Metabolic Bile Acid Profile Impairments in Dogs Affected by Chronic Inflammatory Enteropathy.

Bile acids (BAs), endogenous acidic steroids synthetized from cholesterol in the liver, play a key role in the gut-liver axis physiopathology, including in hepatotoxicity, intestinal inflammatory processes, and cholesterol homeostasis. Faecal Oxo-BAs, relatively stable intermediates of oxidation/epimerization reactions of the BA hydroxyls, could be relevant to investigating the crosstalk in the liver-gut axis and the relationship between diseases and alterations in microbiota composition. A paucity of information currently exists on faecal BA profiles in dogs with and without chronic inflammatory enteropathy (CIE). Comprehensive assessment of 31 molecules among faecal BAs and related microbiota metabolites was conducted with high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). Odds ratios (ORs) for associations of BAs with CIE were estimated using logistic regression. Principal component analysis was performed to find differences between the control and pathological dogs. Higher levels of primary BAs and muricholic acids, and lower levels of secondary BAs were found in pathological dogs. Higher concentrations in faecal oxo-metabolites were associated with the absence of CIE (OR < 1). This study shows a marked difference in faecal BA profiles between dogs with and without CIE. Further research will be needed to better understand the role of oxo-BAs and muricholic acids in CIE dogs.

Extraction method for the multiresidue analysis of legacy and emerging pollutants in marine mussels from the Adriatic Sea.

The release of hazardous chemicals into aquatic environments has long been a known problem, but its full impact has only recently been realized. This study presents a validated liquid chromatography-mass spectrometry (HPLC-MS/MS) method for detecting pharmaceutical and pesticide residues in mussels (Mytilus galloprovincialis). An innovative MS-compatible extraction method was developed and validated, demonstrating successful recovery rates for analytes at three different concentration levels (25-95%). The method detected the target analytes at ng/g concentrations with high accuracy (-7% to 11%) and low relative standard deviation (<10%) for both intra-day and inter-day analyses. After validation, the method was applied to mussel samples collected from a commercial farm near Senigallia, Adriatic Sea, detecting different contaminants in the range of 2-40 ng/g (dry weight). The study provides a valuable tool for investigating the potential threats posed by diverse contaminant classes with high annual tonnage, including analytes with known persistence and/or illegal status.

Seasonal dynamics of the microbiome-host response to pharmaceuticals and pesticides in Mytilus galloprovincialis farmed in the Northwestern Adriatic Sea.

Marine mussels, especially Mytilus galloprovincialis, are well-established sentinel species, being naturally resistant to the exposure to multiple xenobiotics of natural and anthropogenic origin. Even if the response to multiple xenobiotic exposure is well known at the host level, the role of the mussel-associated microbiome in the animal response to environmental pollution is poorly explored, despite its potential in xenobiotic detoxification and its important role in host development, protection, and adaptation. Here, we characterized the microbiome-host integrative response of M. galloprovincialis in a real-world setting, involving exposure to a complex pattern of emerging pollutants, as occurs in the Northwestern Adriatic Sea. A total of 387 mussel individuals from 3 commercial farms, spanning about 200 km along the Northwestern Adriatic coast, and in 3 different seasons, were collected. Multiresidue analysis (for quantitative xenobiotic determination), transcriptomics (for host physiological response), and metagenomics (for host-associated microbial taxonomical and functional features) analyses were performed on the digestive glands. According to our findings, M. galloprovincialis responds to the presence of the complex pattern of multiple emerging pollutants - including the antibiotics sulfamethoxazole, erythromycin, and tetracycline, the herbicides atrazine and metolachlor, and the insecticide N,N-diethyl-m-toluamide - integrating host defense mechanisms, e.g., through upregulation of transcripts involved in animal metabolic activity, and microbiome-mediated detoxification functions, including microbial functionalities involved in multidrug or tetracycline resistance. Overall, our data highlight the importance of the mussel-associated microbiome as a strategic player for the orchestration of resistance to the multixenobiotic exposure at the holobiont level, providing strategic functionalities for the detoxification of multiple xenobiotic substances, as occurring in real world exposure settings. Complementing the host with microbiome-dependent xenobiotic degradative and resistance genes, the M. galloprovincialis digestive gland associated microbiome can have an important role in the detoxification of emerging pollutants in a context of high anthropogenic pressure, supporting the relevance of mussel systems as potential animal-based bioremediation tool.

Determination of Free Amino Acids in Milk, Colostrum and Plasma of Swine via Liquid Chromatography with Fluorescence and UV Detection.

Amino acids are ubiquitous components of mammalian milk and greatly contribute to its nutritional value. The compositional analysis of free amino acids is poorly reported in the literature even though their determination in the biological fluids of livestock animals is necessary to establish possible nutritional interventions. In the present study, the free amino acid profiles in mature swine milk, colostrum and plasma were assessed using a targeted metabolomics approach. In particular, 20 amino acids were identified and quantified via two alternative and complementary reversed-phase HPLC methods, involving two stationary phases based on core-shell technology, i.e., Kinetex C18 and Kinetex F5, and two detection systems, i.e., a diode array detector (DAD) and a fluorescence detector (FLD). The sample preparation involved a de-proteinization step, followed by pre-chromatographic derivatization with 9-fluorenylmethylchloroformate (FMOC-Cl). The two optimized methods were validated for specificity, linearity, sensitivity, matrix effect, accuracy and precision and the analytical performances were compared. The analytical methods proved to be suitable for free amino acid profiling in different matrices with high sensitivity and specificity. The correlations among amino acid levels in different biological fluids can be useful for the evaluation of physio-pathological status and to monitor the effects of therapeutic or nutritional interventions in humans and animals.

Analysis of fecal bile acids and metabolites by high resolution mass spectrometry in farm animals and correlation with microbiota.

There is a growing interest in the named "acidic sterolbiome" and in the genetic potential of the gut microbiome (GM) to modify bile acid (BA) structure. Indeed, the qualitative composition of BAs in feces correlates with the bowel microorganisms and their collective genetic material. GM is responsible for the production of BA metabolites, such as secondary and oxo-BAs. The specific BA profiles, as microbiome-host co-metabolic products, could be useful to investigate the GM-host interaction in animals under physiological conditions, as well as in specific diseases. In this context, we developed and validated an ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry method for the simultaneous analysis of up to 21 oxo-BAs and their 9 metabolic precursors. Chromatographic separation was achieved in 7 min with adequate analytical performance in terms of selectivity, sensitivity (LOQ from 0.05 to 0.1 µg/mL), accuracy (bias% < 5%), precision (CV% < 5%) and matrix effect (ME% < 10%). A fast solvent extraction protocol has been fine-tuned, achieving recoveries > 90%. In parallel, the gut microbiota assessment in farming animals was evaluated by 16S rRNA next-generation sequencing, and the correlation with the BA composition was performed by multivariate analysis, allowing to reconstruct species-specific associations between the BA profile and specific GM components.