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.

Keywords: Bile acid; Conjugation; Host-gut microbial co-metabolism; Liquid chromatography; Oxidative stereochemistry; Tandem mass spectrometry.

Fig. 1

Structural diversity of the unconjugated and conjugated C24 bile acids (BAs) with a cis/5β-H A/B ring fusion stereochemistry

Fig. 2.

Ion chromatograms of unconjugated BAs in the mixed standard samples (A) and the digested urine and serum samples (prepared by T4) from a NASH patient (B) and two healthy subjects (C, D). Peak intensity was normalized to the highest response of MRM transients. Peaks were labeled with the NO. recorded in Table 1

Fig. 3

Ion chromatograms of BAs detected in the mixed standard sample (black) and a representative urine sample (as seen in Fig. 1B) prepared without enzymes (T1), with choloylglycine hydrolase (T2), with sulfatase and β-glucuronidase (T3), with all three enzymes (T4). (A) Trihydroxy-BAs detected by pseudo-MRM transition m/z 407 > 407, (B) Dihydroxy-BAs by pseudo-MRM transition m/z 391 > 391, (C) Monohydroxy-BAs detected by pseudo-MRM transition m/z 375 > 375. (D) Differentiation of the detected trihydroxy-BAs by the val-MRM transients. (E) Differentiation of DBA01 (12-epiDCA) and DCA from the other detected dihydroxy-BAs by the val-MRM transient of m/z 391>345. The detected BA species were labeled with the abbreviations recorded in Table 1

Fig. 4

PCA scores plot (A) and loadings plot (B) of the total levels of the detected BA species in the postprandial serum and urine samples from 7 NASH patients (subject A-G) and 13 healthy volunteers (subject H-T). The total levels of the detected BA species were determined in samples prepared using the enzyme-digestion protocol, T4. The detected BA species were labeled in the loadings plot with the NO. and abbreviations recorded in Table 1

Fig. 5

Conjugation patterns and urinary excretion tendencies of the downstream metabolites of CA (A) and CDCA (B) in serum and urine of NASH patients (n=7) and healthy controls (n=13). Data was shown as MEAN ± SEM. The serum level and urine level were given as the area under curve (AUC) of the 0–2h post-prandial serum levels and the urinary concentration in the 0–2h post-prandial urine. Primary BAs were colored in red and secondary BAs were colored in green. The gradient-ramp demonstrated the tendency of certain BAs to undergo renal excretion. The free unconjugated form (Free), free one plus its aminoacyl amidates linked with either glycine or taurine (G/T+Free), free one plus its sulfates and glucuronides (Free+G/S), and the total level of each BA species were determined using the T1, T2, T3 and T4 protocol, respectively