk of Coronary Heart Disease in the
CAREMA Cohort Study. PLoS ONE 7(7): e41681. doi:10.1371/journal.pone.0041681
Editor: Robert Clarke, University of Oxford, United Kingdom
Received April 15, 2012; Accepted June 24, 2012; Published July 23, 2012
Copyright: ß2012 Lu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by grant 2006B195 of the Netherlands Heart Foundation. The monitoring project on cardiovascular disease risk factors was
financially supported by the Ministry of Public Health, Welfare and Sports of the Netherlands. The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: kevin.lu@wur.nl (YL); edith.feskens@wur.nl (EJMF)
Introduction
Polyunsaturated fatty acids (PUFAs) are generally believed to
reduce coronary heart disease (CHD) risk [1,2,3,4]. Intakes of n-
3 PUFAs, especially eicosapentaenoic acid (EPA, C20:5n-3) and
docosahexaenoic acid (DHA, C22:6n-3) present in fish oil, are
confirmed to prevent fatal CHD and sudden cardiac death in
both observational studies and large-scale randomized controlled
trials (RCTs) [1,3]. However, direct evidence for the preventive
effect of n-3 PUFAs on non-fatal CHD was only recently
observed in some, but not all, large-scale RCTs [5,6,7]. The
replacement of saturated fatty acids by n-6 PUFAs protected
against incident CHD in a recent meta-analysis including 8
RCTs [8]. As some of these RCTs also included n-3 PUFAs in
addition to n-6 PUFAs [2,8], the effects specific to n-6 PUFAs,
however, remain unclear.
The fatty acid profile of various biological tissues is often used
as a biomarker of dietary fatty acid intake. Adipose tissue
reflects the intake of past months to years, while erythrocyte
membranes, and plasma or serum phospholipids or cholesteryl
esters reflect the intake of several weeks [9,10,11]. However, the
PUFA profile in biological tissues does not only reflect dietary
intake, but is also strongly dependent on the endogenous
metabolism of PUFAs [10,12]. Therefore, PUFA biomarkers in
biological tissues mirror the internal PUFA exposure that may
be biologically more relevant. Several PUFAs can be endoge-
nously synthesized by a series of alternate desaturation and
PLoS ONE | www.plosone.org 1 July 2012 | Volume 7 | Issue 7 | e41681
elongation processes [12,13]. The d-5 desaturase and d-6
desaturase are rate-limiting enzymes for synthesizing long-chain
n-3 and n-6 PUFAs (Figure 1) [12,14,15,16]. They are encoded
by the FADS1 and FADS2 genes on chromosome 11 (11q12–
13.1), respectively [12,17]. Potential functional genetic variants
in these genes have been identified including rs174547 [18], and
confirmed in recent genome-wide association studies [19,20,21].
They have an impact on FADS1 mRNA abundance
[22,23,24,25,26], and, as a result, on desaturase activity, plasma
PUFA levels, and endogenous PUFA pools
[18,19,20,21,26,27,28,29]. Since it is impractical to directly
assay the enzyme activities of d-5 and d-6 desaturase in humans
[12,14,15,29], especially in large-scale epidemiological studies,
their activities have traditionally been estimated by using PUFA
product-to-precursor ratios [11,21,27,28].
Although few prospective cohort studies have investigated
PUFA biomarkers in relation to the incidence of CHD [30], the
relation with PUFA product-to-precursor ratios as markers of
desaturase activities has, to the best of our knowledge, never been
evaluated. In this prospective cohort study, we therefore aim to
investigate whether C20:4n-6 to C20:3n-6 and C18:3n-6 to
C18:2n-6 ratios, as respective markers of d-5 and d-6 desaturase
activity, influence CHD risk.
