Comparison of Serum Prolidase Activity in Smokers and Non-Smokers

By Tahire Betul Kor¹, Basri Furkan Dagcioglu², Aygul Tikit³, Salim Neselioglu⁴

Affiliations

1. Department of Family Medicine, Aksaray Community Health Centre, Family Medicine, Aksaray, Turkiye
2. Department of Family Medicine, Ankara Bilkent City Hospital, Faculty of Medicine, Ankara Yildirim Beyazit University, Ankara, Turkiye
3. Department of Medical Biochemistry, Ankara Bilkent City Hospital, Ankara, Turkiye
4. Department of Medical Biochemistry, Ankara Bilkent City Hospital, Faculty of Medicine, Ankara Yildirim Beyazit University, Ankara, Turkiye

doi: 10.29271/jcpsp.2025.07.859

ABSTRACT
Objective: To investigate the effect of smoking on serum prolidase enzyme activity and proline/hydroxyproline levels.
Study Design: A case control study.
Place and Duration of the Study: Department of Family Medicine, Ankara Bilkent City Hospital, Family Medicine, Ankara, Turkiye, from June 2022 to May 2023.
Methodology: A total of 150 participants were included in the study, including 54 smokers, 43 ex-smokers, and 53 non-smokers. Proline and hydroxyproline concentrations were analysed using a liquid chromatography-mass spectrometre (Sciex QTrap 4500-Foster City-CA). Serum prolidase enzyme activity was determined with the Chinard reagent according to the spectrophotometric test. Multinomial logistic regression analysis compared prolidase activity and amino acid levels among smokers, ex-smokers, and non-smokers.
Results: The median values of prolidase enzyme activity were statistically lower in the smoker group 734.2 (643.8-799.6) than in the non-smoker group 836.8 (755.2-884.2, β = 0.003, OR = 0.997, 95% CI = 0.994-1,000, p = 0.025. In multiple comparisons, the median values of hydroxyproline were significantly higher in the non-smoker group 8.99 (5.76-11.22) than in the ex-smoker 6.05 (4.37-8.21, p = 0.008). Still, no significant difference was found in the multinomial logistic regression analysis (β = -0.051, OR = 0.951, CI 95% = 0.848-1.066, p = 0.384).
Conclusion: Serum prolidase enzyme activity was significantly lower in smokers than in non-smokers. The prolidase activity may be a fruitful area of ​​study to understand the pathophysiology of smoking-related diseases better.

Key Words: Smoking, Prolidase, Proline, Hydroxyproline, Collagen synthesis, COPD, Malignancy.

INTRODUCTION

Approximately 23 percent of the world's population smokes, and smoking is associated with significant public health problems such as cardiac disease, chronic obstructive pulmonary disease (COPD), most malignancies, and reduced fertility.¹ The exact mechanisms of tobacco-related diseases are not yet known. However, inhalation of tobacco smoke increases the level of both exogenous and endogenous free radicals in the body, resulting in the development of oxidative stress. Increased oxidative stress results in vasomotor dysfunction, increased prothrombotic factors, decreased fibrinolytic factors, lipid peroxidation, increased adhesive/inflammatory molecules, and smooth muscle proliferation. Carcinogens in cigarette smoke form molecules called DNA adducts by forming covalent bonds with DNA, thus causing mutations by directly damaging DNA and leading to neoplastic tumour formation.²

Prolidase (peptidase D), encoded by the PEPD gene, is a cytosolic metalloproteinase, responsible for cleaving imido dipeptides containing C-terminal hydroxyproline or proline. Prolidase enzyme is the rate-limiting step in collagen recycling and plays essential roles in protein metabolism, collagen turnover, and remodelling of the extracellular matrix. Therefore, the prolidase enzyme has critical functions in inflammation, wound healing, cell proliferation, angiogenesis, and carcinogenesis.³ While nitric oxide (NO) phosphorylation increases prolidase enzyme activity, inhibition of NO phosphorylation decreases prolidase enzyme activity. Proline and hydroxyproline, which are products of prolidase, take part in collagen synthesis, provide stabilisation by preventing the catabolism of hypoxia- inducible factor (HIF)-la, and activate the transforming growth factor (TGF) receptors (R).³ Proline also regulates and maintains redox balance. It plays a biological and pathophysiological role in cirrhotic liver disease, idiopathic pulmonary fibrosis (IPF), and wound healing by participating in protein synthesis and organ fibrosis related to TGF-β.^4,5

