The Journal of NutritionNutrient Requirements and Optimal Nutrition
Intake of Fish Oil, Oleic Acid, Folic Acid, andVitamins B-6 and E for 1 Year Decreases PlasmaC-Reactive Protein and Reduces Coronary HeartDisease Risk Factors in Male Patients in aCardiac Rehabilitation Program1
Juan Jesu´s Carrero,2 Juristo Fonolla´,3 Jose´ Luis Marti,4 Jesu´s Jime´nez,3 Julio J. Boza,3and Eduardo Lo´pez-Huertas3*
2Department of Biochemistry and Molecular Biology, University of Granada, Spain; 3Department of Human Nutrition,
Puleva Biotech, Granada, Spain; and 4Service of Cardiology, University ‘‘San Cecilio’’ Hospital, Granada, Spain
Certain nutrients have been shown to be effective in preventing coronary heart disease. We hypothesized that a daily
intake of low amounts of a number of these nutrients would exert beneficial effects on risk factors and clinical variables in
patients that suffered from myocardial infarction (MI) and were following a cardiac rehabilitation program. Forty male MI
patients were randomly allocated into 2 groups. The supplemented group consumed 500 mL/d of a fortified dairy product
containing eicosapentaenoic acid, docosahexaenoic acid, oleic acid, folic acid, and vitamins A, B-6, D, and E. The control
group consumed 500 mL/d of semi-skimmed milk with added vitamins A and D. The patients received supervised exercise
training, lifestyle and dietary recommendations, and they were instructed to consume the products in addition to their
regular diet. Blood extractions and clinical examinations were performed after 0, 3, 6, 9, and 12 mo. Plasma concentrations
of eicosapentaenoic acid, docosahexaenoic acid, oleic acid, folic acid, vitamin B-6, and vitamin E increased after
supplementation (P , 0.05). Plasma total and LDL-cholesterol, apolipoprotein B, and high-sensitivity C-reactive protein
concentrations decreased in the supplemented group (P , 0.05), and plasma total homocysteine decreased in both
groups. There were no changes in heart rate, blood pressure, or cardiac electrocardiographic parameters in either group.
Therapeutic lifestyle changes, effected through a CR program comprising regular exercise and the intake of a combination
of dietary nutrients, reduced a variety of risk factors in MI patients, which supports the rationale for nutritional programs in
the secondary prevention of coronary heart disease.
who have received a diagnosis of MI. The overall objectives of
Acute myocardial infarction (MI),5 a manifestation of athero-
CR are to optimize patients’ functioning by decreasing disabil-
sclerosis caused by the occlusion of coronary arteries, is the
ities resulting from coronary heart disease (CHD), enhancing the
leading cause of death for both men and women all over the
quality of life, and minimizing the risk of recurrent cardiac
world. It is also a major cause of physical disability, particularly
events and hospitalization (2,3). Comprehensive rehabilitation
in the rapidly growing population of elderly persons (1). Cardiac
programs usually combine supervised exercise training and be-
rehabilitation (CR) programs are recommended for patients
havioral changes (smoking cessation, control of excess weight,and increased physical activity) with lifestyle and nutritionalcounseling, thus aiming to reduce the cardiovascular (CV) riskfactors that are generally increased in these patients (4).
1 Supported in part by a PhD education grant from the University of Granada
Cholesterol-lowering therapies remain the main strategies
(J.J.C.). Dairy products were supplied by Puleva Food S.L., Granada, Spain.
for primary and secondary prevention of CHD (4). Numerous
Abbreviations used: AA, arachidonic acid; ApoB, apolipoprotein B; C, control
studies report a direct relation between levels of total or LDL-
group; CHD, coronary heart disease; CR, cardiac rehabilitation; CV, cardiovas-cular; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; HDL-C, HDL
cholesterol (LDL-C) and the rate of CHD in healthy individuals
cholesterol; hs-CRP, high-sensitivity C-reactive protein LDL-C, LDL cholesterol;
and in people with established CHD (5–8). Moreover, several
MI, myocardial infarction; S, supplemented group; sICAM-1, soluble intercellular
prospective studies indicate that plasma levels of high-sensitivity
cell adhesion molecule 1; sVCAM-1, soluble vascular cell adhesion molecule 1;
C-reactive protein (hs-CRP), a marker of systemic inflammation,
TC, total cholesterol; TG, triacylglycerols; tHcy, total plasma homocysteine.
