Comparative assessment of individual RONS in serum of smokers compared with non-smokers and their correlation with the lipid profile and antioxidant status.

Introduction

Cigarette smoking is a major risk factor for the development and progression of several diseases via diverse underlying mechanisms.[1-3] Among these mechanisms, the free radical-induced oxidative effect has been suggested to play a significant role in the pathogenesis of cardiovascular diseases and cancer.[4,5] It has been argued that the increased production of reactive species associated with smoking may exceed the capacity of the endogenous antioxidant defence system, resulting in oxidative damage and lipid peroxidation.[6-9] Although cigarette smoke is a rich source of free radical and non-radical oxidants, direct exposure to cigarette smoke represents only a portion of the total oxidative stress that is found in living tissues. Additionally, smoking contributes to further endogenous oxidant formation that magnifies the inflammatory immune responses.[10,11]

Reactive oxygen and nitrogen species (RONS) are currently the most widely studied free radical oxidants in living organisms. The most important reactive oxygen species (ROS) generated by cigarette smoking are the superoxide anion (O2–•), the hydroxyl radical (HO•), the singlet oxygen radical (1O2), hypochlorous acid (HClO), hydrogen peroxide (H2O2), and the peroxyl radical (ROO•).[5,7,8,12,13] These ROS species significantly affect the haematological and oxidative stress biomarkers in both active and passive smokers.[14] The most important reactive nitrogen species (RNS) found in cigarette smoke are nitrogen dioxide (ONO•), nitric oxide (NO•), and peroxynitrite (ONOO–).[4] Although endogenous nitric oxide is involved in the endothelial vasodilatory mechanism, its availability is decreased by the effects of oxygen radicals. Additionally, this reaction produces peroxynitrite, which, in turn, enhances oxidative stress.[4,8]

In normal physiology, there is a dynamic equilibrium between ROS activity and antioxidant defence capacity.[15] When this equilibrium shifts in favour of ROS, either by a reduction in antioxidant defences or an increase in ROS production, the oxidative stress increases, leading to potential cellular damage.[16]

Because RONS have a short lifetime, it has been difficult to measure their levels, especially in biologic fluids.[17] However, the introduction of specific fluorescence-based probes has made this type of measurement possible.[17,18] Thus, we measured individual RONS in serum from rheumatoid arthritis patients in a recent study.[19]

To date, most of the studies involving smokers have been concerned with assessing the total oxidative stress without focusing on the individual RONS and their expected pathological roles.[14,16] Therefore, the aim of this study was to comparatively measure the levels of individual RONS in serum from smokers and non-smokers, and to highlight their impact on the lipid profile and the endogenous antioxidant status, which is represented by vitamins C and E. These levels will be correlated with the individual RONS levels to assess the oxidant–antioxidant balance. Our results are expected to shed light on reactive species that play a central role in smoking-induced diseases.

Materials & methods

Study design

This study was conducted between August 2016 and January 2017, and it was approved by the Research Ethics Committee of the Faculty of Pharmacy in Assiut University, Assiut, Egypt (no. 1451/2017). A detailed questionnaire on demography information and smoking status was distributed to visitors of outpatient clinics at Assiut University who attended the clinics for a routine check-up. All patients with infectious diseases and chronic diseases, and those who were on long-term medications were excluded. Additionally, persons who were subjected to any of the following within the 3 months before the start of the study were excluded: antibiotics, steroids, thiazide diuretics, nonsteroidal anti-inflammatory drugs, immunomodulatory drugs, drugs that affect lipid profiles, hospitalisation, surgery, radiotherapy, and previous history of direct smoking (for current non-smokers). Ninety-four healthy volunteers were enrolled and divided into two groups based on their smoking habit. The smokers group included 48 subjects (all male), and the non-smokers group included 46 subjects (32 males and 14 females). Informed consent was obtained from each participant.

Biochemical analysis

Fasting venous blood samples were collected from the study subjects under aseptic conditions. A sample of the fresh whole blood from each subject was analysed using an automatic haematology analyser Celltac G (Nihon Kohden Co., Tokyo, Japan) to determine haemoglobin (Hb), packed cell volume (PCV, haematocrit), red blood cells (RBCs), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), total white blood cells (WBC), neutrophils, eosinophils, lymphocytes, monocytes, and platelets counts.

