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MnSOD and GPx1 polymorphism relationship with coronary heart disease risk and severity

MnSOD and GPx1 polymorphism relationship with coronary heart disease risk and severity Background: Disturbance of the equilibrium between reactive oxygen species (ROS) and anti-oxidants (AOX) has been implicated in various diseases, including atherosclerosis, the most common pathologic process underlying coronary heart disease (CHD). Thus, the defense systems against ROS are critical protecting blood vessel walls against oxidative damage. In this study, we investigate whether Ala16Val MnSOD and Pro198Leu GPx polymorphisms are associated with CHD susceptibility and/or severity. Methods: Both polymorphisms were genotyped in a sample of 203 controls and 164 patients. CHD risk and severity, antioxidant status (enzymatic and/or non enzymatic) and biochemical parameters were assessed and analysed by genotype. Results: A significant association of MnSOD variant to CHD risk was revealed in males. Males harboring the Val/Val genotype were approximately at twofold increased risk of CHD compared to controls (Ala carriers vs Val/Val, adjusted OR 1.89; 95 % CI 1.18‒3.42, p = 0.03). Significant decreases in SOD activity and total antioxidant status ( TAS) were observed in Val carriers and by CHD status. Whereas, no association of GPx variant genotype (Leu/Leu) and activity to cardiopathy events was discerned. CHD severity, as demonstrated by the number of vessel stenosis, was associated with significantly higher frequency of Val allele and LDL levels in CHD subjects. Conclusions: Our results showed a lack of association of Pro198Leu GPx polymorphism to CHD risk and severity. However, they suggest that Ala16Val MnSOD polymorphism and decreased antioxidant defences are likely contrib- uted to CHD risk in Tunisian men. Furthermore, the Val encoding MnSOD allele and decreased SOD activity were significantly correlated with CHD stenosis progression. Keywords: Genetic polymorphism, Coronary heart disease, SOD activity, GPx activity, Total antioxidant status, Atherosclerosis Atherosclerosis, the most common pathologic process Background underlying coronary heart disease, represents a state of Coronary Artery Disease is the major cause of mortality heightened oxidative stress characterized by endothelial and morbidity worldwide [1]. It is independently asso- dysfunction and plaque disruption. Oxidative stress can ciated with various risk factors such as advanced age, occur when the balance is upset, either by an excessive hypertension, smoking habit, diabetes mellitus, hyper- production of reactive oxygen species (ROS), by deficient lipidemia, positive family story, obesity and inactivity [2]. antioxidant defenses, or by the combination of both [3]. In such circumstances, ROS may interact with cellular *Correspondence: [email protected] bio-molecules, leading to modification and potentially Foued Chéour and Kacem Mahdouani contributed equally to this work Laboratory of Biochemistry and Molecular Biology, Hospital of Ibn serious consequences for the cell [4]. Eljazzar of Kairouan, Avenue Ibn Eljazzar, 3140 Kairouan, Tunisia Full list of author information is available at the end of the article © 2016 Souiden et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Souiden et al. Biol Res (2016) 49:22 Page 2 of 12 Several potentially significant genetic variants related The aim of our study was to assess the association of to oxidative stress have been already identified [5, 6]. In MnSOD and GPx polymorphisms and activities with this study, we are interested to polymorphisms of the CHD risk and severity in the Tunisian population. superoxide dismutase (SOD) and glutathione peroxi- dase (GPx) in order to check their possible implication in Methods CHD risk and severity. Study population SOD is the primary antioxidant in the mitochondria One hundred and sixty-four patients with coronary heart that converts ROS into oxygen and hydrogen perox- disease (CHD) and two hundred and three controls were ide [7–10]. There are three SOD isoforms, including the enrolled in this study from June to September 2011. The mitochondrial SOD manganese dependent (MnSOD). healthy subjects were recruited at their annual health MnSOD is encoded by a single gene containing five examination at Hospital Ibn Eljazzar, Kairouan, Tunisia, exons and it is located on chromosome 6q25 [11]. One and did not have any chest symptoms or electrocardio- of the common polymorphisms of MnSOD results in the gram (ECG) abnormalities suggesting CHD, or a medical replacement of alanine 16 (GCT) with a valine (GTT); history for CHD. the Ala16Val polymorphism. This polymorphism affects Patients were admitted to the cardiovascular depart- the import of MnSOD into the mitochondria by altering ment of Hospital Ibn Eljazzar, Kairouan, Tunisia. The the conformation of its leader signal [12]. This mutation diagnosis of Acute CHD was based on the presence of at may reflect a functional polymorphism of mitochondrial least 2 of the following three elements: (1) Ischemic type transport of human MnSOD. It has been shown that the of chest pain (2) changes on serial electrocardiogram 16Ala variant allows efficient targeting of MnSOD to (ECG) tracings (3) Increase in serum cardiac marker the mitochondria, as evidenced by Sutton et al. [13] who [elevated creatine kinase isoenzyme MB (CK-MB) and found that it was 30–40  % more efficiently localized to troponin T within 12  h after the onset of pain]. Patients the mitochondria than the 16Val variant. This is related with associated renal failure, liver disease, lung disease, to the fact that 16Ala variant has an α-helix structure pregnancy, thyroid disease, gastrointestinal disease were that is easily imported and it reaches high levels of mito- excluded. The study protocol was approved by the local chondrial concentration and activity, whereas the 16Val Ethics Committee of the hospital Ibn Eljazzar. Written variant has a partial ß-sheet structure that is partly stuck informed consent was obtained from all subjects before within the narrow inner membrane import pore and is their participation in the study. subsequently degraded by the proteasome. Furthermore, All participants were interviewed, and data on hyper- the mRNA that encodes the 16Val variant is more rapidly tension, diabetes mellitus, dyslipidemia, medical history degraded than the Ala variant [14]. including family history, smoking status and duration of The second line of enzymatic antioxidant defense is CHD were recorded. For coronary risk factors the fol- played by glutathione peroxidase (GPx) isoenzymes. GPx lowing definitions were used: individuals were defined as has 6 isoforms (GPx-1-6) [15]. GPx-1 is the most widely hypertensive if their blood pressure was >140/90 mmHg distributed and abundant form in human cells, includ- or in case of self-reported use of an antihypertensive ing vascular endothelium [16]. GPx-1 is an intracellular drug. Diabetes mellitus was diagnosed if the fasting soluble selenoprotein which converts peroxides such as plasma glucose concentration was  ≥7  mmol/l or if the H2O2 and ROOH into water and alcohol [17]. The gene patient was treated with insulin or oral hypoglycemic coding for GPx-1, is located on chromosome 3p21.3 agents; individuals were deemed dyslipidemic when [18] and it is composed of 2 exons with a 1.42 kb region their total cholesterol concentration was ≥5.68  mmol/l, [19]. Several polymorphisms have been described in the or their triglyceride concentration was ≥2.28 mmol/l, or GPx-1 gene. One of them is located at codon 198 (C > T) they receive lipid-lowering drugs. Smoking was coded as resulting in an amino acid variation from proline (CCC) never and current smoker. Gender and body mass index to leucine (CTC). This amino acid substitution causes a (BMI) were also recorded. change of the structural conformation of the active site region and modifies the enzyme activity [20]. Previous Blood collection studies using aortic endothelial cells demonstrated a 40 % From all subjects, Blood samples were collected in hep- reduction in the GPx-1 activity being associated with arinized (5 ml) and EDTA (5 ml) tubes after 12 h fasting the T allele and showed that the Pro198Leu variant was and immediately centrifuged at 1600×g. After separation, associated with increased carotid intima-to-media thick- washed and lysed erythrocytes as well as plasma were ness, peripheral arterial disease, and increased CAD risk stored at −70  °C until biochemical measurements were [21–24]. performed. Souiden et al. Biol Res (2016) 49:22 Page 3 of 12 Biochemical analyses Genetic analyses Superoxide Dismutase (SOD) Assay Genomic DNA was isolated from 300  µl of periph- The proportioning of erythrocyte enzymatic activ - eral blood leukocytes using Promega DNA isolation kit ity superoxide dismutase (SOD) is based on velocity according to the manufacturer’s recommendations. An measurement of oxidation reaction inhibition of I.N.T. alanine/valine polymorphism in the signal peptide of Mn- (2-(4-Iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazo- SOD gene was evaluated by PCR–RFLP analysis accord- liumn chloride) by SOD. The role of SOD is to acceler - ing to the method described by Ibrahim et  al. [26]. PCR ate the dismutation of toxic superoxide radical (O2−) amplifications were performed in a total volume of 20 μl produced during oxidative energy processes to hydrogen containing 50 ng of genomic DNA, 1.25 U Taq polymer- peroxide and molecular oxygen. This method uses xan - ase, 2 mM dNTP, 2 mM MgCl and 1X PCR buffer in the thine and xanthine oxidase (XOD) to generate superoxide presence of 0.4  μmol/l of each primer (forward 5′-CAG radicals which react with I.N.T. to form a red formazan CCC AGC CTG CGT AGA CGG-3′ and reverse 5′-CTT dye. SOD activity is then measured by the degree of GGC CAA CGC CTC CTG GTA CTT-3′) to amplify a inhibition of this reaction. The results were expressed as 267-bp fragment. The PCR conditions involved an ini - U/g Hb. tial denaturation of DNA at 95  °C for 5  min, followed by 30 cycles of amplification at 95  °C for 45  s (melting), Glutathione peroxidase (GPx) assay 54 °C for 30 s (annealing) and 72 °C for 30  s, and a final The erythrocyte hemolysate GPx1 activity was assessed extension at 72  °C for 5  min. The resulting 267-bp PCR by Paglia and Valentine method [25] using the Ran- product was digested with the restriction endonuclease sel kit (Randox, Antrim, UK) based on the oxidation BsaW1 at 37  °C for 2  h according to the manufacturer’s reduction of glutathione and the measurement of the recommendations and digested products were visualized variations of reduced