Dermatology: Practical and Conceptual Original Article | Dermatol Pract Concept. 2024;14(4):e2024249 1 Evaluation of Thiol Disulfide, Ischemia Modified Albumin, and Prolidase Parameters in Patients with Localized Scleroderma Ayşe Akbaş1, Orhan Şen 2, Fadime Kılınç 1, Salim Neşelioğlu3, Gülhan Aksoy Saraç1, Akın Aktaş4 1 Ankara City Hospital, Dermatology, Ankara, Turkey 2 Yozgat City Hospital, Dermatology, Yozgat, Turkey 3 Ankara City Hospital, Biochemistry, Ankara, Turkey 4 Yildirim Beyazit University, Medical Faculty, Ankara Citation: Akbaş A, Şen O, Kılınç F, Neşelioğlu S, Aksoy Saraç G, Aktaş A. Evaluation of Thiol Disulfide, Ischemia Modified Albumin, and Prolidase Parameters in Patients with Localized Scleroderma. Dermatol Pract Concept. 2024;14(4):e2024249. DOI: https://doi .org/10.5826/dpc.1404a249 Accepted: July 3, 2024; Published: October 2024 Copyright: ©2024 Akbaş et al. This is an open-access article distributed under the terms of the Creative Commons Attribution- NonCommercial License (BY-NC-4.0), https://creativecommons.org/licenses/by-nc/4.0/, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original authors and source are credited. Funding: None. Competing Interests: None. Authorship: All authors have contributed significantly to this publication. Corresponding Author: Ayşe Akbaş MD, Associate professor, Ankara City Hospital, Dermatology, Ankara, orcid.org/0000-0002-1626- 1796, Phone: +905324064994. E-mail: ayseakbas62@gmail.com Introduction: Localized scleroderma is a rare inflammatory skin disease that causes sclerosis in the dermis and subcutaneous tissue. Oxidative stress may play a role in the etiology or be responsible for the chronicity or progression of the disease. Objectives: We aimed to investigate the presence of oxidative stress in patients with localized scleroderma by examining thiol-disulfide balance, ischemia-modified albumin (IMA), and prolidase parameters. Methods: Twenty patients over the age of 18 who were diagnosed with localized scleroderma both clinically and histopathologically and 20 control subjects were included in the study. Age, sex, age at disease onset, duration of the disease, and presence of accompanying systemic diseases were ques- tioned and recorded. Lesion type and modified Rodnan and LoSSI scores were calculated through dermatological examination. CRP, sedimentation rate, total thiol, native thiol, and disulfide levels indicated by the Erel profile, IMA level, and prolidase levels were measured and compared in both the patient and control groups. Results: Levels of native thiol (P=0.958), total thiol (P=0.979), disulfide (P=0.449), (disulfide/ native thiol%) (P=0.368), (disulfide/total thiol%) (P=0.361), (native thiol/total thiol%) (P=0.368), and ABSTRACT 2 Original Article | Dermatol Pract Concept. 2024;14(4):e2024249 Introduction Localized scleroderma (LS, morphea) is a rare inflamma- tory skin disease that causes sclerosis in the dermis and subcutaneous tissue, with a pathogenesis that is not fully understood. Inflammatory phase manifests as erythematous purple patches and plaques, which later become white scle- rotic lesions surrounded by a violet-colored halo [1]. It is more common in females and occurs in children between the ages of 2 and 14; in adults, it is more prevalent in middle age [2]. Environmental factors such as trauma, radiation, genetics, drugs and chemicals, infections, vascular dysregu- lation, autoimmunity, and oxidative stress can all play a role in its etiology [3,4]. Oxidative stress (OS) arises from an imbalance between the oxidant system and the antioxidant system in the organ- ism, either due to excessive production of oxidant radicals or to a decrease in antioxidants or to both. This can lead to cell membrane damage, critical enzymes in intermediary metab- olism and other proteins, and DNA constituents, resulting in tissue damage and necrosis in cells [5] . Consequently, they contribute to the onset or progression of many chronic dis- eases, including skin diseases [6]. Various parameters have been evaluated in studies aimed at understanding the impact of oxidative stress on skin diseases, including thiol-disulfide balance, ischemia-modified albumin (IMA), and prolidase [7-10]. Oxidant substances cause decarboxylation in intra- cellular proteins, hydrolysis of peptide bonds, and formation of disulfide cross-links [11]. Plasma thiols are potent antioxidants that physiologi- cally neutralize free radicals [12]. Thiol groups are