Study of the Relationship between the Nicotine and Lipid Profile with Some Hematology Parameters in Serum of Smoker and Non-Smoker Blood Samples

The effect of cigarette smoking on lipid profile and CBC (Complete Blood Count) of blood was investigated in this study. Spectrophotometric methods were used to estimate the amounts of nicotine, cholesterol, triglycerides, and HDL-cholesterol. The findings revealed that cigarette smokers and the duration of smoking had significantly higher levels of nicotine and cholesterol when compared to non-smokers. The data also revealed differences in the contents of hematological parameters between smokers’ and non-smokers’ blood samples, with high counts of WBC, MCHC, HGB, and PLT clearly visible in the smokers’ samples. However, there was no discernible effect on RBC, MCV, or MCH counts in the trial. Smoker samples had high PLT values (243.8 ± 84.26), while non-smoker samples had lower PLT values (229.3 ± 30.07 on average).


Introduction
High blood levels of total cholesterol (TC 240 mg/dL), low-density lipoprotein cholesterol (LDL-C 160 mg/dL), and triglyceride (TG 200 mg/dL) and a low amount of high-density lipoprotein cholesterol (HDL-C 40 mg/dL) characterize dyslipidemia [1]. Both individually and in combination, serum lipid components are linked to the risk of cardiovascular disease (CVD) [2]. Cigarette smoking is a well-known primary risk factor for atherosclerosis and cardiovascular disease [3]. These lipids that have been damaged may induce atherogenesis and atherosclerosis [4]. However, the findings varied between research, and the impact of smoking on lipid levels is not well established [5].
Dyslipidemia affects about four out of ten (40.5 percent) persons over the age of 30, with the severity being substantially higher in men. The current study aims to explore the independent connection between cigarette smoking and blood lipid levels in smokers and non-smokers, according to the Organization for Economic Cooperation and Development Health Statistics 2017, which is still higher than the average of 23 percent [6]. The aim of this study was to determine the impact of smoking on the lipid profile and a few blood parameters in serum samples taken from a Libyan population.

Sampling and Preparation of Blood Samples
In this study, two separate groups of serum blood samples were used. The first group consisted of fifty samples taken from male smokers, whereas the second was made up of non-smokers. Blood is extracted from veins and placed in blood tubes. The serum was extracted from the cells by centrifugation at 3000 rpm for a few minutes and then stored in the freezer after the Complete Blood Count (CBC) was measured. There were no major health issues among the participants.

Nicotine Content
The nicotine concentration of blood samples was tested using the following method: Based on [7], the extraction processes were carried out with a little modification. A screw-capped glass test tube was used to aliquot 0.5 mL of plasma. 200 l of 2.5 M NaOH was added, and the mixture was stirred for 5 minutes at 3000 rpm. For one-step single extraction, a 10 ml mixture of dichloromethanediethylether (1:1 v/v) was utilized, followed by vortex mixing at 3000 rpm for 5 minutes. After centrifugation at 3000 rpm for 2 -4 minutes, the organic layer was transferred to a fresh glass tube containing 40 l of 0.25 M HCl.
The organic phase was then evaporated at 35˚C until dry, and then reconstituted to 2 ml with a mixture of 0.2973 g KH2PO4, 820 ml distilling water, and 180 ml methanol. The plasma samples were then analyzed using a UV-Visible Spectrophotometer at 258 nm, and the nicotine content of the samples was calculated using the standard curve of standard nicotine solutions.

Lipid Profile Test
Cholesterol, Triglycerides, and HDL-Cholesterol values in the blood were provided. All lipid profile tests and complete blood counts (CBC) were calculated using a kit approach on an automated analyzer in all patients (Spectrophotometer, 4040 V5).

Statistical Treatments
The soft ware computer program of (R program, version, 2013) was used for draw the Figure and

