Arostegui et al. BMC Veterinary Research (2023) 19:33 BMC Veterinary Research https://doi.org/10.1186/s12917-023-03586-4 RESEARCH Open Access Changes in biomarkers of redox status in serum and saliva of dogs with hypothyroidism Luis G. González Arostegui1, Alberto Muñoz Prieto2, Luis Pardo Marín1, Gregorio García López1, Asta Tvarijonaviciute1, Jose Joaquín Cerón Madrigal1 and Camila Peres Rubio3* Abstract Background Hypothyroidism is the most common endocrine disorder diagnosed in dogs, leading to deleterious effects on a dog’s life quality. This study aims to evaluate changes in the redox status in canine hypothyroidism. For this purpose, a comprehensive panel of antioxidants and oxidants biomarkers were measured in serum and saliva of 23 dogs with hypothyroidism, 21 dogs with non-thyroidal illness, and 16 healthy dogs. Among the antioxidants, cupric reducing antioxidant capacity (CUPRAC), ferric reducing ability of plasma (FRAP), Trolox equivalent antioxidant capacity (TEAC), thiol, paraoxonase type 1 (PON-1) and glutathione peroxidase (GPx) were determined in serum and CUPRAC, ferric reducing ability of saliva (FRAS) and TEAC in saliva. The oxidant biomarkers included were total oxidant status (TOS), peroxide-activity (POX-Act), reactive oxygen-derived compounds (d-ROMs), advanced oxidation protein products (AOPP), and thiobarbituric acid reactive substances (TBARS) in serum and AOPP and TBARS in saliva. Results Results showed a significantly higher TEAC, PON-1, GPx, TOS, POX-Act, and d-ROMs, and a significantly lower AOPP in serum of dogs with hypothyroidism. Meanwhile, significantly lower FRAS and AOPP were observed in saliva of dogs with hypothyroidism. Once salivary concentrations were corrected based on their total protein con- centrations, the only analyte showing significant changes was TBARS which was significantly higher in dogs with hypothyroidism. Conclusions Our results show that dogs with hypothyroidism present alterations in the redox status in both serum and saliva. This study should be considered a preliminary study and further research addressing these changes should be made using larger populations. Keywords Antioxidants, Biomarkers, Dogs, Hypothyroidism, Oxidants, Oxidative stress Background *Correspondence: Hypothyroidism is described as a deficiency in the syn- Camila Peres Rubio thesis of thyroid hormones, namely, tetraiodothyro- camila.peres@uab.cat 1 nine (thyroxin, T4) and triiodothyronine (T3), due to an Interdisciplinary Laboratory of Clinical Analysis (Interlab‑UMU), Veterinary School, Campus of Excellence Mare Nostrum, University impairment of the thyroid gland, commonly associated of Murcia, 30100 Murcia, Espinardo, Spain in dogs with idiopathic follicular atrophy or destruction 2 Clinic for Internal Diseases, Faculty of Veterinary Medicine, University of the gland as a consequence of lymphocytic thyroiditis of Zagreb, Heinzelova 55, 1000 Zagreb, Croatia 3 Department of Animal and Food Science, School of Veterinary Science, [1–3]. Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Thyroid hormones have an important association with Barcelona, Spain oxidative status [4]. In physiological conditions, thyroid hormones have positive effects on metabolism, as they © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Arostegui et al. BMC Veterinary Research (2023) 19:33 Page 2 of 11 influence both catabolic and anabolic reactions. How- including antioxidants as, the cupric ion reducing antioxi- ever, these reactions can lead to oxygen consumption, dant capacity (CUPRAC), ferric reducing ability of plasma which is a key factor in the development and synthesis of (FRAP), Trolox equivalent antioxidant capacity (TEAC), reactive oxygen species (ROS). On the other hand, thy- thiol, paraoxonase type-1 (PON-1) and glutathione per- roid hormones also play an important role in the anti- oxidase activity (GPx), and oxidants as, the total oxi- oxidant defense system, as they act as part of this system dant status (TOS), peroxide-activity (POX-Act), reactive being enzymatic and non-enzymatic free radical scaven- oxygen-derived compounds (d-ROMs), advanced oxida- gers [3–10]. tion protein products (AOPP) and TBARS in serum and Markers of oxidative stress associated with hypo- CUPRAC, FRAS, TEAC, AOPP and TBARS in saliva. thyroidism have