Changes in biomarkers of the redox status in whole blood and red blood cell lysates in canine hypothyroidism.

Hypothyroidism is the most commonly diagnosed endocrine disease in dogs. The objective of this study was to evaluate the changes in the redox status in canine hypothyroidism using whole blood (WB) and red blood cell (RBCs) lysates. For this purpose, a panel of five antioxidants and five oxidants biomarkers was measured in WB and RBCs lysates of 30 dogs with hypothyroidism, 26 dogs with non-thyroidal illnesses and 15 healthy dogs. The antioxidants measured were cupric reducing antioxidant capacity (CUPRAC), ferric reducing ability of plasma (FRAP), Trolox equivalent antioxidant capacity (TEAC), thiol and paraoxonase type-1 (PON-1). Oxidants measured include the total oxidant status (TOS), peroxide-activity (POX-Act), reactive oxygen-derived metabolites (d-ROMs), advanced oxidation protein products (AOPP) and thiobarbituric acid reactive substances (TBARS). WB showed a significant decrease of the antioxidants CUPRAC, TEAC and thiol, and also an increase in TBARS and a decrease in AOPP in dogs with hypothyroidism compared to healthy dogs. Meanwhile, RBCs lysates showed a significant increase in FRAP and PON-1 in dogs with hypothyroidism. The changes in the redox biomarkers in this study show that WB in canine hypothyroidism had a higher number of changes in biomarkers of the redox status than RBCs lysates, making it a promising sample type for the evaluation of the redox status in this disease. In addition, WB is easier and simpler to process than RBCs lysates and unlike serum, it does not have any hemolysis interference.

Automated assays for trace elements and ferritin measurement in saliva of pigs: Analytical validation and a pilot application to evaluate different iron status.

The aim of this study was to validate automated methods to measure iron (Fe), zinc (Zn), copper (Cu) and ferritin in pig saliva samples. A complete analytical validation was performed of all assays. In addition, these methods were applied to saliva of Fe supplemented (n = 22) and non-supplemented (n = 20) piglets. All assays were able to measure these biomarkers in pig saliva with adequate precision, accuracy and high sensitivity and, in case of trace elements without needing a deproteinization pre-process. The group of piglets supplemented with Fe presented significantly higher levels of ferritin and Zn in saliva. In conclusion, the automated assays evaluated were able to measure Fe, Zn, Cu and ferritin in saliva of pigs, and in case of trace elements, they have the advantage of not needing a deproteinization pre-treatment and thus these analytes can be measured in a simple and fast manner.

Evaluation of acute phase proteins, adiponectin and endothelin-1 to determine vascular damage in dogs with heartworm disease (Dirofilaria immitis), before and after adulticide treatment.

Previous studies have shown the existence of an acute phase response in dogs with heartworm (Dirofilaria immitis), probably caused by the vascular inflammation that occurs during the pathogenesis of this disease. In addition, it has been seen that this acute phase response persists after finishing treatment, especially in dogs with pulmonary hypertension (PH). Furthermore, echocardiographic studies have shown that PH and endarteritis appear to persist for at least 10 months after completion of adulticide treatment, suggesting that the vascular changes in these dogs may not be reversible. Therefore, the objective of this study was to evaluate the serum concentrations of different positive acute phase proteins (APP) [C reactive-protein (CRP), haptoglobin and ferritin] and negative APP (albumin and paraoxonase-1 (PON-1)), and the usefulness of the endothelin-1 (ET-1) and adiponectin, in dogs infected by D. immitis to evaluate their usefulness as diagnostic biomarkers of vascular damage and PH and their progression throughout therapy up to 7 months after the end of adulticide treatment. Twenty-five heartworm-infected dogs received adulticide treatment, and serum measurements were performed on the day of diagnosis (day 0), day of discharge (day 90), and 6 months after discharge (day 270). In addition, presence or absence of PH was also echocardiographically determined using the Right Pulmonary Artery Distensibility Index. PH was present in 44% of the dogs on day 0 and day 90, and in 48% of dogs on day 270. Alterations were observed in the concentrations of all APP throughout the study, persisting the alterations in PON-1 and ferritin on day 270. Depending on the presence or absence of PH, CRP showed significant differences throughout the study, as did ET-1. On the other hand, adiponectin did not show variations throughout the study, so it did not seem a useful marker in this disease. These results could reflect the possible persistence of vascular inflammation up to 7 months after finishing treatment, whether or not there was PH, and consolidate the study of APP as useful markers in heartworm disease. Moreover, persistent PH could be the consequent clinical manifestation in dogs with more severe vascular alterations so the study of APP, especially CRP, and ET-1 could be especially advantageous in these patients in the early evaluation of the disease, as well as for the determination of disease severity, monitoring therapeutic responses, and predicting outcomes.

