Dynamic Thiol/Disulphide Homeostasis in Acute Urticaria.

BACKGROUND: Urticaria is an unknown, sudden, and itchy skin disease that is recognized with redness, swelling, and is sometimes seen with angioedema. It is classified as acute or chronic, depending on the duration of symptoms. Thiols in plasma are powerful antioxidants that physiologically eliminate free radicals. The mostly and rapidly affected proteins are thiols that contain the sulfhydryl group. In the present study, the thiol/disulfide homeostasis was investigated as a brand new indicator of oxidative stress in patients who had acute urticaria and presented to the emergency department.
OBJECTIVE: In the present study, the thiol/disulfide homeostasis, ischemia-modified albumin (IMA), and and neutrophil lymphocyte ratio (N/L ratio) were investigated in the etiopathogenesis of acute urticaria. MATERIAL AND
METHOD: A total of 37 patients and 40 healthy volunteers were included in the study. Thiol/disulfide homeostasis (TDH) [total thiol-native thiol/disulfide changes] was measured in both groups (patient group and control group) using a brand novel method developed by Erel and Neselioglu. Half of the difference between total thiol and native thiol concentrations gives the amount of disulfide bond.
RESULTS: Total thiol and native thiol levels in blood were found to be low. The levels of total thiol (P = 0.218) and native thiol (P = 0,001) were significantly lower in patients with acute urticaria than in the control group. At the same time, the level of disulfide was significantly higher in the patient group than in the control group (P = <0.001). The level of IMA was higher in the patient group than in the control group (P < 0.001).
CONCLUSION: While total thiol and native thiol are low in acute urticaria, the levels of disulfide and IMA are high. Copyright:

Introduction

Acute urticaria is an unknown, sudden, and itchy skin disease that is recognized by redness, swelling, and is sometimes seen with angioedema.[1] It is classified as acute or chronic, depending on the duration of symptoms. The clinical type lasting up to 6 weeks is called acute urticaria (AU), and the clinical type lasting more than 6 weeks is called chronic urticaria (CU).[123] AU is mostly caused by drugs, food, and local and systemic infections, and the causes are unknown in 45% of AU cases.[34]

Oxidative stress (OS) causes cellular damage when reactive oxygen products (ROP) and reactive nitrogen products (RNS) levels in the cells exposed to endogenous or exogenous oxidants rise above the physiological levels.[56] Oxidizing agents damage the lipids, proteins and DNA in cells. Increased ROP is a cause of the disease but ROP levels may sometimes increase after some diseases.[78]

Thiols in plasma are powerful antioxidants that physiologically eliminate free radicals. The mostly and rapidly affected proteins are thiols that contain the sulfhydryl group. Native and total thiol serum levels are among the indicators of the antioxidant status in the body.[9]

In some studies, the thiol/disulfide homeostasis was investigated. This homeostasis was measured unilaterally until 2014, but it is now measured bilaterally using a brand novel method developed by Erel and Neselioglu.[1011]

In the present study, the thiol/disulfide homeostasis, ischemia-modified albumin (IMA), and N/L ratio were investigated in the etiopathogenesis of acute urticaria.

Procedure

Ethics committee approval was obtained for the present study from Yıldırım Beyazıt University (28.11.2018/250). Informed consent was obtained from all the participants in the study. A total of 77 participants consisting of 37 patients with acute urticaria and uvula edema and 40 healthy volunteers were included in the study. The study included the patients presented to the emergency department within a maximum of 1 h from the onset of symptoms, the patients were not receiving systemic cortisone or other medications in the last month and not receiving antihistamine treatment within the last 7 days. The patients with food and drug allergy, presence of infection, systemic and neoplastic disease, solar urticaria cold urticaria, chronic urticaria, vasculitis, and hereditary angioedema were excluded from the study. Urticarial vasculitis (HUV) is a rare form of vasculitis characterized by inflammation of the small blood vessels. HUV causes recurrent episodes of urticaria and painful skin lesions that itch or burn. We excluded cases with recurrent urticaria attacks or painful lesions on the suspicion of vasculitis. Venous blood samples were taken in the emergency department before starting the treatment.

