The effects of smoking cessation on the ratios of neutrophil/lymphocyte, platelet/lymphocyte, mean platelet volume/lymphocyte and monocyte/high-density lipoprotein cholesterol

INTRODUCTION

Cigarette smoking is an important and independent risk factor for atherosclerosis, coronary artery disease, peripheral vascular disorders, cancer, etc.¹ It is assumed that the negative effects of smoking are due to the changes in inflammation, oxidative stress, antithrombotic and fibrinolysis systems.2 It is known that smoking has negative effects on the haematopoietic system and lipid parameters, and many studies have shown that smoking causes an increase in haemoglobin, haematocrit, leucocyte, eosinophil and platelet levels in peripheral blood and lipid parameters except high-density lipoprotein (HDL).^1,3 Nicotine increases the release of catecholamines and steroids from the adrenal gland, causing an increase in the number of leucocytes. The inflammation caused by the tissue destruction effect of cigarette smoke on the lung parenchyma also contributes to the increase in leucocyte levels.⁴

Studies have shown that the levels and proportions of cells (e.g. leukocytes, neutrophils, thrombocytes) such as complete blood count can be used as a marker in the detection of systemic inflammation, which is known to play a role in the pathogenesis of many diseases. Neutrophil/lymphocyte ratio (NLR) is a combined marker obtained by the use of absolute neutrophil and lymphocyte counts.⁵ Platelet/lymphocyte ratio (PLR), which is the combination of both parameters, is seen as a mortality determinant in cardiovascular and lung diseases and some malignant diseases.⁶ PLR provides insight into aggregation and inflammation pathways and is considered more valuable than either platelet or lymphocyte count alone in predicting coronary atherosclerotic burden. Therefore, these markers are used as effective, simple and inexpensive laboratory parameters for the diagnosis and prognosis of coronary artery disease even in asymptomatic individuals.⁷

Mean platelet volume (MPV) is a potential marker of platelet activation and MPV levels are elevated in thromboembolic diseases.⁸ Many studies have shown that MPV/lymphocyte ratio (MPVLR) is also a negative inflammatory marker in cardiovascular diseases and some malignant diseases.^9,10 Red cell distribution width (RDW) is an indicator of the size of erythrocytes, and it has been reported that, in addition to the differential diagnosis of anaemia, it can be used as a mortality indicator in acute heart failure, pulmonary embolism, acute myocardial infarction, peripheral artery disease and acute renal failure in the general population.¹¹

Monocyte/HDL ratio (MHR) is a new cardiovascular prognostic marker reported in recent studies.¹² It has also been reported that the MHR rate is higher in smokers compared to non-smokers and the degree of this increase correlates positively with the duration and amount of smoking.¹³

Some studies have examined the effects of smoking on the haemostatic, fibrinolytic, antioxidant and haematological systems, and compared smokers and non-smokers in this regard. However, to the best of our knowledge, no study has investigated the effects of cessation of smoking on NLR, PLR, MPVLR and MHR values in individuals who quit smoking. We investigated the changes in haemogram values, blood lipid levels and NLR, PLR, MPVLR and MHR values in smokers after cessation of smoking.

METHODS

In this self-controlled experimental study, we included individuals who enrolled at the smoking cessation outpatient clinic of the Family Medicine Department, Training and Research Hospital, Karabuk University between January and December 2018. Their sociodemographic and clinical characteristics such as age, gender, education, employment status, history of alcohol use and comorbid illness, and the amount of cigarettes they consumed (pack/year), their Fagerstrom test for nicotine dependence (FTND) results, haemogram values before and 6 months after cessation of smoking, NLR, PLR, MPVLR, MHR and blood lipid levels were retrospectively obtained from the patient files and hospital automation system. Those with missing data in their files and blood tests, those who did not come for follow-up and who could not quit smoking were not included in the study. The parameters were evaluated in terms of the amount of cigarettes smoked, the level of addiction, and whether there were any changes before and after cessation of smoking.