NADP. GPx catalysis the oxida- with electrophoresis in 2.5  % agarose gel stained with tion of glutathione (GSH) by cumene hydroperoxide. In ethidium bromide (0.5 µg/ml). Restriction enzyme diges- the presence of glutathione reductase and NADPH oxi- tion results in a 267-bp product (16Ala) or 183 and 84 bp dized glutathione (GSSG) is immediately converted to products (16Val) (Fig. 1). reduced form with a concomitant oxidation of NADPH The GPx1  198Pro/Leu variant was determined using to NADP+. The enzyme activity is measured at a wave - 5′-TCC AGA CCA TTG ACA TCG AG-3′ (forward) length of 340 nm. One unit of GPX activity is defined as and 5′-ACT GGG ATC AAC AGG ACC AG-3′ (reverse) the amount of the enzyme required for oxidizing 1 μmol primers. A 222 base pair DNA fragment containing of NADPH per minute. Results were expressed in units the polymorphic site was amplified in a total volume per gram of hemoglobin. of 20  μl, containing 2  μl 10xPCR buffer, 1.125  mmol/l Total antioxidant status (TAS) assay Total antioxidant status (TAS) is measured by the reduc- ®+ tion in color produced by a radical cation (ABTS ). The assay principle is as follow: the ABTS (2,2′-azino- di-[3-ethylbenzthiazoline sulphonate]) is incubated with metmyoglobin which has peroxidase activity and H O to 2 2 ®+ produce the radical cation ABTS . This has a relatively stable blue-green color measured at 600  nm. Antioxi- dants in the added sample cause suppression of this color production to a degree which is proportional to their concentration. Other analyses Measurements of other biochemical parameters tested during this study included heparinized plasma triglyc- Fig. 1 Restriction fragments of MnSOD gene amplified products. erides, cholesterol, HDL-Cholesterol, were detected by Lane1 DNA molecular weight marker (50 pb); lane 2 negative control; lanes 3 and 7 Val/Val mutated homozygous genotype; lanes 4 and an automate (Konelab 20, Thermo Clinical Labsystems 6 Ala/Ala wildtype genotype; lanes 5 and 8, Ala/Val heterozygous Oy, Finland). LDL-Cholesterol was calculated using the genotype Friedewald equation. Souiden et al. Biol Res (2016) 49:22 Page 4 of 12 MgCl2, 0.15  mmol/l dNTPs, 0.25  μmol/l each primer, permutations. Theoretical power was also calculated 100 ng of genomic DNA and 1.5 U of Taq DNA polymer- using Quanto and parameter values (Odds ratio, allele ase (Promega Madison, USA). The PCR amplification frequencies and sample size) equals to those found in our was performed in 35 cycles at 94  °C for 30  s, 59  °C for study. 30  s and 72  °C for 30  s, preceded by an initial denatura- tion at 94 °C for 8 min and followed by a final elongation Results step at 72 °C for 7 min. The amplicon was then digested Three hundred and sixty-seven subjects were investi - with ApaI (Promega Madison, USA) restriction enzyme gated in this study in order to evaluate the association and resolved in 2  % agarose gel. The digested products of the MnSOD Ala16Val and GPx1 Pro198Leu polymor- showed 2 fragments of 170 and 52  bp for the 198Pro phisms with the coronary heart disease (CHD). wildtype homozygous, 3 fragments of 222, 170 and 52 Characteristics of the enrolled subjects stratified for the 198Pro/Leu (CT) heterozygote, and 1 fragment according to MnSOD and GPx1 polymorphisms are of 222 pb for the 198Leu (T) mutated homozygous geno- illustrated in Table  1. Results observation showed a sig- type (Fig. 2). nificant difference between MnSOD genotype classes associated with male gender and diabetes mellitus. Statistical tests Whereas, a significant difference between GPx1 geno - Statistical analyses were performed using the Statistical type groups was noted only according to LDL level in the Package for the Social Sciences (SPSS) version 17.0 for study population. Windows (SPSS, Chicago, IL), R software version 3.0.2 Biochemical examination of our population was car- and QUANTO program version 1.2. The Chi square test ried out by testing several parameters (Table  1). Results was used to test the distribution of genotypes for devia- showed a significant difference between genotype groups tions from Hardy–Weinberg equilibrium. genotype dis- for antioxidant status. Indeed, SOD2 but not GPx1 activ- tribution of MnSOD and GPx1 gene in all subjects was ity decreased significantly in the presence of the mutated analyzed by Chi square test. Distributions of continuous allele (Val and Leu, respectively). The same is true for variables in groups were expressed as mean ± SD. total antioxidant status (TAS). In fact, significant reduced Comparison between two groups was performed using levels of TAS were detected in variant carrier groups. For two-tailed t tests. Comparison between more than 2 other parameters, no differences were noted by genotype. groups was performed by one-way analysis of variance In the whole population, genotype distribution of the (ANOVA). Simple associations between variables were Mn-SOD Ala16Val (CC/CT/TT: 92/172/103, χ   =  1.40, calculated as the Pearson correlation. p = 0.24) and GPx1 Pro198Leu (CC/CT/TT: 180/152/35, Logistic regression was used to evaluate the effect χ   =  0.13, p  =  0.72) variants satisfied the Hardy–Wein - of MnSOD genotypes and activity, after adjusting for berg equilibrium (p  >  0.05) with a C allele frequency of other potential confounders such as age, gender, BMI 48.50  % and 69.75  % respectively for MnSOD and GPx1 and smoking status. p  <  0.05 was required for statistical genes (Table 1). significance. Relationship between gene polymorphisms and CHD, Empirical power to detect the association provided by as well as CHD risk estimation, were assessed by compar- our sample was computed using an R script with 10,000 ing Mn-SOD Ala16Val and GPx1 Pro198Leu genotype distribution among cases and controls (Tables 2, 3). Taking into account the significant difference detected between Mn-SOD genotype groups associated to some risk factors (Table  1), genotype distribution in control and case groups was studied by gender (Table  2). Several compounds proposed to act as antioxidants (AOX) in vivo [27] were considered in our study. We found a significant decrease in direct and total bilirubin in both male and female cases. Moreover, a significant difference at iron level was observed between controls and cases in the male group. However, in female group, an increase of uric acid was noted. No significant difference at albumin level Fig. 2 Restriction fragments of GPx1 gene amplified products. Lane 1 was observed between cases and controls of male and DNA molecular weight marker (50 pb); lane 2 negative control; lane 3 female groups. SOD activity and TAS were measured in Pro/Pro wildtype genotype; lanes 4 and 6 Leu/Leu mutated homozy- both control and CHD groups. In harmony with the first gous genotype; lane 5 Pro/Leu mutated heterozygous genotype results obtained in the whole population after genotype Souiden et al. Biol Res (2016) 49:22 Page 5 of 12 Table 1 Basic characteristics of the study population by genotype Trait SOD genotype GPx genotype Ala/Ala (CC) Ala/Val (CT) Val/Val (TT) P Pro/Pro (CC) Pro/Leu (CC) Leu/Leu (TT) P (N = 92) (N = 172) (N = 103) (N = 180) (N = 152) (N = 35) Age (years) 60.47 ± 10.88 62.42 ± 11.98 61.51 ± 11.99 0.43 61.57 ± 11.59 62.42 ± 12.24 63.37 ± 10.10 0.64 Body mass index 25.90 ± 4.08 26.26 ± 3.72 26.42 ± 3.49 0.61 26.02 ± 3.63 26.45 ± 3.77 26.17 ± 4.25 0.58 (kg/m ) Male, n (%) 61 (66.30 %) 88 (51.16 %) 67 (65.05 %) 0.02 107 (59.44 %) 91 (59.87 %) 18 (51.43 %) 0.64 Hypertension, 22 (23.91 %) 43 (25.0 %) 31 (30.10 %) 0.55 50 (27.78 %) 37 (24.34 %) 9 (25.72 %) 0.77 n (%) Hyperlipidemia, 16 (17.39 %) 27 (15.70 %) 29 (28.15 %) 0.03 26 (14.45 %) 22 (14.47 %) 10 (28.75 %) 0.09 n (%) Diabetes mellitus, 14 (15.22 %) 20 (11.62 %) 25 (24.27 %) 0.02 30 (16.67 %) 23 (15.13 %) 6 (17.14 %) 0.92 n (%) Current Smokers, 47 (51.08 %) 86 (50.0 %) 49 (47.57 %) 0.88 86 (47.78 %) 78 (51.32 %) 18 (51.43 %) 0.79 n (%) Plasma glucose 5.71 ± 3.68 5.25 ± 2.26 5.62 ± 2.15 0.33 5.70 ± 3.27 5.24 ± 2.51 5.22 ± 2.68 0.32 (mmol/l) LDL cholesterol 2.52 ± 1.09 2.46 ± 1.02 2.48 ± 1.08 0.91 2.68 ± 1.03 2.42 ± 0.89 2.73 ± 1.13 0.04 (mmol/l) Triglycerides 1.57 ± 0.66 1.63 ± 0.70 1.66 ± 0.63 0.64 1.65 ± 0.58 1.64 ± 0.74 1.67 ± 0.66 0.97 (mmol/l) HDL cholesterol 1.32 ± 0.72 1.45 ± 0.74 1.30 ± 0.69 0.18 1.30 ± 0.75 1.40 ± 0.71 1.32 ± 0.72 0.52 (mmol/l) CRP (mg/l) 1.50 ± 0.34 1.61 ± 0.44 1.58 ± 0.46 0.13 1.80 ± 0.53 1.73 ± 0.38 1.87 ± 0.66 0.22 Non enzymatic antioxidant parameters Uric Acid 323.13 ± 120.6 313.69 ± 108.9 314.83 ± 132.6 0.82 319.4 ± 123.7 329.5 ± 123.5 323.1 ± 120.5 0.76 (µmol/l) Total bilirubin 14.12 ± 6.57 13.32 ± 6.52 13.16 ± 7.20 0.56 12.88 ± 6.90 13.50 ± 6.70 13.07 ± 6.32 0.71 (µmol/l) Direct bilirubin 5.34 ± 2.39 5.91 ± 2.09 5.53 ± 2.25 0.11 5.16 ± 2.14 5.37 ± 2.02 4.77 ± 2.71 0.28 (µmol/l) Albumin (g/l) 42.46 ± 6.28 43.06 ± 4.47 41.88 ± 7.33 0.3 40.89 ± 5.99 41.44 ± 4.65 43.15 ± 10.47 0.14 Iron (µmol/l) 18.35 ± 5.07 17.34 ± 5.37 18.34 ± 6.32 0.23 18.58 ± 5.65 17.33 ± 4.91 18.07 ± 7.10 0.12 Enzymatic antioxidant parameters −4 SOD activity 1392.6 ± 219.3 1285.3 ± 188.9 979.4 ± 220.4 <10 – – ‒ ‒ (U/gHb) GPx activity – – – – 40.22 ± 12.79 42.91 ± 13.17 39.01 ± 11.97 0.1 (U/gHb) TAS (mmol/l) 1.51 ± 0.27 1.53 ± 0.31 1.42 ± 0.33 0.006 1.54 ± 0.29 1.50 ± 0.28 1.41 ± 0.35 0.002 *P < 0.05 was required for statistical significance stratification (Table  1), significant differences were the mutation can be overestimated. As shown in Table 2, detected in male and female groups between cases and compared with controls, men with CHD had increased controls. In line with these results, comparison of MnSOD age, higher prevalence of diabetes and hypertension, and genotype distribution by CHD status and gender, enables elevated LDL level. Adjustment for the last possible con- us to detect a significant difference between controls and founders by a multivariate regression analysis demon- cases of male group (controls vs cases: 31.93/45.38/22.69 strated that the association persisted (Ala carriers vs Val/ vs 23.71/37.11/39.18  %, p =  0.03). The statistical analysis Val, adjusted OR 1.89; 95 % CI 1.18‒3.42, p = 0.03). showed an association between the MnSOD polymor- Association between GPx polymorphism and CHD phism and the risk of CHD among men (Ala carriers vs risk was examined. Baseline characteristic stratification Val/Val, OR 2.19, 95 % CI 1.21‒3.97, p = 0.009). according to GPx genotype showed no differences in clin - Since CHD is a multifactor pathology, the association ical, anthropometric and biochemical parameters except of Val variant to the risk of CHD may be influenced by for LDL. An elevation of LDL level was noted among a number of risk factors. Accordingly, the significance of the