Rubio et al. BMC Veterinary Research (2019) 15:144 https://doi.org/10.1186/s12917-019-1875-z RESEARCH ARTICLE Open Access Biomarkers of oxidative stress in saliva in pigs: analytical validation and changes in lactation Camila Peres Rubio1†, Eva Mainau2†, José Joaquín Cerón1*, Maria Dolores Contreras-Aguilar1, Silvia Martínez-Subiela1, Elena Navarro2, Fernando Tecles1, Xavier Manteca2* and Damian Escribano2 Abstract Background: Biomarkers of oxidative stress in pigs have been measured in serum/plasma samples. However, blood collection in pigs can be highly stressful to the animals. Saliva is a biological fluid with several advantages in pigs over blood, since it can be easily collected without stress to the animals, being therefore an ideal sample in this species. The objective of this study was the validation of assays for the evaluation of oxidative stress status in saliva of pigs. For this purpose, three assays commonly used to evaluate the total antioxidant capacity (TAC): trolox equivalent antioxidant capacity (TEAC), cupric reducing antioxidant capacity (CUPRAC), and ferric reducing ability of plasma (FRAP)), one individual antioxidant (uric acid) and two assays to evaluate oxidant concentrations (advanced oxidation protein products (AOPP) and hydrogen peroxide (H2O2)) were measured and validated in porcine saliva. In addition, the possible changes of these assays in sows’ saliva during lactation were be studied. Results: The methods had intra- and inter-assays coefficient of variation lower than 15%. They also showed an adequate linearity and recovery, and their detection limits were low enough to detect the analytes in saliva of pigs. Overall the analytical validation tests showed that the assays used in our study are valid and reliable for the evaluation of oxidative stress in porcine saliva. In addition, it was observed that these salivary biomarkers can change in a situation of oxidative stress such as lactation in sows. Conclusions: All assays for salivary biomarkers of oxidative stress evaluated in this study have demonstrated a high analytical accuracy and low imprecision. In addition, it has been observed that these biomarkers showed significant changes in a situation of oxidative stress such as lactation in sows. Therefore, this study opens a new possibility of using saliva as a non-invasive sample to evaluate oxidative stress in pigs. Keywords: Antioxidant, Oxidant, Reactive oxygen species, Saliva, Sows Background A ROS accumulation results in oxidative damage to lipids, Oxidative stress is a term used to indicate an imbalance proteins and DNA and consequently tissue injury, there- between the production of reactive oxygen species (ROS) fore the oxidative stress is involved in pathogenesis in the organism and the ability of the antioxidant mole- of many diseases. In pigs, oxidative stress has been linked cules to neutralize them [1]. ROS formation leads to the to several diseases and clinical conditions such as experi- accumulation of proinflammatory substances, which fur- mental endotoxemia or septic shock [3], respiratory infec- ther contributes to more generation of these oxidants [2]. tions [4], porcine reproductive and respiratory syndrome (PRSS) [5], and skin disease [6]. ROS accumulation can * Correspondence: jjceron@um.es; Xavier.Manteca@uab.cat also induce damage in the intestinal tissue, which can fa- † Camila Peres Rubio and Eva Mainau contributed equally to this work. cilitate bacterial translocation and compromise the intes- 1 Interdisciplinary Laboratory of Clinical Analysis Interlab-UMU, Faculty of tinal barrier integrity in pigs [2]. Veterinary Medicine, Regional Campus of International Excellence ‘Campus Mare Nostrum’, University of Murcia, Espinardo, Murcia 30100, Spain In addition to diseases, changes in oxidative stress bio- 2 Department of Animal and Food Science, School of Veterinary Science, markers in pigs can occur at different situations during their Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Rubio et al. BMC Veterinary Research (2019) 15:144 Page 2 of 7 productive period which are associated with variations in the Table 1 Mean, standard deviation (SD) and intra- and inter- oxidant balance of the animals. For example, ROS increase assay coefficients of variation (CVs) in each analyte during transportation, resulting in an oxidative stress [7–9]. concentrations of three saliva samples of