* To whom correspondence should be addressed. E-mail: [email protected]
are a strong independent predictor for the risk of future CV
events among individuals, including MI patients (9–11).
0022-3166/07 $8.00 ª 2007 American Society for Nutrition.
Manuscript received 12 October 2006. Initial review completed 13 October 2006. Revision accepted 9 November 2006.
There is a wealth of evidence regarding the benefits produced
or during the cardiac rehabilitation program; 2) endocrine or metabolic
by changing lifestyle habits, dietary patterns, and nutrients in the
disturbances; 3) blood concentrations of LDL-C .3.4 mmol/L, or HDL-C
primary and secondary prevention of CHD. Among those, a
,1 mmol/L, or triacylglycerols .1.7 mmol/L; 4) using a prescription of
Mediterranean dietary pattern, characterized by a high intake of
lipid lowering drugs (statins); 5) liver insufficiency; 6) residence outside
olive oil (rich in oleic acid and antioxidants), fish [rich in (n-3)
the recruitment area of the study; 7) unable to come to the CR unit;8) taking fish oil supplements.
long-chain PUFA], vegetables and fruits (rich in folic acid andother vitamins), has been associated with a lower CHD inci-
Study protocol and diets. We carried out a longitudinal, randomized,
dence and total mortality (12–14).
controlled, double-blind intervention study to investigate the effects of a
In this sense, the AHA recommends to MI patients a daily
nutritional supplement in markers of CHD of a group of men following a
intake of 1 g of eicosapentaenoic acid (EPA) and docosahex-
cardiac rehabilitation program after an episode of MI.
aenoic acid (DHA) from fish or fish oils (15). Dietary intakes of
From June to October 2003, 71 possible candidates were recruited;
folate and/or vitamin B-6 have been described as the main
27 of them did not fulfill the inclusion criteria because of residence
nutrients responsible for lowering hyperhomocysteinemia,
outside the metropolitan area of Granada (n ¼ 7), taking prescription
which is considered an independent risk factor for CVD (16).
statins 1 mo before the time of the inclusion due to high blood LDL-C
Consequently, European Recommended Dietary Allowances for
concentration (n ¼ 4), clinical history of stroke or peripheral vasculardisease (n ¼ 3), or a lack of willingness to participate (n ¼ 13). The
these vitamins have been well established (17). Evidence has
remaining candidates were randomly assigned to 2 intervention groups
been recently gathered by the latest WHO report, which rec-
of 20 subjects each, using a table of random numbers (Fig. 1).
ommends 1) regular fish consumption to provide ;200–500 mg
The patients received tailor-made exercise instructions and attended
of EPA 1 DHA/wk, 2) a replacement of saturated fat by
the CR unit twice/wk for exercise classes. They also were encouraged to
monounsaturated fat (to increase the oleic acid intake), and 3)
exercise on their own every day (walk for 30 min to 1 h). Participating
an increase in the consumption of fruit and vegetables to achieve
subjects gave their written consent. The protocol was approved by the
proper antioxidant and folate status (18). Despite the recom-
Ethical Committee of S. Cecilio University Hospital and it was
mendations, modern Western societies tend to include little fish,
conducted in accordance with the Helsinki Declaration. The study
fruit, and vegetables in their diets (18). In addition, whereas CR
lasted 12 mo, from November 2003 to December 2004. Details of the
programs emphasize the adherence to healthy dietary patterns,
drugs prescribed to the subjects were recorded at baseline (Table 1).