TT genotype group. Moreover, significant decreases Souiden et al. Biol Res (2016) 49:22 Page 6 of 12 Table 2 Basic characteristics of the control and case groups by gender Trait Males (N = 216) Females (N = 151) Controls (N = 119) Cases (N = 97) P Controls (N = 84) Cases (N = 67) P −4 Age (years) 58.68 ± 12.01 64.15 ± 10.71 <10 63.38 ± 12.16 61.28 ± 11.00 0.27 Body mass index (kg/m ) 24.80 ± 3.41 25.23 ± 2.79 0.32 25.64 ± 3.68 24.93 ± 3.66 0.24 −4 Hypertension, n (%) 0 (0.0 %) 65 (67.01 %) <10 13 (15.48 %) 18 (26.87 %) 0.09 −4 Diabetes mellitus, n (%) 0 (0.0 %) 39 (58.21 %) <10 10 (11.90 %) 20 (29.85 %) 0.006 Plasma glucose (mmol/l) 4.56 ± 0.71 5.02 ± 3.09 0.12 4.37 ± 0.70 4.71 ± 2.13 0.17 −4 LDL cholesterol (mmol/l) 2.55 ± 1.24 3.13 ± 1.14 5E−04 2.54 ± 1.14 3.49 ± 1.49 <10 Triglycerides (mmol/l) 1.24 ± 0.73 1.21 ± 0.65 0.75 1.33 ± 0.47 1.47 ± 0.92 0.23 HDL cholesterol (mmol/l) 0.83 ± 0.22 0.84 ± 0.27 0.76 0.82 ± 0.44 0.79 ± 0.26 0.62 Non enzymatic antioxidant parameters Uric Acid (µmol/l) 332.3 ± 92.90 342.38 ± 154.27 0.55 285.21 ± 83.71 326.27 ± 138.95 0.03 −4 −4 Total bilirubin (µmol/l) 18.39 ± 4.21 8.11 ± 4.58 <10 15.89 ± 5.49 7.62 ± 5.04 <10 −4 −4 Direct bilirubin (µmol/l) 7.93 ± 3.14 2.71 ± 1.95 <10 6.83 ± 3.72 2.07 ± 1.74 <10 Albumin (g/l) 41.72 ± 4.23 40.65 ± 4.30 0.07 41.75 ± 7.83 40.95 ± 4.10 0.45 −4 Iron (µmol/l) 18.47 ± 4.54 16.20 ± 3.90 10 17.93 ± 5.41 16.60 ± 3.10 0.075 Enzymatic antioxidant parameters −4 −4 SOD activity, (U/gHb) 1321.4 ± 204.8 1089.2 ± 219.7 <10 1463.8 ± 233.4 1166.5 ± 213.9 <10 −4 −4 TAS (mmol/l) 1.63 ± 0.271 1.40 ± 0.232 <10 1.70 ± 0.267 1.38 ± 0.289 <10 SOD genotypes (%), n (CC/CT/TT ) 31.93/45.38/22.69 % 23.71/37.11/39.18 % 0.03 17.86/55.95/26.19 % 23.88/52.24/23.88 % 0.66 38/54/27 23/36/38 15/47/22 16/35/16 *P < 0.05 was required for statistical significance in TAS levels but not in GPx activities was discerned In the light of our first findings of high LDL levels among T carriers (Table  1). Based on these findings, we among case group, the relationship between MnSOD/ explored the association between T allele, lipid and anti- GPx1 activities, genotype and lipid parameters was stud- oxidant parameters further after stratifying by CHD sta- ied in a group of 106 subjects who underwent coronary tus (Table  3). On one hand, results observation revealed angiography (Table 4). a significant increase of plasma LDL level among T car - CHD group was classified into four subgroups accord - riers in the cases group compared to the control one, on ing to the number of affected coronary arteries (Table  4). the other, a significant decrease in total and direct biliru - Plasma levels of lipid parameters tended to be increased bin, iron and TAS level but not in GPx activity was noted with more severe coronary atherosclerosis. Lower SOD in the same group (p  <  0.05). However, we observed no activity was significantly associated with the severity of difference in genotype distribution between CHD and CHD (p  <  0.05). We noted graded reduced SOD activi- control groups (Controls vs CHD 55.49/37.80/6.71  %; ties in patients presenting 1–3 vessel stenosis. However, 43.84/44.34/11.82  %, p  =  0.051). Statistical analysis no differences in GPx activities were observed between showed a lack of association of GPx1 Pro198Leu poly- stenosis subgroups (p = 0.53). morphism with CHD risk (Pro carriers vs Leu/Leu, OR Pearson correlation study between MnSOD activity 0.54, 95 % CI 0.25‒1.13, p = 0.10). and lipid parameters showed a positive correlation with The assessment of the power provided by our sample size high density lipoprotein (HDL) (r  =  0.143, p  =  0.028), to detect allele association was performed for each vari- and a negative correlation with low density lipoprotein ant. Given a sample size of 164 control subjects and 203 (LDL) (r = −0.210, p = 0.034). Nonetheless, no correla- patients with CHD, our study had an empirical power of 30 tion between the two parameters and GPx activity was and 43 % respectively for MnSOD Ala16Val and GPx1 Pro- observed. 198Leu polymorphisms. Whereas, the current study had a Exploration of the relationship between enzyme activi- theoretical power of 97  % to detect an OR of 1.80 associ- ties and genotypes through CHD pathogenesis showed, ated with each variant, assuming an additive effect of alleles in contrast to GPx activity, an association of decreased and with a 5 % type 1 error rate. In this sample, a size effect SOD activity to the frequent presence of variant geno- of approximately 1.52 is detectable with 80 % power. type (Val/Val) as increased stenosed vessel number. Souiden et al. Biol Res (2016) 49:22 Page 7 of 12 Table 3 GPx polymorphism and  parameter stratification dichotomous age (p = 0.012), hyperlipidemia (p = 0.027) by CHD status effect of the Mn-SOD Ala16Val polymorphism on CHD severity. Trait Controls (203) Cases (164) P Age (years) 60.63 ± 12.27 62.98 ± 11.89 0.07 Discussion −4 Body mass index 24.73 ± 3.52 28.05 ± 3.17 <10 A growing body of evidence supports the concept that (kg/m ) oxidative stress plays critical roles in the initiation and Male, n (%) 119 (58.62 %) 97 (59.15 %) 0.92 progression of numerous diseases including atheroscle- −4 Hypertension, n (%) 13 (6.40 %) 96 (58.54 %) <10 rosis [28]. Since the vulnerability to oxidative stress is −4 Diabetes mellitus, n (%) 10 (4.93 %) 59 (35.98 %) <10 partly determined by genetic background, there have Plasma glucose 4.33 ± 0.71 4.47 ± 2.91 0.51 been several studies examining the association between (mmol/l) −4 functional gene polymorphisms of the key enzymes of LDL cholesterol 2.54 ± 1.12 3.28 ± 1.13 <10 redox regulation and the risk of CAD. (mmol/l) To our knowledge, this is the first study to investi - HDL cholesterol 0.83 ± 0.37 0.82 ± 0.27 0.77 (mmol/l) gate the relationship between Mn-SOD/GPx1 poly- Triglycerides (mmol/l) 1.24 ± 0.64 1.31 ± 0.77 0.34 morphisms, and CHD risk and severity in a Tunisian Non enzymatic antioxidant parameters population. MnSOD and GPx1 genotype distributions Uric Acid (µmol/l) 312.96 ± 92.00 335.82 ± 147.99 0.07 in control and patient groups were examined. The −4 Total bilirubin (µmol/l) 17.04 ± 4.73 7.68 ± 4.81 <10 comparison of genotypic frequencies in our control −4 Direct bilirubin 7.34 ± 3.21 2.36 ± 1.08 <10 group (TT genotype: 26.11 and 9.54  %, respectively) (µmol/l) with those reported in other studies showed that our Albumin (g/l) 41.74 ± 6.05 40.78 ± 4.18 0.09 results agree with those of the Caucasian population −4 Iron (µmol/l) 18.23 ± 4.94 16.38 ± 3.47 <10 (26.11 and 9.54 %, respectively) [29–31] but differ from Enzymatic antioxidant parameters those reported in Asian (66–79.4 and 7–13  %, respec- GPx activity, (U/gHb) 42.17 ± 13.99 40.20 ± 12.25 0.16 tively), Afro-Caribbean (42.9 and 18  %, respectively) −4 TAS (mmol/l) 1.68 ± 0.26 1.37 ± 0.25 <10 and oriental (77.8 and 0.0  %, respectively) population GPx genotypes (%), 43.84/44.34/11.82 % 55.49/37.80/6.71 % 0.051 [21, 31–39]. This clear difference, mostly related to T n (CC/CT/TT ) 89/90/24 91/62/11 0.1 allele, is evident, suggesting that this allele may have a GPx genotypes (%), 88.18/11.82 % 93.29/6.71 differential role in the disease process in these ethni - n (CC + CT/TT ) 179/24 153/11 cally distinct populations. In fact, in the studied control *P < 0.05 was required for statistical significance group, T allele frequencies of MnSOD and GPx1 genes were equal to those mentioned in previous studies of Caucasian population (49  % [48–51  %] and 34  % [31– Multivariate regression analysis stratified by gender 36  %], respectively), but unlike those of Asian popula- and adjusted according to age, body mass index hyperli- tion (13  % [5–21  %] and 23  % [18–29  %], respectively) pidimia and smoking habits as covariates, confirmed the [20, 31, 40–42]. Table 4 Lipid parameters, enzymatic antioxydant activity/polymorphism and CHD severity Parameters 0-vessel (n = 13) 1-vessel (n = 42) 2-vessels (n = 35) 3-vessels (n = 16) Total cholesterol (mmol/L) 4.11 ± 1.03 4.26 ± 1.07 4.59 ± 1.03 5.36 ± 1.41* HDL-cholesterol (mmol/L) 0.86 ± 0.28 0.84 ± 0.26 0.81 ± 0.29 0.79 ± 0.23 LDL-cholesterol (mmol/L) 3.02 ± 0.93 3.15 ± 1.01 3.49 ± 1.04 4.19 ± 1.39* Triglycerides (mmol/L) 1.14 ± 0.63 1.27 ± 0.65 1.34 ± 0.97 1.89 ± 1.00* , ‡ SOD activity, (U/gHb) 1246.9 ± 242.15 1112.4 ± 141.37 1065.6 ± 194.28 864.3 ± 229.08** SOD genotypes, n (%), (CC/CT/TT ) 4/6/3 10/21/11 7/18/10 3/7/6 (30.77/46.15/23.08) (23.81/50.0/26.19) (20.0/51.43/28.57) (18.75/43.75/37.5) GPx activity, (U/gHb) 38.90 ± 7.89 44.09 ± 11.32 42.94 ± 11.15 43.65 ± 12.64 GPx genotypes, n (%), (CC/CT/TT ) 7/8/1 22/17/3 21/12/2 9/6/1 (53.85/38.46/7.69) (52.38/40.47/7.14) (60.0/34.29/5.71) (56.26/37.5/6.25) * P < 0.05 compared with 0-vessel disease and P = 0.001 with 1-vessel disease ** P < 0.001 compared with 0-vessel disease and with 1-vessel disease and P = 0.002 with 2-vessels disease  P < 0.001 compared with 0-vessel disease, 1-vessel disease and 2-vessels disease Souiden et al. Biol Res (2016) 49:22 Page 8 of 12 Given their role in antioxidant defense, SOD2 and in disagreement with our data, other studies performed GPx1 genes are considered as attractive low penetrance on Asian population demonstrated an association of candidate genes for CHD. In this study, we examined GPx1 variant genotypes to CHD risk and carotid artery the impact of SOD2 Ala16Val and GPx1 Pro198Leu intra-media thickness (IMT). In a Japanse population, polymorphisms in CHD risk and severity. Our results Haminichi et al. [21] and Oguri et al. [39] suggested that indicated that the Val/Val genotype of MnSOD envis- functional variants in GPx1 gene are associated with ages in Tunisian men higher risk to CHD as compared increased carotid IMT of cardiovascular and peripheral to controls (Table  2). In fact, Val/Val genotype was sig- vascular diseases in type 2 diabetic patients. Further- nificantly more common among case (39.18  %) than more, data of a study carried out by Tang et al. [48], pro- control (22.69  %) men. Accordingly, we note that SOD2 vide evidence that GPx1 Pro198Leu variant genotypes polymorphism has a significant influence on CHD risk in are significantly associated with CAD risk in a Chinese Tunisian men (Ala carriers vs Val/Val, adjusted OR 1.89, population. In fact, compared to the 198Pro/Pro car- 95 % CI 1.18‒3.42, p = 0.03). Furthermore, the observed riers, subjects with the variant genotypes (198Pro/Leu difference between CHD and control men is unlikely to and 198Leu/Leu) had a significantly higher risk of CAD be the result of biased population sampling. There was (adjusted OR 2.02, 95 % CI 1.27–3.22). In the same way, no significant difference in our frequency estimation of a recent meta-analysis confirms the association of