Contreras-Aguilar et al. BMC Veterinary Research (2019) 15:187 https://doi.org/10.1186/s12917-019-1933-6 RESEARCH ARTICLE Open Access Changes in saliva analytes in equine acute abdominal disease: a sialochemistry approach María Dolores Contreras-Aguilar1, Damián Escribano1,2, Silvia Martínez-Subiela1, María Martín-Cuervo3, Elsa Lamy4, Fernando Tecles1* and Jose Joaquín Cerón1 Abstract Background: The biochemical components of saliva can change in certain pathologies in horses, for example in acute abdominal disease. The aim of this study was (1) to evaluate if a panel of biochemical analytes usually used in serum can be measured in saliva of horses and (2) to study the possible changes of these biochemical analytes in saliva of horses affected by acute abdominal disease. A panel of 23 analytes was analytically validated in saliva of horses and possible changes in these analytes in a pilot study with six healthy horses and six horses with acute abdominal disease were evaluated. The analytes with significant changes were then evaluated in a larger population of 20 healthy and 37 diseased horses. Results: Seven analytes showed significant increases in the pilot study which were confirmed in the larger population. The analytes which showed significant changes, and their median fold increase and significance shown in the larger population were salivary γ-glutamyl transferase (gGT, 2.3 fold, P = 0.001), creatine kinase (CK, 6.2 fold, P < 0.001), urea (2.3 fold, P = 0.001), total bilirubin (2.6 fold, P < 0.001), total proteins (3.2 fold, P < 0.001), phosphorus (P, 4.5 fold, P < 0.001) and alpha-amylase (sAA, 8.5 fold, P < 0.001). Total proteins, P and sAA showed sensitivities higher than 70% at their optimal cut-off points and a specificity of 100% in differentiating between healthy horses and those with acute abdominal disease. Conclusions: A panel of 23 biochemical analytes can be measured in saliva of horses, where gGT, CK, urea, total bilirubin, total protein, P and sAA levels are raised in horses with acute abdominal disease. Keywords: Biomarkers, Colic, Horse, Saliva, Sialochemistry Background (ALP), lactate dehydrogenase (LDH) and creatine kinase Saliva mainly consists of water, which represents up to (CK), among others [3–8]. 99% of its total volume. In addition, studies on human The composition of saliva can be affected by systemic dis- and veterinary species have reported that saliva also orders [9]. Due to this, there has been increased interest over contains compounds such as phosphorus (P), lactate, recent years in the use of saliva as a diagnostic fluid. In cholesterol, fatty acids, glucose, hormones such as addition, saliva has some advantages over blood since it is cortisol, triglycerides, urea and uric acid [1, 2]. In easily collected by non-invasive, non-stressful procedures addition, saliva has enzymes such as salivary alpha- [10, 11] and by non-trained personnel with a minimum of amylase (sAA), lipase, carbonic anhydrase, adenosine material. For these reasons, a new term in human medicine, deaminase (ADA), aspartate aminotransferase (AST), "sialochemistry", has been created, which consists of the alanine aminotransferase (ALT), alkaline phosphatase measurement of analytes included in the routine serum biochemistry profiles in saliva [12, 13]. In animal spe- * Correspondence: ftecles@um.es 1 cies, saliva analytes have been studied in different situations. Interdisciplinary Laboratory of Clinical Analysis (Interlab-UMU), Veterinary School, Campus of Excellence Mare Nostrum, University of Murcia, Campus For example, total esterase (TEA), cholinesterase (ChE) and de Espinardo s/n, 30100 Espinardo, Murcia, Spain ADA increase in pigs affected by an acute stress or by a Full list of author information is available at the end of the article © 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. Contreras-Aguilar et al. BMC Veterinary Research (2019) 15:187 Page 2 of 9 pathologic condition such as lameness [4, 5]. In The healthy population (n = 6) was composed of horses, sAA and cortisol increase