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,4
Department of Biochemistry and Molecular Biology, University of Granada, Granada, Spain;
* Department of Human Nutrition, Puleva Biotech, Granada, Spain;
Service of Angiology and Vascular Surgery, University San Cecilio Hospital, Avda. de Madrid s/n, Granada, Spain; and
** Distrito Sanitario Costa del Sol, Servicio Andaluz de Salud, Málaga, Spain
4To whom correspondence should be addressed. E-mail: eros{at}ugr.es.
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ABSTRACT |
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KEY WORDS: • peripheral vascular disease • intermittent claudication • (n-3) PUFAs • oleic acid • B vitamins.
Peripheral vascular disease (PVD)5 is a manifestation of systemic atherosclerosis caused by the occlusion of the arteries to the legs. Patients with PVD may be asymptomatic or suffer from intermittent claudication (IC), rest pain, and/or gangrene (1), and have a 3- to 5-fold increased risk of cardiovascular mortality (2,3). IC is the most common symptom, present in 
40% of patients with PVD (4). Ischemia of the calf muscles causes exercise-induced lower leg discomfort that classically resolves with rest and is associated with a diminished ability to perform daily activities. The degree of functional impairment of patients with PVD is assayed according to the distance that the patient can walk without pain, or pain-free walking distance (PFWD). Treatment focuses on decreasing functional impairment caused by symptoms (5), and treating the underlying systemic atherosclerosis by targeting risk factors (6). The major risk factors for PVD are age (>40 y), cigarette smoking, and diabetes. Hyperlipidemia, hypertension and hyperhomocysteinemia are also important PVD (7) and cardiovascular disease (CVD) risk factors. In fact, there is a clear association between IC and the risk of developing CVD, which is present in as many as 90% of patients with IC (8). However, several studies demonstrate how risk factors for this disease are often overlooked (9,10), suggesting that guidelines for the specific management of these patients are needed together with strategies to ensure their implementation.
Diet plays a major role in the prevention of CVD, and there is a wealth of evidence regarding the beneficial effects of changing lifestyle habits, dietary patterns, and nutrient consumption on CVD prevention. International health societies have established nutritional guidelines in this regard. The latest WHO report (11) recommends regular consumption of fish to provide 200–500 mg of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) per week, replacement of saturated fat by monounsaturated fat (oleic acid), and increased consumption of fruit and vegetables to achieve proper antioxidant and folate status.
Earlier intervention studies reported discrete beneficial effects on clinical outcomes and risk factors in patients with IC after dietary supplementation with (n-3) polyunsaturated fatty acids (PUFAs) (12), olive oil (13), sunflower oil (14), vitamin E (15), and folic acid or vitamin B-6 (16,17). However, these results were not sufficiently developed to provide specific dietary guidelines for patients with PVD (18).
In this study, we carried out a longitudinal, controlled, randomized, and double-blind 12-mo intervention in which we supplemented the diet of PVD patients with a dairy product containing low amounts of EPA, DHA, oleic acid, folic acid, and vitamins A, B-6, D, and E. We studied the effects of this supplement on cardiovascular risk factors and clinical outcomes.
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MATERIALS AND METHODS |
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Study protocol and diets. We carried out a longitudinal, randomized, controlled, double-blind intervention study to investigate the effects of a nutritional supplement in patients with peripheral vascular disease and intermittent claudication (PVD-IC).
From May 2003 to July 2003, 107 possible candidates were recruited; 34 of them did not fulfil the inclusion criteria because of residence outside the metropolitan area of Granada (n = 11), a previous history of myocardial infarction (n = 12), a current statin prescription before the time of inclusion (n = 6), or a lack of willingness to participate (n = 5). The remaining candidates were randomly assigned to 2 intervention groups of 30 subjects each using a table of random numbers (Fig. 1). Participating subjects gave their informed written consent. The protocol was approved by the Ethical Committee of S. Cecilio University Hospital and conducted according to the Helsinki Declaration.