GPx1 the Val allele (45.38 %) from the Tunisian men of control variants with CAD in people with type 2 diabetes mel- group than those of the Caucasian population (44–48 %) litus of Chinese and Indian populations [49]. reported in other studies [29, 30]. The opposing findings of these reports may be attrib - Our findings concur with those reported by two recent utable to inaccurate genotype frequency estimates result- meta-analyzes in which Crawford et  al. [28] concluded ing from small sample sizes, selection of control group, that the Val/Val or Val allele has been positively associ- case characterization linkage disequilibrium between ated with cardiovascular disease risk and with comor- SOD2 and/or GPx1 alleles with different functionally rel - bidities in type 1 (T1DM) and type 2 diabetes mellitus evant polymorphisms in specific ethnic groups and gene- (T2DM) patients, and Tian et  al. [43] showed a signifi - environment interactions. Further variations in genetic cant association of the C allele with reduced risk of CAD. background, dietary habits and/or environmental factors Furthermore, in agreement with our data, Valenti et  al. between different populations might account for these [44] reported an increased risk of cardiomyopathy in findings. hemochromatosis patients with Val allele compared with In this study, in addition to polymorphism association Ala allele. Evenly, Fujimoto et al. [45] found that individu- to CHD risk, we investigated the SOD and GPx activi- als harboring the valine variant were at increased risk for ties in CHD subjects. Consistent with our results some CAD and acute myocardial infraction. Moreover, in a studies suggested an association between SOD activ- large study of 776 Caucasian subjects with diabetes, Jones ity [34] but not GPx activity [31, 46, 47] with cardiopa- et al. [34] showed an increased risk of CHD associated to thy events. In contrast, others proposed the opposite Val/Val genotype. Besides, in 24 of 100 patients recruited [50–52]. We have observed a significant decrease in the with severe heart failure, Charniot et  al. [46] revealed a SOD activity in patients with CHD, as compared to con- significant correlation of the Val-encoding MnSOD allele trols. We confirmed known fact that erythrocyte SOD with the severity and prognosis of cadiogenic shock. activity is significantly reduced in patients with CHD [51, In contrast to SOD2 polymorphism, previous stud- 53–55]. Lower enzyme activity could be a consequence ies on the association between CHD and GPx1 poly- of the increased oxidative stress induced by the coronary morphism have been highly inconsistent. Polymorphic events. It may be the leading cause of post-translational variation in GPx1 gene at position 198 is associated in covalent modifications in SOD e.g. nitration, phospho - some, but not in all studies. Our observations suggest rylation, glutathionylation and glycation, which results in that Pro198Leu GPx1 polymorphism was not associated decreased enzyme activity [56]. to CHD risk. In line with our findings, previous Cau - Our results also showed no differences in GPx activ - casian population studies revealed no significant asso - ity between genotype classes and according to CHD sta- ciation between allele frequency and risk to suffer from tus. Indeed, normal GPx activity was measured in Leu CAD. Indeed, in a French population, Charniot et  al. carriers. These findings can be explained by the GPx [46] declared that GPx—variants influenced neither GPx upreglation under oxidative stress conditions and/or the activity nor cardiac events. Moreover, in a recent study presence of a compensatory mechanism in other cells of of Iranian population, Najafi et al. [47] showed that GPx1 the body with faster turnover. In addition, through an activity and rs1050450 (Pro198Leu) site are not involved analysis of the predicted tertiary structures, Jones et  al. in the development of coronary artery stenosis. However, [34] provided another possible explanation. They showed Souiden et al. Biol Res (2016) 49:22 Page 9 of 12 that the polymorphic variants have no essential role on allele was significantly correlated with the severity and protein stability and function. They suggested that this prognosis of cardiogenic shock [46]. may be due to their surface location. Predisposing association of the Val allele and high LDL- Antioxidant status of the study population was further cholesterol levels in subjects with two or three stenosis explored by measurement of some non-enzymatic anti- vessels, showed that high LDL levels are more harmful oxidants (AOX) and total antioxidant status (TAS). Sig- in subjects with a diminished antioxidant capacity of nificant decreases in both total and direct bilirubin as MnSOD. Consistent with our data, a number of stud- well as in iron against no differences in albumin and uric ies reported an association between antioxidant enzyme acid levels, were noted in cases compared to controls. activities and progression of stenosis [45, 46, 59, 70, 72]. Taken together, these non-enzymatic antioxidant (AOX) This could be explained on the one hand, by the decline constitute an important aspect of a network essential resistance against ROS produced in the mitochondria for assessing in  vivo AOX status [57]. In our popula- due to the Val isoform of the SOD2, and on the other tion, their reduced values reflect a state of an imbalance hand, it might be due to SOD activity inhibition by lipid between AOX and pro-oxidants and/or free radicals. The peroxidation products [73, 74]. In this context, Botto assessment of the TAS of a biological fluid is a compos - et al. [75] have reported elevated levels of oxidative DNA ite measurement of the combined effects of individual damage in patients with angiographically documented scavenging AOX within the sample and providing insight CAD. In addition, it has been reported that isoprostanes, into the overall prooxidant-antioxidant balance [58]. In markers of lipid peroxidation, and reduced antioxidant our population, a global evaluation of the TAS showed capacity are related to increased risk for cardiovascular a significantly reduced levels in CHD subjects compared disease [62, 76]. to controls. Our result is in agreement with that of Nojiri Since atherosclerosis is a complex process affected by et  al. [59] but not with those of Alamdari et  al. [60] and a network of numerous genes and environmental fac- Rahsepar et  al. [61] who found that the level of prooxi- tors [77–80] and given that the effect of the C47T poly - dant/antioxidant balance (PAB) in patients with stable morphism in MnSOD may vary by exogenous sources of CAD was significantly higher compared to that measured antioxidants and oxidants, multivariate regression was in healthy control subjects. studied in order to identify eventual confounders such as Low TAS levels could reflect either high oxida - age, gender, BMI and smoking status. tive stress or decrease of defense against it. In CHD Multivariate analysis of variance revealed that patients, even in stable cases, a high oxidative stress Ala16  Val polymorphism and low Mn-SOD activity may status has been reported [62–65]. For instance, circulat- be independently associated with CHD severity, a state of ing oxidized low density lipoprotein levels are positively progressive atherosclerosis, documented by the number associated with severity of acute coronary syndromes of vessel stenosis. Our findings point out the main role of [66, 67] and with subclinical CHD [68]. In line with oxidative stress in atherosclerosis. these findings, in our population, the CHD severity, The strong points of the present report are this is the as demonstrated by the number of vessel stenosis, was first study to show the effect of the Ala16Val and Pro - associated with high LDL levels. The observed posi - 198Leu polymorphisms on CHD risk in a Tunisian popu- tive correlation between atherosclerosis progression lation, the uniform mechanism of CHD diagnosis for all and high levels of LDL-cholesterol in CHD patients cases and the homogeneity of our population (all subjects was associated with the SOD2-Val/Val genotype. This are originated from central Tunisia). Nevertheless, the has not been confirmed by another study focused on main limitation of this study is the relatively small sample the role of antioxidant enzymes in determining genetic size. Although there is a significant relationship between susceptibility to the coronary artery disease in patients Ala16Val polymorphism and CHD; low statistical power with T2DM [69]. Nevertheless, Kakko et  al. [70] of the current study is our another limitation. However, reported that carotid artery intima-media thickness, the finding of an association of the Ala16Val MnSOD was greater in women with the Val allele and high levels polymorphism with CHD despite the low power means of low-density lipoprotein (LDL) cholesterol (p = 0.03). that this association is real. Indeed, with insufficient Similarly, in patients with oxidised LDL  <0.5  nmol/ power, we may miss the true association but not to find mg, Gottilieb et  al. [71] revealed an association of Ala/ the one that does not exist. Val and Val/Val genotypes with increased levels of oxi- dised LDL compared with Ala/Ala genotype in the same Conclusion group. Furthermore, in a cohort of patient with car- Although small statistical power of the current study, we diogenic shock due to dilated cardiomyopathy without have demonstrated that in contrast to 198Leu variant, the acute coronary syndrome, the Val-encoding MnSOD 16Val variant was associated with CHD risk in men and Souiden et al. Biol Res (2016) 49:22 Page 10 of 12 enhances myocardial blood flow and oxygen consumption in postisch- the severity of cardiovascular events in the Tunisian pop- emic mouse heart. J Pharmacol Exp Ther. 2008;327:402–10. ulation. Moreover, the SOD activity was inversely related 11. Church SL, Grant JW, Meese EU, Trent JM. Sublocalization of the gene to CHD progression as documented by the number of encoding manganese superoxide dismutase (MnSOD/SOD2) to 6q25 by fluorescence in situ hybridization and somatic cell hybrid mapping. vessel stenosis. 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Urinary 8-iso-prostaglandin F2alpha as a risk marker in patients with coronary heart disease: a matched case-control study. Circulation. 2004;109:843–8. Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Biological Research Springer Journals

MnSOD and GPx1 polymorphism relationship with coronary heart disease risk and severity

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Springer Journals
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Copyright © 2016 by Souiden et al.