been studied in humans, showing a decrease in antioxidants and an increase in lipid peroxi- Results dation [4]. Namely, a decrease in superoxide dismutase Patient characteristics activity (SOD), catalase activity, and an overall increase A complete description of the dogs included in the study in thiobarbituric acid reactive substances (TBARS) were is presented in Table 1. There were no statistically signifi- found in red blood cell lysates of humans with hypothy- cant differences between groups related to age or sex. roidism [11–13], and these changes were attenuated after treatment with levothyroxine [12]. Serum In humans, serum has been traditionally used to evalu- Antioxidant status ate changes in the oxidative status of patients with dif- Results for the serum antioxidant biomarkers are shown ferent thyroidal diseases [14–17]. However, saliva is an in Fig. 1. TEAC and PON-1 showed significantly higher easy-to-obtain, stress-free sample that has been used for values in dogs with hypothyroidism compared to dogs the study of different biomarkers in other diseases and with non-thyroid diseases (P = 0.0486 and P = 0.0069, species [18–22]. To the author’s knowledge, the oxidative respectively) and healthy dogs (P = 0.0420 and P = 0.0074, status has not been evaluated in serum and saliva of dogs respectively). GPx activity showed significantly higher with hypothyroidism previously. values in dogs with hypothyroidism compared to healthy We hypothesized that biomarkers of the redox status dogs (P = 0.0344). No significant differences (P > 0.05) could change in serum and saliva of dogs with hypothy- were found between groups in CUPRAC, FRAP, and thiol. roidism. Thus, the objective of this study was to evaluate biomarkers of the redox status in serum and saliva from Oxidant status dogs with hypothyroidism and compare them with dogs Results for the oxidant biomarkers for serum are shown with non-thyroidal illnesses and healthy controls. A com- in Fig. 2. TOS concentrations were significantly higher prehensive panel of redox biomarkers was measured, in dogs with hypothyroidism compared to dogs with Table 1 Detailed characteristics (breed, mean age, and body condition score (BCS) of the different groups included in the study Hypothyroidism (n = 23) Non-thyroid diseased Control group (n = 16) P-value (n = 21) Breed - 7 Mongrel - 8 Mongrel - 11 Beagle - 5 Beagle - 5 Beagle - 2 Yorkshire Terrier - 2 Golden Retriever - 2 French Bulldog - 1 Mongrel - 1 Teckel - 2 Yorkshire Terrier - 1 Greyhound - 1 Spanish Water dog - 1 Basset Hound - 1 Malinois - 1 Dalmatian - 1 Boxer - 1 Neapolitan Mastiff - 1 Pomeranian - 1 Pinscher - 1 Pug - 1 Yorkshire Terrier - 1 Bodeguero Andaluz - 1 Swiss Shepherd - 1 Pomeranian Age, mean (range) 9.78 years (7 – 14) 8.14 years (2 – 12) 7.35 years (5 – 14.6) > 0.05 BCS, mean (range) 4.08 (3 – 5) 3.81 (2 – 5) 3.31 (2 – 4) 0.013 (Hypo- thyroid vs control) Sex (Female /male) 20 males 13 males 13 males > 0.05 3 females 9 females 3 females Arostegui et al. BMC Veterinary Research (2023) 19:33 Page 3 of 11 Fig. 1 Results of antioxidant biomarkers in serum. Cupric reducing antioxidant capacity (CUPRAC); ferric reducing ability of plasma (FRAP); Trolox equivalent antioxidant capacity (TEAC); thiol, paraoxonase type-1 (PON-1), and glutathione peroxidase (GPx) in dogs with hypothyroidism (HT), non-thyroid diseased (NHT) dogs and controls (CT). Asterisks indicate significant differences between groups. *P ≤ 0.05 non-thyroid diseases P = 0.0327) and healthy dogs Concentrations of AOPP before albumin correction (P = 0.0004). In addition, concentrations of POX-Act were significantly lower in dogs with hypothyroidism in and d-ROMs were significantly higher in dogs with comparison to dogs with non-thyroid diseases (P = 0.031) hypothyroidism (P = 0.0003) and P = 0.0066, respec- and healthy dogs (P = 0.0143). However, no significant tively) and dogs with non-thyroid diseases (P = 0.0070) differences were found (P > 0.05) between groups in and P = 0.0280, respectively) in comparison to healthy AOPP and TBARS when results were corrected by albu- dogs. min concentrations. Arostegui et al. BMC Veterinary Research (2023) 19:33 Page 4 of 11 Fig. 2 Results of oxidant biomarkers in serum. Total oxidant status (TOS); peroxide-activity (POX-Act); reactive oxygen-derived compounds (d-ROMs); advanced oxidation protein products (AOPP), thiobarbituric acid reactive substances (TBARS) and AOPP: albumin ratio in dogs with hypothyroidism (HT), non-thyroid diseased (NHT) dogs and controls (CT). Asterisks indicate significant differences between groups. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 Saliva diseases (P = 0.0026) and healthy dogs (P = 0.0158). Antioxidant status No statistical differences (P > 0.05) were found for Results for the salivary antioxidant biomarkers are CUPRAC and TEAC between groups. When the results shown in Fig. 3. FRAS concentrations before protein were corrected by salivary protein, CUPRAC, FRAS, correction were significantly lower in dogs with hypo- and TEAC showed no significant differences (P > 0.05) thyroidism in comparison to dogs with non-thyroid (Fig. 4). Arostegui et al. BMC Veterinary Research (2023) 19:33 Page 5 of 11 Fig. 3 Results of the measurement of antioxidant biomarkers in saliva. Cupric reducing antioxidant capacity (CUPRAC); ferric reducing ability of saliva (FRAS), and Trolox equivalent antioxidant capacity (TEAC) in dogs with hypothyroidism (HT), non-thyroid diseased (NHT) dogs and controls (CT). Asterisks indicate significant differences between groups. *P ≤ 0.05, **P ≤ 0.01 Fig. 4 Ratio between the salivary antioxidants results and salivary protein concentration. Cupric reducing antioxidant capacity (CUPRAC), ferric reducing ability of saliva (FRAS), and Trolox equivalent antioxidant capacity (TEAC) in dogs with hypothyroidism (HT), non-thyroid diseased (NHT) dogs and controls (CT) Fig. 5 Results of oxidant biomarkers in saliva. Advanced oxidation Fig. 6 Ratio between the salivary antioxidants results and salivary protein products (AOPP), and thiobarbituric acid reactive substances protein or microalbumin concentration. Advanced oxidation protein (TBARS) in dogs with hypothyroidism (HT), non-thyroid diseased products (AOPP), and thiobarbituric acid reactive substances (TBARS) (NHT) dogs, and controls (CT). Asterisks indicate significant in dogs with hypothyroidism (HT), non-thyroid diseased (NHT) dogs, differences between groups. *P ≤ 0.05 and controls (CT). Asterisks indicate significant differences between groups. *P ≤ 0.05 Oxidant status correction were significantly lower in dogs with hypo- Results for the salivary oxidant biomarkers are shown in thyroidism in comparison to dogs with non-thyroid dis- Fig. 5 and 6. AOPP concentrations before microalbumin eases (P = 0.0113) and healthy dogs (P = 0.0113). When Arostegui et al. BMC Veterinary Research (2023) 19:33 Page 6 of 11 Table 2 Correlation coefficients (r) between serum and Discussion saliva, and serum and corrected saliva based on its protein To the author’s knowledge, there are no reports about concentrations in dogs biomarkers of the redox status in serum and saliva of Biomarker Serum VS Saliva Serum VS dogs with hypothyroidism. In this study, changes in Corrected various antioxidant and oxidant biomarkers were found Saliva in hypothyroid dogs that could indicate an altered CUPRAC​ -0.0347 -0.1306 redox status in this disease. FRAP -0.1248 -0.1358 An increase in three antioxidant biomarkers (TEAC, TEAC -0.2512 -0.2230 PON-1, and GPx) was found in serum of dogs with TBARS -0.1782 -0.0275 hypothyroidism. The increases in GPx are in line with what was described in humans with Hashimoto’s thy- CUPRAC​Cupric reducing antioxidant capacity, FRAP Ferric reducing ability of plasma, TEAC Trolox equivalent antioxidant capacity, TBARS thiobarbituric acid roiditis [16, 23–25]. GPx is an important selenoprotein reactive substances that scavenges and detoxifies H ­ 2O2, protecting thyroid cells from oxidative damage [26–28]. We hypothesize that the increase in GPx activity could be associated Table 3 Correlation coefficients between serum and salivary by an increased TSH stimulation, as some reports indi- AOPP concentrations, using obtained values and values cate that this hormone can influence GPx activity by corrected by albumin for serum (CSe) and microalbumin for increasing its concentrations [29]. saliva (CSa) The increase in PON-1 in hypothyroid dogs of our Comparison Correlation study differs from the decreases described in humans coefficient with hypothyroidism; in which low PON-1 values are (r) considered a risk of