Oxidative Stress And Prolidase Enzyme Activity In The Pathogenesis Of Primary Varicose Veins

Abstract

Objectives

Vascular endothelial dysfunction leads to the emerging of free oxygen radicals, deficiency of antioxidant system, forming of oxidative stress, inflammatory processes and release of proinflammatory cytokines. These things play big role in the development of primary varicose veins. Prolidase has been reported as an indicator of oxidative stress in diabetes, diabetic neuropathy, non-ulcerous dyspepsia, osteoporosis, polycystic over syndrome and many other diseases. The aim of this study is to evaluate the oxidative stress at venous insufficiency and to provide preliminary knowledge about the role of prolidase enzyme in varicose vein formation.

Methods

Ninety patients aged between 22 and 80 (47.35 ± 17.69) were included in the study and divided into 3 groups. Group1(n:30)(Serum control group): Patients without venous insufficiency. Group 2(n:30)(Tissue control group(healthy vein group): Patients underwent coronary artery bypass surgery (the remaining portion of great saphenous vein used as coronary artery bypass graft used as normal tissue) . Group 3(n:30)(Varicose vein group): Patients underwent varicose vein surgery (varicose vein and serum of these patients were used for study). Total Oxidant Status (TOS), Total Antioxidant Status (TAS), Oxidative Stress Index (OSI) and Prolidase enzyme levels were detected in tissue and serum samples.

Results

No significant changes were detected between three groups’ serum samples in oxidative stress parameters and in the prolidase enzyme activity. The tissue TOS and OSI were higher in varicose vein group according to normal vein group and this was found statistically significant. And TAC levels in varicose vein group were significantly lower than normal vein group. Prolidase enzyme activity in varicose vein group was found higher according to normal vein group.

Conclusion

Oxidative stress plays a role at the development of primary varicose veins at biochemical level. Prolidase enzyme related with oxidative stress may play an important role in the pathogenesis of primary varicose veins.

Keywords

Oxidative stress varicose vein prolidase

Introduction

Varicose veins are defined as tortuous, wide, palpable veins which are larger than 3 mm in diameter. In developed countries, it has tendency to increase with age and it occurs in 65% of females and 50% of males above 45 years of age.¹ Varicose veins may cause cosmetic problems such as lipodermatosclerosis, pigmentation and also functional limitations in the activities of patients related to pain. At the same time, they may occur with complications such as superficial thrombophlebitis (varicophlebitis), ulcerations and bleeding. Family history, advanced age, female gender, pregnancy, obesity, history of deep venous thrombosis, occupations requiring standing for a long duration are dominant risk factors.² The etiology of primary varicose veins is still unclear. Several factors which play important role in their development have been reported. These include; vascular endothelial dysfunction that leads to the accumulation of free oxygen radicals at high levels, deficiency of antioxidant system, development of oxidative stress, inflammatory processes, the release of proinflammatory cytokines.^3,4 Some researchers investigated the effects of anoxia on the interactions between epithelium and multinuclear macrophages, monocytes and neutrophils and indicated that adhesion of multinuclear cells in varicose and/or insufficient veins was stronger when it was compared to normal veins.⁵ Disruption of the balance between oxidative stress indicators such as malondialdehyde (MDA) formation and superoxide dismutase activity results in overproduction of free oxygen radicals. This can lead to the destruction of lipid membranes, proteins and other molecules within the endothelium. Increased MDA release causes venoconstriction by negatively affecting the relaxation balance in varicose veins. It was also determined that MDA increases the release of free oxygen radicals. These increased free oxygen radicals bring themselves out with endothel dysfunction which is characterized by the loss of protective nitric oxide bioavailability.^5,2

Despite all these studies, the underlying pathogenesis in the development of primary varicose veins has not been entirely elucidated.