The supplement (S) group (n ¼ 20) was supplied with 500 mL/d of a
their implementation is often unsuccessful and usually involves
fortified dairy product (Puleva Omega3, Puleva Food) containing the
An effective approach to increasing the intake of healthy
dietary nutrients that may lead to a decrease of CHD risk is theenrichment of foods that are regularly consumed by the majority
of the population (20–24). We recently conducted a nutritionalintervention with this nutritional approach in patients sufferingfrom peripheral vascular disease, showing improvements inclinical outcomes while undertaking reductions in a variety ofrisk factors (23). With MI being the most frequent manifestationof CV disease, we hypothesized that the inclusion of a sup-plement in the daily diet of MI patients would help improvenutritional status and risk profile in the context of a CR pro-gram, and we carried out a longitudinal, controlled, random-ized, double-blind, 12-mo intervention with a dairy productcontaining low amounts of EPA and DHA, oleic acid, folic acid,and vitamins A, D, E, and B-6.
Subject recruitment. Subject recruitment was conducted at the CardiacRehabilitation Unit of the University Hospital of Granada (Spain).
Hospital records and medical history of the patients were consultedbefore their inclusion in the study. All male patients diagnosed with atleast 1 episode of acute MI and followed and successfully completedphase II of the CR program, and patients already on phase III, werecandidates for inclusion in the study. Phase II of the CR programtypically started after hospital discharge and usually lasted for 3 mo.
During this phase, the patients were assessed for cardiovascular risk andreceived lifestyle and nutritional counseling, supervised exercise training,drug therapy, and psychological support. In phase III, the patients werereferred to their medical centers for follow-up, where general practi-tioners supervised the CR of the patients in accordance with theguidelines received from the CR unit (25). For this study, the patientsincluded in the study were asked to visit the CR unit twice/wk for the12-mo duration of the study. Patients were not admitted to the study ifany of the following criteria were present: 1) at high-risk after hospitaldischarge (New York Heart Association class III and IV), low functionalcapacity, ventricular arrhythmia, severe valvular disease, severe heartfailure, or presenting with additional CV events during the hospital stay
Baseline characteristics of the participating subjects1
program-Phase II, and also 2 wk before the 1st delivery of the study’sdairy beverages (in phase III), the subjects and their partners attended a
dietary counseling session on general aspects of food composition, foodprocessing, adequate portions, the effects of alcohol consumption, and
the beneficial effects of the Mediterranean diet. Subjects received a leaflet
with information about the CV benefits produced by fish consumption
and were also advised to increase the consumption of fruit, legumes, and
vegetables to ensure adequate intake of fiber and vitamins. They wereadvised not to eat fast food or precooked meals and to avoid smoky
places or to stop smoking. Dietary intake was assessed at baseline and
again at the end of the study with a 7-d self-administered food-frequency
questionnaire, following instructions from the principal investigator.
Spanish food composition tables were used to estimate dietary intake
Blood extraction and clinical examination. The patients were
interviewed in the hospital at the beginning of the study (T0) and after3, 6, 9, and 12 mo (T
3, T6, T9, and T12). At every visit, after an overnight
Data are presented as means 6 SEM or n (%); n ¼ 20/group. * Different from C,
fast of at least 10 h, blood samples (30 mL) were collected by
venipuncture into vacutainer tubes containing EDTA. Samples werekept on ice before centrifugation at 1700 3 g for 15 min at 4°C to obtain
plasma. To ensure analytical consistency, plasma samples T0 –T12 from
following nutrients: EPA, DHA (from fish oils), oleic acid, folic acid, and
each subject were processed at the same time and analyzed in 1 batch.
vitamins A, B-6, D, and E. The dairy supplement was prepared by adding
The subjects also received a complete clinical examination, including
a mixture of fish and vegetable oils to skimmed milk, yielding a product
an anamnesis. Blood pressure and heart rate were determined. Electro-
containing a total fat comparable to that of standard semi-skimmed milk
cardiogram tests were performed and recorded at the times of the study.