Materials and Methods
Study Population
We conducted a case-cohort study within the Monitoring
Project on Cardiovascular Disease Risk Factors 1987–1991 [31],
one of the two monitoring studies that were included in the
Cardiovascular Registry Maastricht (CAREMA) study. The
CAREMA study was described in detail before [32,33]. In total,
12,486 men and women, born between 1927 and 1967 and living
in the Maastricht area, participated in the Monitoring Project on
Cardiovascular Disease Risk Factors and had given informed
consent to retrieve information from the municipal population
registries and from the general practitioner and specialist. The
Medical Ethics Committee of the Netherlands Organization for
Applied Scientific Research (TNO) approved the study protocol
and all participants signed an informed consent form.
Cardiological Follow-up
The cardiologic follow-up has been described before [32]. In
brief, 97.6% of the CAREMA members could be found by linking
the cohort to the hospital information system of University
Hospital Maastricht (UHM). They were linked to the cardiology
information system of the Department of Cardiology to obtain
information about the occurrence of myocardial infarction (MI),
unstable angina pectoris (UAP), coronary artery bypass grafting
(CABG), or percutanous transluminal coronary angioplasty
Figure 1. Effect of genotypes of rs174547 on synthesis of PUFAs in the n-3 and n-6 pathways. Measurements of n-3 and n-6
polyunsaturated fatty acid (PUFA) levels in plasma cholesteryl esters in the sub-cohort of CAREMA study (n = 1246, Table 2). The three bars in each of
the smaller plots represent levels of fatty acids (%) in individuals who carry AA, AG and GG genotypes of rs174547, respectively.
doi:10.1371/journal.pone.0041681.g001
FADS1 and CHD Risk
PLoS ONE | www.plosone.org 2 July 2012 | Volume 7 | Issue 7 | e41681
surgery (PTCA). For participants who died, the cause of death was
obtained from Statistics Netherlands. In addition, the CAREMA
cohort was linked to the hospital discharge registry of the UHM to
increase the completeness of the cardiologic follow-up. Follow-up
ended on 31 December 2003 with a median follow-up of 12.1 yrs
(range: 0.0–16.9 yrs).
Subcohort and Incident CHD Selection for Case-cohort
Design
For the present study, participants who were younger than
30 years at baseline (n = 2204), had a history of MI, UAP, CABG,
or PTCA before baseline (n = 118), or were lost to follow-up (n = 2)
were excluded. Thus, the eligible cohort consisted of 10,164
participants. All 620 participants who developed incident CHD
during follow-up (315 MIs, 244 UAPs and 61 CHD deaths) were
included in the case-cohort study. From the eligible cohort, 1483
participants were randomly drawn as a subcohort [34]. By
randomly selecting a subcohort and using the specific statistics for
this type of research design, the results are expected to be
extrapolated to the entire cohort without the need of biomarker
measurements in the entire cohort [11,34,35,36].
Risk Factor Determination
At baseline, all participants filled in a questionnaire on life-
style characteristics, medical history, and parental history of MI.
During a medical examination, information was collected on
blood pressure, height, and weight. In addition, non-fasting blood
samples were collected using EDTA tubes. The blood was
centrifuged for 10 minutes at 1000 rpm and fractioned into
blood plasma, white blood cells and erythrocytes and subse-
quently stored at 220uC. Within three weeks, the plasma
samples were transported to the Lipid Reference Laboratory of
the University Hospital Dijkzigt (LRL) in Rotterdam where the
total and HDL-cholesterol levels were determined using a
CHOD-PAP method [37]. The LRL in Rotterdam is a
permanent member of the International Cholesterol Reference
Method Laboratory Network.
Fatty Acid Determination
Fatty acids from plasma cholesteryl esters were quantified by
gas-liquid chromatography between 2010 and 2011 at the
Department of Human Nutrition of Wageningen University.
