- Email: [email protected]
Fatty acid composition of HDL phospholipids and coronary artery disease
Journal of Clinical Lipidology (2009) 3, 39–44
Fatty acid composition of HDL phospholipids and coronary artery disease Mohammad Noori, PhD, Masoud Darabi, PhD, Ali Rahimipour, PhD*, Mohammad Rahbani, PhD, Naser Aslan Abadi, MD, Maryam Darabi, PhD, Keyhan Ghatrehsamani, PhD Department of Biochemistry (Drs. Noori, Masoud Darabi, Rahimipour, Rahbani, and Ghatrehsamani) and Department of Cardiology (Dr. Abadi) School of Medicine, Tabriz University (Medical Sciences), Golgasht Avenue, Tabriz; and Department of Biochemistry School of Pharmacy and Pharmaceutical Sciences, Isfahan University (Medical Sciences), Isfahan, Iran (Dr. Maryam Darabi) KEYWORDS: CAD; Fatty acids; HDL composition
BACKGROUND: The phospholipid fatty acid content of high-density lipoprotein (HDL) partially reflects that of the diet and has been reported to be associated with several important biological functions that might serve as risk markers for coronary heart disease. OBJECTIVE: To investigate whether fatty acid composition of HDL phospholipids correlates with angiographically documented coronary artery disease (CAD). METHODS: The population included 212 patients who underwent clinically indicated coronary angiography. The patients were classified with significantly diseased arteries (SDA) if one or more coronary arteries had a stenosis .50% and with minimally diseased arteries (MDA) if there was no significant stenosis (,40%) in any artery. The severity of CAD was expressed by the number of affected vessels. The fatty acid composition of HDL phospholipids was determined by gas liquid chromatography. Multivariate analyses were used to test the independence of associations between the presence and severity of CAD as outcome variables and fatty acid composition of HDL phospholipids. RESULTS: Patients with SDA showed significantly lower levels of linoleic acid (P 5 .041), eicosapentaenoic acid (EPA) (P 5 .027), and docosahexaenoic acid (DHA) (P 5 .026) than patients with MDA in univariate analyses. The association of linoleic acid (odds ratio [OR] .90, P , .05), EPA (OR, 0.41, P , .02), and DHA (OR, 0.48, P , .02) remained statistically significant in the multivariate analyses. The content of EPA (b 5 20.23, P , .01) and DHA (b 5 20.17, P , .05) remained inversely significantly associated with the severity of CAD. CONCLUSIONS: This study shows that polyunsaturated fatty acids, including EPA and DHA content of HDL particles, are independently associated with the presence and severity of angiographically documented CAD. Ó 2009 National Lipid Association. All rights reserved.
* Corresponding author. E-mail address: [email protected] Submitted August 10, 2008. Accepted for publication November 29, 2008.
The proportions of specific fatty acids in blood or tissue are known to be useful risk markers for a variety of diseases, especially cardiovascular diseases.1–3 The current evidence shows that n-3 fatty acids are effective in the treatment and management of several diseases, which makes the evaluation of fatty acid status even more important.4,5
1933-2874/$ -see front matter Ó 2009 National Lipid Association. All rights reserved. doi:10.1016/j.jacl.2008.11.010
40 Polyunsaturated fatty acids (PUFA), both n-6 and n-3, have been shown in several studies to decrease the risk of cardiovascular diseases through their beneficial effects on blood lipids, insulin sensitivity, inhibition of thrombosis, and vascular inflammation.6 Diets enriched with n-3 fatty acids protect against coronary artery atherosclerosis, an affect that appears to be independent of plasma lipoproteins.7 Previous studies have suggested that the fatty acid composition of serum phospholipids is an independent risk factor for coronary heart disease (CHD).8,9 High-density lipoprotein (HDL) phospholipids constitute the major part of serum phospholipids. The fatty acid composition of HDL phospholipids partially reflects that of the diet and appears to be an important factor that affects their metabolism and could modulate their antiatherogenic activities.10,11 To our knowledge, no clinical study has yet examined the association between the lipid composition of HDL and CHD risk. We therefore designed this study to evaluate the relationship between fatty acid composition of HDL phospholipids and angiographically documented coronary artery disease (CAD).
Methods Subjects Subjects were recruited from a patient population scheduled for diagnostic coronary angiography at Madani Hospital in Tabriz between February 1, 2007 and February 30, 2008. In the present study, 212 patients (151 men and 61 women) were enrolled. Exclusion criteria were age .60 years, a previous hospital admission related to cardiovascular disease, current lipid-lowering medication, and a previous diagnosis of angina, hypercholesterolemia, or diabetes. Hypercholesterolemia was defined as a total level of cholesterol .280 mg/dl or a low-density lipoprotein (LDL) cholesterol level .190 mg/dl. Exclusion of persons with diabetes mellitus was based on self-reported diabetes as well as on fasting serum glucose .125 mg/dl. Venous blood samples were taken after 12–14 hours fasting in the morning before angiography. Blood was centrifuged and the HDL fraction was obtained by precipitation of lipoproteins containing apolipoprotein B (Apo-B) with phosphotungstic acid and magnesium chloride.12 Serum and HDL supernatant were frozen at 270 C until the analyses were performed. The study was approved by the ethic committee of Tabriz University of Medical Sciences, and all patients gave written informed consent.
Journal of Clinical Lipidology, Vol 3, No 1, February 2009 number of affected vessels (one-, two-, or three-vessel disease). By this classification, 36 patients had three-vessel, 39 patients had two-vessel, and 42 patients had one-vessel disease; 95 patients had MDA.
