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The relationship between paraoxonase1-192 polymorphism and activity with coronary artery disease
Clinical Biochemistry 43 (2010) 553–558
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Clinical Biochemistry j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c l i n b i o c h e m
The relationship between paraoxonase1-192 polymorphism and activity with coronary artery disease Randa H. Mohamed a,⁎, Rasha H. Mohamed b, Rehab A. Karam a, Tarek A. Abd El-Aziz c a b c
Medical biochemistry department, Faculty of medicine, Zagazig University, Zagazig, Egypt Biochemistry department, Faculty of pharmacy, Zagazig University, Zagazig, Egypt Cardiology department, Faculty of medicine, Zagazig University, Zagazig, Egypt
a r t i c l e
i n f o
Article history: Received 17 October 2009 Received in revised form 10 December 2009 Accepted 11 December 2009 Available online 21 December 2009 Keywords: PON1 activity PON1 polymorphism Lipoprotein oxidation susceptibility CAD
a b s t r a c t Objective: We tested the association between PON1 polymorphism, PON1 activity, oxidative susceptibility of LDL and coronary artery disease in Egyptians. Methods: PON1 polymorphism, serum PON1 activity, lipoprotein oxidation susceptibility and lipid profile were measured. Results: Levels of HDL and paraoxonase activity were significantly decreased in CAD patients compared to control group, and in patients with three vessels compared to those of single or two vessels disease. Highactivity allele (R) has a more atherogenic lipid profile than for the low activity allele (Q). PON1 RR genotype has nine fold risks to develop CAD in Egyptians while those with PON1 QR genotype have four fold risks. Conclusion: The PON1 activity is lower in subject with CAD and there is a significant relationship between activity of PON1 and the severity of coronary atherosclerosis. Also, we provide evidence of a significant association between R allele of the PON1 polymorphism and the development of coronary artery disease. © 2010 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction Oxidative stress plays a crucial role in the development of atherosclerosis by oxidation of low-density lipoprotein (LDL) that subsequently leads to formation of foam cells. Conversely, highdensity lipoprotein (HDL) is a well-known anti-oxidant molecule that prevents atherosclerosis [1]. HDLs can protect LDLs from oxidative damage. The antioxidant effect of HDLs is determined by its enzymes, in particular paraoxonase, an HDL-associated enzyme capable of hydrolyzing lipid peroxides [2]. Paraoxonase (PON1) is a 44 kDa Ca2+-dependent glycoprotein, synthesized in the liver and is located on the surface of high density lipoprotein (HDL). Recently, it has been shown that PON1 decreases generation and accumulation of lipoperoxides in LDL. In addition, PON1, by destroying oxidized phospholipids, reduces the ability of oxidized LDL to induce monocyte binding and transmigration and thus, inflammation in the vessel wall [3]. In knockout mice lacking the gene for PON1, atherosclerosis develops more rapidly than in wildtype mice, whereas mice that overexpress human PON1 are resistant to atherosclerosis [4]. Thus, PON1 may be involved in protection against atherosclerosis. The role of PON1 may be particularly meaningful because oxidized LDL promotes secretion of the potent
⁎ Corresponding author: Fax: +20 552366896. E-mail address: [email protected] (R.H. Mohamed).
endothelial constrictor, endothelin, and reverses the impairment of endothelium-mediated vasodilatation in stenotic vessel [5]. Besides its protective effects against LDL peroxidation, HDLassociated paraoxonase has been demonstrated to inhibit the oxidative damage of HDL as well. The oxidation of HDL not only reduces its capability to prevent the oxidative modification of LDL, but also diminishes the ability of HDL to function as a potent acceptor for cholesterol efflux [6]. PON1 enzyme activity for paraoxon as a substrate is modulated by a number of polymorphisms at the PON1 gene located in chromosome 7q21.3, which is clustered with at least two other related genes, paraoxonase2 and paraoxonase3 [7]. Paraoxonase (A/G) polymorphism results in glutamine (Q) to arginine (R) substitution at codon 192. This 192Q isoform has been related to lower paraoxonase (paraoxonhydrolyzing) and arylesterase (phenylacetate-hydrolyzing) activity, and 192 R, an isoform with high activity toward paraoxon hydrolysis [8]. Although PON1 activity and concentration are determined genetically, various factors, such as diet, lifestyle, and environmental factors, can influence PON1 activity and/or concentration. Degraded cooking oil has been reported to lower serum PON1 levels in humans. Dietary polyphenols increase PON1 activity, as does moderate alcohol intake. Smoking is known to decrease serum PON1 activity. Recent evidence shows that exposure to environmental chemicals can inhibit PON1 activity. Furthermore, low serum PON1 activity independent of genotype has been reported in diseases associated with accelerated atherogenesis, such as diabetes mellitus, hypercholesterolemia, and renal failure [4].
0009-9120/$ – see front matter © 2010 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2009.12.015
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Whereas some authors have failed to find a link between the variation in paraoxonase gene and changes in lipoprotein concentrations [9,10], others have found a significant association between paraoxonase-192 genetic variants and changes in HDL-cholesterol levels and in triglyceride concentrations in relatively genetically isolated populations [11,12] Therefore, at present the relationship between paraoxonase genetic polymorphism and atherosclerosis is unclear. Whether the paraoxonase gene can modulate the lipid profile is a matter of conjecture, but remains a possibility in the light of the above population studies. Because PON1-192 genetic polymorphism strongly influences PON1 activity and does vary between different ethnic groups, we tested the association between this polymorphism, PON1 activity, changes in oxidative susceptibility of LDL and coronary artery disease (CAD) in Egyptian patients. Material and methods Subjects Subjects who underwent coronary angiography in the Cardiovascular Center for detection of the presence and extent of stenosis in coronary artery vessels were recruited according to a designed protocol. Forty-three patients had previous history of myocardial infarction, and the mean value of left ventricular ejection fraction (LVEF) for patients was 0.59 ± 0.08 and ranged from 0.43 to 0.80. All patients were treated with statin, nitrates, aspirin, angiotensin converting enzyme inhibitor, 145 patients received β-adrenoceptorblocking drug, 5 were taking a calcium channel blocker and 8 patients were taking diuretics. Participants with hepatic or renal disease, cardiomyopathy, class IV congestive heart failure, significant valvular or congenital heart disease, and recent coronary angioplasty or coronary bypass surgery and acute myocardial infarction within the last three months were excluded from the study. The CAD group included 150 patients who have at least a 70% stenosis in a major epicardial artery and subdivided into three groups: single-vessel disease (SVD), two-vessel disease (2VD) and threevessel disease (3VD). Fifty control subjects age- and sex-matched with the patients were randomly recruited. The control group had neither clinical symptoms nor electrocardiographic changes indicative of CAD and therefore did not undergo coronary angiography. Biochemical measurements Analyses of Lipid Blood samples were drawn from all subjects after an overnight fast. Sera were separated immediately and stored at −20 °C. Total cholesterol and triglycerides were measured by routine enzymatic
