antioxidants Article Measurement of Redox Biomarkers in the Whole Blood and Red Blood Cell Lysates of Dogs Luis G. González-Arostegui 1 , Alberto Muñoz-Prieto 1,2 , Asta Tvarijonaviciute 1 , José Joaquín Cerón 1 and Camila Peres Rubio 3, * 1 Interlab-UMU, Regional Campus of International Excellence “Mare Nostrum”, University of Murcia, 30100 Murcia, Spain; luisgarostegui@gmail.com (L.G.G.-A.); alberto.munoz@um.es (A.M.-P.); asta@um.es (A.T.); jjceron@um.es (J.J.C.) 2 Clinic for Internal Diseases, Faculty of Veterinary Medicine, University of Zagreb, Heinzelova 55, 1000 Zagreb, Croatia 3 Department of Animal and Food Science, School of Veterinary Science, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Spain * Correspondence: camila.peres@um.es Abstract: The evaluation of the biomarkers of oxidative status is usually performed in serum, however, other samples, such as red blood cells (RBCs) lysates or whole blood (WB), can be used. The objective of this study was to evaluate if a comprehensive panel of redox biomarkers can be measured in the WB and RBCs of dogs, and their possible changes “in vitro” after the addition of different concentrations of ascorbic acid. The panel was integrated by biomarkers of the antioxidant status, such as cupric reducing antioxidant capacity (CUPRAC), ferric reducing ability of plasma (FRAP), Trolox equivalent antioxidant capacity (TEAC), thiol and paraoxonase type 1 (PON-1), and  of the oxidant status, such as total oxidant status (TOS), peroxide-activity (POX-Act), reactive oxygen-  derived compounds (d-ROMs), advanced oxidation protein products (AOPP) and thiobarbituric Citation: González-Arostegui, L.G.; Muñoz-Prieto, A.; Tvarijonaviciute, acid reactive substances (TBARS). All the assays were precise and accurate in WB and RBCs lysates. A.; Cerón, J.J.; Rubio, C.P. In addition, they showed changes after ascorbic acid addition that are in line with previously Measurement of Redox Biomarkers published results, being WB more sensitive to detect these changes in our experimental conditions. in the Whole Blood and Red Blood In conclusion, the panel of assays used in this study can be measured in the WB and RBCs of the dog. Cell Lysates of Dogs. Antioxidants In particular, the higher sensitivity to detect changes in our experimental conditions and its easier 2022, 11, 424. https://doi.org/ sample preparation makes WB a promising sample for the evaluation of redox status in dogs, with 10.3390/antiox11020424 also potential applications to other animal species and humans. Academic Editor: Saverio Francesco Retta Keywords: antioxidants; biomarkers; dogs; oxidants; whole blood Received: 14 January 2022 Accepted: 17 February 2022 Published: 19 February 2022 1. Introduction Publisher’s Note: MDPI stays neutral The oxidation–reduction (redox) homeostasis is of high importance to life, being with regard to jurisdictional claims in involved in the most important biological processes [1]. Redox status can be assessed by published maps and institutional affil- the measurement of oxidant substances such as reactive oxygen species (ROS) [2,3] and iations. by the evaluation of the antioxidant status, using biomarkers of the total oxidant capacity (TAC) as the cupric reducing antioxidant capacity (CUPRAC), ferric reducing ability of plasma (FRAP) and Trolox equivalent antioxidant capacity (TEAC), as well as, individual Copyright: © 2022 by the authors. antioxidant compounds such as thiol, superoxide dismutase (SOD), glutathione peroxidase Licensee MDPI, Basel, Switzerland. (GPx) and paraoxonase type-1 (PON-1) [4–7]. Oxidative stress is defined as a disturbance This article is an open access article in the redox balance directed towards oxidation, which can produce cellular and tissue distributed under the terms and damage [8]. conditions of the Creative Commons The evaluation of biomarkers of redox status is usually performed in serum, nonethe- Attribution (CC BY) license (https:// less, other samples, such as red blood cells (RBCs) lysates, whole blood (WB), saliva and creativecommons.org/licenses/by/ urine, can be used. The involvement of RBCs in the measurement of redox analytes have 4.0/). the potential advantage of being more sensitive to oxidative stress due to their role in Antioxidants 2022, 11, 424. https://doi.org/10.3390/antiox11020424 https://www.mdpi.com/journal/antioxidants Antioxidants 2022, 11, 424 2 of 11 oxygen transport, making them one of the first blood components affected by oxidative stress [9,10]. In addition, WB serves as a reflection of the overall redox balance of other tissues [11], making it an interesting sample type to study antioxidants and oxidants [12]. In dogs, the use of RBCs lysates and WB has been proven to be effective in the measure- ment of selected antioxidants and oxidants. In RBCs lysates, SOD was measured to evaluate the antioxidant response of dogs receiving a diet consisting of high polyunsaturated fatty acids [13], as well as in dogs with chronic kidney disease [14], atopic dermatitis [15], par- voviral infection [16], sarcoptic mange [17], leishmaniasis [18] and uncomplicated babesio- sis [19]. In addition, reduced glutathione has been measured in RBCs lysates from dogs with renal azotemia [20] and GPx has been determined in RBCs lysates from dogs infected with Dirofilaria immitis [21] and with multicentric lymphoma [22]. Among oxidants, thio- barbituric acid reactive substances (TBARS) have been used to evaluate lipid peroxidation in the RBCs lysates of dogs with a diet consisting of high polyunsaturated