pigs (A, low The weaning period also has been linked with oxidative stress concentrations; B, medium concentrations; C, high producing an increase of ROS [10]. In addition, the serum concentrations) levels of ROS and thiobarbituric acid reactive substances Intra-assay Inter-assay (TBARS) are higher during gestation (days 90 and 109) and Mean SD CV (%) Mean SD CV (%) lactation (days 1 and 3) than in early gestation (day 10) [11]. TEAC (mmol/L) In addition to the above mentioned biomarkers of oxida- A 0.06 0.00 6.32 0.06 0.01 10.76 tive stress, total antioxidant capacity (TAC) assays, such as B 0.15 0.00 2.40 0.14 0.01 4.35 the ferric reducing ability of plasma (FRAP) has also been C 0.31 0.01 1.78 0.30 0.01 3.56 measured in serum of pigs in studies mainly related with CUPRAC (mmol/L) the evaluation of different dietary supplementations [12, 13]. Moreover, Trolox equivalent antioxidant capacity (TEAC), A 0.04 0.00 3.03 0.04 0.01 14.81 another TAC assay, has been used to evaluate TAC in B 0.11 0.00 0.82 0.11 0.00 3.11 plasma of piglets undergoing weaning-induced stress [14]. C 0.24 0.00 0.31 0.25 0.00 2.02 To the authors' knowledge, all these reports regarding bio- FRAP (mmol/L) markers of oxidative stress in pigs have measured them in A 0.09 0.01 6.37 0.09 0.00 4.25 serum/plasma samples. However, blood collection in pigs B 0.23 0.00 1.43 0.23 0.01 3.17 can be highly stressful to the animals since it implies animal immobilization, and how this stress can affect the levels of C 0.40 0.02 5.11 0.40 0.01 3.28 the different oxidant biomarkers is unknown. Saliva is a bio- Uric acid (mg/dL) logical fluid with several advantages in pigs over blood, since A 0.11 0.01 4.80 0.09 0.01 12.59 it can be easily collected without stress to the animals, being B 0.54 0.01 2.27 0.52 0.01 2.63 therefore an ideal sample in this species. Although bio- C 1.65 0.01 0.79 1.65 0.02 1.25 markers of oxidative stress have been widely measured in sal- AOPP (μmol/L) iva in humans [15–17], to the authors’ knowledge they have not been previously measured in porcine saliva. A 28.06 0.50 1.79 24.08 2.00 8.31 The objective of this study is the validation of assays for B 72.84 1.16 1.59 66.49 8.95 13.46 the evaluation of oxidative stress status in saliva of pigs. In C 213.02 3.23 1.52 185.59 21.25 11.45 addition, the possible changes in oxidative stress bio- H2O2 (μmol/L) markers in sows’ saliva during lactation, which is a period A 9.74 0.69 7.14 8.41 1.21 14.34 in which variations in oxidative stress are known, will be B 14.58 0.48 3.27 13.58 1.50 11.07 studied. For this purpose, three assays commonly used to evaluate the total antioxidant capacity (TAC) (TEAC, cu- C 19.62 1.06 5.40 18.40 0.86 4.66 pric reducing antioxidant capacity (CUPRAC), and FRAP), one individual antioxidant (uric acid) and two assays to Changes of the biomarkers in saliva during lactation evaluate oxidant concentrations (advanced oxidation pro- Figure 1 shows the antioxidant and oxidant concentrations tein products (AOPP) and hydrogen peroxide (H2O2)) will in saliva of sows during lactation. All salivary biomarkers of be included in this report. oxidative stress analyzed in our study were higher at first day of lactation and then decreased during the following Results days of lactation. Salivary TEAC, CUPRAC, FRAP, AOPP Analytical validation and H2O2 were significantly lower at day 9 (P ≤ 0.01) and Intra- and inter-assay coefficients of variation (CVs) were 20 (P ≤ 0.0001) than at day 1 of lactation. Salivary uric acid below 8 and 15%, respectively for all the assays evaluated decreased significantly at day 20 (P ≤ 0.0001) of lactation (Table 1). The mean accuracy assessed by the recovery study compared to day 1. in saliva samples was 90% for TEAC, 98% for CUPRAC, The values of TEAC in saliva were 2.0 and 2.9-fold 89% for FRAP, 97% for uric acid, 70% for AOPP and 83% for lower at 9 and 20 day of lactation, respectively, than H2O2. Linearity under dilution in saliva samples resulted in those of the first day. Salivary CUPRAC and FRAP de- linear regression equations with correlation coefficients creased 1.6 and 1.7-fold, respectively, at day 9, and 2.2 higher than 0.99 in all cases. The limit of detection (LOD) and 2.5-fold, respectively at day 20. The concentrations was 0.09 mmol/L for TEAC, 0.003 mmol/L for CUPRAC, of uric acid in saliva were 2.8 and 3.7-fold lower in the 0.031 mmol/L for FRAP, 0.031 mg/dL