The case and non-case samples were evenly distributed among
the different batches and the assay sequence within each batch
was random. The solid-phase extraction method was used to
separate the cholesteryl ester fraction from total plasma lipid
extracts. Fatty acid methyl esters were prepared by incubating
isolated cholesteryl esters with acidified methanol. Peak retention
times and area percentages of total fatty acids were identified by
using known cholesteryl ester standards (mixture of FAME
components from Sigma (MO) and NuChek (MN)) and analyzed
with the Agilent Technologies ChemStation software (Agilent,
Amstelveen, The Netherlands). For certain fatty acids, the values
were too low to be reliably detected in some subjects, and ‘‘0’’
was assigned to their values. Interassay coefficients of variance in
fatty acids in plasma cholesteryl esters were 1.68% for C16:0,
1.01% for C18:2n-6, 1.88% for C20:4n-6, and 5.02% for
C22:6n-3, respectively. Fatty acid product-to-precursor ratios
were calculated, i.e. C20:4n-6 to C20:3n-6 to reflect d-5
desaturase activity, and C18:3n-6 to C18:2n-6 to reflect d-6
desaturase activity (Figure 1). The 20 subjects with a ‘‘0’’ value
for C20:3n-6 were not included in the analyses for the C20:4n-6
to C20:3n-6 ratio, reflecting d-5 desaturase activity. Information
on plasma fatty acids was available on 1323 subcohort members
and 537 CHD cases.
DNA Extraction and Genotyping
DNA was extracted from the white blood cell fraction (buffy
coats), using a standard procedure [38]. The resulting DNA pellet
was dissolved in TE buffer and DNA concentrations were
determined using the Nanodrop ND1000 Spectrophotometer.
The single nucleotide polymorphism (SNP) of rs174547 in the
FADS1 gene was selected based on its association with blood
cholesterol and triglyceride levels in a genome-wide association
study [23]. This SNP is in high linkage disequilibrium (D9= 1 and
r
2
$0.8) with several other SNPs around the FADS1 and FADS2
gene region, which have an impact on mRNA abundance of
FADS1 [22,23,24,25], desaturase activity, plasma PUFA levels,
and endogenous PUFA pools [18,19,20,21,26,27,28,29].
Rs174547 was genotyped entirely independent of case and non-
case status using the iPLEX Gold chemistry of Sequenom’s
MassARRAY platform (San Diego, CA) at the Leiden University
Medical Center. Sequenom’s MassARRAYHAssay Design 3.1
Software was used for SNP assay design, and Sequenom’s
SpectroTyper 4.0 software was used to call genotypes automat-
ically, followed by manual review. The total genotyping success
rate was 93%. Among the subjects who were measured for plasma
fatty acid levels, information on rs174547 genotype was available
for 1246 subcohort members and 492 CHD cases. The genotype
distribution was consistent with Hardy-Weinberg equilibrium
expectations.
Statistical Analysis
Generalized linear models adjusted for age and sex were used
to study the relations of rs174547 genotypes with PUFAs and
PUFA ratios. Cox proportional hazards models adapted for the
case-cohort design according to the Prentice’s method [35] were
used to calculate hazard ratios (HRs) as measures for relative risk
[36]. All the major predictors satisfied the proportional hazard
assumption (data not shown). We estimated hazard ratios for
quintiles of fatty acids (expressed as the percentage of total fatty
acids present in the chromatogram) and ratios of C20:4n-6 to
C20:3n-6 and C18:3n-6 to C18:2n-6 based on subcohort
distributions, and the respective lowest quintile was used as
reference. The base models included age and sex. Additional
models were further adjusted for covariates from the Third
Report of the National Cholesterol Education Program Expert
Panel on Detection, Evaluation, and Treatment of High Blood
Cholesterol in Adults (ATP III) risk score based on the
Framingham cohort (current smoking, systolic blood pressure,
hypertensive medication use, total and HDL cholesterol levels)
with the addition of a history of diabetes [39]. The models were
also further adjusted for the total percentage of n-3 PUFAs or n-
6 PUFAs in plasma cholesteryl esters where necessary. Additional
covariates studied were parental history of MI, alcohol use and
physical activity. The significance of a linear trend across
quintiles of fatty acids and ratios of C20:4n-6 to C20:3n-6 and
C18:3n-6 to C18:2n-6 was examined by including the exposure
as a continuous variable in the model. Potential interactions
between continuous ratios of C20:4n-6 to C20:3n-6 and C18:3n-
6 to C18:2n-6 and dichotomized rs174547 genotype (homozy-
gous major allele carriers vs. minor allele carriers) were tested by
including interaction terms into the model. Statistical significance
was considered to be met with a Pvalue ,0.05 and all testing
was 2-sided. All statistical analyses were performed with SAS
version 9.1 software (SAS Institute, Cary, NC).
