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Omega-3 polyunsaturated fatty acid in peripheral arterial disease: Effect on lipid pattern, disease severity, inflammation profile, and endothelial function
Clinical Nutrition (2008) 27, 241e247
available at www.sciencedirect.com
http://intl.elsevierhealth.com/journals/clnu
ORIGINAL ARTICLE
Omega-3 polyunsaturated fatty acid in peripheral arterial disease: Effect on lipid pattern, disease severity, inflammation profile, and endothelial function Vittorio Schiano a,*, Eugenio Laurenzano a, Gregorio Brevetti a, Julieta Isabel De Maio a, Simona Lanero a, Francesco Scopacasa b, Massimo Chiariello a a Department of Clinical Medicine and Cardiovascular and Immunological Sciences, University of Naples ‘‘Federico II’’, Naples, Italy b Department of Laboratory Medicine; University of Naples ‘‘Federico II’’, Naples, Italy
Received 2 August 2007; accepted 27 November 2007
KEYWORDS Peripheral arterial disease; Polyunsaturated fatty acids; Endothelial function; Inflammation
Summary Background & aims: Peripheral arterial disease (PAD) is strongly associated with endothelial dysfunction and inflammation, which portend a high cardiovascular risk. Accordingly, we investigated the effects of omega-3 polyunsaturated fatty acid (n-3 PUFA) supplementation on endothelial function and inflammatory status in affected individuals. Methods: PAD patients were randomly divided into two groups. In Group I (n Z 16) preenrollment therapy was not modified, while in Group II (n Z 16) n-3 PUFAs 1 g b.i.d. for 3 months were added to the previous treatment. Endothelial function was assessed by measuring plasma soluble thrombomodulin (sTM) and brachial artery flow-mediated dilation (FMD), and the inflammatory status by measuring high-sensitivity C-reactive protein and myeloperoxidase. Results: In Group II, n-3 PUFAs reduced sTM levels from the median value of 33.0 ng/mL (interquartile range 16.7, 37.2) to 17.0 ng/mL (11.2, 33.7) (p Z 0.04), and improved FMD from 6.7% (3.7, 8.7) to 10.0% (6.2, 14.2) (p Z 0.02). Conversely, these markers did not change in Group I. After 3 months, the levels of inflammatory markers remained unmodified in both groups.
Abbreviations: PAD, peripheral arterial disease; n-3 PUFAs, omega-3 polyunsaturated fatty acids; CAD, coronary artery disease; FMD, flowmediated dilation; sTM, soluble thrombomodulin; CRP, C-reactive protein; MPOx, myeloperoxidase; ABI, ankle/brachial index; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; HDL, high-density lipoprotein; LDL, low-density lipoprotein; hs, high-sensitive. * Corresponding author. Via Pirro Ligorio, 10, 80129 Napoli, Italy. Tel./fax: þ39 081 7462240. E-mail address: [email protected] (V. Schiano). 0261-5614/$ - see front matter ª 2007 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved. doi:10.1016/j.clnu.2007.11.007
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V. Schiano et al. Conclusions: In PAD, n-3 PUFAs induced a marked improvement in endothelial function. Conversely, they did not affect the inflammatory status. In future, large, prospective studies are needed to investigate whether n-3 PUFAs, by improving endothelial function, would reduce the incidence of ischemic events in a population at high risk. ª 2007 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.
Introduction An increased omega-3 polyunsaturated fatty acid (n-3 PUFA) intake has been shown to reduce mortality in both healthy subjects and patients with previous myocardial infarction.1e3 The cardiac benefits of n-3 PUFAs are mainly attributed to their anti-arrhythmic properties,3 however, several additional effects may contribute to the protective cardiovascular action of these molecules. Actually, n-3 PUFAs by reducing triglyceridemia4 and inflammatory status,5,6 and improving the endothelial function,7,8 interferes with fundamental mechanisms promoting atherogenesis, and may inhibit the development of new plaques, retard the growth of established plaques, and contribute to plaque stabilization.9,10 Peripheral arterial disease (PAD), which is characterized by partial or complete obstruction of the arteries of the lower limb, is one of the most common manifestations of atherosclerosis. Its main clinical expression (i.e., intermittent claudication) is characterized by a reduction in walking capacity which limits daily activities. However, another important clinical aspect of this condition is its association with a high risk of developing atherothrombotic events, such as myocardial infarction and stroke.11,12 The role of polyunsaturated fatty acid in PAD is less clear than in coronary artery disease (CAD), however, there is some evidence that low levels of n-3 PUFAs are associated with increased disease.13 A small trial showed little improvement in walking capacity in PAD patients taking gamma-linoleic acid,14 but a review of randomized controlled trials in people with intermittent claudication reported that the only effect of n-3 PUFA supplementation was to reduce triglyceride levels and diastolic blood pressure.15 However, to the best of our knowledge, no study investigated the effect of n-3 PUFAs on inflammation and endothelial function in PAD. This is particularly unfortunate because in these patients, markers of inflammation and endothelial dysfunction are strongly associated with the presence and severity of PAD16,17 and have a profound impact on the risk of future cardiovascular events.18e21 Accordingly, the primary objective of the present study was to assess the effect of n-3 PUFAs on brachial artery flowmediated dilation (FMD), soluble thrombomodulin (sTM), C-reactive protein (CRP) and myeloperoxidase (MPOx) in patients with PAD.
