Introduction

The World Health Organization (WHO) has evaluated that the number of deaths each year from smoking-related disease would increase to ten million within the next 30 years in developing countries [1,2,3]. Smoking is one of the major environmental risk factors in many serious systemic diseases, including cardiovascular diseases (CVD), cerebrovascular pathologies, cancers (especially, lung cancer), respiratory diseases such as chronic obstructive pulmonary disease (COPD), strokes, and sudden death [3,4,5]. According to WHO, every 10 s, a person dies as a result of smoking in the world [6].

Many toxic trace elements are found in cigarette, cigarette paper, cigarette filters, and cigarette smoke which can accelerate injury and inflammation in human body. Smoking deteriorates blood flow and tissue oxygenation directly or indirectly [7, 8]. Trace elements are available in very low quantities in the body that take active roles in all metabolic processes including erythropoiesis in the body [9, 10], synthesis of DNA, intra- and extracellular fluid ion balance [11], inhibition or activation of enzyme reactions [12], initiation of action potential [13], muscle contraction [14], bone and teeth growth and stability [15], nerve conduction, and osmotic homeostasis [16]. However, their biological significance is the result of their key roles in major human metabolic pathways as tissue oxygenation via oxygen transport and components or cofactors of many enzymes. Transport of oxygen to peripheral tissue by blood flow has a crucial role in systemic and pulmonary circulation. Smoking can induce antioxidant enzyme activities directly or indirectly effecting trace element metabolism [17,18,19].

In this study, we focused on serum aluminum (Al) as a toxic element and serum manganese (Mn) and selenium (Se) as antioxidant trace elements in smokers. Although toxic effect of Al is recognized, its biological functions have not been clearly explained [20]. Al which takes abundantly part in cigarette enters into the body through gastrointestinal absorption and respiration [21]. Al can induce several systemic disorders related with all cell membranes especially covering endothelium and airway epithelium. Recent reports suggested that Al decreased red blood cell (RBC) membrane fluidity by stimulating morphological and functional alterations in erythroid series resulting in lipid peroxidation within membrane lipid bilayer [22, 23]. Furthermore, low-density lipoprotein (LDL) oxidation was evaluated to cause atherosclerosis via smoking and circulating level of Al [24]. Mn is a constituent of numerous enzymes playing an important role in a number of physiologic metabolic processes as human nutrition and in protection against lipid peroxidation [25]. For example, Mn containing enzyme manganese superoxide dismutase is a principal antioxidant enzyme holding oxygen [26]. Furthermore, excessive Mn contributes to the generation of free radicals by its pro-oxidative properties [27]. Selenium (Se) is accepted to belong to the group of trace elements involving the mechanisms of cellular antioxidant defense [28]. Se is a component of several selenoproteins with essential biological functions. Major function of Se is being a cofactor for glutathione peroxidase that protects within defense mechanism [29].

Blood circulation is a closed system constituting of heart, vessels, and blood; therefore, blood is closely associated with the endothelium that organizes the internal structure of the vessel. Hemorheology is the science of blood flow throughout the endothelium and deformation behavior of blood and its formed elements including RBCs [5, 30]. Hemorheological and hematological properties display correlativity with smoked cigarette per puff [2, 31]. Hemorheological dysfunctions related with smoking include initiation of endothelial dysfunction, progression of atherosclerotic lesions, and alterations in lipid and hemostatic systems [32,33,34,35]. RBCs specialized biconcave disc shape structure determines oxygen transport capacity within the biological system. RBCs lose their biconcave disc shape resulting in their ability to deform in microcirculation through capillaries tending to aggregate throughout vessels. Oxygen delivery index (ODI), which can be explained with a mathematical formula as the ratio of hematocrit (Hct) and blood viscosity (BV), defines the volume of oxygen delivery to tissues. For that reason, ODI and partial oxygen pressure (pO2) accompanied with spirometric parameters such as forced expiratory volume (FEV1) (%), forced vital capacity (FVC) (%), and FEV1/FVC for lung function are gold markers of respiratory function [5, 36, 37].

Trace-toxic elements, BV, plasma viscosity (PV), fibrinogen, RBC aggregation (RBC Agg), and RBC deformability (Tk) have been associated with smoking in CVD and COPD in numerous studies [2, 3, 5, 9, 31, 33, 38, 39]. The relationship between mechanisms effecting hemorheology and trace-toxic elements for the patients without COPD diagnosis has not been completely elucidated. The novelty of our study is to evaluate the effects of altered serum levels of Al, Mn, and Se on hemorheologic and hematologic alterations in systemic oxygenation in smoking individuals without clinic symptoms of COPD.

