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CLINICAL RESEARCH
Lipids
Trans fatty acids and mortality in patients referred
for coronary angiography: the Ludwigshafen Risk
and Cardiovascular Health Study
Marcus E. Kleber1, 2*, Graciela E. Delgado1, Stefan Lorkowski2, 3, Winfried Ma
¨rz1, 4, 5,
and Clemens von Schacky6,7
1
Vth Department of Medicine, Medical Faculty Mannheim, Heidelberg University, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany;
2
Competence Cluster of Nutrition and
Cardiovascular Health (nutriCARD), Halle-Jena-Leipzig, Germany;
3
Institute of Nutrition, Friedrich Schiller University Jena, Jena, Germany;
4
Clinical Institute of Medical and Chemical
Laboratory Diagnostics, Medical University of Graz, Graz, Austria;
5
Synlab Academy, Synlab Services GmbH, Mannheim, Germany;
6
Omegametrix, Martinsried, Germany; and
7
Department of Preventive Cardiology, Medizinische Klinik und Poliklinik I, Munich University, Munich, Germany
Received 10 November 2014; revised 6 July 2015; accepted 17 August 2015
Aims Trans fatty acids (TFAs) are generated by the food industry and also occur naturally in trace amounts in dairy products.
For the latter, beneficial health effects have been claimed, while there are numerous reports about TFA of industrial
origin being hazardous to human health. Therefore, we aimed to investigate the association of TFAwith mortality in the
Ludwigshafen Risk and Cardiovascular Health (LURIC) study.
Methods
and results
The fatty acid composition of erythrocyte membranes was analysed using the HS-Omega-3 Index
w
methodology in
3259 participants of the LURIC study at baseline. During a median of 10.0 years of follow-up, a total of 975 (29.9%)
study participants died, 614 (18.8%) from cardiovascular causes including 254 (7.8%) sudden cardiac deaths (SCDs).
Association of TFA with clinical outcome was investigated with Cox proportional hazards regression. Total TFAs
were inversely associated with mortality due to cardiovascular causes or SCD. This was mainly driven by the naturally
occurring TFA C16:1n-7t (trans-palmitoleic acid). The reduced risk of SCD associated with C16:1n-7t persisted after
multivariate adjustment with a hazard ratio of 0.63 (0.46– 0.86) for the third tertile compared with the first tertile.
There was no association of any TFA subgroup with an increased risk of adverse outcomes.
Conclusions In contrast to previous findings, the low concentrations of total TFAs found in LURIC were inversely associated with
adverse cardiac outcomes. While the naturally occurring TFA C16:1n-7t was associated with reduced risk, no increased
risk was found for industrially produced TFAs.
-----------------------------------------------------------------------------------------------------------------------------------------------------------
Keywords Trans fatty acids †Trans-palmitoleic acid †Mortality †Cardiovascular mortality †Sudden cardiac death
Introduction
Trans fatty acids (TFAs) are unsaturated fatty acids containing double
bonds in trans configuration. In animals and plants, fatty acids usually
occur in cis configuration. However, in milk, dairy products, and
meat, some trans-isomers occur naturally in small quantities. Other
TFAs are ‘man-made’ by industrial hardening of unsaturated fats. If
oils are only partially hydrogenated, a portion of cis-isomers is con-
verted into trans-isomers (industrially produced TFAs, IP-TFAs).
While beneficial health effects have been claimed for the naturally
occurring TFA vaccenic acid or trans-palmitoleic acid in some
studies,
1,2
there are numerous reports suggesting that IP-TFA may
be hazardous to human health.
3–7
Industrially produced trans fatty
acids have been linked to increased risks for a number of diseases
that are associated with modern Western lifestyle, such as cardio-
vascular disease (CVD), stroke, diabetes, infertility, Alzheimer’s dis-
ease, or certain cancers. Therefore, a range of actions has been
taken to reduce the intake of IP-TFA on a population level. These
measures are effective as demonstrated by decreasing levels of
IP-TFAs in the USA.
8
In Europe, intake of IP-TFA has traditionally
been lower compared with the USA where major sources for
TFAs have been cakes, cookies, pies and pastries.