Apart from its catalytic activity, prolidase also functions as a biological regulatory enzyme in cellular regulation. Human epidermal growth factor receptors HER2/ErbB2 and EGFR/ ErbB2 have a receptor function to which prolidase binds. By binding to the extracellular EGFR receptor, prolidase stimulates essential processes such as cell growth and proliferation through some mediators. In addition, prolidase inhibits signal transmission by binding HER2/ErbB2 receptors at high expression levels.⁶ Independent of its catalytic activity, prolidase directly binds to the p53 tumour suppressor protein and regulates its functions.⁷ In addition, prolidase regulates the IFN-I immune response by playing a role in the maturation and trafficking of the type-I interferon (IFN-I) receptor.⁸

The chemicals in cigarette smoke cause activation of β1 integrin, insulin-like growth factor-1 (IGF-1), and TGF-Rs, together with TP53 mutations, which are associated with the development of various cancers.^9-12 Prolidase affects biological processes related to cell proliferation by altering the balance in the TP53 gene and β1 integrin, IGF-1, and TGF receptors.^13,14 The fact that cigarettes and prolidase have similar pathological or physiological effects on these pathways has been effective in the preparation of this study, investigating the relationship between smoking and prolidase enzyme activity. This study was designed to compare the serum prolidase enzyme activity and proline/hydroxyproline levels, which are prolidase products, in smokers, ex-smokers, and non-smokers. In addition, this study aimed to investigate the correlation between serum prolidase enzyme activity, proline/hydroxyproline levels, and pack-years of smoking.

METHODOLOGY

It was a case control study. Patients aged 18 years and older who applied to the Department of Family Medicine, Ankara Bilkent City Hospital, Family Medicine outpatient clinics with any complaint from June 2022 to May 2023 were included in the study. A total of 150 participants were included in the study, including 54 smokers, 43 ex-smokers, and 53 non-smokers. Although power analysis was used to determine the sample size of this study, factors such as the cost of the study, the waiting time for biochemical samples, and the difficulty of recruiting subjects without comorbid diseases were also adequate. The smoker group included individuals who were active smokers for at least six months and smoked at least one cigarette per day. Those who had not smoked for at least six months were included in the ex-smokers group. In the clinical questioning, those who were passive smokers (exposed to cigarette smoke at home, in the working environment, or in the smoking areas of places such as cafes and restaurants if they go frequently) were not included in the ex-smoker group or the non-smoker group. It was thought that conditions such as comorbid diseases and malignancy may cause subclinical inflammation and that medicines used in the treatment of comorbid diseases may affect the possible pathways responsible for collagen synthesis and alter the prolidase enzyme activityprolinehydroxyproline measurement. Those with any chronic disease (hypertension, COPD, diabetes mellitus, chronic heart disease, etc.), those who use any medication regularly, those with active infection, pregnant women, and those with a diagnosis of any malignancy were not included in the study groups. Individuals with similar demographics, age, gender, and body mass index (BMI) to those in the smoker and ex-smoker groups were selected for the non-smoker group.