Material and Methods

Study Groups

This study was planned under the guidance and approval of the Ethical Committee at Cerrahpasa Medical Faculty of Istanbul University. The study was performed in accordance with the Helsinki Declaration, and written informed consent was obtained from all individuals prior to their admission in the study. Blood samples were taken after the questionnaire administrated by each subject. The study involved 128 subjects (25–65 years) who attended to the Outpatient Clinic of Department of Respiratory Medicine in Haseki Hospital. Individuals were divided in three groups; ex-smokers (ES) (n = 44), smokers (S) 10 pack-years (n = 39), and healthy control (HC) (n = 45). Subjects diagnosed with COPD (n = 3), hypertensive (n = 2), diabetes mellitus (n = 4), metabolic diseases, cancer (n = 2), autoimmune diseases, and pathology in biologic parameters (n = 2) were excluded from the study.

Blood Collection and Preparation of Blood Samples

Blood samples were allowed to coagulate for serum analysis. Blood samples were centrifuged within 20 min, and serum was prepared by centrifugation at 3000×g rpm for 15 min using a Hettich Universal centrifuge. Serum and plasma samples were stored at − 80 °C until analysis for trace elements and hemorheological and biochemical parameters. Hemolyzed samples were excluded.

Serum Aluminum, Selenium, and Manganese Analyses

In order to eliminate adsorbed metals on the glassware being use, all the glassware was kept in 10% (v/v) nitric acid solution before use. These were then cleaned with deionized water and dried in an oven overnight at 100 °C. The analyses of serum Al, Mn, and Se levels of samples have been carried out using inductively coupled plasma-optical emission spectrophotometer (ICP-OES 6000) at the Trace Element Analysis Laboratory at Biophysics Department of Cerrahpasa Medical Faculty. Each measurement was performed three times and averages were used for analysis. Results were expressed in micrograms per milliliter (μg/mL) of serum.

The ICP-OES system used was an ICAP 6000 ICP-OES emission spectrophotometer equipped with the plus autosampler and was controlled by a computer (Thermo Fisher Scientific Inc., Istanbul, Turkey). ICP-OES was operated for suitable wavelengths for Al, Mn, and Se (167.090 nm; 257.610 nm; 196.090 nm, respectively). The plasma-operating conditions for ICP-OES system in this study were 15 L/min plasma gas flow rate, 0.5 argon carrier flow rate, 1.51/min sample flow rate, and elution flow rate. The speed of the peristaltic pump was 100 rpm. Transport lines were made using a 1.25-mm-i.d. polytetrafluoroethylene tubing [40].

Reagents

Test standards for ICP-OES analysis were prepared from proper standard solutions containing 1000 μg/mL for each tested element obtained from Chem Lab NV (Belgium). All reagents were of analytical reagent grade, and deionized water was used. Stock solutions of Al, Mn, and Se were prepared by taking appropriate amounts of standards in deionized water. Test solutions were prepared immediately before use. Doubly distilled deionized water was used in the current study. To reduce the risk of contamination from ambient air and dust, all work was performed on a clean bench. All the glassware used was cleaned by being soaked in with 10% (v/v) nitric acid solution for 1 day before use. These were rinsed thoroughly with deionized water and dried in an oven overnight at 100 °C [40].

Determination of Biochemical Parameters

Hemogram was measured using electro-impedance method with Coulter LH 780 device of Beckman Coulter Company. Corrected Hct was used in order to standardize alterations of Hct among individuals using the equation of Matrai [41]. Glucose, total cholesterol (TC), triglyceride (TG), and high-density lipoprotein (HDL) cholesterol, and creatinine levels were determined on a Hitachi Modular P analyzer using commercial kits (Roche Diagnostics, GmbH, Mannheim). If TG results were lower than 4.5 mmol/L, LDL was analyzed using Friedewald’s formula. Free triiodothyronine (FT3) and free thyroxine (FT4) and thyroid stimulating hormone (TSH) were studied with Advandia Centaur XP immunoassay device of Siemens Company using chemiluminescence method. Other biochemical parameters were assessed using spectrophotometric method with Advia 2400 device of Siemens Company. Fibrinogen was measured by using Clauss method with MDA 180 device of Trinity Biotech Company. Results were expressed as milligrams per deciliter.