9
*Corresponding author. Tel: +49 621 383 3019, Fax: +49 621 383 3804, Email: marcus.kleber@medma.uni-heidelb erg.de
Published on behalf of the European Society of Cardiology. All rights reserved. &The Author 2015. For permissions please email: journals.permissions@oup.com.
European Heart Journal
doi:10.1093/eurheartj/ehv446
European Heart Journal Advance Access published September 22, 2015
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It is unclear whether variations of TFAs at low concentrations are
associated with CVD risk and how the highest concentration of TFA
that may be harmless should be defined.
10
Equally open today is the
question whether the same thresholds should be applied to both
IP-TFA and ruminant TFA. Therefore, it has been the aim of our
study to investigate the association of total TFA concentrations
and individual TFA species measured in the membrane of red blood
cells with CVD and mortality in a large and well-characterized
clinical cohort, the Ludwigshafen Risk and Cardiovascular Health
(LURIC) study.
Methods
Study populations
The LURIC study included 3316 Caucasians hospitalized for coronary
angiography between 1997 and 2000 at a tertiary care centre in south-
western Germany.
11
Clinical indications for angiography were chest
pain or a positive non-invasive stress test suggestive of myocardial is-
chaemia. To limit clinical heterogeneity, individuals suffering from acute
illnesses other than acute coronary syndrome, chronic non-cardiac dis-
eases, and a history of malignancy within the past 5 years were excluded.
The study was approved by the ethics committee at the ‘Landesa¨rzte-
kammer Rheinland-Pfalz’ and was conducted in accordance with the
‘Declaration of Helsinki’. Informed written consent was obtained from
all participants.
Laboratory procedures
Fasting blood samples were obtained by venipuncture in the early morn-
ing. Cholesterol and triglycerides (TG) were measured with enzymatic
reagents from WAKO (Neuss, Germany) on an Olympus AU640 ana-
lyser (Centre Valley, PA). Lipoproteins were separated by a combined
ultracentrifugation– precipitation method (b-quantification) as de-
scribed previously.
11
Erythrocyte fatty acid composition was analysed
according to the HS-Omega-3 Index
w
methodology as described previ-
ously.
12
Fatty acid methyl esters were generated from erythrocytes by
acid transesterification and analysed by gas chromatography using a
GC2010 gas chromatograph (Shimadzu, Duisburg, Germany) equipped
with a 100-m SP2560 column (Supelco, Bellefonte, PA) using hydrogen
as carrier gas. Fatty acids were identified by comparison with a standard
mixture of fatty acids characteristic of erythrocytes. Results are given as
a percentage of total identified fatty acids after response factor correc-
tion. The chromatographic conditions allowed to separate the C16:1
trans-isomers and three trans-isomers of C18:2n6 (C18:2n6tt,
C18:2n6ct, and C18:2n6tc). The individual trans-isomers of C18:1 (i.e.
C18:1
d
6 through C18:1
d
13) could not be separated, but appeared as
two blended peaks, eluting just ahead of oleic acid. The areas of these
two peaks were added and referred to as C18:1t. The sum of the five
TFAs had a coefficient of variation of 7%. The sum of C18:2n6tt,
C18:2n6ct, and C18:2n6tc is referred to as ‘C18:2t’, whereas the sum
of all measured TFAs is referred to as total TFAs.
Clinical definitions
Coronary artery disease (CAD) was defined as the presence of a visible
luminal narrowing (.20% stenosis) in at least one of the 15 coronary
segments according to the classification of the American Heart Associ-
ation. Diabetes mellitus was defined according to 2010 guidelines of the
American Diabetes Association as increased fasting (≥126 mg/dL) and/
or post-challenge (2 h after the 75 g glucose load ≥200 mg/dL) glucose
and/or elevated glycated haemoglobin (≥6.5%) and/or history of dia-
betes. Blood pressure was measured with an automated oscillometric
device (Omron MX4, Omron Healthcare GmbH, Hamburg, Germany)
while supine for at least 10 min. At least three consecutive measure-
ments of systolic and diastolic blood pressures were taken 30 s apart.
