Camila Peres Rubio

Spectrophotometric assays for total antioxidant capacity (TAC) in dog serum: an update.

The aim of this review is to study the main spectrophotometric methods used to evaluate total antioxidant capacity (TAC) in serum samples of dogs. Total antioxidant capacity (TAC) is an analyte frequently used to assess the antioxidant status of biological samples and can evaluate the antioxidant response against the free radicals produced in a given disease. Trolox equivalent antioxidant capacity (TEAC), ferric reducing ability of plasma (FRAP), and cupric reducing antioxidant capacity (CUPRAC) are different assays described to determine TAC of a sample. This review explains the basis of each assay and their application in the determination of TAC in dogs, and also provides selected information about reports in humans for comparative purposes. It is concluded that, ideally, various different assays integrated in a panel should be used for TAC evaluation, since depending on the assay performed TAC results can be markedly different.

Validation of an automated assay for the measurement of cupric reducing antioxidant capacity in serum of dogs.

BackgroundThe objective of the present study was to optimize and validate an automated method to assess the total antioxidant capacity (TAC) in serum of dogs using the cupric reducing antioxidant capacity (CUPRAC) methodology (TACc) with bathocuproinedisulfonic acid disodium salt as chelating agent, evaluating also possible variations due to the use of two different automated analyzers. The method is based on the reduction of Cu2+ into Cu1+ by the action of the non-enzymatic antioxidants that are present in the sample.ResultsImprecision was low in both apparatus utilized, and the results were linear across serial Trolox and canine serum samples dilutions. Lipids did not interfere with the assay; however, hemolysis increased the TACc concentrations. When TACc concentrations were determined in ten healthy (control) dogs and in twelve dogs with inflammatory bowel disease (IBD), dogs with IBD had lower TACc concentrations when compared with the healthy dogs.ConclusionsThe method validated in this paper is precise, simple, and fast and can be easily adapted to automated analyzers.

Evaluation of salivary oxidate stress biomarkers, nitric oxide and C‐reactive protein in patients with oral lichen planus and burning mouth syndrome

OBJECTIVES The aim of this study was to evaluate oxidative stress factors and C-reactive protein in the saliva of patients with oral lichen planus (OLP) and burning mouth syndrome (BMS). METHODS This consecutive, cross-sectional study included 20 patients with OLP, 19 with burning mouth syndrome (BMS), and 31 control subjects. The oral cavity of each patient was examined and patients responded to a quality of life questionnaire (OHIP-14) and the xerostomia inventory. The following parameters were measured in whole non-stimulated saliva: trolox equivalent antioxidant capacity (TEAC); total antioxidant capacity (TAC); cupric reducing antioxidant capacity (CUPRAC); ferric reducing ability of plasma (FRAP); C-reactive protein (CRP); nitric oxide; nitrates; and nitrites. RESULTS The OLP group presented statistically significant differences in reactive oxygen species (ROS) (29 600 cps) in comparison with the control group (39 679 cps) (P < 0.05). In the BMS group, ROS was 29 707 cps with significant difference in comparison with the control group (P < 0.05). Significantly higher salivary nitric oxide (145.7 μmol) and nitrite (141.0 μmol) levels were found in OLP patients in comparison with control group (P < 0.05). CONCLUSION Increases in nitric oxide and C-reactive protein were found in the saliva of OLP patients in comparison with BMS and control patients. Further studies are required to confirm these findings.

Validation of three automated assays for total antioxidant capacity determination in canine serum samples

We performed analytical validation of 3 automated assays of the total antioxidant capacity (TAC) in canine serum and evaluated their use in dogs with inflammatory bowel disease. The assays were based on the generation of a 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radical cation (ABTS•+) in aqueous media, which produces a blue-green color. The antioxidants present in the sample remove the chromogen in proportion to their concentrations. The assays differed mainly in the way in which this radical was produced. All 3 assays produced acceptable results in the analytical validation. However, only 2 of the assays were capable of detecting significantly different TAC values in healthy and diseased animals.

Changes in serum biomarkers of oxidative stress after treatment for canine leishmaniosis in sick dogs

Canine leishmaniosis (CanL) is a zoonotic disease being endemic in several parts of the world. In this study we investigated the behavior of a panel of biomarkers of oxidative stress in 12 sick dogs naturally infected by CanL before and at days 30 and 180 of a successful therapy with a standard treatment. The assays total oxidant status (TOS), trolox equivalent antioxidant capacity (TEAC), ferric reducing ability of plasma (FRAP), cupric reducing antioxidant capacity (CUPRAC), serum thiol and paraoxonase 1 (PON1) were included in the panel. In addition, correlations between biomarkers of oxidative stress and inflammation (C-reactive protein (CRP) and ferritin) and urinary protein:creatinine ratio (UPC) were calculated. Serum CUPRAC, thiol and PON1 significantly increased after treatment and were negatively correlated with CRP, ferritin and UPC. This study demonstrates that biomarkers of oxidative stress, not previously studied in leishmaniosis such as CUPRAC and thiol, can change after a successful treatment for CanL showing a potential for use in monitoring the treatment of this disease.