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The supplement (S) group (n = 30) was supplied with 500 mL/d of a fortified dairy product (Puleva Omega3©, Puleva Food) containing the following nutrients: EPA, DHA, oleic acid, folic acid, and vitamins A, B-6, D, and E. The dairy supplement was prepared by adding a mixture of fish and vegetable oils to skimmed milk, yielding a product containing a total fat content comparable to that of standard semiskimmed milk (1.9 g/100 mL), but with a different fatty acid profile. Folic acid and vitamins A, B-6, D, and E were also added to the final product. The control (C) group (n = 30) was supplied with 500 mL/d of regular semiskimmed milk with added vitamins A and D (Table 1). The dairy products were produced and packaged in white 500-mL Tetra Bricks by Puleva Biotech, so that neither the subjects nor the researchers would know what was consumed. The subjects were instructed to consume the dairy products in 2 x 250-mL doses at the beginning and at the end of the day. The dairy products were home-delivered to the subjects monthly. Compliance with the consumption protocol during the intervention period was ensured and monitored by regular telephone calls and collection of the emptied containers.
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Blood extraction and clinical examination. The subjects were interviewed in the hospital at the beginning of the study (T0) and after 3, 6, 9, and 12 mo (T3, T6, T9 and T12). At every visit, after an overnight fast of at least 10 h, blood samples (30 mL) were collected by venipuncture into vacutainer tubes containing EDTA. Samples were kept on ice before centrifugation at 1700 x g for 15 min at 4°C to obtain plasma. To ensure analytical consistency, plasma samples T0 to T12 from each subject were processed at the same time and analyzed in one batch.
The subjects also received a complete clinical and vascular exploration, including an anamnesis. PFWD was measured using a treadmill set at 3 km/h speed and 10% slope and was expressed as the mean of 2 consecutive tests performed before and after ABI calculation (40 min interval between tests). To calculate the ABI, an air-filled plethysmograph was placed on the lower limbs to record pulse volume and segmental pressure by continuous Doppler. The ABI (ratio of the ankle systolic pressure to the brachial artery systolic pressure) is useful in assessing the severity of vascular disease. An ABI > 0.90 is considered normal; 0.70 to 0.89 is considered mild disease, 0.5 to 0.69 moderate disease, and < 0.5 severe disease (20)
Biochemical measurements. Plasma concentrations of triacylglycerols (TG), total cholesterol (TC), and HDL cholesterol (HDL-C) were measured at the hospital central laboratory by colorimetry, using commercial kits (Biosystems). Analyses were conducted in triplicate and in one batch, following the protocols provided by the manufacturer. Plasma LDL cholesterol (LDL-C) was calculated according to the Friedewald formula (21). Plasma fatty acid profiles were determined by GLC (22). Plasma apolipoprotein B (ApoB) was measured using an immunoturbidimetry test (Olympus Diagnostica). Plasma concentrations of total homocysteine (tHcy), vitamin E, and malondialdehyde (MDA) were quantified by HPLC with fluorescence detection (23–25). Plasma vitamin B-6 concentration was also measured by HPLC, using instructions from a commercial kit (Immundiagnostik). Plasma and RBC folate and plasma vitamin B-12 concentrations were measured using commercial immunoassay kits (ICN Pharmaceuticals). Plasminogen activator inhibitor 1 (PAI-1), E-selectin, soluble vascular adhesion molecule 1 (sVCAM-1), and soluble intercellular cell adhesion molecule 1 (sICAM-1) concentrations were measured by commercial ELISA kits (Biosource International). High-sensitivity C-reactive protein (CRP) concentrations were quantified by immunonephelometry (Dade Behring). Oxidized LDL in plasma was quantified using an ELISA kit (Mercodia). Apo B, CRP, and all the vitamins were measured in one batch at Balagué Center Laboratories.