Laboratory analysis Serum total cholesterol, triglycerides, and LDL cholesterol were determined using standard enzymatic procedures. Apolipoprotein (Apo-A-I) and Apo-B concentrations were measured by immunoturbidometric methods (DiaSys Diagnostics, Holzheim, Germany). LDL cholesterol concentrations were calculated using the Friedewald formula.13 The composition of HDL was estimated after precipitation of lipoprotein containing Apo-B. Total lipids from the supernatant were extracted by the method of Folch et al14 with a 2:1 chloroform:methanol solution and several washes. HDL total phospholipids were separated by one-dimensional thin-layer chromatography using silica gel 60 G plates (E. Merck, Darmstadt, Germany) and an 80:20:1 hexane:ethyl ether:acetic acid (10.005% butylated hydroxytoluene) development solvent.15 Phospholipids fatty acid methyl esters were prepared by transmethylation with methanol during catalysis with acetyl chloride.16 Once the reaction mixture had been neutralized with potassium carbonate, the methyl esters were extracted into hexane and were preserved under nitrogen atmosphere at –70 C. Fatty acid methyl ester derivatives formed from the isolated phospholipid fraction were separated on a 60- ! 0.25-mm capillary column (TR CN100, Tekno Kroma, Barcelona, Spian) using a Buck Scientific model 610 gas chromatograph (SRI Instruments, Torrance, CA) equipped with a split injector and a flame ionization detector. Helium was used as the carrier gas and column linear velocity was set at 20.0 cm/s and the oven temperature at 210 C. The oven temperature program was 170–210 C, 1 C/min, and then isothermal for 45 minutes. Tridecanoic acid (13:0) was used as the internal standard. Peak retention times were identified by injecting known standards (Sigma chemicals). Response factor for fatty acid methyl esters were applied, and area percentage of total fatty acid were analyzed with Peak Simple software version 3.59 (SRI Instruments, Torrance, CA). The laboratory variability of the fatty acid analysis was established by repeated measures on a pooled plasma or pooled HDL fraction. The coefficient of variation was 2.5% for palmitic acid (16:0), 3.8 for linoleic acid (18:2n-6), 18.3 for eicosapentaenoic acid (EPA), and 12.3 for docosahexaennoic acid (DHA).
Statistical analysis Angiographic analysis All patients underwent coronary angiography; minimally diseased arteries (MDA) were defined as those with all stenoses ,40% and significantly diseased arteries (SDA) as those with at least one stenosis .50% or occlusions. The severity of CAD among those with SDAwas expressed by the
The level of significance between groups was calculated according to the t test for continuous variables and c2 test for categorical variables. Multivariate analyses were used to test the independence of associations between the presence and severity of CAD as outcome variables, and HDL cholesterol and fatty acid composition of HDL phospholipids
Noori et al Table 1
HDL composition and coronary artery disease
41
Clinical characteristics of patients with minimal versus severe coronary artery disease*
Age, y Sex, women, % Body mass index, kg/m2 Smokers, % Hypertension, % Family history, % Hydrogenated oil intake, % Cholesterol, mg/dl Triglycerides, mg/dl HDL cholesterol, mg/dl LDL cholesterol, mg/dl Apolipoprotein A-I, mg/dl Apolipoprotein B, mg/dl
MDA (n 5 95)
SDA (n 5 117)
P value
47.1 33 26.7 31 27 11 69 165 176 37 92 123 102
50.6 24 27.7 38 36 15 77 176 189 37 101 119 107
.003 NS NS NS NS NS NS .036 NS NS .046 NS NS
6 9.1 6 4.0
6 6 6 6 6 6
34 96 11 29 19 28
6 6.5 6 4.4
6 6 6 6 6 6
41 95 10 35 17 29
*Values are means 6 standard deviation or percentage with condition. The patients were classified as having significantly diseased arteries (SDA) if one or more coronary arteries had a stenosis .50% and as minimally diseased arteries (MDA) if there was no significant stenosis (.40%) in any artery.
as independent variables. We estimated relative risks as odds ratios (ORs) using multiple logistic regression models. Each multiple logistic regression model included the risk factors for which significant differences were found between the MDA and SDA groups. The values of the n-6:n-3 ratio and arachidonic acid (AA):EPA ratio were converted by logarithmic transformation to normalize distribution of the data. For univariate analysis, the Pearson correlation test was performed to identify risk factors associated with severity of CAD. These factors were then entered into multiple regression models to identify independent associations with severity of CAD. The standard regression coefficient b and P value were calculated for each variable. A P value of , .05 was considered
statistically significant. All analyses were carried out using SPSS for windows version 11.0 (SPSS Inc., Chicago, IL).
Results The clinical details and lipid profiles of both groups of patients are presented in Table 1. Patients with angiographically significant coronary atherosclerosis were 3.5 years older than patients without coronary atherosclerosis (P 5 .003). The mean values of total cholesterol was higher in the patients with SDA than in the patients with MDA (176 6 41 vs 165 6 35 mg/dL), reflecting the higher LDL cholesterol in patients with SDA (101 6 35 vs 92 6 29 mg/dL).
Table 2 High-density lipoprotein phospholipids fatty acid composition in patients with minimal versus significant coronary artery disease* Percent of total fatty acids
16:0 (palmitic acid) 16:1 (palmitoleic acid) 18:0 (stearic acid) 18:1n-9 (oleic acid) 18:2n-6 (linoleic acid) 20:3n-6 (dihomo-g-linolenic acid) 20:4n-6 (AA) 20:5n-3 (EPA) 22:6n-3 (docosahexaenoic acid) Saturated FAs Monounsaturated Fas S:P ratio n-6:n-3 ratio AA:EPA ratio
MDA (n 5 95)
SDA (n 5 117)
P value
31.6 6 3.8 1.3 6 0.5 14.0 6 1.8 10.2 6 1.7 19.9 6 3.4 2.3 6 0.7 8.4 6 1.5 0.63 6 0.48 1.10 6 0.6 45.6 6 4.7 11.5 6 1.5 1.46 6 0.31 17.2 (12.3–26.4) 10.13 (8.0–25.2)
32.0 1.3 13.8 9.9 18.9 2.1 8.1 0.49 0.93 45.8 11.2 1.52 20.8 15.9
NS NS NS NS .041 NS NS .027 .026 NS NS .118 NS NS
6 2.9 6 0.5 6 1.7 6 1.8 6 2.9 6 0.7 6 1.2 6 0.41 6 0.46 6 3.6 6 1.8 6 0.25 (14.9–29.5) (9.9–40.6)
AA, arachidonic acid; EPA, eicosapentaenoic acid; FAs, fatty acids; S:P, saturated to polyunsaturated fatty acids. *Values are mean 6 standard deviation, or if variables did not exhibit normal distribution, medians with 25 and 75 percentiles in brackets. Detection limit was 0.05% of the total area. The patients were classified as having significantly diseased arteries (SDA) if one or more coronary arteries had a stenosis .50% and as having minimally diseased arteries (MDA) if there was no significant stenosis (,40%) in any artery.