Prolidase is a manganese-dependent metalloproteinase enzyme which hydrolyzes the peptide bond of imido-dipeptides and imido-tripeptides (X-Pro, X-Hydroxyproline) that contain proline or hydroxyproline at the carboxyl terminal position. Within the cell, it plays a big role in the catabolism of proteins including procollagen, collagen, proteins containing proline and hydroxyproline. Collagen is abundant in many tissues, especially in the connective tissue.⁶ The presence of prolidase activity is demonstrated in various tissues such as serum, plasma, leukocytes and erythrocytes, amnion fluid, intestinal mucosa, kidney, liver, brain, heart, uterus, and thymus. It was reported as an oxidative stress indicator in diabetes mellitus, diabetic neuropathy, diabetic nephropathy, non-ulcerous dyspepsia, erectile dysfunction, osteoporosis, polycystic over syndrome and many other diseases. It was pointed in various studies that the increase in serum prolidase activity is effective in the oxidant/antioxidant capacity in various diseases and may cause them by this way⁷⁻¹⁰. Ozan et al.¹¹ indicated that prolidase enzyme activity is increased in patients with varicosele.

The aim of this study is to evaluate the oxidative stress in venous insufficiency, which is an inflammatory process and to provide preliminary information regarding the efficacy of prolidase in the progression or even in the diagnosis of venous insufficiency which is still being diagnosed and followed up with imaging modalities and provide pilot data for further follow-up and comprehensive studies if it was determined to be effective.

Materials and methods

The study was commenced after obtaining the approval of Clinical Investigations Ethics Committee of Tokat Gaziosmanpasa University Faculty of Medicine. All patients were informed about the study and their consents were obtained. Ninety patients, whose ages ranged between 22–80 (47,35 ± 17,69), were included in the study. Forty one of them were female and 49 of them were male. Patients were divided into three groups.

Group 1 (n: 30) (Serum Control Group): Patients who were admitted to the Cardiovascular Surgery policlinic with the complaints of leg pain and in whom no pathology was detected in the lower extremity venous Doppler ultrasonography. The remaining serum; from the blood taken for routine hematological examinations of these patients were used as normal serum samples.

Group 2 (n: 30) (Tissue Control (healthy vein) Group): The patients whom underwent elective coronary artery bypass surgery in the cardiovascular surgery clinic. The remaining portion of the large saphenous vein that was taken as graft from these patients was used as a normal tissue sample in the study. Also the remaining serum; from preoperative examinations of these patients were used as negative control group. The reason was that serum prolidase levels were found to be high in the patients with coronary artery disease in the study conducted by Yıldız and colleagues.¹² The patients who did not have deep and superficial venous insufficiency in the preoperative lower extremity venous Doppler ultrasonography and, who had a macroscopically normal saphenous vein in the intraoperative evaluation, were included in the study.

Group 3 (n: 30) (Varicose vein group): These are patients whom underwent varicose vein surgery in the Cardiovascular Surgery clinic. They were at the stage of CEAP C2. Varicose veins removed during surgery and the remaining serum from pre-operative blood samples were used for the study.

The patients who are known with acute-chronic liver disease, acute-chronic lung disease, have autoimmune, neoplastic disease, osteoporosis, infection and surgical intervention within last month were not included in the study.

Biochemical analysis

Total antioxidant capacity (TAC), Total oxidant status (TOS), Oxidative stress index (OSI), Prolidase activity were measured in the serum and tissue samples taken from the patients.

Whole blood samples were centrifuged to obtain serum. All tissues were washed three times with cold isotonic saline solution (0.9%) and their wet tissue weights were determined Afterwards, the tissues were homogenized at 13,000 rpm for 2 minutes via a homogenizer (IKA Ultra-Turrax t 25 Basic, Stanfen, Germany) in the Tris-HCl (50 mM, pH 7.4) buffer solution containing 0.05% Triton X-100. The supernatant was obtained by centrifugating the homogenates at, 3500 rpm for 60 min. All processes were carried out at +4° C. Homogenate protein measurement for each sample was performed according to the method developed by Lowry et al.¹³

The level of prolidase was determined using a quantitative sandwich enzyme immunoassay kit (Cusabio Biotech), in accordance with the producor’s instructions. After the color development was stopped, the optical density of the samples was measured at 450 nm and the prolidase levels were determined using the standard curve. The results were expressed as mU/mg protein for tissue samples and mU/ml for serum samples.