fatty acids [13], as well as in dogs with sarcoptic mange [17] and dirofilariasis [21]. Regarding WB, GPx has been measured in a storage study [23], as well as in dogs with leishmaniasis [18] and heart failure [24]. In vitro studies in which ascorbic acid (vitamin C) is added to the sample have been performed previously as a model to evaluate the ability of different analytes to detect changes in the redox status of the sample [25]. Ascorbic acid is a known chain-breaking antioxidant that can also act as a pro-oxidant under some specific conditions in biological systems [26]. To the author’s knowledge, the assessment of oxidative stress in dogs using samples such as WB and RBCs has been limited to the evaluation of SOD, reduced glutathione, GPx activity and TBARS in RBCs and GPx activity in WB, as previously indicated. Therefore, the objective of this study was to evaluate if a comprehensive panel of redox biomarkers, integrated by 10 analytes that are commonly measured in canine serum, could also be measured in WB and RBCs, and if they could show changes after an “in vitro” addition of an antioxidant. For this purpose, in this report, the following activities were performed: (1) an analytical validation in WB and RBCs lysates of biomarkers of the antioxidant status, such as CUPRAC, FRAP, TEAC, thiol and PON-1, and of the oxidant status, such as total oxidant status (TOS), peroxide-activity (POX-Act), reactive oxygen-derived compounds (d-ROMs), advanced oxidation protein products (AOPP) and TBARS; and (2) an evaluation of these biomarkers after the addition of an antioxidant compound, such as ascorbic acid, at two different concentrations, situations that are known to produce changes in the redox status of the sample. 2. Materials and Methods 2.1. Sample Preparation Blood samples were obtained from clinically normal Beagle dogs that belonged to the Animal Resources Center of the University of Murcia. All samples were collected by jugular venipuncture and placed in EDTA tubes and analyzed for a complete blood count. Each blood sample was then divided into two different aliquots for posterior WB and RBCs preparation and plasma separation. To obtain the WB, one aliquot was kept at −80 ◦ C for at least two hours before the analysis of the oxidative status biomarkers. The other one was used to prepare the RBCs lysate by osmotic shock, as reported previously [25,27,28]. For this purpose, the sample was centrifuged at 3000 rpm × 10 min at 4 ◦ C. Subsequently, plasma was separated and stored in Eppendorf tubes, the buffy coat was discarded, and the RBCs were washed with isotonic saline (NaCl 0.9%) and centrifuged, as previously mentioned. Then, the supernatant was removed, and the process was repeated. After a total of four washes, the packed RBCs were hemolyzed in a 1:4 dilution using ultrapure water and then stored at −80 ◦ C until analysis. Antioxidants 2022, 11, 424 3 of 11 2.2. Assays 2.2.1. Antioxidant Status CUPRAC assay was based on the reduction of Cu2+ into Cu1+ by the nonenzymatic antioxidants in the sample [29]. Evaluation of CUPRAC was made following the procedure previously validated for use in serum of dogs [30]. Results are expressed in millimoles per liter of sample (mmol/L). FRAP assay was based on the reduction of ferric-tripyridyltriazine (Fe3+ -TPTZ) to the ferrous (Fe2+ ) form [31]. Its determination was made following previously described methods [31,32]. Results are expressed in mmol/L. Measurement of TEAC was based on the assay described by Arnao et al. [33], which has been used previously in canine serum samples [34]. Its principle is based on the enzymatic generation of 2,20 -azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS) radical and its reduction by nonenzymatic antioxidants presents in the sample [33]. Results are expressed in mmol/L. The determination of total thiol is based on the reaction of thiols within the sample with 5,50 -dithiobis-(2-nitrobenzoic acid) (DTNB). The assay used was performed according to previously described methods for serum samples [35,36]. Results are expressed in micromoles per liter (µmol/L). Measurement of PON-1 was based on the hydrolysis of phenylacetate into phenol and it was determined as previously described in canine serum [37]. Results are expressed in international units per milliliter of sample (IU/mL). 2.2.2. Oxidant Status The determination of TOS was based on the assay described by Erel [38], which was previously used in dogs [39]. Its reaction is based on the ability of oxidants in the sample to oxidize Fe2+ -o-dianisidine complex to Fe3+ [38]. Results are expressed in µmol/L. The POX-Act assay was based on the determination of total peroxides through a peroxide–peroxidase reaction using tetramethylbenzidine as the chromogenic substrate [40]. Determination of POX-Act was measured following a validated method for human sera [40]. Results are expressed in µmol/L. The determination of d-ROMs assay was based on the reaction of the sample in an acidic medium in the presence of N,N,-diethyl-para-phenylenediamine (DEPPD), and it was made following a previously described method [41]. Results are expressed in Carratelli Units (U.CARR). AOPP determination was based on oxidized albumin and di-tyrosine containing cross- linked proteins, as previously described [42] and measured in canine serum [43]. Results are expressed in µmol/L. Determination of TBARS is based on the reaction of the sample to a trichloroacetic acid, thiobarbituric acid and N hydrochloric acid stock (15% w/v trichloroacetic acid; 0.375% w/v thiobarbituric acid; 0.25 N hydrochloric acid) in heated conditions [44]. TBARS was measured following a previously described method [44] using a microplate reader (Powerwave XS, Biotek Instruments, Winusky, VT, USA). Results are expressed in µmol/L. 2.3. Analytical Validation Analytical performances of the assays were assessed by calculating precision, accuracy and detection limit according to the protocol previously described by Tiwari and Tiwari [45] that has been applied in other analytical validations in dogs [30,34,37]. 