Statistical analysis. The data were analyzed using SPSS software (version 12.0). Data are expressed as means ± SEM; values 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 nongaussian variables. The longitudinal effect of each dairy product within each group at the various time points of the study was analyzed by one-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 product were analyzed using two-way repeated-measures ANOVA. For the nongaussian 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 nutrient concentrations and PFWD improvements were assessed using two-tailed Pearson’s bivariate correlations.
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RESULTS |
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Plasma fatty acid concentrations varied in response to the supplemental dietary fats. In the S group, the levels of oleic acid, DHA, and EPA increased by 10%, 64%, and 21%, respectively, together with total PUFAs. The supplemental vitamins also increased serum and RBC folate and plasma concentrations of vitamins B-6 and E. Studies report similar increases in plasma concentrations of those fatty acids and vitamins when consumed as a supplement in a dairy product (28–30), and reports show good compliance with intake of the supplement. The means of administering the nutrients (in a drink used every day) may have contributed to the very good compliance obtained.
Although the prescription of drugs and the recommendations of lifestyle changes and a Mediterranean dietary pattern yielded PFWD improvements in both groups, the increase in the S group was outstanding. Whereas mean PFWD increased by 44 m in the C group, it increased by 280 m in the S group. The PFWD increased in the S group after consuming the fortified dairy product for only 3 mo, indicating an early response. The correlation of increased PFWD with plasma EPA and RBC folate concentrations suggests that the supplemental nutrients are responsible for the clinical improvements. These results agree with the increase in the ABI, suggesting an improvement in blood flow to the lower limbs in the S group.
Inflammation within the vessel walls is a major contributory factor in atherosclerosis, and an anti-inflammatory effect is reported for DHA and EPA (31). In the present study, the AA:EPA ratio decreased and the plasma EPA and DHA concentrations increased in the S group. EPA and DHA compete with AA for insertion at the sn-2 position of membrane phospholipids, producing less potent eicosanoids. Prostaglandin I3, formed from EPA in the endothelium, is a more active vasodilator and inhibitor of platelet aggregation than prostaglandin I2, formed from AA. Therefore, the decreased plasma AA:EPA ratio may be related to vasodilatation and inhibition of platelet aggregation (32). In addition, EPA and DHA increase RBC deformability (33) and reduce RBC aggregation (34), perhaps as a result of modifying the cell membrane lipid content. Therefore, reduced platelet and RBC aggregation can potentially increase blood flow (35). Recent reports show that atherosclerotic plaques may quickly incorporate dietary EPA and DHA, resulting in increased plaque stability and reduced macrophage infiltration, slowing the progression of the vascular lesion (36) and perhaps the onset of clinical events. These effects are likely to influence PFWD in patients with PVD-IC. Oleic acid, DHA, and EPA are reported to influence endothelial function and the production of endothelial adhesion molecules (31). Neither these nor other markers of inflammation were affected in the S group.
The plasma lipid concentrations of the subjects at the beginning of the study were borderline high (25). Although plasma LDL-C did not change, plasma ApoB clearly decreased in this group. ApoB is reported to be a better index of CVD risk than LDL, as ApoB is a marker for all the potential atherogenic particles (37). In fact, this reduction in ApoB indicates a reduction in the number of proatherogenic small and dense LDL particles that would not be evident by observing only LDL-C. Plasma TC decreased in the S group, but there were major decreases among subjects with high initial TC concentrations (26). These results suggest that the supplemental nutrients might have contributed to blood-lipid stabilization in the context of a blood-lipid imbalance. Previous studies (29,30) describe a similar lipid-lowering effect on TC and LDL-C, but in contrast to those reports, the present work did not record any effect on TG concentration. Almost half the subjects in the present study were smokers (>15 cigarettes/d), which could explain the 7% reduction in plasma HDL-C in both study groups (38,39).