The blood of the individuals in the study groups was taken after 8-12 hours of fasting in the morning. Venous blood samples taken by venipuncture were centrifuged at 1500 G for 10 minutes, and their serums were separated. The obtained sera were then divided into Eppendorf tubes, frozen without waiting for any period, and stored at -80°C until analysis. Since interference caused by oxidation may occur when samples are kept for different periods before freezing, all steps until the investigation of venous blood samples were performed for equal periods for all models. Proline and hydroxyproline concentration analyses were performed using a liquid chromatography (LC) / mass spectrometre (MS) (Sciex QTrap 4500- Foster City-CA). According to this LC/MS method, a precipitation process was first carried out to remove the high molecular substances in determining amino acids. After centrifugation, a supernatant was injected into the HPLC system. HPLC separation was performed using an isocratic method at 20-25°C using an HPLC column (IC4500rp). Each separation took 15 minutes, depending on the column used. Results were calculated using the internal standard method of integrating peak areas or heights measured by the delivered calibrator. A ready-to-use commercial kit (Immuchrom-Heppenheim-Germany) was used to study the samples. The intra-day and inter-day accuracy ranged from 98.8-106.3% for hydroxyproline and 95.3-103.2% for proline. The precision was within 6.20% for hydroxyproline and 3.63% for proline. The detection range of the test was 0.0488-50 µmol/L for hydroxyproline and 24.95- 1247.57 µmol/L for proline.

In the analysis of serum prolidase enzyme activity, Chinard reagent was used according to the spectrophotometric test described by Myara et al.¹⁵ According to this method, briefly, serum samples were diluted 6-fold with 1 mmol/L Mn+2 and 0.45 mmol/L trizma HCl buffer (pH 7.8), and pre-incubated for 24 hours at 37°C. After adding 94 mmol/L glycyl-L proline solution to 100 µL of the diluted and pre-incubated plasma (final dipeptide concentration was 47 mmol/L), the solution was incubated at 37°C for approximately 30 minutes. The incubation reaction was stopped by adding 1 mL of 0.45 mmol/L 20% trichloroacetic acid solution. The supernatant reaction liquid was used to measure proline according to the method proposed by Myara et al.,¹⁵ which uses a modification of the Chinard method.¹⁶ Intra-assay and interassay coefficient variations (CV) of the assay were lower than 10%.

The measurements used a Siemens Advia 1,800 chemical analyzer (Siemens Healthcare-Erlangen- Germany).

In this study, statistical analyses were performed using version 22.0 Statistical Packages for the Social Sciences (SPSS). Kolmogorov Smirnov test/Box Plots/Q-Q Plots/histogram plots were used to determine the normality distribution of continuous variables. In descriptive statistical analyses, mean and standard deviation (mean ± SD) values ​​were used for normal distribution, and median (interquartile range [IQR], [25-75%]) values ​​were used for non-normal distribution. The relationship between prolidase enzyme activity, proline, and hydroxyproline, which were not normally distributed, and some continuous variables were tested with Spearman’s correlation analysis. Comparisons between multiple groups were made after the Bonferroni correction. Quantitative variables showing normal distribution were analysed with one-way ANOVA, while quantitative variables were not analysed with the independent samples Kruskal-Wallis test. Comparisons between categorical variables were made with Fisher's exact / Chi-square tests. The predictive effect of the independent variables of age, prolidase enzyme activity, proline, and hydroxyproline on the categorical dependent variables of smokers, ex-smokers, and non-smokers was tested using multinomial logistic regression analysis. While the cut-off point was <0.05 for the p-value in pairwise comparisons, it was determined by Bonferroni correction for multiple comparisons.
 

RESULTS

Table I compares the laboratory data and demographic charac- teristics between the groups. A total of 53 people (32 women and 21 men) were included in the non-smoker group, 43 people (25 women and 18 men) were included in the ex-smoker group, and a total of 54 people (30 women and 24 men) were included in the smoker group (p = 0.880). The median values of prolidase enzyme activity were the lowest in the smoker group [734.2 (643.8- 799.6)] and the highest in the non-smoker group [836.8 (755.2-884.2)]. In the multiple comparisons made in terms of median values of prolidase enzyme activity between the groups after Bonferroni recovery, it was determined that the statistically significant difference was due to the difference between the non-smoker [836.8 (755.2-884.2)] and smoker [734.2 (643.8- 799.6)] groups (p = 0.002). While the median values of hydroxyproline were highest in the non-smoker group [8.99 (5.76-11.22)], lower values were found in the ex-smoker [6.05 (4.37-8.21)] and smoker [6.45 (4.25-9.17)] groups (p = 0.008). Proline median values ​​were found to be slightly significantly lower in the ex-smoker group than in the other groups (p = 0.044).

