The relationships among the levels of oxidative and antioxidative parameters, FEV1 and prolidase activity in COPD

Abstract

Introduction: Chronic obstructive pulmonary disease (COPD) is a progressive condition characterized by poorly reversible airflow limitations associated with an abnormal inflammatory response of the lung.

Methods: We investigated whether prolidase levels in serum, total antioxidant status, total oxidative status (TOS), and the oxidative stress index (OSI) were associated with the etiopathogenesis of COPD, and whether there is a relationship between prolidase activity and oxidative parameters and carotid artery intima-media thickness (CIMT) in patients with COPD. This study included 91 patients with COPD and 15 control cases. Routine haematological and biochemical parameters were determined in all patients. All subjects were fully informed about the study and provided consent.

Results: The mean age of the patients with COPD was 61.3 ± 10.5 years and that of the control group was 56.2 ± 12.1 years. The control group had a significantly higher plasma prolidase level than that in the COPD group. TOS and OSI levels in the control group were significantly lower than those in the COPD group. However, no significant differences were found in TALs or CIMT levels between the COPD and control groups. A negative correlation was detected between prolidase activity and age; however, no significant difference in age was observed between the two groups.

Conclusion: These results indicate that prolidase activity decreases in patients with COPD.

Keywords:

• COPD
• Oxidative stress
• Prolidase

Introduction

Chronic obstructive pulmonary disease (COPD) is a leading cause of mortality and morbidity worldwide, and its prevalence is expected to increase worldwide.^1,2 Furthermore, it is a progressive condition characterized by a poorly reversible airflow limitation associated with an abnormal inflammatory response of the lung. The prevalence of airflow limitation was 10.9% in the NICE study and 13.9% in another study.^3,4 Prolidase is a cytosolic and multifunctional exopeptidase that degrades iminodipeptidase, which releases the carboxy-terminal proline or hydroxyproline from oligopeptides.⁵ Collagen is an important prolidase substrate due to its high content of amino acid. Prolidase enzyme activity in serum is elevated in conditions characterized by chronic tissue inflammation and/or increased collagen turnover. Prolidase has been detected in various tissues, including the plasma, heart, thymus, brain, and uterus.⁶ Elevated levels in serum are associated with oxidative stress in some organic diseases, such as mitral stenosis, Helicobacter pylori infection, and ovarian cancer.^7,8 Previous studies have investigated prolidase activity under different clinical conditions, such as rheumatic diseases and asthma.^9,10 Carotid atherosclerosis is strongly correlated with coronary atherosclerosis and carotid intima-media thickness (CIMT), as measured by carotid Doppler ultrasound.^11,12 In this study, we investigated whether prolidase levels in serum are associated with the etiopathogenesis of COPD, and whether there are relationships among prolidase activity, oxidative parameters, and CIMT in patients with COPD.

Materials and methods

This study included 91 patients with COPD who applied to the outpatient department at the Pulmonary Medicine Department, Medical Faculty, Yuzuncu Yıl University, and 15 control cases. The male-to-female ratio, age, and number of smokers were similar between the two groups. Smokers had a minimum 10 pack-year smoking history for 20–30 years in both groups. All patients’ routine haematological and biochemical parameters were determined. COPD was diagnosed based on taking a medical history, a physical examination, and spirometric data (i.e., forced expiratory volume in 1 second, FEV1), according to the guidelines of the Global Initiative for Chronic Obstructive Lung Disease. All patients were diagnosed with COPD, and their FEV1/forced vital capacity was <70%. None of the patients had any illness except moderate to severe COPD. All subjects were fully informed about the study and provided informed consent.

Determining CIMT

Bilateral common carotid arteries of the patients were scanned longitudinally with a 7 MHz transducer attached to an available machine (Vivid 3, General Electric, Milwaukee, WI, USA). Images were obtained from the distal portion of the common carotid artery, 1–2 cm proximal to the carotid bulb. The two bright echogenic lines in the arterial wall were identified as the intima and media lines. Intima-media thickness was measured as the distance from the main edge of the first echogenic line to the main edge of the second. All examinations were performed by the same physician. Images showing maximum intima-media thickness were digitally stored and CIMT measurements were made offline. The intima-media thickness of the distal wall of the right common carotid artery on the lengthwise axis was calculated according to the method described by Pignoli et al.¹¹ Each measurement was repeated three times and the mean of the left and right common carotid arteries was used for analysis. Plaques, defined as >50% localized thickening of the intima compared to the rest of the wall or as an endoluminal protrusion of the arterial lumen >0.5 mm, were not included in the CIMT measurements.