Methods Patients Thirty-two consecutive patients, who were referred to our vascular laboratory with a history of intermittent
claudication and fitted with the exclusion/inclusion criteria of the study, were enrolled. All had a resting ankle/brachial index (ABI) < 0.90 and the presence of one or more stenoses >50% in at least one leg artery on duplex scanning. The exclusion criteria were the presence of rest pain and/or trophic lesions in the affected limb; acute coronary syndrome or cerebrovascular events during the previous 6 months; significant renal, hepatic or inflammatory disease; history of hypersensitivity; or other contraindications to n-3 PUFAs. All women were postmenopausal, and none was receiving hormone replacement therapy. Written informed consent was obtained from all patients before the study, which was approved by our institutional ethics committee.
Study protocol This was a single-blind (blind-observer) parallel group study in which patients were randomly assigned to two groups. In Group I (n Z 16) pre-enrollment therapy was not modified, while in Group II (n Z 16) n-3 PUFAs 1 g b.i.d. were added to the previous treatment for 3 months. The capsules used in the study contained at least 85% of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) as ethyl esters in the average ratio of EPA/DHA 0.9:1.5. Concomitant cardiovascular treatments were not discontinued. Measurements of endothelial function, inflammatory status, lipid pattern and severity of peripheral vascular disease were performed at the beginning and end of the study by personnel unaware of group treatment assignment. All studies were carried out in the morning after an overnight fast, in a quiet room at a constant temperature of 21 1 C. All subjects were abstained from smoking and from consuming caffeine-containing food or beverages for at least 12 h before the visits.
Serum lipids Total serum cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL) and triglycerides were measured by enzymatic methods (CHOD-PAP and GPO-PAP; Boehringer Mannheim diagnostic).22
Measurement of ABI PAD severity was evaluated by ABI. After 5 min of supine rest, the systolic pressure in the right and left posterior tibial artery and in the right brachial artery (no patients had a systolic blood pressure difference 10 mmHg between the two arms) was measured twice with Doppler ultrasound probe. The average of the two measurements was used to calculate ABI.
Polyunsaturated fatty acid in peripheral arterial disease
243
Endothelial function
Lipid profile
Plasma levels of sTM were measured by ELISA method (Asserachrom TM, Roche Diagnostics). Brachial artery FMD was assessed according to the recent guidelines,23 using a high-resolution ultrasound system with a 7.5-MHz linear-array vascular transducer. Briefly, a pneumatic tourniquet placed around the forearm was inflated to 50 mmHg above systolic blood pressure for 5 min, and rapidly deflated, resulting in reactive hyperemia. Brachial artery images were obtained at baseline and 1 min after cuff deflation to assess the change in brachial artery diameter in response to reactive hyperemia. End-diastolic frames (coincident with the electrocardiographic R-wave) from four consecutive cardiac cycles at baseline and 1 min after reactive hyperemia were recorded on VHS videotape for subsequent blinded analysis. Responses of vessel diameters to reactive hyperemia were expressed as percent increase versus the baseline diameter. In our laboratory, the intraobserver variability for repeated measurements of resting arterial diameter is 0.01 0.02 mm.
In Group II, n-3 PUFAs induced a significant reduction in triglyceride levels but not in serum concentrations of total cholesterol, LDL and HDL cholesterol. In patients of Group I, serum lipid levels remained unmodified after 3 months. For triglyceride levels we observed a significant difference between the study groups in relative changes from baseline. All these data are shown in Table 2.
Inflammatory status Plasma levels of CRP were measured by high-sensitive (hs) ELISA methods (Dade Behring Diagnostics). Plasma levels of MPOx were measured by ELISA according to procedures recommended by the manufacturer (Calbiochem).
Statistical analysis This was an exploratory phase III study without previous knowledge about effectesize magnitude in PAD. Therefore, no formal sample size computations were performed. Because the parameters evaluated in this study were not normally distributed, continuous values are expressed as median (interquartile range) and group comparisons were made by nonparametric tests. The baseline characteristics of the two groups were compared by the ManneWhitney U test for continuous variables, and by a chi-square test for categorical variables. Within group comparisons of the parameters measured on day 0 and 90 were made by Wilcoxon test. Differences in relative changes from baseline values between Group I and Group II were compared by Manne Whitney U test.
Results At baseline, clinical characteristics and use of cardiovascular drugs were similar in patients of Group I and Group II (Table 1). In particular, no group difference was observed for serum lipid concentrations, ABI and markers of endothelial function and inflammation. All subjects completed the study. No patient took new medications during the study period. No adverse side effects were reported. Compliance with n-3 PUFAs treatment was assessed by count of the capsules returned at the end of the study and was considered satisfactory (>94%).
Endothelial function At the end of the study period, Group II showed a significant decrease in plasma levels of sTM (Table 3). The beneficial effect of n-3 PUFAs on endothelium was confirmed by the increase in brachial artery FMD observed in Group II (Table 3). On the contrary, these endothelial markers remained unmodified in patients who did not receive n-3 PUFAs treatment. As Fig. 1 shows, in Group I, the relative changes from baseline were 0.1 ng/mL (0.2, 10.9) for sTM and 0.2% (1.6, 1.0) for FMD. The corresponding value for the group who received n-3 PUFA supplementation were 4.0 ng/mL (16.0, 0.0) (p Z 0.03), and 2.3% (0.7, 6.2) (p Z 0.02). Reduction in sTM and improvement in FMD did not correlate with the reduction in triglycerides in the n-3 PUFAs group.
Inflammatory status Compared to baseline values, plasma levels of hs-CRP and MPOx remained unmodified after 3 months, in both treated and control group (Table 3).
