|Year : 2012 | Volume
| Issue : 3 | Page : 139-142
High serum peroxynitrite level is an early effect of chronic cigarette smoking
Abdulkadir A Alnakshbandi1, Marwan S. M. Al-Nimer2, Ansam N Alhassani1
1 Department of Pharmacology, College of Pharmacy, Hawler Medical University, Erbil, Iraq
2 Department of Pharmacology, College of Medicine, Al-Mustansiriya University, Baghdad, Iraq
|Date of Web Publication||15-Jan-2013|
Marwan S. M. Al-Nimer
Professor of Pharmacology, Department of Pharmacology, College of Medicine, Al-Mustansiriya University, Baghdad
Source of Support: None, Conflict of Interest: None
Background: Nicotine dependence is a chronic and relapsing disease. Oxidant components of cigarette smoke reduce nitric oxide (NO) production and/or inhibit its function. Objectives: The aim of this study was to assess serum NO, peroxynitrite ONOO and C-reactive protein in smoker individuals using the Fagerström test for nicotine dependence. Materials and Methods: A total number of 50 male smoker subjects and 20 non-smoker subjects serving as controls were recruited in this study. The Fagerström test of nicotine dependence was used to assess the level of nicotine dependence. Venous blood was obtained from each subject and serum was separated for the following tests: C-reactive protein, NO and ONOO. Results: The mean values of C-reactive proteins in the smokers were less than 6 mg/L and there was no significant difference in the C-reactive protein level between levels of nicotine dependence according to the Fagerström test scoring. A non-significant low serum NO level was observed in smokers, which did not correlate with C-reactive protein. A significant high serum ONOO level was observed in smokers compared with non-smoker subjects, which did not correlate with C-reactive protein. Conclusions: The study concludes that serum ONOO level may serve as an initial marker to point the impact of smoking on human beings. Its elevation preceded the decline in serum NO level and the evidence of inflammation.
Keywords: Nicotine, nitric oxide, peroxynitrite
|How to cite this article:|
Alnakshbandi AA, Al-Nimer MS, Alhassani AN. High serum peroxynitrite level is an early effect of chronic cigarette smoking. Saudi J Health Sci 2012;1:139-42
| Introduction|| |
Nicotine dependence is a chronic and relapsing disease. The prevalence of nicotine dependence in the general population is about 13%.  Factors related to the level of nicotine dependence and psychological distress are strongly involved in sustaining cigarette consumption.  These factors include sociodemographic characteristics, smoking history, smoking by parents and peers, parent psychopathology and personality characteristics. There is no doubt that nicotine itself interacts with nitrogen species in humans. Smoking may reduce the synthesis of nitric oxide (NO). Oxidant components of cigarette smoke (e.g., nitrogen oxides hydrogen peroxide, hydrogen cyanide, acrolein) can react with l-arginine to form an l-arginin adduct.  This reaction may have two possible effects: it reduces the amount of l-arginine in the body and thus reduces NO production and/or the adduct bound to the NO itself, inhibiting its function. Also, these components are capable of affecting endothelial nitric oxide synthase (eNOS) expression and thereby reducing NO synthesis.  The reduction of eNOS protein expression was found after long-term cigarette smoking exposure.  Moreover, nicotine was involved in pathogenesis of endothelium dysfunction due to its effect on the synthesis and release of NO. ,, On the other hand, nicotine induced the release of NO from nervous tissue, thereby decreasing the sympathetic output of the brain causing and alleviating the stress. Further interaction between nicotine and NO was observed at the hypothalamic pituitary axis. NO is an inhibitory mediator in the hypothalamus pituitary axis, which, in turn, significantly enhanced the nicotine-induced hypothalamic pituitary axis response.  Few studies explored the oxidative stress status among smokers, while nitrosative stress status in nicotine-dependent individuals was scarce. Miller et al.  found that the mean plasma concentration of nitrates (NOx) from smokers was significantly greater compared with non-smokers. The aim of this pilot study was to investigate the hidden (asymptomatic) cardiovascular events of smoking via determination of NO, peroxynitrite (ONOO) and C-reactive protein in smoker individuals assessed by the Fagerström test for nicotine dependence.
| Materials and Methods|| |
This study was done in the Department of Pharmacology, College of Pharmacy, Hawler Medical University, Erbil in cooperation with the Department of Pharmacology, College of Medicine, Al-Mustansiriya University in Baghdad, Iraq during 2011. This study was approved by the local scientific committee and a consent form was obtained from each subject prior to the beginning of the study. The criteria of inclusion were male gender, history of current active cigarette smoking and of whatever age. The criteria of exclusion were history of recent infection and chronic diseases, e.g. diabetes mellitus, chronic renal failure, hypertension. A total number of 50 male smoker subjects were recruited in this study. Each subject was interviewed with the researchers to complete the questionnaire of Fagerström test of nicotine dependence. This questionnaire was composed of six questions and the status of dependence was classified according to the scores into: 0-2 Very low, 3-4 Low, 5 Medium, 6-7 High and 8-10 Very high dependence. Non-smoker control subjects (n = 20) were also admitted in this study. All control subjects were male and had no past history of smoking, i.e. ex-smoker.
Venous blood was obtained from each subject and serum was separated for the following tests: C-reactive protein, NO and ONOO.