in acute abdominal horses admitted for castration or routine health check in disease, acting as possible pain-induced stress bio- the Veterinary Teaching Hospital of the University of markers [3]. In addition, in a proteomic study [14], Extremadura and of privately owned horses from a acute phase proteins in saliva were observed to stable in the province of Almería (Spain) (Table 1). The change in horses with this disease. However, to the healthy horses showed no clinical signs of abdominal authors’ knowledge, other routine biochemistry ana- pain [15] during the physical examination. In addition, lytes have not been measured in saliva in horses with the heart rate (HR) and respiratory rate (RR) were within acute abdominal disease, so their potential utility as normal limits and they had no haematological or biomarkers is unknown. biochemical abnormalities. The objective of this paper was to evaluate if a panel of biochemical analytes usually used in serum can be Confirmatory study measured in saliva of horses, and also to study the The diseased population (n = 37), diagnosed as having possible changes of these biochemical analytes in saliva acute gastrointestinal disease, came from the Veterinary of horses affected by acute abdominal disease. For this Teaching Hospital of the University of Extremadura purpose, a pilot exploratory evaluation was first made of between January and October 2018. It was composed of healthy horses and those with acute abdominal disease 14 geldings, 14 stallions and 9 mares with 9 ± 4.6 years to assess possible differences between the two groups of of age, including 12 Pure Spanish horses, 11 Crossbreds, animals in a profile of 23 biochemical analytes. Then, 4 Warmbloods breeds, 4 Arabians, 3 Lusitanian horses, those salivary analytes that significantly varied in the 2 Thoroughbreds, and 1 Friesian horse. The diagnosis pilot study were later clinically validated in a larger was performed as in the pilot study and included enter- population. itis (n = 8), strangulating lesion in the small intestine (n = 7), impaction of the pelvic flexure (n = 5), large colon displacement (n = 4), impaction of the right dorsal Methods colon (n = 3), colitis (n = 3), large colon volvulus (n = 2), Pilot exploratory study cecal tympany (n = 1), gastric perforation (n = 1), impac- The diseased population (n = 6), diagnosed as having tion of the small colon (n = 1), gastric ulcer syndrome acute gastrointestinal disease, came from the Veterinary (n = 1) and fecaloma in the small colon (n = 1). Teaching Hospital of the University of Extremadura The healthy population (n = 20) was selected as in the (Table 1). The diagnosis was based on history, physical pilot study. It included 8 stallions, 6 females and 6 examination, haematology and plasma biochemistry geldings, with an average age of 9 ± 4.4 years. The breeds (Olympus Diagnostica GmbH AU 600, Beckman included 14 Pure Spanish horses, 4 Crossbreds, 1 Coulter, Ennis, Ireland), transabdominal ultrasonog- Arabian and 1 Thoroughbred. raphy, and response to treatment by a board-certified internist (MMC). In horses undergoing surgery, the Sampling diagnosis of the acute gastrointestinal disease was based Saliva samples were obtained in the diseased population on laparotomy findings. within 30 min of arrival at the hospital once horses were Table 1 Overview of horses included in the pilot exploratory study Group Age (year) Gender Breed Diagnosis Diseased horses 23 Stallion Pure Spanish horse Enteritis 4 Mare Crossbred Impaction of the pelvic flexure 8 Stallion Arabian Impaction of the right dorsal colon 5 Stallion Pure Spanish horse Strangulated lesion in the small intestine 10 Mare Crossbred Fecaloma with ischemia 7 Gelding Crossbred Large colon displacement Healthy horses 5 Stallion Pure Spanish horse Healthy horse 5 Stallion Pure Spanish horse Healthy horse 15 Gelding Pure Spanish horse Healthy horse 13 Gelding Crossbred Healthy horse 14 Mare Crossbred Healthy horse 4 Mare Pure Spanish horse Healthy horse Contreras-Aguilar et al. BMC Veterinary