PAD severity In Group I, ABI was 0.67 (0.55, 0.77) at baseline and 0.68 (0.55, 0.78) at the end of the study. Also in the treated group ABI remained unmodified, being 0.66 (0.51, 0.74) at the study entry and 0.66 (0.53, 0.69) after n-3 PUFA administration.
Discussion PAD is a major manifestation of atherosclerosis and is associated with a high incidence of ischemic events such as myocardial infarction and stroke.11,12 However, the increased risk of claudicant patients appears to be independent of the traditional risk factors, and only partly explained by the expected association of PAD with coronary and carotid diseases consequent to the systemic nature of atherosclerosis.11,12 This surprising, but consistent observation suggests that other factors may contribute to the excessive atherosclerotic burden and cardiovascular risk of claudicant patients. In recent years it has become increasingly clear that endothelial dysfunction and inflammation play pivotal roles in the initiation, progression, and clinical manifestations of atherosclerosis.24 In PAD, a powerful impact on the cardiovascular risk has been shown for endothelial dysfunction20,21,25 and inflammation,18,19 which thus
244 Table 1
V. Schiano et al. Characteristics in patients of Group I and Group II Group I (n Z 16)
Age (yr) Males, n (%)
Group II (n Z 16)
66 (59, 73) 14 (87)
Risk factors Diabetes mellitus, n (%) Hypercholesterolemia, n (%) Hypertriglyceridemia, n (%) Hypertension, n (%) Smoking, n (%) Body Mass Index
8 11 6 11 15 26.8
Serum lipids Total cholesterol (mg/dL) HDL cholesterol (mg/dL) LDL cholesterol (mg/dL) Triglycerides (mg/dL)
182 44 116 142
Comorbidity Previous MI, n (%) Previous stroke, n (%)
66 (58, 71) 15 (94)
(50) (69) (38) (69) (94) (24.4, 29.7)
7 12 8 14 15 27.0
(141, 227) (30, 47) (87, 148) (123, 181)
210 45 132 171
6 (38) 2 (12)
Medications Antiplatelets, n (%) Beta-blockers, n (%) Calcium antagonists, n (%) ACE-inhibitors, n (%) Statins, n (%)
16 4 7 11 10
P 0.63 0.54
(44) (75) (50) (87) (94) (25.8, 30.0)
0.72 0.69 0.48 0.20 1 0.57
(167, 241) (37, 51) (90, 159) (78, 262)
0.26 0.54 0.47 0.93
7 (44) 1 (6)
(100) (25) (44) (69) (62)
16 5 10 11 11
0.72 0.54
(100) (31) (62) (69) (69)
1 0.69 0.29 1 0.71
PAD severity Ankle/brachial index
0.67 (0.55, 0.77)
0.66 (0.51, 0.74)
0.48
Endothelial function Flow-mediated dilation (%) s-Thrombomodulin (ng/mL)
7.1 (4.0, 9.8) 30.2 (10.0, 37.1)
6.7 (3.7, 8.7) 33.0 (16.7, 37.2)
0.63 0.55
Inflammatory markers C-reactive protein (mg/L) Myeloperoxidase (ng/mL)
3.1 (2.1, 3.9) 20.2 (16.0, 32.3)
2.6 (2.1, 5.8) 18.3 (16.8, 21.2)
0.90 0.48
HDL Z high-density lipoprotein; LDL Z low-density lipoprotein; MI Z myocardial infarction; and ACE Z angiotensin converting enzyme.
the presence of endothelial dysfunction). These findings are in agreement with those of previous papers showing a beneficial effect of n-3 fatty acids on endothelial function in dyslipidemic and CAD patients,7,8,26 although we used a lower dosage of the drug. Consistent with a previous study,7 the improvement in endothelial function achieved by n-3 PUFAs did not correlate with the reduction in triglycerides, thus excluding
could represent a target for therapies aimed at reducing the risk. Results of the present study indicate that, in PAD, n-3 PUFA supplementation for 3 months, not only reduce triglyceride levels, but also improves endothelial function as reflected by the increase in brachial artery FMD and decrease in plasma levels of sTM (a specific constituent of the endothelial cells, which is cleaved away in the plasma in
Table 2
Serum lipids (mg/dL) at baseline and after 3 months in patients of Group I and Group II Group I (n Z 16)
Total cholesterol HDL cholesterol LDL cholesterol Triglycerides
Group II (n Z 16)
PD
Baseline
3 Months
P1
Baseline
3 Months
P2
182 44 116 142
174 39 112 152
0.82 0.97 0.98 0.91
210 45 132 171
169 48 109 108
0.16 0.89 0.11 0.03
(141, 227) (30, 47) (87, 148) (123, 181)
(140, 222) (32, 56) (78, 149) (116, 179)
(167, 241) (37, 51) (90, 159) (78, 262)
(137, 210) (37, 53) (66, 133) (79, 146)
0.27 0.74 0.25 0.03
P1 Z significance between baseline and 3 months in Group I; P2 Z significance between baseline and 3 months in Group II; PD Z significance between group differences in relative changes from baseline (statistical significance was observed only for triglycerides); HDL Z high-density lipoprotein; and LDL Z low-density lipoprotein.
