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ORIGINAL ARTICLE
Year : 2015  |  Volume : 4  |  Issue : 3  |  Page : 159-164

Impact of smoking on erythrocyte indices and oxidative stress in acute myocardial infarction


1 Department of Physiology, Gandhi Medical College, Secunderabad, Telangana, India
2 Department of Biochemistry, Dr. Pinnamaneni Siddhartha Institute of Medical Sciences, Gannavaram, Vijayawada, Andhra Pradesh, India
3 Department of Physiology, Kamineni Institute of Medical Sciences, Narketpally, Nalgonda, Telangana, India
4 Department of Social and Preventive Medicine, Kamineni Institute of Medical Sciences, Narketpally, Nalgonda, Telangana, India

Date of Web Publication15-Sep-2015

Correspondence Address:
Sandhya Metta
Sandhya Metta, Quarter No. 105, KIMS Campus, Narketpally - 508 254, Nalgonda, Andhra Pradesh
India
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Source of Support: First author is recipient of a Research Fellowship from Dr. NTR University of Health Sciences, Vijayawada., Conflict of Interest: There are no conflicts of interest.


DOI: 10.4103/2277-8632.165400

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  Abstract 

Context: The free radicals generated by cigarette smoke are responsible for the production of excessive oxidative stress, causing damage to the cellular and subcellular components in acute myocardial infarction (AMI).
Aims: The present study is aimed to evaluate the impact of smoking on erythrocytic oxidative stress and erythrocyte indices in patients with AMI.
Materials and Methods: Two hundred consecutively admitted male patients with AMI were enrolled in our study and were subsequently divided into two groups, smokers and nonsmokers. All the subjects were evaluated for lipid profile, red blood cell (RBC) indices and the antioxidant enzyme activities-catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPX) were studied.
Statistical Analysis Used: The independent sample t-test was used, and P values were calculated.
Results: We found significantly high (P < 0.001) hematocrit, hemoglobin (Hb) concentration, mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) in smokers. While evaluating the antioxidant enzymes, we observed a reduction in GPX activity in the erythrocytes of smokers in comparison to nonsmokers and this was found to be highly significant (P < 0.001) whereas, CAT and SOD activities even though were reduced, they were not as highly significant (P = 0.023, P = 0.006 respectively) as GPX activity.
Conclusions: Along with altered RBC indices, the erythrocyte GPX activity is more reliable and sensitive indicator of oxidative stress than CAT and SOD activities for the assessment of oxidative stress in AMI patients who are smokers.

Keywords: Acute myocardial infarction (AMI), cigarette smoking, oxidative stress, smokers


How to cite this article:
Metta S, Uppala S, Basalingappa DR, Badeti SR, Gunti SS. Impact of smoking on erythrocyte indices and oxidative stress in acute myocardial infarction. J NTR Univ Health Sci 2015;4:159-64

How to cite this URL:
Metta S, Uppala S, Basalingappa DR, Badeti SR, Gunti SS. Impact of smoking on erythrocyte indices and oxidative stress in acute myocardial infarction. J NTR Univ Health Sci [serial online] 2015 [cited 2020 Mar 28];4:159-64. Available from: http://www.jdrntruhs.org/text.asp?2015/4/3/159/165400


  Introduction Top


Oxidative stress produced by reactive oxygen species (ROS) has been linked to the development of atherosclerosis and ischemic heart disease (IHD).[1] Epidemiological studies have reported cigarette smoke exposure to be an important causative factor for IHD. Clinical and experimental studies indicate that active or passive exposure to cigarette smoke promotes vasomotor dysfunction, atherogenesis, and thrombosis in multiple vascular beds.[2] Although the precise mechanisms responsible remain undetermined, free radical-mediated oxidative stress appears to play a central role in predisposing smokers to acute myocardial infarction (AMI).[2] It has been estimated that 1016 radicals are present in one puff of cigarette smoke.[3] These free radicals affect lipids, DNA, proteins, and the cellular and subcellular antioxidant enzyme system, and impose proteolytic changes to plasma constituents and the circulating RBC in smokers.[2],[4] Human erythrocytes are important targets for the biological oxidative effects of free radicals as they are rich in polyunsaturated fatty acid and contain Hb, which can function as oxidase as well as peroxidase. Therefore, the oxidative effects of oxygen-free radicals on the RBC membrane are greater than on other tissues.[4] The invasion of the RBC membrane by peroxidants, which occurs with hemoglobinopathies, radioactive radiation, and cigarette smoking, alters the cellular metabolic function and can ultimately lead to RBC hemolysis.[5] Presenting a limited biosynthesis capacity, the stressed-out circulating RBC suffers, accumulating physical and chemical changes, which becomes more pronounced with cell aging. High oxidative stress levels in AMI are already well established.[6],[7] Several studies have reported changes in the RBC indices in AMI.[8] However, there are very few studies on the role of smoking in the modulation of the RBC indices and oxidative stress levels in relation to AMI. Therefore, the present study was undertaken to study the impact of smoking on hematological profile and erythrocyte antioxidant enzymes in AMI patients and to assess the utility of these markers along with conventional risk factors for the screening of smokers at risk of AMI.