Research (2019) 15:187 Page 3 of 9 in their box stalls. Upon arrival at the hospital, saliva following a previously described method [6]. In addition, samples were obtained prior to clinical examination or two new analytes were measured in saliva samples: TEA treatment. Saliva was collected by introducing a small activity, which was measured using a previously de- sponge (Esponja Marina, La Griega E. Koronis, Madrid, scribed method [5] in the Olympus chemistry analyser, Spain) into the horse’s mouth toward the side of the and cortisol, which was analysed with a solid-phase cheeks, allowing the sponge to be chewed until it was competitive enzyme-amplified chemiluminescent im- soaked with saliva. The entire procedures took around 1 munoassay by the immunoassay system (Immulite 1000, min. Then, sponge was placed in collection devices Siemens Healthcare Diagnostic, Deerfields, IL) and (Salivette, Sarstedt, Aktiengesellschaft & Co, Nümbrecht, validated in saliva by Escribano et al. [18]. The analytes Germany) and was proceessed as previously reported in saliva were validated by the researchers’ laboratory, [16]. Blood samples were collected just after saliva sam- showing an inter- and intra-variability lower than 10% of pling by jugular venipuncture and transferred into coefficient of variation (CV) and were linear when lithium-heparin tubes (Li Heparin, Aquisel®, Barcelona, applied to serial sample dilutions (R2 = 0.992 ± 0.004) Spain). Horses that yielded plasma samples with gross (Additional file 2). SAA and Hp could not be detected in haemolysis and blood-contaminated saliva samples saliva with the assays used in plasma (LZ SAA, Eiken were excluded from the study. Horses with increased Chemical Co.; and Haptoglobin turbidimetry, Spinreact, digital pulses or evident lameness were excluded in Spain, respectively). Therefore, they were excluded from order to avoid clinical cases combined with laminitis the saliva evaluation. SAA and Hp assays used anti- or musculoskeletal problems (Additional file 1). Infor- human serum antibodies. The SAA assay used has been mation about HR, RR and rectal temperature were demonstrated to have cross-reactivity and adequate ac- also obtained from the diseased and healthy curacy for SAA measurement in horses’ plasma [19, 20], populations. and the antibodies used in the assay for Hp measure- ment have cross-reactivity with plasma Hp in horse by Plasma and saliva biochemistry profile western blot (data no showed), similarly to other human The chemistry profile measured in plasma was inte- antibodies that showed cross-reactivity with Hp of grated by: horses [21].  Enzymes: AST, ALP, γ-glutamyl transferase (gGT), Severity scale lipase, amylase, LDH, CK, butyrilcholinesterase The severity of the acute abdominal disease was assessed (BChE) and ADA. using the systemic inflammatory response syndrome  Metabolites: creatinine, urea, uric acid, total (SIRS) score developed by Roy et al. [22], since it is easy bilirubin, cholesterol, triglycerides, glucose and to apply and because it has been demonstrated in a large lactate. population to be closely associated with the risk of  Proteins: total proteins, albumin, serum amyloid A death, mainly in cases of acute gastrointestinal illness (SAA) and Haptoblobin (Hp). cases. It was calculated as the sum of abnormal results  Minerals: P and total calcium. in HR (> 52 beats/min), RR (> 20 breaths /min), white blood cell (WBC) count (above or below 5.0–12.5 × 109/L) These assays were carried out on an automated and temperature (below or above 37.0–38.5 °C). Horses chemistry analyser (Olympus Diagnostica GmbH AU were considered to have SIRS when the score was equal to 600), using commercial kits from Beckman (Beckman or higher than 2 (4-point-score). Coulter Inc., Fullerton, CA, USA) for all assays with the exception of ADA, which was measured with a Diazyme Statistical analysis kit (ADA-D assay kit, Diazyme Laboratories, Poway, CA, To evaluate if there were significant differences in the USA), and BChE, which was measured according to a analytes