Polyunsaturated fatty acid in peripheral arterial disease Table 3
245
Markers of endothelial function and inflammation at baseline and after 3 months in patients of Group I and Group II Group I (n Z 16)
FMD (%) sTM (ng/mL) MPOx (ng/mL) CRP (mg/L)
Group II (n Z 16)
PD
Baseline
3 Months
P1
Baseline
3 Months
P2
7.1 30.2 20.2 3.1
6.8 30.9 17.0 3.0
0.41 0.31 0.19 0.81
6.7 33.0 18.3 2.6
10.0 17.0 16.9 2.9
0.02 0.04 0.53 0.12
(4.0, 9.8) (10.0, 37.1) (16.0, 32.3) (2.1, 3.9)
(2.5, 7.7) (15.2, 50.1) (14.8, 21.0) (2.6, 7.7)
(3.7, 8.7) (16.7, 37.2) (16.8, 21.2) (2.1, 5.8)
(6.2, 14.2) (11.2, 33.7) (15.7, 25.3) (2.2, 4.6)
0.02 0.03 0.19 0.39
P1 Z significance between baseline and 3 months in Group I; P2 Z significance between baseline and 3 months in Group II; PD Z significance between group differences in relative changes from baseline (statistical significance was observed only for FMD and sTM); FMD Z flow-mediated dilation; sTM Z soluble thrombomodulin; MPOx Z myeloperoxidase; and CRP Z C-reactive protein.
that the latter mechanism may be responsible for the increase in FMD and the reduction in sTM. Further support for a lack of association between hypertriglyceridemia and endothelial dysfunction comes from the previous demonstration that severe hypertriglyceridemia was not associated with significant impairment of the L-arginine/NO pathway in forearm resistance vessels.27 The mechanism(s) underlying the beneficial effect of omega-3 PUFAs on endothelial function remain to be defined. An in vitro artery study28 demonstrated that the greatest improvement in endothelial function occurred in those patients who had the greatest increase in membrane EPA and DHA, as reflected by increase in these fatty acids in the red cell membrane. Hence, it is possible that dietary supplementation with marine omega-3 fatty acids may change the membrane fluidity of endothelial cells, promoting increased synthesis and/or release of NO. In addition, feeding human with fish oils has been shown to reduce oxygen-derived free radical formation, which are harmful for the endothelium, and enhance NO production by cultured human endothelial cells.29 With respect to inflammation, several trials have shown that n-3 PUFAs, by reducing the levels of leukotriene B4, IL-1 and TNF,6 exert anti-inflammatory and immune-modulating effects in a variety of inflammatory diseases such as psoriasis, asthma and rheumatoid arthritis. However, among the plasma inflammatory markers, the analyte of choice to
evaluate the cardiovascular risk is hs-CRP according to the indication of the American Heart Association.30 In particular, CRP plays an important role in the pathophysiology and natural history of PAD.18,19,31 In this regard, the evidence that n-3 PUFAs reduce plasma levels of hs-CRP is not strong. A recent study32 showed that n-3 fatty acids significantly reduced the arachidonic acid:EPA ratio but had no effect on CRP levels in both healthy subjects and CAD patients. Similarly, two prospective studies did not find a significant effect of omega-3 fatty acid on CRP.33,34 In line with these findings, we found that n-3 PUFAs did not modify hs-CRP plasma levels in PAD. To extend the knowledge about the effect of n-3 PUFAs on the inflammatory markers of cardiovascular risk, in addition to hs-CRP, we measured MPOx. When secreted by activated neutrophil, MPOx converts low-density lipoprotein into an atherogenic form, generates numerous reactive oxidants and diffusible radical species, and reduces nitric oxide availability.35 Thus, not surprisingly, elevated levels of MPOx are related to the severity of CAD,36 and entail a worse prognosis in patients with acute coronary syndrome.37 In our study, similar to what was observed for hs-CRP, n-3 PUFAs did not reduce plasma levels of MPOx. The reasons for the discrepancy between the results concerning the effect of n-3 PUFAs on the markers of inflammation and endothelial function may be several. First, different population may respond differently to the
Figure 1 Relative change from baseline value for soluble thrombomodulin (sTM) and flow-mediated dilation (FMD). Results are shown as median (lines) and interquartile ranges (boxes).
246 treatment. Second, the duration of the treatment may affect the results. Indeed, Abe et al.38 found no reduction in the soluble forms of intercellular adhesion molecules-1 and E selectin after 6 weeks of n-3 PUFA supplementation but found a significant reduction in the levels of these molecules after 7 months. Third, the effect of n-3 PUFAs may vary by dose. Consistent with our results, lower doses were reported to decrease some markers of endothelial activation although they did not affect inflammatory cell numbers or function.5 In conclusion, the present study shows that, in PAD, n-3 PUFA administration at the dose of 1 g b.i.d for 3 months improves FMD and reduces sTM levels, but does not reduce the plasma levels of hs-CRP and MPOx. However, in the absence of anti-inflammatory action, the beneficial effect of n-3 PUFAs on endothelial function supports a possible future use of these drugs in PAD patients, in whom endothelial dysfunction portends a high cardiovascular risk.20,21,25 In this regard, it is noteworthy that in a study39 including 120 PAD patients, during the follow-up there were less non-fatal myocardial infarctions and other coronary events in the fatty acid group than in the placebo group (10% vs. 15%). This difference did not reach statistical significance because of the small sample size. However, it is clinically relevant that patients in the placebo group experienced 50% more of coronary events compared to the fatty acid group. Interestingly, this result was obtained without any clinically significant modification in classic risk factor, thus suggesting that other mechanisms may have played a role in the risk reduction. Therefore, there is a need for future, large, prospective studies to confirm the results of the present study and investigate whether n-3 PUFAs, by improving endothelial function, would reduce the incidence of ischemic events in a population at high risk.
Conflict of interest statement The authors have no financial or other relations that could lead to conflict of interest.
Acknowledgement VS, GB and MC designed the study. JIDM and SL collected the data. VS, EL and FS analyzed the data. VS, EL and GB wrote the manuscript. VS, GB and MC revised the manuscript. All authors read and approved the manuscript.