methods (spinreact). HDL-cholesterol was determined after precipitation of the apoB-containing lipoproteins. LDL-cholesterol was calculated using Friedewald formula [13]. Lipoprotein oxidation susceptibility The oxidizability of apo B-containing lipoproteins is as described [14], and this fraction was precipitated from 500 μL of plasma by adding 50 μL of dextran sulfate-magnesium chloride. The pellet was dissolved in 2.5 mL of 4% saline solution. A volume of redissolved precipitate containing 100 μg of non-HDL-cholesterol (calculated from the total minus HDL-cholesterol level) was mixed with 4% sodium chloride to give 500 μL of total solution. Copper solution (0.5 mM Cu Cl2) was added and incubated at 37 °C for 3 h in a shaking water bath. The thiobarbituric acid-reactive substance assay (as an index of oxidation) was conducted by adding 2 mL of the thiobarbituric acid-reactive substance reagent to the 550 μL incubation mixture and heating it in a boiling water bath for 15 min. The pink color was read in a spectrophotometer at 532 against a blank containing 4% saline solution. The nmoles of malonyldialdehyde (MDA) present in the sample were estimated by comparison with a standard curve prepared from 1,1,3,3-tetraethoxypropane (0.5 to 16 nmol/mL). Analysis of PON1 activity PON1 activity toward paraoxon was measured after paraoxon hydrolysis into p-nitrophenol and diethyl phosphate catalyzed by the enzyme. The activity was measured by adding 10 μL serum to 350 μL Tris–HCl buffer (100 mmol/L, pH 8.0) containing 2.0 mmol/L CaCl2 and 2.0 mmol/L paraoxon. The rate of generation of p-nitrophenol was determined at 405 nm on a Milton Roy Spectronic 3000 array at 37 C° over 3 min after 42 s delay [15]. Determination of the paraoxonase 192 genotype DNA was isolated and purified from whole blood (EDTA) using QIAamp-spin-columns according to the protocol provided by the manufacturer (QIAamp Blood Kit; Qiagen GmbH, Hilden, Germany). The 99-bp target region in the paraoxonase gene was amplified by polymerase chain reaction (PCR) using forward 5′-TAT TGT TGC TGT GGG ACC TGA G-3′ and reverse 5′-CAC GCT AAA CCC AAA TAC ATC TC-3′ primers. The PCR reaction mix contained 10 μg genomic DNA, 0.5 μmol/L of each primer, 200 μmol/L of each dNTP, 5 μL of 10× reaction buffer, and 1.25 U Taq DNA polymerase. After the DNA was denatured at 95 °C for 3 min, the reaction mixture was subject to 30 cycles, each cycle comprising denaturation at 94 °C for 60 s, annealing at 61 °C for 30 s, and extension at 72 °C for 60 s, with a final extension time of 5 min. The
Table 1 Study participants characteristics. Parameter
SVD
2VD
3VD
CAD
Controls
n Age (years) Sex (m/f) Hypertension, n (%) Diabetes, n (%) Smoker, n (%) Cholesterol (mg/dL) Triglycerides (mg/dL) HDL (mg/dL) LDL (mg/dL) Lipoprotein oxidation susceptibility (nmol/mg non-HDLc) Paraoxonase (U/mL)
50 53.5 ± 8.1 29/21 41 (82%) 32 (64%) 17 (34%) 230.3 ± 26.8a 170.1 ± 10.1a 43 ± 4.3a 153.3 ± 25.3a 45.3 ± 12.6a 194.5 ± 41.5a
50 59.9 ± 7.9 40/10 27 (54%) 9 (18%) 18 (36%) 242.8 ± 27.9a,b 176.6 ± 15.7a,b 42.4 ± 4.1a 165 ± 25.8a,b 46.6 ± 9.3a 178.6 ± 25.5a,b
50 53.4 ± 5.8 50/0 14 (28%) 14 (28%) 43 (86%) 261.7 ± 13.9a,b,c 188 ± 11.7a,b,c 39.3 ± 2.9a,b,c 184.8 ± 14.6a,b,c 58.2 ± 22.3a,b,c 152.8 ± 25.1a,b,c
150 55.5 ± 8.1 119/31 82 (54%) 55 (36.6%) 78 (52%) 240.5 ± 27.4a 175.7 ± 13.9a 42.1 ± 4.2a 163.3 ± 26.1a 48.3 ± 14.6a 181.2 ± 37a
50 50.7 ± 9.5 26/24 – – 18 (36%) 185.9 ± 8.4 132 ± 14.4 54.7 ± 3.2 106.2 ± 6.2 35.8 ± 6.9 225.7 ± 58.3
a b c
Significant difference from control. Significant difference from SVD. Significant difference from 2VD.
R.H. Mohamed et al. / Clinical Biochemistry 43 (2010) 553–558
PCR products were digested with 2.5 U AlwI restriction endonuclease for 1 h, and the digested products were separated by electrophoresis on a 3% agarose gel. Then the gel was visualized under UV transilluminator with a 50 base pair ladder. Individuals homozygous for the 192 Gln allele present a 99-bp PCR product, those homozygous for the 192 Arg allele present 69- and 30-bp products, and those heterozygous present 90-, 69-, and 30-bp products [6].
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Table 3 ORs for CAD and three-vessel disease.
Q/Q Q/R R/R R allele
Presence of CAD
Severity of CAD
OR
Confidence interval
OR
Confidence interval
1 4.2 9.7 3.8
1.7–9.5 4.2–23.9 2.4–6.1
1 1.7 1.1 1.1
0.5–5.3 0.4–3.1 0.6–1.9
Statistical analysis The results for continuous variables are expressed as means ± SD. The means of the three genotype groups were compared in a one-way analysis of variance. The correlation coefficients were calculated using spearman correlation. The statistical significances of differences in frequencies of variants between the groups were tested using the chisquare (χ2) test. In addition, the odds ratios (ORs) and 95% CIs were calculated as a measure of the association of the paraoxonase 192 genotypes with CAD and its severity. A difference was considered significant at P b 0.05. All data were evaluated using SPSS version 10.0 of windows. Results Distribution of clinical characteristics of the study subjects in relation to the severity of CAD (Table 1) Levels of total cholesterol, triglyceride, LDL-cholesterol and lipoprotein oxidation susceptibility were significantly increased in CAD patients compared to control group. Furthermore, levels of HDLcholesterol and paraoxonase activity were significantly decreased in CAD patients compared to control group. In order to evaluate the relationship between the risk factors and the severity of coronary artery disease, patients with CAD were divided into three categories according to the number of diseased coronary vessels. The levels of total cholesterol, triglyceride, LDLcholesterol and lipoprotein oxidation susceptibility were significantly increased in patients with three vessels compared to those of single or two-vessel disease. Levels of HDL-cholesterol and paraoxonase activity were significantly decreased in patients with three vessels compared to those of single or two-vessel disease. PON1 polymorphisms genotypes and alleles distribution between patients and controls (Tables 2 and 3) In the CAD group, the frequencies of PON1 RR genotype were significantly increased compared to control group (50.6% versus 20%) resulting in a significantly increased OR in the CAD subject bearing this gene (OR = 9.7, 95% CI 3.9–23.9); also, the R allele was significantly increased in the CAD group compared to the control group (69% versus 37%) resulting in a significantly increased OR in the CAD subject bearing this allele ( OR = 3.8, 95% CI 2.4–6.1). The PON1 genotypes did not exhibit any significant differences between patients with single-, double-, or triple-vessel disease. Table 2 Distribution frequency of genotypes and alleles of PON1 polymorphisms between patients and controls.
Q/Q, n (%) Q/R, n (%) R/R, n (%) R allele a b c
SVD
2VD
3VD
CAD
6 (12)a 23 (46)b 21 (42)a,b 65 (65)a
5 (10) a 18 (36)b 27 (54)a,b 72 (72)a
7 (14)a 15 (30) 28 (56)a,b,c 71 (71)a
18 56 76 208
Significant difference from control. Significant difference from QQ. Significant difference from QR.