evaluated in saliva between the healthy and previously reported assay [17]. All these assays showed diseased populations in the pilot exploratory study, an low inter and intra-assay imprecision (< 10%) and unpaired Student’s t test (2-tailed) was performed. high linearity under dilution (R2 > 0.995) in serum. Previously, the values were transformed by log applying The same chemistry profile, methods and apparatus the formula ln x = ln (x + 1) [23], since all showed non- were also used in saliva samples, with the exception of normal distribution when checked using the Shapiro- two analytes in which the analytical method was Wilk test. The analytes that showed significant changes different: total proteins, which was evaluated using a were studied in the confirmatory population using the commercial colorimetric kit to measure urine and Low- statistical analysis described above. A stand-alone power Complexity Region (LCR) proteins (protein in urine and program for statistical testing (G-Power) [24] was CSF, Spinreact, Spain), and BChE, which was measured employed using the means and standard deviations of Contreras-Aguilar et al. BMC Veterinary Research (2019) 15:187 Page 4 of 9 the selected analytes initially calculated in the pilot abdominal disease. All statistical analyses were performed exploratory study, to establish the minimum number of using a spreadsheet (Excel 2000, Microsoft Corporation, individuals necessary to be included in the confirmatory Redmond, Washington, USA) and the commercial study in each group for reaching a significance level of statistics package Graph Pad Prism 6 (GraphPad Software α = 5% (P < 0.05) and a power of 80%. Additionally, a Inc., La Jolla, CA, USA). Values of P ≤ 0.05 were selected post hoc analysis was performed with the number of in- to indicate significance in all analyses. dividuals used in the confirmatory study to guarantee that the significance level and power required were Results correctly obtained. Differences in salivary analytes between diseased and Spearman correlation was performed on the analytes healthy populations of the confirmatory study, in order to evaluate a correl- Medians and interquartile ranges (IQR) from all the ation between them in saliva and plasma, and between analytes evaluated in the pilot exploratory study are the SIRS score. An r value from 0.90 to 1 was considered shown in Table 2. There were seven analytes that to have very high correlation, 0.70 to 0.90 high correl- showed a significant increase in horses with acute ab- ation, 0.50 to 0.70 moderate correlation, 0.30 to 0.50 low dominal disease compared to healthy horses: gGT correlation and less than 0.30 little if any correlation (showing a 1.8 difference between log means, 95% confi- [25]. A receiver operator characteristic (ROC) curve dence interval [CI] 0.6–3.0, P = 0.008), CK (1.5 difference analysis was also performed to obtain the optimal cut-off between log means, 95% CI 0.1–2.9, P = 0.04), urea (0.9 point to maximise sensitivity and specificity for deter- difference between log means, 95% CI 0.2–1.6, P = 0.02), mining the healthy and diseased population with acute total bilirubin (0.2 difference between log means, 95% CI Table 2 Medians, interquartile ranges (IQR, 25–75%), minimal (Min) and maximal (Max) and values of a biochemical analysis performed in saliva from 6 healthy horses and 6 horses diagnosed with acute abdominal disease Healthy horses (n = 6) Diseased horses (n = 6) Median Min Max IQC Median Min Max IQC AST (IU/L) 101.8 45.5 205.8 47.8–146.3 212.8 28.1 1216.9 68.5–615.7 ALP (IU/L) 58.5 1.0 79.8 30.3–74.4 131.6 48.0 355.7 48.9–198.2 gGT (IU/L) 31.1 2.5 62.8 19.3–51.8 199.5** 55.9 359.0 65.9–331.5 Lipase (IU/L) 49.8 10.0 100.1 13.23–75.3 26.4 6.7 91.6 16.3–76.8 LDH (IU/L) 270.7 0.0 1195.0 41.9–662.3 688.6 42.3 2896.0 43.9–1795.0 CK (IU/L) 11.1 0.0 23.5 2.4–15.4 30.1* 7.0 140.7 13.5–88.9 BChE (nmol/mL/min) 19.2 5.6 31.1 12.1–27.5 18.6 12.5 62.1 13.6–57.7 ADA (IU/L) 14.5 8.2 85.2 12.4–36.2 41.1 11.8 216.3 26.9–111.6 Creatinine (μmol/L) 28.3 8.8 285.5 13.3–114.0 