The study also addressed the question of whether the regular intake of small amounts of PUFAs, together with vitamin E, would make plasma and LDL particles more prone to oxidation. Plasma and LDL oxidation potentials did not change during the intervention period, but the S-group plasma vitamin E concentration and vitamin E:TC ratio increased to values > 5.2 µmol/L. This ratio is considered more useful when describing vitamin E status, and values > 5.2 µmol/L are considered optimal (40). Increased RBC deformability produced by antioxidant protection of PUFAs at the cell membranes and amelioration of the ischemia-induced oxidative stress at the lower limbs are possible beneficial effects of vitamin E in patients with PVD-IC described in a recent meta-analysis (15).
The dietary supplement contributed >70% of the European Recommended Dietary Allowances for folic acid and vitamin B-6 (41). Plasma folate concentration increased from suboptimal levels (<15 nmol/L) (42) at T0 to optimal status at T12 in the S group, as opposed to the C group. Responses of plasma vitamin B-6 and RBC folate concentrations were similar. Folate and B-6 intake are independent predictors of PVD in men aged > 50 y (17), and are the main factors responsible for lowering hyperhomocysteinemia, which is itself considered an independent risk factor for PVD-IC, present in 30% of PVD patients (43). In the present study, plasma tHcy decreased (15%) only in the S-group subjects with hyperhomocysteinaemia (>15 µmol/L) (27). Folate and vitamin B-6 status, together with decreased plasma tHcy, are associated with changes in the coagulation response, reduced endothelium-dependent relaxation, reduced nitrous oxide synthesis, and prostacyclin production [(44) and references therein] and may affect endothelium-derived hyperpolarizing factor, a major determinant of vascular tone in small resistance vessels (45).
The isolated effects of the nutrients used in the supplement are described in the literature, but no dietary interventions using them in combination are reported. Previous studies with olive oil in patients with IC (13,14) report only protection against LDL oxidation. The intake of (n-3) PUFAs appears to have some beneficial effects in patients with PVD-IC, but no clear evidence for improved clinical outcomes is reported (12).
Although Adult Treatment Panel III recommendations emphasize lifestyle and dietary changes in CVD prevention, recent reports suggest that more attention should be paid to dietary approaches in the management of patients with PVD: after a hospital discharge, only 50% of patients with PVD would modify their diet for lipid control (9), and only 18% of general practitioners would consider cholesterol-lowering therapy to be primary prevention (10).
The present study reported that the inclusion of specific nutrients (EPA, DHA, oleic acid, folic acid, and vitamins B-6 and E) in the everyday diet of a group of male patients with PVD-IC improved clinical outcomes and reduced a variety of risk factors, providing new evidence for the potential role of nutrition in the reduction of PVD-IC symptoms.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported in part by a PhD educational grant from the University of Granada (J.J.C.). Milk products, reagents, and kits were supplied by Puleva Food S.L., Granada, Spain; E.L.-H. is an employee of Puleva Biotech.
3 Supplemental Tables 1–3 are available with the online posting of this paper at www.nutrition.org.
5 Abbreviations used: AA, arachidonic acid; ABI, ankle-brachial index; ApoB, apolipoprotein B; C, control group; CRP, C-reactive protein; CVD, cardiovascular disease; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; HDL-C, HDL cholesterol; IC, intermittent claudication; LDL-C, LDL cholesterol; MDA, malondialdehyde; PAI-1, plasminogen activator inhibitor 1; PFWD, pain-free walking distance; PVD, peripheral vascular disease; PVD-IC, peripheral vascular disease and intermittent claudication; S, supplement group; sICAM-1, soluble intercellular cell adhesion molecule 1; sVCAM-1, soluble vascular cell adhesion molecule 1; TC, total cholesterol; TG, triacylglycerols; tHcy, total plasma homocysteine.
6 Supplemental Tables 1–3 are available with the online posting of this paper at www.nutrition.org.
Manuscript received 12 January 2005. Initial review completed 1 February 2005. Revision accepted 3 March 2005.
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