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Life Sciences; Life Sciences, general; Cell Biology; Neurosciences; Developmental Biology; Microbiology; Plant Sciences
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10.1186/s40659-016-0083-6
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27067415
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Abstract

Background: Disturbance of the equilibrium between reactive oxygen species (ROS) and anti-oxidants (AOX) has been implicated in various diseases, including atherosclerosis, the most common pathologic process underlying coronary heart disease (CHD). Thus, the defense systems against ROS are critical protecting blood vessel walls against oxidative damage. In this study, we investigate whether Ala16Val MnSOD and Pro198Leu GPx polymorphisms are associated with CHD susceptibility and/or severity. Methods: Both polymorphisms were genotyped in a sample of 203 controls and 164 patients. CHD risk and severity, antioxidant status (enzymatic and/or non enzymatic) and biochemical parameters were assessed and analysed by genotype. Results: A significant association of MnSOD variant to CHD risk was revealed in males. Males harboring the Val/Val genotype were approximately at twofold increased risk of CHD compared to controls (Ala carriers vs Val/Val, adjusted OR 1.89; 95 % CI 1.18‒3.42, p = 0.03). Significant decreases in SOD activity and total antioxidant status ( TAS) were observed in Val carriers and by CHD status. Whereas, no association of GPx variant genotype (Leu/Leu) and activity to cardiopathy events was discerned. CHD severity, as demonstrated by the number of vessel stenosis, was associated with significantly higher frequency of Val allele and LDL levels in CHD subjects. Conclusions: Our results showed a lack of association of Pro198Leu GPx polymorphism to CHD risk and severity. However, they suggest that Ala16Val MnSOD polymorphism and decreased antioxidant defences are likely contrib- uted to CHD risk in Tunisian men. Furthermore, the Val encoding MnSOD allele and decreased SOD activity were significantly correlated with CHD stenosis progression. Keywords: Genetic polymorphism, Coronary heart disease, SOD activity, GPx activity, Total antioxidant status, Atherosclerosis Atherosclerosis, the most common pathologic process Background underlying coronary heart disease, represents a state of Coronary Artery Disease is the major cause of mortality heightened oxidative stress characterized by endothelial and morbidity worldwide [1]. It is independently asso- dysfunction and plaque disruption. Oxidative stress can ciated with various risk factors such as advanced age, occur when the balance is upset, either by an excessive hypertension, smoking habit, diabetes mellitus, hyper- production of reactive oxygen species (ROS), by deficient lipidemia, positive family story, obesity and inactivity [2]. antioxidant defenses, or by the combination of both [3]. In such circumstances, ROS may interact with cellular *Correspondence: [email protected] bio-molecules, leading to modification and potentially Foued Chéour and Kacem Mahdouani contributed equally to this work Laboratory of Biochemistry and Molecular Biology, Hospital of Ibn serious consequences for the cell [4]. Eljazzar of Kairouan, Avenue Ibn Eljazzar, 3140 Kairouan, Tunisia Full list of author information is available at the end of the article © 2016 Souiden et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Souiden et al. Biol Res (2016) 49:22 Page 2 of 12 Several potentially significant genetic variants related The aim of our study was to assess the association of to oxidative stress have been already identified [5, 6]. In MnSOD and GPx polymorphisms and activities with this study, we are interested to polymorphisms of the CHD risk and severity in the Tunisian population. superoxide dismutase (SOD) and glutathione peroxi- dase (GPx) in order to check their possible implication in Methods CHD risk and severity. Study population SOD is the primary antioxidant in the mitochondria One hundred and sixty-four patients with coronary heart that converts ROS into oxygen and hydrogen perox- disease (CHD) and two hundred and three controls were ide [7–10]. There are three SOD isoforms, including the enrolled in this study from June to September 2011. The mitochondrial SOD manganese dependent (MnSOD). healthy subjects were recruited at their annual health MnSOD is encoded by a single gene containing five examination at Hospital Ibn Eljazzar, Kairouan, Tunisia, exons and it is located on chromosome 6q25 [11]. One and did not have any chest symptoms or electrocardio- of the common polymorphisms of MnSOD results in the gram (ECG) abnormalities suggesting CHD, or a medical replacement of alanine 16 (GCT) with a valine (GTT); history for CHD. the Ala16Val polymorphism. This polymorphism affects Patients were admitted to the cardiovascular depart- the import of MnSOD into the mitochondria by altering ment of Hospital Ibn Eljazzar, Kairouan, Tunisia. The the conformation of its leader signal [12]. This mutation diagnosis of Acute CHD was based on the presence of at may reflect a functional polymorphism of mitochondrial least 2 of the following three elements: (1) Ischemic type transport of human MnSOD. It has been shown that the of chest pain (2) changes on serial electrocardiogram 16Ala variant allows efficient targeting of MnSOD to (ECG) tracings (3) Increase in serum cardiac marker the mitochondria, as evidenced by Sutton et al. [13] who [elevated creatine kinase isoenzyme MB (CK-MB) and found that it was 30–40  % more efficiently localized to troponin T within 12  h after the onset of pain]. Patients the mitochondria than the 16Val variant. This is related with associated renal failure, liver disease, lung disease, to the fact that 16Ala variant has an α-helix structure pregnancy, thyroid disease, gastrointestinal disease were that is easily imported and it reaches high levels of mito- excluded. The study protocol was approved by the local chondrial concentration and activity, whereas the 16Val Ethics Committee of the hospital Ibn Eljazzar. Written variant has a partial ß-sheet structure that is partly stuck informed consent was obtained from all subjects before within the narrow inner membrane import pore and is their participation in the study. subsequently degraded by the proteasome. Furthermore, All participants were interviewed, and data on hyper- the mRNA that encodes the 16Val variant is more rapidly tension, diabetes mellitus, dyslipidemia, medical history degraded than the Ala variant [14]. including family history, smoking status and duration of The second line of enzymatic antioxidant defense is CHD were recorded. For coronary risk factors the fol- played by glutathione peroxidase (GPx) isoenzymes. GPx lowing definitions were used: individuals were defined as has 6 isoforms (GPx-1-6) [15]. GPx-1 is the most widely hypertensive if their blood pressure was >140/90 mmHg distributed and abundant form in human cells, includ- or in case of self-reported use of an antihypertensive ing vascular endothelium [16]. GPx-1 is an intracellular drug. Diabetes mellitus was diagnosed if the fasting soluble selenoprotein which converts peroxides such as plasma glucose concentration was  ≥7  mmol/l or if the H2O2 and ROOH into water and alcohol [17]. The gene patient was treated with insulin or oral hypoglycemic coding for GPx-1, is located on chromosome 3p21.3 agents; individuals were deemed dyslipidemic when [18] and it is composed of 2 exons with a 1.42 kb region their total cholesterol concentration was ≥5.68  mmol/l, [19]. Several polymorphisms have been described in the or their triglyceride concentration was ≥2.28 mmol/l, or GPx-1 gene. One of them is located at codon 198 (C > T) they receive lipid-lowering drugs. Smoking was coded as resulting in an amino acid variation from proline (CCC) never and current smoker. Gender and body mass index to leucine (CTC). This amino acid substitution causes a (BMI) were also recorded. change of the structural conformation of the active site region and modifies the enzyme activity [20]. Previous Blood collection studies using aortic endothelial cells demonstrated a 40 % From all subjects, Blood samples were collected in hep- reduction in the GPx-1 activity being associated with arinized (5 ml) and EDTA (5 ml) tubes after 12 h fasting the T allele and showed that the Pro198Leu variant was and immediately centrifuged at 1600×g. After separation, associated with increased carotid intima-to-media thick- washed and lysed erythrocytes as well as plasma were ness, peripheral arterial disease, and increased CAD risk stored at −70  °C until biochemical measurements were [21–24]. performed. Souiden et al. Biol Res (2016) 49:22 Page 3 of 12 Biochemical analyses Genetic analyses Superoxide Dismutase (SOD) Assay Genomic DNA was isolated from 300  µl of periph- The proportioning of erythrocyte enzymatic activ - eral blood leukocytes using Promega DNA isolation kit ity superoxide dismutase (SOD) is based on velocity according to the manufacturer’s recommendations. An measurement of oxidation reaction inhibition of I.N.T. alanine/valine polymorphism in the signal peptide of Mn- (2-(4-Iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazo- SOD gene was evaluated by PCR–RFLP analysis accord- liumn chloride) by SOD. The role of SOD is to acceler - ing to the method described by Ibrahim et  al. [26]. PCR ate the dismutation of toxic superoxide radical (O2−) amplifications were performed in a total volume of 20 μl produced during oxidative energy processes to hydrogen containing 50 ng of genomic DNA, 1.25 U Taq polymer- peroxide and molecular oxygen. This method uses xan - ase, 2 mM dNTP, 2 mM MgCl and 1X PCR buffer in the thine and xanthine oxidase (XOD) to generate superoxide presence of 0.4  μmol/l of each primer (forward 5′-CAG radicals which react with I.N.T. to form a red formazan CCC AGC CTG CGT AGA CGG-3′ and reverse 5′-CTT dye. SOD activity is then measured by the degree of GGC CAA CGC CTC CTG GTA CTT-3′) to amplify a inhibition of this reaction. The results were expressed as 267-bp fragment. The PCR conditions involved an ini - U/g Hb. tial denaturation of DNA at 95  °C for 5  min, followed by 30 cycles of amplification at 95  °C for 45  s (melting), Glutathione peroxidase (GPx) assay 54 °C for 30 s (annealing) and 72 °C for 30  s, and a final The erythrocyte hemolysate GPx1 activity was assessed extension at 72  °C for 5  min. The resulting 267-bp PCR by Paglia and Valentine method [25] using the Ran- product was digested with the restriction endonuclease sel kit (Randox, Antrim, UK) based on the oxidation BsaW1 at 37  °C for 2  h according to