0.579 24.442 0.485 20.724 0.406
24.305
.014
1.065 1.004 1.001 .004 .616 .952 0.063 0.004 0.001
22.445
20.920 0.899 .041 20.106
24.698
.015
1.067 1.006 1.001 .003 .500 .975 0.065 0.006 0.002 1.056 1.007 0.997 .014 .402 .760 0.054 0.007 -0.003 1.065 1.006 1.006 0.992
25.897
.003 .467 .569 .605 0.063 0.006 0.006 -0.008
Age Cholesterol LDL cholesterol HDL cholesterol 18:2n-6 (linoleic acid) 20:5n-3 (eicosapentaenoic acid) 22:6n-3 (docosahexaenoic acid) S:P ratio Constant
HDL, high-density lipoprotein; LDL, low-density lipoprotein; S:P, saturated to polyunsaturated fatty acids. *Values are regression coefficients B, the level of significance (P value) and the odds ratio (OR), which shows the association between parameters and the existence of coronary artery disease. The patients were classified as significantly diseased arteries if one or more coronary arteries had a stenosis .50% and as minimally diseased arteries if there was no significant stenosis (,40%) in any artery.
0.266
1.784
1.064 1.001 1.008 .002 .97 .342 0.062 0.001 0.008
B B B B
P value
OR
Model B
OR P value B
Model C
P value
OR
Model D
P value
OR
Model E
P value
OR
Journal of Clinical Lipidology, Vol 3, No 1, February 2009
Model A
Table 3 Multiple logistic regression analysis of HDL cholesterol and fatty acids of HDL phospholipids, found to be different in univariate analysis, with respect to the presence of significant coronary artery disease as dependent variable*
42
Palmitic acid was the major fatty acid in HDL phospholipids in both groups (Table 2). Patients with SDA showed significantly lower levels of linoleic acid (P 5 .041), EPA (P 5 .027), and docosahexaenoic acid (DHA) (P 5 .026) than patients with MDA. A trend was also seen for higher saturated to polyunsaturated fatty acids (S:P) ratios in patients with SDA. In a multiple logistic regression analysis that included age, total cholesterol, and LDL cholesterol, the inverse association of SDA with linoleic acid (OR, 0.90, P 5 .041), EPA (OR, 0.41, P 5 .015), and DHA (OR, 0.49, P 5 .014) remained significant (Table 3). These associations were independent of each other and HDL cholesterol. An association trend of SDA with a higher S:P ratio (OR, 1.784) was not statistically significant. Correlation coefficients between potential risk factors and severity of coronary stenosis in SDA patients are shown in Table 4. The number of vessels with significant stenoses had positive correlations with age, smoking, hypertension, total cholesterol, LDL cholesterol, and AA:EPA ratio, and had negative correlations with both EPA and DHA content of HDL phospholipids. In a multiple regression analysis (Table 5), the number of diseased vessels (.50% stenosis) was used as the dependent variable and all parameters that correlated significantly with disease severity as independent variables. Age, smoking, hypertension, total cholesterol, and LDL cholesterol were entered as constant covariates. In different models, the independence of the relationship of the HDL cholesterol, EPA, DHA, and AA:EPA ratio was analyzed. Table 5 shows that the EPA (b 5 20.23, P , .01) and DHA (b 5 20.17, P , .05) content of HDL were inversely and independently associated with severity of CAD. The association of the AA:EPA ratio with the severity of CAD was not independent of the level of EPA (data not shown).
Discussion In this cross-sectional study, we investigated the strength and independence of association between angiographically significant CAD and fatty acid composition of HDL phospholipids. The results showed that higher n-6 and n-3 PUFA percentages of HDL phospholipids are associated with reduced incidence of stenoses .50%. These associations were independent of risk factors found to be correlated with CAD in univariate analysis. These findings are consistent with results from the Nurses’ Health Study17 and the Western Electric Study,18 which showed that intake of PUFA was inversely related to risk of coronary heart disease. Although there have been reports that high adipose tissue linoleic acid level have not been associated with increased risk of CHD,19 we observed an inverse relation with plasma phospholipid content of linoleic acid in this population. Similarly, Miettinen et al8 found an inverse association between the linoleic acid level in serum phospholipids and the risk of myocardial infarction. The negative
Noori et al
HDL composition and coronary artery disease
43 properties that suppress the atherogenic activation of vascular endothelial cells.21–23 In line with our study, Ferrucci et al24 reported that higher plasma levels of u-6 and u-3 fatty acids were both associated with decreased levels of serum proinflammatory markers particularly interleukin-6 and interleukin-1, and increased levels of anti-inflammatory markers, particularly tumor growth factor-b. Baker et al25 have found that a high linoleic acid content of phospholipids in HDL diminished the concentration of adhesion molecules that play a major role in the development of atherosclerosis compared to palmitic acid. Taken together, the data suggest that even without significant changes in HDL cholesterol, the fatty acid content of HDL phospholipids is an important risk marker of CAD. The lipid composition of HDL is determined by interaction of various processes, eg, dietary intake, metabolism, storage, and exchange among compartments.26 The n-6:n-3 ratio, which is highly dependent on the dietary intake, has been shown to be associated with the risk of coronary atherosclerosis27 In contrast, the ratio of AA to EPA in tissue is believed to be under metabolic control, which would not be influenced by the dietary n-6:n-3 ratio. Our results showed that there was no significant difference in both n-6:n-3 and AA:EPA ratios between patients with significant versus minimal stenoses in the coronary arteries. Although there was a significant association between the AA:EPA ratio and severity of CAD, this association was not independent of the EPA levels. It is of note that Harris et al,28 in a meta-analysis of 25 studies examining the association between risk for CHD events and tissue fatty acid composition, found that the AA:EPA ratio in phospholipid rich samples did not distinguish cases from controls. This study focused only on cases with no history of heart disease and with no interfering medication, and it used a reliable fatty acid biomarker instead of dietary recalls. However, because of the lack of an accurate and
Table 4 Correlations of parameters with the severity of coronary artery disease
Age Gender Body mass index Smoking Hypertension Cholesterol Triglycerides LDL cholesterol HDL cholesterol Apolipoprotein A-I Apolipoprotein B 18:2n-6 (linoleic acid) 20:4 (AA) 20:5n-3 (EPA) 22:6n-3 (docosahexaenoic acid) Saturated FAs Monounsaturated FAs S:P ratio ln n-6:n-3 ratio ln AA:EPA ratio
R
P value
0.211 20.112 0.108 0.146 0.154 0.137 0.069 0.136 20.040 20.104 0.066 20.059 20.056 20.189 20.146 0.034 20.028 0.078 0.130 0.192
.002 NS NS .034 .025 .049 NS .049 NS NS NS NS NS .006 .035 NS NS NS NS .007
AA, arachidonic acid; EPA, eicosapentaenoic acid; FAs, fatty acids; HDL, high-density lipoprotein; LDL, low-density lipoprotein; S:P, saturated to polyunsaturated fatty acids. *The Pearson correlation coefficients (r values) are displayed. The severity of CAD was determined by the number of significantly stenosed (.50%) coronary arteries.