Serum samples were obtained from the blood samples by centrifugation at 2000 × g for 10 minutes at 4°C. The plasma was analysed using a Cobas c 311 analyzer (Roche Diagnostics GmbH, Mannheim, Germany) to determine the total cholesterol, triglyceride, and high-density, low-density, and very low-density lipoprotein cholesterol (HDL-C, LDL-C, and VLDL-C, respectively) levels. Serum vitamin C and E levels were analysed using reversed-phase high performance liquid chromatography (HPLC) methods.[20,21]

Serum RONS levels were measured using specific fluorescence-based reagents. The superoxide anion level was determined by measuring the level of fluorogenic ethidium (E+), which forms in an oxidation reaction of superoxide anion with hydroethidine (HE) (Sigma-Aldrich, Seelze, Germany). Potassium superoxide (Sigma-Aldrich) was used as a reference standard for the superoxide anion.[22] The Singlet Oxygen Sensor Green (SOSG) reagent (Molecular Probes, Eugene, Oregon, USA) was used for the specific fluorescence determination of singlet oxygen using 5,10,15,20-tetrakis(N-methyl-4-pyridyl)-21H,23H-porphine (TMPyP), a photosensitising agent, to produce singlet oxygen as a reference standard for calibration purposes.[23] The selective monitoring of hydroxyl radical was performed through fluorescence determination, which was achieved by coumarin-3-carboxylic acid (3-CCA) (Sigma-Aldrich), where the hydroxyl radical was generated through Fenton’s reaction of a mixture containing hydrogen peroxide, ferrous ammonium sulfate, and phosphate buffer (pH 7.4).[24] Fluorescence detection using Amplex® Red Hydrogen Peroxide/Peroxidase kit (Molecular Probes) was used for the specific detection of the hydrogen peroxide level.[25] The hypochlorite anion was selectively determined using 2-[6-(4ʹ-amino) phenoxy-3H-xanthen-3-on-9-yl] benzoic acid (APF) reagent (Sigma-Aldrich), followed by fluorescence detection using sodium hypochlorite (Sigma-Aldrich) as a standard.[26] The lipid peroxidation sensor, 4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4adiaza-s-indacene-3-undecanoic acid (BODIPY581/591 C11) (Molecular Probes) was used for the fluorescence detection of the peroxyl radical using tert-butyl hydroperoxide (Sigma-Aldrich) to generate peroxyl radicals for calibration.[27] To measure nitric oxide radical, 4,5-diamino-fluorescein (DAF-2) (Sigma-Aldrich) was used as a specific fluorescence reagent, and spermine nonoate (Sigma-Aldrich) was used as the nitric oxide radical donor.[28] A specific fluorescence kit of 2,3-diaminonaphthalene (DAN) (Sigma-Aldrich) was used to detect nitrite.[29] Finally, peroxynitrite was reduced by nitrate reductase (Sigma-Aldrich) to nitrite, and was detected using DAN. Sodium nitrate and sodium nitrite (Sigma-Aldrich) were used as calibration reference standards for the nitrate and nitrite radicals, respectively.

Statistical analysis

Statistical Package for the Social Sciences (SPSS), version 20 (IBM Corp., Armonk, NY, USA) was used for statistical analysis. RONS levels were expressed as the mean and standard error of the mean (SEM). One-way analysis of variance (ANOVA) was used to compare means, with Tukey’s test used for post-hoc analysis. Differences were considered to be highly significant, significant, or nonsignificant for P ≤ 0.001, P ≤ 0.05, or P > 0.05, respectively. Pearson’s correlation coefficient (r) was used to determine correlations between variables. The correlation strength was strictly defined using Evans’ method, and was considered to be very strong, strong, moderate, weak, or very weak if (0.8 ≤ r ≤ 1.0), (0.6 ≤ r < 0.8), (0.4 ≤ r < 0.6), (0.2 ≤ r < 0.4), or (0.0 ≤ r < 0.2), respectively.[30]

Results

Participant features and biochemical analyses

As shown in Table 1, there were no significant differences in age, height, weight, body mass index (BMI), and systolic and diastolic blood pressures (BP) between the smokers and non-smokers groups.

