Bile acids (BAs) are important signaling molecules in the gastrointestinal (GI) tract and are associated with health and disease in humans and animals. Intestinal bacteria transform BA through deconjugation, dehydroxylation, and epimerization reactions, producing various isoforms, many of which have not been investigated in companion animal diseases. We aimed to develop and analytically validate a novel liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for the quantification of 30 BAs in dog feces, with a simple extraction procedure and on-line solid-phase extraction. Validation demonstrated good accuracy, precision, sensitivity, spiking recovery, dilution, and stability for 29 BAs. The method was applied to fecal samples from healthy dogs (H; n = 121) and dogs with chronic enteropathy (CE; n = 58). The immediate and downstream products of bacterial 7α-dehydroxylation reactions with cholic acid were lower in concentration in dogs with CE when compared to healthy dogs (deoxycholic acid, 3-oxo-deoxycholic acid, and 12-oxo-lithocholic acid; q < 0.001). Across all fecal samples, the products of hydroxysteroid dehydrogenase (including oxo- and iso-BA) made up an average of 30% of the total measured fecal BA pool (glycine-BA, 0.1%; taurine-BA, 2.2%; unconjugated BA, 53%).

Amino acids play an important role in metabolism. Comprehensive analytical validation of an assay for the concurrent measurement of a large number of amino acids in dogs is lacking, which precludes its usefulness in a clinical setting. Amino acids are often measured in plasma or whole blood. However, serum is commonly used for gastrointestinal diagnostic testing in dogs and is therefore convenient to use. This study aimed to analytically validate an assay for the concurrent measurement of amino acids in dog serum and to evaluate differences in amino acid concentrations in whole blood, plasma, and serum in dogs. Analytical validation of the assay (Biochrom 30+ Amino Acid Analyzer) was performed on fresh or banked serum samples from dogs. Whole blood, plasma, and serum from 36 healthy dogs were analyzed, and concentrations of the three sample types were compared. The assay was demonstrated to be precise, reproducible, accurate, linear, and stable for the measurement of the majority of compounds detected in dog serum. Cystine, glutamic acid, and ethanolamine were shown to be unstable at conditions commonly encountered in clinical settings. Significant differences in concentrations were identified between whole blood, plasma, and serum for 33 of 42 compounds. Amino acid profiles in serum and plasma were more similar to each other than to those in whole blood. While some amino acids are present in similar concentrations in whole blood, plasma, and serum, others are highly dependent on the type of biofluid, and measurements warrant strict adherence to sample type-based reference intervals.

Isoprostanes are stable end products of lipid peroxidation that can be used as markers of oxidative stress. It was previously reported that a cohort of dogs with various liver diseases had increased urinary isoprostane concentrations compared to healthy control (HC) dogs. The aim of this study was to measure and report urinary isoprostane concentrations in dogs with different types of liver diseases. Urine was collected from 21 HC dogs and from 40 dogs with liver disease, including 25 with chronic hepatitis (CH), 7 with steroid hepatopathy (SH), and 8 with a congenital portosystemic shunt (CPSS). In this prospective, observational study, urinary 15-F_2t-isoprostane (F₂-IsoP) concentrations were measured by liquid chromatography/mass spectrometry and normalized to urinary creatinine concentrations. Concentrations were compared between groups using a Kruskal–Wallis test followed by Dunn’s multiple comparisons tests. Significance was set at p < 0.05. The median (range) urinary F₂-IsoP to creatinine ratios (ng/mg UCr) were 3.6 (2.2–12.4) for HC dogs, 5.7 (2.4–11.3) for dogs with CH, 4.8 (2.4–8.6) for dogs with SH, and 12.5 (2.9–22.9) for dogs with CPSS. CPSS dogs had significantly higher urinary F₂-IsoP concentrations than HC dogs (p = 0.004), suggesting increased oxidative stress among this cohort.