Basics for the potential use of saliva to evaluate stress, inflammation, immune system, and redox homeostasis in pigs.

The use of saliva as a biological sample has many advantages, being especially relevant in pigs where the blood collection is highly stressful and painful, both for the animal and the staff in charge of the sampling. Currently one of the main uses of saliva is for diagnosis and detection of infectious diseases, but the saliva can also be used to measure biomarkers that can provide information of stress, inflammation, immune response and redox homeostasis. This review will be focused on the analytes that can be used for such evaluations. Emphasis will be given in providing data of practical use about their physiological basis, how they can be measured, and their interpretation. In addition, some general rules regarding sampling and saliva storage are provided and the concept of sialochemistry will be addressed. There is still a need for more data and knowledge for most of these biomarkers to optimize their use, application, and interpretation. However, this review provides updated data to illustrate that besides the detection of pathogens in saliva, additional interesting applicative information regarding pigs´ welfare and health can be obtained from this fluid. Information that can potentially be applied to other animal species as well as to humans.

Insulin in the saliva of pigs: Validation of an automated assay and changes at different physiological conditions.

This study aimed to evaluate whether insulin could be measured in the saliva of pigs and if its concentration changes in some physiological conditions. For this purpose, a validation of an automated heterologous immunoassay for measuring insulin in the saliva of pigs was performed. In addition, the possible changes of salivary insulin concentration in sows after food intake and during gestation and lactation were studied. The evaluated immunoassay was able to detect insulin in the saliva of pigs in a precise and accurate way when species-specific calibrators were used. There was no correlation in insulin concentrations between serum and saliva. Insulin concentrations showed a significant increase in the saliva of sows after feeding. Sows at farrowing and lactation presented higher salivary insulin levels as compared with those in gestation. In conclusion, the results showed that insulin could be measured in the saliva of pigs, and changes in its concentration can be detected due to food intake and different physiological conditions.

Changes in saliva proteins in cows with mastitis: A proteomic approach.

This study aimed to evaluate the possible saliva proteome changes in cows with mastitis using a Tandem Mass Tags (TMT) proteomics approach. For this purpose, the salivary proteomes from healthy cows and cows with mastitis were analysed, and their serum proteomes were also studied for comparative purposes. A total of eight saliva and serum paired samples for each group were used for the proteomic study, and eight additional samples for each group were analysed in the analytical and overlap performance studies. In saliva samples, 2192 proteins were identified, being sixty-three differentially modulated in mastitis. In serum, 1299 proteins were identified, being twenty-nine differentially modulated in mastitis. Gamma glutamyl transferase (γGT) in saliva and serum amyloid A (SAA) were validated by commercially available automated assays. In conclusion, there are changes in protein expression and metabolic pathways in saliva and serum proteomes of cows with mastitis, showing different response patterns but complementary information.

Changes in the salivary proteome of beagle dogs after weight loss.

Being overweight or obese represents an important health issue in humans and pets. The aim of this study was to investigate changes in the salivary proteome of overweight beagles after induced weight loss to better understand the physiological changes involved in this process. Five overweight/obese neutered males of pure breed beagles were evaluated. During the 3-mo period of weight loss, each animal received a strictly controlled amount of a low fat commercial diet per day. Body condition scores (BCS), body weight (BW), and serum biochemical parameters (total cholesterol, triglycerides, and C-reactive protein) were assessed weekly. Quantitative proteomics analysis by SWATH was used to evaluate the salivary proteome changes induced by weight loss treatment. BCS, BW, serum total cholesterol concentration, and abundances of 23 salivary proteins differed significantly between before and after treatment. Some of the altered protein amounts, namely of peptidyl-prolyl cis-trans isomerase, fructose-bisphosphate aldolase C, and 78-kDa glucose-regulated protein, increased after weight loss. These proteins are related with the immune system, inflammatory status, oxidative stress, and glucose metabolism. The results obtained suggest a potential use of salivary proteins in monitoring physiological changes in dogs subjected to weight loss. Moreover, the type of changes identified reinforces the postulated physiological improvements, which weight loss induces. Further research is needed to determine whether the changes observed in this study are due to weight loss, dietary changes, or a combination of both.

Use of proteases for the evaluation of the different adiponectin isoforms in the dog.