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available at www.sciencedirect.com
http://intl.elsevierhealth.com/journals/clnu
ORIGINAL ARTICLE
Omega-3 polyunsaturated fatty acid in peripheral arterial disease: Effect on lipid pattern, disease severity, inflammation profile, and endothelial function Vittorio Schiano a,*, Eugenio Laurenzano a, Gregorio Brevetti a, Julieta Isabel De Maio a, Simona Lanero a, Francesco Scopacasa b, Massimo Chiariello a a Department of Clinical Medicine and Cardiovascular and Immunological Sciences, University of Naples ‘‘Federico II’’, Naples, Italy b Department of Laboratory Medicine; University of Naples ‘‘Federico II’’, Naples, Italy
Received 2 August 2007; accepted 27 November 2007
KEYWORDS Peripheral arterial disease; Polyunsaturated fatty acids; Endothelial function; Inflammation
Summary Background & aims: Peripheral arterial disease (PAD) is strongly associated with endothelial dysfunction and inflammation, which portend a high cardiovascular risk. Accordingly, we investigated the effects of omega-3 polyunsaturated fatty acid (n-3 PUFA) supplementation on endothelial function and inflammatory status in affected individuals. Methods: PAD patients were randomly divided into two groups. In Group I (n Z 16) preenrollment therapy was not modified, while in Group II (n Z 16) n-3 PUFAs 1 g b.i.d. for 3 months were added to the previous treatment. Endothelial function was assessed by measuring plasma soluble thrombomodulin (sTM) and brachial artery flow-mediated dilation (FMD), and the inflammatory status by measuring high-sensitivity C-reactive protein and myeloperoxidase. Results: In Group II, n-3 PUFAs reduced sTM levels from the median value of 33.0 ng/mL (interquartile range 16.7, 37.2) to 17.0 ng/mL (11.2, 33.7) (p Z 0.04), and improved FMD from 6.7% (3.7, 8.7) to 10.0% (6.2, 14.2) (p Z 0.02). Conversely, these markers did not change in Group I. After 3 months, the levels of inflammatory markers remained unmodified in both groups.
Abbreviations: PAD, peripheral arterial disease; n-3 PUFAs, omega-3 polyunsaturated fatty acids; CAD, coronary artery disease; FMD, flowmediated dilation; sTM, soluble thrombomodulin; CRP, C-reactive protein; MPOx, myeloperoxidase; ABI, ankle/brachial index; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; HDL, high-density lipoprotein; LDL, low-density lipoprotein; hs, high-sensitive. * Corresponding author. Via Pirro Ligorio, 10, 80129 Napoli, Italy. Tel./fax: þ39 081 7462240. E-mail address: [email protected] (V. Schiano). 0261-5614/$ - see front matter ª 2007 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved. doi:10.1016/j.clnu.2007.11.007
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V. Schiano et al. Conclusions: In PAD, n-3 PUFAs induced a marked improvement in endothelial function. Conversely, they did not affect the inflammatory status. In future, large, prospective studies are needed to investigate whether n-3 PUFAs, by improving endothelial function, would reduce the incidence of ischemic events in a population at high risk. ª 2007 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.
Introduction An increased omega-3 polyunsaturated fatty acid (n-3 PUFA) intake has been shown to reduce mortality in both healthy subjects and patients with previous myocardial infarction.1e3 The cardiac benefits of n-3 PUFAs are mainly attributed to their anti-arrhythmic properties,3 however, several additional effects may contribute to the protective cardiovascular action of these molecules. Actually, n-3 PUFAs by reducing triglyceridemia4 and inflammatory status,5,6 and improving the endothelial function,7,8 interferes with fundamental mechanisms promoting atherogenesis, and may inhibit the development of new plaques, retard the growth of established plaques, and contribute to plaque stabilization.9,10 Peripheral arterial disease (PAD), which is characterized by partial or complete obstruction of the arteries of the lower limb, is one of the most common manifestations of atherosclerosis. Its main clinical expression (i.e., intermittent claudication) is characterized by a reduction in walking capacity which limits daily activities. However, another important clinical aspect of this condition is its association with a high risk of developing atherothrombotic events, such as myocardial infarction and stroke.11,12 The role of polyunsaturated fatty acid in PAD is less clear than in coronary artery disease (CAD), however, there is some evidence that low levels of n-3 PUFAs are associated with increased disease.13 A small trial showed little improvement in walking capacity in PAD patients taking gamma-linoleic acid,14 but a review of randomized controlled trials in people with intermittent claudication reported that the only effect of n-3 PUFA supplementation was to reduce triglyceride levels and diastolic blood pressure.15 However, to the best of our knowledge, no study investigated the effect of n-3 PUFAs on inflammation and endothelial function in PAD. This is particularly unfortunate because in these patients, markers of inflammation and endothelial dysfunction are strongly associated with the presence and severity of PAD16,17 and have a profound impact on the risk of future cardiovascular events.18e21 Accordingly, the primary objective of the present study was to assess the effect of n-3 PUFAs on brachial artery flowmediated dilation (FMD), soluble thrombomodulin (sTM), C-reactive protein (CRP) and myeloperoxidase (MPOx) in patients with PAD.