2.3.1. Precision Precision was expressed as the coefficient of variation (CV; mean divided by standard deviation [SD] and multiplied by 100) and was calculated as inter- and intra-assay variation. Intra-assay precision was determined as the CV between five replicates from two WB and RBCs lysates samples (one with high and one with low concentration) in a single assay run. Inter-assay precision was determined as the CV between five replicates from two WB and Antioxidants 2022, 11, 424 4 of 11 RBCs lysates samples (one with high and one with low concentration) measured on five separate days. Samples used for the evaluation of the intra-assay CV were aliquoted in five different Eppendorf tubes and used for the determination of the inter-assay CV. Each aliquot was measured on a different day. 2.3.2. Accuracy and Limit of Detection Accuracy was evaluated through linearity under dilution and spiking recovery. To study linearity under dilution, the canine RBCs lysate samples were diluted with a Phos- phate buffer pH 7.5 at 1:2, 1:4, 1:8, 1:16 and 1:32, and WB samples were diluted at the same dilutions and 1:25, 1:50, 1:100, 1:200 and 1:400. In order to study spike recovery, one sample containing high concentrations of each analyte and one sample containing low concentrations of each analyte were selected and mixed at different percentages (87.5%, 75%, 50%, 25% and 12.5% of the sample with high concentration with 87.5%, 75%, 50%, 25% and 12.5% of the sample with low concentration of each method, respectively). The ratios of the measured values to the expected values of each method were then calculated. The limit of detection (LOD) for each assay was evaluated based on the data from 20 replicate determinations of phosphate buffer pH 7.5. 2.4. “In Vitro” Test In brief, 5 mL of blood samples were drawn from the jugular vein of 7 male adult Beagle dogs (aged between 4–9 years) and collected in EDTA tubes. Each sample was divided into three groups: (1) control; (2) ascorbic acid 10 mM (VC10); and (3) ascorbic acid 60 mM (VC60). All plastic vials of all groups contained 940 µL of blood. To the control group vials, 60 µL of NaCl 0.9% were added. To the VC10 group vials, 60 µL of a 166.67 mM ascorbic acid solution were added. In the case of the VC60 group vials, 60 µL of 1 M ascorbic acid solution were added. The content of the vials was mixed and incubated for 2 h at 4 ◦ C. After that, the WB, RBCs lysates and plasma samples were prepared as described in “sample preparation”. For the analysis, the WB and RBCs lysates were diluted with a Phosphate buffer pH 7.5 to achieve a concentration that allows the measurement of each analyte. Final concentrations of both WB and RBCs lysates were corrected by the dilution factor which varied depending on the analyte measured (ranging from 1:10 to 1:30) and were expressed in concentration or activity per volume of sample. The procedures were approved by the University of Murcia’s ethics committees and the Ministry of Agriculture, Livestock, Fishing, and Aquaculture of the Region of Murcia (A13170503). 2.5. Statistical Analysis Data were analyzed using GraphPad Prism software (GraphPad Software Inc., version 9.3 for MacOS). Arithmetic means, medians, intra- and inter-assay CVs were calculated by use of routine descriptive statistical procedures and computer software (Excel 2020, Microsoft; GraphPad Statistics Guide). Linearity under dilution was investigated by linear regression. The Shapiro-Wilk test was first used to assess whether data of the “in vitro” test were normally distributed. Differences in the concentrations between groups, when data were normally distributed, were assessed using a 2-way ANOVA followed by Tukey’s range test, and non-normally distributed datasets were assessed employing a 2-way ANOVA followed by Kruskal–Wallis test. Statistical differences were considered for p-values < 0.05. 3. Results 3.1. Analytical Validation For WB samples, all assays showed an intra-assay CV between 0.01% and 15.89% and an inter-assay CV between 0.01% and 13.62% (Table S1). Serial dilution of WB resulted in linear regression higher than 0.9667 (Figure S1). Recovery in all cases was between 92.77% and 122.49%. Antioxidants 2022, 11, 424 5 of 11 Antioxidants 2021, 10, x FOR PEER REVIEW 5 of 12 For RBCs lysates, all assays showed an intra-assay CV between 1.02% and 15.98% (Table 3. ResultsS2) and an inter-assay CV between 0.58% and 12.6%. Serial dilution of RBCs lysates resulted in linear regression higher than 0.96 for all assays studied (Figure S2). Recovery in 3.1. Analytical all cases Validation was between 87.5% and 113.9%. For TheWB samples, limit all assays of detection for showed CUPRAC, an FRAP, intra-assay thiol,CV between PON-1, 0.01% TOS, and 15.89% POX-Act, and d-ROMs, an AOPP inter-assay and TBARS CV between was of0.01% 0.021 and 13.62%0.013 mmol/L, (Table S1). Serial mmol/L, dilution 47.67 of WB0.12 µmol/L, resulted IU/mL,in linear regression higher than 0.9667 (Figure S1). Recovery in all 11.92 µmol/L, 5.28 