Table I: Comparison between groups in terms of laboratory data and demographic characteristics.
 

Variables	Non-smoker	Ex-smoker	Smoker	+p
Gender (female/male)	32/21	25/18	30/24	0.880
Age, mean ± SD (years)	35.6 ± 11.3	39.1 ± 12.8	35.8 ± 11.8	0.064
Body mass index, mean ± SD.	26.5 ± 6.7	28.6 ± 7.3	25.8 ± 6.4	0.980
Cigarette pack-year, median (IQR)	0	0	10 (5-20)	-
Haemoglobin, mean ± SD [x10^9/L]	16.8 ± 5.8	14.4 ± 4.6	14.9 ± 5.1	0.277
Platelets, mean ± SD [x10^9/L]	266.2 ± 60.7	250.9 ± 47.4	265.9 ± 58.2	0.333
White blood cell, mean ± SD [x10^9/L]	6.42 ± 2.5	6.33 ± 2.34	5.92 ± 2.51	0.309
Neutrophil, mean ± SD [x10^9/L]	3.54 ± 1.8	4.17 ± 2.2	3.9 ± 2.1	0.341
Lymphocyte, mean ± SD [x10^9/L]	1.91 ± 0.7	1.66 ± 0.68	1.8 ± 0.89	0.343
Glucose, median (IQR) [mg/dL]	88 (83-92)	89 (85-93)	87 (80.25-90)	0.082
Creatinine, mean ± SD [mg/dL]	0.72 ± 0.13	0.75 ± 0.13	0.74 ± 0.18	0.141
Urea, mean ± SD [mg/dL]	25.6 ± 6.6	28.3 ± 8.2	27.7 ± 8.3	0.196
Alanine aminotransferase, median (IQR) [U/L]	18 (15-22)	22 (16-30)	19 (15-28)	0.093
Aspartate aminotransferase, mean ± SD [U/L]	20.8 ± 5.7	23.06 ± 5.6	22.7 ± 9.7	0.262
Thyroid stimulating hormone, mean ± SD	1.59 ± 0.54	1.36 ± 0.11	1.74 ± 0.46	0.229
C-reactive protein, median (IQR) [mg/L]	5 (5-18)	7 (5-14)	5 (5-16)	0.761
Prolidase, median (IQR) [U/L]	836.8 (755.2-884.2)	762.6 (638.8-842.6)	734.2 (643,8-799.6)*	0.002
Hydroxyproline, median (IQR) [µmol/L]	8.99 (5.76-11.22)	6.05 (4.37-8.21)*	6.45 (4.25-9.17)	0.008
Proline, median (IQR) [µmol/L]	214.3 (166.8-265.1)	186.7 (120.6-226.1)*	200.3 (138.2-234.1)	0.044
+Chi-square test, one-way ANOVA, and independent samples Kruskal-Wallis test were used to determine p-values. After bonferroni correction, hypotheses with p <0.01667 were accepted as significant. *The group is different from the non-smoker group.

Table II: Results of multinomial logistic regression analysis for comparison between groups.

Groups			β	Std. Error	Wald	OR (CI %95)	+p
	Smoker	Constant	1.693	1.253	1.825	-	0.177
		Age	0.047	0.019	1.406	1.023 (0.985-1.062)	0.236
		Prolidase activity	-0.003	0.001	5.051	0.997 (0.994-1.000)	0.025
		Proline	-0.002	0.003	0.329	0.998 (0.992-1.004)	0.566
		Hydroxyproline	0.011	0.03	0.121	1.011 (0.952-1.072)	0.728
Non-smokera	Ex-smoker	Constant	0.421	1.226	0.118	-	0.734
		Age	0.047	0.019	2.925	1.001 (0.998-1.071)	0.550
		Prolidase activity	-0.001	0.001	0.906	0.999 (0.996-1.001)	0.341
		Proline	-0.005	0.003	1.877	0.995 (0.989-1.002)	0.171
		Hydroxyproline	-0.051	0.058	0.756	0.951 (0.848-1.066)	0.384
Smokera	Ex-smoker	Constant	-1.272	1.273	0.998	-	0.318
		Age	0.304	2.209	0.019	1.024 (0.988-1.062)	0.190
		Prolidase activity	0.002	0.001	1.950	1.002 (0.999-1.004)	0.163
		Proline	-0.003	0.003	0.697	0.997 (0.990-1.004)	0.404
		Hydroxyproline	-0.061	0.058	1.109	0.941 (0.839.1.054)	0.292
N = 150, Model X 2(8) = 17.165, R2 = 0.122 (Cox-Snell), R2 = 0.137 (Nagelkerke), p = 0.028, aReference category, +Multinomial logistic regression analysis was used to determine p-values.