Background/Objectives: A functional liver is vital for normal protein metabolism. Alterations of circulating amino acid (AA) concentrations have previously been reported in dogs with hepatocellular carcinoma, chronic hepatitis, and hepatocutaneous syndrome. The purpose of this study was to compare serum AA profiles between dogs with a congenital portosystemic shunt (CPSS) and healthy control dogs. Methods: Serum samples were collected from 50 dogs with an extrahepatic congenital portosystemic shunt (eCPSS) and 10 dogs with an intrahepatic congenital portosystemic shunt (iCPSS) at time of surgical intervention and from 21 healthy control dogs. Serum AA and other nitrogenous compounds were measured with a dedicated amino acid analyzer. The concentration of each AA was compared between groups using a Kruskal–Wallis test followed by Dunn’s multiple comparisons tests, as appropriate. The Benjamini–Hochberg procedure was used to control for false discovery. Significance was set at q < 0.05. Results: Compared to healthy controls, dogs with a CPSS had significantly increased serum concentrations of ammonia, asparagine, glutamic acid, histidine, phenylalanine, serine, and tyrosine and had significantly decreased concentrations of isoleucine, leucine, threonine, urea, and valine. There were no significant differences in serum AA concentrations between dogs with an eCPSS and dogs with an iCPSS. Conclusions: Dogs with a CPSS had altered serum AA concentrations compared to healthy control dogs, including decreased branched-chain amino acids (BCAAs) and increased aromatic amino acids (AAAs). In summary, serum AA profiles can differentiate dogs with a CPSS from healthy dogs but not dogs with an eCPSS from dogs with an iCPSS.

Objective: To determine whether microRNAs 122, 1275, 21, 222, and 181A serve as noninvasive biomarkers of canine chronic hepatitis (CH); can distinguish between dogs with idiopathic CH (iCH) and copper-associated CH (CuCH); and correlate with serum ALT activity, grade of inflammation, fibrosis, and degree of copper accumulation. Methods: From May 2019 through October 2023, dogs with CH, histologically characterized as either iCH or CuCH, and healthy control (HC) dogs without liver biopsies were enrolled in this prospective uncontrolled study. Serum samples were collected, and serum microRNAs were quantified by real-time quantitative PCR. Differences in gene fold expression between groups were calculated. Correlations between microRNA gene fold expression and serum ALT, hepatic copper quantification, grade of necroinflammatory activity, fibrosis, and histological copper score were assessed. Results: A total of 18 dogs with iCH, 14 dogs with CuCH, and 42 HC dogs were enrolled. No differences between iCH and CuCH dogs were observed for any microRNA. Differences between HC and overall CH dogs were observed for microRNAs 122, 1275, and 21. Positive correlations with serum ALT activity between microRNAs 122 and 21 were observed. Conclusions: None of the studied microRNAs were able to differentiate iCH from CuCH and only correlated with ALT activity. Serum microRNAs 122, 1275, and 21 appear to be nonspecific makers of hepatocellular injury. Clinical Relevance: Our results do not support the utility of these microRNAs as markers for distinguishing between iCH and CuCH in dogs.

Objective: To adapt and partially validate the accuracy and precision of serum exchangeable copper and 3-kDa ultrafiltered urinary copper concentrations as an analytical prerequisite to investigate the clinical utility of relative exchangeable copper and urinary small copper carrier-to-creatinine ratio as noninvasive biomarkers in dogs with copper-associated hepatitis. Methods: Serum exchangeable copper and 3-kDa ultrafiltered urinary copper concentrations of surplus samples of dogs submitted for diagnostic purposes from October 2023 through March 2024 were measured by adapting a previously described ultracentrifugation method. Copper concentrations were quantified using inductively coupled plasma mass spectrometry. The optimal relative centrifugal force was selected based on minimizing the variability among replicates. Analytical performance was evaluated through spike recovery and intra- and interassay repeatability. Results: Optimal parameters were identified as 5 g/L EDTA and 5,000 X g for serum exchangeable copper and 4,000 X g for 3-kDa ultrafiltered urinary copper. Spike recovery ranged from 97.9% to 108.5% for serum exchangeable copper and from 96.7% to 120% for ultrafiltered urinary copper. The median intra- and interassay coefficients of variation were 8.5% and 10.8% for exchangeable copper and 2.6% and 5.6% for ultrafiltered urinary copper, respectively. Conclusions: Ultrafiltration-based methods for measuring serum exchangeable copper and 3-kDa ultrafiltered urinary copper concentrations were successfully adapted for dogs, with spike recovery and intra- and interassay coefficients of variation within predefined acceptance criteria. Clinical Relevance: The results provide the analytical foundation for future investigation of the diagnostic utility of relative exchangeable copper and urinary small copper carrier protein as noninvasive biomarkers for copper-associated hepatitis in dogs.