Table 1.

Selected features of smoker and non-smoker healthy participants.

Feature	Smokers group*(n = 48)	Non-smokers group*(n = 46)	P Value
Age (years)	32.6 ± 9.4	30.9 ± 10.5	NS
Weight (kg)	63.8 ± 12.7	61.4 ±11.8	NS
Height (cm)	165.7 ± 12.0	163.3 ± 12.7	NS
BMI (kg/m2)	23.2 ± 4.4	22.9 ± 4.1	NS
Number of cigarettes smoked/day	16.7 ± 6.3	–	–
Smoking duration (years)	8.5 ± 4.8	–	–
Systolic BP (mmHg)	116.9 ± 8.5	119.1 ± 7.7	NS
Diastolic BP (mmHg)	77.4 ± 6.9	79.2 ± 5.3	NS

*Mean ± SEM.

BMI, body mass index; BP, blood pressure; NS, non-significant; SEM, standard error of the mean.

Table 2 shows the results of the haematology tests, lipid profiling, and endogenous serum antioxidant levels between the smokers and non-smokers groups. For the haematology tests, there were no significant differences in the RBC count, MCV, and total and differential WBC count. However, Hb, PCV, MCH, MCHC, and platelet count were significantly higher (P ≤ 0.05) in the smokers group. The lipid profile for smokers showed a highly significant increase (P ≤ 0.001) in the levels of total cholesterol, LDL-C, VLDL-C, and triglycerides, but a highly significant decrease in HDL compared with the non-smokers. There was a highly significant decrease in serum endogenous antioxidants (vitamin C and vitamin E) in the smokers group compared with the non-smokers group.

Table 2.

Comparison of haematology tests, lipid profile and endogenous serum antioxidants between smokers and non-smokers groups.

Parameter	Smokers group*(n = 48)	Non-smokers group*(n = 46)	P Value
Haematological parameters
Hb (g/dL)	14.4 ± 1.2	11.3 ± 0.9	≤0.01 (S)
PCV (%)	44.5 ±1.4	38.9 ± 1.1	≤0.01 (S)
RBCs (106/µL)	4.9 ± 0.3	4.6 ± 0.2	>0.05 (NS)
MCV (fL)	85.7 ± 2.6	83.4 ± 2.4	>0.05 (NS)
MCH (pg)	31.2 ± 0.9	27.9 ± 1.2	≤0.05 (S)
MCHC (g/dL)	32.8 ± 1.4	29.4 ± 1.0	≤0.01 (S)
Total WBCs (103/µL)	7.4 ± 0.4	6.7 ± 0.2	>0.05 (NS)
Neutrophils (%)	62.5 ± 2.3	62.0 ± 3.1	>0.05 (NS)
Eosinophils (%)	5.1 ± 0.05	5.7 ± 0.06	>0.05 (NS)
Lymphocytes (%)	28.5 ± 1.7	28.1 ± 1.3	>0.05 (NS)
Monocytes (%)	3.9 ± 0.04	4.2 ± 0.05	>0.05 (NS)
Platelets (103/µL)	265.2 ± 15.7	215.9 ± 14.3	≤0.01 (S)
Lipid profile
Total Cholesterol (mg/dL)	197.7 ± 25.2	155.2 ± 18.5	≤0.001 (HS)
HDL-Cholesterol (mg/dL)	35.6 ± 4.6	42.5  ± 5.1	≤0.001 (HS)
LDL-Cholesterol (mg/dL)	128.7 ± 8.8	88.4 ± 7.6	≤0.001 (HS)
VLDL-Cholesterol (mg/dL)	33.4 ± 3.7	24.3 ± 2.7	≤0.001 (HS)
Triglycerides (mg/dL)	166.8 ± 15.3	121.7 ± 13.1	≤0.001 (HS)
Serum antioxidants
Vitamin C (mg/dL)	0.75 ± 0.15	1.19 ± 0.22	≤0.001 (HS)
Vitamin E (mg/dL)	1.08 ± 0.33	1.98 ± 0.41	≤0.001 (HS)

*Mean ± SEM.