FTND is a widely used scale for determining nicotine addiction. It consists of 6 questions which yield a total score where 0–2 points refer to very low addiction, 3–4 points to low addiction, 5 points to medium addiction, 6–7 points to high addiction and 8–10 points to very high addiction.¹⁴

IBM SPSS software package (v.22.0) was used for statistical analysis. Paired t-test was used for normally distributed values, and Wilcoxon signed-rank test for non-parametric data. The chi-square test was used for comparison of categorical data. Statistical significance was set at p<0.05.

The study was conducted with the approval of the Karabuk University Non-Interventional Clinical Studies Ethics Committee (Approval No: 2020/198, 27/02/2020).

RESULTS

Between January and December 2018, 457 people applied to the smoking cessation outpatient clinic of the Family Medicine Department, Training and Research Hospital, Karabuk University. Of these, 74 could not quit smoking, 98 did not come for follow-up and 46 had incomplete examinations. Hence, 239 individuals who met the study criteria were included. Of these, 55.2% (132) individuals were women. Their mean (SD) age was 41.7 (10.9) years, 66.1% of them were in the age group of 31–50 years, 54.9% were college graduates and 77% were working in a job. The alcohol use rate was 5% and 27.7% had comorbid conditions. The mean FTND score of the study group was 7.06 (2.0), and 47.7% of them were highly addicted. The mean number of cigarettes consumed was 18.1 (11.2) packs/year and 42.3% had been smoking for 11–20 years (Table I).

When the individuals’ blood tests before and 6 months after quitting smoking were compared, a significant decrease was observed in their haemoglobin, haematocrit, mean corpuscular volume (MCV), white blood cells (WBC), red blood cells (RBC), neutrophil, platelet, MPV, RDW, platelet distribution width (PDW), total cholesterol, triglyceride, low-density lipoprotein (LDL) levels; and a significant increase in their HDL levels compared to those before quitting smoking. It was also observed that there was a statistically significant decrease in their NLR, PLR, MPVLR and MHR after cessation of smoking compared with before cessation (Table II).

We found a negative correlation between the cigarette pack/year and FTND addiction levels and the change in HDL after cessation of smoking. There was a positive correlation between the cigarette pack/year and FTND levels and the changes in haemogram values, other blood lipid parameters, NLR, PLR, MPVLR and MHR after smoking cessation (Table III).

DISCUSSION

We found that, after smoking cessation, there was a significant decrease in haemoglobin, haematocrit, MCV, WBC, neutrophil, platelet, MPV, RDW, PDW, cholesterol, triglyceride, LDL levels, NLR, PLR, MPVLR, and MHR, and a significant increase in HDL levels. We also observed that the amount of smoking and the level of dependence were negatively correlated with HDL, and positively correlated with haemogram values, other blood lipid parameters, NLR, PLR, MPVLR and MHR.

Cigarette smoke contains more than 4000 toxic agents that have negative effects on human health. The carbon monoxide formed by smoking causes a decrease in the oxygen-carrying capacity of haemoglobin by binding to haemoglobin faster than oxygen, and this leads to impaired oxygenation of tissues and changes in haematological parameters. Hence, haemoglobin levels increase to compensate for the reduced oxygen-carrying capacity in smokers.¹⁵ Free radicals and peroxides from cigarette smoke affect the synthesis of prostaglandins and thromboxane and play a role in the pathogenesis of various diseases, including atherosclerosis, carcinoma and inflammatory processes.¹⁶ Fricker et al. reported that cigarette consumption reduced tissue oxygen supply and caused systemic hypoxia.¹⁷ Malenica et al. reported that haemoglobin and MCV levels were significantly higher in smokers compared to non-smokers, but there was no difference in RDW level.⁴ While Alkhedaide reported that smoking caused a significant increase in RBC, haemoglobin and haematocrit levels,¹⁸ Whitehead et al. found a significant increase in haemoglobin, RDW, haematocrit and neutrophil values in smokers compared to non-smokers.19 Similarly, in our study, a significant decrease was found in the individuals’ haemoglobin, haematocrit, MCV, RBC and RDW levels 6 months after cessation of smoking.