atherosclerosis associated with Serum VS CSe 0.4175 *** this disease [30–33]. Dogs carry cholesterol in HDL Saliva VS CSa 0.7336 where PON-1 is bounded protecting it from oxidation CSe VS Saliva 0.1268 [34] instead of in LDL, as it occurs in humans. There- CSe VS CSa 0.9393 fore, the elevation of cholesterol that usually appears in canine hypothyroidism could imply an increase in Asterisks indicate significant differences *** HDL and subsequently in PON-1. This could explain P ≤ 0.0002 the increase of PON-1 found in dogs with hypothyroid- ism in our study, and this increase in PON-1 could also be one of the reasons why dogs have lower risks of ath- the AOPP results were corrected by salivary microalbu- erosclerosis than humans [35–37]. In humans, TEAC min no significant differences (P > 0.05) were found. No was found to be decreased in various thyroid disorders, statistical differences (P > 0.05) were found for TBARS including multinodular goitre, contrary to our results before protein correction; however, when the results [38]. Since TEAC is an analyte that represents the were corrected by salivary protein concentration TBARS effect of various antioxidants, we believe further studies values were significantly higher in dogs with hypothy- should be made to evaluate if PON-1 and/or GPx influ- roidism in comparison to dogs with non-thyroid diseases ence the concentrations of TEAC. In addition, alpha- (P = 0.0210). tocopherol, one of the molecules measured by TEAC, is known to be transported through lipoproteins which Correlation study are increased in hypothyroidism. Although it was not Correlations between results obtained in serum and evaluated in this study, increases in this antioxidant are saliva, and serum and corrected saliva are found in associated with increases in lipoproteins which could Table 2. Correlations between the results of serum AOPP explain the high TEAC levels found in this study [39]. and corrected serum AOPP based on its albumin concen- On the other hand, in serum, three oxidant biomark- trations, and salivary AOPP and corrected salivary AOPP ers were found to be increased in dogs with hypothy- based on its microalbumin concentrations are found in roidism, namely, TOS, POX-Act, and d-ROMs. TOS Table 3. As shown in Table 3, serum AOPP was corre- measures the total oxidant status, as it is integrated lated to serum AOPP corrected by albumin (r = 0.4175, by different oxidant molecules [40]. In humans, serum P = 0.0006), nonetheless, no significant correlation was TOS concentrations were found to be increased in sub- found between any of the biomarkers measured in both clinical hypothyroidism [41] and Hashimoto’s thyroidi- samples. tis [42]. The increases in POX-Act and d-ROMs could be related to the increase in TOS concentrations, as Arostegui et al. BMC Veterinary Research (2023) 19:33 Page 7 of 11 both oxidants are part of the whole oxidant status of an if the presence of changes found in serum and saliva in organism [40, 43, 44]. dogs with clinical signs compatible with hypothyroidism In our study, serum AOPP concentrations without could possibly help on the suspicion of this disease and albumin correction were decreased in hypothyroid also evaluate if the normalization of these biomarkers patients, however, when values were corrected, no signif- could be a sign of an adequate treatment. icant changes were found, as reported in humans [1, 45]. In general, it is recommended to correct serum AOPP Conclusions concentrations by albumin concentrations, as AOPP are Biomarkers of redox status show changes in serum and carried by oxidized plasma proteins like albumin [46, 47], saliva in canine hypothyroidism compared with both so it could be postulated that the corrected AOPP values healthy and non-thyroid-diseased dogs. Namely, there should be considered for studies. was an increase of antioxidants (TEAC, PON-1, and In saliva, FRAS and AOPP were decreased in hypo- GPx) and oxidants (TOS, POX-Act, and d-ROMs) in thyroid dogs. However, when the salivary biomarkers serum of dogs and an increase in TBARS: protein ratio were corrected by protein, no significant changes were in dogs with hypothyroidism in comparison to healthy found in these analytes. On the other hand, the salivary dogs. However, further studies should be made to con- TBARS: protein ratio increased in dogs with hypothy- firm our results in larger populations of dogs and evalu- roidism. Although this is a topic that should be discussed ate the practical potential of these analytes as biomarkers more deeply in the future, it seems that the results of for this disease. saliva after correction would be more in line with what has been described in humans, since TBARS concentra- Methods tions have been shown to be increased in plasma [23] Study design and erythrocytes [48], possibly due to an increase in lipid This case–control study involved client-owned dogs peroxidation that is associated with the hyperlipidae- attending different Veterinary Clinics of Murcia Region mia consistently seen in hypothyroidism [49, 50]. In our (Spain) between March 2021 and May 2022. study, no significant changes were observed in TBARS Dogs were divided into three groups. A group of dogs serum or saliva without correction in dogs with hypothy- with hypothyroidism (n = 23) following the subsequent roidism. Therefore, it could be suggested that for TBARS, criteria: (1) being adult dogs (over a year old), (2) not the correction of salivary results by protein could be presenting other diseases, (3) not having received any more sensitive to detect the oxidant overproduction treatment six months before diagnosis, (4) presence of occurring in dogs with hypothyroidism, which is evi- symptoms related to the disease that justify the specific denced by the increases in TOS, POX-Act, and d-ROMs diagnosis tests (lethargy, tiredness, weight gain) [55] and in serum. However, further studies should be performed (5) having the disease confirmed through specific diag- using a larger number of animals and ideally also in dogs nostic tests (determination of T4 and thyroid stimulating going through levothyroxine replacement therapy to con- hormone (TSH)) [56]. Secondly, a group of non-thyroid firm the results of our report. The divergences observed diseased dogs (n = 21) with the same criteria mentioned between serum and salivary TBARS are in line with the previously was included, except for having negative lack of correlation shown, as well as in the other serum results for specific thyroid diagnostic tests [56], and lastly, and saliva biomarkers of redox status of our study, as a control group of healthy adult dogs (n = 16) that were divergencies in the composition of both fluids have been ruled out of any disease were included. In all cases, the described in other reports in dogs [51], pigs [52] and nutritional status was reported based on the body condi- humans [53]. tion score (BCS) 5-scale (1-thin; 2-underweight; 3-opti- This report should be considered a preliminary study, mal (lean); 4-overweight; 5-obese) [57]. and, as previously mentioned, further trials with larger Samples were obtained after 12 h of fasting. Blood populations should be made to evaluate the changes in samples were obtained by jugular venipuncture after the the oxidative status analytes used in this study through- saliva collection. In brief, saliva was obtained by intro- out treatment. Also, an evaluation of the biomarkers in ducing a sponge into the dog’s mouth for one to two other sample types, such as whole blood and red blood minutes until wet and then placed into a Salivette tube cell lysates should be performed, since in a previous study (Salivette, Sarstedt, Aktiengesellschaft & CO., Nüm- these samples provided additional interesting informa- brecht, Germany). All tubes with blood and saliva were tion about the redox status in dogs [54]. In addition, fur- stored with ice until taken to the laboratory, where they ther studies should be made to evaluate the applicability were centrifuged at 3000 × g for 20 min at 4 ºC. Obtained of these biomarkers in the diagnosis of this disease and serum and saliva were collected and transferred into treatment monitorization. These studies should assess 1.5 mL tubes and stored at -80ºC until analysis. All Arostegui et al. BMC Veterinary Research (2023) 19:33 Page 8 of 11 samples were obtained by the clinicians and were sent to Evaluation of the POX-Act assay was based on the our laboratory for further analysis. None of the dogs used determination of total peroxides through a peroxide- in this study showed any clinical signs of periodontitis. peroxidase