oxidized by reactive oxygen species (ROS), resulting in the reversible formation of disulfide bonds. Total thiol groups are highly sensitive to oxidations and are the first to be consumed during oxidative stress, thus neutralizing free radicals and preventing oxidative stress. These can be reduced back to thiol groups, depending on the situation [5,6,11]. This dy- namic balance of thiol-disulfide is essential for critical cellu- lar functions such as the antioxidant defense system, cellular transcription, cellular signal transduction, detoxification, enzymatic pathways, and apoptotic pathways [13]. Ischemia- modified albumin is a modified type of serum albumin that forms under oxidative stress conditions. This protein arises due to the reduced binding capacity of albumin’s N-terminal region for cobalt, copper, and nickel following cellular events caused by ischemia [9,14]. Although primarily a marker for cardiac ischemia, it has been reported in various studies that IMA levels can also increase in various inflammatory conditions such as psoriasis and rheumatoid arthritis and in disorders related to oxidative stress [14-17]. Prolidase is an exopeptidase important in the collagen cycle abundantly found in human and animal tissues, playing a significant role in collagen turnover and cell growth [10,18]. It is involved in the synthesis of collagen within cells and the catabolism of proteins containing proline or hydroxyproline. It has been suggested for the detection of disorders in collagen metabo- lism. Due to its widespread tissue distribution, abnormalities in prolidase enzyme activity could be associated with many clinical conditions and play a role in disease progression, particularly in fibrotic diseases and chronic injuries, where serum prolidase activity varies and is affected by oxidative stress [10,18-21]. Recent studies have continued to investigate the role of oxidative stress in the pathogenesis of skin diseases, including scleroderma, which is associated with the fibrosis of connec- tive tissue [8-10]. Various oxidant and antioxidant parame- ter levels have been measured in systemic scleroderma, and numerous hypotheses have been proposed [22-31]. However, these parameters have not been sufficiently studied in local- ized scleroderma, which has a similar pathogenesis [32,33]. Therefore, in this study, we aimed to investigate the presence of oxidative stress in LS by simultaneously measuring the levels of oxidative markers such as thiol-disulfide, IMA, and prolidase, and comparing them with a control group. Methods This a single-center prospective case-control study was approved by the local ethics committee, conducted in ac- cordance with the principles of the Declaration of Helsinki (approval number: E2-21-476 /02.06.2021) and was carried out ethically. Written informed consent forms were obtained from patients and volunteers. prolidase (P=0.121) were similar in both patient and control groups. Only IMA was significantly dif- ferent. No significant relationship was found between the levels of native thiol, total thiol, disulfide, (disulfide/native thiol), (disulfide/total thiol), IMA, prolidase, and Rodnan and LoSSI scores. Conclusion: According to the data obtained from this study, we can say that the thiol-disulfide balance is not disrupted and that prolidase levels are not affected in localized scleroderma; however, IMA is negatively affected. Original Article | Dermatol Pract Concept. 2024;14(4):e2024249 3 Twenty patients over the age of 18, diagnosed with LS clinically and histopathologically and followed up at our clinic, and 20 control subjects were included in the study. Age, sex, age at disease onset, duration of the disease, and presence of accompanying systemic diseases were ques- tioned and recorded. Lesion type, modified Rodnan and LoSSI scores were calculated through dermatological exam- ination [34,35]. (In the modified Rodnan score, the skin area is divided into a total of 17 different regions, and the score is calculated by manual palpation. Scoring is done accord- ing to the degree of thickness of the skin between 0 and 3; 0=Normal, 1=Mild thickening, 2=Moderate thickening, and 3=Severe thickening, and the maximum score is 51. The modified LoSSI score evaluates the skin erythema, thickness, and development of lesions or lesion enlarge- ment in 18 anatomical skin areas of the body. It is calcu- lated by adding scores between 0 and 3. The total score is between 0 and 162.) Individuals with