The following chemicals were used in the study: phenol (Fisher Scientific, NJ, USA), sodium chloride (BDH, UK), disodium phosphate hydrogen phosphate (BDH), sodium hydroxide (BDH), zinc sulfate (EM Sciences, UK), glycine (BDH), cadmium granules, copper sulfate (Sigma-Aldrich Chemicals Co., USA), hydrochloric acid (Merck, Germany), sulfuric acid (Merck), sulfanilic acid, N-naphthylethyelenediamine (Borochem, India) and C-reactive protein kit (Biolis 24i, Tokyo, Japan).
Determination of C-reactive protein
It was determined quantitatively using the enzyme-linked immunosorbent assay (ELISA) technique. Serum C-reactive protein level of ≥ 6 mg/L was considered as an inflammatory marker.
Determination of serum nitric oxide
Serum (NO) was determined according to the method described by Navarro-Gonzalvez et al.,  which measured the nitrate concentration. This method was based on the deproteinization of serum followed by reduction of nitrate to nitrite by cadmium and then the reaction of nitrites with a Griess reagent to form a Griess chromophore. The procedure was performed in a dark room and the absorbance Griess chromophore was measured at 340 nm by a UV-visible spectrophotometer.
Determination of serum peroxynitrite (ONOO)
ONOO-mediated nitration of phenol was measured as described by van Uffelen et al.  In brief, 50 μL of serum was added to 5 mM phenol in 50 mM sodium phosphate buffer (pH 7.4) to get a final volume of 3 mL. After 2 h of incubation in the dark at 37°C, 25 μL of 0.1 M sodium hydroxide was added and the absorbance was immediately measured at a wavelength of 412 nm. The yield of nitrophenol was calculated from e = 4400/M/cm.
The results are expressed as absolute number, percentages and, whenever possible, mean ± SD. The data were analyzed using unpaired two-tailed Student's "t" test and simple correlation test taking the P ≤ 0.05 as the lowest limit of significance.
| Results|| |
The characteristics of the study [Table 1] revealed that the mean age of the patients was 33.7 years, and that 15 of them were single. The mean number of consumed cigarettes per day was 17.68 and the duration of smoking ranged from two 2 to 25 years. All subjects attempted to quit smoking but failed to achieve complete cessation. Seventeen patients (34%) were classified as highly dependent with a score more than 6 [Table 2].
|Table 2: Distribution of smokers according to the Fagerström test scoring, C-reactive protein and nitrogen species |
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The mean values of C-reactive proteins were less than 6 mg/L and there was no significant difference in C-reactive protein level between levels of nicotine dependence according to the Fagerström test scoring. Two subjects had C-reactive protein more than 6 mg/L and scored 3 and 4, i.e. low dependence. There was no significant correlation between C-reactive protein and scores of nicotine dependence (r = 0.072).
Low serum NO level was observed in subjects with low or medium nicotine dependence compared with non-smokers (75 ± 23.4 μmol) or with high nicotine dependence subjects who did not reach a significant level. Serum NO level did not correlate with C-reactive protein (r = 0.061) and scores of nicotine dependence (r = 0.083).
Serum ONOO level in smoker subjects of whatever nicotine dependence level were significantly (P < 0.001) higher than non-smoker subjects (2.9 ± 1.3 μmol). Serum ONOO non-significantly correlated with C-reactive protein (r = -0.099) and scores of nicotine dependence (r = -0.202) and significantly (P < 0.001) with serum NO (r = 0.496), as shown in [Figure 1].
|Figure 1: Correlation between serum peroxynitrite and nicotine dependence score (a), C-reactive protein (b), and serum nitric oxide (c)|
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| Discussion|| |
The results of this study show that the significantly high serum ONOO level correlated with serum NO in the absence of biochemical evidence of inflammation in chronic smokers. Previous studies investigated the effect of smoking on endothelium functions. Heitzer and Meinertz  reported that smoking causes endothelial dysfunction due to inactivation of NO by oxygen-derived free radicals and induces a systemic inflammatory response presented with high C-reactive protein level. Cigarette smoke activates leucocytes to release reactive oxygen and nitrogen species, secrets proinflammatory cytokines, increases the adherence of monocytes to the endothelium and elicits inflammation.  Smoking reduces NO bioavailability, which leads to the initial step of plaque formation and promotes local and systemic inflammation, which contributes to plaque progression and maturation.  Campos et al.  found a significant increase of oxidative stress biomarkers, 8-hydroxy-2΄-deoxyguanosine, 15-F (2t)-IsoP and advanced glycation end-products in healthy smokers. The others found a significant high plasma and red cell lysates nitrite/nitrate levels in chronic smokers.  The results of this study add further information to the previous studies by demonstrating significant high serum ONOO in the absence of significant changes in serum NO or evidences of inflammation. This indicates that the increment in ONOO level preceded the decreased NO level and the turnover of NO presented by ONOO/NO ratio is accelerated in smokers (0.305) compared with non-smokers (0.038). Furthermore, the significant positive correlation between serum ONOO and NO indicates higher generation of NO and higher formation of ONOO in smokers. These changes did not relate to the number of cigarettes, duration of smoking and the level of dependence. Small sample size and the data deficient in measurements of oxidative stress biomarkers are the limitations of this study. Further studies are recommended to measure simultaneously the biomarkers related to reactive oxygen and nitrogen species. In conclusion, serum ONOO level may serve as an initial marker to point the impact of smoking on human beings. Its elevation preceded the decline in serum NO level and the evidence of inflammation.
| Acknowledgment|| |
The authors wish to thank Shahin H. Mustafa for his assistance in obtaining the samples.
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[Table 1], [Table 2]