reaction using tetramethylbenzidine as the Informed consent was taken from the dog owners. chromogenic substrate [44]. Determination of POX-Act was measured following a validated method for human Assays sera [44]. Results are expressed in µmol/L. POX-Act was Antioxidant status measured in serum but could not be measured in saliva. The determination of the CUPRAC assay was based on Measurement of the d-ROMs assay was based on the the reduction of ­Cu2+ into ­Cu1+ by the nonenzymatic reaction of the sample in an acidic medium in the pres- antioxidants in the sample [58]. Evaluation of CUPRAC ence of N,N,-diethyl-para-phenylenediamine (DEPPD), was made following the procedure previously validated and it was made following a previously described method for use in serum of dogs [59]. Results are expressed in [43]. Results are expressed in Carratelli Units (U.CARR). millimoles per liter (mmol/L). CUPRAC was measured in d-ROMs was measured in serum but could not be meas- serum and saliva. ured in saliva. Determination of the FRAP/FRAS assay was based on Determination of AOPP was based on oxidized albu- the reduction of ferric-tripyridyltriazine (­Fe3+-TPTZ) to min and di-tyrosine containing cross-linked proteins, as the ferrous (­Fe2+) form [60] by the sample. Its determi- previously described [47], and measured in canine serum nation was made following previously described methods [70]. Results are expressed in µmol/L. AOPP was meas- [60, 61]. Results are expressed in mmol/L. This assay was ured in serum and saliva. measured in serum (FRAP) and saliva (FRAS). Determination of TBARS is based on the reaction Measurement of TEAC was based on the assay of the sample to a Trichloroacetic acid, thiobarbituric described by Arnao et al. [62] that has been used previ- acid, and N hydrochloric acid in heated conditions [71]. ously in canine serum samples [63]. Its principle is based TBARS was measured following a previously described on the enzymatic generation of 2,2’-azino-bis(3-ethylb- method [71] using a microplate reader (Powerwave XS, enz-thiazoline-6-sulfonic acid) (ABTS) radical and its Biotek Instruments). Results are expressed in µmol/L. reduction by non-enzymatic antioxidants present in the TBARS was measured in serum and saliva. sample [62]. Results are expressed in mmol/L. TEAC was measured in serum and saliva. Proteins The determination of total thiol is based on the reac- Salivary protein concentration was measured using a tion of thiols within the sample with 5,5’-dithiobis-(2-ni- commercially available colorimetric kit (protein in urine trobenzoic acid) (DTNB). The assay used was performed and CSF, Spinreact, Spain) as done in previous studies according to previously described methods for serum [72]. Results are expressed in milligrams per milliliter samples [64, 65]. Results are expressed in micromoles per (mg/mL). liter (µmol/L). Thiol was measured in serum but could Microalbumin was measured in saliva using a commer- not be detected in saliva. cially available (Microalbumin spectrophotometry kit, Measurement of PON-1 was based on the hydroly- REF 22,324, Biosystems, Barcelona, Spain). Results are sis of phenyl acetate into phenol and it was determined expressed in milligrams per deciliter (mg/dL). as previously described in canine serum [66]. Results Salivary values for all assays were corrected accord- are expressed in units per milliliter (IU/ml). PON-1 was ing to total protein concentrations found on each saliva measured in serum but could not be measured in saliva. sample as performed previously [73], meanwhile, salivary Measurement of GPx was based on the use of com- AOPP values were corrected using salivary microalbu- mercially available assays following the manufacturer’s min concentrations, as well as serum AOPP values were instructions (Randox, Crumlin, UK), as used in previous corrected using serum albumin concentrations, accord- studies [67, 68]. Results are expressed in IU/ml. GPx was ing to previous reports [46, 47]. measured in serum but could not be measured in saliva. Statistical analysis Oxidant status Data were analyzed using GraphPad Prism software Determination of TOS was based on the assay described (GraphPad Software Inc., version 9.3 for MacOS). The by Erel [40] that was previously used in dogs [69]. Its Shapiro–Wilk test was