µmol/L, 20.31 U.CARR, 16.16 µmol/L and 1.06 µmol/L, respectively. cases was between 92.77% and In the122.49%. case of TEAC, values obtained were all negative and the limit of detection could not For RBCs lysates, all assays showed an intra-assay CV between 1.02% and 15.98% be calculated. (Table S2) and an inter-assay CV between 0.58% and 12.6%. Serial dilution of RBCs lysates 3.2. “In Vitro” resulted Testregression higher than 0.96 for all assays studied (Figure S2). Recovery in linear 3.2.1. in Antioxidant all cases Status87.5% and 113.9%. was between The limitforofantioxidant Results detection for CUPRAC,inFRAP, biomarkers the “inthiol, vitro”PON-1, test areTOS, shownPOX-Act, in Figured-ROMs, 1. After AOPP and TBARS the addition was of of ascorbic 0.021 acid mmol/L, at 10 mM, the 0.013 threemmol/L, TAC assays 47.67FRAP, µmol/L, TEAC0.12and IU/mL, 11.92 CUPRAC, µmol/L, showed 5.28 µmol/L, 20.31 significantly higher U.CARR, 16.16the levels than µmol/L control and 1.06 when group µmol/L, respectively. measured in WBIn and the plasma case (exceptvalues of TEAC, for CUPRAC obtainedin WB). were In allRBCs lysates, negative andno significant the differences limit of detection werenot could found be in any assay. In plasma, the ascorbic acid at 10 mM produced a significant decrease in thiol. calculated. With ascorbic acid at 60 mM, in WB the three TAC assays showed significantly higher 3.2. “Inthan levels Vitro” control, Test and the magnitude of increases was significantly higher than with 10 mM. 3.2.1. The TAC Status Antioxidant assays also showed significantly increased concentrations in plasma. However, in RBCs lysates, the three TAC assays were significantly lower than in the control Results for antioxidant biomarkers in the “in vitro” test are shown in Figure 1. After group. Thiol was significantly lower than control in the three types of samples. PON1 was the addition of ascorbic acid at 10 mM, the three TAC assays FRAP, TEAC and CUPRAC, lower than control and VC10 in RBCs lysate. showed significantly higher levels than the control group when measured in WB and plasma (exceptStatus 3.2.2. Oxidant for CUPRAC in WB). In RBCs lysates, no significant differences were found in any assay. In plasma, the ascorbic acid at 10 mM produced a significant decrease Results for antioxidant biomarkers in the “in vitro” test are shown in Figure 2. After in thethiol. addition of ascorbic acid at 10 mM, POX-Act and d-ROMs were significantly lower than With control in ascorbic WB. No acid at 60 mM, significant in WB changes the three were foundTAC assays in RBCs. Inshowed plasma,significantly TBARS was higher higher levels than than control. control, and the magnitude of increases was significantly higher than with 10 mM. WithThe TAC assaysacid ascorbic alsoatshowed 60 mM, significantly POX-Act and increased d-ROMs, concentrations in plasma. and in addition, TOSHow- were ever, in RBCs lysates, the three TAC assays were significantly lower significantly lower than control in WB. In RBCs lysate, d-ROMs values were not different than in the control group. Thiolbut to control, was significantly POX-Act lower than and AOPP werecontrol in the three significantly lowertypes thanofcontrol samples.andPON1 VC10. was In lower than control and VC10 in RBCs lysate. plasma, POX-Act was significantly lower and TBARS was significantly higher than control. 70 120 *# 60 *# 100 CUPRAC (mmol/L) 80 FRAP (mmol/L) 50 40 * 60 30 40 * * 20 20 10 2.0 *# 8.5 * 1.5 * * 1.0 0.5 2 0.0 1 0 B s a m C W Cs a B B as m R W Pl B as R Pl (a) (b) Figure 1. Cont. Antioxidants 2021, 10, x FOR PEER REVIEW 6 of 12 Antioxidants 2022, 11, 424 6 of 11 60 *# 20000 40 15000 *# TEAC (mmol/L) Thiol (µmol/L) * * 10000 20 *# 5000 * * 500 2.0 400 1.5 300 1.0 200 0.5 100 * * 0.0 0 B s a B s a m C m C W W B B as as R R Pl Pl (c) (d) 5 4 PON-1 (IU/mL) 3 *# # * 2 1 0 B s a W C m B as R l P (e) Figure Figure1.1. Results Resultsof ofthe the “in “in vitro” vitro”test teston onantioxidant antioxidantbiomarkers. biomarkers.(a) (a)Cupric Cupricreducing reducingantioxidant antioxidant capacity (CUPRAC); (b) ferric reducing ability of plasma (FRAP); (c)Trolox equivalent antioxidant capacity (CUPRAC); (b) ferric reducing ability of plasma (FRAP); (c)Trolox equivalent antioxidant capacity (TEAC); (d) thiol, and (e) paraoxonase type 1 (PON-1) results obtained during the “in vitro” capacity (TEAC); (d) thiol, and (e) paraoxonase type 1 (PON-1) results obtained during the “in vitro” study for whole blood (WB), red blood cells (RBCs) lysates and plasma samples. Probability levels ofstudy for whole p < 0.05 Antioxidants 2021, 10, x FOR PEER REVIEW blood (WB), were regarded red blood and as significant cellsmarked (RBCs) lysates with anand plasma asterisk (*: samples. Probability vs. control) levels and a hashtag 7 of 12 of p < 0.05 were regarded as significant and marked with an asterisk (*: vs. control) (#: VC10 vs. VC60). Red box: control group; green box: VC10 group; and blue box: VC60 group. and a hashtag (#: VC10 vs. VC60). Red box: control group; green box: VC10 group; and blue box: VC60 group. 3.2.2. Oxidant Status 1500 Results for antioxidant biomarkers in the “in vitro” test are shown in Figure 2. After 6000 the addition of ascorbic acid at 10 mM, POX-Act and d-ROMs were significantly lower 5000 1250 POX-Act (µmol/L) than control in WB. No significant changes were found in RBCs. In plasma, TBARS was TOS (µmol/L) 4000 higher than control. 