We found that there were significant changes in WBC levels after quitting smoking. It is known that the leucocyte and neutrophil counts are also higher in smokers and it has been assumed that the atherogenic effect of smoking may be partially mediated by leucocytes.⁴ The total leukocyte count encompasses several cell types such as granulocytes, lymphocytes and monocytes, all of which potentially play a different role. While lymphocytes represent a more favourable immune response, neutrophils cause a destructive inflammatory reaction. NLR, a composite marker derived from the use of absolute neutrophil and lymphocyte counts, is a well-studied marker for survival in patients with cancer and cardiovascular diseases.^20,21 Aula and Qadir showed that there was a significant increase in the smokers’ leucocyte, neutrophil, eosinophil, basophil, lymphocyte and monocyte levels compared to non-smokers.²² In a similar study by Kumar et al., a significant increase in neutrophil and eosinophil counts was reported in smokers.23 We found that, 6 months after cessation of smoking, neutrophil and lymphocyte levels and consequently the NLR decreased significantly. Fest et al. found that NLRs were higher in smokers, elderly patients and those with a history of diabetes, cancer or cardiovascular disease. However, they reported that smoking was an important confounder and after adjustment for this factor, only a part of the association between NLR and mortality was explained by smoking.⁵

Among the markers of inflammation involved in atherosclerosis, increasing importance has been given to circulating blood components such as subtype leucocyte and platelet counts, and future cardiovascular events in healthy subjects.^5,7,24 Similar to NLR, PLR is also increasingly being referred in clinical cardiovascular practice as a marker of inflammation, since these ratios can reflect an acute episode of inflammation and acute physiological stress.^24–26 In a study on secondary polycythaemia and leucocytosis in smokers, it was emphasized that tobacco smoking could cause inflammatory conditions in both alveolar tissues or a vascular damage that leads to an immune response demonstrated by leucocytosis and lymphopoiesis.¹⁸ According to recent studies, PLR is seen as a determinant of mortality in heart, lung diseases and some oncological diseases, while studies on atherosclerotic coronary artery disease, which is accepted as a chronic subclinical systemic inflammatory disease, reveal a significant relationship between cardiovascular risk and NLR and PLR.⁶ It was also reported that increased platelet activation plays an important role in the initiation and progression of atherosclerosis.²⁷ High MPV, indicating platelet activation, indicates large platelets. Large platelets have more granules and higher thromboxane A2 levels, and aggregate faster with collagen.²⁵ Since large platelets are more active metabolically and enzymatically, platelet diameter reflects platelet activity better than platelet number.^28,29 Braekkan et al. reported that the platelet count had no effect on the risk of deep vein thrombosis, but the risk increased with high MPV levels, and that identification of MPV as a risk factor for deep- vein thrombosis suggested that platelet size may be a common risk factor for both arterial and venous thrombosis.²⁶ There are many studies showing that chronic smoking causes platelet activation.^30,31 Gumus et al. reported that, in the smoker group, they found an increase in the NLR, a decrease in the PLR, and an increase in the MPV/platelets ratio which indicates risk of thromboembolism.³ Tulgar et al. found that NLR and PLR were significantly higher in smokers, but there was no difference in the MPV level.³² Many studies have reported increased MPV levels especially in coronary artery patients and asserted that high MPV levels are an independent risk factor for myocardial infarction and stroke.^11,12,26 We found that the levels of platelets, PLR and MPV significantly decreased after cessation of smoking. Varol et al. reported that MPV levels were higher in smokers compared to non-smokers, and the MPV levels of those who quit smoking during the study period decreased significantly compared to the basal levels 3 months after quitting.²⁵