Controls (12)a (37.3)b (50.6)a,b,c (69)a
23 17 10 37
(46) (34) (20)b (37)
Association between paraoxonase 192 genotype and clinical characteristics of the study subjects (Table 4) The risk factors for CAD (diabetes, hypertension, dyslipidemia) were significantly associated with PON1 RR. Levels of total cholesterol, triglyceride, LDL-cholesterol, paraoxonase activity and lipoprotein oxidation susceptibility were significantly increased in PON1 RR than in PON1 QQ. Levels of HDL-cholesterol were significantly decreased in PON1 RR than in PON1 QQ. Association between paraoxonase activity and lipid profile (Table 5) Paraoxonase activity had significant positive correlation with HDLcholesterol and significant negative correlation with total cholesterol, triglyceride, LDL-cholesterol and lipoprotein oxidation susceptibility. Multiple regression analysis (Table 6) The difference in severity of CAD was tested for independence from other variables by multiple regression analysis. The model included sex, age, hypertension, diabetes, smoker, total cholesterol, triglycerides, LDL, HDL, Lipoprotein oxidation susceptibility, PON1 activity, and the 192 polymorphisms. The difference in severity of CAD was found to be dependent on diabetes only. Discussion The oxidative damage to vital biological systems can lead to enhanced expression of inflammatory genes that ultimately contribute to the development of several chronic diseases, including CAD, cancer, and diabetes, and that contribute to aging. The balance between oxidants and antioxidants basically affects all biological systems and, ultimately, the clinical course. The oxidation of LDL and its involvement in the development of foam cell-laden fatty streaks in the arterial wall are believed to initiate the atherosclerotic process [16]. In vitro studies indicate that HDL-associated PON1 prevents LDL oxidation and can destroy biologically active lipids in mildly oxidized
Table 4 Study participants' characteristics according to the PON1 192 Q/R genotypes.
n Age (years) Sex (m/f) Hypertension, n (%) Diabetes, n (%) Smoker, n (%) Cholesterol (mg/dL) Triglycerides (mg/dL) HDL (mg/dL) LDL (mg/dL) Lipoprotein oxidation susceptibility (nmol/mg non-HDLc) Paraoxonase Activity(U/mL) a b
Significant difference from QQ. Significant difference from QR.
Q/Q
Q/R
R/R
41 53 ± 9.7 25/13 12 (25.5) 7 (14.8) 15 (31.2) 205.2 ± 32.8 146.8 ± 22.9 52.7 ± 6.1 125.3 ± 32.6 39.3 ± 16.9
73 51.3 ± 8.5 52/21 30 (40) 22(30) 23 (31.5) 223.4 ± 33.1a 160 ± 22.6a 48.2 ± 6a 143.2 ± 34.1a 40.9 ± 9.8a
86 56.5 ± 8.3a,b 59/21 40 (50)a 26 (32.5)a 40 (50) 227.3 ± 35.7a 167.4 ± 26.9a 41.8 ± 5.6a,b 152.1 ± 34.2a 48.8 ± 13.7a,b
164.9 ± 34.5 185.2 ± 34.6a 227.6 ± 54.5a,b
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Table 5 Correlation between paraoxonase activity and lipid profile, Lipoprotein oxidation susceptibility. Paraoxonase activity
Cholesterol Triglycerides HDL LDL Lipoprotein oxidation susceptibility
r
p
−0.469 −0.482 0.29 −0.457 −0.299
b 0.001 b 0.001 b 0.05 b 0.001 b 0.05
LDL [17], and PON1 may thus affect the process of atherosclerosis. It has also been demonstrated that PON1 has the capacity to reduce oxidized lipids in human atherosclerotic lesions derived from either coronary artery or carotid specimens [18]. Previous studies that have investigated the relationship between the PON1-192 polymorphism and CAD have produced inconsistent results. Some studies have shown the PON1-192R genotype to be present at a higher frequency in CAD, leading to the hypothesis that the PON1-192 polymorphism might be a risk factor for atherosclerosis [6,19], but some studies have failed to find such a relationship [20,21]. Study population characteristics in relation to the severity of CAD The present study demonstrates that levels of total cholesterol, triglycerides, LDL-cholesterol and lipoprotein oxidation susceptibility are significantly increased in CAD patients compared to control group. These parameters were also elevated in patients who had more severe disease compared to those with less severe disease. In accordance with our data, De Rijke et al. [22] studied the association between oxidation parameters and progression of coronary artery stenosis and found a higher susceptibility of LDL to oxidation in CAD patients who had shown progression in stenosis. In another study, Regnström et al. [23] described an inverse association between resistance of LDL to oxidation and severity of coronary stenosis; however, the study of Croft et al.[24] did not reveal a difference in oxidation parameters between coronary atherosclerotic patients and control subjects. In this study, we demonstrated that levels of HDL-cholesterol and paraoxonase activity were significantly decreased in CAD patients compared to control group, and in patients with 3VD compared to those of one or 2VD. Mackness et al. [25] showed that PON1 activities toward paraoxon are lower in subjects with CHD than in control subjects regardless of the PON1 genotype. Azarsıza et al. [26] found that PON1 activity in patients with CAD were lower than control and is depleted during the propagation phase of the atherosclerotic process; however, this difference was not found statistically significant.
Paraoxonase 192 genotype and CAD We found that the risk factors for CAD (diabetes, hypertension, dyslipidemia) are significantly associated with PON1 RR. While several studies showed a positive association between the R allele and the risk for CAD [6,10,27], others showed no association [28,29]. We demonstrated that levels of HDL-cholesterol were significantly decreased in PON1 RR than in PON1 QQ. Previous studies have investigated the association between PON1 192 polymorphism and blood lipids. HDL may play a significant role in the effect of PON1 on coronary disease. In one study, the PON1-192 R allele was associated with lower HDL-cholesterol levels [11], but this was not confirmed in other studies [2,30]. We demonstrated that levels of total cholesterol, triglyceride, and LDL-cholesterol were significantly increased in PON1 RR than in PON1 QQ. In accordance with our data, Hegele et al. [11] observed that homozygotes for the low activity allele (Q) have a less atherogenic lipid profile than heterozygotes and homozygotes for the highactivity allele (R). Several lines of evidence have been provided for a potential biological link between the Q/R192 polymorphism and the antiatherogenic effect. Mackness et al. [31] demonstrated that HDL containing R192 PON1 was less effective in protecting LDL from oxidative modification than HDL with Q192 PON1. Aviram et al. [32] showed that recombinant PON1 encompassing R192 alleles were less effective in blocking LDL oxidation. These results indicate that 192R PON1 have lower activity for hydrolyzing lipid peroxide, thus lower anti-atherogenic property. These findings may explain why the paraoxonase R allele has been found to be present at an increased frequency in coronary heart disease (CAD) leading to the hypothesis that the PON1-192 polymorphism might be a risk factor for atherosclerosis. We demonstrated that individuals with PON1 RR genotype have 9fold risks to develop CAD in Egyptians while those with the PON1 QR genotype have 4-fold risks. Serrato and Marian [33] found that the R allele was clearly associated with coronary heart disease in the United States. Sanghera et al. [10] reported that Q/R192 gene polymorphism was associated with CAD in Indians, but not in Chinese. Ko et al. [34] compared the Q/R192 genotype distribution between CAD patients and age- and sex-matched control subjects in Taiwan, which showed no difference. This variability in results suggests that gene–environment and/or gene–gene interactions might modulate the relationship between paraoxonase polymorphism and coronary heart disease. Inconsistent association of PON1 with CAD may be attributed to differences in several factors between studies, including ethnic and environmental factors, and methodological factors such as sampling scheme and trial size. Among these factors, of note is the fact that allele frequencies of the three polymorphisms do vary between different ethnic groups; for example, we found that PON1 the QQ