first used to assess whether the reaction is based on the ability of oxidants in the sam- results for each analyte were normally distributed. Dif- ple to oxidize Fe 2+-o-dianisidine complex to ­Fe3+ [40]. ferences in the concentrations between groups, when Results are expressed in µmol/L. TOS was measured in data were normally distributed, were assessed using serum but could not be detected in saliva. a One-way ANOVA followed by Tukey’s multiple Arostegui et al. BMC Veterinary Research (2023) 19:33 Page 9 of 11 comparisons range test, and non-normally distributed Declarations data were assessed with a One-way ANOVA followed Ethics approval and consent to participate by the Kruskal–Wallis test. Correlation between saliva This project has been approved by the Murcia University Ethics Commit- and serum was assessed using Pearson’s and Spearman’s tee with the number CEEA 288/2017. Informed consent was taken from all correlation tests, according if the data were normally dog owners. All experiments were performed in accordance with relevant guidelines in regulations, as well as with ARRIVE guidelines for animal distributed or not. Statistical differences were consid- experimentation. ered for P-values < 0.05. Parametric data is shown as mean ± standard deviation (SD), meanwhile, non-para- Consent for publication Not applicable. metric data is shown as median and interquartile range (IQR). Competing interests None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper. Abbreviations T4 Tetraiodothyronine T3 Triiodothyronine Received: 28 September 2022 Accepted: 23 January 2023 ROS Reactive oxygen species SOD Superoxide dismutase activity TBARS Thiobarbituric acid reactive substances CUPRAC​ Cupric reducing antioxidant capacity FRAP Ferric reducing ability of plasma References FRAS Ferric reducing ability of saliva 1. Cheserek MJ, Wu G-R, Ntazinda A, Shi Y-H, Shen L-Y, Le G-W. Association PON-1 Paraoxonase type-1 Between Thyroid Hormones, Lipids and Oxidative Stress Markers in Sub- GPx Glutathione peroxidase activity clinical Hypothyroidism / Povezanost Izme\U Tireoidnih Hormona, Lipida TOS Total oxidant status I Markera Oksidativnog Stresa U SubkliniĉKoj Hipotireozi. J Med Biochem. POX-Act Peroxide-activity 2015;34:323–31. d-ROMs Reactive oxygen-derived compounds 2. Jaiswal M, Tiwari A, Gupta D, Pavan Kumar M, Singh B, Maravi P, et al. 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L-GA, A-MP, and L-PM Physiol Rev. 2006;86:435–64. collected the data. L-GA, G-GL, and CPR were involved in the chemical analy- 11. Reddy S, Swapna LA, Ramesh T, Singh TR, Pradeep K. Influence of ciga- sis. L-GA, A-MP, AT, JJC, and CPR were involved in the data interpretation. L-GA rette smoking on blood and salivary super oxide dismutase levels among drafted the manuscript and A-MP, AT, JJC and CPR critically read and edited smokers and non-smokers. J Investig Clin Dent. 2012;3:298–303. the manuscript. The author(s) read and approved the final manuscript. 12. Masullo LF, Magalhães RA, Lemes RPG, de Almeida Filho TP, de Castro MF, Maia Filho PA, et al. Levothyroxine Replacement Improves Oxidative Status in Funding Primary Hypothyroidism. Front Endocrinol (Lausanne). 2018;9 November:1–5. This study was supported by the Seneca Foundation-Agency of Science and 13. Dumitriu L, Bartoc R, Ursu H, Purice M, Ionescu V. Significance of high Technology of the Region of Murcia through the Subprogram to the Scientific levels of serum malonyl dialdehyde (MDA) and ceruloplasmin (CP) in Leadership and the Transition to the Independent Investigation (20649/JLI/18). hyper- and hypothyroidism. Endocrinologie. 1988;26:35–8. LG-A was funded by 21453/FPI/20, Fundación Séneca, Región de Murcia, 14. Ates I, Yilmaz FM, Altay M, Yilmaz N, Berker D, Güler S. The relationship Spain. AM-P was funded by the University of Murcia through a post-doctoral between oxidative stress and autoimmunity in Hashimoto’s thyroiditis. grant (Margarita Salas) within the mark of “Ayudas en el marco del Programa Eur J Endocrinol. 2015;173:791–9. de Recualificación del Sistema Universitario Español” through the European 15. Torun AN, Kulaksizoglu S, Kulaksizoglu M, Pamuk BO, Isbilen E, Tutuncu Union Next Generation Funds. CP-R has a post-doctoral fellowship “Juan de la NB. 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