FADS1 and CHD Risk
PLoS ONE | www.plosone.org 3 July 2012 | Volume 7 | Issue 7 | e41681
Results
The general characteristics of the study population by
subcohort-case status are shown in Table 1. As expected, cases
were older, more frequently male, had higher blood pressure and
total cholesterol levels, lower HDL cholesterol levels, smoked more
often, and more often reported to have diabetes and a parental
history of MI.
Carrying the minor G allele of rs174547 was associated with
higher levels of substrates for desaturases (C18:2n-6, C20:3n-6,
and C18:3n-3) and lower levels of products from desaturases
(C18:3n-6, C20:4n-6, C20:5n-3, and C22:6n-3) in the plasma
cholesteryl esters. Consequently, lower C20:4n-6 to C20:3n-6 and
C18:3n-6 to C18:2n-6 ratios, as markers of d-5 and d-6 desaturase
activity, respectively, were observed in carriers of the G allele as
compared to those with the AA genotype (Table 2 and Figure 1).
A high baseline C20:4n-6 to C20:3n-6 ratio was associated with
reduced CHD risk (Table 3). A 30% reduction in CHD risk was
observed among the participants in the second, third, fourth and
fifth quintile of C20:4n-6 to C20:3n-6 ratio compared with those
in the first quintile after adjustment for age, sex, systolic blood
pressure, hypertensive medication use, current smoking, diabetes,
total cholesterol, and high-density lipoprotein cholesterol (Pfor
trend = 0.02). Although the statistical interaction between
rs174547 and d-5 desaturase activity was not significant
(P= 0.56), the protective effect of high d-5 desaturase activity
was mainly confined to subjects with the AA genotype (Table S1).
In this group, the effect was stronger with a 65% risk reduction for
the subjects in the fifth quintile compared with the first quintile (P
for trend = 0.02). Rs174547 itself was not associated with CHD
risk, the age- and sex-adjusted HR per G-allele being 0.99 (95%
CI 0.84–1.16, Table S2).
No association was observed between d-6 desaturase activity
and CHD risk (Table 3), also not after stratification by rs174547
genotype (data not shown).
The results for the four n-6 PUFAs that determine d-5 and d-6
desaturase activity are shown in Table S3. No associations with
CHD were observed for the C20 precursor (C20:3n-6) and
product (C20:4n-6, arachidonic acid) of d-5 desaturase (Figure 1),
or for the C18 precursor (C18:2n-6, linoleic acid) and product
(C18:3n-6) of d-6 desaturase (Figure 1) after adjustment for age,
sex, systolic blood pressure, hypertensive medication use, current
smoking, diabetes, total cholesterol, and high-density lipoprotein
cholesterol (Pfor trend .0.16).
Regarding the n-3 PUFAs affected by desaturases, a significant
inverse association was observed between C22:6n-3 (DHA) and
CHD risk. This association became stronger after adjustment for
plasma total and HDL cholesterol levels, and the percentages of n-
6 PUFA in plasma cholesteryl esters (Pfor trend = 0.027, Table
S4). The proportion of plasma C20:5n-3 (EPA) was not associated
with incident CHD (Pfor trend = 0.724, Table S4). No association
Table 1. Baseline characteristics of sub-cohort subjects and cases of incident coronary heart disease in the CAREMA cohort study
1
.