Analyses of Blood Viscosity and Plasma Viscosity

BV was measured in uncentrifuged EDTA containing blood samples within 1 h from sample collection, utilizing a digital cone and plate viscometer (Brookfield LVDV III viscometer, Stougthon, MA) with a CP 42 spindle. Viscometer was standardized with a Newtonian silicon standard (4.9 m.Pas). Temperature was kept constant at 37 °C [42]. PV was measured by utilizing Harkness Capillary Viscometry (Coulter Electronics LTD Serial Number 6083, England) at 37 °C. The flow rate, measured in seconds (s), of each plasma sample was compared with distilled water to obtain relative PV [31]. PV measurements were repeated three times. PV was expressed as in millipascal × seconds (mPa s) (Formula 1).

$$ {P}_v={\eta}_w\frac{T_p(S)}{T_w(S)}\kern0.5em {\eta}_w=0.693\kern0.1em \mathrm{mPa}.\mathrm{s} $$
(1)

Dintenfass’ “Tk” erythrocyte deformability was calculated as follows [43]. Tk characterizes the deformation of RBC (Formula 2).

$$ \mathrm{H}.\mathrm{Tk}=\left({\mathrm{nr}}^{0.4}-1\right)/{\mathrm{nr}}^{0.4} $$
(2)

H being Hct given as volume fraction; nr is the relative viscosity of blood (BV derived by PV; at shear rate sufficient disaggregation of red blood cells). An increase in the rigidity index indicates a decrease in the RBC deformability. BV is used for calculation of the ODI (Formula 3) [43].

$$ ODI=\frac{\mathrm{Hematocrit}}{\ \mathrm{Blood}\ \mathrm{viscosity}} $$
(3)

Every individual has different Hct values; we calculated Tk45 and ODI45 with Quemada rheological model to avoid these differences [44].

Arterial Blood Gas Analysis

The assessment of arterial blood gas parameters was analyzed at rest using Radiometer ABL 800 with heparinized 0.2–0.5-mL injection from radial artery.

Spirometric Analysis

Spirometric analysis was assessed using ZAN 100 USB flow handy device over all individuals being rested for 15 min. Forced percentual vital capacity (FVC %), forced percentual expiratory volume (FEV1%), and FEV1/FVC declared as Tiffeneau index were evaluated.

Statistical Analysis

Statistical analysis was performed using SPSS 21 statistical software for Windows. Results were presented as means ± standard deviation (SD). Variables with normal distribution were analyzed with the ANOVA parametric test to evaluate the mean from three groups and Pearson’s test to analyze correlations. Variables without normal distribution were analyzed by means of non-parametric tests; Kolmogorov and Tamhane test to compare the median among many groups. The mean and median values were presented along with their 95% confidence interval. All the analyses were assumed statistically significant at p < 0.05.

Results

Demographic data and biochemical parameters of three groups are given in Table 1. Individuals in our study were divided into the following three groups: ES (n = 44), S 10 p/year (n = 39), and HC (n = 45) groups. No significant differences were found between the groups of age and sex except body mass index (BMI). BMI values of HC were statistically higher than those of the ES and S groups (p < 0.05). There was statistically significance between ES and S versus HC in terms of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (p < 0.01; p < 0.05, respectively). There were statistically lower TG values and statistically higher HDL values in HC compared with those in the other groups (p < 0.05) (Table 1).

Table 1 Demographic and biochemical parameters of study groups
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It was observed that Al levels were also significantly higher in S than those in HC (p < 0.05), whereas no significant difference was present in ES and S. Mn levels were statistically significantly higher in S and ES than those in HC (p < 0.001). Also, Mn levels in S were higher than those in ES (p < 0.05). Se levels in S and ES ranged from 0.22 ± 0.07 to 0.26 ± 0.16 μg/mL whereas serum Se in HC ranged from 0.49 ± 0.33 μg/mL. These results indicated that there was a significant difference between Se levels in S and ES with HC (p < 0.05) (Table 2).

Table 2 Serum aluminum, manganese, and selenium levels of study groups
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Hemoglobin (Hgb) and Hct values were higher in S and ES compared with those in HC (p < 0.001; p < 0.001, respectively). S had statistically higher fibrinogen values compared with ES and HC (p < 0.005). BV was 4.32 ± 0.42, 4.37 ± 0.16, and 4.04 ± 0.54 mPa s in study groups, respectively. There were significant differences in BV between ES and S with HC (p < 0.05). When the values of PV compared, there was statistically significantly difference between S and HC (p < 0.001). Furthermore, PV values of ES were statistically higher than those of HC (p < 0.001) (Tables 2 and 3).

