New Oxidative Stress Markers Useful in the Diagnosis ...

05.12.2017

New Ox dat ve Stress Markers Useful n the D agnos s of Acut... : Ped atr c Emergency Care

New Oxidative Stress Markers Useful in the Diagnosis of Acute Appendicitis in Children: Thiol/Disulfide Homeostasis and the Asymmetric Dimethylarginine Level Elmas, Bahri MD; Yildiz, Turan MD; Yazar, Hayrullah MD; Ilçe, Zekeriya MD; Bal, Ceylan MD; Özbek, Betül MD; Yürümez, Yusuf MD

Pediatric Emergency Care: Post Author Corrections: November 14, 2017 doi: 10.1097/PEC.0000000000001339 Original Article: PDF Only Abstract Objectives The aim of this study was to evaluate 2 new oxidative stress markers, thiol/disulfide homeostasis status and the asymmetric dimethylarginine (ADMA) level, in children with acute appendicitis (AA) and to evaluate their diagnostic utility. Methods This case-control study included 45 patients with AA and 35 healthy children. Age, sex, white blood cell count, neutrophil-to-lymphocyte ratio, high-sensitivity C-reactive protein (hs-CRP) level, ultrasonographic findings, thiol/disulfide homeostasis parameters (native and total thiol levels, native thiol/total thiol ratios [antioxidant parameters], and disulfide, disulfide/native thiol, and disulfide/total thiol ratios [oxidant parameters]), and the ADMA level were compared between the 2 groups. Results The native and total thiol levels, and the native thiol/total thiol ratio, were significantly lower, and the disulfide level and disulfide/native thiol and disulfide/total thiol ratios significantly higher, in the AA compared with the control group (all P < 0.001). The ADMA level was significantly higher in a perforated versus nonperforated subgroup of AA patients, but the thiol/disulfide homeostasis parameters did not differ significantly between the two subgroups. In addition, the hs-CRP level and appendiceal wall thickness were higher in the perforated subgroup. The thiol/disulfide antioxidant parameters and ADMA level correlated negatively with the white blood cell count, the neutrophil-to-lymphocyte ratio, and the hs-CRP level, in the AA group, but correlated positively with oxidant parameters. The sensitivity and specificity of the disulfide/native thiol and disulfide/total thiol ratios were high when used to diagnose AA, whereas the sensitivity of the ADMA level was high when used to diagnose perforated appendicitis. Conclusions Thiol/disulfide homeostasis and the ADMA level, together with certain other parameters, may be useful biomarkers of AA in children. Disclosure: The authors declare no conflict of interest. Reprints: Bahri Elmas, MD, Department of Pediatrics, Faculty of Medicine, Sakarya University, Adnan Menderes Cad, Sağlik Sok No:195, Adapazari, Sakarya, Turkey. (e- mail: [email protected]). Copyright © 2017 Wolters Kluwer Health, Inc. All rights reserved. http://journals.lww.com/pec-onl ne/Abstract/publ shahead/New_Ox dat ve_Stress_Markers_Useful_ n_the.98565.aspx