chronic systemic diseases, neoplastic diseases, pregnant women, and those who had received systemic or topical drug treatment in the preceding three months were excluded from the study. The control group was selected from healthy individuals coming in for general check-ups. Laboratory tests included CRP sedimentation rate, total thiol, native thiol, and di- sulfide levels indicated by the Erel profile, IMA level, and prolidase levels. Participants provided venous blood samples after 12 hours of fasting, which were then centrifuged at 1500 rpm for 10 minutes. The separated serum samples were frozen and stored at -80 °C until analysis. All oxidative parameters were studied in the same samples. Serum native and total thiol concentrations and disulfide to native and total thiol ratios were determined by the spec- trophotometric Erel method using automatic clinical chem- ical analyzer (Roche, Cobas 501, Mannheim, Germany) [13]. Functional thiol groups were exposed using sodium borohydride. Unused reduced sodium borohydride was in- hibited by the reduction of 5,5’-dithiobis-(2-nitrobenzoic) acid (DTNB) with formaldehyde. The total thiol groups formed by reduced and native thiol groups were determined after reaction with DTNB. The dynamic disulfide amount was found by dividing the difference between total thiol and native thiol by two. Ratios of disulfide/total thiol, disulfide/ native thiol, and native thiol/total thiol were calculated, and the results were reported in absorbance units (ABSU) [36]. The Bar-Or method measures the IMA content with a 0.1% cobalt binding. After 10 minutes of incubation with cobalt, dithiothreitol was added. Finally, a 0.9% sodium chloride solution was added, and the absorbance was measured using a spectrophotometer. Prolidase activities were measured by Myara method [37]. A commercial kit (Human Xaa-Pro Dipeptidase/Prolidase Enzyme-linked Immunosorbent Assay kit (Cusabio Biotech Co Ltd) was used with ELISA method. (Myara method: The enzyme was activated by preincubation with MnCl2 for 2 hours (step 1), incubated with Gly-Pro substrate (Km = 2.9 mM) for 30 minutes (step 2), enzyme activity was calculated by measuring the amount of proline released (Ninhidrin rxn) (step 3).) Statistics Statistical analysis presented data as counts, arithmetic means, and standard deviations. The differences in average values between two groups were investigated using the Stu- dent’s t-test for parametric values and the Mann-Whitney U test for non-parametric values. The relationship between patients’ parameters and their demographic and clinical features was evaluated using Pearson correlation analysis. A  p-value of <0.05 was considered statistically significant in all tests. Data were analyzed using the SPSS 20.0 software package. Results The average age was 40.24 ± 14.77 in patients and 37.86 ± 10.98 in controls, and 85% of both the patient and the control groups were female (n=17). The patient and con- trol groups were similar in terms of age and sex (P=0.706). Comparison of serum parameter levels between patient and control group. Levels of native thiol (P=0.958), total thiol (P=0.979), disulfide (P=0.449), disulfide/native thiol% (P=0.368), disulfide/total thiol % (P=0.361), native thiol/ total thiol % (P=0.368), and prolidase (P=0.121) were sim- ilar in both patient and control groups. Only IMA was sig- nificantly different (Table 1). While a negative correlation was observed between native thiol, total thiol, and age, no significant correlation was observed between disulfide, disulfide/native thiol rates, disulfide/total thiol rates, IMA, and prolidase levels and age. While a negative correlation was observed between native thiol, total thiol, and disease duration, no significant correlation was observed with di- sulfide, disulfide/native thiol rates, disulfide/total thiol rates, IMA, and prolidase levels. No significant relationship was found between the levels of native thiol, total thiol, disul- fide, disulfide/native thiol rates, disulfide/total thiol rates, IMA, prolidase, and Rodnan and LoSSI scores. Native thiol (P<0.001, r= -0.538) and total thiol (P<0.001, r= -0.553) were negatively correlated with age. Native thiol and total thiol levels decreased with increasing age. Native thiol (P=0.008, r= -0.574) and total thiol (P=0.007, r= -0.583) were nega- tively correlated with disease duration. Native thiol and to- tal thiol levels decreased with increasing disease duration. Native thiol and total thiol levels decreased with increasing disease duration (Table 2). 