Table 6 Multiple regression analysis. Variable
Age Sex Hypertension Diabetes Smoker Cholesterol Triglycerides HDL LDL Lipoprotein oxidation susceptibility Paraoxonase Q192R
Unstandardized coefficients
Standardized coefficients
B
Standard error
β
3.9E−03 5.38E−02 −0.125 −0.438 0.276 2.02E−02 6.95E−03 −4.44E−02 −8.38E−03 −3.2E−03 −6.4E−03 0.283
0.189 0.008 0.16 0.179 0.172 0.016 0.005 0.017 0.027 0.007 0.003 0.224
0.024 0.033 −0.059 −0.193 0.132 0.671 0.169 −0.28 −0.31 −0.042 −0.308 0.214
95% CI
t
p
−0.013 to 0.021 −0.44 to 0.198 −0.8 to −0.08 −0.07 to 0.62 −0.013 to 0.053 −0.004 to 0.018 −0.043 to 0.026 −0.099 to 0.01 −0.02 to 0.01 −0.013 to 0.00 −0.17 to 0.73
0.472 −0.78 −2.444 1.61 1.234 1.308 −0.494 −1.649 −0.474 −1.98 1.26
0.639 0.44 0.019 0.115 0.224 0.198 0.624 0.107 0.638 0.054 0.215
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genotype is common in Egyptians which has also been found in Caucasians [28,29]; however, R allele frequencies have proven to be relatively common in Japanese [35,36] and Chinese [34]. Paraoxonase activity and CAD We demonstrated that paraoxonase activity and lipoprotein oxidation susceptibility were significantly increased in PON1 RR than in PON1 QQ. Activity of PON1 can vary up to 40-fold in human populations. Part of this variability is explained by the polymorphism of PON1 gene because of an amino acid substitution at 192. The R allele (arginine at position 192) displays several-fold higher activity toward paraoxon hydrolysis than the Q allele (glutamine at position 192) [4,37,38]. Regarding lipid peroxidation, preliminary studies suggest that the R/R isoform is less effective in hydrolyzing lipid peroxides than the Q/Q isoform [39,40]. Moreover, Regieli et al. [41] stated that 192R isoforms of PON1 have relatively low peroxidase activity toward copperinduced oxidated LDL while 192Q isoforms were more capable of protecting against LDL oxidation. This has been confirmed by other workers and indicates that the efficacy of the two allozymes toward lipid peroxides is opposite that toward paraoxon [25,17]. We have shown that the paraoxonase activity has significant positive correlation with HDL-cholesterol and significant negative correlation with total cholesterol, triglyceride, LDL-cholesterol and lipoprotein oxidation susceptibility. Navab et al. [42] also suggested that low paraoxonase activity has reduced the capacity of HDL to prevent the oxidation of LDL and therefore may lead to CAD. Putative mechanisms leading to decreased PON1 activity can be the inactivation of enzyme by increased oxidative stress [32] or accompanying acute phase response that inhibits hepatic synthesis of PON1 [43]. The decrease in PON1 activity could be the result of lower HDL concentrations in patients with CAD. Lipid peroxides, which are substrates for PON1 and which have been shown to be raised in people with CAD, are inhibitors of PON1. The role of oxidative stress in decreased PON1 activity might be confirmed by the inverse association between lipid peroxidation and PON1 activity [25]. Aviram et al. [32] clearly demonstrated that PON1 inactivation by oxidized-LDL resulted in the reduction of paraoxonase activity and its ability to protect LDL from oxidation; the enzyme is time-dependently inactivated during the formation of Ox-LDL. Oxidized LDL appears to inactivate PON1 through interactions between the enzyme-free sulfhydryl group and oxidized lipids, which are formed during LDL oxidation. This effect can be possibly related to displacement of calcium ions (which are required for PON1 activities) by copper ion (in the copper ion oxidative system). In conclusion, our results indicate that PON1 activity is lower in Egyptian patients with CAD and there is a significant relationship between activity of PON1 and the severity of coronary atherosclerosis. Also, we provide evidence of a significant association between the R allele of the PON1 polymorphism and the development of coronary artery disease in Egyptian patients. However, PON1 polymorphism is not related to severity of coronary atherosclerosis. References [1] Carew TE. Role of biologically modified low-density lipoprotein in atherosclerosis. Am J Cardiol 1989;64:18G–22G. [2] Sen-Banerjee S, Siles X, Campos H. Tobacco smoking modifies association between Gln-Arg192 polymorphism of human paraoxonase gene and risk of myocardial infarction. Arterioscler Thromb Vasc Biol 2000;20:2120–6. [3] Turban S, Fuentes F, Ferlic L, et al. A prospective study of paraoxonase gene Q/R192 polymorphism and severity, progression and regression of coronary atherosclerosis, plasma lipid levels, clinical events and resPON1se to fluvastatin. Atherosclerosis 2001;154:633–40. [4] Granér M, James RW, Kahri J, Nieminen MS, Syvänne M, Taskinen MR. Association of paraoxonase-1 activity and concentration with angiographic severity and extent of coronary artery disease. J Am Coll Cardiol 2006;47:2429–35.
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[23] Regnström J, Nilsson J, Tornvall P, Landou C, Hamsten A. Susceptibility to low density lipoprotein oxidation and coronary atherosclerosis in man. Lancet 1992;339:1183–6. [24] Croft KD, Dimmitt SB, Moulton C, Beilin LJ. Low density lipoprotein composition and oxidizability in coronary disease-apparent favourable effect of beta blockers. Atherosclerosis 1992;97:123–30. [25] Mackness B, Davies GK, Turkie W, Lee E, Roberts DH, Hill E. Paraoxonase status in coronary heart disease: are activity and concentration more important than genotype? Arterioscler Thromb Vasc Biol 2001;21:1451–7. [26] Azarsıza E, Kayıkcıo, lub M, Payzınb S, Sözmen EY. PON1 activities and oxidative markers of LDL in patients with angiographically proven coronary artery disease. Int J Cardiol 2003;91:43–51. [27] Ruiz J, Blanche' H, James RW, et al. Gln-Arg 192 polymorphism of paraoxonase and coronary heart disease in type 2 diabetes. Lancet 1995;346:869–72. [28] Herrmann SM, Blanc H, Poirier O, et al. The Gln/Arg polymorphism of human paraoxonase (PON1 192) is not related to myocardial infarction in the ECTIM study. Atherosclerosis 1996;126:299–303. [29] Rice GI, Ossei-Gerning N, Stickland MH, Grant PJ. The paraoxonase Gln–Arg 192 polymorphism in subjects with ischaemic heart disease. Coron Artery Dis 1997;8: 677–82. [30] Cao H, Girard-Globa A, Berthezene F, Moulin P. Paraoxonase protection of LDL against peroxidation is independent of its esterase activity towards paraoxon and is unaffected by the Q-R genetic polymorphism. J Lipid Res 1999;40: 133–9. [31] Mackness B, Mackness MI, Arrol S, Turkie W, Durrington PN. Effect of the human serum paraoxonase 55 and 192 genetic polymorphisms on the protection by high density lipoprotein against low density lipoprotein oxidative modification. FEBS Lett 1998;423:57–60.