Nicotine
For the smokers and non-smokers blood tests, respectively, the nicotine substance was modified within the ranges of (11 -52 µg/ml) and (0.9 -9.07 µg/ml), as shown in Table 1 & Table 2 and Figure 1. The findings revealed high levels of nicotine in smokers' blood tests, which can be attributed to a variety of factors such as cigarette type, age, nicotine intake, and certain disorders. This study used the smoking period as the most important factor in comparing the nicotine substance in the various tests. As a result, the study recorded the relative impact of the smoking period on the nicotine substance. On the other hand, in nonsmokers' tests, moo substance of nicotine emerged, as did the proximity of high amounts of nicotine [8].
It has been claimed that meals or foods may have an impact on nicotine levels in the human body; nonetheless, Because of the high degree of hepatic extraction, nicotine clearance should be influenced by liver blood flow. As a result, physiological events such as meals, posture, exercise, or medicines that disrupt hepatic blood flow are expected to alter nicotine metabolism. Meals ingested during a steady state nicotine infusion result in a continuous decrease in nicotine concentrations, with the maximum effect occurring 30 -60 minutes after the meal ends [9]. After a meal, hepatic blood flow increases by around 30%, and nicotine clearance increases by about 40%.
The kidney is also one of the most important components that has a significant impact on nicotine levels in human body tissues. According to some research, nicotine is expelled by glomerular filtration and tubular secretion, with varying re-absorption depending on pH. Renal clearance of nicotine ranges between 35 and 90 ml min1 when pH is not regulated, accounting for around 5% of total clearance. Nicotine is mainly ionized in acid urine, and tubular re absorption is reduced; renal clearance can reach 600 ml min1 depending on urinary flow rate [10]. A greater proportion of nicotine is unionized in alkaline urine, allowing net tubular re absorption with a renal clearance as low as 17 ml min 1 (urine pH 7.0).
Furthermore, several researches have found a link between nicotine and genetic variables, with hereditary contributions to nicotine and cotinine renal clearances being assessed in a twin study. These data show that, even in the presence of net re absorption, nicotine and cotinine re absorption are active processes regulated by the genetics of re absorptive transporters, or that the active secretor component of renal clearance has a significant impact on clearance. It's possible that the hereditary component of nicotine re absorption clearance is determined by the matching variable in re absorption transporters. Renal dysfunction, as previously indicated, affects overall renal clearance as well as nicotine and cotinine metabolic clearance [9].
In this investigation, the contents of nicotine differed by smoking group and smoking period, with significant amounts of nicotine detected in several samples after ten years of smoking (Table 1), and a high level (52 g/ml) detected after thirty years of smoking (Table 2). Low levels of nicotine have also been linked to disorders such as (nicotine metabolism), which may be exacerbated in the elderly by lower liver blood flow, despite the fact that no decrease in liver protein levels or nicotine metabolism in liver microsomes has been identified as a result of age. The nicotine levels in non-smoking blood samples indicated no variations in steady nicotine plasma levels, as illustrated in Figure 1, Box blot, where the nicotine contents were between (1 and 2).
According to some research, dietary sources of nicotine may be a potential confounder of cotinine (one of nicotine's isomers) levels utilized in secondhand smoke exposure testing. Nicotine can be found in a variety of foods [8]. Nicotine levels in meals, on the other hand, are fairly modest. The amounts of cotinine produced by a diet high in nicotine-containing foods are lower than those reported in individuals exposed to moderate levels of secondhand smoke, according to nicotine levels in foods and the normal daily consumption of various nicotine-containing meals. In general, the new study supports prior research that found a direct link between smoking and nicotine levels in blood samples [10].

The Hematological Parameters
The collected findings showed that the contents of various CBC values in the smokers blood samples were ranged as follows:  Table 4).

WBC
Increases in WBC and platelet (PLT) counts have been seen in several studies as one of the acute effects of smoking on the hematological system [11]. A relationship was discovered between smoking and WBC levels. WBC counts were found to be greater in smokers' blood samples. This was also observed in this investigation, where greater WBC (9.82 4.43) values were found in smokers' blood samples compared to WBC (6.78 1.59) in non-smokers' blood samples, (Table 3). Additionally, the correlation matrix revealed a relative positive connection between nicotine and WBC (0.12), Table 5. Many studies have concluded that the WBC count is arguably the most useful, inexpensive, and straightforward biomarker for endothelium injury, and the current findings are in line with that conclusion. In certain research, it was also discovered that the median total leukocyte.  According to several research, nicotine, which is a component of cigarette smoke, promotes catecholamine release and causes cortisol levels to rise [12]. Direct damage resulting from modifications in epithelial and endothelial surfaces and/or cytokine levels (particularly IL-6) produced by components of cigarette smoke can cause increases in peripheral blood WBC counts and alterations in WBC function (Smith et al., 2003). The link between smoking and the number of white blood cells in the blood has been well documented [13]. Smokers have larger white blood cell counts than nonsmokers, according to a number of studies [14].

Hemoglobin (HGb)
In this study, the hemoglobin values for smokers and non-smokers blood samples varied between (10.1% -17.5%) and (13.1% -16.1%). However, many smokers' blood samples showed relative higher hemoglobin values, such as 17. 5, 16.80, 16.50, 16.45, and 16.20, whereas most non-smokers' samples showed hemoglobin values below 16 percent. The high HGb values are due to smoking, and the correlation matrix showed a relative positive (0.07) relationship between nicotine and hemoglobin (Table 5), which is consistent with many studies [15].
Smoking active tobacco cigarettes, both acute and chronic, has been shown to raise white blood cell counts. In addition, past epidemiological researchers have found that long-term passive tobacco cigarette smoking can raise white blood cell counts.
WBC, red blood cells, Hb, and HTC levels were shown to be significantly higher in a study on the effects of smoking on hematological parameters, but MCV and MCH counts were reduced [16]. These changes have been linked to atherosclerosis, polycythemia vera, chronic obstructive pulmonary disease, and cardiovascular disorders, as well as an increased risk of atherosclerosis, polycythemia, COPD, and cardiovascular disease in smokers [17].