4 Original Article | Dermatol Pract Concept. 2024;14(4):e2024249 hyperactivation that exhibits abnormal profibrotic activ- ity, increases collagen synthesis, and decreases collagenase synthesis [1,3,23,38]. ROS in the environment directly or indirectly mediate extracellular and intracellular oxidative processes and stimulate the accumulation of matrix proteins by affecting endothelial cells and fibroblasts [24,28,29]. Murrel revealed the effect of OS in the pathogenesis of scleroderma for the first time [39]. Several subsequent studies Discussion In morphea, the pathological process starts in the vascular endothelium. Excessive release of various adhesion mole- cules on the endothelial cell surface triggers an inflammatory cascade involving proinflammatory cytokines and chemok- ines as well as growth factors with fibrogenetic potential. Transforming growth factor β (TGF-β) stimulates fibroblast Table 1. Native Thiol, Total Thiol, Disulfide, Disulfide/Native Thiol (%), Disulfide/Total Thiol (%), IMA, Investigation of the Relationship between Prolidase Levels and Demographic and Clinical Characteristics of Patients. Comparison of mean parameters between patient and control groups Parameters Patient Mean ± sd Control Mean ± sd p-value (Mann-Whitney U Test) Native thiol (μmol/) 424.61 ± 71.57 427.88 ± 62.84 0.958 Total thiol (μmol/L) 468.99 ± 77.19 473.55 ± 66.46 0.979 Disulfide (μmol/L/) 22.19 ± 8.72 . 0.449 Disulfide/Native thiol (%) 5.28 ± 2.15 5.39 ± 1.18 0.368 Disulfide/Total thiol (%) 4.71 ± 1.71 4.84 ± 0.98 0.361 Native Thiol/Total thiol (%) 90.56 ± 3.43 90.30 ± 1.97 0.368 IMA(ABSU) 0.63 ± 0.18 0.88 ± 0.22 0.001 Prolidase(U/L) 1015.87 ± 31.34 994.91 ± 50.78 0.121 Table 2. The Correlation between Parameters and Age, Disease Duration, and Scoring. Age r value p-value Duration of disease r value p-value Rodnan r value p-value LoSSI r value p-value Native thiol (μmol/L) -0.538 <0.001 -0.574 0.008 -0.051 0.833 0.056 0.814 Total thiol (μmol/L) -0.553 <0.001 -0.583 0.007 -0.011 0.963 0.110 0.645 Disulfide (μmol/L) -0.252 0.112 -0.222 0.346 0.158 0.506 0.255 0.278 Disulfide/native thiol (%) -0.007 0.966 0.011 0.963 0.136 0.567 0.193 0.414 Disulfide/total thiol (%) 0.001 0.993 -0.014 0.954 0.135 0.571 0.198 0.403 Native thiol/total thiol (%) -0.001 0.995 0.014 0.954 -0.135 0.571 -0.198 0.403 IMA (ABSU) -0.188 0.240 0.074 0.756 0.219 0.354 0.338 0.145 Prolidase (U/L) -0.265 0.094 -0.089 0.709 0.053 0.823 0.013 0.958 Rodnan 0.231 0.313 -0.065 0.778 LoSSI 0.100 0.666 -0.198 0.390 Abbreviations: R: correlation coefficient. Original Article | Dermatol Pract Concept. 2024;14(4):e2024249 5 Riccieri et al. suggested that the levels of carbonyl groups, which are biomarkers of protein oxidation, are inversely cor- related with the modified Rodnan skin score used for skin symptoms in systemic scleroderma [43]. In this study, no sig- nificant correlation was found between the LoSSI and Rod- nan scales showing lesion severity in LS and thiol, IMA, and prolidase levels. This may be influenced by the low patient scores in our study. In addition, we think that the low IMA levels in the study may be due to the absence of ischemia in the LS. In conclusion, this evaluation shows that LS is not affected by oxidative stress as much as systemic scleroderma due to its limited involvement or it can limit itself with other antioxidant systems. It could be argued that the aging process potentially af- fects OS levels. For example, several studies have shown that aging cells accumulate increasing levels of oxidant-damaged DNA, which can further increase the level of OS. The mean age of the patients in this study was 40.24 years. It was ob- served that native and total thiol values decreased with in- creasing age. The decrease in these values with age supports that while the production of free radicals increases with aging, endogenous defense mechanisms decrease and the balance shifts to the OS side [44]. There was no significant difference between IMA and prolidase levels and age. We can say that these enzymes are not affected by age. In this study, a negative correlation was observed between native thiol and total thiol and disease duration. Native thiol and total thiol levels decreased with increasing disease. This may indicate that OS increases as the disease duration in- creases, or that the disease becomes chronic as OS increases. This negative correlation suggests that the decrease