for uric acid, 9th and 20th day of lactation, respectively, than the first 3.67 μmol/L for AOPP and 0.001 μmol/L for H2O2. day. Salivary AOPP were 2.1 and 3.4-fold lower at day 9 Rubio et al. BMC Veterinary Research (2019) 15:144 Page 3 of 7 Fig. 1 Results for saliva concentrations of trolox equivalent antioxidant capacity (TEAC), cupric reducing antioxidant capacity (CUPRAC), ferric reducing ability of plasma (FRAP), uric acid, advanced oxidation protein products (AOPP) and hydrogen peroxide (H2O2) in sows during the first, 9th and 20th day of lactation (median with interquartile range). Asterisks indicate significant differences between the first day of lactation. * P ≤ 0.01, ** P ≤ 0.0001 and 20 of lactation, respectively. The H2O2 concentra- antioxidant status in saliva [19]. Finally, two oxidant bio- tions in the saliva of the sows were 107 and 216-fold markers were included: AOPP which is a marker of pro- lower in the 9th and 20th day of lactation, respectively, tein oxidation [20] and H2O2 which is a product of than the first day. superoxide anion [21]. The analytical validation tests for assay sensitivity, re- Discussion producibility, and accuracy showed that the assays used To the best of authors’ knowledge, no validation studies in our study are valid and reliable for determination of of assays for measurement of oxidative stress in saliva of biomarkers of oxidative stress in porcine saliva. They pigs have been performed. We included in the panel of were precise, linear, accurate and reproducible, which is biomarkers three analytes that measure the antioxidant in accordance with previous validation studies with oxi- capacity in a global way, such as TEAC, CUPRAC and dative stress markers in human saliva [22]. FRAP. It is recommended to measure these analytes to- All assays evaluated in this study were automated. The gether since they show different changes depending of automated assays presented here have considerable advan- the physiopathological situation [18]. We also included tages over the manual ones: set-up time is reduced which an analyte, the acid uric, that in humans is indicated as makes possible a hundreds of measurements within time one of the more appropriate biomarkers to evaluate the periods ranging from minutes to hours, require reduced Rubio et al. BMC Veterinary Research (2019) 15:144 Page 4 of 7 volume of sample and reagents, operator attendance is of oxidative stress occurs in the early lactation, the balance not needed once the samples are placed in the automated would be tried to be reestablished. analyser and operator error is reduced leading to a higher Based on the results of our experimental model, TEAC precision and accuracy [23]. Although these assays are would be the recommended TAC assay to evaluate changes adapted to automated analysers, they could be used in of antioxidant capacity in saliva of sows in lactation, since other formats such as ELISA plates or in manual spectro- showed higher changes than CUPRAC and FRAP between photometric measurements. the samples taken at the beginning and end of the lactation, It is known that in peripartum, particularly the deliv- and also showed significant changes between the day 1 of ery, and early lactation, energy and consequently oxygen lactation and the other samples times, contrarily to uric acid. are extremely required. The rapid differentiation of Therefore, a profile including TEAC as a biomarker of anti- secretory parenchyma, intense mammary gland growth, oxidant status and H2O2 and AOPP as oxidant biomarkers and the onset of copious milk synthesis and secretion might be used for an evaluation of oxidative stress status in are accompanied by high metabolic demand and ele- saliva of sows during the period of lactation. However, fur- vated requirements for tissue oxygen [24]. This physio- ther studies evaluating the behaviour of these biomarkers in logical situation can lead to an oxidative stress state due saliva samples in different situations of stress or diseases to the overproduction of oxidants [25]. In this regard, should be made. there are clinical and experimental evidences that in the Saliva is predominantly composed by watery fluid, periparturient period in sows there is an antioxidant/oxi- however also contains a complex mixture of proteins, dant imbalance [11, 26]. This lead us to use the lactation ions and other organic compounds