Table 3 Hemorheologic parameters of study groups
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S had lower RBC Agg values than ES and HC. Also, there was significance between S and HC (p < 0.05). The values of Tk in S were higher than those of the other study groups. However, when the Tk45 values of all three groups were analyzed, Tk45 of S versus HC showed statistically higher values (p < 0.05). ODI values were lower in S versus ES and HC with no significance. When the values of ODI45 of all groups compared, values of S (9.72 ± 0.89) were significantly higher than those of HC (8.57 ± 1.46) (p < 0.01) (Table 3).

When arterial blood gas results were examined at rest with heparinized 0.2–0.5-mL injection from radial artery, pO2 levels were analyzed as 115 ± 6, 110 ± 10, and 119 ± 12 mmHg in the groups, respectively. Pulmonary blood flow rate (PBFR) was 1.03 ± 0.17, 0.98 ± 0.15, and 1.12 ± 0.34 m/sn in ES, S, and HC, respectively. There were significantly lower PBFR levels for ES and S than those for HC (p < 0.05; p < 0.01, respectively). When the spirometric values of groups were determined, FEV1 (%) and FVC (%) were statistically lower for S than those for HC (p < 0.01). S had lower FEV1/FVC values compared with ES and HC with no significance (Table 4).

Table 4 Arterial blood gas parameters and PBFR and spirometric values of study groups
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The correlations utilizing Pearson correlation analysis between the parameters of all groups were displayed in Tables 5, 6, and 7.

Table 5 Correlation between changes in variables in ex-smokers (n = 44)
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Table 6 Correlation between changes in variables in smokers (n = 39)
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Table 7 Correlation between changes in variables in healthy controls (n = 45)
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Discussion

Smoking has been demonstrated to be the most significant risk factor for CVD, lung cancer, and COPD [4, 6]. It is also known that smoking has both acute and chronic effects on trace-toxic elements and hemorheologic parameters via alterations of systemic blood flow [2, 3, 5, 9, 31, 33, 38, 39]. Smokers and people living in polluted areas were reported to have a higher level of toxic elements in their blood and urine, and it was also mentioned that smokers had higher levels of toxic elements more than twice when compared with non-smokers [45, 46]. The effects of smoking on trace-toxic elements on hemorheologic parameters have not been clearly evaluated yet. Our study included the individuals that were randomized in Outpatient Clinic of Department of Respiratory Medicine. Also, the individuals in current study were characterized with statistically higher BMI, ALT, AST, creatinine, TG, and with no meaning in white blood cell (WBC) and C-reactive protein (CRP) values, and statistically lower HDL compared with HC. These results have not been reflected to clinical findings, but biochemical alterations can be evaluated as obvious data. For this reason, we aimed as a pilot study to investigate particular effects of Se and Mn as trace and Al as a toxic element on hemorheologic parameters (including BV, PV, fibrinogen, RBC Agg, Tk, and ODI), transmission of oxygen to tissues, and progression of atherosclerotic pathway via blood flow in smokers before the onset of COPD.

Se and Mn as protective and anti-inflammatory elements and Al as a toxic element interact with lipid profile, BV, PV, fibrinogen, Tk, RBC Agg, ODI, and respiratory function. Smokers tend to generate atherosclerosis via disrupted blood flow and poor tissue oxygenation. Dyslipidemia can be defined as an anti-inflammatory defense marker besides its effects in hemorheology. Recent studies pointed that PV was positively correlated with high TC and TG and inversely correlated with HDL levels in dyslipidemia [31, 47]. However, the fact that there was no correlation between PV and lipid profile in our smoker individuals, may be related with our study groups diagnosed without COPD. In accordance with these results, other 25 analyzed higher serum TG levels and lower HDL levels in nicotine-treated rats compared with those in control groups [38, 48].