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[32] Aviram M, Rosenblat M, Billecke S, Erogul J, Sorenson R, Bisgaier CL. Human serum paraoxonase (PON1) is inactivated by oxidized low density lipoprotein and preserved by antioxidants. Free Radic Biol Med 1999;26:892–904. [33] Serrato M, Marian AJ. A variant of human paraoxonase/arylesterase (HUMPON1A) gene is a risk factor for coronary artery disease. J Clin Invest 1995;96:3005–8. [34] Ko YL, Ko YS, Wang SM, et al. The Gln–Arg191 polymorphism of the human paraoxonase gene is not associated with the risk of coronary artery disease among Chinese in Taiwan. Atherosclerosis 1998;141:259–64. [35] Zama T, Murata M, Matsubara Y, et al. A 192Arg variant of the human paraoxonase (HUMPON1A) gene polymorphism is associated with an increased risk for coronary artery disease in the Japanese. Arterioscler Thromb Vasc Biol 1997;17: 3565–9. [36] Odawara M, Tachi Y, Yamashita K. Paraoxonase polymorphism (Gln192Arg) is associated with coronary artery disease in Japanese non insulin-dependent diabetes mellitus. J Clin Endocrinol Metab 1997;82:2257–60. [37] Humbert R, Adler DA, Disteche CK, Hassett C, Omiecinski CJ, Furlong CE. The molecular basis of the human serum paraoxonase activity polymorphism. Nat Genet 1993;3:73–6.
[38] Adkins S, Gan KN, Mody M, LaDu BN. Molecular basis for the polymorphic forms of human serum paraoxonase: arylesterase: glutamine or arginine at position 191, for the respective A or B allozymes. Am J Hum Genet 1993;52:598–608. [39] Mackness MI, Arrol S, Mackness B, Durrington PN. Alloenzymes of paraoxonase and effectiveness of high-density lipoproteins in protecting low-density lipoprotein against lipid peroxidation. Lancet 1997;349:851–2. [40] Mackness MI, Durrington PN, Mackness B. Polymorphisms of paraoxonase genes and low-density lipoprotein lipid oxidation. Lancet 1999;353:468–9. [41] Regieli JJ, Jukema JW, Doevendans PA, Zwinderman AH, Kastelein JJ, Grobbee DE, Graaf YV. Paraoxonase variants relate to 10-year risk in coronary artery disease impact of a high-density lipoprotein–bound antioxidant in secondary prevention. J Am Coll Cardiol 2009;54:1238–45. [42] Navab M, Hama SY, Cooke CJ, et al. Normal high density lipoprotein inhibits three steps in the formation of mildly oxidized low density lipoprotein: step I. J Lipid Res 2000;41:1481–94. [43] Feingold KR, Memon RA, Moser AH, Grunfeld C. Paraoxonase activity in the serum and hepatic mRNA levels decrease during the acute phase response. Atherosclerosis 1998;139:307–15.
Contents lists available at ScienceDirect
Clinical Biochemistry j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c l i n b i o c h e m
The relationship between paraoxonase1-192 polymorphism and activity with coronary artery disease Randa H. Mohamed a,⁎, Rasha H. Mohamed b, Rehab A. Karam a, Tarek A. Abd El-Aziz c a b c
Medical biochemistry department, Faculty of medicine, Zagazig University, Zagazig, Egypt Biochemistry department, Faculty of pharmacy, Zagazig University, Zagazig, Egypt Cardiology department, Faculty of medicine, Zagazig University, Zagazig, Egypt
a r t i c l e
i n f o
Article history: Received 17 October 2009 Received in revised form 10 December 2009 Accepted 11 December 2009 Available online 21 December 2009 Keywords: PON1 activity PON1 polymorphism Lipoprotein oxidation susceptibility CAD
a b s t r a c t Objective: We tested the association between PON1 polymorphism, PON1 activity, oxidative susceptibility of LDL and coronary artery disease in Egyptians. Methods: PON1 polymorphism, serum PON1 activity, lipoprotein oxidation susceptibility and lipid profile were measured. Results: Levels of HDL and paraoxonase activity were significantly decreased in CAD patients compared to control group, and in patients with three vessels compared to those of single or two vessels disease. Highactivity allele (R) has a more atherogenic lipid profile than for the low activity allele (Q). PON1 RR genotype has nine fold risks to develop CAD in Egyptians while those with PON1 QR genotype have four fold risks. Conclusion: The PON1 activity is lower in subject with CAD and there is a significant relationship between activity of PON1 and the severity of coronary atherosclerosis. Also, we provide evidence of a significant association between R allele of the PON1 polymorphism and the development of coronary artery disease. © 2010 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction Oxidative stress plays a crucial role in the development of atherosclerosis by oxidation of low-density lipoprotein (LDL) that subsequently leads to formation of foam cells. Conversely, highdensity lipoprotein (HDL) is a well-known anti-oxidant molecule that prevents atherosclerosis [1]. HDLs can protect LDLs from oxidative damage. The antioxidant effect of HDLs is determined by its enzymes, in particular paraoxonase, an HDL-associated enzyme capable of hydrolyzing lipid peroxides [2]. Paraoxonase (PON1) is a 44 kDa Ca2+-dependent glycoprotein, synthesized in the liver and is located on the surface of high density lipoprotein (HDL). Recently, it has been shown that PON1 decreases generation and accumulation of lipoperoxides in LDL. In addition, PON1, by destroying oxidized phospholipids, reduces the ability of oxidized LDL to induce monocyte binding and transmigration and thus, inflammation in the vessel wall [3]. In knockout mice lacking the gene for PON1, atherosclerosis develops more rapidly than in wildtype mice, whereas mice that overexpress human PON1 are resistant to atherosclerosis [4]. Thus, PON1 may be involved in protection against atherosclerosis. The role of PON1 may be particularly meaningful because oxidized LDL promotes secretion of the potent
⁎ Corresponding author: Fax: +20 552366896. E-mail address: [email protected] (R.H. Mohamed).
endothelial constrictor, endothelin, and reverses the impairment of endothelium-mediated vasodilatation in stenotic vessel [5]. Besides its protective effects against LDL peroxidation, HDLassociated paraoxonase has been demonstrated to inhibit the oxidative damage of HDL as well. The oxidation of HDL not only reduces its capability to prevent the oxidative modification of LDL, but also diminishes the ability of HDL to function as a potent acceptor for cholesterol efflux [6]. PON1 enzyme activity for paraoxon as a substrate is modulated by a number of polymorphisms at the PON1 gene located in chromosome 7q21.3, which is clustered with at least two other related genes, paraoxonase2 and paraoxonase3 [7]. Paraoxonase (A/G) polymorphism results in glutamine (Q) to arginine (R) substitution at codon 192. This 192Q isoform has been related to lower paraoxonase (paraoxonhydrolyzing) and arylesterase (phenylacetate-hydrolyzing) activity, and 192 R, an isoform with high activity toward paraoxon hydrolysis [8]. Although PON1 activity and concentration are determined genetically, various factors, such as diet, lifestyle, and environmental factors, can influence PON1 activity and/or concentration. Degraded cooking oil has been reported to lower serum PON1 levels in humans. Dietary polyphenols increase PON1 activity, as does moderate alcohol intake. Smoking is known to decrease serum PON1 activity. Recent evidence shows that exposure to environmental chemicals can inhibit PON1 activity. Furthermore, low serum PON1 activity independent of genotype has been reported in diseases associated with accelerated atherogenesis, such as diabetes mellitus, hypercholesterolemia, and renal failure [4].