correlation of EPA and DHA with the severity of CAD agrees with recent data that linked reduced progression of coronary atherosclerosis in patients with higher levels of plasma DHA.20 Clinical studies have demonstrated multiple cardioprotective benefits from the use of PUFA. It has been shown that both u-6 and u-3 fatty acids have anti-inflammatory
Table 5 Multivariate regression analysis of HDL cholesterol and fatty acids of HDL phospholipids, found to be correlated in univariate analysis, with respect to the severity of coronary artery disease as a dependent variable* Model A
Age Smoking Hypertension Cholesterol LDL cholesterol HDL cholesterol 20:5n-3 (EPA) 22:6n-3 (docosahexaenoic acid) ln AA:EPA ratio r2
Model B
b
P value
0.191 0.170 0.166 -0.002 0.027 0.048
.010 .024 .024 .991 .844 .527
0.093
b
Model C P value
0.210 0.181 0.171 0.037 0.034
.004 .011 .017 .783 .798
-0.234
.001
0.143
Model D P value
b
P value
0.199 0.165 0.163 0.011 0.032
.006 .022 .025 .935 .810
0.180 0.192 0.199 0.020 0.023
.016 .010 .008 .887 .870
-0.171
.016 0.236 0.115
.001
b
0.120
AA, arachidonic acid; EPA, eicosapentaenoic acid; FAs, fatty acids; HDL, high-density lipoprotein; LDL, low-density lipoprotein. *Values are standard regression coefficients b and the level of significance (P values) that shows the association between parameters and the severity of coronary artery disease. The severity of coronary artery disease was determined by the number of significantly stenosed (.50%) coronary arteries.
44 comprehensive database on the nutrient content of foods, we could not measure all aspects of diet, and the possibility of confounding cannot be eliminated.
Conclusion This study shows that PUFA, including linoleic acid, EPA, and DHA content of HDL particles, are inversely associated with the severity of stenoses in coronary arteries. Moreover, EPA and DHA content of HDL particles are inversely associated with the number of lesions producing .50% stenosis in those with significant CAD. These correlations appear to be independent of serum lipoprotein concentrations. Further investigation, while controlling for other risk factors, namely inflammatory markers and dietary intake, is warranted to look for the independent relationship between HDL phospholipid fatty acid content and cardiovascular risk.
Financial disclosure This work was supported by a grant from the Tabriz University of Medical Sciences.
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Journal of Clinical Lipidology, Vol 3, No 1, February 2009 10. Rye K, Duong M, Psaltis MK, et al. Evidence that phospholipids play a key role in high density lipoprotein remodeling. Biochemistry. 2002; 41:12538–12545. 11. Chen MF, Wang TD, Yeh HT, Hsu HC, Lee YT. Gemfibrozil treatment potentiates oxidative resistance of high density lipoprotein in hypertriglyceridemic patients. Eur J Clin Invest. 2001;31:707–713. 12. Assmann G, Schriewer H, Schmitz G, Hagele EO. Quantification of high-density-lipoprotein cholesterol by precipitation with phosphotungstic acid/MgCl2. Clin Chem. 1983;29:2026–2030. 13. Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem. 1972;18:499–502. 14. Folch J, Lees M, Sloane Stanley GH. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem. 1957;226:497–509. 15. Brekke OL, Espevik T, Bardal T, Bjerve KS. Effects of n-3 and n-6 fatty acids on tumor necrosis factor cytotoxicity in WEHI fibrosarcoma cells. Lipids. 1992;27:161–168. 16. Lepage G, Roy CC. Direct transesterification of all classes of lipids in a one-step reaction. J Lipid Res. 1986;27:114–120. 17. Oh K, Hu FB, Manson JE, Stampfer MJ, Willett WC. Dietary fat intake and risk of coronary heart disease in women: 20 years of followup of the nurses’ health study. Am J Epidemiol. 2005;161:672–679. 18. Shekelle RB, Shryock AM, Paul O, et al. Diet, serum cholesterol, and death from coronary heart disease. The Western Electric study. N Engl J Med. 1981;304:65–70. 19. Kark JD, Kaufmann NA, Binka F, Goldberger N, Berry EM. Adipose tissue n-6 fatty acids and acute myocardial infarction in a population consuming a diet high in polyunsaturated fatty acids. Am J Clin Nutr. 2003;77:796–802. 20. Erkkila AT, Matthan NR, Herrington DM, Lichtenstein AH. Higher plasma docosahexaenoic acid is associated with reduced progression of coronary atherosclerosis in women with CAD. J Lipid Res. 2006; 47:2814–2819. 21. De CR, Liao JK, Libby P. Fatty acid modulation of endothelial activation. Am J Clin Nutr. 2000;71:213S–223S. 22. Zhao G, Etherton TD, Martin KR, et al. Anti-inflammatory effects of polyunsaturated fatty acids in THP-1 cells. Biochem Biophys Res Commun. 2005;336:909–917. 23. Weldon SM, Mullen AC, Loscher CE, Hurley LA, Roche HM. Docosahexaenoic acid induces an anti-inflammatory profile in lipopolysaccharide-stimulated human THP-1 macrophages more effectively than eicosapentaenoic acid. J Nutr Biochem. 2007;18:250–258. 24. Ferrucci L, Cherubini A, Bandinelli S, et al. Relationship of plasma polyunsaturated fatty acids to circulating inflammatory markers. J Clin Endocrinol Metab. 2006;91:439–446. 