Hypertension was defined as a systolic and/or diastolic blood pressure
.140 and/or .90 mmHg or a significant history of hypertension. The
glomerular filtration rate was estimated by using the 2012 CKD-EPI
eGFRcreat-cys equation.
13
Self-reported physical exercise was mea-
sured on a scale ranging from 25 (extremely low) to +5 (extremely
high, athlete).
Definition of clinical endpoints
Information on vital status was obtained from local registries. Death cer-
tificates and medical records of local hospitals and autopsy data were
reviewed independently by two experienced clinicians who were
blinded to patient characteristics and who classified the causes of death.
In cases of disagreement or uncertainty concerning the coding of a spe-
cific cause of death, the decision was made by a principal investigator
(W.M.). During a median follow-up of 10.0 years (range: 0.1–11.9
years), 995 (30%) participants died. Cardiovascular mortality (CVM) in-
cluded the following categories: sudden cardiac death [SCD; n¼259
(12.8%)], fatal myocardial infarction [n¼106 (3.20%)], death due to
congestive heart failure [n¼148 (4.46%)], death immediately after
intervention to treat CAD [n¼26 (0.78%)], fatal stroke [n¼61
(1.84%)], and other causes of death due to CAD [n¼19 (0.57%)]. Infor-
mation for mortality is complete for all participants. The cause of death
of 23 individuals was unknown and these patients were included in cal-
culations of all-cause mortality but not in calculations considering differ-
ent causes of death. Trans fatty acid measurements were available for
3259 study participants.
Statistical analyses
The primary aim of our study was to examine the association of TFA
with fatal endpoints, namely all-cause mortality, CVM, and SCD, which
we did by building Cox proportional hazard models. As the distribution
of individual TFA was slightly skewed to the left, wechose to stratify our
patients into tertiles of TFAs. The first tertile of total TFAs included all
values ≤0.83% of all fatty acids in the membrane of erythrocytes, the
second tertile encompassed all values from 0.84 to 1.08%, and the third
tertile included all values ≥1.09%. The functional form of covariates was
analysed by calculating Martingale residuals and the proportional hazard
assumption was checked by examination of scaled Schoenfeld residuals.
Adjustments were carried out including known confounding variables
for CVM.
We also adjusted the data distribution by inverse probability weight-
ing so that each tertile’s weighted distribution matched that of the whole
cohort, thereby balancing the subgroups for the confounding variables.
We plotted the reweighted distribution of confounding variables for
each C16:1n7t tertile to check whether the balancing worked. A
weighted Cox model was calculated and we report the result of the ro-
bust score test as implemented in the coxph function in R that corre-
sponds to a log-rank test corrected for weighting.
The distribution of all variables was examined visually by viewing his-
tograms and by comparing mean and median values. Continuous data
are presented as the mean +standard deviation when normally distrib-
uted or as the median and 25th and 75th percentile for non-normally
distributed variables. Categorical data are presented as percentages.
Statistical differences between groups and continuous variables were
determined using analysis of variance. Non-normally distributed vari-
ables were log-transformed before entering analysis in order to achieve
an approximately normal distribution. The x
2
test was used for categor-
ical variables. Correlation and partial correlation adjusted for age and
M.E. Kleber et al.Page 2 of 7
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sex between TFAs and biomarkers were analysed by Spearman’s r. All
tests were two sided and Bonferroni-adjusted thresholds for signifi-
cance were calculated as indicated for the respective tables to correct
for multiple testing. All analyses were carried out using the SPSS 21.0
statistical package (IBM SPSS, USA) and R v3.1.1 (http://www.r-project.
org).
Results
Trans fatty acids were measured in 3259 participants of the LURIC
study who all underwent coronary angiography. They ranged from
0.27 to 2.40% of total fatty acids in erythrocyte membranes with a
mean of 0.96 +0.26%. Total TFAs were composed of 61.8 +0.1%
C18:1t isomers, 22.4 +0.1% C18:2t isomers, and 15.8 +0.1%
C16:1n-7t isomers. Study demographics are shown in Table 1.