Subcohort
(n = 1323)
2
Cases (n = 537) Crude HR (95% CI)
3
Adjusted HR (95% CI)
4
Age (y) 45.268.5 49.767.3 1.07 (1.06–1.09) 1.05 (1.04–1.07)
Male sex 608 (46.0%) 392 (73.0%) 3.34 (2.69–4.15) 2.22 (1.66–2.99)
Total cholesterol (mmol/L) 5.761.1 6.461.2 1.71 (1.56–1.87) 1.42 (1.26–1.60)
HDL cholesterol (mmol/L) 1.260.3 1.060.2 0.04 (0.03–0.06) 0.09 (0.05–0.16)
Systolic blood pressure (mmHg) 119.2614.9 128.0616.9 1.03 (1.02–1.04) 1.02 (1.01–1.03)
Hypertensive medication use 67 (5.1%) 58 (10.8%) 2.34 (1.63–3.35) 1.27 (0.79–2.05)
Diabetes mellitus 13 (1.0%) 20 (3.7%) 5.33 (2.74–10.36) 2.83 (1.39–5.78)
Current smoking 551 (41.8%) 304 (56.7%) 1.81 (1.49–2.21) 1.72 (1.33–2.22)
Parental history of MI 452 (34.3%) 228 (42.5%) 1.40 (1.14–1.71) 1.51 (1.16–1.95)
1
Data are expressed as mean 6SD or n (%) unless otherwise indicated. HDL: high-density lipoprotein; MI: myocardial infarction; and HR (95% CI): hazard ratio and 95%
confidence interval.
2
Including 84 cases.
3
Hazard ratios were calculated per unit increase in total cholesterol, HDL cholesterol, and systolic blood pressure, and for the presence of the categorical traits.
4
All variables were added into one multivariable Cox proportional hazards model.
doi:10.1371/journal.pone.0041681.t001
Table 2. Association of rs174547 in FADS1 with baseline
PUFAs in plasma cholesteryl esters and desaturase activities in
the sub-cohort (n = 1246)
1
.
PUFA
Rs174547 P
value
2
AA (545) AG (569) GG (132)
n-6 PUFA
C18:2n-6 (%) 44.3060.27
2
44.8860.26 46.6060.54 7.48610
24
C18:3n-6 (%) 0.6060.009 0.4860.009 0.4060.019 6.87610
228
C20:3n-6 (%) 0.4260.005 0.4360.005 0.4460.010 0.051
C20:4n-6 (%) 4.2960.05 3.5660.05 2.8960.09 3.92610
246
n-3 PUFA
C18:3n-3 (%) 0.4060.005 0.4160.005 0.4560.010 3.28610
24
C18:4n-3 (%)
3
0.1860.007 0.1860.007 0.1760.014 0.708
C20:5n-3 (%) 0.5660.01 0.4660.01 0.4060.03 8.71610
28
C22:6n-3 (%) 0.3460.006 0.3160.006 0.3060.013 0.005
d-5
4
10.6560.09 8.5960.09 6.8660.19 6.40610
285
d-6
4
0.01460.0002 0.01160.0002 0.00960.0005 2.51610
227
1
77 subjects in the subcohort had missing values for rs174547. PUFAs:
polyunsaturated fatty acids.
2
General linear models were used, and all values are mean 6SEM, adjusted for
age and sex.
3
Only few subjects were successfully measured (AA = 161, AG = 185, and
GG = 42).
4
d-5 and d-6 desaturase activities were assessed by the ratio of C20:4n-6 to
C20:3n-6 and C18:3n-6 to C18:2n-6 in plasma cholesteryl esters, respectively.
doi:10.1371/journal.pone.0041681.t002
FADS1 and CHD Risk
PLoS ONE | www.plosone.org 4 July 2012 | Volume 7 | Issue 7 | e41681
was observed between C18:3n-3 (a-linolenic acid) and CHD risk
(data not shown). To explore whether there is any independent
effect of C20:4n-6 to C20:3n-6 ratio on CHD beyond DHA, we
additionally adjusted the models in Table 3 for percentages of
DHA. The association between C20:4n-6 to C20:3n-6 ratio and
CHD risk attenuated, but remained highly significant, especially
among the AA carriers of rs174547 (HR:95% CI = 0.44:0.19–1.04
for comparing the extreme quintiles, Table S1).