  Materials and Methods Top


The study included 200 male patients aged 30-65 years and consecutively admitted to the Intensive Cardiac Care unit of the Cardiology Department of Gandhi Medical College and Hospital, Secunderabad, India over a period of 1 year. The patients were subsequently categorized as smokers (n = 100) and nonsmokers (n = 100) based on their individual history of smoking, the mean duration being 15 ± 1 pack years (20 cigarettes per day for 1 year constitutes 1 pack year).[9] All the patients were diagnosed according to the cardiologist's diagnostic criteria: Chest pain lasting for >3 h, electrocardiographic (ECG) changes (ST elevation >2 mm in at least two leads) and elevation of the enzymatic activity of serum creatine phosphokinase and asparatate aminotransferase. The study was approved by the Institutional Ethics Committee. Informed consent was obtained from all the patients before collection of the blood samples. Patients with diabetes mellitus, renal diseases, hepatic diseases, and any other neurodegenerative disorders were excluded from the study. Clinical parameters were documented in a well-designed proforma prior to the collection of the blood samples. From each of these patients, 10 mL of blood was collected for carrying out hematological and biochemical investigations.

Study of erythrocyte indices

Whole blood [ethylenediaminetetraacetic acid (EDTA) as anticoagulant] was used for determining RBC count; count, Hb, MCV, MCH, MCHC and RI by an automated cell counter (Autocounter AC 970, Lab Life H3D, Mindray).

Erythrocyte osmotic fragility

The osmotic fragility was measured by saline solutions buffered to pH 7.4, which were used at different concentrations (1.0-9.0 g/L). Heparinized blood was added to these test tubes in the proportion of 1:100; hemolysis was evaluated after 30 min of incubation at room temperature, by measuring the absorbance at 540 nm of the supernatant obtained after centrifugation (1500 g for 5 min).

Erythrocyte antioxidant defenses

Heparinized blood was centrifuged at 1000× for 10 min at 4°C, the buffy coat was discarded, and the isolated RBC pellet was hemolyzed in four times its volume of ice-cold high-performance liquid chromatography (HPLC)-grade water and again centrifuged at 4°C. The erythrocyte lysate was then used to evaluate the catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPX) activities.

GPX activity assay

GPX activity was determined with Cayman kits (item no. 703102, Cayman Chemical, Ann Arbor, MI, USA) at 25°C by colorimetry at 340 nm, based on the method of Paglia and Valentine, which requires cumene hydroperoxide as a substrate.[10] Before analysis, the erythrocyte lysates were diluted to 20-fold with the sample buffer. The final concentration of reagents in the assay was as recommended by the manufacturer. The GPX activity was measured in IU/g of Hb.

SOD activity assay

SOD activity was determined with Cayman kits (item no. 706002, Cayman Chemical, Ann Arbor, MI, USA) at 25°C by colorimetry at 340 nm, based on the method of Marklund.[11] This method employs xanthine and xanthine oxidase to generate superoxide radicals that react with 2-(-4-iodophenyl)-3-(4-nitrophenol)-5-phenyl tetrazolium chloride to form red formazan dye. The SOD activity was then measured by the degree of inhibition of this reaction. SOD units were obtained from the standard curve expressed in IU/g of Hb.