Correlation of total trans fatty acids with
biomarkers at study baseline
Study characteristics according to tertiles of total TFAs as well as
specific subgroups of TFAs are shown in Supplementary material
online, Tables S1 – S4. Partial correlation coefficients adjusted for
age and gender are shown in Table 2.
Percentages of total TFAs were directly correlated with LDL-C
and inversely correlated with body mass index (BMI), waist-to-hip
ratio, physical exercise, blood pressure, TG, and markers of glucose
metabolism (fasting glucose, fasting insulin, HbA1c, and homeostasis
model assessment (HOMA) index). These correlations were largely
similar for the subgroups of C16:1n-7 and C18:1t isomers, but not
for the subgroup of C18:2t isomers (Supplementary material online,
Table S5). About 50% of the LURIC participants were receiving
lipid-lowering therapy (mostly statins) at baseline with a lower per-
centage in the higher tertiles of total TFAs. We, therefore, repeated
the analyses restricted to those patients not receiving lipid-lowering
therapy (Supplementary material online, Table S6). Several associa-
tions then turned insignificant, which is likely due to the reduced
power following the approximate bisection of the sample. Most
notably, however, the strong associations of TFAs with BMI, TG,
blood pressure, markers of glucose metabolism, and diabetes melli-
tus remained almost unchanged.
Trans fatty acids and mortality
We examined the association of TFA with all-cause mortality, CVM,
and SCD by means of Cox regression adjusted for age and gender
(Model 1) or additionally adjusted for traditional risk factors and
markers significantly associated with TFA, namely BMI, LDL-C,
HDL-C, TG, fibrinogen, smoking, hypertension, diabetes, estimated
glomerular filtration rate, and lipid-lowering therapy (Model 2). In-
creasing total TFAs were associated with lower CVM and SCD in
Model 1 (Table 3). C16:1n-7t was associated with reduced all-cause
mortality, CVM as well as SCD in Model 1.
In Model 2, only the association of the highest tertile with de-
creased risk of SCD remained statistically significant at a hazard ratio
(HR; 95% confidence interval) of 0.63 (0.46 – 0.86), and Pfor trend
across tertiles was also significant (Table 3). Adjusted survival curves
are shown in Figure 1. The distribution of confounding variables was
balanced by inverse variance weighting. Resulting HRs were similar
to the ones obtained by simple adjustment with HR of 0.82 (0.61–
1.12) and 0.67 (0.48– 0.93) for the second and the third tertile, re-
spectively. The C18:1t and C18:2t isomers did not show any associ-
ation with endpoints except for an association of the middle tertile
of C18:1t with SCD. Of note, we did not observe any increased risk
for any of the investigated endpoints for any of the TFAs. This was
also true for the individual C18:2t isomers C18:2n6tt, C18:2n6ct,
and C18:2n6tc (Supplementary material online, Table S7).
Table 1 Study demographics
Age ( years) 62.7 +10.6
Male sex (%) 69.7
BMI (kg/m
2
) 27.5 +4.07
Waist-to-hip ratio 0.96 +0.08
Systolic BP (mmHg) 141 +23.6
Diastolic BP (mmHg) 81.0 +11.5
LDL-C (mg/dL) 116 +34.3
HDL-C (mg/dL) 38.8 +10.8
TG (mg/dL) 147 (109 – 201)
Fasting glucose (mg/dL) 102 (93.6–118)
Coronary artery disease (%) 77.9
Hypertension (%) 72.9
Diabetes (%) 39.8
Lipid-lowering therapy (%) 48.6
Shown are mean +standard deviation or median (25th –75th percentile).
BMI, body mass index; BP, blood pressure.
........................
................................................................................