25. Baker PW, Rye KA, Gamble JR, Vadas MA, Barter PJ. Phospholipid composition of reconstituted high density lipoproteins influences their ability to inhibit endothelial cell adhesion molecule expression. J Lipid Res. 2000;41:1261–1267. 26. Rise P, Eligini S, Ghezzi S, Colli S, Galli C. Fatty acid composition of plasma, blood cells and whole blood: relevance for the assessment of the fatty acid status in humans. Prostaglandins Leukot Essent Fatty Acids. 2007;76:363–369. 27. Griffin BA. How relevant is the ratio of dietary n-6 to n-3 polyunsaturated fatty acids to cardiovascular disease risk? Evidence from the OPTILIP study. Curr Opin Lipidol. 2008;19:57–62. 28. Harris WS, Poston WC, Haddock CK. Tissue n-3 and n-6 fatty acids and risk for coronary heart disease events. Atherosclerosis. 2007;193:1–10.
Fatty acid composition of HDL phospholipids and coronary artery disease Mohammad Noori, PhD, Masoud Darabi, PhD, Ali Rahimipour, PhD*, Mohammad Rahbani, PhD, Naser Aslan Abadi, MD, Maryam Darabi, PhD, Keyhan Ghatrehsamani, PhD Department of Biochemistry (Drs. Noori, Masoud Darabi, Rahimipour, Rahbani, and Ghatrehsamani) and Department of Cardiology (Dr. Abadi) School of Medicine, Tabriz University (Medical Sciences), Golgasht Avenue, Tabriz; and Department of Biochemistry School of Pharmacy and Pharmaceutical Sciences, Isfahan University (Medical Sciences), Isfahan, Iran (Dr. Maryam Darabi) KEYWORDS: CAD; Fatty acids; HDL composition
BACKGROUND: The phospholipid fatty acid content of high-density lipoprotein (HDL) partially reflects that of the diet and has been reported to be associated with several important biological functions that might serve as risk markers for coronary heart disease. OBJECTIVE: To investigate whether fatty acid composition of HDL phospholipids correlates with angiographically documented coronary artery disease (CAD). METHODS: The population included 212 patients who underwent clinically indicated coronary angiography. The patients were classified with significantly diseased arteries (SDA) if one or more coronary arteries had a stenosis .50% and with minimally diseased arteries (MDA) if there was no significant stenosis (,40%) in any artery. The severity of CAD was expressed by the number of affected vessels. The fatty acid composition of HDL phospholipids was determined by gas liquid chromatography. Multivariate analyses were used to test the independence of associations between the presence and severity of CAD as outcome variables and fatty acid composition of HDL phospholipids. RESULTS: Patients with SDA showed significantly lower levels of linoleic acid (P 5 .041), eicosapentaenoic acid (EPA) (P 5 .027), and docosahexaenoic acid (DHA) (P 5 .026) than patients with MDA in univariate analyses. The association of linoleic acid (odds ratio [OR] .90, P , .05), EPA (OR, 0.41, P , .02), and DHA (OR, 0.48, P , .02) remained statistically significant in the multivariate analyses. The content of EPA (b 5 20.23, P , .01) and DHA (b 5 20.17, P , .05) remained inversely significantly associated with the severity of CAD. CONCLUSIONS: This study shows that polyunsaturated fatty acids, including EPA and DHA content of HDL particles, are independently associated with the presence and severity of angiographically documented CAD. Ó 2009 National Lipid Association. All rights reserved.
* Corresponding author. E-mail address: [email protected] Submitted August 10, 2008. Accepted for publication November 29, 2008.
The proportions of specific fatty acids in blood or tissue are known to be useful risk markers for a variety of diseases, especially cardiovascular diseases.1–3 The current evidence shows that n-3 fatty acids are effective in the treatment and management of several diseases, which makes the evaluation of fatty acid status even more important.4,5
1933-2874/$ -see front matter Ó 2009 National Lipid Association. All rights reserved. doi:10.1016/j.jacl.2008.11.010
40 Polyunsaturated fatty acids (PUFA), both n-6 and n-3, have been shown in several studies to decrease the risk of cardiovascular diseases through their beneficial effects on blood lipids, insulin sensitivity, inhibition of thrombosis, and vascular inflammation.6 Diets enriched with n-3 fatty acids protect against coronary artery atherosclerosis, an affect that appears to be independent of plasma lipoproteins.7 Previous studies have suggested that the fatty acid composition of serum phospholipids is an independent risk factor for coronary heart disease (CHD).8,9 High-density lipoprotein (HDL) phospholipids constitute the major part of serum phospholipids. The fatty acid composition of HDL phospholipids partially reflects that of the diet and appears to be an important factor that affects their metabolism and could modulate their antiatherogenic activities.10,11 To our knowledge, no clinical study has yet examined the association between the lipid composition of HDL and CHD risk. We therefore designed this study to evaluate the relationship between fatty acid composition of HDL phospholipids and angiographically documented coronary artery disease (CAD).