33.6 0.9 106.1 3.5–64.53 Urea (mmol/L) 7.9 4.1 10.4 5.4–9.7 18.8* 8.2 44.9 9.9–33.7 Uric acid (μmol/L) 59.5 51.8 103.5 52.3–86.8 38.7 0.6 88.6 10.7–71.4 Total bilirubin (μmol/L) 2.9 1.0 10.3 1.7–6.2 7.5* 3.6 13.5 4.3–10.4 Cholesterol (μmol/L) 35.4 0.00 249.8 0.0–169.7 93.4 0.0 263.8 0.00–187.0 Triglycerides (mmol/L) 0.34 0.05 0.68 0.06–0.67 0.21 0.02 3.64 0.04–1.17 Glucose (mmol/L) 0.54 0.01 1.80 0.13–1.05 0.51 0.00 2.99 0.16–2.24 Lactate (mmol/L) 1.73 0.02 3.44 0.42–2.70 0.39 0.01 20.91 0.09–9.32 Total protein (g/L) 1.65 0.96 4.23 1.01–2.72 5.77** 3.47 6.18 3.53–5.99 Albumin (g/L) 0.5 0.1 0.9 0.3–0.8 0.8 0.1 2.6 0.4–1.5 Phosphorus (mmol/L) 0.26 0.04 0.48 0.12–0.34 0.59* 1.20 2.04 0.24–1.72 Calcium (mmol/L) 4.7 4.2 7.0 4.2–7.0 6.4 4.1 9.4 4.8–9.0 sAA (IU/L) 13.5 1.0 23.1 4.2–20.3 109.3** 43.7 544.3 51.7–446.5 Cortisol (μg/dL) 0.93 0.56 1.12 0.79–1.05 0.91 0.6 1.08 0.60–1.02 TEA (IU/L) 141.7 77 165.5 120.7–159.4 133 39.8 184.9 44.2–133.0 AST aspartate aminotransferase, ALP alkaline phosphatase, gGT γ-glutamyl transferase, LDH lactate dehydrogenase, CK creatine kinase, BChE butyrilcholinesterase, ADA adenosine deaminase, sAA alpha-amylase salivary, TEA total esterase Asterisk indicates statistically significant differences between groups (*: P < 0.05; **: P < 0.01) Contreras-Aguilar et al. BMC Veterinary Research (2019) 15:187 Page 5 of 9 0.1–0.4, P = 0.05), total protein (1.0 difference between P = 0.003), P (r = 0.50, P = 0.002) and sAA (r = 0.35, log means, 95% CI 0.4–1.6, P = 0.004), P (0.6 differ- P = 0.05). ence between log means, 95% CI 0.2–1.3, P = 0.05) and sAA (2.6 difference between log means, 95% CI Discussion 1.3–3.8, P = 0.001). This report describes for the first time that a profile in- The power study calculated with the data from the tegrated by various analytes that are used in routine pilot exploratory study showed that a minimum number serum biochemistry analysis can be measured in saliva of 11 healthy horses and 21 horses with acute abdominal in horses, and that some of these analytes can change in disease would be needed to get appropriate results for situations of acute abdominal disease. the selected analytes. The post hoc analysis performed in In this report, we followed a similar approach that is the confirmatory study computed a power 96.4 ± 0.1% made in proteomics studies when changes in a profile of for the gGT, CK, urea, total bilirubin, total protein, P proteins are studied in a clinical situation [26–28]. First, and sAA results. When these analytes were evaluated in an exploratory study in a small population was the study made in the larger population, horses with performed in order to detect possible analytes that can acute abdominal disease had values statistically higher change in the situation being studied, in our case acute than the healthy ones for all the analytes (Fig. 1). abdominal disease. This population, as described in The optimal cut-off point in saliva for predicting previous studies, comprised horses with different ages, disease due to acute abdominal disease in the selected sexes, and diagnoses in order to be representative of the analytes was gGT ≥ 82.7 IU/L (AUC = 0.79; 95% CI: different clinical conditions that can appear in this 0.67–0.92; sensitivity 70% [51–85]; specificity 80% [56– disease [14, 29]. Then, the analytes showing changes 94]), CK ≥ 30.1 IU/L (AUC = 0.85; 95% CI: 0.74–0.95; were validated in a larger population. sensitivity 64% [45–81]; specificity 100 [83–100]), urea Seven analytes were detected as being raised in saliva ≥5.3 mmol/L (AUC = 0.79; 95% CI: 0.67–0.92; sensitivity of horses with acute abdominal disease. Two of them 66% [46–82]; specificity 95% [75–100]), total bilirubin were gGT and total bilirubin, which in serum are associ- ≥4.4 μmol/L (AUC = 0.73; 95% CI: 0.60–0.86; sensitivity ated with liver dysfunction [30, 31]. Elevations in serum 56% [38–72]; specificity 95% [75–100]), total proteins activity and concentration of both analytes have been as- ≥4.24 g/L (AUC = 0.95; 95% CI: 0.90–1.01; sensitivity sociated with