Determining prolidase activity

Prolidase activity was determined by a photometric method based on the measurement of proline levels produced by prolidase.¹³ Plasma samples (100 µl) were mixed with 100 µl of serum physiological. A total of 25 µl of the mixture was preincubated with 75 µl of the preincubation solution (50 mmol/l Tris–HCl buffer pH 7.0 containing 1 mmol/l GSH, 50 mmol/l MnCl₂) at 37°C for 30 minutes. The reaction mixture containing 144 mmol/l gly-pro, pH 7.8 (100 ml) was incubated with 100 µl of preincubated sample at 37°C for 5 minutes. To stop the incubation reaction, 1 ml glacial acetic acid was added. After adding 300 µl Tris–HCl buffer, pH 7.8 and 1 µl of ninhydrin solution (3 g/dl ninhydrin was melted in 0.5 mol/l orthophosphoric acid) the mixture was incubated at 90°C for 20 minutes and cooled with ice and subsequently its absorbance was measured at a wavelength of 515 nm for determining proline level as proposed by Myara et al.¹⁴ This method is a modification of Chinard's method.¹⁵ Intra- and inter-assay coeffecient variations of the assay were lower than 10%.¹⁰

Measurement of total oxidative status (TOS)

The TOS of serum was determined using a novel automated method, developed by Erel.¹⁶ Oxidants present in the sample oxidize the o-dianisidine–ferrous ion complex to ferric ions. The oxidation reaction is enhanced by glycerol, which is abundant in the reaction medium. Ferric ions form a coloured complex with xylenol orange in the acidic medium. Colour intensity measured spectrophotometrically is related to the total amount of oxidant molecules present in the sample. The assay is calibrated with hydrogen peroxide, and the results are expressed as micromolar hydrogen peroxide equivalents per litre (mmol H₂O₂ equiv./l).

Oxidative stress index (OSI)

The ratio of percent TOS to total antioxidant status (TAS) was accepted as the OSI and was calculated as follows: TOS (mmol trolox equiv./l)/TAS (mmol trolox equiv./l)] × 100.¹⁷

Ethics

This study was approved by the local ethics committee in accordance with the Declaration of Helsinki and written informed consent was received from all patients and control subjects before enrolment in the study. The patient and control cohorts were recruited at the Pulmonary Medicine Department, Medical Faculty, Yuzuncu Yıl University. Blood samples were analysed at the Biochemistry Laboratory of Harran University Medical Faculty.

Statistical analysis

Statistical analyses were performed using SPSS 16 software (SPSS Inc., Chicago, IL, USA). Continuous data were expressed as means ± standard deviations. Associations between variables were identified using Pearson's correlation analysis, and Pearson's correlation coefficients were calculated. A P-value ≤0.05 was considered significant.

Results

The study was conducted on 91 patients with COPD and 15 control cases. The mean age of patients with COPD was 61.3 ± 10.5 years, and that of the control group was 56.2 ± 12.1 years. No differences in age and sex distribution were detected between the two groups. The control group had a significantly higher prolidase levels in plasma compared to COPD patients (P ≤ 0.01). FEV1 and prolidase activity were positively correlated (Fig. 1). TOS and OSI levels were significantly lower in the control group (P ≤ 0.05). However, no differences were found in TAS or CIMT levels between the two groups. No association was found between prolidase activity and CIMT values in the two groups.

Figure 1 FEV1 (%) and prolidase activity (U/l) were positively correlated (r = 0.768).

A negative correlation was observed between prolidase activity and age; however, no difference in age was found between the COPD and control groups. The clinical characteristics of the patients and controls are shown in Table 1. The parameters compared between the two groups are shown in Table 2.

Table 1 Demographic characteristics of the patients with COPD and controls

Parameters	COPD	Control	P
Case, n	91	15
Age (years)	61.3 ± 10.5	56.2 ± 12.1	0.10
Female/total, n	40/91	9/15	0.22
Smoker/total, n	67/91	8/15	0.11
FEV1 (% predicted)	55.6 ± 9.2	88.6 ± 9.7	0.001

Values are given as mean ± standard deviation.