Table 2 Partial correlation of total trans fatty acids
with biomarkers adjusted for age and sex
Total TFAs
RP*
BMI (kg/m
2
)20.137 ,0.001
Waist-to-hip ratio 20.042 0.018
Physical exercise 20.057 0.001
Systolic BP (mmHg) 20.065 ,0.001
Diastolic BP (mmHg) 20.075 ,0.001
LDL-C (mg/dL) 0.042 0.016
HDL-C (mg/dL) 20.012 0.507
TG (mg/dL) 20.132 ,0.001
Fasting glucose (mg/dL) 20.147 ,0.001
Fasting insulin (mmol) 20.052 0.003
HbA1c (%) 20.192 ,0.001
HOMA index 20.083 ,0.001
High-sensitivity C-reactive protein (mg/dL) 20.004 0.839
Fibrinogen (mg/dL) 20.069 ,0.001
SAA (mg/L) 0.009 0.589
NT-proBNP (ng/mL) 0.027 0.129
BMI, body mass index; BP, blood pressure; TFAs, trans fatty acids; SAA, serum
amyloid A; NT-proBNP, N-terminal of the prohormone brain natriuretic peptide.
*After Bonferroni adjustment for 16 tests, a Pvalue of 0.0031 could be regarded as
significant.
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Table 3 Association of tertiles of trans fatty acids with mortality, cardiovascular mortality, and sudden cardiac death
N
events
Total TFAs C16:1n-7t C18:1t C18:2t
Model 1 Model 2 Model 1 Model 2 Model 1 Model 2 Model 1 Model 2
HR (95% CI) PHR (95% CI) PHR (95% CI) PHR (95% CI) PHR (95% CI) PHR (95% CI) PHR (95% CI) PHR (95% CI) P
All-cause mortality
First 292 1
reference
1
reference
1
reference
1
reference
1
reference
1
reference
1
reference
1
reference
Second 399 0.87 (0.74 –
1.01)
0.065 0.91 (0.78 –
1.06)
0.241 0.85 (0.73 –
0.99)
0.037 0.89 (0.76 –
1.04)
0.130 0.87 (0.74 –
1.01)
0.073 0.88 (0.75 –
1.03)
0.106 0.89 (0.76 –
1.03)
0.139 0.92 (0.79 –
1.08)
0.315
Third 284 0.86 (0.73–
1.01)
0.072 0.92 (0.78 –
1.09)
0.345 0.81 (0.69 –
0.94)
0.005 0.88 (0.76 –
1.03)
0.116 0.90 (0.77 –
1.05)
0.186 0.94 (0.81 –
1.11)
0.476 0.96 (0.81 –
1.14)
0.630 1.00 (0.84 –
1.19)
1.000
P
trend
0.117 0.473 0.012 0.189 0.176 0.269 0.300 0.463
Cardiovascular mortality
First 196 1
reference
1
reference
1
reference
1
reference
1
reference
1
reference
1
reference
1
reference
Second 240 0.77 (0.64 –
0.93)
0.008 0.82 (0.68 –
1.00)
0.051 0.87 (0.71 –
1.05)
0.140 0.92 (0.76 –
1.12)
0.398 0.82 (0.67 –
0.99)
0.042 0.83 (0.68 –
1.01)
0.069 0.86 (0.71 –
1.05)
0.139 0.91 (0.75 –
1.11)
0.344
Third 178 0.79 (0.64–
0.98)
0.029 0.87 (0.70 –
1.08)
0.194 0.75 (0.62 –
0.91)
0.003 0.85 (0.70 –
1.04)
0.106 0.89 (0.74 –
1.09)
0.257 0.95 (0.78 –
1.16)
0.603 0.88 (0.71 –
1.08)
0.222 0.93 (0.75 –
1.15)
0.501
P
trend
0.019 0.142 0.013 0.268 0.125 0.174 0.301 0.631
Sudden cardiac death
First 100 1
reference
1
reference
1
reference
1
reference
1
reference
1
reference
1
reference
1
reference
Second 80 0.52 (0.39–
0.71)
,0.001 0.56 (0.42 –
0.76)
,0.001 0.74 (0.55 –
1.00)
0.050 0.81 (0.60 –
1.09)
0.157 0.70 (0.51 –
0.96)
0.027 0.71 (0.52 –
0.98)
0.034 0.85 (0.63 –
1.14)
0.270 0.90 (0.67 –
1.22)
0.490
Third 74 0.68 (0.50–
0.92)
0.013 0.74 (0.54 –
1.02)
0.063 0.55 (0.41 –
0.76)
,0.001 0.63 (0.46 –
0.86)
0.004 0.92 (0.70 –
1.24)
0.594 1.00 (0.74 –
1.36)
0.981 0.87 (0.63 –
1.22)
0.426 0.94 (0.67 –
1.31)
0.703
P
trend
,0.001 0.001 0.001 0.015 0.075 0.055 0.532 0.788
Model 1: adjusted for age and gender. Model 2: additionally adjusted for body mass index, LDL-C, HDL-C, logTG, log-fibrinogen, smoking, hypertension, diabetes, lipid-lowering therapy, and estimated glomerular filtration rate. A P-value of
,0.004 would be regarded as significant after Bonferroni correction for 12 tests (three outcomes and four TFA metrics).