Hb, haemoglobin; HS, highly significant; HDL, high density lipoprotein; LDL, low density lipoprotein; MCH, mean corpuscular haemoglobin; MCHC, mean corpuscular haemoglobin concentration; MCV, mean corpuscular volume; NS, nonsignificant; PCV, packed cell volume (haematocrit); RBCs, red blood cells; S, significant; SEM, standard error of the mean; VLDL, very low-density lipoprotein; WBCs, white blood cells.

Individual serum RONS levels in smokers and non-smokers groups

Serum levels of individual ROS and RNS in the study subjects are presented in Table 3. Except for singlet oxygen, which was found to be significantly higher in the smokers group (P ≤ 0.05), all other radical levels were highly significantly increased in the smokers group compared with the non-smokers group (P  ≤  0.001). The mean serum levels of the quantified RONS in the smokers and non-smokers groups are shown in Figure 1.

Table 3.

Serum levels of reactive oxygen and nitrogen species in smokers and non-smokers.

Reactive species levels	Smokers group*(n=48)	Non-smokers group*(n=46)	P Value
Superoxide anion (nM)	259.6 ± 63.1	145.3 ± 52.7	≤0.001 (HS)
Hydroxyl radical (nM)	275.3 ± 57.2	149.5 ± 38.1	≤0.001 (HS)
Singlet oxygen (nM)	116.0 ± 47.9	92.1 ± 12.7	≤0.05 (S)
Hydrogen peroxide (nM)	891.4 ± 153.7	235.1 ± 64.8	≤0.001 (HS)
Hypochlorite radical (nM)	107.2 ± 18.6	53.4 ± 9.5	≤0.001 (HS)
Peroxyl radical (nM)	138.1 ± 21.5	76.2 ± 11.9	≤0.001 (HS)
Nitric oxide (µM)	25.5 ± 8.3	13.1 ± 6.6	≤0.001 (HS)
Nitrogen dioxide (µM)	3.36 ± 1.0	1.8 ± 0.8	≤0.001 (HS)
Peroxynitrite (µM)	4.72 ± 1.25	2.54 ± 0.7	≤0.001 (HS)

*Mean ± SEM.

HS, highly significant; S, significant; SEM, standard error of the mean.

Figure 1.

Mean serum levels of (a) reactive oxygen species and (b) reactive nitrogen species in smokers and non-smokers groups.

Correlations between individual RONS and serology markers in smokers

As shown in Table 4, the correlations between individual ROS serum levels and lipid profiles can be summarised as follows: moderate to strong positive correlation between total cholesterol and O2–•, HO•, H2O2, and ClO•; a moderate positive correlation between triglycerides and O2–•, HO•, and H2O2; a moderate positive correlation between both LDL and VLDL and HO•; a moderate negative correlation between HDL and both O2–•and HO•; and very weak to weak positive correlations for the rest of the ROS and lipid profiles.

Table 4.

Correlation of serum lipid profile and endogenous antioxidant levels with serum levels of individual reactive oxygen and nitrogen species in the smokers group.

Parameter	O2–•(r)	HO•(r)	[1]O2(r)	H2O2(r)	ClO•(r)	ROO•(r)	NO•(r)	ONO•(r)	ONOO– (r)
Lipid profile
Total Cholesterol	0.54	0.69	0.31	0.57	0.51	0.48	0.43	0.47	0.43
HDL-Cholesterol	−0.44	−0.50	−0.31	−0.34	−0.31	−0.29	−0.30	−0.35	−0.38
LDL-Cholesterol	0.37	0.41	0.28	0.35	0.28	0.24	0.27	0.25	0.27
VLDL-Cholesterol	0.39	0.41	0.15	0.27	0.26	0.30	0.26	0.24	0.21
Triglycerides	0.40	0.54	0.28	0.42	0.39	0.31	0.36	0.38	0.37
Serum antioxidants
Vitamin C	−0.54	−0.62	−0.24	−0.41	−0.31	−0.23	−0.44	−0.46	−0.39
Vitamin E	−0.51	−0.50	−0.34	−0.42	−0.28	−0.23	−0.40	−0.38	−0.39