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ORIGINAL ARTICLE

New Oxidative Stress Markers Useful in the Diagnosis of Acute Appendicitis in Children Thiol/Disulfide Homeostasis and the Asymmetric Dimethylarginine Level Bahri Elmas, MD,* Turan Yıldız, MD,† Hayrullah Yazar, MD,‡ Zekeriya İlçe, MD,† Ceylan Bal, MD,§ Betül Özbek, MD,§ and Yusuf Yürümez, MD|| Objectives: The aim of this study was to evaluate 2 new oxidative stress markers, thiol/disulfide homeostasis status and the asymmetric dimethylarginine (ADMA) level, in children with acute appendicitis (AA) and to evaluate their diagnostic utility. Methods: This case-control study included 45 patients with AA and 35 healthy children. Age, sex, white blood cell count, neutrophil-tolymphocyte ratio, high-sensitivity C-reactive protein (hs-CRP) level, ultrasonographic findings, thiol/disulfide homeostasis parameters (native and total thiol levels, native thiol/total thiol ratios [antioxidant parameters], and disulfide, disulfide/native thiol, and disulfide/total thiol ratios [oxidant parameters]), and the ADMA level were compared between the 2 groups. Results: The native and total thiol levels, and the native thiol/total thiol ratio, were significantly lower, and the disulfide level and disulfide/native thiol and disulfide/total thiol ratios significantly higher, in the AA compared with the control group (all P < 0.001). The ADMA level was significantly higher in a perforated versus nonperforated subgroup of AA patients, but the thiol/disulfide homeostasis parameters did not differ significantly between the two subgroups. In addition, the hsCRP level and appendiceal wall thickness were higher in the perforated subgroup. The thiol/disulfide antioxidant parameters and ADMA level correlated negatively with the white blood cell count, the neutrophil-tolymphocyte ratio, and the hs-CRP level, in the AA group, but correlated positively with oxidant parameters. The sensitivity and specificity of the disulfide/native thiol and disulfide/total thiol ratios were high when used to diagnose AA, whereas the sensitivity of the ADMA level was high when used to diagnose perforated appendicitis. Conclusions: Thiol/disulfide homeostasis and the ADMA level, together with certain other parameters, may be useful biomarkers of AA in children. Key Words: appendicitis, asymmetric dimethylarginine, thiol/disulfide homeostasis (Pediatr Emer Care 2017;00: 00–00)

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cute appendicitis (AA) is the most common reason for emergency abdominal surgery in children.1 Although the incidence varies with age, the annual frequency is 1.9 to 2.8 per 1000 in children aged 4 to 14 years.2,3 Acute appendicitis pathophysiology is characterized by an inflammatory response that commences with an increase in the permeability of the appendiceal mucosal barrier, attributable to occlusion of the lumen.4 Microscopically, changes in appendiceal tissue range from minimal focal inflammation to total wall necrosis.5 Clinical, laboratory, and imaging methods are used for diagnosis. However, diagnosis is difficult because children do not usually present with a From the Departments of *Pediatrics, †Pediatric Surgery, and ‡Biochemistry, Faculty of Medicine, Sakarya University, Sakarya; §Department of Biochemistry, Faculty of Medicine, Yıldırım Beyazıt University, Ankara; and ||Department of Emergency, Faculty of Medicine, Sakarya University, Sakarya, Turkey. Disclosure: The authors declare no conflict of interest. Reprints: Bahri Elmas, MD, Department of Pediatrics, Faculty of Medicine, Sakarya University, Adnan Menderes Cad, Sağlık Sok No:195, Adapazarı, Sakarya, Turkey. (e- mail: [email protected]). Copyright © 2017 Wolters Kluwer Health, Inc. All rights reserved. ISSN: 0749-5161

Pediatric Emergency Care • Volume 00, Number 00, Month 2017

typical anamnesis, pathognomonic symptoms, or signs. Furthermore, the cutoff values of the relevant laboratory tests vary by age and have low predictive utility.6–8 Despite the availability of many scoring systems and modern imaging modalities, initial misdiagnosis rates range from 28% to 57% for children younger than 12 years, and reaches almost 100% for those younger than 2 years.8,9 Delay in diagnosis increases mortality and morbidity, attributable to complications such as perforation, peritonitis, and abscess formation. The risk of early complications is higher in younger children.9,10 Although the pathophysiology of AA has been described in detail, factors influencing AA progression remain under investigation.11 Many authors have suggested that an increase in oxidative status (both total oxidant and antioxidant status) is related to progression of the AA inflammatory process.4,12 Therefore, certain biochemical markers, such as those used to identify oxidative stress and inflammation, have been proposed to be useful indicators of the presence and extent of AA.13 It is known that plasma thiols play physiological roles as free radical scavengers and may act as antioxidants in several ways. It has been reported that measurement of plasma total thiols, and evaluation of thiol/ disulfide homeostasis, are good indicators of excess free radical generation in humans.11,13 Thus, the levels of serum thiol and nitric oxide (NO) metabolites, oxidative stress markers, and lipid peroxidation markers have been investigated recently in the context of AA.4,11,13 Asymmetric dimethylarginine (ADMA) is a toxic nonproteinogenic amino acid formed via posttranslational modification and is involved in the pathophysiology of oxidative stress and inflammation.14 Asymmetric dimethylarginine increases inflammation by decreasing NO synthesis and enhances oxidative stress by shifting the NO-reactive oxygen species (ROS) balance toward ROS, which effectively increase ADMA levels either by enhancing the activity of an enzyme that plays a role in ADMA production or by decreasing the level of an enzyme that plays a role in ADMA breakdown.15–19 Thus, interaction between ROS and ADMA increases the levels of both.17 We hypothesized that oxidative stress might play a role in AA pathogenesis. Therefore, we aimed to investigate thiol/ disulfide homeostasis and the ADMA level in children with AA and to correlate the levels of the new markers with those of other inflammatory markers, including high-sensitivity C-reactive protein (hs-CRP), as well as the white blood cell (WBC) count and neutrophil-to-lymphocyte ratio (NLR). To the best of our knowledge, no previous study has investigated thiol/disulfide homeostasis and the ADMA level as new and sensitive markers of oxidative stress in children with AA compared with healthy controls.