HR, hazard ratio; TFAs, trans fatty acids; CI, confidence interval.
M.E. Kleber et al.Page 4 of 7
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We repeated the analyses for only those patients not on
lipid-lowering therapy. While the inverse association of total TFAs
with CVM became insignificant, the association of C16:1n-7t with
a reduced risk of all-cause mortality and SCD remained statistically
significant in Model 2 (Supplementary material online, Table S8).
Additional adjustment for antihypertensive medication, glycaemic
status, and alcohol intake only slightly attenuated the association
(Supplementary material online, Table S9).
Discussion
Main findings
We analysed the association of the TFA content in erythrocyte
membranes with total mortality and a number of biomarkers for
cardiovascular risk in the well-characterized LURIC cohort including
patients of Caucasian origin scheduled for coronary angiography.
Our main findings are as follows: First, the concentrations of TFAs
in erythrocyte membranes were low compared with concentrations
reported for the USA at a similar time period (e.g. mean 0.96 +
0.3% in LURIC vs. 2.68 +0.8inHarriset al.
14
). Second, none of
the TFAs showed any association to adverse outcome. Finally, there
were statistically significant inverse associations with the risk of
CVM and SCD, especially for trans-palmitoleic acid.
Trans fatty acids, coronary heart disease,
and mortality
Initially, TFAs were considered as a safe replacement for saturated
fat. However, it has soon been noted that TFA increase LDL-C while
decreasing HDL-C.
15
Many studies consistently reported
associations between TFA consumption and coronary heart disease
(CHD).
6,16
A recent meta-analysis confirmed the direct association
of dietary TFA intake with coronary outcomes while there was no
association for circulating TFA.
17
Consequently, organizations like
the World Health Organization recommended reducing TFA diet-
ary intake to ,4% and several countries like Denmark introduced
legal bans. The German Society for Nutrition went even further by
recommending a daily TFA intake of ,1% of energy.
18
Furthermore,
the FDA has concluded in June 2015 that partially hydrogenated oils
are no longer ‘Generally Recognized As Safe’.
19
While TFA concentrations in food products have declined mark-
edly in most industrialized countries over the last decades,
8,14,20,21
the decline was smaller in Eastern Europe
22
and concentrations re-
main high in several developing countries.
23
Furthermore, many
food products labelled as free of TFA still contain significant
amounts of TFAs.
24,25
Most studies suggesting increased risks associated with TFAs have
recruited patients decades ago when TFA concentrations were
higher than today.
26 –28
Furthermore, most studies were conducted
in the USA, where TFA concentrations have traditionally been high-
er compared with Europe,
29
and used questionnaires to assess TFA
intake. These estimates may not be accurate because of incomplete
or inaccurate nutrient databases and the common under-reporting
of unhealthy foods like sugar or fat.
30,31
We, therefore, investigated
the association of TFA with all-cause mortality, CVM, and SCD in
the German LURIC cohort. Trans fatty acids were measured in
erythrocyte membranes using the Omega-3 Index technology that
allows a more objective way to estimate individual nutrient intake.