CAT assay

CAT activity was assayed based on the method of Johansson and Borg,[12] using the Cayman kits (item no. 707002, Cayman Chemical, Ann Arbor, MI, USA). The method is based on the reaction of the enzyme with methanol in the presence of an optimal concentration of H2O2. The formaldehyde produced is measured colorimetrically with purpald as the chromogen at 340 nm. CAT activity is calculated as the amount of enzyme that will cause the formation of 1.0 nmol of formaldehyde per minute expressed in nmol/min/mL.

Lipid profile

Serum total cholesterol (TC), triglycerides (TG), high-density lipoprotein (HDL), and low-density lipoprotein (LDL) were done using the autoanalyzer (Hitachi 912). Very low-density lipoprotein (VLDL) was calculated by Friedewald's equation.[13]

Blood pressure

The systolic blood pressure (SBP) and diastolic blood pressure (DBP) were recorded in the morning hour, after collection of blood samples.

Body mass index (BMI)

The BMI of all the subjects was calculated by the accepted formula: Weight (kg)/[height (meter)2]

Statistical analysis

The data was expressed as mean ± standard deviation. Comparison of data was done using the independent sample t-test, and P values were calculated using the Open Epi 6 software (Open Epi Version 2.3.1 from the Department of Epidemiology, Rollins School of Public Health, Emory University, Atlanta, GA, USA). A P value of <0.05 was considered statistically significant.


  Results Top


All the 200 patients included in the study were analyzed for clinical and biochemical parameters. The clinical details and lipid profiles of the AMI cases are given in [Table 1]. The erythrocyte indices are compared in [Table 2]. The antioxidant status is given in [Table 3]. We did not find any significant difference in SBP and DBP between the two groups. While comparing the different parameters of RBC indices, the total RBC count, Hb concentration, PCV, MCH, and MCHC were found to be significantly increased and the osmotic fragility significantly decreased in smoker patients in comparison to nonsmoker patients (P< 0.001). The MCV was found to be increased, but not significantly in the smokers group. While comparing the lipid profiles, TC, TG and VLDL were significantly higher and HDL was found to be significantly lower in the smoker group. The antioxidant enzyme activity of GPX was found to be significantly reduced in the erythrocytes of smokers (P< 0.001). Even though the activities of CAT and SOD were reduced, this was not as highly significant as the GPX activity (P< 0.05).
Table 1: Baseline Characteristics and Lipid Profiles of Nonsmoker and Smoker Patients

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Table 2: Hematological Indices in Nonsmoker and Smoker Patients

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Table 3: Erythrocyte Antioxidant Enzyme Activity in Nonsmoker and Smoker Patients

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  Discussion Top


Smoking is a potent risk factor for cardiovascular morbidity and mortality by causing atherosclerosis. In myocardial infarction, the formation of an occlusive thrombus at the site of rupture of a plaque in the coronary arteries leads to reduced circulation to that part of the myocardium, compromising its contractility and eventually leading to heart failure.

Cigarette smoke contains toxic active molecules such as aldehydes, heavy metals, hydrogen cyanide, low-molecular-weight phenols, nitrosamines, and polycyclic aromatic hydrocarbons.[14],[15] Cigarette smoke is also a source of metallic ions and hydroxyl radicals.[14] The large volume of oxidants from the smoke phase as well as the tar phase of cigarette smoke produces copious amounts of ROS, which are responsible for increased lipid peroxidation and atherosclerosis.[16] This explains the significantly lower age of onset of myocardial infarction: 52.62 ± 5.72 years in smokers compared to nonsmokers (P< 0.001) [Table 1]. Our study agrees with that of Panwar et al. (2011), who have reported the association of smoking with lower age of onset and higher mortality from IHD in Indian smokers.[17] Surprisingly, in our study we observed higher BMI in smokers than in nonsmokers, which is contrary to earlier studies.[17],[18],[19] However, Chieloro et al. (2008) have suggested that heavy smokers tend to have greater body weight as there is a likelihood of clustering unhealthy lifestyle characteristics in smokers, such as a low degree of physical activity, inappropriate diet, and alcohol consumption, which is responsible for weight gain.[20]