The toxic effects of Al on respiratory system have been published by several studies; nevertheless, its effects on biological system remain under uncertainty. Al content of the cigarette is considered a high risk for smokers [20, 49, 50]. The most noticeable effect of Al toxicity is loss of flexibility of the RBC membrane. Moreover, because of its high polarity, Al extensively locates in the water-lipid interface inducing a redistribution of the lipids and reacts with phospholipid heads in the external membrane changing RBC shape and limiting the surface area. As a result, smoking-induced Al increment causes disturbed blood flow, RBC Agg, Tk, and oxygen transport via lipid peroxidation in RBC membrane [23]. Our results pointed out that serum levels of Al, BV and PV values, and RBC Agg in S statistically increased compared with those in HC (p < 0.05; p < 0.05; p < 0.001; p < 0.05, respectively) (Tables 2 and 3). A study held by Norton and Rand [51] revealed increment in viscosity utilizing filtration method in smokers versus control. Moreover, Lowe and coworkers [52] confirmed a significant reduced blood flow after smoking resulted from high BV and PV. In a RBC membrane fluidity study, Van Rensburg SJ et al. [53] showed that the RBC membrane viscosity was raised with increasing Al levels. On a further study, Bazzoni et al. [20] established significance of the relative BV and Tk by demonstrating the augmentation of the RBC membrane rigidity. This research expressed the most obvious effect of Al toxication in the loss of flexibility of the RBC membrane [20]. Al was negatively correlated with TG and BV in S and positively correlated with RBC Agg in HC. According to our results, we might conclude that smoking induces the increment of serum Al levels resulting in RBC membrane disruption and disturbed blood flow.

Mn is one of the most important cellular antioxidant defense mechanisms, against damaging superoxide radicals, formed by aerobic metabolism and inflammatory disease [46, 48]. Uz et al. [3] demonstrated that significantly decreased serum Mn levels in smokers versus controls could be due to interactions between toxic and trace elements. Besides, Bocca et al. [18] reported that low levels of serum Mn in smoker men could be related to low absorption of Mn due to high levels of ferritin. Consistently, Meltzer et al. [29] determined the negative relation between serum Mn levels and anemia in women could be due to decrement of Mn absorption. In a general population study, Kim et al. [54] concluded that iron deficiency anemia increased serum Mn levels. In this study, we revealed that serum Mn levels in ES and S were statistically significantly higher than those in HC (p < 0.001). Hgb and Hct levels in ES and S were significantly higher than those in HC (p < 0.001; p < 0.05; p < 0.01, respectively); moreover, Hgb and Hct values were observed near the upper limit in ES and S. Overall, increase of Mn levels in ES and S can be pointed out as the specificity of Mn in cellular defense mechanism to scavenge free radicals. The cause of the decrease of Mn levels in ES that quitted smoking might be related with improving of defense mechanism in systemic metabolism. In their study related with Mn and dyslipidemia in smokers, Rivera-Mancía et al. [55] determined that dyslipidemia and smoking were statistically associated with increased serum Mn levels consistently with our study. The reason of the positive correlation between Mn and TC and TG and LDL and PV in S might be resulted from the inflammation in dyslipidemia leading atherosclerosis and pathologically disrupted blood flow effected from high BV and PV. Deteriorated blood flow may induce RBC deformability and cellular and systemic oxygenation terminating with poor respiratory function. Moreover, Mn levels in HC were negatively correlated with hemorheologic parameters including BV, Hct, RBC Agg, and Tk45 supporting our postulate related with Mn cellular defense mechanism. These results in the current study by means of Mn pointed out that lipid profile, blood flow, and respiratory function deteriorated in S.

Another inflammation marker besides lipid profile is fibrinogen which is an acute phase reactant behaves like an adsorbed molecule on RBCs stimulating bridging forces and RBC Agg [5]. Several studies have detailed increased PV and fibrinogen levels in smokers correlating with smoking intensity [5, 31, 39], although quitting smoking by using nicotine replacement therapy has been determined to increase in PV, Hgb, and fibrinogen [39]. Moreover, there are also data suggesting that fibrinogen might be one of the earliest variables of coagulation that is increased in smokers [56]. Almarshad et al. [2] demonstrated that BV and PV levels for smokers were slightly higher than those for controls. Several studies have detailed increased PV and fibrinogen levels in smokers correlating with smoking intensity, although quitting smoking by using nicotine replacement therapy has been determined to increase in PV, Hgb, and fibrinogen [39]. Moreover, there are also data suggesting that fibrinogen might be one of the earliest variables of coagulation that is increased in smokers [56]. It is feasible that higher fibrinogen levels and enhanced RBC Agg—without any alteration in WBC and CRP—in ES and S versus HC (p < 0.01 and p < 0.01, respectively) in the current study might support endothelial dysfunction including arterial wall inflammation and effect Htc through BV [5, 57]. However, ES and S had statistically higher PV compared with HC (p < 0.001 and p < 0.001, respectively), because higher fibrinogen indicated the effect of fibrinogen over PV as a plasma protein and higher PV concentration was due to free radical accumulation in the plasma.