(19 g/L), but with a different fatty acid profile. Folic acid and vitamins A,
P-wave, QRS complex, and T-wave abnormalities were measured and
B-6, D, and E were also added to the final product. The control (C) group
(n ¼ 20) was supplied with 500 mL/d of regular semi-skimmed milk withadded vitamins A and D (Table 2). The dairy products were produced
Biochemical measurements. The plasma concentrations of triacyl-
and packaged in white 500 mL Tetra Bricks by Puleva Biotech S.A., so
glycerols (TG), total cholesterol (TC), and HDL cholesterol (HDL-C)
that neither the patients nor the researchers would know what was
were measured at the hospital central laboratory by colorimetry, using
consumed. The patients were instructed to consume the dairy products,
commercial kits (Biosystems). Analyses were conducted in triplicate and
in addition to their regular diets, in 2 3 250 mL doses at the beginning
in 1 batch, following the protocols provided by the manufacturer. Plasma
and at the end of the day. The dairy products were home-delivered to the
LDL-C was calculated according to the Friedewald formula (27). Plasma
patients monthly. Compliance with the consumption protocol during the
fatty acid profiles were determined by GLC (28). Plasma apolipoprotein
intervention period was ensured and monitored by regular telephone
B (ApoB) was measured using an immuno-turbidimetry test (Olympus
calls and collection of the emptied containers. At the beginning of the CR
Diagnostica). Plasma concentrations of total homocysteine (tHcy), vita-min E, and malondialdehyde were quantified by HPLC with fluorescencedetection (29–31, respectively). Plasma vitamin B-6 concentration wasalso measured by HPLC, using instructions from a commercial kit
(Immundiagnostik). Plasma and RBC folate concentrations were mea-sured using commercial immunoassay kits (ICN Pharmaceuticals).
Soluble vascular adhesion molecule 1 (sVCAM-1) and soluble intercel-lular cell adhesion molecule 1 (sICAM-1) concentrations were measured
by ELISA commercial kits (Biosource International). Hs-CRP concen-
trations in plasma were quantified by an immuno-nephelometric com-
mercial kit (Dade Behring). Oxidized LDL in plasma was quantifiedusing an ELISA kit (Mercodia). ApoB, hs-CRP, and the vitamins
described above were measured in 1 batch at Balague´ Center Labora-
Statistical analysis. The data were analyzed using SPSS software
(version 12.0). Data are expressed as means 6 SEM, and differences of
P , 0.05 were considered significant. Normality was assessed by the
Kolmogorov-Smirnov test. Between-group comparisons at the beginning
of the study were assessed by an independent t test or Mann-Whitney test
for the non-Gaussian variables. The longitudinal effect of each dairy
product within each group at the various time points of the study was
analyzed by 1-way repeated-measures ANOVA followed by Tukey’s
honestly significant difference post hoc test (within-group comparison).
Statistical differences produced by the consumption of each dairy prod-
uct were analyzed using 2-way repeated-measures ANOVA. For the non-
Gaussian variables, Wilcoxon and Krustal-Wallis comparisons were
performed to assess differences within and between groups, respectively.
When between-group comparisons showed significant differences, an
independent t test or Mann-Whitney test was applied to determine the
time points at which the groups differed. The relations among increased
plasma nutrients concentrations and CHD risk-factor improvement were
assessed using two-tailed Pearson’s bivariate correlations.
(Table 5). Plasma tHcy concentrations at baseline were withinthe normal range (,15 mmol/L) in both study groups (32). The S
Baseline characteristics of the subjects included in the groups
group exhibited a within-group reduction in the plasma tHcy
(Table 1) did not change from the beginning to the end of the
concentration beginning at T3 that was sustained throughout the
study (not shown). At entry, 50% of patients were overweight
intervention, whereas the C group had only a modest reduction
(BMI $25 and ,30 kg/m2) and 33% were obese ($30 kg/m2).
at T12. The vitamin E in the supplemented dairy product
The dairy products used were well accepted and compliance was
increased the plasma vitamin E concentration and the vitamin
good. Dietary intake of nutrients did not differ between the
beginning and the end of the study (not shown). None of the
The plasma hs-CRP concentration at baseline was above
patients included suffered further CV events during the study.
high-risk values (.3 mg/L; 32) in both study groups. In the S
One patient in the S group did not finish the study due to taking
group, this concentration decreased 20% at T6 and 48% at T12
a prescription of statins (n ¼ 1). The rest of the patients
(Table 5). The reductions were more pronounced when the
successfully completed the study (Fig. 1).
