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

Abstract

Host-gut microbiota metabolic interactions are closely associated with health and disease. A manifestation of such co-metabolism is the vast structural diversity of bile acids (BAs) involving both oxidative stereochemistry and conjugation. Herein, we describe the development and validation of a LC-MS-based method for the analysis of human C24 BA metabolome in serum and urine. The method has high throughput covering the discrimination of oxidative stereochemistry of unconjugated species in a 15-min analytical cycle. The validated quantitative performance provided an indirect way to ascertain the conjugation patterns of BAs via enzyme-digestion protocols that incorporated the enzymes, sulfatase, β-glucuronidase, and choloylglycine hydrolase. Application of the method has led to the detection of at least 70 unconjugated BAs including 27 known species and 43 newly found species in the post-prandial serum and urine samples from 7 nonalcoholic steatohepatitis patients and 13 healthy volunteers. Newly identified unconjugated BAs included 3α, 12β-dihydroxy-5β-cholan-24-oic acid, 12α-hydroxy-3-oxo-5β-cholan-24-oic acid, and 3α, 7α, 12β-trihydroxy-5β-cholan-24-oic acid. High-definition negative fragment spectra of the other major unknown species were acquired to facilitate future identification endeavors. An extensive conjugation pattern is the major reason for the "invisibility" of the newly found BAs to other common analytical methods. Metabolomic analysis of the total unconjugated BA profile in combination with analysis of their conjugation patterns and urinary excretion tendencies have provided substantial insights into the interconnected roles of host and gut microbiota in maintaining BA homeostasis. It was proposed that the urinary total BA profile may serve as an ideal footprint for the functional status of the host-gut microbial BA co-metabolism. In summary, this work provided a powerful tool for human C24 BA metabolome analysis that bridges the gap between GC-MS techniques in the past age and LC-MS techniques currently prevailing in biomedical researches. Further applications of the present method in clinical, translational research, and other biomedical explorations will continue to boost the construction of a host-gut microbial co-metabolism network of BAs and thus facilitate the decryption of BA-mediated host-gut microbiota crosstalk in health and diseases. Graphical abstract ᅟ.

Continuum of Host-Gut Microbial Co-metabolism: Host CYP3A4/3A7 are Responsible for Tertiary Oxidations of Deoxycholate Species

“…As shown in Fig. 1, A-C, we found five unknown tri-hydroxyl BA species (TBA05, TBA09, TBA10, TBA13 and TBA18) in the Tertiary Bile Acids Metabolism 285 at ASPET Journals on July 10, 2020 dmd.aspetjournals.org post-prandial urine and serum of 13 healthy volunteers collected at UNC-CH Hospitals (Zhu et al, 2018). Enzyme digestion techniques demonstrated that they were extensively conjugated, thus, explaining their "invisibility" to those direct analysis techniques without consideration of BA conjugation.…”

“…Enzyme digestion techniques demonstrated that they were extensively conjugated, thus, explaining their "invisibility" to those direct analysis techniques without consideration of BA conjugation. The Quad time-of flight MS/MS spectra captured in the digested urine samples showed that they had fragment ions possibly associated with 12-hydroxylation on the skeleton (Lan et al, 2016;Zhu et al, 2018). They were therefore supposed to be structurally associated with DCA and CA, the two major BAs with 12-hydroxylation.…”

“…Sample Preparation for BAs Analysis. Analysis of BAs metabolome were performed using the enzyme digestion techniques published in our recent work (Zhu et al, 2018). For the postprandial human serum and urine samples from 13 healthy adults, aliquot (50 ml) was transferred to 700-ml round-well 96-well plate in quadruplicate.…”

“…Quantitative LC-MS/MS Analysis. Quantitative analysis of unconjugated BAs was performed using the method published in our recent work on ACQUITY ultraperformance LC coupled to a Xevo TQ-S MS (Waters, Milford, MA) (Yin et al, 2017;Zhu et al, 2018). The mobile phases consisted of 0.01% formic acid in water (mobile phase A) and acetonitrile (mobile phase B).…”

“…The "unusual" gradient used a fast elution with 0.5-1.0 minute to ensure a 15-minute throughput. The decreased elution within 1.0-6.0 minutes was purposefully optimized to separate bHCA, isoCA, and TBA13 (DCA-1b-ol), as discussed in our recent publications (Yin et al, 2017;Zhu et al, 2018). It was because of the slowest elution within 4.0-6.0 minutes that the peaks eluted approximately within the time window demonstrated a slightly broadened peak compared with the other peaks.…”