the manufacturer’s reduction of glutathione and the measurement of the recommendations and digested products were visualized variations of reduced NADP. GPx catalysis the oxida- with electrophoresis in 2.5  % agarose gel stained with tion of glutathione (GSH) by cumene hydroperoxide. In ethidium bromide (0.5 µg/ml). Restriction enzyme diges- the presence of glutathione reductase and NADPH oxi- tion results in a 267-bp product (16Ala) or 183 and 84 bp dized glutathione (GSSG) is immediately converted to products (16Val) (Fig. 1). reduced form with a concomitant oxidation of NADPH The GPx1  198Pro/Leu variant was determined using to NADP+. The enzyme activity is measured at a wave - 5′-TCC AGA CCA TTG ACA TCG AG-3′ (forward) length of 340 nm. One unit of GPX activity is defined as and 5′-ACT GGG ATC AAC AGG ACC AG-3′ (reverse) the amount of the enzyme required for oxidizing 1 μmol primers. A 222 base pair DNA fragment containing of NADPH per minute. Results were expressed in units the polymorphic site was amplified in a total volume per gram of hemoglobin. of 20  μl, containing 2  μl 10xPCR buffer, 1.125  mmol/l Total antioxidant status (TAS) assay Total antioxidant status (TAS) is measured by the reduc- ®+ tion in color produced by a radical cation (ABTS ). The assay principle is as follow: the ABTS (2,2′-azino- di-[3-ethylbenzthiazoline sulphonate]) is incubated with metmyoglobin which has peroxidase activity and H O to 2 2 ®+ produce the radical cation ABTS . This has a relatively stable blue-green color measured at 600  nm. Antioxi- dants in the added sample cause suppression of this color production to a degree which is proportional to their concentration. Other analyses Measurements of other biochemical parameters tested during this study included heparinized plasma triglyc- Fig. 1 Restriction fragments of MnSOD gene amplified products. erides, cholesterol, HDL-Cholesterol, were detected by Lane1 DNA molecular weight marker (50 pb); lane 2 negative control; lanes 3 and 7 Val/Val mutated homozygous genotype; lanes 4 and an automate (Konelab 20, Thermo Clinical Labsystems 6 Ala/Ala wildtype genotype; lanes 5 and 8, Ala/Val heterozygous Oy, Finland). LDL-Cholesterol was calculated using the genotype Friedewald equation. Souiden et al. Biol Res (2016) 49:22 Page 4 of 12 MgCl2, 0.15  mmol/l dNTPs, 0.25  μmol/l each primer, permutations. Theoretical power was also calculated 100 ng of genomic DNA and 1.5 U of Taq DNA polymer- using Quanto and parameter values (Odds ratio, allele ase (Promega Madison, USA). The PCR amplification frequencies and sample size) equals to those found in our was performed in 35 cycles at 94  °C for 30  s, 59  °C for study. 30  s and 72  °C for 30  s, preceded by an initial denatura- tion at 94 °C for 8 min and followed by a final elongation Results step at 72 °C for 7 min. The amplicon was then digested Three hundred and sixty-seven subjects were investi - with ApaI (Promega Madison, USA) restriction enzyme gated in this study in order to evaluate the association and resolved in 2  % agarose gel. The digested products of the MnSOD Ala16Val and GPx1 Pro198Leu polymor- showed 2 fragments of 170 and 52  bp for the 198Pro phisms with the coronary heart disease (CHD). wildtype homozygous, 3 fragments of 222, 170 and 52 Characteristics of the enrolled subjects stratified for the 198Pro/Leu (CT) heterozygote, and 1 fragment according to MnSOD and GPx1 polymorphisms are of 222 pb for the 198Leu (T) mutated homozygous geno- illustrated in Table  1. Results observation showed a sig- type (Fig. 2). nificant difference between MnSOD genotype classes associated with male gender and diabetes mellitus. Statistical tests Whereas, a significant difference between GPx1 geno - Statistical analyses were performed using the Statistical type groups was noted only according to LDL level in the Package for the Social Sciences (SPSS) version 17.0 for study population. Windows (SPSS, Chicago, IL), R software version 3.0.2 Biochemical examination of our population was car- and QUANTO program version 1.2. The Chi square test ried out by testing several parameters (Table  1). Results was used to test the distribution of genotypes for devia- showed a significant difference between genotype groups tions from Hardy–Weinberg equilibrium. genotype dis- for antioxidant status. Indeed, SOD2 but not GPx1 activ- tribution of MnSOD and GPx1 gene in all subjects was ity decreased significantly in the presence of the mutated analyzed by Chi square test. Distributions of continuous allele (Val and Leu, respectively). The same is true for variables in groups were expressed as mean ± SD. total antioxidant status (TAS). In fact, significant reduced Comparison between two groups was performed using levels of TAS were detected in variant carrier groups. For two-tailed t tests. Comparison between more than 2 other parameters, no differences were noted by genotype. groups was performed by one-way analysis of variance In the whole population, genotype distribution of the (ANOVA). Simple associations between variables were Mn-SOD Ala16Val (CC/CT/TT: 92/172/103, χ   =  1.40, calculated as the Pearson correlation. p = 0.24) and GPx1 Pro198Leu (CC/CT/TT: 180/152/35, Logistic regression was used to evaluate the effect χ   =  0.13, p  =  0.72) variants satisfied the Hardy–Wein - of MnSOD genotypes and activity, after adjusting for berg equilibrium (p  >  0.05) with a C allele frequency of other potential confounders such as age, gender, BMI 48.50  % and 69.75  % respectively for MnSOD and GPx1 and smoking status. p  <  0.05 was required for statistical genes (Table 1). significance. Relationship between gene polymorphisms and CHD, Empirical power to detect the association provided by as well as CHD risk estimation, were assessed by compar- our sample was computed using an R script with 10,000 ing Mn-SOD Ala16Val and GPx1 Pro198Leu genotype distribution among cases and controls (Tables 2, 3). Taking into account the significant difference detected between Mn-SOD genotype groups associated to some risk factors (Table  1), genotype distribution in control and case groups was studied by gender (Table  2). Several compounds proposed to act as antioxidants (AOX) in vivo [27] were considered in our study. We found a significant decrease in direct and total bilirubin in both male and female cases. Moreover, a significant difference at iron level was observed between controls and cases in the male group. However, in female group, an increase of uric acid was noted. No significant difference at albumin level Fig. 2 Restriction fragments of GPx1 gene amplified products. Lane 1 was observed between cases and controls of male and DNA molecular weight marker (50 pb); lane 2 negative control; lane 3 female groups. SOD activity and TAS were measured in Pro/Pro wildtype genotype; lanes 4 and 6 Leu/Leu mutated homozy- both control and CHD groups. In harmony with the first gous genotype; lane 5 Pro/Leu mutated heterozygous genotype results obtained in the whole population after genotype Souiden et al. Biol Res (2016) 49:22 Page 5 of 12 Table 1 Basic characteristics of the study population by genotype Trait SOD genotype GPx genotype Ala/Ala (CC) Ala/Val (CT) Val/Val (TT) P Pro/Pro (CC) Pro/Leu (CC) Leu/Leu (TT) P (N = 92) (N = 172) (N = 103) (N = 180) (N = 152) (N = 35) Age (years) 60.47 ± 10.88 62.42 ± 11.98 61.51 ± 11.99 0.43 61.57 ± 11.59 62.42 ± 12.24 63.37 ± 10.10 0.64 Body mass index 25.90 ± 4.08 26.26 ± 3.72 26.42 ± 3.49 0.61 26.02 ± 3.63 26.45 ± 3.77 26.17 ± 4.25 0.58 (kg/m ) Male, n (%) 61 (66.30 %) 88 (51.16 %) 67 (65.05 %) 0.02 107 (59.44 %) 91 (59.87 %) 18 (51.43 %) 0.64 Hypertension, 22 (23.91 %) 43 (25.0 %) 31 (30.10 %) 0.55 50 (27.78 %) 37 (24.34 %) 9 (25.72 %) 0.77 n (%) Hyperlipidemia, 16 (17.39 %) 27 (15.70 %) 29 (28.15 %) 0.03 26 (14.45 %) 22 (14.47 %) 10 (28.75 %) 0.09 n (%) Diabetes mellitus, 14 (15.22 %) 20 (11.62 %) 25 (24.27 %) 0.02 30 (16.67 %) 23 (15.13 %) 6 (17.14 %) 0.92 n (%) Current Smokers, 47 (51.08 %) 86 (50.0 %) 49 (47.57 %) 0.88 86 (47.78 %) 78 (51.32 %) 18 (51.43 %) 0.79 n (%) Plasma glucose 5.71 ± 3.68 5.25 ± 2.26 5.62 ± 2.15 0.33 5.70 ± 3.27 5.24 ± 2.51 5.22 ± 2.68 0.32 (mmol/l) LDL cholesterol 2.52 ± 1.09 2.46 ± 1.02 2.48 ± 1.08 0.91 2.68 ± 1.03 2.42 ± 0.89 2.73 ± 1.13 0.04 (mmol/l) Triglycerides 1.57 ± 0.66 1.63 ± 0.70 1.66 ± 0.63 0.64 1.65 ± 0.58 1.64 ± 0.74 1.67 ± 0.66 0.97 (mmol/l) HDL cholesterol 1.32 ± 0.72 1.45 ± 0.74 1.30 ± 0.69 0.18 1.30 ± 0.75 1.40 ± 0.71 1.32 ± 0.72 0.52 (mmol/l) CRP (mg/l) 1.50 ± 0.34 1.61 ± 0.44 1.58 ± 0.46 0.13 1.80 ± 0.53 1.73 ± 0.38 1.87 ± 0.66 0.22 Non enzymatic antioxidant parameters Uric Acid 323.13 ± 120.6 313.69 ± 108.9 314.83 ± 132.6 0.82 319.4 ± 123.7 329.5 ± 123.5 323.1 ± 120.5 0.76 (µmol/l) Total bilirubin 14.12 ± 6.57 13.32 ± 6.52 13.16 ± 7.20 0.56 12.88 ± 6.90 13.50 ± 6.70 13.07 ± 6.32 0.71 (µmol/l) Direct bilirubin 5.34 ± 2.39 5.91 ± 2.09 5.53 ± 2.25 0.11 5.16 ± 2.14 5.37 ± 2.02 4.77 ± 2.71 0.28 (µmol/l) Albumin (g/l) 42.46 ± 6.28 43.06 ± 4.47 41.88 ± 7.33 0.3 40.89 ± 5.99 41.44 ± 4.65 43.15 ± 10.47 0.14 Iron (µmol/l) 18.35 ± 5.07 17.34 ± 5.37 18.34 ± 6.32 0.23 18.58 ± 5.65 17.33 ± 4.91 18.07 ± 7.10 0.12 Enzymatic antioxidant parameters −4 SOD activity 1392.6 ± 219.3 1285.3 ± 188.9 979.4 ± 220.4 <10 – – ‒ ‒ (U/gHb) GPx activity – – – – 40.22 ± 12.79 42.91 ± 13.17 39.01 ± 11.97 0.1 (U/gHb) TAS (mmol/l) 1.51 ± 0.27 1.53 ± 0.31 1.42 ± 0.33 0.006 1.54 ± 0.29 1.50 ± 0.28 1.41 ± 0.35 0.002 *P < 0.05 was required for statistical significance stratification (Table  1), significant differences were the mutation can be overestimated. As shown in Table 2, detected in male and female groups between cases and compared with controls, men with CHD had increased controls. In line with these results, comparison of MnSOD age, higher prevalence of diabetes and hypertension, and genotype distribution by CHD status and gender, enables elevated LDL level. Adjustment for the last possible con- us to detect a significant difference between controls and founders by a multivariate regression analysis demon- cases of male group (controls vs cases: 31.93/45.38/22.69 strated that the association persisted (Ala carriers vs Val/ vs 23.71/37.11/39.18  %, p =  0.03). The statistical analysis Val, adjusted OR 1.89; 95 % CI 1.18‒3.42, p = 0.03). showed an association between the MnSOD polymor- Association between GPx polymorphism and CHD phism and the risk of CHD among men (Ala carriers vs risk was examined. Baseline characteristic stratification Val/Val, OR 2.19, 95 % CI 1.21‒3.97, p = 0.009). according to GPx genotype showed no differences in clin - Since CHD is a multifactor pathology, the association ical, anthropometric and biochemical parameters except of Val variant to