Methods Subjects Subjects were recruited from a patient population scheduled for diagnostic coronary angiography at Madani Hospital in Tabriz between February 1, 2007 and February 30, 2008. In the present study, 212 patients (151 men and 61 women) were enrolled. Exclusion criteria were age .60 years, a previous hospital admission related to cardiovascular disease, current lipid-lowering medication, and a previous diagnosis of angina, hypercholesterolemia, or diabetes. Hypercholesterolemia was defined as a total level of cholesterol .280 mg/dl or a low-density lipoprotein (LDL) cholesterol level .190 mg/dl. Exclusion of persons with diabetes mellitus was based on self-reported diabetes as well as on fasting serum glucose .125 mg/dl. Venous blood samples were taken after 12–14 hours fasting in the morning before angiography. Blood was centrifuged and the HDL fraction was obtained by precipitation of lipoproteins containing apolipoprotein B (Apo-B) with phosphotungstic acid and magnesium chloride.12 Serum and HDL supernatant were frozen at 270 C until the analyses were performed. The study was approved by the ethic committee of Tabriz University of Medical Sciences, and all patients gave written informed consent.
Journal of Clinical Lipidology, Vol 3, No 1, February 2009 number of affected vessels (one-, two-, or three-vessel disease). By this classification, 36 patients had three-vessel, 39 patients had two-vessel, and 42 patients had one-vessel disease; 95 patients had MDA.
Laboratory analysis Serum total cholesterol, triglycerides, and LDL cholesterol were determined using standard enzymatic procedures. Apolipoprotein (Apo-A-I) and Apo-B concentrations were measured by immunoturbidometric methods (DiaSys Diagnostics, Holzheim, Germany). LDL cholesterol concentrations were calculated using the Friedewald formula.13 The composition of HDL was estimated after precipitation of lipoprotein containing Apo-B. Total lipids from the supernatant were extracted by the method of Folch et al14 with a 2:1 chloroform:methanol solution and several washes. HDL total phospholipids were separated by one-dimensional thin-layer chromatography using silica gel 60 G plates (E. Merck, Darmstadt, Germany) and an 80:20:1 hexane:ethyl ether:acetic acid (10.005% butylated hydroxytoluene) development solvent.15 Phospholipids fatty acid methyl esters were prepared by transmethylation with methanol during catalysis with acetyl chloride.16 Once the reaction mixture had been neutralized with potassium carbonate, the methyl esters were extracted into hexane and were preserved under nitrogen atmosphere at –70 C. Fatty acid methyl ester derivatives formed from the isolated phospholipid fraction were separated on a 60- ! 0.25-mm capillary column (TR CN100, Tekno Kroma, Barcelona, Spian) using a Buck Scientific model 610 gas chromatograph (SRI Instruments, Torrance, CA) equipped with a split injector and a flame ionization detector. Helium was used as the carrier gas and column linear velocity was set at 20.0 cm/s and the oven temperature at 210 C. The oven temperature program was 170–210 C, 1 C/min, and then isothermal for 45 minutes. Tridecanoic acid (13:0) was used as the internal standard. Peak retention times were identified by injecting known standards (Sigma chemicals). Response factor for fatty acid methyl esters were applied, and area percentage of total fatty acid were analyzed with Peak Simple software version 3.59 (SRI Instruments, Torrance, CA). The laboratory variability of the fatty acid analysis was established by repeated measures on a pooled plasma or pooled HDL fraction. The coefficient of variation was 2.5% for palmitic acid (16:0), 3.8 for linoleic acid (18:2n-6), 18.3 for eicosapentaenoic acid (EPA), and 12.3 for docosahexaennoic acid (DHA).
Statistical analysis Angiographic analysis All patients underwent coronary angiography; minimally diseased arteries (MDA) were defined as those with all stenoses ,40% and significantly diseased arteries (SDA) as those with at least one stenosis .50% or occlusions. The severity of CAD among those with SDAwas expressed by the
The level of significance between groups was calculated according to the t test for continuous variables and c2 test for categorical variables. Multivariate analyses were used to test the independence of associations between the presence and severity of CAD as outcome variables, and HDL cholesterol and fatty acid composition of HDL phospholipids
Noori et al Table 1
HDL composition and coronary artery disease
41
Clinical characteristics of patients with minimal versus severe coronary artery disease*
Age, y Sex, women, % Body mass index, kg/m2 Smokers, % Hypertension, % Family history, % Hydrogenated oil intake, % Cholesterol, mg/dl Triglycerides, mg/dl HDL cholesterol, mg/dl LDL cholesterol, mg/dl Apolipoprotein A-I, mg/dl Apolipoprotein B, mg/dl
MDA (n 5 95)
SDA (n 5 117)
P value
47.1 33 26.7 31 27 11 69 165 176 37 92 123 102
50.6 24 27.7 38 36 15 77 176 189 37 101 119 107
.003 NS NS NS NS NS NS .036 NS NS .046 NS NS
6 9.1 6 4.0
6 6 6 6 6 6
34 96 11 29 19 28
6 6.5 6 4.4
6 6 6 6 6 6
41 95 10 35 17 29
*Values are means 6 standard deviation or percentage with condition. The patients were classified as having significantly diseased arteries (SDA) if one or more coronary arteries had a stenosis .50% and as minimally diseased arteries (MDA) if there was no significant stenosis (.40%) in any artery.
as independent variables. We estimated relative risks as odds ratios (ORs) using multiple logistic regression models. Each multiple logistic regression model included the risk factors for which significant differences were found between the MDA and SDA groups. The values of the n-6:n-3 ratio and arachidonic acid (AA):EPA ratio were converted by logarithmic transformation to normalize distribution of the data. For univariate analysis, the Pearson correlation test was performed to identify risk factors associated with severity of CAD. These factors were then entered into multiple regression models to identify independent associations with severity of CAD. The standard regression coefficient b and P value were calculated for each variable. A P value of , .05 was considered
statistically significant. All analyses were carried out using SPSS for windows version 11.0 (SPSS Inc., Chicago, IL).