originated from sal- time as an experimental model to evaluate the possible ivary glands and blood [30]. Its availability, easy collec- changes in analytes of our study in situations of oxida- tion and possibility of repeated non-invasive sampling tive stress. makes it ideal for screening, diagnosis, or monitoring of Our results showed increased antioxidant and oxidant many diseases [31]. Because of that, salivary markers are concentrations in saliva of sows at the first day of lactation very attractive and are becoming a promising tool for which decreased during the 20 days of lactation. It is possible the research. In addition, the collection of saliva leads to that the overproduction of oxidants and increased response minimal discomfort to the animal, being considered to of the antioxidant system have been induced not only by be an ideal material for evaluating the stress condition in early lactation but also due to farrow. Results of this study pigs [32]. This study opens the possibility of use saliva to were in agreement with a previous report [27] which de- evaluate the oxidative status in pigs. It would be of inter- scribed increased concentrations of various oxidant com- est to perform further studies about how oxidative bio- pounds in serum of sows such as TBARS, markers change in saliva in different physiological 8-hydroxy-deoxyguanosine (8-OHdG), and ROS during early conditions or situations of stress or diseases, and also to lactation. These oxidants reached the highest values at first evaluate if those sows with higher values of antioxidant day of lactation and decreased progressively between the and lower values of oxidant biomarkers in saliva at day 1 third and 21th day of lactation with exception of 8-OHdG of lactation can have a higher performance as described that decreased from day 7 and remained with the same con- previously in serum [16]. centrations at day 21 [11]. Similar results with increases in antioxidant and oxidant Conclusions biomarkers in serum at the beginning of lactation were It can be concluded that the panel of assays for 6 bio- also found in other species. In cows it was reported that markers of oxidative stress in saliva of pigs that have plasma FRAP levels were high immediately, and 1 and 2 been evaluated in this study demonstrated a high analyt- days after parturition and decreased at 6 and 12 days after ical accuracy and low imprecision, and therefore are delivery [28]. In addition, they indicated an increase in valid from an analytical point of view. In addition, it has protein oxidation at beginning of lactation. Also a report been observed that these biomarkers can change in a [29] showed that serum TEAC in cows was higher at be- situation of oxidative stress such as lactation in sows. ginning of calving when compared with two weeks after. This study opens a new possibility of using saliva as a In our study, the antioxidants and oxidants evaluated non-invasive sample to evaluate oxidative stress in pigs. had a similar evolution during the 20 days of lactation, which could imply that an increase in antioxidants is pro- Methods duced in order to compensate the overproduction of oxi- Spectrophotometric methods dant compouds that occur during this period. An increase TEAC in the antioxidant response in order to reach a balance be- This assay is based on the enzymatically generation of 2,2′-azi- tween antioxidant and oxidant compounds has been de- no-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical scribed in lactation [28]. Which means that, even a state [33]. The assay measures the ability of the non-enzymatic Rubio et al. BMC Veterinary Research (2019) 15:144 Page 5 of 7 antioxidants present in the sample in reducing the preformed Reproducibility radical. Its disappearance was estimated by decrease in absorb- Intra-assay precision was determined by the analysis of ance at 700 nm and Trolox (6-hydroxy-2,5,7,8-tetramethyl- three saliva samples (one with high, one with medium, chroman-2-carboxylic acid), an α-tocopherol analogue, was and one with low analyte concentration) five times in the used as standard. The assays results were expressed in milli- same analytical run (same day). Inter-assay precision was moles of Trolox equivalents per liter. determined by the analysis of the same samples on five different days within 1 week. The CV was determined by dividing the SD of the parallel measurements