several causes of colic in horses, including 77% [60–90]; specificity 100% [83–100]), P ≥ 0.50 cholelithiasis, infectious colitis, large colon displacement, mmol/L (AUC = 0.94; 95% CI: 0.89–1.00; sensitivity ulcerative duodenitis, proximal enteritis and neoplasia 75% [58–88]; specificity 100% [83–100]) and sAA ≥ [32]. Total bilirubin in saliva correlated moderately with 23.3 IU/L (AUC = 0.92; 95% CI: 0.86–1.00; sensitivity plasma; therefore it could be postulated that part of the 73% [56–86]; specificity 100% [83–100]). bilirubin that appears in saliva comes from plasma. In addition, total bilirubin was correlated moderately with Correlations the SIRS score, so it could be associated with the The correlations between the different analytes selected severity of the disease. in saliva, and between these analytes in saliva and CK, which in serum is associated with muscle damage plasma are shown in Table 3. High correlations in saliva [33], was raised in our study in the saliva of diseased were observed between gGT and urea (r = 0.70, P < horses. Increases in CK in saliva in situations of muscle 0.001), CK and phosphorus (r = 0.71, P < 0.001) and total damage have been described in dogs [34] and humans proteins and P (r = 0.75, P < 0.001). Moderate correla- [7] and, therefore, the high values of CK in saliva of tions in saliva were observed between gGT and total horses with acute abdominal disease could indicate the proteins (r = 0.67, P < 0.001), P (r = 0.61, P < 0.001) and existence of muscle damage, which has been described CK (r = 0.68, P < 0.001); between CK and total proteins as occurring in this disease by ischemic injuries [35, 36]. (r = 0.69, P < 0.001), urea (r = 0.66, P < 0.001), total biliru- One source of saliva CK could be the CK from bin (r = 0.57, P < 0.001) and gGT (r = 0.68, P < 0.001); plasma, since significant correlations were found be- between urea and total proteins (r = 0.61, P < 0.001) and tween CK in plasma and saliva in our study, similarly P (r = 0.52, P < 0.001); and between total proteins and to that reported in dogs [34]. In our study, CK in sal- sAA (r = 0.61, P < 0.001). iva had a significant although low correlation with Positive correlations between saliva and plasma SIRS status and therefore it could be an indicator of were observed in CK (r = 0.34, P = 0.05), urea (r = the severity of the process. 0.49, P = 0.002), total bilirubin (r = 0.37, P = 0.02) and Increases in serum urea with normal values of creatin- total proteins (r = 0.34, P = 0.03). The SIRS score ine are associated with reduced hydration status [37] or correlated in saliva with CK (r = 0.40, P = 0.03), total bleeding in the gastrointestinal tract [38]. Therefore, it bilirubin (r = 0.41, P = 0.02), total proteins (r = 0.49, could be postulated that similar causes could produce Contreras-Aguilar et al. BMC Veterinary Research (2019) 15:187 Page 6 of 9 Fig. 1 Results of γ-glutamyl transferase (gGT) (a), creatine kinase (CK) (b), urea (c), total bilirubin (d), total protein (e), phosphorus (f) and salivary alpha-amylase (sAA) (g) in saliva of a healthy horses population (n = 20) and a diseased horses population (n = 37) with acute abdominal disease. The plot shows median (line within box), 25th–75th percentiles (box), 5th and 95th percentiles (whiskers) and outliers (•). The cross inside the box shows the mean. Asterisk indicates statistically significant differences between groups (***: P < 0.001; ****: P < 0.0001) the higher salivary urea found in our study in horses correlation has also been reported in dogs with chronic with acute abdominal disease. A moderate correlation kidney disease [39]. The higher concentration of total between urea in saliva and plasma was observed. This proteins observed in saliva in horses with acute Contreras-Aguilar et al. BMC Veterinary Research (2019) 15:187 Page 7 of 9 Table 3 Spearman correlation coefficients between gGT, CK, urea, total bilirubin, total protein, phosphorus and sAA in saliva and plasma in healthy (n = 20) and diseased horses (n = 37) with acute abdominal