Measurement of Thiol/Disulfide Homeostasis Parameters

Thiol/disulfide homeostasis tests were measured by the automated spectrophotometric method described by Erel and Neselioglu. Briefly, disulfide bonds were first reduced to form free functional thiol groups with sodium borohydride. Unused reductant sodium borohydride was consumed and removed with formaldehyde to prevent reduction of DTNB (5,5′-dithiobis-(2-nitrobenzoic) acid), and all of the thiol groups including reduced and native thiol groups were determined after the reaction with DTNB. Half of the difference between the total thiols and native thiols provides the dynamic disulfide amount. After the determination of native and total thiols, disulfide amounts, Index-1 disulfide/native thiol percent ratios ([SS]/[SH] × 100) Index-2: Disulfide/total thiol percent ratios (SS]/[SH + SS] × 100) and Index-3: Native thiol/total thiol percent ratios([SH]/[SS + SH] × 100) were calculated.[10]

Measurement of the Ischemia- Modified Albumin (IMA)

Measurement of IMA levels was obtained using venous blood samples on admittance within 1 h. Specimens were stored for 30 min at room temperature and then centrifuged at 3500 rpm for 5 min. Later, the samples were transferred to Eppendorf tubes and stored at –80°C until analysis. Albumin cobalt binding test was used to detect the presence of IMA. This test was performed by adding 50 mL 0.1% cobalt (II) chloride (CoCl2, 6H2O) (Sigma-Aldrich Chemie GmbH Riedstrasse 2, Steinheim, Germany) to the patient serum. After mixing, followed by 10 min of incubation to allow for albumin cobalt binding, 50 mL of 1.5 mg/mL dithiothreitol was added. After mixing followed by 2 min of incubation, 1.0 mL of 0.9% sodium chloride solution was added in order to reduce the binding capacity. The blank was prepared similarly with distilled water instead of dithiothreitol. The absorbance of samples was measured at 470 nm using a spectrophotometer. The results were expressed as absorbance units (ABSU)

Statistical analysis

The Statistical Package for Social Sciences for Windows, Version 22 (IBM, Armonk, NY, USA) was used for the statistical analyses. The Kolmogorov-Smirnov test was used for the normality of the variables. Mean ± standard deviation (SD) was used for the parameters with normal distribution, and median (interquartile range) (IQR) was used for the parameters not consistent with the normal distribution. One-Way analysis of variance (ANOVA) test was used for the parameters that were normally distributed. Those with not normal distribution were evaluated with the Kruskal-Wallis Test. Comparisons for categorical variables were performed using the Chi-Square Test or the Fisher's Exact Test. A P value < 0.01 was considered to be statistically significant.

Conclusion

The mean age was calculated as 37 in the acute urticaria and 40 in the control group. It was found that there was no statistically significant difference between the 37 patients with acute urticaria (patient group) and the 40 healthy individuals (control group) in terms of age.

The levels of total thiol (P = 0.218) and native thiol (P = 0,001) were significantly lower in patients with acute urticaria than in the control group [Figures 1 and 2]. At the same time, the level of disulfide was significantly higher in the patient group than in the control group (P = <0.001). Considering the comparison of IMA parameters in the control and acute urticaria groups, IMA levels were high in the patient group as seen in Table 1 (P < 0.001).

Figure 1

Comparison (a) native thiol, (b) disulphide, (c) total thiol and )d) IMA levels of control and urticaria groups. *P values less than .05 were considered significant

Figure 2

Correlation graphs of N/L with Native thiol (a), disulphide (b), tottotal thiol (c) and Index-1 levels (d)

Table 1

Parameters of urticaria and control groups

Parameters	Control group (n=40)	Urticaria group (n=37)	P
Age, mean±SD, range, years	36.7±12.1, 18-59	35.1±12.9, 18-68	0.576
Gender, female/male	17/23	21/16	0.370
WBC, mean±SD, 109/L	9.9±4.5	9.9±3.2	0.958
Neutrophil, mean±SD, 109/L	6.9±4.5	6.9±3.1	0.995
Lymphocytes, mean±SD, 109/L	2.4±1.6	2.7±1.1	0.432
Hemoglobin, mean±SD, g/dL	13.5±1.3	14.3±1.7	0.033
N/L, mean±SD	3.7±3.3	3.4±2.7	0.299
PLT, mean±SD, 109/L	260.5±66.6	275.1±60.1	0.302
Native Thiol [SH], mean±SD, µmol/L	447.7±48.7	404.4±64.8	0.001
Total Thiol [SH+SS], mean±SD, µmol/L	480.5±51.8	464.7±59.0	0.218
Disulphide [SS], mean±SD, µmol/L	13.6±7.7	30.1±16.6	<0.001
Index-1 [SS]/[SH] × 100, mean±SD	3.1±1.7	7.9±5.8	<0.001
Index-2 [SS]/[SH+SS] × 100, mean±SD	2.8±1.5	6.4±3.8	<0.001
Index-3 [SH]/[SS+SH] × 100, mean±SD	91.9±9.2	87.1±7.6	0.016
IMA, mean±SD, ABSU	58.9±7.5	68.7±4.4	<0.001

P<0.001 were considered significant highlighted in asterisk

There was a significant high in the calculated Index-1 and Index-2 values [Table 1]. There was no statistically significant difference in the N/L ratio between groups (P = 0.299). The correlation between thiol/disulfide/IMA values and the N/L ratio is shown in Figure 2 and Table 2.