the risk of CHD may be influenced by for LDL. An elevation of LDL level was noted among a number of risk factors. Accordingly, the significance of the TT genotype group. Moreover, significant decreases Souiden et al. Biol Res (2016) 49:22 Page 6 of 12 Table 2 Basic characteristics of the control and case groups by gender Trait Males (N = 216) Females (N = 151) Controls (N = 119) Cases (N = 97) P Controls (N = 84) Cases (N = 67) P −4 Age (years) 58.68 ± 12.01 64.15 ± 10.71 <10 63.38 ± 12.16 61.28 ± 11.00 0.27 Body mass index (kg/m ) 24.80 ± 3.41 25.23 ± 2.79 0.32 25.64 ± 3.68 24.93 ± 3.66 0.24 −4 Hypertension, n (%) 0 (0.0 %) 65 (67.01 %) <10 13 (15.48 %) 18 (26.87 %) 0.09 −4 Diabetes mellitus, n (%) 0 (0.0 %) 39 (58.21 %) <10 10 (11.90 %) 20 (29.85 %) 0.006 Plasma glucose (mmol/l) 4.56 ± 0.71 5.02 ± 3.09 0.12 4.37 ± 0.70 4.71 ± 2.13 0.17 −4 LDL cholesterol (mmol/l) 2.55 ± 1.24 3.13 ± 1.14 5E−04 2.54 ± 1.14 3.49 ± 1.49 <10 Triglycerides (mmol/l) 1.24 ± 0.73 1.21 ± 0.65 0.75 1.33 ± 0.47 1.47 ± 0.92 0.23 HDL cholesterol (mmol/l) 0.83 ± 0.22 0.84 ± 0.27 0.76 0.82 ± 0.44 0.79 ± 0.26 0.62 Non enzymatic antioxidant parameters Uric Acid (µmol/l) 332.3 ± 92.90 342.38 ± 154.27 0.55 285.21 ± 83.71 326.27 ± 138.95 0.03 −4 −4 Total bilirubin (µmol/l) 18.39 ± 4.21 8.11 ± 4.58 <10 15.89 ± 5.49 7.62 ± 5.04 <10 −4 −4 Direct bilirubin (µmol/l) 7.93 ± 3.14 2.71 ± 1.95 <10 6.83 ± 3.72 2.07 ± 1.74 <10 Albumin (g/l) 41.72 ± 4.23 40.65 ± 4.30 0.07 41.75 ± 7.83 40.95 ± 4.10 0.45 −4 Iron (µmol/l) 18.47 ± 4.54 16.20 ± 3.90 10 17.93 ± 5.41 16.60 ± 3.10 0.075 Enzymatic antioxidant parameters −4 −4 SOD activity, (U/gHb) 1321.4 ± 204.8 1089.2 ± 219.7 <10 1463.8 ± 233.4 1166.5 ± 213.9 <10 −4 −4 TAS (mmol/l) 1.63 ± 0.271 1.40 ± 0.232 <10 1.70 ± 0.267 1.38 ± 0.289 <10 SOD genotypes (%), n (CC/CT/TT ) 31.93/45.38/22.69 % 23.71/37.11/39.18 % 0.03 17.86/55.95/26.19 % 23.88/52.24/23.88 % 0.66 38/54/27 23/36/38 15/47/22 16/35/16 *P < 0.05 was required for statistical significance in TAS levels but not in GPx activities was discerned In the light of our first findings of high LDL levels among T carriers (Table  1). Based on these findings, we among case group, the relationship between MnSOD/ explored the association between T allele, lipid and anti- GPx1 activities, genotype and lipid parameters was stud- oxidant parameters further after stratifying by CHD sta- ied in a group of 106 subjects who underwent coronary tus (Table  3). On one hand, results observation revealed angiography (Table 4). a significant increase of plasma LDL level among T car - CHD group was classified into four subgroups accord - riers in the cases group compared to the control one, on ing to the number of affected coronary arteries (Table  4). the other, a significant decrease in total and direct biliru - Plasma levels of lipid parameters tended to be increased bin, iron and TAS level but not in GPx activity was noted with more severe coronary atherosclerosis. Lower SOD in the same group (p  <  0.05). However, we observed no activity was significantly associated with the severity of difference in genotype distribution between CHD and CHD (p  <  0.05). We noted graded reduced SOD activi- control groups (Controls vs CHD 55.49/37.80/6.71  %; ties in patients presenting 1–3 vessel stenosis. However, 43.84/44.34/11.82  %, p  =  0.051). Statistical analysis no differences in GPx activities were observed between showed a lack of association of GPx1 Pro198Leu poly- stenosis subgroups (p = 0.53). morphism with CHD risk (Pro carriers vs Leu/Leu, OR Pearson correlation study between MnSOD activity 0.54, 95 % CI 0.25‒1.13, p = 0.10). and lipid parameters showed a positive correlation with The assessment of the power provided by our sample size high density lipoprotein (HDL) (r  =  0.143, p  =  0.028), to detect allele association was performed for each vari- and a negative correlation with low density lipoprotein ant. Given a sample size of 164 control subjects and 203 (LDL) (r = −0.210, p = 0.034). Nonetheless, no correla- patients with CHD, our study had an empirical power of 30 tion between the two parameters and GPx activity was and 43 % respectively for MnSOD Ala16Val and GPx1 Pro- observed. 198Leu polymorphisms. Whereas, the current study had a Exploration of the relationship between enzyme activi- theoretical power of 97  % to detect an OR of 1.80 associ- ties and genotypes through CHD pathogenesis showed, ated with each variant, assuming an additive effect of alleles in contrast to GPx activity, an association of decreased and with a 5 % type 1 error rate. In this sample, a size effect SOD activity to the frequent presence of variant geno- of approximately 1.52 is detectable with 80 % power. type (Val/Val) as increased stenosed vessel number. Souiden et al. Biol Res (2016) 49:22 Page 7 of 12 Table 3 GPx polymorphism and  parameter stratification dichotomous age (p = 0.012), hyperlipidemia (p = 0.027) by CHD status effect of the Mn-SOD Ala16Val polymorphism on CHD severity. Trait Controls (203) Cases (164) P Age (years) 60.63 ± 12.27 62.98 ± 11.89 0.07 Discussion −4 Body mass index 24.73 ± 3.52 28.05 ± 3.17 <10 A growing body of evidence supports the concept that (kg/m ) oxidative stress plays critical roles in the initiation and Male, n (%) 119 (58.62 %) 97 (59.15 %) 0.92 progression of numerous diseases including atheroscle- −4 Hypertension, n (%) 13 (6.40 %) 96 (58.54 %) <10 rosis [28]. Since the vulnerability to oxidative stress is −4 Diabetes mellitus, n (%) 10 (4.93 %) 59 (35.98 %) <10 partly determined by genetic background, there have Plasma glucose 4.33 ± 0.71 4.47 ± 2.91 0.51 been several studies examining the association between (mmol/l) −4 functional gene polymorphisms of the key enzymes of LDL cholesterol 2.54 ± 1.12 3.28 ± 1.13 <10 redox regulation and the risk of CAD. (mmol/l) To our knowledge, this is the first study to investi - HDL cholesterol 0.83 ± 0.37 0.82 ± 0.27 0.77 (mmol/l) gate the relationship between Mn-SOD/GPx1 poly- Triglycerides (mmol/l) 1.24 ± 0.64 1.31 ± 0.77 0.34 morphisms, and CHD risk and severity in a Tunisian Non enzymatic antioxidant parameters population. MnSOD and GPx1 genotype distributions Uric Acid (µmol/l) 312.96 ± 92.00 335.82 ± 147.99 0.07 in control and patient groups were examined. The −4 Total bilirubin (µmol/l) 17.04 ± 4.73 7.68 ± 4.81 <10 comparison of genotypic frequencies in our control −4 Direct bilirubin 7.34 ± 3.21 2.36 ± 1.08 <10 group (TT genotype: 26.11 and 9.54  %, respectively) (µmol/l) with those reported in other studies showed that our Albumin (g/l) 41.74 ± 6.05 40.78 ± 4.18 0.09 results agree with those of the Caucasian population −4 Iron (µmol/l) 18.23 ± 4.94 16.38 ± 3.47 <10 (26.11 and 9.54 %, respectively) [29–31] but differ from Enzymatic antioxidant parameters those reported in Asian (66–79.4 and 7–13  %, respec- GPx activity, (U/gHb) 42.17 ± 13.99 40.20 ± 12.25 0.16 tively), Afro-Caribbean (42.9 and 18  %, respectively) −4 TAS (mmol/l) 1.68 ± 0.26 1.37 ± 0.25 <10 and oriental (77.8 and 0.0  %, respectively) population GPx genotypes (%), 43.84/44.34/11.82 % 55.49/37.80/6.71 % 0.051 [21, 31–39]. This clear difference, mostly related to T n (CC/CT/TT ) 89/90/24 91/62/11 0.1 allele, is evident, suggesting that this allele may have a GPx genotypes (%), 88.18/11.82 % 93.29/6.71 differential role in the disease process in these ethni - n (CC + CT/TT ) 179/24 153/11 cally distinct populations. In fact, in the studied control *P < 0.05 was required for statistical significance group, T allele frequencies of MnSOD and GPx1 genes were equal to those mentioned in previous studies of Caucasian population (49  % [48–51  %] and 34  % [31– Multivariate regression analysis stratified by gender 36  %], respectively), but unlike those of Asian popula- and adjusted according to age, body mass index hyperli- tion (13  % [5–21  %] and 23  % [18–29  %], respectively) pidimia and smoking habits as covariates, confirmed the [20, 31, 40–42]. Table 4 Lipid parameters, enzymatic antioxydant activity/polymorphism and CHD severity Parameters 0-vessel (n = 13) 1-vessel (n = 42) 2-vessels (n = 35) 3-vessels (n = 16) Total cholesterol (mmol/L) 4.11 ± 1.03 4.26 ± 1.07 4.59 ± 1.03 5.36 ± 1.41* HDL-cholesterol (mmol/L) 0.86 ± 0.28 0.84 ± 0.26 0.81 ± 0.29 0.79 ± 0.23 LDL-cholesterol (mmol/L) 3.02 ± 0.93 3.15 ± 1.01 3.49 ± 1.04 4.19 ± 1.39* Triglycerides (mmol/L) 1.14 ± 0.63 1.27 ± 0.65 1.34 ± 0.97 1.89 ± 1.00* , ‡ SOD activity, (U/gHb) 1246.9 ± 242.15 1112.4 ± 141.37 1065.6 ± 194.28 864.3 ± 229.08** SOD genotypes, n (%), (CC/CT/TT ) 4/6/3 10/21/11 7/18/10 3/7/6 (30.77/46.15/23.08) (23.81/50.0/26.19) (20.0/51.43/28.57) (18.75/43.75/37.5) GPx activity, (U/gHb) 38.90 ± 7.89 44.09 ± 11.32 42.94 ± 11.15 43.65 ± 12.64 GPx genotypes, n (%), (CC/CT/TT ) 7/8/1 22/17/3 21/12/2 9/6/1 (53.85/38.46/7.69) (52.38/40.47/7.14) (60.0/34.29/5.71) (56.26/37.5/6.25) * P < 0.05 compared with 0-vessel disease and P = 0.001 with 1-vessel disease ** P < 0.001 compared with 0-vessel disease and with 1-vessel disease and P = 0.002 with 2-vessels disease  P < 0.001 compared with 0-vessel disease, 1-vessel disease and 2-vessels disease Souiden et al. Biol Res (2016) 49:22 Page 8 of 12 Given their role in antioxidant defense, SOD2 and in disagreement with our data, other studies performed GPx1 genes are considered as attractive low penetrance on Asian population demonstrated an association of candidate genes for CHD. In this study, we examined GPx1 variant genotypes to CHD risk and carotid artery the impact of SOD2 Ala16Val and GPx1 Pro198Leu intra-media thickness (IMT). In a Japanse population, polymorphisms in CHD risk and severity. Our results Haminichi et al. [21] and Oguri et al. [39] suggested that indicated that the Val/Val genotype of MnSOD envis- functional variants in GPx1 gene are associated with ages in Tunisian men higher risk to CHD as compared increased carotid IMT of cardiovascular and peripheral to controls (Table  2). In fact, Val/Val genotype was sig- vascular diseases in type 2 diabetic patients. Further- nificantly more common among case (39.18  %) than more, data of a study carried out by Tang et al. [48], pro- control (22.69  %) men. Accordingly, we note that SOD2 vide evidence that GPx1 Pro198Leu variant genotypes polymorphism has a significant influence on CHD risk in are significantly associated with CAD risk in a Chinese Tunisian men (Ala carriers vs Val/Val, adjusted OR 1.89, population. In fact, compared to the 198Pro/Pro car- 95 % CI 1.18‒3.42, p = 0.03). Furthermore, the observed riers, subjects with the variant genotypes (198Pro/Leu difference between CHD and control men is unlikely to and 198Leu/Leu) had a significantly higher risk of CAD be the result of biased population sampling. There was (adjusted OR 