Results The clinical details and lipid profiles of both groups of patients are presented in Table 1. Patients with angiographically significant coronary atherosclerosis were 3.5 years older than patients without coronary atherosclerosis (P 5 .003). The mean values of total cholesterol was higher in the patients with SDA than in the patients with MDA (176 6 41 vs 165 6 35 mg/dL), reflecting the higher LDL cholesterol in patients with SDA (101 6 35 vs 92 6 29 mg/dL).
Table 2 High-density lipoprotein phospholipids fatty acid composition in patients with minimal versus significant coronary artery disease* Percent of total fatty acids
16:0 (palmitic acid) 16:1 (palmitoleic acid) 18:0 (stearic acid) 18:1n-9 (oleic acid) 18:2n-6 (linoleic acid) 20:3n-6 (dihomo-g-linolenic acid) 20:4n-6 (AA) 20:5n-3 (EPA) 22:6n-3 (docosahexaenoic acid) Saturated FAs Monounsaturated Fas S:P ratio n-6:n-3 ratio AA:EPA ratio
MDA (n 5 95)
SDA (n 5 117)
P value
31.6 6 3.8 1.3 6 0.5 14.0 6 1.8 10.2 6 1.7 19.9 6 3.4 2.3 6 0.7 8.4 6 1.5 0.63 6 0.48 1.10 6 0.6 45.6 6 4.7 11.5 6 1.5 1.46 6 0.31 17.2 (12.3–26.4) 10.13 (8.0–25.2)
32.0 1.3 13.8 9.9 18.9 2.1 8.1 0.49 0.93 45.8 11.2 1.52 20.8 15.9
NS NS NS NS .041 NS NS .027 .026 NS NS .118 NS NS
6 2.9 6 0.5 6 1.7 6 1.8 6 2.9 6 0.7 6 1.2 6 0.41 6 0.46 6 3.6 6 1.8 6 0.25 (14.9–29.5) (9.9–40.6)
AA, arachidonic acid; EPA, eicosapentaenoic acid; FAs, fatty acids; S:P, saturated to polyunsaturated fatty acids. *Values are mean 6 standard deviation, or if variables did not exhibit normal distribution, medians with 25 and 75 percentiles in brackets. Detection limit was 0.05% of the total area. The patients were classified as having significantly diseased arteries (SDA) if one or more coronary arteries had a stenosis .50% and as having minimally diseased arteries (MDA) if there was no significant stenosis (,40%) in any artery.
0.579 24.442 0.485 20.724 0.406
24.305
.014
1.065 1.004 1.001 .004 .616 .952 0.063 0.004 0.001
22.445
20.920 0.899 .041 20.106
24.698
.015
1.067 1.006 1.001 .003 .500 .975 0.065 0.006 0.002 1.056 1.007 0.997 .014 .402 .760 0.054 0.007 -0.003 1.065 1.006 1.006 0.992
25.897
.003 .467 .569 .605 0.063 0.006 0.006 -0.008
Age Cholesterol LDL cholesterol HDL cholesterol 18:2n-6 (linoleic acid) 20:5n-3 (eicosapentaenoic acid) 22:6n-3 (docosahexaenoic acid) S:P ratio Constant
HDL, high-density lipoprotein; LDL, low-density lipoprotein; S:P, saturated to polyunsaturated fatty acids. *Values are regression coefficients B, the level of significance (P value) and the odds ratio (OR), which shows the association between parameters and the existence of coronary artery disease. The patients were classified as significantly diseased arteries if one or more coronary arteries had a stenosis .50% and as minimally diseased arteries if there was no significant stenosis (,40%) in any artery.
0.266
1.784
1.064 1.001 1.008 .002 .97 .342 0.062 0.001 0.008
B B B B
P value
OR
Model B
OR P value B
Model C
P value
OR
Model D
P value
OR
Model E
P value
OR
Journal of Clinical Lipidology, Vol 3, No 1, February 2009
Model A
Table 3 Multiple logistic regression analysis of HDL cholesterol and fatty acids of HDL phospholipids, found to be different in univariate analysis, with respect to the presence of significant coronary artery disease as dependent variable*
42
Palmitic acid was the major fatty acid in HDL phospholipids in both groups (Table 2). Patients with SDA showed significantly lower levels of linoleic acid (P 5 .041), EPA (P 5 .027), and docosahexaenoic acid (DHA) (P 5 .026) than patients with MDA. A trend was also seen for higher saturated to polyunsaturated fatty acids (S:P) ratios in patients with SDA. In a multiple logistic regression analysis that included age, total cholesterol, and LDL cholesterol, the inverse association of SDA with linoleic acid (OR, 0.90, P 5 .041), EPA (OR, 0.41, P 5 .015), and DHA (OR, 0.49, P 5 .014) remained significant (Table 3). These associations were independent of each other and HDL cholesterol. An association trend of SDA with a higher S:P ratio (OR, 1.784) was not statistically significant. Correlation coefficients between potential risk factors and severity of coronary stenosis in SDA patients are shown in Table 4. The number of vessels with significant stenoses had positive correlations with age, smoking, hypertension, total cholesterol, LDL cholesterol, and AA:EPA ratio, and had negative correlations with both EPA and DHA content of HDL phospholipids. In a multiple regression analysis (Table 5), the number of diseased vessels (.50% stenosis) was used as the dependent variable and all parameters that correlated significantly with disease severity as independent variables. Age, smoking, hypertension, total cholesterol, and LDL cholesterol were entered as constant covariates. In different models, the independence of the relationship of the HDL cholesterol, EPA, DHA, and AA:EPA ratio was analyzed. Table 5 shows that the EPA (b 5 20.23, P , .01) and DHA (b 5 20.17, P , .05) content of HDL were inversely and independently associated with severity of CAD. The association of the AA:EPA ratio with the severity of CAD was not independent of the level of EPA (data not shown).