Methods Patients Thirty-two consecutive patients, who were referred to our vascular laboratory with a history of intermittent
claudication and fitted with the exclusion/inclusion criteria of the study, were enrolled. All had a resting ankle/brachial index (ABI) < 0.90 and the presence of one or more stenoses >50% in at least one leg artery on duplex scanning. The exclusion criteria were the presence of rest pain and/or trophic lesions in the affected limb; acute coronary syndrome or cerebrovascular events during the previous 6 months; significant renal, hepatic or inflammatory disease; history of hypersensitivity; or other contraindications to n-3 PUFAs. All women were postmenopausal, and none was receiving hormone replacement therapy. Written informed consent was obtained from all patients before the study, which was approved by our institutional ethics committee.
Study protocol This was a single-blind (blind-observer) parallel group study in which patients were randomly assigned to two groups. In Group I (n Z 16) pre-enrollment therapy was not modified, while in Group II (n Z 16) n-3 PUFAs 1 g b.i.d. were added to the previous treatment for 3 months. The capsules used in the study contained at least 85% of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) as ethyl esters in the average ratio of EPA/DHA 0.9:1.5. Concomitant cardiovascular treatments were not discontinued. Measurements of endothelial function, inflammatory status, lipid pattern and severity of peripheral vascular disease were performed at the beginning and end of the study by personnel unaware of group treatment assignment. All studies were carried out in the morning after an overnight fast, in a quiet room at a constant temperature of 21 1 C. All subjects were abstained from smoking and from consuming caffeine-containing food or beverages for at least 12 h before the visits.
Serum lipids Total serum cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL) and triglycerides were measured by enzymatic methods (CHOD-PAP and GPO-PAP; Boehringer Mannheim diagnostic).22
Measurement of ABI PAD severity was evaluated by ABI. After 5 min of supine rest, the systolic pressure in the right and left posterior tibial artery and in the right brachial artery (no patients had a systolic blood pressure difference 10 mmHg between the two arms) was measured twice with Doppler ultrasound probe. The average of the two measurements was used to calculate ABI.
Polyunsaturated fatty acid in peripheral arterial disease
243
Endothelial function
Lipid profile
Plasma levels of sTM were measured by ELISA method (Asserachrom TM, Roche Diagnostics). Brachial artery FMD was assessed according to the recent guidelines,23 using a high-resolution ultrasound system with a 7.5-MHz linear-array vascular transducer. Briefly, a pneumatic tourniquet placed around the forearm was inflated to 50 mmHg above systolic blood pressure for 5 min, and rapidly deflated, resulting in reactive hyperemia. Brachial artery images were obtained at baseline and 1 min after cuff deflation to assess the change in brachial artery diameter in response to reactive hyperemia. End-diastolic frames (coincident with the electrocardiographic R-wave) from four consecutive cardiac cycles at baseline and 1 min after reactive hyperemia were recorded on VHS videotape for subsequent blinded analysis. Responses of vessel diameters to reactive hyperemia were expressed as percent increase versus the baseline diameter. In our laboratory, the intraobserver variability for repeated measurements of resting arterial diameter is 0.01 0.02 mm.
In Group II, n-3 PUFAs induced a significant reduction in triglyceride levels but not in serum concentrations of total cholesterol, LDL and HDL cholesterol. In patients of Group I, serum lipid levels remained unmodified after 3 months. For triglyceride levels we observed a significant difference between the study groups in relative changes from baseline. All these data are shown in Table 2.
Inflammatory status Plasma levels of CRP were measured by high-sensitive (hs) ELISA methods (Dade Behring Diagnostics). Plasma levels of MPOx were measured by ELISA according to procedures recommended by the manufacturer (Calbiochem).
Statistical analysis This was an exploratory phase III study without previous knowledge about effectesize magnitude in PAD. Therefore, no formal sample size computations were performed. Because the parameters evaluated in this study were not normally distributed, continuous values are expressed as median (interquartile range) and group comparisons were made by nonparametric tests. The baseline characteristics of the two groups were compared by the ManneWhitney U test for continuous variables, and by a chi-square test for categorical variables. Within group comparisons of the parameters measured on day 0 and 90 were made by Wilcoxon test. Differences in relative changes from baseline values between Group I and Group II were compared by Manne Whitney U test.
Results At baseline, clinical characteristics and use of cardiovascular drugs were similar in patients of Group I and Group II (Table 1). In particular, no group difference was observed for serum lipid concentrations, ABI and markers of endothelial function and inflammation. All subjects completed the study. No patient took new medications during the study period. No adverse side effects were reported. Compliance with n-3 PUFAs treatment was assessed by count of the capsules returned at the end of the study and was considered satisfactory (>94%).
Endothelial function At the end of the study period, Group II showed a significant decrease in plasma levels of sTM (Table 3). The beneficial effect of n-3 PUFAs on endothelium was confirmed by the increase in brachial artery FMD observed in Group II (Table 3). On the contrary, these endothelial markers remained unmodified in patients who did not receive n-3 PUFAs treatment. As Fig. 1 shows, in Group I, the relative changes from baseline were 0.1 ng/mL (0.2, 10.9) for sTM and 0.2% (1.6, 1.0) for FMD. The corresponding value for the group who received n-3 PUFA supplementation were 4.0 ng/mL (16.0, 0.0) (p Z 0.03), and 2.3% (0.7, 6.2) (p Z 0.02). Reduction in sTM and improvement in FMD did not correlate with the reduction in triglycerides in the n-3 PUFAs group.
Inflammatory status Compared to baseline values, plasma levels of hs-CRP and MPOx remained unmodified after 3 months, in both treated and control group (Table 3).
PAD severity In Group I, ABI was 0.67 (0.55, 0.77) at baseline and 0.68 (0.55, 0.78) at the end of the study. Also in the treated group ABI remained unmodified, being 0.66 (0.51, 0.74) at the study entry and 0.66 (0.53, 0.69) after n-3 PUFA administration.