in native and total thiols may be associated with the chronicization of the disease and that these parameters may be a marker. However, there was no significant difference between IMA and prolidase levels and disease duration. IMA is related to OS caused by free radicals during isch- emia and/or reperfusion. Although most biomarkers are neg- ative in ischemia, IMA is highly sensitive and can be detected at an early stage [14,45]. Therefore, it has been suggested that serum IMA levels may be used as a marker of systemic oxidative stress. Elevated IMA levels have been reported in various clinical pathologic conditions including vascular endothelial cell disorder, diabetes mellitus, liver and kidney diseases, some cancers, and systemic sclerosis in which oxi- dative status is affected [ 9,46,47]. Some investigators have shown that vasculopathy in scleroderma causes tissue hy- poxia, resulting in increased OS, fibrotic changes, and skin thickening [24]. It has been suggested that it may be dis- tinctive especially during the active period of the disease. In our study, IMA levels were significantly lower compared to controls. This may suggest that LS is not affected by ischemia-induced OS. have supported this hypothesis for both localized and sys- temic scleroderma, but the specific role of OS in the patho- genesis of vascular injury and fibrosis has yet to be elucidated [23,27,31,40-42]. ROS are considered to play a background role in the pathology of this disease and are considered to be instrumental in the manifestation of clinical symptoms. ROS generated by various reactions can stimulate the pro- duction of proinflammatory cytokines. Fibrotic cytokines in- duce proliferation and activation of fibroblasts, increase type I collagen synthesis, and cause vascular dysfunction. Skin changes typical for scleroderma have been demonstrated in vivo by intradermal injection of ROS-producing compounds in several animal studies [23]. There are studies  showing that antioxidant capacity decreases in patients with systemic scleroderma [27,30,42]. Bozkurt et al. found that serum to- tal oxidant capacity and oxidative stress index (OSI) were high in patients with systemic scleroderma. They also re- ported that prolidase activity is also high in fibrotic diseases, and this may be an important marker for OS [18]. LS, which is a localized form of scleroderma, is thought to have a common pathogenesis; therefore, similar studies on OS have also been conducted in LS, albeit to a lesser extent. The researchers investigated OS parameters in both tissue and plasma [32,33]. In these studies, it was attempted to prove the presence of OS in LS, with significant changes in OS parameters such as serum S glutathione transferase and total oxidant capacity compared to controls. Based on the same hypothesis, we investigated thiol/ disulfide, IMA, and prolidase enzymes, which are among the OS parameters thought to play a role in the pathogenesis of LS. Unlike other studies, these parameters were measured si- multaneously in patient sera and compared with the control group. Thiols are antioxidants that react quickly in oxidative environments. In various studies, it has been reported that di- sulfide levels increase in inflammatory diseases and decrease in malignant diseases [6,8,26]. Therefore, it has been stated that the increase in disulfide concentrations is associated with OS, and the increase in native thiol levels may be an indicator of a reaction against the oxidative environment [6]. Kılınç et al. investigated total oxidant capacity (TOC), OSI, and arylesterase as OS markers in LS patients in a pre- vious study using a control group; they found a significant increase in the patient group [32]. Uzun Çakmak et al. found that glutathione S-transferase isoenzymes were significantly higher in tissue biopsies obtained from patients with LS com- pared to control tissues [33]. In this study, when we compared LS patients with the control group, we found that the native thiol, total thiol, di- sulfide values, and their ratios to each other did not change, i.e., the thiol-disulfide balance was not impaired. Prolidase activity levels were similar to controls. IMA levels were lower in the patient group (P<0.05). 6 Original Article | Dermatol Pract Concept. 2024;14(4):e2024249 13;10:1108623. DOI: 10.3389/fmed.2023.1108623. PMID: 3686 0340; PMCID: PMC9969991. 2. Florez-Pollack S, Kunzler E, Jacobe HT. Morphea: Current concepts. Clin Dermatol. 