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

Abstract

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.

New Insights into Bile Acids Related Signaling Pathways in the Onset of Colorectal Cancer

“…Conjugate BAs are not directly metabolized to secondary BAs and should be firstly deconjugated by BSH, which largely functions as a detoxification mechanism to reduce the bile salts levels in the colonic environment ( Figure 3 and Figure 4 ). Then, anaerobes of the genera Bacteroides, Clostridium, Eubacterium, Lactobacillus and Escherichia convert the unconjugated primary BAs into the secondary BAs LCA and DCA through 7α dehydroxylation by CYP7A1 [ 34 , 35 ]. Lately, six new microbial enzymes involved the conversion of CA into DCA have been identified, establishing a complete roadmap from a primary BA to its secondary derivatives [ 36 ].…”

Fecal biomarkers and micromorphological indicators of sheep penning and flooring at La Fortaleza pre-Hispanic site (Gran Canaria, Canary Islands)

“…The bile acid ratio in fecal matter has also been recently used by Porru et al. (2023) 31 as an identification tool. Bile acids are produced during cholesterol metabolism in the liver, excreted in the intestine, and then transformed into secondary bile acids by microorganisms.…”

“…Bile acids are produced during cholesterol metabolism in the liver, excreted in the intestine, and then transformed into secondary bile acids by microorganisms. 30 , 31 Only a small percentage of these secondary bile acids are excreted, as the vast majority are reabsorbed in the intestine. Almost all feces contain appreciable amounts of lithocholic and deoxycholic acids, according to the studies.…”

“…Almost all feces contain appreciable amounts of lithocholic and deoxycholic acids, according to the studies. 29 , 31 , 55 , 56 Hyodeoxycholic and ursodeoxycholic acids have only been detected in pig feces, while chenodeoxycholic acid points to horse, goat, or goose. Concentrations of chenodeoxycholic acid in horse and goat feces are lower than that of deoxycholic acid but similar to that of lithocholic acid.…”

“… 28 These studies are based on the identification of: 1) sterols, which are steroids typical of plant biomass (mainly stigmasterol and β-sitosterol) and animal tissues (predominantly cholesterol), 29 2) stanols (5α-stanols, 5β-stanols, and epi-5β-stanols), which are biomarkers derived from the reduction of cholesterol and phytosterols in the intestinal tract of most mammals, 29 , 30 and 3) bile acids, modified by microbial flora in the liver and intestines of mammals. 29 , 30 , 31 These molecules appear in different proportions depending on the source animal, which can be ascertained using algorithmic equations that help discern between the presence/absence of feces, 29 , 32 different dietary sources (herbivore, carnivore, or omnivore) 33 , 34 and different herbivorous animals. 26 , 29 …”