New markers of oxidative stress in dogs. Technical and applicative issues

Abstract

The objectives of this PhD thesis were to increase the knowledge about the possible use and applications of the different assays that can be used for Total Antioxidant Capacity (TAC) determination in dogs and to validate new different spectrophotometric assays to evaluate TAC in serum of dogs. In addition, we wanted to provide an overview of how TAC measured by different assays and others biomarkers of oxidative stress can behave in different diseases in dogs. Finally, the stability of the different TAC assays as well as other oxidative stress biomarkers in canine serum was studied. First, the main spectrophotometric assays (Trolox equivalent antioxidant capacity [TEAC], cupric reducing antioxidant capacity [CUPRAC] and ferric reducing ability of plasma [FRAP]) used in dogs to evaluate the TAC in fluid samples were investigated by reviewing the basis of each one, their practical application in the determination of TAC in dogs, and also providing selected information about reports in humans for comparative purposes. Subsequently, an automated assay for CUPRAC measurement, based on the reduction of Cu2+ into Cu1+ by the action of the non-enzymatic antioxidants presented in the sample, three automated assays for TEAC measurements, based on the ability of a sample to reduce ABTSo+, and one automated assay for FRAP measurement, based on the ability of the antioxidants present in the sample to reduce ferric (Fe3+) to its ferrous (Fe2+) form, were validated for use in serum of dogs. The behaviour of the different biomarkers of oxidative stress in different canine diseases was assessed by using sick dogs naturally infected by canine leishmaniosis, dogs with inflammatory bowel disease (IBD), dogs with clinical and subclinical ehrlichiosis (CME), and dogs with non-food and food-induced atopic dermatitis (AD) were used. Last, six serum pools from healthy dogs were divided in various aliquots and stored at 4º C and 25 ºC for a period of 6, 24 and 72 h, and at -20 ºC and -80 ºC for a period of 7, 14, 30, 60, 180, and 360 days. In addition, the effect of repeated freeze-thaw cycle was evaluated. The methods validated allowed the TAC determination in a precise, accurate and sensitive way and detected decreased concentrations of this analyte in inflammation and infection disease. When individual biomarkers were evaluated in different diseases, serum TEAC, CUPRAC, and total thiol were decreased in dogs with leishmaniosis. In addition, CUPRAC, and thiol increased after treatment against this disease. Dogs with IBD had decreased serum TEAC, CUPRAC, and total thiol, and increased oxidant biomarkers in serum such as FOX, TBARS and ROS. Dogs with clinical CME showed decreased CUPRAC, FRAP, TEAC and total thiol and increased ROS. In the case of subclinical CME all the biomarkers were also different from the healthy dogs with exception of the TEAC. Oxidative stress also may play a role in the pathogenesis of AD in dogs, which had decreased concentrations of serum CUPRAC, TEAC, and total thiol and increased FRAP, and increased FOX when compared with healthy dogs. In relation to the stability study, most of the analytes were stable for up 24 h at 4 ºC. In case of long-term storage, the use of -80 ºC provides a better stability than at -20 ºC. In addition, all biomarkers are affected by three freeze-thaw cycles. The automated assays validated in this Thesis for TAC determination in canine serum samples are simple, fast, and easy to perform, and can be easily adapted to automated biochemical analysers. A panel integrated by a combination of TAC assays should be used for evaluation of antioxidant status of a sample, because the results of TAC could be markedly different depending on the assay performed. When oxidative stress biomarkers were evaluated in different diseases of the dog such as leishmaniosis, IBD, CME and AD, it was observed that each disease had particular changes in the biomarkers evaluated. In addition, in the case of canine leishmaniosis some biomarkers such as CUPRAC and thiol were useful for treatment monitoring. As individual oxidative biomarkers can show differences in stability, it is important to consider the storage conditions of each analyte when stored samples are used.