1O2, singlet oxygen; ClO•, hypochlorite radical; H2O2, hydrogen peroxide; HDL-C, high-density lipoprotein; HO•, hydroxyl radical; LDL, low-density lipoprotein; NO•, nitric oxide; O2–•, superoxide anion; ONO•, nitrogen dioxide; ONOO–, peroxynitrite; r, correlation coefficient; ROO•, peroxyl radical; VLDL, very-low-density lipoprotein.

For RNS, the correlations were as follows: a moderate positive correlation between total cholesterol and NO•, ONO•, and ONOO–; a weak positive correlation between triglycerides, LDL, VLDL, NO•, ONO•, and ONOO–; and a weak negative correlation between HDL and NO•, ONO•, and ONOO–.

Generally, positive correlations were found between total cholesterol, triglycerides, LDL, VLDL, and all RONS. The strength of the correlations were in the following descending order: HO•, H2O2, O2–•, ClO•, ROO•, ONO•, ONOO–, NO•, and [1]O2, whereas the only negative correlation was between HDL and all assessed reactive species. Figure 2 summarises the correlations between individual RONS and the lipid profiles for the smokers group.

Figure 2.

Correlations between individual reactive oxygen and nitrogen species and lipid profiles in serum of the smoker group. HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; VLDL-C, very low-density lipoprotein cholesterol. Correlation strength: very strong, ± (0.8 ≤r ≤ 1.0); strong, ± (0.6 ≤ r < 0.8); moderate, ± (0.4 ≤ r < 0.6); weak, ± (0.2 ≤ r < 0.4); very weak, ± (0.0 ≤ r < 0.2).

For the endogenous antioxidant status (Table 4), we found that smokers had moderate to strong negative correlations between both vitamins C and E and the oxygen radicals O2–•, HO•, and H2O2, as well as weak negative correlations with [1]O2 and ClO•. For RNS, moderate negative correlations were found in smokers between vitamin C and NO• and ONO•, while a weak negative correlation was found with ONOO–. Finally, a moderate negative correlation was found between vitamin E and NO•, as well as a weak negative correlation between vitamin E and ONO• and ONOO–.

Discussion

Chronic exposure to cigarette smoke affects a wide range of immunological and haematological parameters in humans.[31,32] The main objective of the current study was to measure the serum levels of individual RONS in smokers and non-smokers to increase our understanding of the possible roles of these radicals in the pathology of various diseases. These radicals are either endogenously produced or acquired from exogenous sources. The endogenous sources include immune cell activation, inflammation, excessive exercise, ischaemia, infection, cancer, and rheumatoid arthritis, while the exogenous sources include cigarette smoke, air pollution, alcohol, heavy metals, and certain drugs.[33] Cigarette smoke is a major source of exogenous oxidants, which can initiate redox cycling reactions that generate new free radicals. Moreover, cigarette smoke promotes the activation of neutrophils and macrophages that act as endogenous sources of free radicals.[34] In this study, a highly significant increase in the serum levels of O2–•, HO•, H2O2, ClO•, and ROO• was observed in the smokers group, as well as a significant elevation in [1]O2, as shown in Figure 1.

For RNS, a highly significant elevation was observed for NO•, ONO•, and ONOO– in the smokers group. Although the NO• radical is much less reactive compared with the other studied serology markers, its effects appear to result from a slow conversion to ONO• and ONOO– by oxidation, and it can be present in amounts as high as 500 ppm in cigarette smoke.[35] Cigarette smoke may also affect levels of vascular endothelial nitric oxide, and can also modulate inflammatory reactions through inflammatory cytokines.[36] Additionally, ONO• can react with other gaseous components of cigarette smoke (such as isoprene) to form alkoxyl radicals. These peroxyl radicals then react with either NO• or ONOO– to form peroxynitrate radicals.[37]