Discussion In this cross-sectional study, we investigated the strength and independence of association between angiographically significant CAD and fatty acid composition of HDL phospholipids. The results showed that higher n-6 and n-3 PUFA percentages of HDL phospholipids are associated with reduced incidence of stenoses .50%. These associations were independent of risk factors found to be correlated with CAD in univariate analysis. These findings are consistent with results from the Nurses’ Health Study17 and the Western Electric Study,18 which showed that intake of PUFA was inversely related to risk of coronary heart disease. Although there have been reports that high adipose tissue linoleic acid level have not been associated with increased risk of CHD,19 we observed an inverse relation with plasma phospholipid content of linoleic acid in this population. Similarly, Miettinen et al8 found an inverse association between the linoleic acid level in serum phospholipids and the risk of myocardial infarction. The negative
Noori et al
HDL composition and coronary artery disease
43 properties that suppress the atherogenic activation of vascular endothelial cells.21–23 In line with our study, Ferrucci et al24 reported that higher plasma levels of u-6 and u-3 fatty acids were both associated with decreased levels of serum proinflammatory markers particularly interleukin-6 and interleukin-1, and increased levels of anti-inflammatory markers, particularly tumor growth factor-b. Baker et al25 have found that a high linoleic acid content of phospholipids in HDL diminished the concentration of adhesion molecules that play a major role in the development of atherosclerosis compared to palmitic acid. Taken together, the data suggest that even without significant changes in HDL cholesterol, the fatty acid content of HDL phospholipids is an important risk marker of CAD. The lipid composition of HDL is determined by interaction of various processes, eg, dietary intake, metabolism, storage, and exchange among compartments.26 The n-6:n-3 ratio, which is highly dependent on the dietary intake, has been shown to be associated with the risk of coronary atherosclerosis27 In contrast, the ratio of AA to EPA in tissue is believed to be under metabolic control, which would not be influenced by the dietary n-6:n-3 ratio. Our results showed that there was no significant difference in both n-6:n-3 and AA:EPA ratios between patients with significant versus minimal stenoses in the coronary arteries. Although there was a significant association between the AA:EPA ratio and severity of CAD, this association was not independent of the EPA levels. It is of note that Harris et al,28 in a meta-analysis of 25 studies examining the association between risk for CHD events and tissue fatty acid composition, found that the AA:EPA ratio in phospholipid rich samples did not distinguish cases from controls. This study focused only on cases with no history of heart disease and with no interfering medication, and it used a reliable fatty acid biomarker instead of dietary recalls. However, because of the lack of an accurate and
Table 4 Correlations of parameters with the severity of coronary artery disease
Age Gender Body mass index Smoking Hypertension Cholesterol Triglycerides LDL cholesterol HDL cholesterol Apolipoprotein A-I Apolipoprotein B 18:2n-6 (linoleic acid) 20:4 (AA) 20:5n-3 (EPA) 22:6n-3 (docosahexaenoic acid) Saturated FAs Monounsaturated FAs S:P ratio ln n-6:n-3 ratio ln AA:EPA ratio
R
P value
0.211 20.112 0.108 0.146 0.154 0.137 0.069 0.136 20.040 20.104 0.066 20.059 20.056 20.189 20.146 0.034 20.028 0.078 0.130 0.192
.002 NS NS .034 .025 .049 NS .049 NS NS NS NS NS .006 .035 NS NS NS NS .007
AA, arachidonic acid; EPA, eicosapentaenoic acid; FAs, fatty acids; HDL, high-density lipoprotein; LDL, low-density lipoprotein; S:P, saturated to polyunsaturated fatty acids. *The Pearson correlation coefficients (r values) are displayed. The severity of CAD was determined by the number of significantly stenosed (.50%) coronary arteries.
correlation of EPA and DHA with the severity of CAD agrees with recent data that linked reduced progression of coronary atherosclerosis in patients with higher levels of plasma DHA.20 Clinical studies have demonstrated multiple cardioprotective benefits from the use of PUFA. It has been shown that both u-6 and u-3 fatty acids have anti-inflammatory
Table 5 Multivariate regression analysis of HDL cholesterol and fatty acids of HDL phospholipids, found to be correlated in univariate analysis, with respect to the severity of coronary artery disease as a dependent variable* Model A
Age Smoking Hypertension Cholesterol LDL cholesterol HDL cholesterol 20:5n-3 (EPA) 22:6n-3 (docosahexaenoic acid) ln AA:EPA ratio r2
Model B
b
P value
0.191 0.170 0.166 -0.002 0.027 0.048
.010 .024 .024 .991 .844 .527
0.093
b
Model C P value
0.210 0.181 0.171 0.037 0.034
.004 .011 .017 .783 .798
-0.234
.001
0.143
Model D P value
b
P value
0.199 0.165 0.163 0.011 0.032
.006 .022 .025 .935 .810
0.180 0.192 0.199 0.020 0.023
.016 .010 .008 .887 .870
-0.171
.016 0.236 0.115
.001
b
0.120
AA, arachidonic acid; EPA, eicosapentaenoic acid; FAs, fatty acids; HDL, high-density lipoprotein; LDL, low-density lipoprotein. *Values are standard regression coefficients b and the level of significance (P values) that shows the association between parameters and the severity of coronary artery disease. The severity of coronary artery disease was determined by the number of significantly stenosed (.50%) coronary arteries.
44 comprehensive database on the nutrient content of foods, we could not measure all aspects of diet, and the possibility of confounding cannot be eliminated.
Conclusion This study shows that PUFA, including linoleic acid, EPA, and DHA content of HDL particles, are inversely associated with the severity of stenoses in coronary arteries. Moreover, EPA and DHA content of HDL particles are inversely associated with the number of lesions producing .50% stenosis in those with significant CAD. These correlations appear to be independent of serum lipoprotein concentrations. Further investigation, while controlling for other risk factors, namely inflammatory markers and dietary intake, is warranted to look for the independent relationship between HDL phospholipid fatty acid content and cardiovascular risk.
Financial disclosure This work was supported by a grant from the Tabriz University of Medical Sciences.
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