Additional adjustment for parental history of MI, alcohol use or
physical activity did not materially change any of the aforemen-
tioned associations (data not shown).
Discussion
In this prospective cohort study, we observed an inverse
association between C20:4n-6 to C20:3n-6 ratio, as the marker
of d-5 desaturase activity, and incident CHD risk, but no
association with C18:3n-6 to C18:2n-6 ratio, as the marker of d-
6 desaturase activity. This association was partly mediated by
DHA. Furthermore we confirmed associations of rs174547 in the
FADS1 gene with plasma PUFA levels and C20:4n-6 to C20:3n-6
ratio [18,19,20,21,27,28]. Consistent with the established cardio-
vascular protective effects of n-3 PUFAs [1,3], and especially tissue
DHA [4,30], high DHA in plasma cholesteryl esters was associated
with a reduced CHD risk. However, no association was observed
between arachidonic acid or other n-6 PUFAs related to d-5 or d-6
desaturase activity in plasma cholesteryl esters and CHD risk.
Common genetic variants (including rs174547) in the FADS
gene region have been associated with plasma lipid levels (total,
LDL and HDL cholesterol, triglycerides, phospholipids and
sphingolipids) [19,21,23,40,41], glycemic traits (fasting glucose
and beta-cell function) [26], and resting heart rate [42] in recent
genome-wide association studies. However, none of them have
been associated with CHD risk directly [40,43]. This was also the
case in our relatively large prospective study. In contrast, when
using the estimated d-5 desaturase activity based on the fatty acid
proportion in plasma cholesteryl esters, we found a significant
inverse association with incident CHD. This seems contradictory,
as a strong association between rs174547 genotypes and estimated
d-5 desaturase activities was observed. However, the reduced risk
was already observed with relatively low d-5 desaturase activities
(the second quintile) and remained constant over the following
quintiles. Therefore, the majority of the participants with the GG
genotype of rs174547 might have sufficient d-5 desaturase activity
to protect them from CHD. This might explain why no association
between rs174547 genotypes and CHD risk was found. Both
rs174547 genotypes and C20:4n-6 to C20:3n-6 ratio reflect
endogenous d-5 desaturase activity, but from two different
perspectives. The former can be regarded as the desaturase effect
conferred by a single common genetic variant in the FADS1 gene
[20,26,27,28,29], and the latter as an approximate estimation of
full desaturase activity [21,27,28]. Their combination might
provide the most accurate estimate of d-5 desaturase activity.
This might explain the stronger CHD risk reduction with high d-5
desaturase activity in the subjects who inherited the AA genotype.
The exact biological mechanisms that link d-5 desaturase
activity with CHD risk are still not well understood. Arachidonic
acid, EPA, and DHA are currently considered to be potentially
involved directly in the pathogenesis of CHD through thrombotic,
inflammatory, arrhythmic and/or lipid regulatory pathways
[1,3,12,13,44,45,46]. d-5 Desaturase is the key enzyme synthesiz-
ing these PUFAs, while d-6 desaturase is important at the
beginning of the n-3 and n-6 PUFA synthetic pathways [14,15].
Therefore, it is biologically plausible that CHD risk could be
influenced by d-5 desaturase activity, but not by d-6 desaturase
activity [12,13] as was shown in our data. The non-significance of
Table 3. Association between baseline d-5 and d-6 desaturase activity and incident coronary heart disease (CHD).