A significant increase in the levels of total cholesterol, LDL-C, VLDL-C, and triglycerides was observed in smokers compared with non-smokers, while a significant decrease in HDL-C levels was seen in smokers compared with non-smokers. These results are similar to previously reported observations.[32,38] Dyslipidaemia is significantly worsened by an increase in smoking duration. This is partly because nicotine in cigarette smoke stimulates the release of adrenaline from the adrenal medulla, which increases serum levels of free fatty acids. These fatty acids, in turn, stimulate the liver to secrete cholesterol, VLDL-C, and triglycerides.[38] Smoking may induce sub-endothelial oedema with lipid accumulation and changes in vascular permeability. Additionally, nicotine decreases vascular activity, worsens endothelium dysfunction, and induces the formation of coronary artery clots.[32] However, LDL-C level is positively correlated with the VLDL-C level and triglycerides, and the HDL-C level is inversely proportional to the LDL-C level.[39,40] Cigarette smoking also induces insulin resistance, which may lead to hyperinsulinaemia, as observed by a marked decrease in lipoprotein lipase and hepatic lipase that transforms VLDL to LDL.[41] Insulin resistance was shown to negatively affect the lipid profile, inducing endothelial dysfunction and oxidative stress and driving the formation of atheroma and the development of cardiovascular diseases.[40] In our study, moderate to strong positive correlations were found between both total cholesterol and triglycerides and O2–•, HO•, and H2O2, whereas moderate positive correlations were observed between both LDL and VLDL and HO•. However, moderate negative correlations were seen between HDL and O2–•and between HDL and HO•.

As a rich source of active oxidants, cigarette smoke may cause an imbalance in the endogenous antioxidant defences that are associated with increased production of free ROS.[42] Vitamin C, a strong antioxidant, has a hydrophilic nature and a unique structure that makes it an excellent electron donor for scavenging free RONS.[7] However, vitamin E has a lipophilic nature that enables it to pass through the cell membrane to scavenge free reactive species. The biological activity of vitamin E is almost entirely a result of its antioxidant properties that aid in membrane stabilisation and effectively prevent lipid peroxidation.[43] In this study, a marked decrease in serum levels of antioxidant vitamins C and E in smokers was observed, which resulted in an increased lipid peroxidation rate. Our results also show that a moderate to strong negative correlation exists between vitamin C and E and O2–•, HO•, and H2O2. Cigarette smoking depletes these serum antioxidants, which are required to scavenge excess free radicals, thereby increasing the rate of lipid peroxidation.[43]

The most significant radicals that affected the serological markers in our study were the hydroxyl and superoxide anion radicals. This result may be explained by the activation of pulmonary alveolar macrophages by cigarette smoke. During this process, O2–• and H2O2 are produced, and these species are converted through an iron-catalysed reaction into HO• radical, leading to biological damage.[12,44]

The significant elevation of Hb concentration and its related markers that occurred in the smokers group in this study was consistent with other reports.[32,38] This elevation is best explained as a compensatory mechanism that increases Hb concentration because of a lack of oxygen-carrying capacity in carboxy-Hb, which results from continuous exposure of the smoker to carbon monoxide that is generated in the cigarette burning process.[45] Finally, although the effect of smoking on platelet count is controversial, the smokers in our study showed a significant increase in platelet count, which could be attributed to increased platelet aggregation.[46-48]

We acknowledge that there are several limitations to this study. We included subjects from only one geographical region (i.e., Assiut, Egypt), so we cannot generalise the results to other populations. Additionally, we were not able to include female smokers because smoking disclosure for women is socially unacceptable in Egypt.

In summary, cigarette smoking generates several oxidative and nitrative stressors that may act as serious health hazards. To the best of our knowledge, the current study is the first to monitor individual RONS in smokers. The specific effect of the monitored reactive species on lipid markers, combined with practical insight into serum antioxidant status, were confirmed. Some of the reactive species, such as HO• and O2–•, play predominant roles in cellular damage caused by smoking. Consequently, early intervention to promote smoking cessation may reverse these harmful effects and prevent future health problems. However, further insights into the molecular mechanisms of these reactive species in the pathology of smoking-related diseases will be of a great value.