by their CUPRAC mean and then multiplied by 100. CUPRAC assay is based on the generation of a complex con- taining Cu2+ and one chelating agent, in this case the batho- cuproinedisulfonic acid disodium salt, and its reduction to Accuracy Cu1+ by the non-enzymatic antioxidants present in a sample Linearity under dilution and spiking recovery were used to [34]. Results were compared with a standard curve achieved assess accuracy. To linearity, two saliva samples were serially with Trolox and were also expressed in millimoles of Trolox diluted with ultrapure water and assayed. The results were equivalents per liter. compared with those expected by linear regression analysis. To spiking recovery, two saliva samples containing different analyte concentrations were selected and mixed at different FRAP percentages. The percentages of recovery were calculated for The FRAP measurement is based on the assay described each dilution as (observed result/expected result) × 100. by Benzie and Strain [37]. A reaction mixture containing ferric-tripyridyltriazine (Fe3+) is reduced to the ferrous (Fe2+) form by the non-enzymatic antioxidants provided Assay sensitivity by the sample. Ferric chloride hexahydrate (FeCl3·6H2) so- The assay sensitivity was calculated based on the LOD. lution was used to produce a standard curve and compare It was calculated as 2 times the SD above the mean with sample results that were expressed in millimoles of blank sample value, which was obtained from 20 repli- Fe2+ equivalents per liter. cate measurements of the assay buffer or water. Biomarkers performance Uric acid Animals and sampling The uric acid concentrations were measured according The experimental protocol was approved by the Ethical Com- to the manufacturer’s instructions of a commercially mittee of the Autonomous University of Barcelona (UAB). available spectrophotometric method (Beckman) based A total of 14 multiparous (Landrace × Duroc) sows on a previously described assay [38]. around farrowing were included in the study. They were subjected to a clinical examination prior to and through- AOPP out the study, and no clinical signs of disease were Concentrations of AOPP were determined according a found. All of them were under the same local conditions. previously described method [35]. The assay was cali- They were allocated in farrowing crates with regulated brated with Chloramine-T solutions that absorb at 340 nm temperature (approximately 20 °C). The lights in the in the presence of potassium iodide in acidic conditions. room were on from 7 am to 5 pm. The sows ate the same diet at 7 am and 3 pm (a total of 2.6 kg) and had water ad libitum. These sows were from a commercial H2O2 farm (l’Heura S.L., Santa Perpetua de Mogoda, Barce- The H2O2 assay was based on the method described by lona, Spain) and were maintained in their productive Rhee et al. [36]. In this assay, 3,5,3′5′-tetramethylbenzi- system after the experiment. dine (TMB) reacts with horseradish peroxidase (HRP) and The sows were induced to farrow by using Cloproste- H2O2 and a TMB cation free radical is originated. Further nol 0.092 mg/ml (1 ml at 7 am and other at 11 am) at oxidation of this free radical produces the diimine, a day 113 of gestation. Treatments and manual interven- yellow-colored oxidation product with maximal absorb- tions during farrowing were performed by the same per- ance at 450 nm. H2O2 solution was used to produce a cali- son following the usual routine of the farm. bration curve and compare with sample results. Saliva samples were taken at day 1, 9 and 20 of lactation using Salivette® tubes (Sarstedt AG& Co., Germany), contain- Analytical validation ing a cotton swab which was clipped through a Kocher clip, For the analytical validation of the assay, the following and was kept in the sows’ mouth during 1–2 min. The cot- parameters were determined: ton swab was then placed in the tube and centrifuged at Rubio et al. BMC Veterinary Research (2019) 15:144 Page 6 of 7 6000 rpm for 13 min. Finally, saliva samples were stored in Received: 24 January 2019 Accepted: 18 April 2019 Eppendorf tubes and frozen at − 80° until analysis. Statistical analysis References 1. Sordillo LM, Aitken SL. Impact of oxidative stress on the health and immune Analysis were performed using routine descriptive statistical function of dairy cattle. 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