Quintile of d-5 desaturase activity
1
P
value for
trend
2
First (6.45) Second (7.93) Third (9.07) Fourth (10.32) Fifth (12.52)
Incident CHD, n 155 117 94 93 67
Model 1
3
1 0.70 (0.51–0.97) 0.60 (0.42–0.83) 0.60 (0.43–0.83) 0.49 (0.34–0.70) ,0.0001
Model 2
4
1 0.75 (0.54–1.06) 0.66 (0.46–0.94) 0.57 (0.39–0.82) 0.51 (0.35–0.75) ,0.0001
Model 3
5
1 0.68 (0.47–0.98) 0.66 (0.45–0.96) 0.69 (0.46–1.01) 0.68 (0.45–1.02) 0.0249
Model 4
6
1 0.71 (0.49–1.03) 0.70 (0.48–1.04) 0.74 (0.50–1.09) 0.77 (0.50–1.18) 0.1114
Quintile of d-6 desaturase activity
1
P
value for
trend
2
First (0.0055)Second (0.0084) Third (0.0104) Fourth (0.0132) Fifth (0.019)
Incident CHD, n 92 99 93 122 131
Model 1
3
1 0.99 (0.69–1.42) 0.87 (0.60–1.25) 1.09 (0.76–1.55) 1.03 (0.73–1.45) 0.606
Model 2
4
1 1.03 (0.70–1.51) 0.89 (0.61–1.31) 1.07 (0.73–1.58) 0.93 (0.63–1.36) 0.627
Model 3
5
1 1.07 (0.71–1.63) 0.86 (0.55–1.33) 1.11 (0.73–1.69) 0.96 (0.63–1.47) 0.897
1
d-5 and d-6 desaturase activities were assessed by the ratio of C20:4n-6 to C20:3n-6 and the ratio of C18:3n-6 to C18:2n-6 in plasma cholesteryl esters, respectively and
median ratios in each quintile are listed between brackets.
2
From models with desaturase activity included as a continuous variable.
3
Model 1 was adjusted for age and sex.
4
Model 2 was adjusted for age, sex, systolic blood pressure, hypertensive medication use, current smoking, and diabetes.
5
Model 3 was adjusted for all covariates in model 2, total cholesterol, and high-density lipoprotein cholesterol.
6
Model 4 was adjusted for all covariates in model 3 and percentages of C22:6n-3 (DHA) in plasma cholesteryl esters.
doi:10.1371/journal.pone.0041681.t003
FADS1 and CHD Risk
PLoS ONE | www.plosone.org 5 July 2012 | Volume 7 | Issue 7 | e41681
d-6 desaturase activity on CHD risk is perhaps, also compatible
with the reported normal viability and life span of d-6 desaturase
knockout mice [47]. Increased d-5 desaturase activity might
contribute to the intracellular increase of EPA and especially
arachidonic acid levels [16]. In non-fish eating populations,
arachidonic acid is the predominant tissue very-long-chain PUFA,
reaching 80% of the total very-long-chain PUFA [30,44]. Despite
the potential pro-coagulant and pro-inflammatory effects of
increased exposures to arachidonic acid and its derived eicosanoid
metabolites [2,13,44,45,46,48,49], there is no evidence of
increased CHD risk with <5–7 times habitual arachidonic acid
intake based on short-term small-scale controlled feeding studies
[2,50,51,52,53,54]. Tissue arachidonic acid levels are generally
not associated with CHD risk [30]. This was supported by our
finding based on the fatty acid profile in plasma cholesteryl esters,
which suggests that arachidonic acid does not mediate the
observed association between C20:4n-6 to C20:3n-6 ratio, as the
marker of d-5 desaturase activity, and CHD risk.
Increased d-5 desaturase activity (C20:4n-6 to C20:3n-6 ratio)
was associated with increased plasma levels of EPA and DHA.
Our results showed that a possible protective effect of increased d-
5 desaturase activity on CHD may partly be mediated by
increased endogenous exposure to DHA. The observation that
increased DHA levels associated with increased d-5 desaturase
activity protect against CHD is consistent with the established
cardiovascular protective effect of increased n-3 PUFA exposure
(EPA and/or DHA) [1,3]. Accumulating evidence from observa-
tional studies suggests that DHA might be more protective for
CHD than EPA [4,30], which is consistent with our findings.