The antioxidant capacity was analyzed using the ImAnOx colorimetric assay (Immunodiagnostic, Bensheim, Germany). The determination of antioxidative capacity by this test was carried out by making reaction at certain amounts of exogenous hydrogen peroxide (H2O2) with the antioxidants in the samples. The antioxidants in the samples eliminated a certain amount of H2O2. The remaining H2O2 was photometrically detected by an enzymatic reaction which enabled tetramethybenzidin to turn into a colored material. After addition of the stop solution, samples were measured at 450 nm. The results were expressed as nmol/mg protein for tissue samples and nmol/ml for serum samples.

The PerOx colorimetric test was used to detect oxidation level (Immunodiagnostic, Bensheim, Germany). Determination of peroxides in this test was accomplished by the conversion of tetramethylbenzidin to a colored product following the reaction of a peroxidase with the peroxides in the samples. After addition of the stop solution, samples were measured at 450 nm. The results were expressed as nmol/mg protein for tissue samples and nmol/ml for serum samples.

For the determination of oxidative stress index for the tissues and serum samples the commercial kits of Total Oxidant Status and Total Antioxidant Status were used (Rel Assay Diagnostics, Gaziantep, Turkey), in accordance with the instructions of the producer.

Statistical method

Descriptive analyses were performed to provide information on general characteristics of the study population. Quantative data was obtained related to the arithmetic mean and standard deviation. Chi-square test was used to compare the qualitative variables among groups. Independent samples such as; t test or one way analysis of variance were used to compare the continuous data between/among groups. Lower than 0,05 a p-value was accepted as significant. Analyses were done with using SPSS 19 (IBM SPSS Statistics 19, SPSS inc., an IBM Co., Somers, NY).

Results

Demographic characteristics of all cases are summarized in Table 1. Except age and diabetes mellitus, no statistically significant differences were identified among the demographic data (gender, hypertension, smoke,) of groups (p > 0.05). All means are different from each other for age variable (p < 0,001). A significant difference were detected between healthy vein group and control group according to diabetes mellitus (p = 0,019) (Table 1). The patients in healthy vein (tissue control group) were selected from the patients whom underwent elective coronary artery bypass surgery in the cardiovascular surgery clinic. In our opinion; because of diabetes mellitus is a risk factor for coronary artery disease, a significant difference was detected in this group.

Table 1.

Distributions of variables by groups.

Variables		Groups			p
Variables		Varicose Vein (n = 30)	Healthy Vein (n = 30)	Control (n = 30)	p
Age		31,03±8,59	67,1±8,81	43,73±10,8	<0,001
Gender	Female	13(43,3)	16(53,3)	12(40)	0,559
Gender	Male	17(56,7)	14(46,7)	18(60)	0,559
Hypertension	Absent	26(86,7)	18(60)	24(80)	0,055
Hypertension	Present	4(13,3)	12(40)	6(20)	0,055
Smoking	Absent	21(70)	19(63,3)	20(66,7)	0,861
Smoking	Present	9(30)	11(36,7)	10(33,3)	0,861
Diabetes Mellitus	Absent	26(86,7)ab	19(63,3)b	27(90)a	0,019
Diabetes Mellitus	Present	4(13,3)ab	11(36,7)b	3(10)a	0,019

Data are shown as mean ± standard deviation or frequency and percentage. Chi-square test or one way variance analysis test were used for comparisons. Each subscript letter denotes a subset of Group categories whose column proportions do not differ significantly from each other at the, 05 level. All means are different from each other for age variable.

Total oxidant status was detected as 488,56 ± 107,10 nmol/mg protein in the tissue samples taken from the patients in the varicose vein group and 379,64 ± 64,34 nmol/mg protein in the tissue samples taken from the patients in the healthy vein (tissue control) group. There was a statistically significant increase in favor of the varicose vein group between two groups (p <0.001). Considering the total antioxidant capacity, it was measured as 449,19 ± 38,70 nmol/mg protein in the varicose vein group and 479,01 ± 35,45 nmol/mg protein in the tissue control group and a significant decrease was observed in the varicose vein group (p = 0.03). The oxidative stress index was found to be significantly higher in the varicose vein group (p <0.001) (Table 2). When the levels of prolidase were evaluated, it was 142,22 ± 35,6 mU/mg protein in healthy vein while it was found that this value increased to 168,61 ± 39,20 mU/mg protein in the varicose vein group and this increase was statistically significant (p = 0,008) (Table 2).