subjects with baseline values .3 mg/L were considered sepa-
The amounts of oleic acid, DHA, and EPA daily supple-
rately and the changes were independent of weight reduction.
mented in 500 mL of the enriched product were 5.12 g, 0.13 g,
Decreases in the hs-CRP of the subjects were directly correlated
and 0.2 g, respectively, whereas the semi-skimmed milk con-
with the increases in plasma EPA 1 DHA (r ¼ 20.52, P ¼ 0.03)
tained only 1.82 g oleic acid/500 mL and undetectable levels of
and EPA alone (r ¼ 20.51; P ¼ 0.04) and tended to be correlated
DHA and EPA. The plasma fatty acid profile did not change in
with decreases plasma tHcy (r ¼ 0.42; P ¼ 0.08).
patients from the C group, but in the S group, consumption of
The plasma concentration of sICAM-1 increased slightly in
the fortified dairy product significantly increased the propor-
the C group at T6 (276 6 14 mg/L; P , 0.05) and T12 (285 6 13
tions of EPA, DHA, and the decreased ratio of arachidonic acid
mg/L; P , 0.05) compared with initial values (247 6 13 mg/L).
(AA) to EPA (Table 3). The plasma oleic acid level was greater in
In contrast, the plasma slCAM-1 concentration tended to
the S group at T6 and T12 compared with baseline, but at no time
decrease (P ¼ 0.06) in the S group (from 289 6 22 at T0, to
did the S and C groups differ. Plasma total saturated fatty acid
269 6 15 at T6, and 254 6 18 mg/L at T12). Other markers of
levels in the C group and monounsaturated fatty acid levels in
endothelial dysfunction, such as sVCAM-1, and of plasma
the S group tended to increase (P ¼ 0.06). Other plasma fatty
oxidizability, such as malondialdehyde, and oxidized LDL did
not change in either group (data not shown). Blood pressure,
The plasma TC and LDL-C concentrations decreased in the S
heart rate, and results of the electrocardiogram tests did not
group at T9 and T12. The TC, but not the LDL-C concentration,
change in either group throughout the study (data not shown).
differed between the S and C groups at these times. The plasma
HDL-C and TG concentrations did not change in either groupduring the study, whereas the plasma concentration of ApoBsignificantly decreased at T
The amounts of folic acid and vitamin B-6 daily supple-
We found that the inclusion of a combination of nutrients in the
mented in 500 mL of enriched product were 150 mg and 1.5 mg,
daily diet of a group of male MI patients following a CR
respectively. Plasma vitamin B-6 and plasma and RBC folate
program may improve nutritional status and reduce CHD risk
concentrations increased in the S group but not in the C group
factors. The way the nutrients were administered (in a beverageconsumed daily) may have contributed to the good complianceobtained with the protocol, as shown by the percentages of the
Relevant plasma fatty acids in the C and S groups
plasma fatty acids and plasma concentration of vitamins that
at the beginning of the study (T0), after 6 mo (T6),
varied in response to the nutrients supplemented, which is in
agreement with similar studies (21–24). In fact, the absorptionof EPA and DHA from fish oil is improved when associated with
other fats and spread out in small doses during the day (33), and
the fact that milk fat is highly dispersed in very small micelles,
increasing the surface of absorption of fats and lipid-soluble
compounds (20), may explain the metabolic effects achieved
when only small amounts were administered.
Although the prescription of the drugs, exercise, recommen-
dations for healthier lifestyle, and the Mediterranean dietary
pattern of the CR program resulted in the ability of C-group
patients to maintain their lipid concentrations within the de-
sirable range, the inclusion of the supplement in the diet reduced
the total cholesterol concentrations by 10% and LDL-C by
14%, both of which have been associated with a lower risk for
second MI events (4). Reduced ApoB in the S group suggests
further beneficial effects in the reduction of CHD risk. ApoB has
been reported to be a better index of CVD risk than LDL-C, as
ApoB is a marker for all the potential atherogenic particles
(34,35). Such reductions have been reported previously with the
consumption of higher doses of fish oils (36,37) and similar food
supplements (23,24). The amounts of lipid-lowering nutrients
Values are means 6 SEM, n ¼ 19 for S or n ¼ 20 for C. *Different from T0, P , 0.05.