As all the AMI patients were under similar antihypertensive therapy and were constantly monitored for stable blood pressure in the cardiac care unit, we did not find any significant differences in blood pressures between the groups. While comparing the lipid profiles, it was observed that the TC, VLDL, and TG were increased significantly (P< 0.001) [Table 1] and that the HDL was decreased significantly (P< 0.001) in smokers, whereas no difference was observed in LDL. This pattern had been established by several earlier studies.[21]

While comparing the RBC indices among the the two groups, we observed significantly high RBC count, Hb concentration, hematocrit, MCH, and MCHC (P< 0.001) [Table 2] in AMI patients who smoked in comparison to nonsmoker patients. Our study agrees with that of Ugbebor et al., who found significantly higher hematocrit and Hb concentration in smokers.[22] The high Hb concentration in smokers may be due to the fact that smoking causes excessive production of carbon monoxide (CO), which leads to formation of carboxy Hb (COHb). Because Hb has 200 times more affinity to CO than O2, there is a stark unavailability of Hb for O2 carriage, shifting the Hb-O2 dissociation curve to the left.[23] As a compensatory mechanism, the body tries to produce more Hb by increasing the rate of erythropoiesis. The high RBC count and Hb concentration are responsible for the higher PCV and MCHC observed in smokers with acute AMI. Our study is concurrent with that of Goel et al., who reported that smokers with a minimum history of 10 pack years of smoking showed considerably high Hb concentrations.[24] We did not observe any difference in the RI in smokers. The erythrocyte osmotic fragility was observed to be significantly lower in smokers. This can be attributed to the higher membrane peroxidation and altered RBC volume coupled with the cellular oxidative stress, making the RBC more fragile in smokers with AMI.

The erythrocyte is at increased risk from oxidative processes for a variety of reasons. It is continually exposed to high oxygen tensions, and Hb is susceptible to autoxidation.[22] RBCs are unable to repair damaged components by resynthesis. Therefore, the RBC is completely dependent on the antioxidant enzymes CAT, SOD, and GPX throughout its 120 days of lifespan. These components build up an efficient defense system and are particularly involved in the detoxification of the cell from oxygen-free radicals.[25]

In the present study, we observed significantly reduced activity of GPX (P< 0.001). [Table 3] in smokers, whereas the activities of CAT and SOD were not highly significant even though they were also decreased (P = 0.023, P = 0.006 respectively) [Table 3]. Therefore, we suggest that erythrocyte GPX activity is more sensitive and a better indicator of smoking-induced oxidative stress in AMI patients.[26] GPX detoxifies the H2O2 produced by SOD action and also converts lipid hydroperoxides to nontoxic alcohols. Hence, GPX is considered to act as a chain-breaking antioxidant.[27] Our findings support the results of Bolzan et al., who have studied the influence of sex, age, and cigarette smoking on antioxidant enzymes and have reported significantly reduced GPX activity in smokers.[28] Orhan et al. reported significantly lower activity of GPX and SOD in the erythrocytes of smokers, while CAT activity remains unchanged.[29] Muzakova et al. have reported erythrocyte GPX and SOD activities as potent markers of antioxidant defense in smoking and coronary artery disease.[30] However, in the present study we observed the reduced activity of only GPX as the most sensitive oxidative stress indicator of smoking, having a significantly higher association with myocardial infarction.

The present study states that smoking may cause an increase in erythropoiesis by producing more COHb and making Hb unavailable for oxygen transport. This could be responsible for the altered erythrocyte indices observed in smokers with AMI. As AMI is a condition characterized by high oxidative stress, the copious generation of free radicals due to smoking further aggravates it. The present study thus reports that the erythrocytic activity of GPX is the most significant indicator of oxidative stress in smokers in association with AMI.


  Conclusion Top


Chronic cigarette smoking alters the erythrocyte indices in AMI. Along with conventional lipid markers, the erythrocyte GPX activity can be considered a better indicator of oxidative stress than CAT and SOD activities in screening chronic smokers for the risk of AMI.

Acknowledgments

The first author is the recipient of a Research Fellowship from Dr. NTR University of Health Sciences, Vijayawada, Andhra Pradesh, India. The authors are grateful to the faculty members of the Department of Cardiology, Gandhi Medical College for their support with clinical resources.



 
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    Tables

  [Table 1], [Table 2], [Table 3]



 

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