Table 2.

Distributions of oxidative stress parameters and prolidase enzyme levels at tissue samples according to groups.

Variables	Tissue		p
Variables	Varicose vein	Healthy vein	p
TAC (nmol/mg protein)	449,19±38,70	479,01±35,45	0,003
TOS (nmol/mg protein)	488,56±107,10	379,64±64,34	<0,001
OSI	1,09±0,23	0,8±0,14	<0,001
Prolidase (mU/mg protein)	168,61±39,20	142,22±35,6	0,008

Independent samples t test was used.

TAC: Total Antioxidant Capacity, TOS: Total Oxidant Status, OSI: Oxidative Stress Index.

Total oxidant status was determined to be 151,12 ± 58,98 nmol/ml in the serum samples taken from the patients in the varicose vein group, 145,93 ± 54,89 nmol/ml in the serum samples taken from the patients in the healthy vein (tissue control) group and 145,05 ± 44,39 nmol/ml in the serum samples taken from the patients in the serum control group . It was observed that there was no significant difference among all three groups (p = 0.892). Total antioxidant capacity was measured as 1305,25 ± 18,87 nmol/ml in the varicose vein group, 145,93 ± 54,89 nmol/ml in the tissue control group and 145,05 ± 44,39 nmol/ml in the serum control group and no significant difference was found among them (p = 0.892). When oxidative stress index was calculated, again no significant difference was detected between these three groups. Prolidase enzyme levels were found to be 112.89 ± 41.81 mU/ml in the serum of the tissue control group patients’ and 152.95 ± 135.82 mU/ml in serum samples of the patients in the varicose vein group. Although it was observed that there was a numerical increase, it was not found to be statistically significant (p = 0.079). Also, when the prolidase enzyme levels in the serum samples taken from the patients with coronary artery disease who were defined as healthy vein group and the serum samples taken from the patients without coronary artery disease in the normal serum group were compared, it was observed that the serum prolidase enzyme level in the serum of the patients with coronary artery disease was higher than the people in the control group but it was not found statistically significant (p = 0.079) (Table 3).

Table 3.

Distributions of oxidative stress parameters and prolidase enzyme levels at serum samples according to groups.

Variables	Serum			p
Variables	Varicose Vein	Healthy Vein	Control	p
TAC(nmol/ml)	305,25±18,87	306,9±17,56	307,19±14,2	0,892
TOS (nmol/ml)	151,12±58,98	145,93±54,89	145,05±44,39	0,892
OSI	0,49±0,19	0,48±0,18	0,48±0,16	0,887
Prolidase (mU/ml)	152,95±135,82	112,89±41,81	107,73±29,43	0,079

One way ANOVA test was used.

TAC: Total Antioxidant Capacity, TOS: Total Oxidant Status, OSI: Oxidative Stress Index.

Discussion

The varicose disease is characterized by excessive dilatation and tortuosity of the superficial veins of the lower extremity. Varicose veins are characterized by abnormal collagen deposition, a decrease in elastin content and leukocyte infiltration. The dysfunction of endothelial cells in the intimal layer is revealed by demonstrating the endothelial damage indicators and these are high in the patients with varicose veins. Although oxidative stress was pointed to be a key factor in the formation of varicose veins due to the stimulatory effect of oxidative modification of cellular and matrix molecules, the underlying pathology has not been fully elucidated.^1,3