supplemented each day were quite low: 1 g of fish oil (0.33 g of
EPA 1 DHA) and 5.2 g of oleic acid. Still, the effects observed
Plasma lipid and ApoB concentrations in the C and S groups at the beginning of the study (T0) and after 3 (T3), 6 (T6),9 (T9) and 12 (T12) mo of intervention1
1 Values are means 6 SEM, n ¼ 19 for S or n ¼ 20 for C. *Different from T0, P , 0.05. yDifferent from C, P , 0.05.
2 ND, not determined.
were rather remarkable compared with other studies that in-
increased production of EPA-derived eicosanoids, which possess
volved capsules and/or tablets, but were in agreement with
less activity than those derived from AA (39).
previous studies using this supplement (21–24) and could be
At the present time, proven therapies to reduce hs-CRP levels
attributed to the method of administration. Previous short-term
include weight reduction (40) and, particularly, statin therapy
studies using this food supplement (22) or a similar one (20,24)
(41,42). However, in our study, we found reductions of hs-CRP
demonstrated a comparable lipid-lowering effect on TC and
to be independent of those. A recent report showed that exercise
LDL-C, but, in contrast to those reports, our work did not find
training and a Mediterranean dietary pattern during phase II of
any effect on TG concentrations. A plausible explanation could
CR produced a 41% median reduction of hs-CRP that was also
be that, in those studies, TG concentrations were above normal
independent of statin therapy and weight variation (43). The
at baseline, whereas, in our study, TG concentrations were
reductions of hs-CRP in the S group of our study agree with that
normal (,1.7 mmol/L) (25). Indeed, in 2 other of our interven-
report, and both were of similar or greater magnitude to those
tion studies with this food supplement, subjects with normal TG
observed in numerous studies using statin therapy, with reduc-
at baseline did not have TG reductions (21,23). These results
tions in hs-CRP between 15 and 20% (41,42). Whereas statin
suggest that the supplemented nutrients might contribute to
therapy constitutes the major treatment option available for
blood-lipid stabilization in the context of a blood-lipid imbal-
the reduction of hs-CRP and LDL-C (44), the combination of
the supplemented beverage and statins are likely to produce
Local production of proinflammatory cytokines in the
further CV benefits and deserves to be explored. The CRP reduc-
atheromatous plaque increases systemic markers of inflamma-
tion observed with the administering of low amounts of anti-
tion such as hs-CRP (38). In the present study, the hs-CRP
inflammatory nutrients could be attributed, again, to the increased
reductions in the S group suggest a reduced risk of further
bioavailability of the nutrients through the dairy product and
coronary events by amelioration of the inflammatory status. In
perhaps to a pleiotropic effect of such nutrients combined.
addition, the decreases of the AA:EPA ratio and increases of the
Finally, at the end of the study, the S group of patients had
plasma EPA and DHA concentrations in the S group suggest a
improved vitamin status, increasing their vitamin E:TC ratio to
less proinflammatory and thrombotic environment due to the
optimal values (.5.2 mmol/L) (45) and changing their serum
Plasma vitamins, tHcy, and CRP concentrations in the C and S groups at the beginning of the study (T0) and after3 (T3), 6 (T6), 9 (T9), and 12 (T12) mo of intervention1
1 Data are presented as means 6 SEM, n ¼ 19 for S or n ¼ 20 for C. *Different from T0, P , 0.05. yDifferent from C, P , 0.05.
2 ND, not determined.
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PROHIBITED CLASSES OF SUBSTANCES AND PROHIBITED METHODS (art. 5 of the Antidoping Examination Regulations/ Adopted by the UCI President on the proposal of the Antidoping Commission of the UCI. UCI List No.01/2002 Entry into effect: 1stMay 2002 2002 PROHIBITED CLASSES OF SUBSTANCES 1. Androgenic anabolising steroids 2. Non-steroidal anabolic agents Peptide hormones, mimetics
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