0009-9120/$ – see front matter © 2010 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2009.12.015
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Whereas some authors have failed to find a link between the variation in paraoxonase gene and changes in lipoprotein concentrations [9,10], others have found a significant association between paraoxonase-192 genetic variants and changes in HDL-cholesterol levels and in triglyceride concentrations in relatively genetically isolated populations [11,12] Therefore, at present the relationship between paraoxonase genetic polymorphism and atherosclerosis is unclear. Whether the paraoxonase gene can modulate the lipid profile is a matter of conjecture, but remains a possibility in the light of the above population studies. Because PON1-192 genetic polymorphism strongly influences PON1 activity and does vary between different ethnic groups, we tested the association between this polymorphism, PON1 activity, changes in oxidative susceptibility of LDL and coronary artery disease (CAD) in Egyptian patients. Material and methods Subjects Subjects who underwent coronary angiography in the Cardiovascular Center for detection of the presence and extent of stenosis in coronary artery vessels were recruited according to a designed protocol. Forty-three patients had previous history of myocardial infarction, and the mean value of left ventricular ejection fraction (LVEF) for patients was 0.59 ± 0.08 and ranged from 0.43 to 0.80. All patients were treated with statin, nitrates, aspirin, angiotensin converting enzyme inhibitor, 145 patients received β-adrenoceptorblocking drug, 5 were taking a calcium channel blocker and 8 patients were taking diuretics. Participants with hepatic or renal disease, cardiomyopathy, class IV congestive heart failure, significant valvular or congenital heart disease, and recent coronary angioplasty or coronary bypass surgery and acute myocardial infarction within the last three months were excluded from the study. The CAD group included 150 patients who have at least a 70% stenosis in a major epicardial artery and subdivided into three groups: single-vessel disease (SVD), two-vessel disease (2VD) and threevessel disease (3VD). Fifty control subjects age- and sex-matched with the patients were randomly recruited. The control group had neither clinical symptoms nor electrocardiographic changes indicative of CAD and therefore did not undergo coronary angiography. Biochemical measurements Analyses of Lipid Blood samples were drawn from all subjects after an overnight fast. Sera were separated immediately and stored at −20 °C. Total cholesterol and triglycerides were measured by routine enzymatic
methods (spinreact). HDL-cholesterol was determined after precipitation of the apoB-containing lipoproteins. LDL-cholesterol was calculated using Friedewald formula [13]. Lipoprotein oxidation susceptibility The oxidizability of apo B-containing lipoproteins is as described [14], and this fraction was precipitated from 500 μL of plasma by adding 50 μL of dextran sulfate-magnesium chloride. The pellet was dissolved in 2.5 mL of 4% saline solution. A volume of redissolved precipitate containing 100 μg of non-HDL-cholesterol (calculated from the total minus HDL-cholesterol level) was mixed with 4% sodium chloride to give 500 μL of total solution. Copper solution (0.5 mM Cu Cl2) was added and incubated at 37 °C for 3 h in a shaking water bath. The thiobarbituric acid-reactive substance assay (as an index of oxidation) was conducted by adding 2 mL of the thiobarbituric acid-reactive substance reagent to the 550 μL incubation mixture and heating it in a boiling water bath for 15 min. The pink color was read in a spectrophotometer at 532 against a blank containing 4% saline solution. The nmoles of malonyldialdehyde (MDA) present in the sample were estimated by comparison with a standard curve prepared from 1,1,3,3-tetraethoxypropane (0.5 to 16 nmol/mL). Analysis of PON1 activity PON1 activity toward paraoxon was measured after paraoxon hydrolysis into p-nitrophenol and diethyl phosphate catalyzed by the enzyme. The activity was measured by adding 10 μL serum to 350 μL Tris–HCl buffer (100 mmol/L, pH 8.0) containing 2.0 mmol/L CaCl2 and 2.0 mmol/L paraoxon. The rate of generation of p-nitrophenol was determined at 405 nm on a Milton Roy Spectronic 3000 array at 37 C° over 3 min after 42 s delay [15]. Determination of the paraoxonase 192 genotype DNA was isolated and purified from whole blood (EDTA) using QIAamp-spin-columns according to the protocol provided by the manufacturer (QIAamp Blood Kit; Qiagen GmbH, Hilden, Germany). The 99-bp target region in the paraoxonase gene was amplified by polymerase chain reaction (PCR) using forward 5′-TAT TGT TGC TGT GGG ACC TGA G-3′ and reverse 5′-CAC GCT AAA CCC AAA TAC ATC TC-3′ primers. The PCR reaction mix contained 10 μg genomic DNA, 0.5 μmol/L of each primer, 200 μmol/L of each dNTP, 5 μL of 10× reaction buffer, and 1.25 U Taq DNA polymerase. After the DNA was denatured at 95 °C for 3 min, the reaction mixture was subject to 30 cycles, each cycle comprising denaturation at 94 °C for 60 s, annealing at 61 °C for 30 s, and extension at 72 °C for 60 s, with a final extension time of 5 min. The
Table 1 Study participants characteristics. Parameter
SVD
2VD
3VD
CAD
Controls
n Age (years) Sex (m/f) Hypertension, n (%) Diabetes, n (%) Smoker, n (%) Cholesterol (mg/dL) Triglycerides (mg/dL) HDL (mg/dL) LDL (mg/dL) Lipoprotein oxidation susceptibility (nmol/mg non-HDLc) Paraoxonase (U/mL)
50 53.5 ± 8.1 29/21 41 (82%) 32 (64%) 17 (34%) 230.3 ± 26.8a 170.1 ± 10.1a 43 ± 4.3a 153.3 ± 25.3a 45.3 ± 12.6a 194.5 ± 41.5a
50 59.9 ± 7.9 40/10 27 (54%) 9 (18%) 18 (36%) 242.8 ± 27.9a,b 176.6 ± 15.7a,b 42.4 ± 4.1a 165 ± 25.8a,b 46.6 ± 9.3a 178.6 ± 25.5a,b
50 53.4 ± 5.8 50/0 14 (28%) 14 (28%) 43 (86%) 261.7 ± 13.9a,b,c 188 ± 11.7a,b,c 39.3 ± 2.9a,b,c 184.8 ± 14.6a,b,c 58.2 ± 22.3a,b,c 152.8 ± 25.1a,b,c
150 55.5 ± 8.1 119/31 82 (54%) 55 (36.6%) 78 (52%) 240.5 ± 27.4a 175.7 ± 13.9a 42.1 ± 4.2a 163.3 ± 26.1a 48.3 ± 14.6a 181.2 ± 37a
50 50.7 ± 9.5 26/24 – – 18 (36%) 185.9 ± 8.4 132 ± 14.4 54.7 ± 3.2 106.2 ± 6.2 35.8 ± 6.9 225.7 ± 58.3
a b c
Significant difference from control. Significant difference from SVD. Significant difference from 2VD.
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PCR products were digested with 2.5 U AlwI restriction endonuclease for 1 h, and the digested products were separated by electrophoresis on a 3% agarose gel. Then the gel was visualized under UV transilluminator with a 50 base pair ladder. Individuals homozygous for the 192 Gln allele present a 99-bp PCR product, those homozygous for the 192 Arg allele present 69- and 30-bp products, and those heterozygous present 90-, 69-, and 30-bp products [6].
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Table 3 ORs for CAD and three-vessel disease.