2.02, 95 % CI 1.27–3.22). In the same way, no significant difference in our frequency estimation of a recent meta-analysis confirms the association of GPx1 the Val allele (45.38 %) from the Tunisian men of control variants with CAD in people with type 2 diabetes mel- group than those of the Caucasian population (44–48 %) litus of Chinese and Indian populations [49]. reported in other studies [29, 30]. The opposing findings of these reports may be attrib - Our findings concur with those reported by two recent utable to inaccurate genotype frequency estimates result- meta-analyzes in which Crawford et  al. [28] concluded ing from small sample sizes, selection of control group, that the Val/Val or Val allele has been positively associ- case characterization linkage disequilibrium between ated with cardiovascular disease risk and with comor- SOD2 and/or GPx1 alleles with different functionally rel - bidities in type 1 (T1DM) and type 2 diabetes mellitus evant polymorphisms in specific ethnic groups and gene- (T2DM) patients, and Tian et  al. [43] showed a signifi - environment interactions. Further variations in genetic cant association of the C allele with reduced risk of CAD. background, dietary habits and/or environmental factors Furthermore, in agreement with our data, Valenti et  al. between different populations might account for these [44] reported an increased risk of cardiomyopathy in findings. hemochromatosis patients with Val allele compared with In this study, in addition to polymorphism association Ala allele. Evenly, Fujimoto et al. [45] found that individu- to CHD risk, we investigated the SOD and GPx activi- als harboring the valine variant were at increased risk for ties in CHD subjects. Consistent with our results some CAD and acute myocardial infraction. Moreover, in a studies suggested an association between SOD activ- large study of 776 Caucasian subjects with diabetes, Jones ity [34] but not GPx activity [31, 46, 47] with cardiopa- et al. [34] showed an increased risk of CHD associated to thy events. In contrast, others proposed the opposite Val/Val genotype. Besides, in 24 of 100 patients recruited [50–52]. We have observed a significant decrease in the with severe heart failure, Charniot et  al. [46] revealed a SOD activity in patients with CHD, as compared to con- significant correlation of the Val-encoding MnSOD allele trols. We confirmed known fact that erythrocyte SOD with the severity and prognosis of cadiogenic shock. activity is significantly reduced in patients with CHD [51, In contrast to SOD2 polymorphism, previous stud- 53–55]. Lower enzyme activity could be a consequence ies on the association between CHD and GPx1 poly- of the increased oxidative stress induced by the coronary morphism have been highly inconsistent. Polymorphic events. It may be the leading cause of post-translational variation in GPx1 gene at position 198 is associated in covalent modifications in SOD e.g. nitration, phospho - some, but not in all studies. Our observations suggest rylation, glutathionylation and glycation, which results in that Pro198Leu GPx1 polymorphism was not associated decreased enzyme activity [56]. to CHD risk. In line with our findings, previous Cau - Our results also showed no differences in GPx activ - casian population studies revealed no significant asso - ity between genotype classes and according to CHD sta- ciation between allele frequency and risk to suffer from tus. Indeed, normal GPx activity was measured in Leu CAD. Indeed, in a French population, Charniot et  al. carriers. These findings can be explained by the GPx [46] declared that GPx—variants influenced neither GPx upreglation under oxidative stress conditions and/or the activity nor cardiac events. Moreover, in a recent study presence of a compensatory mechanism in other cells of of Iranian population, Najafi et al. [47] showed that GPx1 the body with faster turnover. In addition, through an activity and rs1050450 (Pro198Leu) site are not involved analysis of the predicted tertiary structures, Jones et  al. in the development of coronary artery stenosis. However, [34] provided another possible explanation. They showed Souiden et al. Biol Res (2016) 49:22 Page 9 of 12 that the polymorphic variants have no essential role on allele was significantly correlated with the severity and protein stability and function. They suggested that this prognosis of cardiogenic shock [46]. may be due to their surface location. Predisposing association of the Val allele and high LDL- Antioxidant status of the study population was further cholesterol levels in subjects with two or three stenosis explored by measurement of some non-enzymatic anti- vessels, showed that high LDL levels are more harmful oxidants (AOX) and total antioxidant status (TAS). Sig- in subjects with a diminished antioxidant capacity of nificant decreases in both total and direct bilirubin as MnSOD. Consistent with our data, a number of stud- well as in iron against no differences in albumin and uric ies reported an association between antioxidant enzyme acid levels, were noted in cases compared to controls. activities and progression of stenosis [45, 46, 59, 70, 72]. Taken together, these non-enzymatic antioxidant (AOX) This could be explained on the one hand, by the decline constitute an important aspect of a network essential resistance against ROS produced in the mitochondria for assessing in  vivo AOX status [57]. In our popula- due to the Val isoform of the SOD2, and on the other tion, their reduced values reflect a state of an imbalance hand, it might be due to SOD activity inhibition by lipid between AOX and pro-oxidants and/or free radicals. The peroxidation products [73, 74]. In this context, Botto assessment of the TAS of a biological fluid is a compos - et al. [75] have reported elevated levels of oxidative DNA ite measurement of the combined effects of individual damage in patients with angiographically documented scavenging AOX within the sample and providing insight CAD. In addition, it has been reported that isoprostanes, into the overall prooxidant-antioxidant balance [58]. In markers of lipid peroxidation, and reduced antioxidant our population, a global evaluation of the TAS showed capacity are related to increased risk for cardiovascular a significantly reduced levels in CHD subjects compared disease [62, 76]. to controls. Our result is in agreement with that of Nojiri Since atherosclerosis is a complex process affected by et  al. [59] but not with those of Alamdari et  al. [60] and a network of numerous genes and environmental fac- Rahsepar et  al. [61] who found that the level of prooxi- tors [77–80] and given that the effect of the C47T poly - dant/antioxidant balance (PAB) in patients with stable morphism in MnSOD may vary by exogenous sources of CAD was significantly higher compared to that measured antioxidants and oxidants, multivariate regression was in healthy control subjects. studied in order to identify eventual confounders such as Low TAS levels could reflect either high oxida - age, gender, BMI and smoking status. tive stress or decrease of defense against it. In CHD Multivariate analysis of variance revealed that patients, even in stable cases, a high oxidative stress Ala16  Val polymorphism and low Mn-SOD activity may status has been reported [62–65]. For instance, circulat- be independently associated with CHD severity, a state of ing oxidized low density lipoprotein levels are positively progressive atherosclerosis, documented by the number associated with severity of acute coronary syndromes of vessel stenosis. Our findings point out the main role of [66, 67] and with subclinical CHD [68]. In line with oxidative stress in atherosclerosis. these findings, in our population, the CHD severity, The strong points of the present report are this is the as demonstrated by the number of vessel stenosis, was first study to show the effect of the Ala16Val and Pro - associated with high LDL levels. The observed posi - 198Leu polymorphisms on CHD risk in a Tunisian popu- tive correlation between atherosclerosis progression lation, the uniform mechanism of CHD diagnosis for all and high levels of LDL-cholesterol in CHD patients cases and the homogeneity of our population (all subjects was associated with the SOD2-Val/Val genotype. This are originated from central Tunisia). Nevertheless, the has not been confirmed by another study focused on main limitation of this study is the relatively small sample the role of antioxidant enzymes in determining genetic size. Although there is a significant relationship between susceptibility to the coronary artery disease in patients Ala16Val polymorphism and CHD; low statistical power with T2DM [69]. Nevertheless, Kakko et  al. [70] of the current study is our another limitation. However, reported that carotid artery intima-media thickness, the finding of an association of the Ala16Val MnSOD was greater in women with the Val allele and high levels polymorphism with CHD despite the low power means of low-density lipoprotein (LDL) cholesterol (p = 0.03). that this association is real. Indeed, with insufficient Similarly, in patients with oxidised LDL  <0.5  nmol/ power, we may miss the true association but not to find mg, Gottilieb et  al. [71] revealed an association of Ala/ the one that does not exist. Val and Val/Val genotypes with increased levels of oxi- dised LDL compared with Ala/Ala genotype in the same Conclusion group. Furthermore, in a cohort of patient with car- Although small statistical power of the current study, we diogenic shock due to dilated cardiomyopathy without have demonstrated that in contrast to 198Leu variant, the acute coronary syndrome, the Val-encoding MnSOD 16Val variant was associated with CHD risk in men and Souiden et al. Biol Res (2016) 49:22 Page 10 of 12 enhances myocardial blood flow and oxygen consumption in postisch- the severity of cardiovascular events in the Tunisian pop- emic mouse heart. J Pharmacol Exp Ther. 2008;327:402–10. ulation. Moreover, the SOD activity was inversely related 11. Church SL, Grant JW, Meese EU, Trent JM. Sublocalization of the gene to CHD progression as documented by the number of encoding manganese superoxide dismutase (MnSOD/SOD2) to 6q25 by fluorescence in situ hybridization and somatic cell hybrid mapping. vessel stenosis. 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Urinary 8-iso-prostaglandin F2alpha as a risk marker in patients with coronary heart disease: a matched case-control study. Circulation. 2004;109:843–8. Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit

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Published: Apr 11, 2016

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