Smoking leads to 30% of all cancer deaths in developed countries (1). Lung cancer is the major malignancy caused by smoking (2). Smoking is also the most significant factor associated with chronic obstructive pulmonary disease (COPD) (3). Tobacco contains many free radicals and oxidants that damage airway epithelium, causing airway inflammation. Moreover, reactive oxygen species such as hydroxyl radicals, hydrogen peroxide, superoxide, and nitric oxide are produced during inflammation (4). DNA, proteins, carbohydrates, and lipids are oxidatively damaged by these reactive oxygen radicals (5). The cumulative effect of all oxidants is defined as TOS (6). Cells have developed antioxidant defense mechanisms to remove these harmful oxidative reactions. These mechanisms consist of enzymes such as catalase, superoxide dismutase, paraoxonase, and glutathione peroxidase and vitamins such as ascorbic acid, β-carotene, and tocopherols (7). TAS measures all these serum antioxidants (8). The ratio of TOS to TAS levels is indicated as the oxidative stress index (OSI).

Several studies suggested that cigarette smoking affects collagen metabolism and tissue remodeling (9, 10). It causes inflammation of the airways and then thickening of the walls due to scarring and remodeling. Further progression leads to the narrowing of the small airways. Therefore, tissue remodeling and controlled alterations of extracellular matrix (ECM), in which matrix metallopeptidases (MMPs) are involved, are necessary. Prolidase (EC. 3.4.13.9), a member of the MMP family, hydrolyzes the imidodipeptides at the C-terminal proline or hydroxyproline (11). It plays a very important role in collagen metabolism and matrix remodeling. Prolidase activity was analyzed in various diseases such as cardiac hypertrophy (12), type 2 diabetes mellitus (13), osteoarthritis (14), chronic liver disease (15), chronic uremia (16), and bronchial asthma (17). In addition to an imbalance in the oxidant-antioxidant status, prolidase might also be associated with inflammation caused by cigarette smoking. To date, serum prolidase levels in smokers have not been investigated adequately. In the present study, serum prolidase levels of smokers were compared to nonsmokers to elucidate its role in inflammation associated with cigarette smoking. The correlation between the oxidative-antioxidative status and serum prolidase levels in these patients was also examined.

The present study involved 38 healthy smokers (19 male and 19 female) and 38 healthy nonsmokers (18 male and 20 female) with an average age of 30.05 ± 7.05 years. There was no statistically significant difference between the groups in terms of age. The male group had a higher BMI than the female group (p = 0.013). The mean number of packages smoked per year was 8.73 ± 6.02 packages/year. As expected, the male group smoked more packages on average than the female group (p = 0.02) (Table 1). Serum cotinine levels were analyzed to determine the degree of cigarette smoke exposure and the amount of nicotine ingested. Higher levels of cotinine were found in the smoker group in comparison to the nonsmokers (p < 0.001). Plasma prolidase level was significantly lower in the smoker group than in the control group (p < 0.001) (Figure 1).

Smokers were found to have considerably lower serum prolidase and TAS levels than non-smokers (p < 0.001, p = 0.012 respectively) (Table 3). However, no differences were determined in TOS levels between the two groups. OSI levels were significantly higher in the smoker group (p < 0.001).

Both smokers and nonsmokers showed a positive correlation between serum prolidase and TAS levels. Moreover, serum prolidase and TOS levels were found to have a negative correlation. However, the difference was not statistically significant (Table 4). A statistically significant negative correlation was determined between prolidase and OSI levels (r = −0.629). This negative correlation was more evident in the male smokers (r = −0.601). Moreover, prolidase and cotinine levels also had a statistically significant and negative correlation (r = −0.739) (Figure 2). Even though this correlation was not statistically significant in nonsmokers, the smokers showed a significant negative correlation (r = −0.380). Furthermore, the negative correlation between prolidase and cotinine levels was higher in the male smokers when compared to the female smokers (r = −0.435).

No significant correlation could be found between prolidase levels and spirometric values FEV₁, FVC, PEF, and FEF25-75%. However, the correlation between FEV1/FVC and prolidase levels was negative (r = −0.293, p00.01). This negative correlation was at a higher level in the nonsmokers than the smokers (r = −0.361). Particularly, it was more evident among the male nonsmokers (r = −0.549, p = 0.016) (Table 4).

There are 10¹⁷ oxidant molecules in every cigarette puff (21). Exposure to these oxidants in cigarette smoke causes airway epithelial injury. Numerous health issues, including cancer, cardiovascular disease, and pulmonary disorders, are influenced by this elevated oxidative stress (21). Ceylan et al. demonstrated that one of the main causes of COPD is the increase in oxidative stress (22). Gencer et al. detected high levels of plasma lipid peroxidation in COPD patients (23). Moreover, an imbalance in the oxidant-antioxidant activity leads to pathological processes affecting airways. As stated by Beck-Speier et al., free radical-mediated pathologies such as ischemia–reperfusion and asthma are often influenced by the imbalance between oxidative stress and antioxidant system (24). In the present study, TAS levels of smokers were lower than those of nonsmokers (p = 0.012). The smokers had significantly higher OSI levels (p < 0.002). The results achieved in the present study are consistent with those reported by Mahmood et al., who observed that TAS values in smokers were significantly lower than those in nonsmokers (25). Tavilani et al. observed that plasma TAS levels of patients with COPD and smokers were significantly lower than the nonsmoker group (4). This decrease in the total antioxidant capacity may be related to the increased oxidant production that consumes antioxidants. Gilks et al. reported that antioxidant enzyme levels may decrease severely during exposure to oxidants repeatedly (26). However, serum TOS levels did not differ between the smokers and the nonsmokers in the present study. In a study carried out by Aslan et al., the difference between smokers and nonsmokers in terms of serum TOS levels was not statistically significant (5). Zinellu et al. reported that the bronchoalveolar lavage fluid of COPD patients showed an increase in several oxidative stress biomarkers in contrast to healthy controls. On the other hand, the analysis of serum oxidant levels did not yield any difference (27). The higher concentrations of these oxidants in bronchoalveolar lavage fluid may be observed in the lungs because of direct exposure to oxidants of cigarette smoke and their short lifespan may not be sufficient to affect systemic levels, as shown in the present study.

