Key Role for the 12-Hydroxy Group in the Negative Ion Fragmentation of Unconjugated C24 Bile Acids

Abstract

Host-gut microbial interactions contribute to human health and disease states and an important manifestation resulting from this co-metabolism is a vast diversity of bile acids (BAs). There is increasing interest in using BAs as biomarkers to assess the health status of individuals, and therefore, an increased need for their accurate separation and identification. In this study, the negative ion fragmentation behaviors of C24 BAs were investigated by UPLC-ESI-QTOF-MS. The step-by-step fragmentation analysis revealed a distinct fragmentation mechanism for the unconjugated BAs containing a 12-hydroxyl group. The unconjugated BAs lacking 12-hydroxylation fragmented via dehydration and dehydrogenation. In contrast, the 12-hydroxylated ones, such as deoxycholic acid (DCA) and cholic acid (CA), employed dissociation routes including dehydration, loss of carbon monoxide or carbon dioxide, and dehydrogenation. All fragmentations of the 12-hydroxylated unconjugated BAs, characterized by means of stable isotope labeled standards, were associated with the rotation of the carboxylate side-chain and the subsequent rearrangements accompanied with proton transfer between 12-hydroxyl and 24-carboxyl groups. Compared to DCA, CA underwent further cleavages of the steroid skeleton. Accordingly, the effects of stereochemistry on the fragmentation pattern of CA were investigated using its stereoisomers. Based on the knowledge gained from the fragmentation analysis, a novel BA, 3β, 7β, 12α-trihydroxy-5β-cholanic acid, was identified in the postprandial urine samples of patients with nonalcoholic steatohepatitis. The analyses used in this study may contribute to a better understanding of the chemical diversity of BAs and the molecular basis of human liver diseases that involve BA synthesis, transport, and metabolism.

Analysis of human C24 bile acids metabolome in serum and urine based on enzyme digestion of conjugated bile acids and LC-MS determination of unconjugated bile acids

“…The LM and HM resolutions were set at 25.0 and 15.0, respectively. Such settings confined the quadrupole transmission window, excluded isotopic contributors and facilitated data analysis [28]. …”

“…The enzyme-digestion protocols were developed and validated to achieve optimal selectivity and efficacy of the enzymes capable of hydrolyzing conjugated BAs, as well as, achieving acceptable unconjugated BA stability during incubation. Based on our previous work, which focused on negative fragmentation and chromatographic separation of BAs by LC-MS techniques [28,29], the fine-tuned chromatographic gradient and advanced multiple reaction monitor (MRM) were jointly employed to separate and detect the unconjugated BAs in human serum and urine. The validated method developed here allows for analysis of a more complete structural diversity of the human BAs metabolome associated not only with the oxidative stereochemistry but also with varying conjugation patterns.…”

Planar bile acids in health and disease

“…This study examined the BA content in liver and bile at three different time points and in different hepatic compartments during hepatocarcinogenesis development: the foci stage (12 weeks following chemical induction), hepatoma stage (20 weeks), and carcinoma stage (32 weeks) in rat hepatocyte nuclei, homogenate, and in bile [5]. The allo -BAs, predominantly allo -cholic acid, reached a peak concentration in bile at the hepatoma stage (20 weeks), whereas Δ 4 -unsaturated BAs were elevated at the carcinoma stage (32 weeks) [3, 5]. The authors noted that increased abundance of Δ 4 -unsaturated BAs approximately coincided with the maximal loss of hepatocyte differentiation at the carcinoma stage, whereas the allo -BAs’ increased secretion began much earlier in hepatocarcinogenesis and was maintained throughout disease progression [5].…”

“…While BAs have been studied for nearly a century, human BAs encompass nearly 50 species synthesized by the human body alone, and modifications made by intestinal bacteria result in almost 400 derivatives of the C 24 core structure [3]. Most of these species are still poorly characterized [3].…”

“…Most of these species are still poorly characterized [3]. One of the least studied subspecies are the “flat” BAs, a group of C 24 cholanoic acids that retain the planar structure of their cholesterol predecessor instead of the “bent” or “twisted” structure of the typical mammalian BAs [2, 4–11].…”