We observed rather low concentrations of TFA in our patients
and found no association with mortality. On the contrary, we found
that total TFAs were inversely associated with CVM and SCD in
age- and gender-adjusted models. After adjustment for other car-
diovascular risk factors, only the association with SCD remained
statistically significant.
Individual trans fatty acid and mortality
Looking at specific subgroups of TFAs, we did not find an association
with mortality for any of the TFA species that we were able to sep-
arate. For the mostly ruminant-derived C16:1n7t, there was a strong
inverse association with CVM and SCD with significant Pfor trend
values. For the C18:1t isomers, which are a mixture of ruminant-
derived and industrially produced TFA, Pvalues for trend were
almost nominally significant, while for the exclusively industrially
produced C18:2t isomers, there was no significant trend. This could
point towards an inverse association of ruminant-derived TFA only,
with industrially produced TFA showing no association (at least at
the concentrations found here).
C16:1n-7t, or trans-palmitoleic acid, is relatively specific for dairy
products, although it can be produced by the partial hydrogenation
of vegetable oils as well. Mozaffarian et al.
1,2
have recently reported
an inverse association of C16:1n-7t with incident diabetes, which is
consistent with our findings for prevalent diabetes (Supplementary
material online, Table S2), and animal models showed that the
cis-isomer C16:1n-7c is a major signalling lipid associated with im-
proved insulin sensitivity.
32
Similarly, the trans-isomer may act as a
signalling molecule, but so far no physiologic function has been
shown for C16:1n-7t.
Figure 1 Adjusted survival curves for sudden cardiac death.
Tertiles of C16:1n7t were balanced for body mass index, LDL-C,
HDL-C, logTG, log-fibrinogen, smoking, hypertension, diabetes,
lipid-lowering therapy, and estimated glomerular filtration rate
by inverse variance weighting. The inset shows the same data on
atruncatedyaxis. Hazard ratios (95% confidence interval) for
the second and third tertile compared with the first tertile were
0.82 (0.61 – 1.12) and 0.67 (0.48 – 0.93), respectively. The P-value
of the robust score test was 0.043.
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A number of reports examined the effect of dairy fats on CHD
(reviewed in 33). Animal studies have shown beneficial effects of
trans-vaccenic acid (C18:1n-11t), which constitutes 50– 80% of
TFAs in ruminant-derived fats,
34
on post-prandial lipid metabolism
and dyslipidaemia.
35
Interestingly, a recent report has shown that
C16:1n-7t may be synthesized in humans from C18:1n-11t with
conversion rates of 17%.
36
This conversion may explain the simi-
lar albeit mostly weaker associations for the C18:1t isomers com-
pared with C16:1n-7t because C18:1n-11t constitutes one of the
major TFA summarized in this variable. Alternatively, these associa-
tions could be due to direct effects of C18:1n-11t or another
isomer.
A recent study has shown that circulating C18:2n-t/t was asso-
ciated with higher total mortality, CVM, and total CHD, whereas
C18:2n-t/c was positively related to total mortality, and nonfatal
CHD only after mutual adjustment.
37
Another study showed each
1 SD increase of plasma trans 18:2 to be associated with a 22% lower
risk of heart failure.
38
The differing results for TFA subspecies were
exploited by Liu et al.
39
to calculate a TFA index by dividing the sum
of industrially derived TFA by the sum of ruminant-derived TFA.
This TFA index was associated with 10-year CHD risk, but only
when the TFAs were measured in erythrocyte membranes.
Implications for public health
The TRANSFAIR study reported that the average TFA intake is
2.4 g/d for Germany, which is below 1% of total energy intake,
and that 79% of TFA intake was derived from milk and ruminant
fat.
40
The data had been collected in the 1990s and might, therefore,
be comparable with our LURIC patients, although the methodology
used in this study has been questioned.
41
Nevertheless, we also ob-
serve rather low TFA levels of only 0.96% of total fatty acids of
erythrocyte membranes. Our results are also in line with findings
by Kro¨ger et al.