MPVLR has been proposed as a new biomarker for inflammatory responses and thrombosis.²⁸ As mentioned above, large platelets are active metabolically and enzymatically. The larger-sized platelets do not only accelerate the formation of coronary artery thrombosis but also exacerbate the inflammatory response of the body.³³ A lymphocyte is one of the earliest cells involved in the formation of atherosclerotic plaque, and the reduction of lymphocytes contributes to the growth and rupture of the plaque and expansion of the infarcted area.²³ Hudzik et al.³⁴ found that patients with elevated MPVLR had worse angiographic features, which may indicate a greater thrombus burden. They concluded that elevated MPVLR was an independent risk factor of early and late mortality following ST- segment elevation myocardial infarction. Chen et al. found a significant negative correlation between MPVLR and NLR levels and left ventricular ejection fraction.³⁵ Sahinli et al. found that both disease-free and overall survival of patients with elevated MPVLR were worse than those with low MPVLR.³⁶ We found that MPVLR levels, which were reported to be a negative inflammatory marker, especially in cardiovascular and some malignant diseases, significantly decreased after cessation of smoking.^9,10 These results support the effect of smoking on inflammation, oxidative stress and alterations of antithrombotic and fibrinolysis systems.

Inflammation and oxidative stress are well-known mechanisms in the development and progression of atherosclerosis. Monocytes and macrophages play a key role in the development and progression of atherosclerosis. Following endothelial dysfunction, monocytes adhere to the endothelium tightly and migrate into the subendothelial layer. They then maturate into macrophages that ingest oxidized LDL cholesterol and form dangerous foam cells. Foam cells secrete pro-inflammatory cytokines that induce the local inflammatory response around the atherosclerotic lesion.^12,37 Contrarily, HDL molecules counteract the migration of macrophages and promote efflux of oxidized cholesterol from these cells.³⁷ There is also an interaction between HDL cholesterol and monocytes, which limits the inflammatory response that occurs while monocytes differentiate into macrophages.¹² Therefore, monocytes show a pro-inflammatory effect, but HDL cholesterol functions as a reversal factor during this process. MHR, the ratio of monocyte count to the HDL cholesterol level, was defined to be an easy calculable cardiovascular prognostic marker indicating the extent of the inflammation and oxidative stress.³⁸ Cigarette smoking is an important and independent risk factor for atherosclerosis, coronary artery disease, peripheral vascular disorders, etc. and several studies provide evidence that tobacco is strongly associated with altering the normal status of the lipid profile.^39,40 In addition, many studies have shown that smoking is associated with systemic inflammatory response and vascular endothelial damage.^13,41,42 Anandha Lakshmi et al. reported higher levels of total cholesterol, triglyceride, LDL and VLDL; but lower HDL levels in smokers compared to non-smokers.¹ Ito et al. reported low levels of HDL in smokers.⁴³ Y1 lmaz and Kayançiçek, found negative changes in blood lipid parameters and increased MHRs in smokers, and they asserted that MHR may be used as a surrogate marker of inflammation and endothelial dysfunction in smokers.³⁸ In our study, 6 months after quitting smoking, a significant decrease was found in total cholesterol, triglyceride, LDL levels and MHRs, and a significant increase in HDL levels. These results are consistent with the results of similar studies and may show that the inflammatory and atherosclerotic effects improve upon quitting smoking.

In our study, high levels of cigarette pack/year and FTND were positively correlated with changes in haemogram values, blood lipid parameters, NLR, PLR, MPVLR and MHR after smoking cessation. Anandha Lakshmi et al.¹ found haematocrit and haemoglobin levels to be significantly higher in smokers, and among smokers the RBC count was found to significantly increase as the intensity of smoking increased. Similarly, in the study by Y1 lmaz and Kayançiçek, a positive correlation was found between plasma lipid parameters and MHRs and cigarette pack/year.³⁸ These results show that the more the cigarettes consumed, the greater the negative effects due to smoking.

The limitation of our study is that it is a single-centre and retrospective study. As this is a self-controlled and retrospective clinical trial, the results were not compared with age- and gender-matched non-smoking normal controls and the clinical implications of observations and potential for intervention has not been assessed. The strength of our study lies in that we studied the changes in NLR, PLR, MPVLR and MHR together after quitting smoking.