There are studies showing that the imbalance between oxidative stress and antioxidant systems causes damage to the proteins and other molecules in the lipid membranes of endothelium. In a study conducted by Krzyściak et al.,⁵ it was indicated that the MDA levels which are indicative of oxidative stress on the wall of the varicose vein, were higher than that of the normal venous wall and that superoxide dismutase levels, a marker of antioxidant capacity, were low. In another study conducted by the same researcher, the MDA values of the blood samples taken from the varicose vein and the blood samples taken from the peripheral vein of upper extremity were compared and it was found higher in the blood samples taken from the varicose vein while the value of FRAP (Ferric reducing ability), which is used to measure the total antioxidant capacity, was found as low.¹⁴ In another study conducted by Condezo-Hoyos et al.¹⁵ total thiol, uric acid, catalase, total antioxidant capacity, which are related to the antioxidant defense system and as oxidative damage markers, MDA, carbonyl proteins and advanced oxidation products were evaluated in the blood samples of the patients at the stage of CEAP C2, When the result was compared with the control group, it was determined that oxidative stress was high in the patients with venous insufficiency and that the parameters indicating antioxidant defense system were low.

There are several studies similar to this research in the literature and indicate that reactive oxygen radicals, which may be involved in the etiology of varicose veins, were high, as well as there was a decrease in the parameters indicating antioxidant defense system.^16–18

In addition to the studies that show the increase in oxidative capacity and the decrease in antioxidant capacity are involved in the physiopathogenesis of varicose veins and chronic venous insufficiency there are also studies indicating the increase in the antioxidant capacity which may contrast them. In the study conducted by Krzyściak et al.¹⁹ and published in 2012, oxidative stress and antioxidants were measured in the normal venous wall and varicose venous wall. As an oxidative stress marker, thiobarbituric acid reactive substances (TBARs) and as antioxidants, SOD and GPx levels were measured. Both oxidative stress and antioxidants were indicated that they increased in the wall of the varicose vein.

In a study by Budzyn et al.,²⁰ on 35 patients and 27 normal subjects, plasma oxidant and antioxidant balance in chronic venous insufficiency; it was observed that both oxidant and antioxidant parameters showed a significant increase in the serum of patients with venous insufficiency compared to serum of normal people.

In our study, TOS values of the tissue samples of the patients with varicose vein were higher than those of healthy vein group. When the total antioxidant capacity was examined, it was observed that there was a statistically significant decrease in the varicose vein group compared to the healthy vein group. Oxidative stress index was found high in the varicose vein group.

In recent years it is accepted that oxidative stress plays important roles in etiopathogenesis of most diseases. In vascular surgery the pathophysiological process in primary varicose veins is still unclear. There are many reports that oxidative stress may cause the varicose vein development as we mentioned above. There are several markers that shows oxidant and antioxidant status. Malondialdehyde (MDA), superoxide dismutase, catalase are some of them. In our study we used Total Oxidant Status (TOS) as an oxidant, Total Antioxidant Capacity (TAC) as an antioxidant marker. The statistically significant changes at these markers levels at varicose vein wall suggest that oxidative stress may play role in the etiopathogenesis of primary varicose veins. But the mechanism is unclear.

Prolidase is a manganese-dependent metalloproteinase which hydrolyzes the peptide bond of imido-dipeptides and imido-tripeptides (X-Pro, X-Hydroxyproline) that contain proline or hydroxyproline at the carboxyl terminal position. Within the cell, it plays role in the catabolism of proteins including procollagen, collagen, proteins containing proline and hydroxyproline. Collagen is abundant in many tissues, especially in the connective tissue.⁶

Prolidase enzyme activity was also pointed out to be associated with oxidative stress in many studies.

In a study conducted by Duygu et al.²¹ on the cases with chronic hepatitis C, they found that antioxidant levels were low while prolidase and oxidative stress parameters were high. They proposed that here might be an association between prolidase enzyme and oxidative damage.

Walker and colleagues²² showed that increased prolidase enzyme activity in bladder cancer was associated with increased oxidative stress and decreased antioxidant capacity.