42
who reported very similar erythrocyte concentra-
tions of C16:1n-7t and C18:1t isomers in the EPIC-Potsdam study. A
large percentage of total TFAs seems to be of natural origin and is
likely not derived from industrial processes. We observe no direct
association with mortality or CVM for any of the investigated TFA,
suggesting no need for efforts to further decreasing TFA in food in
Germany. Based on our data, it seems that an upper limit for the sum
of the trans-isomers of 18:1 and 18:2 of the mean of the upper ter-
tile, i.e. 1.04%, might be regarded as safe. However, while well within
the framework of our data, this concept also needs to be substan-
tiated by more research.
In contrast, the beneficial associations of C16:1n-7t with biomar-
kers and long-term cardiovascular outcome in our patients might al-
low speculating about the potential benefit of interventions to
increase the C16:1n-7t content of milk products or perhaps direct
supplementation. Of course, this concept has to be investigated in
more detail.
Strengths and limitations
All LURIC participants were of German origin and were recruited at
a tertiary referral centre. Therefore, our findings may not be repre-
sentative for a random population sample or applicable to other
ethnicities. Furthermore, TFAs were only measured once in baseline
samples and concentrations may vary over time due to dietary
changes, lifestyle changes, or diseases. As TFA concentrations in
our study were rather low, we could not examine the effect of high-
er concentrations (like they have been reported by previous stud-
ies) on mortality. Data on the dietary intake of fatty acids were
not available; we, therefore, cannot exclude that low TFA concen-
trations are caused by changed dietary patterns on physician advice
in patients with severe disease. Some potential confounding vari-
ables like socio-economic status, lifestyle, or drug treatment during
follow-up were also not available. The major strengthsof the LURIC
cohort are, however, the precise clinical and metabolic character-
ization of the participants including the availability of coronary
angiograms and its cross-sectional and prospective design.
Furthermore, fatty acids were measured in erythrocyte cell mem-
branes that represent fatty acid intake over the last months, as
erythrocytes have a lifespan of 120 days.
43
Conclusion
We examined the association of TFA measured in erythrocyte
membranes with plasma biomarkers and long-term outcome in
the LURIC cohort. In contrast to previous findings, we observed
that at generally low concentrations,TFAs were inversely associated
with adverse cardiac outcomes. Especially ruminant-derived TFAs
were associated with a reduced risk of CVM and SCD. Furthermore,
high TFA concentrations in our study (which are low relative to
other populations) were associated with mostly favourable meta-
bolic profiles with lower TG, lower fasting glucose, and lower blood
pressure.
Authors’ contributions
M.E.K. and G.E.D. performed statistical analysis; C.v.S. and W.M.
handled funding and supervision; C.v.S. acquired the data; C.v.S.
and W.M. conceived and designed the research; M.E.K. drafted the
manuscript; and G.E.D., S.L., W.M., and C.v.S. made critical revision
of the manuscript for key intellectual content.
Supplementary material
Supplementary material is available at European Heart Journal online.
Acknowledgements
We extend our appreciation to the participants of the LURIC study
and thank the LURIC study team who were either temporarily or
permanently involved in patient recruitment as well as sample and
data handling, in addition to the laboratory staff at the Ludwigshafen
General Hospital and the Universities of Freiburg and Ulm,
Germany.
Funding
This work was supported by the 7th Framework Program (Athero-
Remo, grant agreement number 201668 and RiskyCAD, grant
agreement number 305739) of the EU and by the INTERREG-IV-
Oberrhein-Program (Project A28, Genetic mechanisms of cardiovascu-
lar diseases) with support from the European Regional Development
Fund (ERDF) and the Wissenschaftsoffensive TMO. M.E.K., S.L., and
W.M. are supported by the German Federal Ministry of Education and
M.E. Kleber et al.Page 6 of 7
by guest on November 7, 2015http://eurheartj.oxfordjournals.org/Downloaded from
Research as part of the Competence Cluster of Nutrition and Cardio-
vascular Health (nutriCARD).
Conflict of interest: C.v.S. has founded Omegametrix, a laboratory
performing fatty acid analysis, which analysed LURIC samples free of
charge. W.M. is employed with synlab Services GmbH and holds shares
of synlab Holding GmbH. The other authors declare no conflict of
interest.
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