No 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.

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.

The gut microbiota modifies endogenous primary bile acids (BAs) to produce exogenous secondary BAs, which may be further metabolized by cytochrome P450 enzymes (P450s). Our primary aim was to examine how the host adapts to the stress of microbe-derived secondary BAs by P450-mediated oxidative modifications on the steroid nucleus. Five unconjugated tri-hydroxyl BAs that were structurally and/or biologically associated with deoxycholate (DCA) were determined in human biologic samples by liquid chromatographytandem mass spectrometry in combination with enzyme-digestion techniques. They were identified as DCA-19-ol, DCA-6b-ol, DCA-5bol, DCA-6a-ol, DCA-1b-ol, and DCA-4b-ol based on matching in-laboratory synthesized standards. Metabolic inhibition assays in human liver microsomes and recombinant P450 assays revealed that CYP3A4 and CYP3A7 were responsible for the regioselective oxidations of both DCA and its conjugated forms, glycodeoxycholate (GDCA) and taurodeoxycholate (TDCA). The modification of secondary BAs to tertiary BAs defines a host liver (primary BAs)-gut microbiota (secondary BAs)-host liver (tertiary BAs) axis. The regioselective oxidations of DCA, GDCA, and TDCA by CYP3A4 and CYP3A7 may help eliminate host-toxic DCA species. The 19-and 4b-hydroxylation of DCA species demonstrated outstanding CYP3A7 selectivity and may be useful as indicators of CYP3A7 activity. 283

The treatment of common multi-factorial, system-wide diseases using multi-component therapeutics, such as herbal medicines, can be regarded as a "system to system" therapeutic approach. The pharmacokinetics (PK) of multi-component therapeutics is a great technical challenge, which has led to significant limitations in understanding the efficacies and toxicities of combination drugs and herbal medicines. Metabolomics has clearly demonstrated its value in elucidating the interaction of the biological system's genome with its environments, and recently been used in the PK analysis of drugs, xenobiotics and several nutrients. The metabolomics strategy can also be used in pharmacokinetic study of plant derived agents to demonstrate their biological fates, thereby linking the plant metabolome to human/animal metabolome, and bridging the gap between multi-component agents and molecular pharmacology. This paper reviews the current progress of metabolomics based PK studies, presents the potentials and challenges of such a strategy for herbal medicines, as well as looks into the future of the role of metabolomics in the relationship between PK, PD and TOX of herbal medicines.

Polyphenols, a ubiquitous group of secondary plant metabolites sharing at least one aromatic ring structure with one or more hydroxyl groups, represent a large group of natural antioxidants abundant in fruits, vegetables, and beverages, such as grape juice, wine, and tea, and are widely considered to contribute to health benefits in humans. However, little is yet known concerning their bioactive forms in vivo and the mechanisms by which they may alter our metabolome, which ultimately contribute toward disease prevention. Here we report a study to determine the metabolic fate of polyphenolic components in a Chinese tea (Pu-erh) in human subjects using a metabonomic profiling approach coupled with multivariate and univariate statistical analysis. Urine samples were collected at 0 h, 1 h, 3 h, 6 h, 9 h, 12 h, and 24 h within the first 24 h and once a day during a 6 week period including a 2 week baseline phase, a 2 week daily Pu-erh tea ingestion phase, and a 2 week "wash-out" phase, and they were analyzed by gas chromatography mass spectrometry and liquid chromatography mass spectrometry. The dynamic concentration profile of bioavailable plant molecules (due to in vivo absorption and the hepatic and gut bacterial metabolism) and the human metabolic response profile were measured and correlated with each other. This study demonstrates that the metabonomic strategy will enable us to integrate the overwhelming amount of metabolic end points as a systems' response to the absorption, metabolism, and disposition of a multicomponent botanical intervention system, leading to a direct elucidation of their mechanisms of action.

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Determination of pharmacokinetics (PKs) of multicomponent pharmaceuticals and/or nutraceuticals (polypharmacokinetics, poly-PKs) is difficult due to the vast number of compounds present in natural products, their various concentrations across a wide range, complexity of their interactions, as well as their complex degradation dynamics in vivo. Metabolomics coupled with multivariate statistical tools that focus on the comprehensive analysis of small molecules in biofluids is a viable approach to address the challenges of poly-PK. This paper discusses recent advances in the characterization of poly-PK and the metabolism of multicomponent xenobiotic agents, such as compound drugs, dietary supplements, and herbal medicines, using metabolomics strategy. We propose a research framework that integrates the dynamic concentration profile of bioavailable xenobiotic molecules that result from in vivo absorption and hepatic and gut bacterial metabolism, as well as the human metabolic response profile. This framework will address the bottleneck problem in the pharmacological evaluation of multicomponent pharmaceuticals and nutraceuticals, leading to the direct elucidation of the pharmacological and molecular mechanisms of these compounds.