disease Saliva gGT (IU/L) CK (IU/L) Urea (mmol/L) Total bilirubin (μmol/L) Total proteins (g/L) Phosphorus (mmol/L) sAA (IU/L) gGT saliva 0.68*** 0.70*** 0.24 0.67*** 0.61*** 0.26 (IU/L) plasma 0.24 0.15 0.08 0.03 0.41* 0.36* 0.09 CK saliva 0.68*** 0.66*** 0.57*** 0.69*** 0.71*** 0.45** (IU/L) plasma 0.22 0.34* 0.36* 0.3 0.31 0.34* 0.55*** Urea saliva 0.70*** 0.66*** 0.34* 0.61*** 0.52*** 0.37** (mmol/L) plasma 0.19 0.24 0.49** 0.36* 0.35* 0.3 0.26 Total bilirubin saliva 0.24 0.57*** 0.34* 0.36** 0.37** 0.49*** (μmol/L) plasma 0.19 0.3 0.08 0.37* 0.51*** 0.60*** 0.39* Total proteins saliva 0.67*** 0.69*** 0.61*** 0.36** 0.75*** 0.61*** (g/L) plasma 0.32* 0.34* 0.35* 0.18 0.34* 0.29 0.26 Phosphorus saliva 0.61*** 0.71*** 0.52*** 0.37** 0.75*** 0.48*** (mmol/L) plasma 0.06 −0.08 0.07 −0.16 0.18 0.1 0.19 Amylase saliva 0.26 0.45** 0.37** 0.49*** 0.61*** 0.48*** (IU/L) plasma −0.30 −0.24 −0.45* −0.02 0.26 0.14 0.24 Asterisk indicates statistically significant difference between comparisons gGT γ-glutamyl transferase, CK creatine kinase, sAA salivary alpha-amylase Asterisk indicates statistically significant correlations (*: P < 0.05; **: P < 0.01; ***: P < 0.001) abdominal disease could also indicate reduced hydration larger population to evaluate if changes in analytes could status, as it has previously been described in serum [40]. be related with outcome (survivors vs. non-survivors), P in serum has been considered a marker of disease diagnosis (ischaemic/strangulating vs. non-ischaemic/ severity and likelihood of mortality in hospitalised foals non-strangulating) and treatment needed (medical [41]. Although in this study P in saliva did not correlate treatment vs. surgery). In addition, it would have been with P in plasma, it correlated in saliva with the SIRS interesting to include an analysis of electrolytes in saliva score. Further studies should be performed in order to in this study, which was not possible due to volume clarify the undergoing mechanisms of P in saliva. constraints. The increases in sAA activity found in our study on diseased horses were in agreement with other studies Conclusions performed on horses with acute abdominal disease [3, A panel of 23 analytes can be measured in saliva in 16]. In addition, a low but significant correlation of sAA horses, and from these analytes, gGT, CK, urea, total with SIRS status was observed in our study, as previ- bilirubin, total protein, phosphorus and sAA can be ously reported [3, 16]. The lack of correlation with amyl- raised in horses with acute abdominal disease. Further ase in plasma confirms that, as humans, the sAA that studies should be made to evaluate and refine the pos- appears in saliva is secreted by the salivary glands [23]. sible application of these analytes in clinical situations. From all the analytes that showed differences between the horses with acute abdominal disease and the healthy Additional files ones, total proteins, P and sAA showed sensitivities higher than 70% at their optimal cut-off points and a Additional file 1: Flow chart describing horses included and excluded specificity of 100% in detecting acute abdominal disease. in the diseased populations. Description of horses included or excluded Ideally, further studies in which these analytes are evalu- from the study in the diseased populations. (PDF 225 kb) ated in other diseases should be performed, in order to Additional file 2: Linearity under dilution in saliva of aspartate aminotransferase (AST), alkaline phosphatase (ALP), γ-glutamyl transferase evaluate their specificity not only in the case of healthy (gGT), lipase, lactate dehydrogenase (LDH), creatine kinase (CK), butyril- individuals but also in individuals with diseases other cholinesterase (BChE), adenosine deaminase (ADA), creatinin, urea, than acute abdominal disease. uric acid, total bilirubin, cholesterol, triglycerides, glucose concentration, total proteins, albumin, lactate, phosphorus (P), calcium and total esterase The power analysis guaranteed adequate statistical (TEA). Linearity under