Table III: Spearman’s correlation analysis results between prolidase and some continuous variables.

r (p)	Prolidase	Proline	Hydroxyproline	Creatinine	C-reactive protein	Body mass index	Fasting Glucose
Age	0.080 (0.360)	0.046 (0.599)	0.072 (0.413)	-0.038 (0.648)	0.166 (0.078)	0.161 (0.050)	0.058 (0.489)
Prolidase	-	0.130 (0.137)	0.147 (0.093)	-0.071 (0.419)	0.019 (0.853)	0.012 (0.891)	0.005 (0.953)
Proline	-	-	0.495 (<0.001)	-0.091 (0.299)	0.013 (0.901)	-0.056 (0.523)	-0.023 (0.801)
Hydroxyproline	-	-	-	-0.092 (0.295)	-0.007 (0.944)	0.040 (0.654)	0.082 (0.356)
Creatinine	-	-	-	-	0.165 (0.079)	0.090 (0.272)	-0.031 (0.714)
C-reactive protein	-	-	-	-	-	0.027 (0.773)	0.037 (0.701)
Body mass index	-	-	-	-	-	-	0.180 (0.030)

Figure 1: Distribution of serum prolidase activity among groups.

Figure 2: Pairwise comparison between groups in terms of prolidase enzyme activity.

Figure 1 shows the boxplot graph, and Figure 2 shows the pairwise comparison of the groups regarding prolidase enzyme activity. Median serum prolidase activity values ​​were higher in the non-smoker group than in the smoker group.

The multinomial logistic regression analysis results of the models created with prolidase enzyme activity, proline, hydroxyproline, and age variables are shown in Table II. The median values of prolidase enzyme activity were statistically lower in the smoker group than in the non-smoker group (β = 0.003, OR = 0.997, 95% CI = 0.994-1,000, p = 0.025). While the median values of hydroxyproline were found to be significantly higher in the non-smoker group compared to the ex-smoker group in the independent samples Kruskal-Wallis test (8.99 (5.76-11.22), 6.05 (4.37-8.21), respectively, p = 0.008, Table I), no significant difference was found in the multinomial logistic regression analysis (β = -0.051, OR = 0.951, 95% CI = 0.848-1.066), p = 0.384).

Table III shows no significant correlation between prolidase enzyme activity and other continuous variables.

DISCUSSION

Results of this study have shown that prolidase enzyme activity is lower in smokers than in non-smokers. Serum hydroxyproline levels were significantly lower in the ex-smokers group compared to the non-smokers group in multiple comparisons. In contrast, multinomial logistic regression analysis showed no significant difference between the groups regarding hydroxyproline levels.

To the authors’ knowledge, there is only one previous study investigating the relationship between smoking and prolidase enzyme activity in humans. Although ex-smokers were not included in this study, serum prolidase activity was found to be significantly lower in the smoking group than in the non-smoking group, similar to the present study’s results.¹⁷ The relationship between prolidase enzyme activity and many diseases is well-known. Previous studies have reported that prolidase enzyme activity is high in liver cirrhosis, alcoholic liver disease, non-alcoholic liver disease, and hepatitis C infection, and prolidase supports fibrotic processes in these diseases.¹⁸ Similarly, prolidase activity was highly associated with fibrosis in the histopathological examination of the lung tissue of animals with idiopathic pulmonary fibrosis.¹⁹ Prolidase deficiency, which has an autosomal recessive inheritance, is a rare genetic disease characterised by abnormal skeletal and facial structures, cardiovascular abnormalities, and mental developmental delays. Among the mechanisms responsible for the pathogenesis of the clinical abnormalities seen in prolidase deficiency, the decrease in the proline pool due to the prolidase defect, insufficient collagen synthesis turnover, and necrosis-like conditions occurring in fibroblasts due to the undigested prolidase substrates have been shown.³

