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Assessment of oxidative stress with thiol disulfide homeostasis and ischemia-modifıed albumin level in acute urticaria

Objective: There is no study in the literature investigating ischemia-modified albumin (IMA) values and thiol disulfide homeostasis (TDH) parameters in acute urticaria patients. This study aimed assessment of TDH parameters and IMA in acute urticaria patients. Methods: The study included a total of 68 cases, with 35 acute urticaria patients and 33 healthy volunteers. Patients who presented to Ordu University Hospital and were diagnosed with acute urticaria between January 2019 and June 2019 and healthy individuals as the control group were included in the study. Serum albumin, IMA, native thiol, total thiol, and disulfide thiol levels were measured, and the results were compared between the groups. Results: IMA values of 0.93±0.09 in the study group were significantly high compared to 0.8±0.10 in the control group (p<0.01). Native thiol (SH) level was 353.66±87.5 in the study group, 393.62±47.7 in the control group (p:0.022), and total thiol (TSH) level was 385.46±86.6 in the study group and 433.53±56.06 in the control group (p:0.008). In the patient group there was a significant negative correlation between SH levels and IMA levels (r=-0.626, p<0.001). Conclusion: In acute urticaria, IMA increases while SH and TSH levels reduce. However, TDH does not change. The lack of change in the balance may be explained by acute urticaria being an acute event and not being a chronic inflammatory process.

angioedema (AE) linked to deep dermis or subcutis involvement or development of one or the other. Clinical tableau lasting less than six weeks is called acute urticaria (AU), while clinical tableau lasting six weeks or longer is called chronic urticaria (CU). Nearly 15-20% of people are determined to experience one AU attack during a period of their lives (1).
The most common triggers of AU attacks are infections, medications, and foods; however, in 50% of patients the situation remains idiopathic. Detailed history and physical examination are required for assessment of AU patients (2).
The balance between oxidants and antioxidants in the organism is the basis of preserving cellular and biochemical functions. Oxidants damage lipids, proteins and DNA in cells and even cause death (3,4). The most common and rapidly affected proteins are thiols containing sulfhydryl. Plasma thiols are strong antioxidants physiologically removing free radicals (ROS). Serum levels of plasma thiols are counted among markers showing antioxidant levels in the body (5). Normally there is a balance between thiols and disulfides, and these play a protective role for cellular redox homeostasis. This is called dynamic thiol/disulfide homeostasis. Defects in this balance may be associated with a variety of diseases (6). There is increasing evidence that ROSs play a role in the pathogenesis of a variety of inflammatory and allergic diseases including urticaria (7,8).
In recent times, another marker known as an oxidative stress marker is ischemia-modified albumin (IMA). Recent studies have shown ischemiamodified albumin (IMA) levels increase in free oxygen radicals formed because of ischemia, acidosis, or oxidative stress. The most common use is early myocardial injury. There are studies showing IMA levels increase in diseases causing oxidative stress like non-cardiac hypoxia, chronic renal disease, hypercholesterolemia, systemic sclerosis, and type 2 diabetes mellitus. In recent years, high levels of IMA are associated with a variety of diseases linked to oxidative stress (9).
There are very few studies about the correlation with oxidative stress in urticaria (4,10). There are a variety of studies reporting contradictory information about the oxidative stress status of chronic idiopathic urticaria patients. However, the number of studies investigating the thiol disulfide homeostasis in urticaria is very low and the majority are associated with chronic urticaria (4,(10)(11)(12). There is no study in the literature investigating IMA values and TDH parameters in acute urticaria patients. In this sense, our study is the first to investigate IMA and TDH parameters together in acute urticaria patients. With this aim, the new oxidative stress markers of thiol disulfide homeostasis and ischemia-modified albumin and the place of oxidative stress in acute urticaria pathogenesis was investigated.

Methods
The study included a total of 68 cases, with 35 being acute urticaria patients and 33 healthy volunteers. Patients who presented to Ordu University Hospital and were diagnosed with acute urticaria between January 2019 and June 2019 and healthy individuals as the control group were included in the study. Patients with physical urticaria and urticarial vasculitis were excluded from the study.
Serum albumin, ischemia-modified albumin, native thiol, total thiol and disulfide thiol levels were measured, and the results were compared between the groups. Patients' demographic data such as age and gender, vital signs, symptoms, lesion onset time, and urticaria activity score according to Turkey's urticaria diagnosis and treatment guidelines 2016 were recorded.

