An investigation of thiol/disulfide homeostasis and ischemia-modified albumin levels to assess the oxidative stress in dogs with canine distemper

Abstract

This study aimed to investigate the diagnostic value of thiol/disulfide homeostasis (TDH) and ischemia-modified albumin (IMA) levels in evaluating oxidative stress in dogs with canine distemper. The study materials consisted of 24 cross-breed dogs: 18 dogs with canine distemper (CD group) and 6 healthy dogs as the control. Blood samples for analysis of antioxidant-oxidant parameters were taken from both groups. In addition to TDH parameters (native thiol [NT], total thiol [TT], and disulfide [Ds] levels and Ds/NT, Ds/TT, and NT/TT ratios), IMA and albumin (ALB) levels were also determined in the blood serum of the groups. The IMA levels of the CD group were significantly higher than the control group (P<0.05). Antioxidant parameters (NT, TT) and oxidant parameters (Ds, Ds/NT, and Ds/TT) both exhibited a significant positive correlation among themselves (P<0.01). The areas under the ROC curve of IMA, Ds/ NT, and Ds/TT were 0.78, 0.62, and 0.62 respectively. The high levels of IMA, Ds, Ds/NT, and Ds/TT and low levels of NT, TT, NT/TT and ALB in dogs with canine distemper can be used to assess the oxidative stress caused by the disease. Overall, the diagnostic performances of IMA, Ds/NT, and Ds/TT for detection of increased oxidative stress were similar, but IMA was superior. key words: canine distemper; oxidative stress; thiol/disulfide homeostasis; ischemia-modified albumin _____________________________________________________________________________________________ DOI: 10.24099/vet.arhiv.0867 ________________ *Corresponding author: Assist. Prof. Şükrü Değirmençay, PhD, DVM, Atatürk University, Faculty of Veterinary Medicine, Department of Internal Medicine, Erzurum, Turkey, Fax: +90 442 231 7244; E-mail: [email protected] APPEL, 1994; THULIN et al., 1992; WRIGHT et al., 1974), gastrointestinal, respiratory and nervous system signs (WILLIAMS, 2001). In this context, the clinical findings may include leukopenia (especially lymphopenia), serous nasal discharge, mucopurulent ocular discharge, lethargy, myoclonus, anorexia and secondary bacterial infections (WILLIAMS, 2001). introduction Canine distemper is an infectious, multisystemic and fatal disease of dogs caused by the canine distemper virus (CDV), a paramyxovirus of the genus Morbillivirus (BEINEKE et al., 2009; CARPENTER et al., 1998). The disease occurs most commonly in young dogs (3-6 months old) (GREENE and APPEL, 1998; MARTELLA et al., 2008; TAYLOR, 2009) and it is mostly characterized by a biphasic fever (SUMMERS and S. Değirmençay et al.: Thiol/disulfide homeostasis in canine distemper Vet. arhiv 91 (1), 39-49, 2021 40 et al., 2013). The thiol disulfide ratio has emerged as a new biomarker used in human patients with diabetes, cardiovascular diseases, cancer and some other diseases (ATES et al., 2016; ELMAS et al., 2017; HANİKOGLU et al., 2016). In veterinary medicine, oxidative stress has also been reported to take part in the pathogenesis of various infections of dogs, such as Babesia gibsoni (MURASE et al.,1996), Leishmaniasis (BİLDIK et al., 2004), concurrent infection of Babesiosis and Ehrlichiosis (KUMAR et al., 2006), canine parvoviral enteritis (PANDA et al., 2009) and canine distemper (KARADENIZ et al., 2008). However, the rate of thiol-disulfide, a novel biomarker for oxidative stress, is a recently studied parameter in the field of veterinary medicine. Albumin, the most abundant protein in the body, is in charge of the elimination of ROS (LIPPI and MONTAGNANA, 2009). KINOSHITA et al. (2017) reported that increased oxidative stress was associated with decreased albumin levels. When ischemia is present, changes in the amino acid sequence occur at the N-terminus of the albumin, and the new emerging protein is called ischemiamodified albumin (IMA). The increase in ROS, ischemia, or the hypoxic process is closely linked to IMA (LIPPI and MONTAGNANA, 2009). The aim of this study was to evaluate the oxidative stress level in dogs with canine distemper in terms of thiol-disulfide ratio and IMA. A further aim was to investigate the compatibility of thioldisulfide balance with IMA and albumin in the evaluation of oxidative stress. This study was also designed to determine for the first time how successful the thiol-disulfide ratio is as an indicator of oxidative stress in dogs with canine distemper. Materials and methods Animals and protocol design. The study material included 24 dogs of 2 to 6 months old, different breeds and both genders. The dogs were divided into two groups as control and CD groups. Six healthy dogs were included in the control group. These 6 dogs were selected from animals brought by their owners to Atatürk University Faculty of Veterinary Medicine Animal Hospital for a routine health check-up or vaccination. They were considered to Oxidative stress plays an indicative role in the pathogenesis of many diseases and inflammatory processes. It