2018;36(4):475-486. DOI: 10.1016/j.clindermatol. 2018.04.005. Epub 2018 Apr 10. PMID: 30047431. 3. Torok KS, Li SC, Jacobe HM, et al. Immunopathogenesis of Pediatric Localized Scleroderma. Front Immunol. 2019;10:908. Published 2019 Apr 30. DOI:10.3389/fimmu.2019.00908. 4. Abbas L, Joseph A, Kunzler E, et al. Morphea: progress to date and the road ahead. Ann Transl Med.2021;9(5):437. DOI: 10.21037 /atm-20-6222. PMID: 33842658; PMCID: PMC8033330. 5. Baba SP, BhatnAGAr A. Role of tiyols ın oxidative stres. Curr Opin Toxicol.2018 ;7:133-139. DOI: 10.1016/j.cotox.2018.03 .005. PMID: 30338308. 6. Erel Ö, Erdoğan S Thiol-disulfide homeostasis: an integrated ap- proach with biochemical and clinical aspects. Turk J Med Sci .2020; 50: 1728-1738 © Tübitak. DOI:10.3906/sag-2003-64. 7. Nakai, K. Tsuruta, D. What Are Reactive Oxygen Species, Free Radicals, and Oxidative Stress in Skin Diseases?  Int. J. Mol. Sci.  2021  ;22: 10799. DOI: 10.3390/ijms221910799. PMID: 34639139; PMCID: PMC8509443. 8. Georgescu SR, Mitran CI, Mitran MI, et al. Thiol-Disulfide Ho- meostasis in Skin Diseases. J Clin Med. 2022 9;11(6):1507. DOI: 10.3390/jcm11061507. PMID: 35329832; PMCID: PMC8954849 9. Tampa M, Mitran CI, Mitran MI, Amuzescu A, Matei C, Georgescu SR. Ischemia-Modified Albumin-A Potential New Marker of Oxidative Stress in Dermatological Diseases. Medicina (Kaunas). 2022 May 17;58(5):669. DOI: 10.3390/medicina 58050669. PMID: 35630086; PMCID: PMC9147831. 10. Eni-Aganga I, Lanaghan ZM, Balasubramaniam M, Dash C, Pandhare J. PROLIDASE: A Review from Discovery to its Role in Health and Disease. Front Mol Biosci. 2021;8:723003. Published 2021 Aug 31. DOI:10.3389/fmolb.2021.723003. 11. Radi R, Trujillo M. Special issue on “Free Radical and Redox Biochemistry of Thiols”.  Free Radic Res. 2016;50(2):123-125. DOI:10.3109/10715762.2015.1121254. 12. Chianeh YR, Prabhu K. Protein Tiyols as an indication of oxida- tive stres. Arch. Med. Res. 2014; 23(3): 443-56. DOI:10.17827 /aktd.72428. 13. Erel O, Neselioglu S. A novel and automated assay for hiol- disulphide homeostasis. Clin Biochem. 2014;47(18):326-332. DOI: 10.1016/j.clinbiochem.2014.09.026 PMID: 25304913. 14. Sbarouni E, Georgiadou P, Voudris V. Ischemia modified albumin changes - review and clinical implications. Clin Chem Lab Med 2011;49:177-84. DOI: 10.1515/CCLM.2011.037. Epub 2010 Nov 18. PMID: 21083441. 15. Guntas G, Sahin A, Duran S, et al. Evaluation of ischemia- modified albumin in patients with inflammatory bowel disease. Clin Lab. 2017; 63(2):341-7. DOI: 10.7754/Clin.Lab.2016 .160623. PMID: 28182340. 16. Aşkın L, Serçelik A, Tanrıverdi O, Süsenbük A, Türkmen S, Aktürk E. Kardiyovasküler İskemik Olayların Yeni Bir Sistemik Göstergesi: İskemi modifiye albumin levels. MN Kardiyoloji 2019;26(3):198-202. 17. Ozdemir M, Kiyici A, Balevi A. Assessment of ischaemia- modified albumin level in patients with psoriasis. Clin Exp Dermatol. 2012; 37:610–4. DOI: 10.1111/j.1365-2230.2012.04384.x. Epub 2012 May 21. PMID: 22607558. 18. Bozkurt M, Yüksel H, Em S, et al. Serum prolidase enzyme activity and oxidative status in patients with Behçet’s disease. Redox Rep. Prolidase is an enzyme involved in collagen metabolism. It is widely distributed in many tissues such as bone, con- nective tissue, and blood cells. Various studies suggest that prolidase could play a significant role in many physiological and pathophysiological processes, including wound healing, inflammation, and angiogenesis through the regulation of growth factors and transcription factors [10,38]. Therefore, it has been proposed that abnormalities in prolidase enzyme activity could play a role in the progression of many dis- eases [10]. Yıldız et al. have indicated that serum prolidase activity is directly related to oxidative stress (OS) and can be used as an indicator of oxidative stress [20]. Oxidative stress leads to the degradation of collagen, a process regu- lated by prolidase [19]. Additionally, the severity of OS is directly linked to the inhibition of collagen structure, and prolidase is considered the main enzyme in this process [10]. Various studies to date have shown that collagen, being widely found in many tissues, undergoes changes during the course of various diseases [18]. Particularly in scleroderma with organ involvement and fibrosis, prolidase levels have been found to be high [38]. However, Çelik et al. reported no difference in serum prolidase activity between patients with scleroderma and controls [40]. Savaş et al., in their