dilution study in three pools of saliva from two analysis for evaluating differences between healthy and specimen of saliva each. The ‘x’ expressed activity or concentration diseased horses in the confirmatory population. How- measured and ‘y’ expected level at the particular dilution. R2 = coefficient of determination of linear correlation. (PDF 2190 kb) ever, it would be of interest to make future studies in a Contreras-Aguilar et al. BMC Veterinary Research (2019) 15:187 Page 8 of 9 Abbreviations Received: 9 February 2019 Accepted: 23 May 2019 ADA: Adenosine deaminase; ALP: Alkaline phosphatase; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; BChE: Butyrilcholinesterase; ChE: Cholinesterase; CI: Confidence interval; CK: Creatine kinase; CV: Coefficient of variation; gGT: γ-glutamyl transferase; Hp: Haptoblobin; HR: Heart rate; References IQR: Interquartile ranges; LCR: Low-complexity region; LDH: Lactate 1. Lindner A, Marx S, Kissenbeck S, Mosen H. Saliva collection and relationship dehydrogenase; P: Phosphorus; ROC: Receiver operator characteristic; between lactate concentration in blood and saliva of exercising horses. J RR: Respiratory rate; sAA: Salivary apha-amylase; SAA: Serum amyloid A; Equine Vet Sci. 2000;20:52–4. SIRS: Systemic inflammatory response syndrome; TEA: Total esterase activity; 2. Bilancio G, Cavallo P, Lombardi C, Guarino E, Cozza V, Giordano F, et al. WBC: White blood cells Salivary levels of phosphorus and urea as indices of their plasma levels in nephropathic patients. J Clin Lab Anal. 2018;32:1–6. 3. Contreras-Aguilar MD, Escribano D, Martín-cuervo M, Tecles F, Cerón JJ. Acknowledgements Salivary alpha-amylase activity and cortisol in horses with acute abdominal The authors gratefully acknowledge the intern veterinarians from the disease : a pilot study. BMC Vet Res. 2018;14:1–7. Veterinary Teaching Hospital of the University of Extremadura for collecting 4. Tecles F, Rubio CP, Contreras-Aguilar MD, Lopez-Arjona M, Martinez-Miro S, saliva samples. We wish to thank Juan Jesús Carreño Martínez for offering Martinez-Subiela S, et al. Adenosine deaminase activity in pig saliva: their horses for the healthy population and to Costel Ursache for helping in analytical validation of two spectrophotometric assays. J Vet Diagn Investig. samples collection. D. Escribano was granted by the postdoctoral program 2018;30:175–9. ‘Juan de la Cierva’ of the ‘Ministerio de Economía y Competitividad’, Spain. 5. Tecles F, Contreras-Aguilar MD, Martínez-Miró S, Tvarijonaviciute A, Martínez- M. Contreras-Aguilar was granted by the predoctoral contract ‘FPU’ of Subiela S, Escribano D, et al. Total esterase measurement in saliva of pigs: University of Murcia (R- 605/2016), Spain. Funding was additionally provided validation of an automated assay, characterization and changes in stress by the FCT – Portuguese Science Foundation: research contract IF/01778/ and disease conditions. Res Vet Sci. 2017;114:170–6. 2013 (E. Lamy) and by the Seneca Foundation of Murcia Regional 6. Tecles F, Escribano D, Martínez-Miró S, Hernández F, Contreras MD, Cerón JJ. Government, Spain (grant number 19894/GERM/15). Cholinesterase in porcine saliva: analytical characterization and behavior after experimental stress. Res Vet Sci. 2016;106:23–8. Authors’ contributions 7. Barranco T, Tvarijonaviciute A, Tecles F, Carrillo JM, Sanchez-Resalt C, MDCA and JJC conceived and designed the study. MDCA and MMC Jimenez-Reyes P, et al. Changes in creatine kinase, lactate dehydrogenase collected the data. MDCA were involved in the chemical analysis. MDCA, DE, and aspartate aminotransferase in saliva samples after an intense exercise: a SMS, MMC, EL, FT and JJC were involved in the data interpretation. MDCA pilot study. J Sports Med Phys Fit. 2018;58:910–6. drafted the manuscript and DE, SMS, MMC, EL, FT and JJC critically read and 8. Contreras-Aguilar MD, Escribano D, Quiles A, López-Arjona M, Cerón JJ, edited the manuscript. All authors read and approved the final manuscript. 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