In another study performed in the patients with renal cancer, prolidase enzyme activity and MDA levels were detected to be significantly higher, while SOD and GSHPx levels which were antioxidant enzymes found to be low.²³

Bozkurt et al.²⁴ examined serum prolidase levels and OSI, TOS and TAS levels which were oxidative stress parameters in the patients with Behcet's disease. They reported that serum prolidase activity had a negative correlation with TAS and a positive correlation with MDA, TOL, and OSI in the patients with Behcet's disease

In a study performed by Uzar et al.⁸ on prolidase and oxidative stress in diabetic neuropathy, serum prolidase and TOS levels in the diabetic neuropathic patients were higher when it was compared to the patients with diabetes without neuropathy and control group. However, Sayın et al.²⁵ found that serum NO and MDA levels were high in the diabetic patients with neuropathy while prolidase activity was significantly low in prolidase activity, in other words in the presence of a negative correlation, they indicated a positive correlation between TAC and prolidase levels. In another study conducted on asthmatic patients, it was reported that serum prolidase activity was significantly higher in this group of patients, and it correlated positively with TAC.²⁶

Fitowska et al²⁷ demonstrated a significantly higher SOD and glutamate dehydrogenase activity in patients with hip osteoarthritis, also prolidase activity has been increased in this patients group compared with the control one.

Aslan et al.²⁸ showed that while the serum TAC levels were low in obese patients, serum TOS and prolidase activity were high. So they commented that increased serum prolidase activity and decreased antioxidant levels seem to be a result of increased of oxidative stres levels in obese patients.

As it is understood from all of these quotations there is a relationship between prolidase enzyme activity and oxidative stress, but there is no consensus on whether this relationship has a positive or negative correlation. In our study, it was found that the activity of prolidase enzyme in the serum of patients in the varicose vein group was numerically high but statistically insignificant when it was compared to the control group. It was observed that prolidase enzyme activity in the tissue samples of the varicose vein group was found to be statistically significant in the tissue samples of the healthy vein group. In a recent study by Kocarslan et al.²⁹ varicose veins and intact venous samples were compared immunohistochemically with respect to prolidase enzyme. Varicose veins showed a stronger staining with prolidase enzyme than normal venous tissue, suggesting that the increase of prolidase enzyme level may play a role in the pathogenesis of primary varicose veins.

Yıldız et al.¹² performed a study about the relationship between prolidase enzyme and coronary artery disease and they determined that prolidase enzyme levels were increased in the patients with severe coronary artery disease. So we did not use the patients’ serum in healthy vein group for serum control group. In our study, there was no significant increase in the prolidase enzyme levels when compared with the serum samples from coronary bypass patients who were defined as normal vein group.

Conclusion

In addition to the known mechanical factors in the development of primary varicose veins, the role of oxidative stress at biochemical level is apparent in the light of our study and literature knowledge. In our oppinion, prolidase enzyme related with oxidative stress may play an important role in etiopathogenesis of primary varicose veins. The exact mechanism of increased prolidase activitiy in varicose vein patients is unknown but oxidative stress may lead to increase prolidase activity in these patients. Relation between activity of prolidase and oxidative stress should be considered in large scale prospective studies that will investigate the pathogenesis of varicose veins.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The study was financially supported by Gaziosmanpasa University Scientific Research Projects with 2015/24 project number.

References

Robertson

Evhalen Ans

Fowkes

FG.

Epidemiology of chronic venous disease. Phlebology. 2008; 23: 103–111.

Guzik

Chwała

Matusik

et al . Mechanisms of increased vascular superoxide production in human varicose veins. Pol Arch Med Wewn 2011; 121: 279–286.

Yasim

Kilinc

Aral

et al . Serum concentration of procoagulant, endothelial and oxidative stress markers in early primary varicose veins. Phlebology 2008; 23: 15–20.

Bergan

Pascarella

SchmiSchönbein

DW.

Pathogenesis of primary chronic venous disease: Insights from animal models of venous hypertension. JVAsc Surg 2008; 47: 183–192.

Krzyściak

Kózka

Generation of reactive oxygen species by a sufficient, insufficient and varicose vein wall. Acta Biochim Pol 2011; 58: 89–94.

Surazynski

Miltyk

Palka

et al . Prolidase-dependent regulation of collagen biosynthesis. Amino Acids 2008; 35: 731–738.

Hilali

Vural

Camuzcuoglu

et al . Increased prolidase activity and oxidative stress in PCOS. Clin Endocrinol 2013; 79: 105–110.