Table 2 Prolidase activity, TAS, TOS, OSI, CIMT levels of the subjects with COPD and controls

	COPD	Control	P
Prolidase (U/l)	460.4 ± 139	716.9 ± 135.4	0.001
TAS (mmol trolox equiv./l)	1.68 ± 0.37	1.84 ± 0.35	0.12
TOS (mmol trolox equiv./l)	73.3 ± 22.5	55.6 ± 16.6	0.01
OSI (ratio)	4.5 ± 1.81	3.0 ± 0.85	0.001
CIMT (mm)	0.72 ± 0.08	0.76 ± 0.07	0.06

Values are given as mean ± standard deviation; TAS, total antioxidant capacity; TOS, total oxidant capacity; OSI, oxidative stress index; CIMT, carotid intima-media thickness.

Discussion

Prolidase activity in serum, TOS of serum, TAS, and OSI are recognized as markers of oxidative stress. A delicate balance exists between oxidative and antioxidative status and this balance breaks down in patients with some chronic diseases, such as COPD. We evaluated this oxidative/antioxidative balance, prolidase activity, and CIMT between patients with COPD and a control group. Increased oxidative stress plays an important role in the pathogenesis of COPD. An active inflammatory response with infiltration of neutrophils is induced in patients with chronic diseases such as COPD. Neutrophils, macrophages, and/or monocytes produce free oxygen radicals that can cause lipid–protein oxidation and oxidative DNA damage to adjacent cells. The DNA damage provoked by reactive oxygen species (ROS) has very harmful consequences, leading to potentially carcinogenic gene modifications. COPD has been associated with ROS generation and increased nitric oxide levels, which lead to oxidative stress in the lung. Increased oxidative stress has an important role in COPD pathogenesis.^18,19 Because several oxidants and antioxidants are likely to be involved in the pathogenesis of the inflammatory response in patients with COPD, we investigated TAS, TOS, OSI, and prolidase activity to determine how oxidative and antioxidative status is affected by COPD.

Gencer et al.¹⁰ found that prolidase activity and TAS are significantly lower, and LPO level is significantly higher, in COPD patients than in controls. Significant correlations were detected between plasma prolidase activity and TAS and LPO levels in the patient group.

Verma et al.²⁰ reported that prolidase activity, TOS, and OSI were significantly higher in diabetic nephropathy and end-stage renal disease group than in controls, whereas TAS were significantly lower. We also found in this study that prolidase activity and TOS levels were significantly lower and OSI levels were significantly higher in patients with COPD than in controls. No significant differences were found in TAS. We also identified positive correlations between OSI and TOS, CIMT and FEV1 and FEV1 and prolidase activity, but negative correlations between TAS and OSI, FEV1 and OSI, age and FEV1, and prolidase and age.

Duygu et al.²¹ showed that prolidase activity and oxidative stress increase in patients with hepatitis C virus (HCV) infection and that the prolidase enzyme and oxidative damage may be associated with the HCV infection becoming chronic. However, Şen et al.²² reported a significant increase in prolidase activity in children with chronic hepatitis.

Uzar et al.²³ studied prolidase activity and oxidative status in patients with diabetic neuropathy (DN) and found that TALs were lower while TOS, OSI, and prolidase activity were higher in the diabetic control group than in the healthy control group. Similarly, TALs were lower, whereas TOS, OSI, and prolidase activity were higher in the DN group. No differences were observed between the diabetic control and DN group with respect to TAS, TOS, or OSI. However, prolidase activity was higher in the DN group than that in the diabetic control group. Another study on prolidase activity and oxidative stress in patients with DN and end-stage renal disease reported similar results.²³ But there are other studies that decreased prolidase activity seems to be associated with increased NO levels and oxidative stress along with decreased antioxidant levels in DN.²⁴ Patients with bronchial asthma reportedly have significantly lower prolidase levels than controls, and TOS levels are significantly higher in patients with asthma.⁹ In contrast to our results, that study found significantly higher TAS in patients.

Prolidase activity may increase or decrease in some chronic illnesses.^9,10,21–24 Other studies have shown increased prolidase activity for a particular disease, whereas others have shown a decrease for the same disease. Studies on COPD and asthma show a consistent decrease in prolidase activity. TAS, TOS, and OSI are similarly affected by the same illness. TAS usually decreases as TOS levels increase. We found that TAS and prolidase activity were low and TOS levels were high in patients with COPD compared to a control group.

Disclaimer statements

Contributors All authors contributed equally.

Funding None.

Conflict of interest Any author does not have any conflict of interest.

Ethics approval Our paper has received ethics approval.