Serum biomarkers of oxidative stress in dogs with idiopathic inflammatory bowel disease

The objective of this work was to study and compare a panel of various serum biomarkers evaluating both the antioxidant response and oxidative damage in dogs with idiopathic inflammatory bowel disease (IBD). Eighteen dogs with IBD and 20 healthy dogs were enrolled in the study. Trolox equivalent antioxidant capacity (TEAC), cupric reducing antioxidant capacity (CUPRAC), ferric reducing ability of the plasma (FRAP), total thiol concentrations, and paraoxonase 1 (PON1) activity were evaluated in serum to determine antioxidant response. To evaluate oxidative status, ferrous oxidation-xylenol orange (FOX), thiobarbituric acid reactive substances (TBARS) and reactive oxygen species production (ROS) concentrations in serum were determined. Mean concentrations of all antioxidant biomarkers analyzed, with exception of FRAP, were significantly lower (P < 0.0001) in the sera of dogs with IBD than in healthy dogs. The oxidant markers studied were significantly higher (P < 0.0001) in sera of dogs with IBD than in healthy dogs. These findings support the hypothesis that oxidative stress could play an important role in the pathogenesis of canine IBD.

Serum antioxidant capacity and oxidative damage in clinical and subclinical canine ehrlichiosis

The objective of this study was to evaluate and compare the antioxidant response and the products of oxidative damage analysed by various assays in clinical and subclinical canine monocytic ehrlichiosis (CME). For this purpose, four assays to measure the total serum antioxidant capacity (TAC), such as the cupric reducing antioxidant capacity (CUPRAC), ferric reducing ability of plasma (FRAP), trolox equivalent antioxidant capacity (TEAC) using acidic medium (TEACA), and the TEAC using the horseradish peroxidase (TEACH) were used. In addition, the serum thiol concentrations were analysed. Reactive oxygen species (ROS), thiobarbituric acid reactive substances (TBARS), and ferrous oxidation-xylenol orange (FOX) were measured to determine the concentrations of free radical and the products of oxidative damage as result of the disease. All antioxidant markers were significantly lower in the dogs on clinical ehrlichiosis when compared with healthy dogs; however only the CUPRAC, FRAP and thiol were significantly lower in subclinical CME compared with healthy dogs. TBARS and FOX showed no significant differences between dogs with CME and healthy dogs; however, a significant increased ROS concentration was observed in dogs with clinical and subclinical CME when compared with healthy dogs. Results showed that in CME there is a state of oxidative stress with significant changes in markers of antioxidant defence and in concentrations of free radicals. However, the detection of these changes would depend of the assay used.

Analytical validation of an automated assay for ferric-reducing ability of plasma in dog serum

We performed analytical validation of an automated ferric-reducing ability of plasma (FRAP) assay in the serum of dogs. Intra- and interassay precision, accuracy, detection limit, and effects of hemolysis and lipemia were evaluated. Intra- and interassay coefficients of variation were <1% and <13%, respectively. The assay showed a high correlation with a FRAP assay described previously, and results were linear when serial sample dilutions were tested. The detection limit was lower than the values observed in sera from healthy dogs; decreased serum FRAP was found in dogs with leishmaniosis. Lipemia and hemolysis caused a significant increase in the results of the assay.

Adenosine deaminase activity in pig saliva: analytical validation of two spectrophotometric assays

We validated 2 assays for the measurement of adenosine deaminase (ADA) activity in the saliva of pigs: the Giusti–Galanti manual method (ADA-GG) and a commercial automated assay (Diazyme Laboratories; ADA-D). Intra-assay coefficients of variation (CVs) were <7 and 9%, and interassay CVs were <12 and 5%, for ADA-GG and ADA-D, respectively. Accuracy was measured by 2 methods: recovery and linearity-under-dilution. Recovery was 82.4–106.8% for ADA-GG, and 92.8–107.9% for ADA-D. Serial dilutions showed R2 > 0.95 and 0.99 for ADA-GG and ADA-D, respectively. Linear regression between the methods gave R2 = 0.997 (p < 0.0001), and a Bland–Altman plot showed a proportional bias of 112 IU/L (95% confidence interval of −99 to 322 IU/L) for ADA-D. No significant differences were observed between the results obtained by either method in saliva or serum. ADA activity was much higher in porcine saliva than in serum. Salivary ADA activity was significantly higher in lame pigs compared to healthy animals. However, serum ADA activity was significantly lower in lame pigs.

New potential biomarkers of oxidative stress in Mytilus galloprovincialis: Analytical validation and overlap performance

The aim of the present report was to develop and validate new automated spectrophotometric assays for measurement of total antioxidant capacity (TAC), thiols, and advanced oxidation protein products (AOPP) in mussel gills, digestive gland and hemolymph samples, and to evaluate their possible utility in biomonitoring programs. Total antioxidant capacity (TAC) was measured by different methods: trolox equivalent antioxidant capacity (TEAC1 and TEAC2), cupric reducing antioxidant capacity (CUPRAC), and ferric reducing ability of plasma (FRAP). The assays were precise, accurate and provided low limits of detection. When oxidative stress was promoted by inducing hypoxia and the behaviour of these biomarkers between hypoxic and controls mussels were compared, statistically significant differences were observed in all biomarkers and tissues evaluated. The results of the present study demonstrated that these biomarkers, not previously studied in mussels, show a potential use as biomarkers of oxidative stress in this species since they were validated and showed changes under a state of oxidative stress.