However, EPA and DHA are usually correlated with each other in
tissues, and their potential effects cannot be easily discerned. More
research on this issue is therefore warranted. In addition to blood
triglyceride lowering and HDL cholesterol increasing effects of
EPA and DHA, n-3 PUFAs have long been observed to have anti-
thrombotic, anti-inflammatory, anti-arrhythmic, and blood pres-
sure-lowering effects in humans even though the underlying
mechanisms for these effects are incompletely understood
[1,3,12,13,46]. Interestingly, the protective effects on fatal CHD
and sudden cardiac death have been shown to level off with a
modest intake of EPA and/or DHA (250 mg/day), and little
additional benefit was observed with higher intakes [1]. This is also
consistent with our results for C20:4n-6 to C20:3n-6 ratio as the
marker of d-5 desaturase activity. Nevertheless, there might be
other unidentified pleiotropic cardiovascular protective effects of
increased d-5 desaturase activity. For example, these desaturases
are also significantly expressed in immune cells [55,56] that play
important roles in atherosclerotic CHD progression.
Our results should be interpreted in the context of several
limitations. First, our analyses were based on a single baseline
measurement of fatty acid levels in plasma cholesteryl esters that
may not accurately reflect long-term fatty acid exposures.
However, we did detect the established protective effect of DHA
against CHD [1,3,4,12,13,30]. Second, we estimated d-5 and d-6
desaturase activities based on n-6 PUFAs, while d-5 and d-6
desaturases are not only involved in n-6 PUFA conversion, but
also in n-3 PUFA conversion. However, in comparison to n-6
PUFA conversion, the amount of n-3 PUFA conversion is
relatively small [16], which should not have affected our results.
Third, other potential unmeasured environmental or physiological
factors could have confounded the observed associations. Howev-
er, the relatively large magnitude of the protective effect of
increased d-5 desaturase activity relative to the effect of other risk
factors for CHD makes confounding with other unknown risk
factors unlikely. Finally, our models that included total and HDL
cholesterol may have been over-adjusted, as these are probably
intermediates in the metabolic pathway between desaturase and
CHD risk (Note S1).
In conclusion, in this prospective cohort study, we observed a
reduced CHD risk with increased C20:4n-6 to C20:3n-6 ratio that
was partly mediated by DHA. These results suggest that d-5
desaturase activity plays a role in protecting us from CHD.
Supporting Information
Table S1 Association between baseline d-5 desaturase activity
and incident coronary heart disease according to rs174547
genotypes.
(DOCX)
Table S2 Association of rs174547 with incident coronary heart
disease (CHD) risk.
(DOCX)
Table S3 Association between baseline n-6 PUFA in plasma
cholesteryl esters (precursors and products of d5- or d6-desaturase)
and incident coronary heart disease (CHD).
(DOCX)
Table S4 Association of baseline C20:5n-3 (EPA) and C22:6n-3
(DHA) in plasma cholesteryl esters with incident coronary heart
disease (CHD).
(DOCX)
Note S1 Analysis of intermediate factors of coronary heart
disease (CHD).
(DOCX)
Acknowledgments
The authors wish to thank D Kromhout for supervision of the project and
the project steering committee consisting of (not mentioning co-authors of
this article) H. B. Bueno de Mesquita, H. A. Smit, and J. C. Seidell (project
leader). Furthermore, the authors thank epidemiologists and field-workers
of the Municipal Health Services in Maastricht for their contribution to
baseline data collection and those involved in the logistic management (A.
Jansen and J. Steenbrink) and the data management (A. Blokstra, A. van
Kessel, P. Steinberger, E. den Hoedt, I. Toxopeus, J. van der Laan). The
authors further wish to thank D. Jaspers, A. Hilton, V. Visser, P. Erkens, S.
Philippens, J. Bremen, B. Bleijlevens, T. van Moergastel, S. van de
Crommert for assistance in clinical data collection, and E. Vijge and A.
Engelen for preparing the plasma samples. Statistics Netherlands is
acknowledged for providing data on causes of death.
Author Contributions
Conceived and designed the experiments: YL JMAB EJMF. Performed the
experiments: AV AHHM METD RH SI LJS BTH MM PES PAvdB
APMG JMAB EJMF. Analyzed the data: YL AV RH MM JMAB EJMF.
Wrote the paper: YL AV AHHM METD LJS BTH PES JMAB EJMF.
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