Table 2

Correlations between N/L and other parameters

Parameters	Control group (n=40)		Urticaria group (n=37)
	r	P	r	P
Native thiol, µmol/L	-0.017	0.920	-0.321	0.059
Total thiol, µmol/L	-0.054	0.750	-0.148	0.395
Disulfide, µmol/L	-0.017	0.920	0.357	0.035
Index-1 [SS]/[SH] ×100	-0.013	0.939	0.368	0.027
Index-2 [SS]/[SH+SS] ×100	0.042	0.805	0.315	0.061
Index-3 [SH]/[SS+SH] ×100	0.156	0.357	-0.314	0.062
IMA, ABSU	-0.241	0.151	0.133	0.466

P<0.05 were considered significant highlighted in bold

Total thiol groups are highly sensitive to oxidation and are the first antioxidants consumed in the case of oxidative stress. Thiols, also known as “native thiols,” are a class of organic compounds containing a sulfhydryl group (Native thiol: [-SH], Disulphide bond: [- SS -], Total thiol: [-SH] + [-SS]). Plasma thiols are mainly composed of albumin and to a lesser extent cysteine and glutathione.[1011] Normally, there is homeostasis between thiols and disulfides, and they also play a protective role in cellular redox homeostasis. This is called the dynamic thiol/disulfide homeostasis. The assertion that ROP play a role in the pathogenesis of various inflammatory and allergic diseases including urticaria is more remarkable.

There is homeostasis between the production of free radicals and the antioxidant system which suppresses the ROP increase in the body. If this homeostasis is impaired and the antioxidant system remains incapable, oxidative stress occurs. Free radicals arising from normal metabolism or pathological processes cause deterioration of the structure and function of thiol-dependent enzymes and change in the ratio of thiol/disulfide in the cellular environment. Reduction in plasma thiol concentration indicates high levels of free radicals.[910]

In some studies, it was found that TDH in the case of oxidative stress deteriorated by a decrease of thiol and increase of disulfide.[91213] In our present study, we investigated the role of the thiol and disulfide in the etiopathogenesis of acute urticaria by measuring the blood thiol and disulfide levels in the early stage of acute urticaria. The low level of thiol shows us that the antioxidant system is dominant. Thiol level was found to be low in AU. The increase in disulfide level at the very early stage of acute urticaria while significantly decrease in total thiol and native thiol levels, makes us think that oxidative stress exists in acute urticaria.

In a previous study investigating the thiol and disulfide levels in patients who had acute urticaria or chronic urticaria and were admitted to the outpatient clinic, it was found there was no significant difference in acute urticaria. In addition to this, it was shown that while total/native thiol levels were high in chronic urticaria, the homeostasis was disrupted in favor of disulfide.[14]

In AU, it was seen that there was a decrease in total thiol ratios, and there was a significant increase in the level of disulfide. This shows that the thiol/disulphide homeostasis shifts in favor of disulfide. The system shifts towards the formation of disulfide. These findings revealed the presence of inflammatory response in AU. The important factor here is the change in the rates showing the general homeostasis. This change may be associated with the immune reaction of thiol in inflammation. The lower plasma thiol concentration is evidence for the production of free radicals. In urticaria, mast cells, basophils, and eosinophils are activated, and ultimately, the immunological activation and inflammation of oxidative stress together with increased ROP occur.[1516]

Ischemia-modified albumin (IMA), protein carbonyl, and superoxide dismutase are other markers for oxidative stress. IMA is an altered type of serum albumin that forms under conditions of oxidative stress, and it increases due to oxidative stress after acute ischemia. IMA returns to the normal levels within hours after reperfusion. IMA is a sensitive marker in myocardial infarction, peripheral vascular diseases, chronic renal disease, and diabetes mellitus.[171819] We also evaluated the significance of IMA for AU. We detected that the IMA has the potential to be used as a marker similar to thiol/disulfide homeostasis for showing oxidative stress in AU.

N/L ratio has been investigated previously in chronic urticarial.[2021] In our study, the correlation between N/L ratio and thiol-disulfide and IMA was examined in acute urticaria. The positive correlation between N/L and thiol-disulfide and IMA parameters makes us think that it has an effect on the etiopathogenesis of the disease.

Considering all the results of our study, serial thiol-disulfide parameters have the potential to be used in response to treatment in acute urticaria. There is a need for comprehensive studies on this subject.

While total thiol and native thiol are low in acute urticaria, the level of disulfide is increased.

Limitations

The study population is small. Dynamic thiol disulfide homeostasis is affected by various diseases and conditions. In patients with unknown etiology of acute urticaria, there may be conditions causing oxidative stress. Does giving thiol sources as treatment contribute to the reduction of oxidative stress in patients with acute urticaria? Does it have a role in etiopathogenesis? Further studies are needed to find answers to these questions.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.