study, found low serum prolidase levels, suggesting this could be due to a decrease in collagen turnover, and possibly a result of increased synthesis and decreased degradation. They also proposed that this could affect the reduced physical func- tions in patients [48]. We also investigated prolidase levels in LS, a localized form of systemic scleroderma, and found no change. This could be due to the localized nature of the disease, not in- volving systemic spread, or possibly due to the small number of patients. Conclusion Localized scleroderma is a multifactorial disease with an un- known cause. Based on the data obtained from our study related to OS, we can say that the thiol-disulfide balance is not disrupted and prolidase levels are not affected in LS. However, IMA is negatively affected. Despite factors such as age and duration, the oxidative balance is not disturbed, suggesting that the organism can correct itself with other antioxidant systems. Further studies are needed to support these findings. We believe that new and comprehensive studies in the future may elucidate the pathogenesis of LS. References 1. Papara C, De Luca DA, Bieber K, Vorobyev A, Ludwig RJ. Morphea: The 2023 update. Front Med (Lausanne). 2023; Original Article | Dermatol Pract Concept. 2024;14(4):e2024249 7 sclerosis.  Clin Rheumatol  30, 921–925 (2011). DOI 10.1007/ s10067-011-1695-4. 32. Kilinc F, Sener S, Akbas A, et al. Oxidative stress parameters in localized scleroderma patients. Arch Dermatol Res. 2016;308: 625-629. DOI: 10.1007/s00403-016-1682-3. Epub 2016 Sep 3. PMID: 27591994. 33. Uzuncakmak TK, Koska MC, Ozkanli S, et al. Comparison of the tissue expressions of glutathione S transferase isoen- zymes among patients with morphea and healthy controls: A preliminary study. Dermatol Ther. 2020 Nov;33(6):e14363. DOI: 10.1111/dth.14363. Epub 2020 Oct 8. PMID: 33002252. 34. Khanna D, Furst DE, Clements PJ, et al. Standardization of the modified Rodnan skin score for use in clinical trials of sys- temic sclerosis. J Scleroderma Relat Disord. 2017. DOI:10.5301 /jsrd.5000231 35. Arkachaisri T, Vilaiyuk S, Torok KS, Medsger TA Jr. Develop- ment and initial validation of the localized scleroderma skin damage index and physician global assessment of disease dam- age: A proof-of-concept study. Rheumatology (Oxford) 2010;49: 373-81. DOI: 10.1093/rheumatology/kep361. Epub 2009 Dec 14. PMID: 20008472; PMCID: PMC3498950. 36. Bar-Or D, Lau E, Winkler JV. A novel assay for cobalt- albumin binding and its potential as a marker for myocardial ischemia-a preliminary report. J Emerg Med. 2000; 19(4):311-5. DOI: 10.1016/s0736-4679(00)00255-9. PMID: 11074321. 37. Myara I, Myara I, Marcon P, Lemonnier A, Chateliér B, Mangeot M. Determination of prolinase activity in plasma. Application to liver disease and its relation with prolidase activity. Clin Biochem. 1985 Aug;18(4):220-3. DOI: 10.1016/s0009-9120(85)80043-6. PMID: 4042319. 38. Surazynski A, Surazynski A, Miltyk W, Prokop I, Palka J. Prolidase-dependent regulation of TGF β (corrected) and TGF β receptor expressions in human skin fibroblasts. Eur J Pharmacol. 2010 Dec 15;649(1-3):115-9. DOI: 10.1016 /j.ejphar.2010.09.034. Epub 2010 Sep 21. PMID: 20868675. 39. Murrell DF. A radical proposal for the pathogenesis of sclero- derma. J Am Acad Dermatol. 1993;28:78-85. DOI: 10.1016 /0190-9622(93)70014-k. PMID: 8425975. 40. Celik A, Birer MN, Kilinc M. Serum prolidase activity in sys- temic sclerosis. Clin Rheumatol. 2017 Aug;36(8):1827-1832. DOI: 10.1007/s10067-017-3677-7. Epub 2017 May 22. PMID: 28534076. 41. Sambo P, Baroni SS, Luchetti M, et al. Oxidative stress in sclero- derma: maintenance of scleroderma fibroblast phenotype by the constitutive up-regulation of reactive oxygen species gener- ation through the NADPH oxidase complex pathway. Arthritis Rheum. 2001;44(11):2653–64. DOI: 10.1002/1529-0131(200111) 44:11<2653::aid-art445>3.0.co;2-1. PMID: 11710721. 42. Devrim E, Erten S, Erguder IB, Namuslu M, Turgay M, Durak I. Malondialdehyde and nitric oxide levels in erythrocytes from patients with systemic sclerosis. Med Princ Pract. 2008;17(4): 349-350. DOI:10.1159/000129620. 43. Riccieri V, Spadaro A, Fuksa L, Firuzi O, Saso L, Valesini G. Specific oxidative stress parameters differently correlate with nailfold capillaroscopy changes and organ involvement in systemic sclerosis. Clin Rheumatol. 2008;27:225-230. DOI: 10.1007/s10067-007-0769-9. Epub 2007 Oct 27. PMID: 17965907. 