1000 *# 3000 *# With ascorbic acid at 60 mM, POX-Act and d-ROMs, * and in addition, TOS were sig- 2000 750 * nificantly lower than control in WB. In RBCs lysate, d-ROMs values were not different to 1000 control, but POX-Act and AOPP were significantly lower than control and VC10. In 500 15 plasma, POX-Act was significantly lower and TBARS was significantly higher than con- 10 trol. 250 *# 5 0 0 B s a B s a m C W C W sm B as B R R la Pl P (a) (b) 4000 Figure 2. Cont. 40000 30000 *# (U.CARR) (µmol/L) 3000 20000 10000 2000 15 PO 10 250 *# 5 0 0 B s a B s a m C W C W sm B Antioxidants 2022, 11, 424 7 of 11 as B R R la Pl P (a) (b) 4000 40000 30000 *# d-ROMs (U.CARR) AOPP (µmol/L) 3000 20000 10000 2000 600 1000 * 400 * 200 0 0 B s a m C s a B W m C B W as B R as Pl R Pl (c) (d) 300 250 TBARS (µmol/L) 200 70 60 50 40 30 10.0 7.5 * * 5.0 2.5 0.0 B s a W C m B as R Pl (e) Figure Figure 2. 2. Results Resultsfor forthe the“in “invitro” test vitro” onon test oxidant biomarkers. oxidant biomarkers.(a) Total oxidant (a) Total status oxidant (TOS); status (b) (TOS); peroxide-activity (POX-Act); (c) reactive oxygen-derived compounds (d-ROMs); (d) advanced oxi- (b) peroxide-activity (POX-Act); (c) reactive oxygen-derived compounds (d-ROMs); (d) advanced dation protein products (AOPP), and (e) thiobarbituric acid reactive substances (TBARS) results ob- oxidation protein products (AOPP), and (e) thiobarbituric acid reactive substances (TBARS) results tained during the “in vitro” study for whole blood (WB), red blood cells (RBCs) lysates and plasma obtained Probability samples. during the “in vitro” levels of pstudy < 0.05for whole were blood (WB), regarded red bloodand as significant cells (RBCs)with marked lysates an and plasma asterisk (*: samples. Probability levels of p vs. control) and a hashtag (#: VC10 vs. VC60). Red box: control group; green box: VC10 group;(*: < 0.05 were regarded as significant and marked with an asterisk vs. and control) blue box:and a hashtag VC60 group. (#: VC10 vs. VC60). Red box: control group; green box: VC10 group; and blue box: VC60 group. 4. Discussion In this study, a panel of assays that can be used for the evaluation of oxidative stress was validated in WB and RBCs lysate samples from dogs. Some of the validated analytes, to our knowledge, have not been previously studied in these types of samples. Therefore, to the author’s knowledge, this is the first study to report the use of CUPRAC, FRAP, TEAC, thiol, PON-1, TOS, d-ROMs, POX-Act and AOPP in WB and RBCs lysate samples from dogs. The results of the analytical validation showed that all methods included in this study demonstrated adequate intra- and inter-assay CVs, as well as good linearity under dilution [45], similar to results obtained previously in serum of dogs [30,32,34,37]. In addition, all methods showed a high recovery. This would indicate that the assays validated in this study are precise and accurate and could be used to evaluate biomarkers of oxidative stress in WB and RBCs lysate samples from dogs. The use of automated assays leads to faster results and reduces error range. All the assays in this study were validated using an automatic biochemistry analyzer, except for TBARS. Nonetheless, the assays that Antioxidants 2022, 11, 424 8 of 11 were used through an automated analyzer can also be adapted to microplate readers and manual spectrophotometers. As a second part of the study, the assays were used for the measurement of analytes in WB and RBCs in a situation that can change the oxidative status of the sample, as the addition of ascorbic acid. In this experiment, the analytes were also measured in plasma for comparative purposes. Ascorbic acid acts as an antioxidant since it participates in the reduction of O2 •− and lipid peroxyl radicals in both RBCs membranes and plasma [46–49] and it can inhibit the damage of RBCs [50]. However, in some specific conditions, ascorbic acid can lead to the production of H2 O2 via reactions with metals such as Cu2+ and Fe2+ as well as with oxygen or by its self-oxidation [26]. The fact that TACs assays such as CUPRAC, FRAP and TEAC measure ascorbic acid could be the reason why these assays showed increased values in WB when ascorbic acid was added, with the increase being in a dose-dependent manner. However, this event was not evident when TACs were measured in RBCs samples, in which even a decrease was observed when 60 mM of ascorbic was added. This is in line with previous findings reporting lower CUPRAC values in erythrocytes at high concentrations of ascorbic acid [25]. We hypothesize that during the sample processing the compounds present in lysate samples, especially those released from lysed cells such as Fe, interact with the high concentrations of ascorbic acid, leading to a degradation of antioxidants present in the RBCs lysate sample. On the other hand, the concentrations of PON-1 in RBCs lysates and plasma and thiol in all sample types decreased when incubated with higher concentrations of ascorbic acid. It has been demonstrated that acid ascorbic can oxidize thiols by its auto-oxidation to dehydroascorbic acid, and, by consequence, the decreased PON1 activity due to the reduction in its free SH groups number could explain the findings of this study [51,52]. Regarding the oxidant status, it was found that the addition of 60 mM of ascorbic acid led to lower levels of POX-Act in all three types of samples, being indicative of the inhibition of peroxides formation by the added antioxidant. The concentrations of TOS and d-ROMs were also reduced in high ascorbic acid concentrations, but only when measured in WB. Overall, this would indicate that