Biochemical analyses
Venous blood samples of cases attending with acute urticaria attack symptoms were taken in smooth gel tubes for biochemical tests (Becton Dickinson and Company, New Jersey, USA) and plasma samples for TDH tests were taken in EDTA tubes (Becton Dickinson and Company, New Jersey, USA). All samples were centrifuged at 1600xg for 10 minutes and stored at -70 °C until study. Biochemical tests were studied with an AU 2700 autoanalyzer (Beckman Coulter Inc. USA) with spectrophotometric methods.
Thiol measurements were assessed with spectrometry (Roche, cobas 501, Mannheim, Germany) using Erel et al.'s"modified Ellman method". Disulfide bonds (-S-S) were broken with sodium borohydride (NaBH4) to create free functional thiol groups (-SH). Unused sodium borohydride waste was removed with formaldehyde. In this way, reduction of any disulfide bond created by the DTNB reaction with 5,5'-dithiobis-(2nitrobenzoic) acid (DTNB) was prevented. As a result of the DTNB reaction, total thiol groups were determined as reduced and native thiols. Disulfide parameter is a parameter calculated as half of the SH and TSH content. After SH and TSH were determined, SS, SS/SH+SS %, SH/SH+SS % and SS/SH % were calculated.
Using a spectrophotometer at 470 nm, the absorbance of samples was measured (Jenway 6315 UV/visible Scanning Spectrophotometers, United Kingdom). Results are given as mg/dL. The albumin concentration was calculated by using the bromocresol green staining method (Biolabo, LesHautesRives, 02160, Maizy, France).

Statistical analysis
All data analyses were conducted using the SPSS v25 (IBM Inc., Chicago, IL, USA) statistical software package. Prior to the statistical analyses, the data were tested for normality using the Shapiro-Wilks test and for homogeneity of variance using the Levene's test. Independent samples t-test was used to assess differences between two groups. Crosstabulations were generated to describe the relationship between categorical variables, and the independence check was performed on the crosstabulations using a chi-square test (χ2) and Contingency Coefficient (CC). Likelihood Ratio Chisquare values (LR χ2) were calculated for frequencies below 5. The Pearson's correlation analysis test was used to determine the relation between continuous variables. All comparisons were two-tailed and Pvalue less than 5% was considered statistically significant.

Power analysis
The sample size for this study was estimated by a priori power analysis using GPower 3.1 (Universität Düsseldorf, Düsseldorf) statistical software; assuming a large effect size (d=0.80), α=0.05 and 1-β=0.80, a minimum sample size of 26 in each group was required to detect the significance of the independent groups in the t-test.

Ethics of the Study
This study was approved by the local ethic committee of Ordu University Medical Faculty with the 2018/234 numbered decision. All participants were informed in detail about the objective of the study and gave written consent. The study was performed in accordance with the ethical principles of the Declaration of Helsinki.

Results
A total of 35 patients with urticaria (patient group) and 33 healthy subjects (control group) participated in this study. IMA values were significantly higher in the patient group than in the control group (p<0.01). Native thiol (SH), Total thiol values were significantly lower in the patient group than in the control group (p<0.01). No other differences regarding study variables were observed between the patient group and the control group (p>0.05) ( Table  1). In the patient group, there was a significant negative correlation between the native thiol (SH) levels and the IMA levels (r=-0.626, p<0.001). In the patient group, there was a significant positive correlation between the SS/SH (%) levels and the IMA levels (r=-0.626, p<0.001). Native thiol decreased significantly as IMA increased in the patient group (r=-0.684, p<0.001; r=-0.626, p<0.001, respectively) ( Table 2  and Table 3).

