All experimental procedures were approved by the Local Ethical Committee of University of Murcia, and were performed in compliance with laws RD32/2007 and RD1201/2005 concerning animal experimentation in Spain.
Assays
CK was measured by a commercial kit (Beckman Coulter, Brea, USA) based on the quantification of the NADPH formation derived from the action of CK on creatin phosphate as recommended by the International Federation of Clinical Chemistry (IFCC) [18]. AST was measured by a commercial kit (Beckman Coulter, Brea, USA) based on the quantification of the NADH consumption derived from the action of AST over aspartate and oxoglutarate as recommended by the IFCC [19]. Specimen volume used was 3 μl for CK and AST in the case of serum and 25 μl for CK and 3 μl for AST in case of saliva. A correction factor of [3/(3 + RV)]: [(25 + RV)/25] (where RV is reagent volume) was applied to the results of CK in saliva in order to take in consideration the different volume used. All the assays were performed in an automated biochemistry analyzer (Olympus AU600, Beckman Coulter, Brea, USA) at 37 °C. CK and AST assays showed an inter-assay imprecision and an inaccuracy of less than 5% in the daily quality control analysis done during the study. Manufacturer’s control solutions of two different values were used for the quality control analysis (Beckman-coulter, Lot 0037 and 0038).
Analytical validation
For analytical validation of both methods the following parameters were calculated.
Precision
The intra-assay coefficient of variation (CV) was calculated after analysis of 2 saliva specimens with different CK and AST concentration 5 times in a single assay run. The inter-assay CV was determined by analyzing the same specimens in 5 separate runs, carried out on different days, being the specimens stored at −80 °C.
Accuracy
It was evaluated indirectly by linearity under dilution. For this purpose, two canine saliva specimens were serially diluted with bidistilated water.
Limit of detection
This was calculated on the basis of data from 10 replicate determinations of the zero standard (bidistilated water) as the mean value plus 3 standard deviations.
Animals
A total of 27 dogs were included in the present study. Thirteen of these animals were healthy dogs belonging to staff and students of Murcia University and were used as controls. None of the dogs presented abnormalities at physical examination, or in the CBC and biochemical profile, and did not have evidence of periodontal disease. In particular, serum activities of AST and CK were lower than 50 UI/L and 250 U/L respectively, values which represent the higher limit of the reference interval of our laboratory.
All animal were adults with a mean (range) age of 5.1 (2.0-8.0) years and a median (range) body weight (BW) of 19.3 (7.0-29.0) kg. Six dogs were mongrels, three were Beagles, two were Labrador Retrievers, one was Golden Retriever and one was Poodle.
In order to evaluate if CK and AST increased in saliva in dogs with muscle damage, fourteen dogs with increased serum CK and AST due to diverse causes such as traumas or surgery were included in the study. The saliva and blood specimens were taken at time of admission. In this group of dogs age ranged between 0.5 and 10.0 years (median 6.4 years), and BW was between 1.5 and 40.0 kg (median 25.5 kg). Seven dogs were mongrels, three were Yorkshire terriers and there was one dog of each of the following breeds: San Bernardo, Boxer, German Shepherd and Spanish water dog. In all cases, the exclusion criteria were: the saliva specimens obtained had not enough volume for measurements; and presence of periodontal disease since salivary CK and AST was shown to increase in humans with periodontal disease [20].
Saliva and blood sampling
Saliva specimens were obtained by placing a sponge in dog’s mouth for 1–2 min as previously described [5, 21]. Then the sponge was placed into the Salivette (Salivette®, Sarstedt AG &Co., Nümbrecht, Germany) device for centrifugation (P Selecta®, JP Selecta S.A, Barcelona, Spain) at 3000 x g 10 min [5, 22]. After saliva collection, venous blood specimens (2 mL) were collected from the jugular vein into plain tubes (Vacutainer®, Plymouth, United Kingdom). Tubes were let to clot at room temperature for 30 min and centrifuged (2000 x g, 10 min) for careful removal of the serum.
The saliva and blood specimens were processed and measured in less than 1 h after collection, and serum and centrifuged saliva samples were keep at 4 °C until were measured.
Statistical analysis
Normality of the data distribution was evaluated with a Kolmogorov-Smirnov test and, since data was not normally distributed, non-parametric tests were used. Differences in serum and salivary CK and AST between healthy controls and dogs muscle enzymes in serum out of reference interval were evaluated using the Mann–Whitney test. Correlations between serum and saliva were calculated using the Spearman correlation coefficient. The level of significance was set at P < 0.05. Statistical analyses were performed with computer software (Graph Pad Prism Version 7 for Windows, Graph Pad software, La Jolla, CA).