Q/Q Q/R R/R R allele
Presence of CAD
Severity of CAD
OR
Confidence interval
OR
Confidence interval
1 4.2 9.7 3.8
1.7–9.5 4.2–23.9 2.4–6.1
1 1.7 1.1 1.1
0.5–5.3 0.4–3.1 0.6–1.9
Statistical analysis The results for continuous variables are expressed as means ± SD. The means of the three genotype groups were compared in a one-way analysis of variance. The correlation coefficients were calculated using spearman correlation. The statistical significances of differences in frequencies of variants between the groups were tested using the chisquare (χ2) test. In addition, the odds ratios (ORs) and 95% CIs were calculated as a measure of the association of the paraoxonase 192 genotypes with CAD and its severity. A difference was considered significant at P b 0.05. All data were evaluated using SPSS version 10.0 of windows. Results Distribution of clinical characteristics of the study subjects in relation to the severity of CAD (Table 1) Levels of total cholesterol, triglyceride, LDL-cholesterol and lipoprotein oxidation susceptibility were significantly increased in CAD patients compared to control group. Furthermore, levels of HDLcholesterol and paraoxonase activity were significantly decreased in CAD patients compared to control group. In order to evaluate the relationship between the risk factors and the severity of coronary artery disease, patients with CAD were divided into three categories according to the number of diseased coronary vessels. The levels of total cholesterol, triglyceride, LDLcholesterol and lipoprotein oxidation susceptibility were significantly increased in patients with three vessels compared to those of single or two-vessel disease. Levels of HDL-cholesterol and paraoxonase activity were significantly decreased in patients with three vessels compared to those of single or two-vessel disease. PON1 polymorphisms genotypes and alleles distribution between patients and controls (Tables 2 and 3) In the CAD group, the frequencies of PON1 RR genotype were significantly increased compared to control group (50.6% versus 20%) resulting in a significantly increased OR in the CAD subject bearing this gene (OR = 9.7, 95% CI 3.9–23.9); also, the R allele was significantly increased in the CAD group compared to the control group (69% versus 37%) resulting in a significantly increased OR in the CAD subject bearing this allele ( OR = 3.8, 95% CI 2.4–6.1). The PON1 genotypes did not exhibit any significant differences between patients with single-, double-, or triple-vessel disease. Table 2 Distribution frequency of genotypes and alleles of PON1 polymorphisms between patients and controls.
Q/Q, n (%) Q/R, n (%) R/R, n (%) R allele a b c
SVD
2VD
3VD
CAD
6 (12)a 23 (46)b 21 (42)a,b 65 (65)a
5 (10) a 18 (36)b 27 (54)a,b 72 (72)a
7 (14)a 15 (30) 28 (56)a,b,c 71 (71)a
18 56 76 208
Significant difference from control. Significant difference from QQ. Significant difference from QR.
Controls (12)a (37.3)b (50.6)a,b,c (69)a
23 17 10 37
(46) (34) (20)b (37)
Association between paraoxonase 192 genotype and clinical characteristics of the study subjects (Table 4) The risk factors for CAD (diabetes, hypertension, dyslipidemia) were significantly associated with PON1 RR. Levels of total cholesterol, triglyceride, LDL-cholesterol, paraoxonase activity and lipoprotein oxidation susceptibility were significantly increased in PON1 RR than in PON1 QQ. Levels of HDL-cholesterol were significantly decreased in PON1 RR than in PON1 QQ. Association between paraoxonase activity and lipid profile (Table 5) Paraoxonase activity had significant positive correlation with HDLcholesterol and significant negative correlation with total cholesterol, triglyceride, LDL-cholesterol and lipoprotein oxidation susceptibility. Multiple regression analysis (Table 6) The difference in severity of CAD was tested for independence from other variables by multiple regression analysis. The model included sex, age, hypertension, diabetes, smoker, total cholesterol, triglycerides, LDL, HDL, Lipoprotein oxidation susceptibility, PON1 activity, and the 192 polymorphisms. The difference in severity of CAD was found to be dependent on diabetes only. Discussion The oxidative damage to vital biological systems can lead to enhanced expression of inflammatory genes that ultimately contribute to the development of several chronic diseases, including CAD, cancer, and diabetes, and that contribute to aging. The balance between oxidants and antioxidants basically affects all biological systems and, ultimately, the clinical course. The oxidation of LDL and its involvement in the development of foam cell-laden fatty streaks in the arterial wall are believed to initiate the atherosclerotic process [16]. In vitro studies indicate that HDL-associated PON1 prevents LDL oxidation and can destroy biologically active lipids in mildly oxidized
Table 4 Study participants' characteristics according to the PON1 192 Q/R genotypes.
n Age (years) Sex (m/f) Hypertension, n (%) Diabetes, n (%) Smoker, n (%) Cholesterol (mg/dL) Triglycerides (mg/dL) HDL (mg/dL) LDL (mg/dL) Lipoprotein oxidation susceptibility (nmol/mg non-HDLc) Paraoxonase Activity(U/mL) a b
Significant difference from QQ. Significant difference from QR.
Q/Q
Q/R
R/R
41 53 ± 9.7 25/13 12 (25.5) 7 (14.8) 15 (31.2) 205.2 ± 32.8 146.8 ± 22.9 52.7 ± 6.1 125.3 ± 32.6 39.3 ± 16.9
73 51.3 ± 8.5 52/21 30 (40) 22(30) 23 (31.5) 223.4 ± 33.1a 160 ± 22.6a 48.2 ± 6a 143.2 ± 34.1a 40.9 ± 9.8a
86 56.5 ± 8.3a,b 59/21 40 (50)a 26 (32.5)a 40 (50) 227.3 ± 35.7a 167.4 ± 26.9a 41.8 ± 5.6a,b 152.1 ± 34.2a 48.8 ± 13.7a,b
164.9 ± 34.5 185.2 ± 34.6a 227.6 ± 54.5a,b
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Table 5 Correlation between paraoxonase activity and lipid profile, Lipoprotein oxidation susceptibility. Paraoxonase activity
Cholesterol Triglycerides HDL LDL Lipoprotein oxidation susceptibility
r
p
−0.469 −0.482 0.29 −0.457 −0.299
b 0.001 b 0.001 b 0.05 b 0.001 b 0.05
LDL [17], and PON1 may thus affect the process of atherosclerosis. It has also been demonstrated that PON1 has the capacity to reduce oxidized lipids in human atherosclerotic lesions derived from either coronary artery or carotid specimens [18]. Previous studies that have investigated the relationship between the PON1-192 polymorphism and CAD have produced inconsistent results. Some studies have shown the PON1-192R genotype to be present at a higher frequency in CAD, leading to the hypothesis that the PON1-192 polymorphism might be a risk factor for atherosclerosis [6,19], but some studies have failed to find such a relationship [20,21]. Study population characteristics in relation to the severity of CAD The present study demonstrates that levels of total cholesterol, triglycerides, LDL-cholesterol and lipoprotein oxidation susceptibility are significantly increased in CAD patients compared to control group. These parameters were also elevated in patients who had more severe disease compared to those with less severe disease. In accordance with our data, De Rijke et al. [22] studied the association between oxidation parameters and progression of coronary artery stenosis and found a higher susceptibility of LDL to oxidation in CAD patients who had shown progression in stenosis. In another study, Regnström et al. [23] described an inverse association between resistance of LDL to oxidation and severity of coronary stenosis; however, the study of Croft et al.[24] did not reveal a difference in oxidation parameters between coronary atherosclerotic patients and control subjects. In this study, we demonstrated that levels of HDL-cholesterol and paraoxonase activity were significantly decreased in CAD patients compared to control group, and in patients with 3VD compared to those of one or 2VD. Mackness et al. [25] showed that PON1 activities toward paraoxon are lower in subjects with CHD than in control subjects regardless of the PON1 genotype. Azarsıza et al. [26] found that PON1 activity in patients with CAD were lower than control and is depleted during the propagation phase of the atherosclerotic process; however, this difference was not found statistically significant.