Adiponectin (ADP) is an adipokine secreted by adipose tissue with anti-inflammatory, antiatherogenic, and antidiabetic properties. In human serum, it is presented as three different forms: low molecular weight (LMW), medium molecular weight (MMW), and high molecular weight (HMW). High molecular weight isomer is the most active form of ADP and is more closely related with obesity-induced insulin resistance and metabolic syndrome than total ADP. Selective protease treatment can be used in humans to isolate the different ADP isoforms but this has not been applied in any veterinary species. Therefore, the objective of this study was to evaluate if the selective protease digestion is able to differentiate serum ADP isomers in dog samples, and if these isomers could change in obese dogs after a weight loss program. A Western blotting analysis confirmed that digestion with protease K showed only the HMW forms of ADP, whereas the use of protease A showed the HMW and MMW forms. This specific protease digestion was applied to serum obtained from 14 obese beagle dogs before and after a weight loss program and total ADP, HMW, and LMW forms increased significantly after the weight reduction. In conclusion, the use of selective protease digestion can be applied in canine serum as a procedure for detecting the different ADP isomers. In addition, by this procedure, it was showed that the HMW and LMW forms were increased after a weight loss program in our experimental conditions.

A new highly sensitive immunoassay for the detection of adiponectin in serum and saliva of dogs and its application in obesity and canine leishmaniosis.

Adiponectin is an adipokine that exerts insulin-sensitizing and antiinflammatory properties. The aim of this study was to develop and validate a new heterologous ultrasensitive assay based on amplified luminescent technology for adiponectin determination in serum and saliva of dogs. A complete analytical validation of the assay was made in these fluids, and also this assay was applied to quantify adiponectin in serum and saliva of obese and lean dogs and dogs with leishmaniosis. These conditions were selected because in obesity there is a controversy about how adiponectin concentrations change in dogs, and in case of canine leishmaniosis, although it is described a decrease in serum adiponectin, there are not studies about how adiponectin changes after treatment. A total of 11 dogs were used in the validation and 26 dogs with different body condition and 8 with canine leishmaniosis were used for the clinical evaluation of the new assay for adiponectin quantification in serum and saliva of dogs. The analytical evaluation showed that the developed method could measure adiponectin in serum and saliva of dogs with high repeatability and sensitivity, adding a limit of quantification lower than commercially available ELISAs. In addition, significantly higher adiponectin concentrations were found in lean dogs and a correlation between serum and saliva was observed (P < .01). Moreover, dogs with leishmania presented reduced levels of adiponectin in serum. In conclusion, a new assay has been developed for adiponectin measurements which is more sensitive and faster than the traditional ELISA assays requiring less sample volume.

Identification of changes in serum analytes and possible metabolic pathways associated with canine obesity-related metabolic dysfunction.

The main objective of this study was to identify analytes that could change and that could help to clarify the metabolic and physiopathological changes related to canine obesity-related metabolic dysfunction (ORMD). For this, serum from 35 overweight/obese dogs, with and without ORMD, was submitted to a comprehensive panel of biochemistry analysis, a gel-free tandem mass tag isobaric label-based proteomic analysis, and, finally, selected proteins were used as a starting point for creating a protein interaction network. Dogs with ORMD showed significantly higher serum concentrations of alanine aminotransferase (ALT), alkaline phosphatase (ALP), Ca, total proteins, albumin, total cholesterol, triglycerides, glucose, and butyrylcholinesterase (BChE) activity in comparison with dogs without ORMD. Proteomic analysis revealed that 23 proteins related to lipid metabolism, the complement factor system, cellular adhesion and functionality, inflammation, and coagulation were altered in dogs with ORMD. Finally, the obtained protein interaction network highlighted that the central term of this network was the negative regulation of the immune response. These data suggest that canine ORMD is associated with changes in analytes that reflect altered lipid metabolism, and liver and immune function impairment and suggests the potential for a prothrombotic state and lung function alterations.

Glucose, fructosamine, and insulin measurements in saliva of dogs: variations after an experimental glucose administration.

The aim of this study was to evaluate if glucose, fructosamine, and insulin levels can be measured in saliva of dogs and assess the changes in these compounds after an experimental glucose administration. Automated spectrophotometric assays for glucose and fructosamine and an ELISA assay for insulin measurements were validated in saliva of dogs, by evaluating precision, accuracy, and limits of detection. In addition, an intravenous glucose bolus was administrated to 10 beagles and fasting serum and saliva samples were obtained immediately before and 5, 10, 20, 30, and 45 min after glucose infusion. The results of the between-run imprecision gave mean CVs of 6.16, 9.40, and 3.10% for glucose, fructosamine, and insulin, respectively. Linearity under dilution showed coefficient of correlation of 0.999, 0.994, and 0.990 for glucose, fructosamine, and insulin, respectively. The LDs were 0.04 mg/dL, 4.08 µmol/L, and 0.02 µg/mL for glucose, fructosamine, and insulin, respectively. The glucose administration caused an increase in serum and salivary levels of glucose with a peak in salivary levels at 30 min and of insulin with a peak in salivary levels at 45 min, while fructosamine did not change. No correlations between serum and salivary concentrations were found for any compound. It is concluded that glucose, fructosamine, and insulin can be measured in saliva of dogs, and an experimental administration of glucose in this species can lead to increases in glucose and insulin in saliva.