METHODS Study Design and Patients Study Population The study was performed in the Sakarya University Research and Training Hospital Pediatric Emergency Clinic between www.pec-online.com

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Elmas et al

January and April 2016. A total of 80 subjects, consisting of 45 patients and 35 healthy volunteers (the control group), were included in the present case-control study. It was performed in accordance with the principles of the Declaration of Helsinki and was approved by our local ethics committee. All participants or their parents provided written informed consent. The principles of good clinical practice were followed during the entire study period.

total thiol ratios were calculated after the dynamic disulfide, native thiol, and total thiol levels had been determined. An automated clinical chemistry analyzer (Cobas 501; Roche Diagnostics, Indianapolis, Ind) was used to this end, and the results are presented in μmol/L. The laboratory staff performing these measurements were blinded to patient clinical data and outcomes; the laboratory results were not available to the treating physicians, study staff, or investigators during the study period.

Inclusion Criteria

Plasma ADMA Levels

The control group consisted of healthy patients similar to the patient group with AA in terms of demographic characteristics. The patient group consisted of children younger than 17 years who underwent operations after AA diagnosis.

Plasma ADMA levels were determined using a quantitative sandwich enzyme-linked immunoassay (ELISA) (Human ADMA ELISA Kit; Cusabio Biotech, Wuhan, China; catalog number CSB-E09298h) employing an automated analyzer (BioTek Gen5 ELx800; BioTek, Burlington, Vt); the results are presented in nmol/mL.

Exclusion Criteria All patients and control cases were excluded if they had any chronic disease, used drugs, or exhibited symptoms of infection during the 2 weeks before inclusion in the study. The other exclusion criterion was any history of abdominal trauma within 7 days of hospital admission.

Acute Appendicitis Definition All patients with AA were diagnosed on the basis of clinical symptoms, physical examination, WBC count, NLR, hs-CRP, abdominal ultrasonography (used to measure appendiceal diameter and wall thickness), and other findings indicative of appendicitis. Patients with an appendiceal diameter greater than 6 mm and a wall thickness greater than 2 mm were considered to have appendicitis.20 All patients with AA were confirmed by examination of pathological specimens after the operation.

Biochemical Parameters Collection of Biological Samples Peripheral venous blood samples from AA patients were taken on admission to the pediatric emergency clinic, ie, before surgical intervention. Peripheral venous blood samples were also taken from healthy volunteers. All blood samples were immediately centrifuged at 1500 rpm for 10 minutes to separate plasma and serum; the serum samples were stored at −80°C until all samples had been collected.

Complete Blood Count and hs-CRP Levels Complete blood counts were determined with the aid of an automated analyzer (Celldyn 3400; Abbott Diagnostics, Chicago, Ill) within 1 hour of blood collection. The NLR was given by the number of neutrophils divided by the number of lymphocytes. Serum hsCRP concentrations were determined with the aid of the Siemens CardioPhase hs-CRP particle-enhanced immunonephelometric assay running on a BNII analyzer (Siemens Healthcare Diagnostics Products GmbH, Marburg, Germany). The hs-CRP level of a healthy individual is less than 3.0 mg/L.