ascorbic acid, in the concentrations used, can inhibit oxidant compounds in biological samples, with POX-Act being more sensitive in detecting this effect. The increase in the oxidant compounds due to the liberation from lysed cells could be the cause of the lack of reduction of these markers in RBCs lysates, therefore RBCs lysates would be less sensitive than WB to detect the changes produced by ascorbic acid. During the experimental conditions, the use of WB was easier when compared with the RBCs lysates and plasma/serum, which are the samples traditionally used. For the obten- tion of plasma or serum, centrifugation and aspiration are needed. In addition, the prepara- tion of lysate takes around one or two hours, depending on the chosen procedure [16,53]. In contrast, the preparation of WB samples is far more simple, since no centrifugation and washing procedures are needed. In addition, the higher concentration of most analytes in WB compared to plasma, possibly due to the intracellular antioxidants present in RBC and other blood cells, and the antioxidants bound to their surfaces [12].would be in favor of using this type of sample. Also, the “in vitro” study indicated that WB reflected better the expected changes after ascorbic acid addition than RBCs lysates or plasma. However, overall, this work should be considered as a pilot study since only an “in vitro” test was performed, and further studies involving larger populations of dogs should be performed to evaluate the changes in these analytes in the different type samples in physiological conditions and different diseases. As a part of these studies, a comparison of the analytes between serum and plasma would be of interest. The results of these studies will determine which type of sample is more adequate for the measurement of the different redox analytes in the dog. In addition, in our conditions, the hemoglobin concentration of the samples did not affect the results obtained in this work, therefore no corrections were made, and the values were only expressed in concentration or activity of the analyte per volume of Antioxidants 2022, 11, 424 9 of 11 the original sample. However, it would be interesting to confirm these findings in disease conditions in which alterations of hemoglobin and/or RBCs concentrations would occur. 5. Conclusions A panel of assays for evaluation of the redox status integrated by CUPRAC, FRAP, TEAC, thiol, PON-1, TOS, POX-Act, d-ROMs, AOPP and TBARS can be measured in WB and RBCs of the dog. Changes in the redox status assays were observed in WB and RBCs lysates after the addition of ascorbic acid, being analytes in WB more sensitive to detect these changes in our experimental conditions. This higher sensitivity to the detection of changes and its easier sample preparation makes WB a promising sample for the evaluation of redox status in dogs. Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/antiox11020424/s1. Figure S1. Linear regression of the antioxidant (a) and oxidant (b) biomarkers validated in whole blood (WB). Regression line showing two WB samples at various dilutions. Regression equation and coefficient of determination (r2 ) are shown. Table S1. Mean, standard deviation (SD) and intra- and inter-assay coefficients of variation (CVs, given in %) for antioxidant and oxidant biomarkers obtained in the precision study of assays for validation in two whole blood (WB) samples from dogs (A = Low concentrations, B = High concentrations). Figure S2. Linear regression of the antioxidant (a) and oxidant (b) biomarkers validated in red blood cells (RBCs) lysates. Regression line showing two WB samples at various dilutions. Regression equation and coefficient of determination (r2 ) are shown. Table S2. Mean, standard deviation (SD) and intra- and inter-assay coefficients of variation (CVs, given in %) for antioxidant and oxidant biomarkers obtained in the precision study of assays for validation in two red blood cells (RBCs) lysate samples from dogs (A = Low concentrations, B = High concentrations). Author Contributions: Conceptualization, L.G.G.-A., J.J.C. and C.P.R.; data curation, L.G.G.-A.; formal analysis, A.T.; funding acquisition, J.J.C.; investigation, L.G.G.-A.; methodology, L.G.G.-A. and C.P.R.; project administration, J.J.C. and C.P.R.; supervision, C.P.R.; validation, L.G.G.-A. and C.P.R.; visualization, A.M.-P. and J.J.C.; writing—original draft, L.G.G.-A.; writing—review and editing, A.M.-P., A.T., J.J.C. and C.P.R. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Seneca Foundation-Agency of Science and Technology of the Region of Murcia through the Subprogram to the Scientific Leadership and the Transition to the Independent Investigation (20649/JLI/18). L.G.G.-A. was funded by the Seneca Foundation (21453/FPI/20). A.M.-P. was funded by the University of Murcia through a post-doctoral grant (Margarita Salas) within the mark of “Ayudas en el marco del Programa para la Recualificación del Sistema Universitario Español” through the European Union Next Generation Funds. C.P.-R. has a post-doctoral fellowship “Juan de la Cierva Formación” supported by the “Ministerio de Economía y Competitividad” (FJC2019-042475-I). Institutional Review Board Statement: The procedures were approved by the University of Murcia’s ethics committees and the Ministry of Agriculture, Livestock, Fishing, and Aquaculture of the Region of Murcia (A13170503). Informed Consent Statement: Not applicable. Data Availability Statement: The data supporting the conclusions of this article are included within the manuscript. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. References 1. Sies, H.; Berndt, C.; Jones, D.P. Oxidative Stress. Annu. Rev. Biochem. 2017, 86, 715–748. [CrossRef] [PubMed] 2. Nita, M.; Grzybowski, A. The Role of the Reactive Oxygen Species and Oxidative Stress in the Pathomechanism of the Age-Related Ocular Diseases and Other Pathologies of the Anterior and Posterior Eye Segments in Adults. Oxid. Med. Cell. Longev. 2016, 2016, 3164734. [CrossRef] [PubMed] Antioxidants 2022, 11, 424 10 of 11 3. Tharmalingam, S.; Alhasawi, A.; Appanna, V.P.; Lemire, J.; Appanna, V.D. Reactive Nitrogen Species (RNS)-resistant microbes: Adaptation and medical implications. Biol. Chem. 2017, 398, 1193–1208. [CrossRef] [PubMed] 4. Rubio, C.P.; Hernández-Ruiz, J.; Martinez-Subiela, S.; Tvarijonaviciute, A.; Ceron, J.J. Spectrophotometric assays for total antioxidant capacity (TAC) in dog serum: An update. BMC Vet. Res. 2016, 12, 1–7. [CrossRef] 5. Bissinger, R.; Bhuyan, A.A.M.; Qadri, S.M.; Lang, F. Oxidative stress, eryptosis and anemia: A pivotal mechanistic nexus in systemic diseases. FEBS J. 2019, 286, 826–854. [CrossRef] 6. Lang, F.; Abed, M.; Lang, E.; Föller, M. Oxidative stress and suicidal erythrocyte death. Antioxid. Redox Signal. 2014, 21, 138–153. [CrossRef] [PubMed] 7. Sies, H. Oxidative Stress: Concept and Some Practical Aspects. Antioxidants 2020, 9, 852. [CrossRef] [PubMed] 8. Harwell, B. Biochemistry of oxidative stress. Biochem. Soc. Trans. 2007, 35, 1147–1150. [CrossRef] 9. Hermann, P.B.; Pianovski, M.A.D.; Henneberg, R.; Nascimento, A.J.; Leonart, M.S.S. Marcadores de estresse oxidativo em eritrócitos de crianças com doença falciforme. J. Pediatr. 2016, 92, 394–399. [CrossRef] [PubMed] 10. Maurya, P.K.; Kumar, P.; Chandra, P. Biomarkers of oxidative stress in erythrocytes as a function of human age. World J. Methodol. 2015, 5, 216. [CrossRef] [PubMed] 11. Margaritelis, N.V.; Veskoukis, A.S.; Paschalis, V.; Vrabas, I.S.; Dipla, K.; Zafeiridis, A.; Kyparos, A.; Nikolaidis, M.G. Blood reflects tissue oxidative stress: A systematic review. Biomarkers 2015, 20, 97–108. [CrossRef] [PubMed] 12. Koren, E.; Kohen, R.; Ginsburg, I. Polyphenols enhance total oxidant-scavenging capacities of human blood by binding to red blood cells. Exp. Biol. Med. 2010, 235, 689–699. [CrossRef] [PubMed] 13. Pacheco, G.; Bortolin, R.; Chaves, R.; Moreira, J.; Kessler, A.; Trevizan, L. Effects of the consumption of polyunsaturated fatty acids on the oxidative status of adults dogs. J. Anim. Sci. 2018, 96, 4590–4598. [CrossRef] 14. Kogika, M.M.; Lustoza, M.D.; Hagiwara, M.K.; Caragelasco, D.S.; Martorelli, C.R.; Mori, C.S. Evaluation of oxidative stress in the anemia of dogs with chronic kidney disease. Vet. Clin. Pathol. 2015, 44, 70–78. [CrossRef] [PubMed] 15. Kapun, A.P.; Salobir, J.; Levart, A.; Kotnik, T.; Svete, A.N. Oxidative stress markers in canine atopic dermatitis. Res. Vet. Sci. 2012, 92, 469–470. [CrossRef] [PubMed] 16. Panda, D.; Patra, R.C.; Nandi, S.; Swarup, D. Oxidative stress indices in gastroenteritis in dogs with canine parvoviral infection. Res. Vet. Sci. 2009, 86, 36–42. [CrossRef] [PubMed] 17. Singh, S.K.; Dimri, U.; Sharma, M.C.; Swarup, D.; Sharma, B. Determination of oxidative status and apoptosis in peripheral blood of dogs with sarcoptic mange. Vet. Parasitol. 2011, 178, 330–338. [CrossRef] 18. Britti, D.; Sconza, S.; Morittu, V.M.; Santori, D.; Boari, A. Superoxide dismutase and Glutathione peroxidase in the blood of dogs with Leishmaniasis. Vet. Res. Commun. 2008, 32, 251–254. [CrossRef] 19. Teodorowski, O.; Winiarczyk, S.; Tarhan, D.; Dokuzeylül, B.; Ercan, A.M.; Erman Or, M.; Staniec, M.; Adaszek, L. Antioxidant status, and blood zinc and copper concentrations in dogs with uncomplicated babesiosis due to Babesia canis infections. J. Vet. Res. 2021, 65, 169–174. [CrossRef] 20. Buranakarl, C.; Trisiriroj, M.; Pondeenana, S.; Tungjitpeanpong, T.; Jarutakanon, P.; Penchome, R. Relationships between oxidative stress markers and red blood cell characteristics in renal azotemic dogs. Res. Vet. Sci. 2009, 86, 309–313. [CrossRef] [PubMed] 21. Dimri, U.; Singh, S.K.; Sharma, M.C.; Behera, S.K.; Kumar, D.; Tiwari, P. Oxidant/antioxidant balance, minerals status and apoptosis in peripheral blood of dogs naturally infected with Dirofilaria immitis. Res. Vet. Sci. 2012, 93, 296–299. [CrossRef] [PubMed] 22. Winter, J.L.; Barber, L.G.; Freeman, L.; Griessmayr, P.C.; Milbury, P.E.; Blumberg, J.B. Antioxidant Status and Biomarkers of Oxidative Stress in Dogs with Lymphoma. J. Vet. Intern. Med. 2009, 23, 311–316. [CrossRef] [PubMed] 23. van Zelst, M.; Hesta, M.; Gray, K.; Janssens, G.P.J. Storage of Heparinised Canine Whole Blood for the Measurement of Glutathione Peroxidase Activity. Biol. Trace Elem. Res. 2016, 172, 361–363. [CrossRef] 24. Verk, B.; Nemec Svete, A.; Salobir, J.; Rezar, V.; Domanjko Petrič, A. Markers of oxidative stress in dogs with heart failure. J. Vet. Diagn. Investig. 2017, 29, 636–644. [CrossRef] [PubMed] 25. Soumya, R.; Vani, R. CUPRAC-BCS and antioxidant activity assays as reliable markers of antioxidant capacity in erythrocytes. Hematology 2015, 20, 165–174. [CrossRef] [PubMed] 26. Yen, G.C.; Duh, P.D.; Tsai, H.L. Antioxidant and pro-oxidant properties of ascorbic acid and gallic acid. Food Chem. 2002, 79, 307–313. [CrossRef] 27. Cohen, G.; Dembiec, D.; Marcus, J. Measurement of catalase activity in tissue extracts. Anal. Biochem. 1970, 34, 30–38. [CrossRef] 28. Gidske, G.; Sølvik, U.