Discussion PAD is a major manifestation of atherosclerosis and is associated with a high incidence of ischemic events such as myocardial infarction and stroke.11,12 However, the increased risk of claudicant patients appears to be independent of the traditional risk factors, and only partly explained by the expected association of PAD with coronary and carotid diseases consequent to the systemic nature of atherosclerosis.11,12 This surprising, but consistent observation suggests that other factors may contribute to the excessive atherosclerotic burden and cardiovascular risk of claudicant patients. In recent years it has become increasingly clear that endothelial dysfunction and inflammation play pivotal roles in the initiation, progression, and clinical manifestations of atherosclerosis.24 In PAD, a powerful impact on the cardiovascular risk has been shown for endothelial dysfunction20,21,25 and inflammation,18,19 which thus
244 Table 1
V. Schiano et al. Characteristics in patients of Group I and Group II Group I (n Z 16)
Age (yr) Males, n (%)
Group II (n Z 16)
66 (59, 73) 14 (87)
Risk factors Diabetes mellitus, n (%) Hypercholesterolemia, n (%) Hypertriglyceridemia, n (%) Hypertension, n (%) Smoking, n (%) Body Mass Index
8 11 6 11 15 26.8
Serum lipids Total cholesterol (mg/dL) HDL cholesterol (mg/dL) LDL cholesterol (mg/dL) Triglycerides (mg/dL)
182 44 116 142
Comorbidity Previous MI, n (%) Previous stroke, n (%)
66 (58, 71) 15 (94)
(50) (69) (38) (69) (94) (24.4, 29.7)
7 12 8 14 15 27.0
(141, 227) (30, 47) (87, 148) (123, 181)
210 45 132 171
6 (38) 2 (12)
Medications Antiplatelets, n (%) Beta-blockers, n (%) Calcium antagonists, n (%) ACE-inhibitors, n (%) Statins, n (%)
16 4 7 11 10
P 0.63 0.54
(44) (75) (50) (87) (94) (25.8, 30.0)
0.72 0.69 0.48 0.20 1 0.57
(167, 241) (37, 51) (90, 159) (78, 262)
0.26 0.54 0.47 0.93
7 (44) 1 (6)
(100) (25) (44) (69) (62)
16 5 10 11 11
0.72 0.54
(100) (31) (62) (69) (69)
1 0.69 0.29 1 0.71
PAD severity Ankle/brachial index
0.67 (0.55, 0.77)
0.66 (0.51, 0.74)
0.48
Endothelial function Flow-mediated dilation (%) s-Thrombomodulin (ng/mL)
7.1 (4.0, 9.8) 30.2 (10.0, 37.1)
6.7 (3.7, 8.7) 33.0 (16.7, 37.2)
0.63 0.55
Inflammatory markers C-reactive protein (mg/L) Myeloperoxidase (ng/mL)
3.1 (2.1, 3.9) 20.2 (16.0, 32.3)
2.6 (2.1, 5.8) 18.3 (16.8, 21.2)
0.90 0.48
HDL Z high-density lipoprotein; LDL Z low-density lipoprotein; MI Z myocardial infarction; and ACE Z angiotensin converting enzyme.
the presence of endothelial dysfunction). These findings are in agreement with those of previous papers showing a beneficial effect of n-3 fatty acids on endothelial function in dyslipidemic and CAD patients,7,8,26 although we used a lower dosage of the drug. Consistent with a previous study,7 the improvement in endothelial function achieved by n-3 PUFAs did not correlate with the reduction in triglycerides, thus excluding
could represent a target for therapies aimed at reducing the risk. Results of the present study indicate that, in PAD, n-3 PUFA supplementation for 3 months, not only reduce triglyceride levels, but also improves endothelial function as reflected by the increase in brachial artery FMD and decrease in plasma levels of sTM (a specific constituent of the endothelial cells, which is cleaved away in the plasma in
Table 2
Serum lipids (mg/dL) at baseline and after 3 months in patients of Group I and Group II Group I (n Z 16)
Total cholesterol HDL cholesterol LDL cholesterol Triglycerides
Group II (n Z 16)
PD
Baseline
3 Months
P1
Baseline
3 Months
P2
182 44 116 142
174 39 112 152
0.82 0.97 0.98 0.91
210 45 132 171
169 48 109 108
0.16 0.89 0.11 0.03
(141, 227) (30, 47) (87, 148) (123, 181)
(140, 222) (32, 56) (78, 149) (116, 179)
(167, 241) (37, 51) (90, 159) (78, 262)
(137, 210) (37, 53) (66, 133) (79, 146)
0.27 0.74 0.25 0.03
P1 Z significance between baseline and 3 months in Group I; P2 Z significance between baseline and 3 months in Group II; PD Z significance between group differences in relative changes from baseline (statistical significance was observed only for triglycerides); HDL Z high-density lipoprotein; and LDL Z low-density lipoprotein.