Serum Thiol/Disulfide Homeostasis Parameters Thiol disulfide status was evaluated using the method of Erel and Neselioglu.21 Briefly, disulfide bonds were reduced to free functional thiol groups. Formaldehyde was used to remove unused sodium borohydride. The levels of all thiol (native and reduced) groups were measured after reaction with DTNB (5,5’-dithiobis2-nitrobenzoic acid). The dynamic disulfide level was calculated as half of the difference between the total and native thiol levels. The disulfide/native thiol, disulfide/total thiol, and native thiol/

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Statistical Analysis Descriptive statistics were used to compare the general features of all participants. The Kolmogorov-Smirnov and ShapiroWilk tests were used to compare the distributions of numerical variables. We calculated means ± standard deviation of variables that were normally distributed, and medians (with interquartile range) of those that were not normally distributed. Categorical variables are shown as numbers (n) with percentages (%). Student t test, the Mann-Whitney U test, analysis of variance, and the Kruskal-Wallis H test were used as appropriate to compare data between groups. Categorical variables were compared using the χ2 or Fisher exact χ2 test. We assessed relationships between numerical variables by calculating linear correlation coefficients using the method of Pearson. Receiver operating characteristic (ROC) curve analysis was used to assess the sensitivity and specificity of the ADMA level and thiol/disulfide parameters in terms of the prediction of AA and perforated appendicitis. The SPSS for Windows software (ver. 20.0; IBM SPSS Inc., Chicago, Ill) was used for all statistical analyses. A P value less than 0.05 was considered to reflect statistical significance.

RESULTS We evaluated a total of 80 individuals (45 AA patients and 35 healthy controls). The groups did not differ significantly in terms of either age or sex (both P > 0.05). The WBC count, NLR, hs-CRP and disulfide levels, and disulfide/native thiol and disulfide/total thiol ratios were higher in the AA group than in the control group (all P < 0.001). Similarly, the ADMA level was higher in the AA group than in the control group (P = 0.024). However, the native and total thiol levels, and the native thiol/total thiol ratio, were significantly lower (all P < 0.001) in AA patients than controls (Table 1). In AA patients, the mean appendiceal diameter (measured by ultrasound) was 10.1 ± 2.6 mm and the wall thickness was 2.1 ± 0.6 mm. We divided AA patients into 2 subgroups: those with nonperforated appendicitis (71%; n = 32) and those with perforated appendicitis (29%; n = 13). The results of subgroup analysis are shown in Table 2. We found no significant between-subgroup difference in sex (P = 0.745). However, the mean age of the perforated group was lower than that of the nonperforated group (P < 0.001). The appendiceal diameter did not differ significantly between the 2 subgroups (P < 0.05), but the appendiceal wall thickness was higher in the perforated than in the nonperforated subgroup (P = 0.047). The WBC count, NLR, native thiol, total thiol and disulfide levels, and disulfide/native thiol, disulfide/ total thiol, and native thiol/total thiol ratios did not differ significantly between the 2 subgroups (all P > 0.05). However, the © 2017 Wolters Kluwer Health, Inc. All rights reserved.

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TABLE 1. The Demographic and Laboratory Findings of Acute Appendicitis and Control Groups

Control (n = 35) Sex, n (%) Male Female Age (y) WBC count (μL) NLR hs-CRP (mg/dL) Native thiol (μmol /L) Total thiol (μmol /L) Disulfide (μmol /L) Disulfide/native thiol (%) Disulfide/total thiol (%) Native thiol/total thiol (%) ADMA (nmol/mL)

Acute Appendicitis (n = 45)

P 0.76

19 (54) 16 (46) 13.3 ± 4.2 8.3 (2.4) 1.7 (2.0) 0.3 (0.01) 413.2 ± 51.7 448.0 ± 54.1 17.4 ± 2.5 4.3 ± 0.6 3.9 ± 0.5 92.2 ± 1.1 1.4 (1.4)

26 (58) 19 (42) 11.9 ± 3.8 17.3 (8.9) 9.4 (12.0) 2.3 (3.7) 334.1 ± 62.7 382.3 ± 76.5 24.1 ± 9.0 7.1 ± 2.1 6.1 ± 1.6 87.7 ± 3.3 2.0 (2.4)

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