44. Rinnerthaler M, Bischof J, Streubel MK, Trost A, Richter K. Oxidative stresi in aging human skin. Biomolecules. 2015 Apr 2014; 19(2):59-64. DOI: 10.1179/1351000213Y.0000000072. Epub 2013 Nov 12. PMID: 24225260; PMCID: PMC6837555. 19. Altindag O, Erel O, Aksoy N, Selek S, Celik H, Karaoglanoglu M. Increased oxidative stress and its relation with collagen me- tabolism in knee osteoarthritis. Rheumatol Int. 2007;27:339–44. DOI: 10.1007/s00296-006-0247-8. Epub 2006 Nov 10. PMID: 17096092. 20. 20.Yildiz A, Demirbag R, Yilmaz R, et al. The association of serum prolidase activity with the presence and severity of cor- onary artery disease. Coron Artery Dis. 2008;19(5):319–25. DOI: 10.1097/MCA.0b013e32830042ba. PMID: 18607169. 21. 21.Aciksari G, Demir B, Uygun T, et al. Serum prolidase ac- tivity in patients with cardiac syndrome X. North Clin Istanb 2020;7(5):471–477. DOI: 10.14744/nci.2020.09086. PMID: 3316 3883; PMCID: PMC7603856. 22. 22.Luo JY, Liu X, Jiang M, Zhao HP, Zhao JJ. Oxidative stress markers in blood in system in blood in systemic sclero- sis: a meta-analysis. Mod Rheumatol. 2017;27:306-314. DOI: 10.1080/14397595.2016.1206510. Epub 2016 Jul 18. PMID: 27425641. 23. Vona R, Giovannetti A, Gambardella L, Malorni W, Pietraforte D, Straface E. Oxidative stress in the pathogenesis of systemic scleroderma: an overview. J Cell Mol Med. 2018;22:3308- 3314. DOI: 10.1111/jcmm.13630. Epub 2018 Apr 17. PMID: 29664231; PMCID: PMC6010858. 24. Piera-Velazquez S, Jimenez SA. Oxidative Stress Induced by Reactive Oxygen Species (ROS) and NADPH Oxidase 4 (NOX4) in the Pathogenesis of the Fibrotic Process in Systemic Sclerosis: A Promising Therapeutic Target. J Clin Med. 2021;10(20):4791. Published 2021 Oct 19. DOI:10.3390/jcm10204791. 25. Gabrielli A, Svegliati S, Moroncini G, Amico D. New insights into the role of oxidative stress in scleroderma fibrosis. Open Rheu- matol J. 2012;6:87-95. DOI: 10.2174/1874312901206010087. Epub 2012 Jun 15. PMID: 22802906; PMCID: PMC3395898. 26. Abdulle AE, van Roon AM, Smit AJ, et al. Rapid free thiol rebound is a physiological response following cold-induced vaso- constriction in healthy humans, primary Raynaud and systemic sclerosis. Physiol Rep. 2019 Mar;7(6):e14017. DOI: 10.14814 /phy2.14017. PMID: 3091. 27. Kundu D, Abraham D, Black CM, Denton CP, Bruckdorfer KR. Reduced levels of S-nitrosothiols in plasma of patients with sys- temic sclerosis and Raynaud’s phenomenon. Vascul Pharmacol. 2014;63: 178-181. DOI: 10.1016/j.vph.2014.09.003. Epub 2014 Oct 15. PMID: 25446164; PMCID: PMC4265732. 28. Grygiel-Gorniak B, Puszczewicz M. Oxidative damage and an- tioxidative therapy in systemic sclerosis. Mediators Inflamm. 2014;2014:389582. DOI: 10.1155/2014/389582. Epub 2014 Sep 8. PMID: 25313270; PMCID: PMC4172878. 29. Svegliati S, Spadoni T, Moroncini G, Gabrielli A. NADPH oxidase, oxidative stress and fibrosis in systemic sclerosis. Free Radic Biol Med. 2018 Sep;125:90-97. DOI: 10.1016/j.freeradbiomed .2018.04.554. Epub 2018 Apr 22. PMID: 29694853. 30. Dziedzic R, Wójcik K, Olchawa M, et al. Increased oxidative stress response in circulating blood of systemic sclerosis patients - relation to disease characteristics and inflammatory blood biomarkers. Semin Arthritis Rheum. 2023 Oct;62:152228. DOI: 10.1016/j.semarthrit.2023.152228. Epub 2023 Jun 9. PMID: 37429138. 31. Ogawa F, Shimizu K, Muroi E, et al.  Increasing levels of se- rum antioxidant status, total antioxidant power, in systemic 8 Original Article | Dermatol Pract Concept. 2024;14(4):e2024249 47. Kirmit A, Kader S, Aksoy M, Bal C, Nural C, Aslan O. Trace Elements and Oxidative Stress Status in Patients with Psoria- sis. Adv. Dermatol. Allergol. 2020, 37, 333–39. DOI: 10.5114 /ada.2020.94265. Epub 2020 Apr 7. PMID: 32792872; PMCID: PMC7394161. 48. Savas E, Aksoy N, Pehlivan Y, et al. Evaluation of oxidant and antioxidant status and relation with prolidase in systemic scle- rosis. Wien Klin Wochenschr. 2014 Jun;126(11-12):341-6. DOI: 10.1007/s00508-014-0534-4. Epub 2014 May 14. PMID: 24825595 21;5(2):545-89. DOI: 10.3390/biom5020545. PMID: 25906193; PMCID: PMC4496685. 45. Apple FS. Clinical and analytical review of ischemia-modified albumin measured by the albumin cobalt binding test. Adv Clin Chem. 2005;39:1-10. DOI: 10.1016/s0065-2423(04)39001-3. PMID: 16013666. 46. Kor A, Maraş Y, Atalar E, Fırat Oğuz E, Gök K, Erel Ö. Ischemia-modified albumin levels are elevated, and thiol/disulfite homeostasis is impaired in Behçet’s disease. Ulus Romatol Derg 2022;14(1):15-22.