In summary, the data from the literature show that prolidase activity increases and supports fibrosis in diseases where fibrotic processes predominate in the pathophysiology. Low prolidase activity causes irregular collagen turnover in insufficient collagen synthesis conditions. Results of this study showed that smokers had significantly lower serum prolidase activity than non-smokers. Considering that smoking creates an environment with decreased collagen synthesis,²⁰ this study supports the pathophysiological roles of prolidase in diseases with irregular collagen turnover.

Data obtained from previous publications investigating the effect of smoking on collagen synthesis suggest that the chemicals in cigarette smoke may interfere with the biological effects of prolidase on collagen synthesis by some pathophysiological mechanisms. Jorgensen et al. have found that subcutaneous collagen synthesis is inhibited in smokers and that these people have lower subcutaneous hydroxy-proline levels, so smoking causes impaired wound healing.²¹ In addition, different studies reported suppression of collagen synthesis and liver protein synthesis due to chemicals in cigarette smoke.²⁰ However, in this study, the authors found that serum prolidase enzyme activity was significantly lower in smokers than in non-smokers. Although further studies are needed to confirm these findings, this study’s results suggest that the adverse effects of smoking on collagen synthesis and wound healing may develop due to the interference of chemicals in cigarette smoke with physiolo-gical processes, in which, prolidase plays a role.

Smoking is considered as the most critical factor in the development of COPD. It has been shown that methylation is induced in the lung tissue and peripheral circulation due to chemicals in cigarette smoke. Smoking is probably involved in xenobiotic metabolism, along with other processes. It has been reported that cigarette smoke up-regulates epidermal growth factor and its ligands, stimulates differentiation of squamous epithelium and goblet cells, respectively, and thus causes changes in small airway epithelial cells and dysfunc-tion of the epithelial barrier and mucociliary clearance.²² Data obtained from previous studies investigating prolidase enzyme activity in patients with COPD suggest that prolidase may play a role in COPD with its mechanism of action similar to smoking. These studies showed that prolidase functions as a ligand for epidermal growth factor,⁴ and prolidase enzyme activity is significantly lower in the serum of COPD patients.²³ In the present study, serum prolidase enzyme activity was significantly lower in active smokers than in non-smokers. This result suggested that prolidase may represent an essential step in the pathophysiology of smoking-induced destructive airways in COPD. Further studies at the molecular level are needed in this area.

It is known that there are at least 17 types of cancer in humans caused by chemicals in cigarette smoke.¹ Although many mutations can develop due to smoking, TP53 mutation, which can be detected positively at 53%, is the most common in smoking-related cancers.⁹ Interestingly, Yang et al. reported that prolidase binds directly to the p53 tumour suppressor protein and suppresses its activity both transcription-dependently and independently of transcription.⁶ It has been argued that prolidase enzyme activity increases in gastric, lung, endometrial, and oesophagal cancers and malignant melanoma, which may support tumour metastasis by increasing collagen synthesis.³ There is no prospective study evaluating serum prolidase enzyme activity in premalignant periods of patients with malignancy. Since patients with malignancy were the exclusion criteria in the present study, the relationship between prolidase and smoking-related cancers could not be evaluated. However, it seems possible that the positive effects of prolidase that support wound healing by regulating collagen turnover may be transformed into a pathogenic mechanism that promotes increased collagen synthesis or inhibition of p53 tumour suppressor protein with the development of malignancy.