Ø.; Sandberg, S.; Kristensen, G.B.B. Hemolysis interference studies: Freeze method should be used in the preparation of hemolyzed samples. Clin. Chem. Lab. Med. 2018, 56, e220–e222. [CrossRef] [PubMed] 29. Campos, C.; Guzmán, R.; López-Fernández, E.; Casado, Á. Evaluation of the copper(II) reduction assay using bathocuproinedisul- fonic acid disodium salt for the total antioxidant capacity assessment: The CUPRAC-BCS assay. Anal. Biochem. 2009, 392, 37–44. [CrossRef] 30. Rubio, C.; Tvarijonaviciute, A.; Martinez-Subiela, S.; Hernández-Ruiz, J.; Cerón, J.J. Validation of an automated assay for the measurement of cupric reducing antioxidant capacity in serum of dogs. BMC Vet. Res. 2016, 12, 1–8. [CrossRef] [PubMed] 31. Benzie, I.F.F.; Strain, J.J. The Ferric Reducing Ability of Plasma (FRAP) as a Measure of “Antioxidant Power”: The FRAP Assay. Anal. Biochem. 1996, 239, 70–76. [CrossRef] [PubMed] Antioxidants 2022, 11, 424 11 of 11 32. Rubio, C.P.; Martinez-Subiela, S.; Hernández-Ruiz, J.; Tvarijonaviciute, A.; Ceron, J.J. Analytical validation of an automated assay for ferric-reducing ability of plasma in dog serum. J. Vet. Diagnostic Investig. 2017, 29, 574–578. [CrossRef] [PubMed] 33. Arnao, M.B.; Cano, A.; Hernández-Ruiz, J.; García-Cánovas, F.; Acosta, M. Inhibition by L-Ascorbic Acid and Other Antioxi- dants of the 2,20 -Azino-bis(3-ethylbenzthiazoline-6-sulfonic Acid) Oxidation Catalyzed by Peroxidase: A New Approach for Determining Total Antioxidant Status of Foods. Anal. Biochem. 1996, 236, 255–261. [CrossRef] [PubMed] 34. Rubio, C.P.; Hernández-Ruiz, J.; Martinez-Subiela, S.; Tvarijonaviciute, A.; Arnao, M.B.; Ceron, J.J. Validation of three automated assays for total antioxidant capacity determination in canine serum samples. J. Vet. Diagnostic Investig. 2016, 28, 693–698. [CrossRef] 35. Da Costa, C.M.; Dos Santos, R.C.C.; Lima, E.S. A simple automated procedure for thiol measurement in human serum samples. J. Bras. Patol. Med. Lab. 2006, 42, 345–350. [CrossRef] 36. Jocelyn, P.C. Spectrophotometric Assay of Thiols. Methods Enzymol. 1987, 143, 44–67. [CrossRef] [PubMed] 37. Tvarijonaviciute, A.; Tecles, F.; Caldin, M.; Tasca, S.; Cerón, J. Validation of spectrophotometric assays for serum paraoxonase type-1 measurement in dogs. Am. J. Vet. Res. 2012, 73, 34–41. [CrossRef] [PubMed] 38. Erel, O. A new automated colorimetric method for measuring total oxidant status. Clin. Biochem. 2005, 38, 1103–1111. [CrossRef] 39. Rubio, C.P.; Martinez-Subiela, S.; Tvarijonaviciute, A.; Hernández-Ruiz, J.; Pardo-Marin, L.; Segarra, S.; Ceron, J.J. Changes in serum biomarkers of oxidative stress after treatment for canine leishmaniosis in sick dogs. Comp. Immunol. Microbiol. Infect. Dis. 2016, 49, 51–57. [CrossRef] [PubMed] 40. Tatzber, F.; Griebenow, S.; Wonisch, W.; Winkler, R. Dual method for the determination of peroxidase activity and total peroxides- iodide leads to a significant increase of peroxidase activity in human sera. Anal. Biochem. 2003, 316, 147–153. [CrossRef] 41. Alberti, A.; Bolognini, L.; Macciantelli, D.; Caratelli, M. The radical cation of N,N-diethyl-para-phenylendiamine: A possible indicator of oxidative stress in biological samples. Res. Chem. Intermed. 2000, 26, 253–267. [CrossRef] 42. Witko-Sarsat, V.; Friedlander, M.; Capeillère-Blandin, C.; Nguyen-Khoa, T.; Nguyen, A.T.; Zingraff, J.; Jungers, P.; Descamps- Latscha, B. Advanced oxidation protein products as a novel marker of oxidative stress in uremia. Kidney Int. 1996, 49, 1304–1313. [CrossRef] [PubMed] 43. Rubio, C.P.; Tvarijonaviciute, A.; Caldin, M.; Hernández-Ruiz, J.; Cerón, J.J.; Martínez-Subiela, S.; Tecles, F. Stability of biomarkers of oxidative stress in canine serum. Res. Vet. Sci. 2018, 121, 85–93. [CrossRef] [PubMed] 44. Buege, J.A.; Aust, S.D. Biomembranes—Part C: Biological Oxidations. Methods Enzymol. 1978, 52, 302–310. 45. Tiwari, G.; Tiwari, R. Bioanalytical method validation: An updated review. Pharm. Methods 2010, 2, 25. [CrossRef] [PubMed] 46. Kennett, E.C.; Kuchel, P.W. Redox Reactions and Electron Transfer Across the Red Cell Membrane. IUBMB Life 2003, 55, 375–385. [CrossRef] [PubMed] 47. Bielski, B.H.; Cabelli, D.E. Highlights of current research involving superoxide and perhydroxyl radicals in aqueous solutions. Int. J. Radiat. Biol. 1991, 59, 291–319. [CrossRef] [PubMed] 48. Çimen, M.Y.B. Free radical metabolism in human erythrocytes. Clin. Chim. Acta 2008, 390, 1–11. [CrossRef] [PubMed] 49. Chakraborthy, A.; Ramani, P.; Sherlin, H.J.; Premkumar, P.; Natesan, A. Antioxidant and pro-oxidant activity of Vitamin C in oral environment. Indian J. Dent. Res. Off. Publ. Indian Soc. Dent. Res. 2014, 25, 499–504. [CrossRef] [PubMed] 50. Niki, E.; Yamamoto, Y.; Takahashi, M.; Yamamoto, K.; Yamamoto, Y.; Miki, M.; Yasuda, H.; Komuro, E.; Mino, A.M. Free Radical-Mediated Damage of Blood and Its Inhibition by Antioxidants. J. Nutr. Sci. Vitaminol. 1988, 34, 507–512. [CrossRef] [PubMed] 51. Barbosa, N.B.V.; Lissner, L.A.; Klimaczewski, C.V.; Colpo, E.; Rocha, J.B.T. Ascorbic acid oxidation of thiol groups from dithiotreitol is mediated by its conversion to dehydroascorbic acid. EXCLI J. 2012, 11, 604–612. [CrossRef] [PubMed] 52. Jaouad, L.; de Guise, C.; Berrougui, H. Age-related decrease in high-density lipoproteins antioxidant activity is due to an alteration in the PON1’s free sulfhydyl groups. Atherosclerosis 2006, 185, 191–200. [CrossRef] [PubMed] 53. Salam, S.; Arif, A.; Mahmood, R. Thiram-induced cytotoxicity and oxidative stress in human erythrocytes: An in vitro study. Pestic. Biochem. Physiol. 2020, 164, 14–25. [CrossRef] [PubMed]