Se is a component of several selenoproteins with essential biological functions. Also, Se is required in small amounts as components of antioxidant enzymes that are actively involved in defense mechanisms in the body [28]. It has been demonstrated that smoking has a negative effect on Se in smokers [58]. Different studies have detected no significance between plasma-Se and total blood-Se in smokers [59, 60]. Consistently, Bocca and coworkers [18] determined that smoking did not change Se levels of the Sardinian population. Besides, Massadeh et al. [61] revealed that Se levels were higher in smokers than those in non-smokers with a positive correlation with toxic elements. They focused on the affinity of toxic elements in bounding RBCs to selenoproteins [61]. On the contrary, smoking decreased the bioavailability of Se within the daily diet [62]. The decrement of Se level could be related with the inflammatory response attributed to smoking via toxic elements and additives [38, 63]. In this study, the mean serum Se level was also significantly lower in S and ES groups than that in HC (p < 0.05). The results of this study showed a negative correlation between serum Mn and Se levels in ES and HC. This negative correlation may be related with amelioration of cellular defense mechanism and poor oxygenation. Vankatesan et al. [64] reported that serum levels of dietary Se increased TC and TG, but decreased HDL levels in smokers. Our results confirmed the relationship between serum Se, serum lipid profile, and hemorheologic parameters. ES and S had lower Se levels and higher BV and PV compared with HC (p < 0.05; p < 0.05; p < 0.001, respectively). There was a negative correlation between Al and Se; on the contrary, Se was positively correlated with PV in S. The negative correlation between Al and Se besides a positive correlation between Se and PV might explain the role of Se in scavenge mechanisms associated with free radicals. Se had a negative correlation with Mn, versus a positive correlation with ODI in ES. Increased Se and decreased Mn and improvement in ODI levels in ES might postulate the beneficial effect of quitting smoking on defense mechanism and tissue oxygenation.

Carbon monoxide (CO) connects reversibly with oxygen carrying parts, on Hgb with an affinity aligning between 210 and 240 times higher than that of oxygen. CO also modifies the dissociation of oxygen in Hgb and places the oxygen in the tissue in risk. The increase in Hgb and Hct is due to the inspired CO which is one of the inhaled constituents of smoking [33, 65]. In our study, Hgb and Hct values in S were significantly higher than those in HC (p < 0.001 and p < 0.05, respectively). There was no significance in ODI values among S and HC, but ODI45 and PBFR values in S were statistically lower versus those in HC (p < 0.01). FEV1 (%) and FVC (%) values in S were significantly lower than those in HC (p < 0.05) with no significance in decreased pO2 and FEV1/FVC in S. Our study results were consistent with several studies [5, 31, 33]. Decrement of ODI45 and PBFR might induce free radical formation at tissue level. The decrease in ODI45 that defined poor tissue oxygenation was balanced with an increase in Hgb and Hct leading in polycythemia prior to higher BV values.

In conclusion, our study remarked the deterioration of oxygenation and blood flow accompanied with dyslipidemia leading atherosclerosis in smokers. Increased serum Al and Mn levels in smokers worsened tissue oxygenation. Furthermore, decreased serum Se levels in smokers effected blood flow by means of increasing blood and plasma viscosity, fibrinogen, and RBC Agg and inability of RBCs to deform. Overall, alterations of all these three elements caused endothelial dysfunction, disturbed defense mechanism, and respiratory dysfunction via pathologic metabolic pathways.

Smoking did not only alter the hemorheological parameters, but also altered the trace (Se and Mn) and toxic (Al) element levels in both ex-smokers and smokers. To the best of our knowledge, it was very important to research whether trace and also toxic element levels and hemorheological, hematological, respiratory parameters associated with smoking were reversible after quitting smoking. The results of our study introduce the fact that serum levels of Al, Mn, and Se and hemorheological parameters might be valuable markers for early microhemodynamic and respiratory alterations before the diagnosis of COPD.

Limitations of the Study

The limitation of the current study is that our research was held in one center in smoking cigarette as a pilot study. Our plans for future are multi-centric and wide-based studies conducted with varied brands of cigarette and tobacco.