Prolidase enzyme activity activates the β1 integrin receptor,¹³ IGF-1R, and TGF.¹⁴ Activation of these receptors by IGF1 and TGFβ1 affects biological processes related to prolidase enzyme activity and cell proliferation, respectively. Chemicals in cigarette smoke can affect the pathways responsible for prolidase activity through different mechanisms. β1 integrin is mainly involved in cytoskeletal remodelling and cell migration. It has been reported that β1 integrin is associated with various tumour cells' migration capacity and invasion tendency. It has been shown that nicotine in cigarette smoke changes the balance of β1 integrin expressed in the cell membrane.¹⁰

Although IGF-1 plays a vital role in the physiology of normal osteogenesis, it contributes to the development and prog-ression of malignancy. Numerous studies have reported that high serum IGF-1 levels may be a risk factor for the develop-ment of colon, breast, prostate, and lung malig-nancies.^11,12 It has been found that there is a positive correlation between cigarette pack-years, serum IGF-1 levels, and higher IGF-1 levels in children exposed to cigarette smoke at prenatal or early ages.²⁴ It has been shown that chemicals in cigarette smoke increase TGF-β1 secretion in mesenchymal cells, and high TGF-β1 levels cause an increase in collagen I and III deposition in the lung, worsening the clinical findings of COPD.²⁵ These data, showing that cigarette smoke affects the pathways that regulate prolidase enzyme activity, support the possible roles of prolidase in conditions such as musculo-skeletal diseases, COPD, and malignancies that may develop due to smoking.

This study found significantly lower serum prolidase activity in smokers compared to non-smokers. However, although this study found that the median values of serum proline and hydroxyproline were lower in smokers than in non-smokers, logistic regression analysis did not show a significant diffe-rence in proline and hydroxyproline between the two groups. In addition, the results of this study did not detect a signifi-cant correlation between prolidase activity and proline and hydroxyproline levels. This study is the first to share the correlation between serum prolidase enzyme activity and proline and hydroxyproline levels. An explanation for these results can be that proline or hydroxyproline, formed due to prolidase activity, are used in protein synthesis according to the body's needs under physiological conditions, and the levels of these two amino acids decrease. Another hypo-thetical explanation is that since smoking does not have a suppressor effect on collagen synthesis, more proline and hydroxyproline are consumed by collagen synthesis in non-smokers than in smokers. Thus, the serum levels of these amino acids may decrease in non-smokers. However, these results need to be confirmed with further research.

One of the limitations of this study was its small sample size and a cross-sectional design. Another limitation of the study is that dietary proline intake and physical activity were not standardised between groups, which may have caused a bias in the results. However, since the number of studies investigating the relationship between smoking and prolidase enzyme activity in the literature is minimal and the findings of this article do not sufficiently support these relationships, causality cannot be established between prolidase enzyme activity and smoking-related diseases. Longitudinal data and animal studies are needed in this area. This study, however, showed that serum prolidase activity or hydroxyproline/ proline levels were significantly higher in non-smokers than in ex-smokers or smokers. Future studies investigating the effects of prolidase activity on the pathophysiological processes of smoking-related diseases, such as improper wound healing and COPD, especially in malignancies, will provide a better understanding of the role of prolidase activity in these diseases.

CONCLUSION

Serum prolidase enzyme activity decreases in smokers. The prolidase activity may be a fruitful area of ​​study to understand the pathophysiology of smoking-related diseases better. Smokers may be exposed to potential clinical effects asso-ciated with low prolidase enzyme activity, such as impaired wound healing and COPD.

FUNDING:
Supported by Ankara Yildirim Beyazit University Scientific Research Projects Coordinatorship with (Project. No: TTU-2022- 2413).

ETHICAL  APPROVAL:
This research was approved by the Ankara City Hospital’s 1^st Ethics Committee (No: E1-22-2809; Dated: 24.8.2022).

PATIENTS’ CONSENT:
Written informed consent was obtained from all subjects participating in this study for the use and publication of study data on the condition that their data remain anonymous.

COMPETING INTEREST:
The authors declared no conflict of interest.

AUTHORS’ CONTRIBUTION:
TBK: Analysis of the study data, interpretation, and article writing.
BFD: Draft and revision of the article.
TBK, BFD: Design of the study, analysis, and collection of the clinical data.
TBK, AT, SN: Analysis and collection of the laboratory data.
All authors approved the final version of the manuscript to be published.

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