Smoking also down-regulates MMPS concentration. Raitio et al. showed that smoking decreased the amount of MMP-9 in skin biopsy samples (28). Smokers have less hydroxyproline accumulation. Prolidase, a MMP family member, is essential for the remodeling of the ECM and the metabolism of collagen and proline recycling is crucial for collagen synthesis. Collagen is an important component of lung tissue and disruption of the collagen cycle can impair the structural integrity and function of the airways. Gencer et al. reported significantly reduced prolidase activity in COPD patients when compared to controls (23). In the study carried out by Ekin et al., prolidase activity decreased in patients with COPD (29). Ergin et al. showed that prolidase activity in patients with COVID-19 was statistically significantly lower when compared to healthy individuals (30). Uysal et al. demonstrated that prolidase activity might contribute to determining the severity of COPD (31). However, in a study carried out by Kaleli et al., prolidase activity was found to be at a higher level in bronchial asthma (17). Türkbeyler et al. detected enhanced prolidase activity in an experimental lung fibrosis model (32). As stated by Gumus et al., pulmonary tuberculosis was associated with increased serum prolidase activity and patients with cavitary disease had higher serum prolidase activity than patients with noncavitary disease (33). In the present study, significantly lower serum prolidase levels were found in the smokers (Figure 1). This decrease in prolidase levels suggests a potential alteration in collagen recycling and ECM remodeling processes in smokers, which may contribute to the pathogenesis of lung diseases, especially those associated with smoking.

The control group was formed by questioning the passive smoking status of the volunteers and blood cotinine level was studied to confirm this. None of the groups in this study was using electronic cigarette. A popular technique for substituting tobacco consumption is the electronic cigarette. Electronic cigarette smoke contains nicotine. It induces changes in oxidative stress and serum prolidase levels (34). Moreover, thirdhand smoke refers to the residual tobacco smoke contaminants that remain on surfaces and in dust after the cigarette has been extinguished. This can include substances such as nicotine, heavy metals, and various chemicals present in tobacco smoke. Even though the visible smoke is gone, these harmful substances can linger on surfaces such as clothing, furniture, walls, and carpets. Third-hand smoking was overlooked in the present study. After the small number of cases, this is one of the biggest limitations of the present study. It’s important to acknowledge the limitations of this study, such as its cross-sectional design and the potential influence of unmeasured confounders. Future research should explore the longitudinal effects of thirdhand smoking on oxidative stress markers and prolidase activity. Cotinine levels were at the upper limit of normality in all cases and were statistically significant in smokers (p < 0.001) (Table 3). Moreover, a strong negative relationship was discovered between prolidase and cotinine levels (r = −0.739). This negative correlation is more pronounced in male smokers, suggesting a potential gender difference in the effect of smoking on collagen metabolism and oxidative stress (r = −0.435) (Figure 2). This also suggests that prolidase levels and oxidative stress parameters change even in the case of passive smoking and thirdhand smoking. In the present study, both smokers and nonsmokers showed negative correlations between TOS levels and prolidase levels and positive correlations between TAS levels. However, these correlations were not statistically significant.

Even though no significant correlation was found between spirometric values (FEV1, FVC, PEF, FEF25-75%) and prolidase levels, there was a poor and negative correlation between FEV1/FVC ratio and prolidase levels, particularly in nonsmokers. This suggests that changes in collagen turnover, as reflected by changes in prolidase activity, may affect airway function. Further studies with larger sample sizes are necessary to elucidate the relationship between prolidase levels and lung function parameters.

Despite several studies in recent years, there are many conflicting data on prolidase levels in diseases. To date, serum prolidase levels have not been investigated adequately in smokers. The present study compared serum prolidase levels between smokers and nonsmokers to elucidate its role in inflammation associated with smoking. It was aimed to show whether serum prolidase levels can be regarded as a marker of inflammation in cigarette smoking and to determine the relationship between prolidase and oxidative-antioxidative status. The results achieved in this study indicate that cigarette smoking decreased serum prolidase levels. This may be interpreted as decreased collagen turnover. Smoking led to an imbalance in the activity of antioxidants and oxidants. However, there was no statistically significant correlation between serum prolidase levels, TAS, and TOS. Further studies with larger groups may be useful to clarify the effects of cigarette smoking.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving humans were approved by the Ethics Committee of Baskent University Medical School (Project no. KA21/390). The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.

BY: Investigation, Writing – original draft, Writing – review & editing. SK: Validation, Writing – original draft, Writing – review & editing.