Hematocrit (HCT) %
Ulmonary disease and cardiovascular disease are two of the most common dis-eases. The hematocrit values of smokers' blood samples ranged from 28.7 to 52.5%, while non-smokers' blood samples had hematocrit values ranging from 39.0 to 47. The effects of smoking on HCT levels were also observed in certain smokers' blood samples, with values such as 52.5, 51.4, 47.3, 46.9, 46.7, and 44.9 being reported. On the other hand, the HCT of non-smoker blood samples was often below 45 percent, with the exception of one sample (50 percent). In addition, there is an increased risk of atherosclerosis. Some studies indicated that smoking has a direct effect on hematocrit levels in blood samples; it was also noted that smokers' hematocrit values were significantly higher than non-smokers', which was consistent with prior findings [18]. Carbon monoxide (CO) is formed by incomplete combustion of carbon-containing materials in cigarette smoking, according to reports. In comparison to oxygen, CO has a very high affinity for hemoglobin. In smokers, higher levels of hematocrit and hemoglobin have been seen, and these increases are likely compensatory for CO exposure. Smokers have higher hematocrit and hemoglobin levels, which may lead to a hypercoagulable state [19].

RBC, PLT and other CBC Parameters
For smoker blood samples, RBC and PLT values varied from (3.5 -6.6) and (139 -603), respectively, while non smoker blood samples had RBC and PLT values of (4.9 -5.9) and (168 -285). Smoking has been found to raise neutrophil, lymphocyte, monocyte, platelet counts (PLT), and RBC indexes in both men and women. This is consistent with the results of the current study, which show that high PLT values were found in many samples (243.8 ± 84.26), while non-smokers' PLT values fell on average (229.3 ± 30.07).
The correlation matrix (Table 5) revealed that PLT and nicotine (0.12) had relative positive values. In addition, the length of time spent smoking has an effect on some RBC levels in smokers' blood samples. When comparing smokers to nonsmokers, RBC values are generally higher in smokers [20]. The current CBC parameters agree with many studies on the effect of smoking on hematology blood analysis, where some studies comparing smokers and nonsmokers have shown increases in Hb, HCT, RBC, MCV, WBC, neutrophil, lymphocyte, eosinophil, and monocyte counts in both groups [21], investigated the impact of smoking on RBC, WBC, and Hb, and indicated increases in WBC counts and decreases in RBC, With a small reduction in the daily number of cigarettes smoked, the detrimental effects of smoking on hematological parameters improve. The correlations matrix between nicotine and the examined hematological parameters revealed that nicotine had a positive association with WBC, HGB, and MCHC, but was somewhat negative with RBC, MCV, and MCA. Different correlation coefficient values between nicotine and hematology blood measures show the presence of additional chemicals in cigarettes that may influence hematology blood parameters.

The Lipid Profile Parameters Results
The contents of lipid profile parameters of smokers and non-smokers are listed

Effect of Cigarette Smoking on Lipid Profile
Lipids are involved in practically every element of biological existence. Serving as hormones or hormone precursors that aid digestion, provide energy, storage function, and metabolic fuels; operating as functional and structural chemicals in bio membranes; and producing insulation to facilitate nerve conduction or prevent heat loss are just a few of these roles. . Cigarette smokers had significantly higher serum triglycerides than non-smokers, according to the current study. This was also discovered in a study, which discovered a significant decrease in HDL levels in smokers' blood samples. According to this study, nicotine raises the quantity of bad fats (total cholesterol (TC) and triglycerides (TG) circulating in the blood arteries while lowering the amount of good fat (high-density lipoprotein cholesterol (HDL-C) available. It has been claimed that smoking has a negative impact on plasma lipid concentrations and lipoprotein levels [22] [23]. In addition, total cholesterol, HDL C and TG levels in smokers are significantly higher than in nonsmokers, and the correlation matrix (Table 8) revealed a significant positive correlation between cholesterol and nicotine (0.25), as well as positive correlations with TG and HDL with values (0.06 and 0.04), respectively. It was shown that cigarette smokers have a higher risk of cancer. When the results are analyzed in terms of cigarette smoking duration, it is discovered that there is a significant increase in serum cholesterol levels as the duration and intensity of cigarette smoking increases. Our findings are contradicted by the HDL-Cholesterol In this study, smokers had significantly higher TC and TG levels than nonsmokers, while smokers had significantly lower blood HDL levels than nonsmokers. Our findings are consistent with those of many other researchers. The difference in serum cholesterol and lipoprotein levels were more pronounced as the number of years spent smoking increased. This conclusion has been supported by [23] [24]. Contrary to the findings of the previous study [24], no significant changes in serum TG, or HDL levels were seen between smokers and nonsmokers. In several studies, no significant differences in triglycerides or total cholesterol were reported between smokers and nonsmokers. Previous studies [25] have suggested that these disparities are linked to ethnic variance in the population.

Conclusion
The data revealed that the contents of hematological parameters differed between smokers' and nonsmokers' blood samples, with high counts of WBC, MCHC, HGB, and PLT plainly visible in smokers' samples. However, there was no discernible effect on RBC, MCV, or MCH counts in the trial. Our research clearly demonstrates a robust link between serum cholesterol and HDL levels and cigarette smoking. It also emphasizes that as the duration and intensity of smoking increase, so do the changes in serum lipids.