Discussion
There is a balance between free radical production and the antioxidant system suppressing the increase in ROS in the body. If this balance is disrupted, oxidative stress (OS) occurs. There are studies assessing the oxidative stress in urticaria patients. Some studies have shown an increase in OS, while some studies have chosen no change in OS (13)(14)(15)(16)(17).
Oxidative stress has been studied in acute urticaria (AU) and chronic urticaria patient groups with different markers such as malondialdehyde (MDA), glutathione peroxidase (GSH-Px) and catalase (CAT). Studies by Kalkan  MDA levels were high and plasma GSH-PX activity reduced (1,10,14). In allergic diseases, especially asthma and to a lesser degree atopic dermatitis, there is increasing literature about the role of oxidative stress.
Free radicals occurring due to normal metabolism or pathologic processes disrupt the structure and functions of thiol dependent enzymes and cause changes in the thiol/disulfide ratio in the cell environment. Reduced plasma thiol concentration shows increased production of free radicals (6). In this study native thiol and total thiol levels reduced but disulfide thiol levels did not increase. But another oxidant parameter, IMA increased. Reduced native thiol levels are not definitely associated with increasing disulfide levels and similarly increased native thiol levels are not definitely associated with reduced disulfide levels. This may be associated with the immune response of thiols to inflammation. Reduced plasma thiol concentration is proof of free radical production. Reduced thiol levels are also associated with leukocyte activation. Urticaria mast cells are associated with immunologic activation and inflammation with oxidative stress (OS) activation of basophils and eosinophils and finally increased ROS (18). Stimulation of all inflammatory cells produces a significant amount of ROS. Eosinophils have higher peroxidase levels compared to other inflammatory cells and have a unique role in formation of oxidative stress (19).
In the acute urticaria patient group in our study, we identified reduced SH and TSH levels compared to the healthy control group. In the literature there are studies of dynamic thiol/disulfide homeostasis in different diseases. Eren et al. (20) showed total thiols were clearly reduced in migraine patients compared to a control group. Ates et al. (21) showed primary hypertension patients had total disulfide balance moving toward disulfide. Kundi et al. (22) identified reduced native and total thiol levels in patients with acute myocardial infarction compared to the control group and stated that thiol/disulfide homeostasis may be a new oxidative stress (OS) marker for acute myocardial infarction patients. A study by Ozyazıcı et al. (23) stated that the total disulfide balance had slid toward disulfide linked to thiol oxidation in a patient group with acute appendicitis and that this balance may be an OS marker for acute appendicitis patients. Yilmaz et al. (24) reported research on the thiol/disulfide homeostasis in asphalt workers exposed to polycyclic aromatic hydrocarbons.
In ischemic conditions, the shape of the amino terminal tip (N-terminal) of albumin changes and metal binding capacity reduces. As a result, this new form is a new ischemia marker called ischemiamodified albumin (IMA). Though IMA levels were initially considered specific to ischemia, superoxide radical damage, and exposure to free iron and copper have been shown to cause IMA formation (25). Based on these factors, in addition to ischemia-associated diseases, IMA values were shown to increase in some inflammatory diseases (26)(27)(28). In our study, a significant increase was identified in IMA levels in the acute urticaria patients compared to the control group.
Ozdemir et al. (29) reported increased IMA levels in patients with psoriasis due to an adaptive response to oxidative stress and systemic inflammation. However, Erem et al. (30) found the IMA irrelevant to oxidative stress.
Mast cells are known to play an important role in allergic diseases. As a result of antigen exposure, mast cells are sensitized and release many mediators causing allergic symptoms (31). Oxidative stress may ease degranulation of mast cells in response to allergens contributing to the development of allergic reactions. In the literature, there are some studies showing an association between oxidative stress and a variety of allergic diseases (3,13,14,19,31,32). Patella et al. (32) found that hymenoptera venom allergy levels of advanced oxidation protein products were consistently high during immunotherapy. They suggested that an oxidative stress state occurs in patients with hymenoptera allergy.
The colorimetric test performed on serum is based on decreased binding of exogenous cobalt to albumin which occurs in tissue damage caused by free radicals. (33). This issue was supported by the reports suggesting that IMA is strongly related with oxidative stress rather than being a myocardial ischemia marker (34,35).

Limitations
The lack of follow-up data, single blood sampling and small sample size are the limitations of our study. This study does not allow any conclusion about the causative correlation between acute urticaria and underlying interactions. To clarify the underlying mechanisms, it is necessary to confirm these findings in future studies with larger sample sizes.

Conclusions
Acute urticaria is a disease without definitely known cause. There are studies related to OS as a part of studies about etiopathogenesis. In our study, our objective data identified that SH and TSH levels reduce, and IMA values increase in AU. In

Oxidative Stress in Acute Urticaria
Mid Blac Sea J Health Sci 2021;7(1):115-121 conclusion, based on our data and literature information, we think increased IMA levels and reduced SH and TSH levels may be markers of oxidative stress in acute urticaria.