Polyunsaturated fatty acid in peripheral arterial disease Table 3
245
Markers of endothelial function and inflammation at baseline and after 3 months in patients of Group I and Group II Group I (n Z 16)
FMD (%) sTM (ng/mL) MPOx (ng/mL) CRP (mg/L)
Group II (n Z 16)
PD
Baseline
3 Months
P1
Baseline
3 Months
P2
7.1 30.2 20.2 3.1
6.8 30.9 17.0 3.0
0.41 0.31 0.19 0.81
6.7 33.0 18.3 2.6
10.0 17.0 16.9 2.9
0.02 0.04 0.53 0.12
(4.0, 9.8) (10.0, 37.1) (16.0, 32.3) (2.1, 3.9)
(2.5, 7.7) (15.2, 50.1) (14.8, 21.0) (2.6, 7.7)
(3.7, 8.7) (16.7, 37.2) (16.8, 21.2) (2.1, 5.8)
(6.2, 14.2) (11.2, 33.7) (15.7, 25.3) (2.2, 4.6)
0.02 0.03 0.19 0.39
P1 Z significance between baseline and 3 months in Group I; P2 Z significance between baseline and 3 months in Group II; PD Z significance between group differences in relative changes from baseline (statistical significance was observed only for FMD and sTM); FMD Z flow-mediated dilation; sTM Z soluble thrombomodulin; MPOx Z myeloperoxidase; and CRP Z C-reactive protein.
that the latter mechanism may be responsible for the increase in FMD and the reduction in sTM. Further support for a lack of association between hypertriglyceridemia and endothelial dysfunction comes from the previous demonstration that severe hypertriglyceridemia was not associated with significant impairment of the L-arginine/NO pathway in forearm resistance vessels.27 The mechanism(s) underlying the beneficial effect of omega-3 PUFAs on endothelial function remain to be defined. An in vitro artery study28 demonstrated that the greatest improvement in endothelial function occurred in those patients who had the greatest increase in membrane EPA and DHA, as reflected by increase in these fatty acids in the red cell membrane. Hence, it is possible that dietary supplementation with marine omega-3 fatty acids may change the membrane fluidity of endothelial cells, promoting increased synthesis and/or release of NO. In addition, feeding human with fish oils has been shown to reduce oxygen-derived free radical formation, which are harmful for the endothelium, and enhance NO production by cultured human endothelial cells.29 With respect to inflammation, several trials have shown that n-3 PUFAs, by reducing the levels of leukotriene B4, IL-1 and TNF,6 exert anti-inflammatory and immune-modulating effects in a variety of inflammatory diseases such as psoriasis, asthma and rheumatoid arthritis. However, among the plasma inflammatory markers, the analyte of choice to
evaluate the cardiovascular risk is hs-CRP according to the indication of the American Heart Association.30 In particular, CRP plays an important role in the pathophysiology and natural history of PAD.18,19,31 In this regard, the evidence that n-3 PUFAs reduce plasma levels of hs-CRP is not strong. A recent study32 showed that n-3 fatty acids significantly reduced the arachidonic acid:EPA ratio but had no effect on CRP levels in both healthy subjects and CAD patients. Similarly, two prospective studies did not find a significant effect of omega-3 fatty acid on CRP.33,34 In line with these findings, we found that n-3 PUFAs did not modify hs-CRP plasma levels in PAD. To extend the knowledge about the effect of n-3 PUFAs on the inflammatory markers of cardiovascular risk, in addition to hs-CRP, we measured MPOx. When secreted by activated neutrophil, MPOx converts low-density lipoprotein into an atherogenic form, generates numerous reactive oxidants and diffusible radical species, and reduces nitric oxide availability.35 Thus, not surprisingly, elevated levels of MPOx are related to the severity of CAD,36 and entail a worse prognosis in patients with acute coronary syndrome.37 In our study, similar to what was observed for hs-CRP, n-3 PUFAs did not reduce plasma levels of MPOx. The reasons for the discrepancy between the results concerning the effect of n-3 PUFAs on the markers of inflammation and endothelial function may be several. First, different population may respond differently to the
Figure 1 Relative change from baseline value for soluble thrombomodulin (sTM) and flow-mediated dilation (FMD). Results are shown as median (lines) and interquartile ranges (boxes).
246 treatment. Second, the duration of the treatment may affect the results. Indeed, Abe et al.38 found no reduction in the soluble forms of intercellular adhesion molecules-1 and E selectin after 6 weeks of n-3 PUFA supplementation but found a significant reduction in the levels of these molecules after 7 months. Third, the effect of n-3 PUFAs may vary by dose. Consistent with our results, lower doses were reported to decrease some markers of endothelial activation although they did not affect inflammatory cell numbers or function.5 In conclusion, the present study shows that, in PAD, n-3 PUFA administration at the dose of 1 g b.i.d for 3 months improves FMD and reduces sTM levels, but does not reduce the plasma levels of hs-CRP and MPOx. However, in the absence of anti-inflammatory action, the beneficial effect of n-3 PUFAs on endothelial function supports a possible future use of these drugs in PAD patients, in whom endothelial dysfunction portends a high cardiovascular risk.20,21,25 In this regard, it is noteworthy that in a study39 including 120 PAD patients, during the follow-up there were less non-fatal myocardial infarctions and other coronary events in the fatty acid group than in the placebo group (10% vs. 15%). This difference did not reach statistical significance because of the small sample size. However, it is clinically relevant that patients in the placebo group experienced 50% more of coronary events compared to the fatty acid group. Interestingly, this result was obtained without any clinically significant modification in classic risk factor, thus suggesting that other mechanisms may have played a role in the risk reduction. Therefore, there is a need for future, large, prospective studies to confirm the results of the present study and investigate whether n-3 PUFAs, by improving endothelial function, would reduce the incidence of ischemic events in a population at high risk.
Conflict of interest statement The authors have no financial or other relations that could lead to conflict of interest.
Acknowledgement VS, GB and MC designed the study. JIDM and SL collected the data. VS, EL and FS analyzed the data. VS, EL and GB wrote the manuscript. VS, GB and MC revised the manuscript. All authors read and approved the manuscript.
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