Paraoxonase 192 genotype and CAD We found that the risk factors for CAD (diabetes, hypertension, dyslipidemia) are significantly associated with PON1 RR. While several studies showed a positive association between the R allele and the risk for CAD [6,10,27], others showed no association [28,29]. We demonstrated that levels of HDL-cholesterol were significantly decreased in PON1 RR than in PON1 QQ. Previous studies have investigated the association between PON1 192 polymorphism and blood lipids. HDL may play a significant role in the effect of PON1 on coronary disease. In one study, the PON1-192 R allele was associated with lower HDL-cholesterol levels [11], but this was not confirmed in other studies [2,30]. We demonstrated that levels of total cholesterol, triglyceride, and LDL-cholesterol were significantly increased in PON1 RR than in PON1 QQ. In accordance with our data, Hegele et al. [11] observed that homozygotes for the low activity allele (Q) have a less atherogenic lipid profile than heterozygotes and homozygotes for the highactivity allele (R). Several lines of evidence have been provided for a potential biological link between the Q/R192 polymorphism and the antiatherogenic effect. Mackness et al. [31] demonstrated that HDL containing R192 PON1 was less effective in protecting LDL from oxidative modification than HDL with Q192 PON1. Aviram et al. [32] showed that recombinant PON1 encompassing R192 alleles were less effective in blocking LDL oxidation. These results indicate that 192R PON1 have lower activity for hydrolyzing lipid peroxide, thus lower anti-atherogenic property. These findings may explain why the paraoxonase R allele has been found to be present at an increased frequency in coronary heart disease (CAD) leading to the hypothesis that the PON1-192 polymorphism might be a risk factor for atherosclerosis. We demonstrated that individuals with PON1 RR genotype have 9fold risks to develop CAD in Egyptians while those with the PON1 QR genotype have 4-fold risks. Serrato and Marian [33] found that the R allele was clearly associated with coronary heart disease in the United States. Sanghera et al. [10] reported that Q/R192 gene polymorphism was associated with CAD in Indians, but not in Chinese. Ko et al. [34] compared the Q/R192 genotype distribution between CAD patients and age- and sex-matched control subjects in Taiwan, which showed no difference. This variability in results suggests that gene–environment and/or gene–gene interactions might modulate the relationship between paraoxonase polymorphism and coronary heart disease. Inconsistent association of PON1 with CAD may be attributed to differences in several factors between studies, including ethnic and environmental factors, and methodological factors such as sampling scheme and trial size. Among these factors, of note is the fact that allele frequencies of the three polymorphisms do vary between different ethnic groups; for example, we found that PON1 the QQ
Table 6 Multiple regression analysis. Variable
Age Sex Hypertension Diabetes Smoker Cholesterol Triglycerides HDL LDL Lipoprotein oxidation susceptibility Paraoxonase Q192R
Unstandardized coefficients
Standardized coefficients
B
Standard error
β
3.9E−03 5.38E−02 −0.125 −0.438 0.276 2.02E−02 6.95E−03 −4.44E−02 −8.38E−03 −3.2E−03 −6.4E−03 0.283
0.189 0.008 0.16 0.179 0.172 0.016 0.005 0.017 0.027 0.007 0.003 0.224
0.024 0.033 −0.059 −0.193 0.132 0.671 0.169 −0.28 −0.31 −0.042 −0.308 0.214
95% CI
t
p
−0.013 to 0.021 −0.44 to 0.198 −0.8 to −0.08 −0.07 to 0.62 −0.013 to 0.053 −0.004 to 0.018 −0.043 to 0.026 −0.099 to 0.01 −0.02 to 0.01 −0.013 to 0.00 −0.17 to 0.73
0.472 −0.78 −2.444 1.61 1.234 1.308 −0.494 −1.649 −0.474 −1.98 1.26
0.639 0.44 0.019 0.115 0.224 0.198 0.624 0.107 0.638 0.054 0.215
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genotype is common in Egyptians which has also been found in Caucasians [28,29]; however, R allele frequencies have proven to be relatively common in Japanese [35,36] and Chinese [34]. Paraoxonase activity and CAD We demonstrated that paraoxonase activity and lipoprotein oxidation susceptibility were significantly increased in PON1 RR than in PON1 QQ. Activity of PON1 can vary up to 40-fold in human populations. Part of this variability is explained by the polymorphism of PON1 gene because of an amino acid substitution at 192. The R allele (arginine at position 192) displays several-fold higher activity toward paraoxon hydrolysis than the Q allele (glutamine at position 192) [4,37,38]. Regarding lipid peroxidation, preliminary studies suggest that the R/R isoform is less effective in hydrolyzing lipid peroxides than the Q/Q isoform [39,40]. Moreover, Regieli et al. [41] stated that 192R isoforms of PON1 have relatively low peroxidase activity toward copperinduced oxidated LDL while 192Q isoforms were more capable of protecting against LDL oxidation. This has been confirmed by other workers and indicates that the efficacy of the two allozymes toward lipid peroxides is opposite that toward paraoxon [25,17]. We have shown that the paraoxonase activity has significant positive correlation with HDL-cholesterol and significant negative correlation with total cholesterol, triglyceride, LDL-cholesterol and lipoprotein oxidation susceptibility. Navab et al. [42] also suggested that low paraoxonase activity has reduced the capacity of HDL to prevent the oxidation of LDL and therefore may lead to CAD. Putative mechanisms leading to decreased PON1 activity can be the inactivation of enzyme by increased oxidative stress [32] or accompanying acute phase response that inhibits hepatic synthesis of PON1 [43]. The decrease in PON1 activity could be the result of lower HDL concentrations in patients with CAD. Lipid peroxides, which are substrates for PON1 and which have been shown to be raised in people with CAD, are inhibitors of PON1. The role of oxidative stress in decreased PON1 activity might be confirmed by the inverse association between lipid peroxidation and PON1 activity [25]. Aviram et al. [32] clearly demonstrated that PON1 inactivation by oxidized-LDL resulted in the reduction of paraoxonase activity and its ability to protect LDL from oxidation; the enzyme is time-dependently inactivated during the formation of Ox-LDL. Oxidized LDL appears to inactivate PON1 through interactions between the enzyme-free sulfhydryl group and oxidized lipids, which are formed during LDL oxidation. This effect can be possibly related to displacement of calcium ions (which are required for PON1 activities) by copper ion (in the copper ion oxidative system). In conclusion, our results indicate that PON1 activity is lower in Egyptian patients with CAD and there is a significant relationship between activity of PON1 and the severity of coronary atherosclerosis. Also, we provide evidence of a significant association between the R allele of the PON1 polymorphism and the development of coronary artery disease in Egyptian patients. However, PON1 polymorphism is not related to severity of coronary atherosclerosis. References [1] Carew TE. Role of biologically modified low-density lipoprotein in atherosclerosis. Am J Cardiol 1989;64:18G–22G. [2] Sen-Banerjee S, Siles X, Campos H. Tobacco smoking modifies association between Gln-Arg192 polymorphism of human paraoxonase gene and risk of myocardial infarction. Arterioscler Thromb Vasc Biol 2000;20:2120–6. [3] Turban S, Fuentes F, Ferlic L, et al. A prospective study of paraoxonase gene Q/R192 polymorphism and severity, progression and regression of coronary atherosclerosis, plasma lipid levels, clinical events and resPON1se to fluvastatin. Atherosclerosis 2001;154:633–40. [4] Granér M, James RW, Kahri J, Nieminen MS, Syvänne M, Taskinen MR. Association of paraoxonase-1 activity and concentration with angiographic severity and extent of coronary artery disease. J Am Coll Cardiol 2006;47:2429–35.
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