occurs when redox homeostasis changes in the cell, either due to the overproduction of reactive oxygen species (ROS) or the lack of an antioxidant system (SEMBA and TANG, 1999). Total oxidant and antioxidant capacity, ceruloplasmin and ischemia-modified albumin (IMA) are some of the important biomarkers of oxidative stress (ATES et al., 2016; ELMAS et al., 2017; HANİKOGLU et al., 2016). EREL and NESELIOGLU (2014) have developed a new, fully-automated method in the evaluation of TDH as a new marker of oxidative stress. Plasma thiols have antioxidant and pro-oxidant features, and contain a sulfhydryl group (-SH) (ATMACA, 2004; EREL and NESELIOGLU, 2014). The plasma thiol pool consists of albumin, protein thiols and low molecular weight thiols (cysteine, cysteinylglycine, glutathione, homocysteine and p-glutamylcysteine) (ATMACA, 2004). The dynamic TDH in the organism can be affected by oxidative stress in disease processes. When thiol compounds undergo oxidation reactions, they form disulfide bonds. Therefore, increasing oxidative stress has an impact on the redox state, which can be explained by the metabolization of thiol groups into disulfide groups (ASOĞLU et al., 2018). The dynamic TDH has a critical role in metabolism and homeostasis (BISWAS et al., 2006; CIRCU and AW, 2010; EREL and NESELIOGLU, 2014). The parameters of this homeostasis include native thiol (NT) and total thiol (TT); disulfide (Ds); disulfide/native thiol (Ds/NT), native thiol/ total thiol (NT/TT), and disulfide/total thiol (Ds/ TT) ratios (ELMAS et al., 2017). TDH levels are measured one-by-one and cumulatively using a novel and automated method which allows a complete evaluation of the oxidant and antioxidant status (OZLER et al., 2015). The antioxidant effect of TDH has a significant function in enzymatic reactions, signal transmission, detoxification, transcription and apoptosis mechanisms. Under normal conditions, TDH has a dynamic structure and it may be adversely affected by pathologies that cause an increase in the level of oxidative stress (EREL and NESELIOGLU, 2014; TURELL S. Değirmençay et al.: Thiol/disulfide homeostasis in canine distemper Vet. arhiv 91 (1), 39-49, 2021 41 be healthy on the basis of physical examination, complete blood count findings and the negative result of a CDV Antigen (Ag) rapid test kit (CDV Ag Antigen Rapid Test Kit®, Bionote Korea, Cat. No: RG1303DD). The CD group consisted of 18 dogs. The dogs with at least two of these complaints: nasal discharge, ocular discharge, fever, anorexia, and cough, were tested with a CDV antigen (Ag) rapid test kit. Those that were tested positive were included in the CD group. The study was approved by the Atatürk University Faculty of Veterinary Medicine Ethics Committee for compliance with ethical rules (2019/08). Blood sampling. Blood samples from all the dogs included in this study were taken from the vena cephalica antebrachii and collected into sterile tubes without an anticoagulant (Becton Dickinson Co., USA). After leaving for 1 hour at room temperature for clotting, sera were obtained by centrifugation at 3000 rpm for 10 minutes, and stored at -80 °C until they were analysed. Chemicals used in the assays. Sodium borohydride (NaBH4), 5,5’-dithiobis-2-nitrobenzoic acid (DTNB), formaldehyde, cobalt (II) chloride (CoCl2*6H2O) and dithiothreitol (DTT) were acquired from Sigma‐Aldrich Chemical Co. (Sigma-Aldrich Chemie GmbH, Riedstrasse 2, Steinheim, Germany). Thiol-disulfide balance. The thiol-disulfide balance (native thiol [-SH] vs. disulfide [-S-S] alteration) in both groups was determined using the automatic measurement method described by EREL and NESELIOGLU (2014), using a Shimadzu UV-1800 spectrophotometer (Shimadzu Scientific Instruments, Kyoto, Japan) and a Roche Cobas c501 automated analyzer (Roche Diagnostics, Indianapolis, Indiana, United States). Firstly, NaBH4 was used to form free functional thiol groups which were reduced from dynamic disulfide bonds present in the serum samples. To avoid any extra reduction in DTNB and further reduction of the already-formed disulfide bonds, formaldehyde was used to clear away the unused NaBH4 reductant. Following the reaction with DTNB, the thiol groups (including reduced and native ones) were measured spectrophotometrically at 415 nm. Serum TDH values were presented in μmol/L. The following formulas were used to calculate the disulfide concentration and Ds/NT, Ds/TT, and NT/TT percentages: Disulfide levels (μmol/L) = (total thiolnative thiol) / 2 Disulfide/native thiol percentage (%) = (disulfide × 100)/ native thiol Disulfide/total thiol percentage (%) = (disulfide × 100) / total thiol Native thiol percentage (%)/total thiol percentage (%) = (native thiol × 100) / total thiol Ischemia-modified albumin. Serum ischemiamodified albumin (IMA) level was measured by a method described by DAŞ et al. (2016) based on the spectrophotometric measurement (470 nm) of colour obtained as a result of the reaction of albumin-cobalt with DTT. Firstly, the reaction was carried out by adding 50 μL 0.1% c