Uzar

Tamam

Evliyaoglu

et al . Serum prolidase activity and oxidative status in patients with diabetic neuropathy. Neurol Sci 2012; 33: 875–880. doi: 10.1007/s10072-011-0857-0. Epub 2011 Nov 27

Savas

Yeni

Celik

et al . The association of serum prolidase activity and erectile dysfunction. J Androl 2010 Mar-Apr; 31: 146–154.

10.

Verma

Chandra

Singh

et al . Serum prolidase activity and oxidative stress in diabetic nephropathy and end stage renal disease: a correlative study with glucose and creatinine. Biochem Res Int 2014; 2014: 291458. doi: 10.1155/2014/291458.

11.

Ozcan

Malkoc

Cosar

et al . Prolidase enzyme activity in varicose venous walls related to sperm count in patients with varicocele. Scand J Clin Lab Invest 2013; 73: 97–101.

12.

Yildiz

Demirbag

Yilmaz

et al . The association of serum prolidase activitiy with the presence and severity of coronary artery disase. Coron Arter Dis 2008; 19: 319–325.

13.

Lowry

Rosebrough

Farr

et al . Protein measurement with the folin phenol reagent. J Biol Chem 1951; 193: 265–275.

14.

Krzyściak

Kózka

Kazek

et al . Selected indicators of the antioxidant system in the blood of patients with lower limb varicose veins. Acta Angiol 2009; Vol. 15: 10–19.

15.

Condezo-Hoyos

Rubio

Arribas

et al . A plasma oxidative stress global index in early stages of chronic venous insufficiency. J Vasc Surg 2013; 57: 205–213.

16.

Flore

Santoliquido

Antonio

et al . Long saphenous vein stripping reduces local level of reactive oxygen metabolites in patients with varicose disease of the lower limbs. World J Surg 2003; 27: 473–475.

17.

Glowiński

Generation of reactive oxygen metabolite by the varicose vein wall. Eur J Vasc Endovasc Surg 2002; 23: 550–555.

18.

Tryankina

Kolobova

Varshavsky

BY.

Lipid peroxidation in pathogenesis of varix dilatation. Kliniczna Diagdnostyka Laboratoryjna 2003; 6: 19–20.

19.

Krzyściak

Kowalska

Kózka

et al . Iron content (PIXE) in competent and incompetent veins is related to the vein wall. Bioelectrochemistry 2012; 87: 114–123.

20.

Budzyń

Iskra

Krasiński

et al . Serum iron concentration and plasma oxidant-antioxidant balance in patients with chronic venous insufficency. Med Sci Monit 2011; 17: 719–727.

21.

Duygu

Koruk

Karsen

et al . Prolidase and Oxidative Stress in Chronic Hepatitis C. J Clin Lab Anal 2012; 26: 232–237.

22.

Gecit

Aslan

Gunes

et al . Serum prolidase activity, oxidative stress, and nitric oxide levels in patients with bladder cancer. J Cancer Res Clin Oncol 2012; 138: 739–743.

23.

Pirinçci

Kaba

Geçit

et al . Serum prolidase activity, oxidative stress, and antioxidant enzyme levels in patients with renal cell carcinoma. Toxicol Ind Health 2013; 30: 1–7.

24.

Bozkurt

Yüksel

et al . Serum prolidase enzyme activity and oxidative status in patients with Behçet's disease. Redox Rep 2014; 19: 59–64.

25.

Sayin

Aslan

Kucukoglu

et al . Serum prolidase enzyme activity and oxidative stress levels in patients with diabetic neuropathy. Endocrine 2014; 47: 146–151.

26.

Cakmak

Zeyrek

Atas

et al . Serum prolidase activity and oxidative status in patients with bronchial asthma. J Clin Lab Anal 2009; 23: 132–138.

27.

Fitowska

Ostałowska

Dobrakowski

et al . Protein metabolism in the synovial membrane in the hip osteoarthritis. Pol Orthop Traumatol 2012; 77: 21–26.

28.

Aslan

Duzenli

Esen

et al . Serum prolidase enzyme activity in obese subjects and its relationship with oxidative stress markers. Clin Chim Acta 2017; 473: 186–190.

29.

Koçarslan

What is the role of prolidase in pathogenesis of primary varicose veins?

Turk Gogus Kalp Dama 2017; 25: 68–73