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.

中文翻译：

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基于共轭胆汁酸的酶消化和LC-MS法测定未结合胆汁酸的血清和尿液中人C24胆汁酸代谢组的分析

宿主肠道菌群的代谢相互作用与健康和疾病密切相关。这种共代谢的表现是胆汁酸（BAs）的巨大结构多样性，涉及氧化立体化学和共轭作用。在本文中，我们描述了基于LC-MS的分析和分析人C24 BA代谢组在血清和尿液中的方法的开发和验证。该方法具有高通量，涵盖了在15分钟的分析循环中对未结合物种的氧化立体化学的判别。经过验证的定量性能提供了一种间接途径，可通过酶消化方案（包括酶，硫酸酯酶，β-葡糖醛酸糖苷酶和胆酰甘氨酸水解酶）来确定BA的偶联模式。该方法的应用已导致从7名非酒精性脂肪性肝炎患者和13名健康志愿者的餐后血清和尿液样品中检测出至少70种未结合的BA，包括27种已知物种和43种新发现的物种。新近鉴定出的未结合的BA包括3α，12β-二羟基-5β-cholan-24-oic酸，12α-羟基-3-氧代5β-cholan-24-oic酸和3α，7α，12β-三羟基-5β-cholan- 24-油酸。获取了其他主要未知物种的高清负片段光谱，以方便将来的鉴定工​​作。广泛的共轭模式是新发现的BA对其他常见分析方法“不可见”的主要原因。代谢组学分析总的未结合的BA概况，结合其共轭模式和尿液排泄趋势的分析，提供了对宿主和肠道菌群在维持BA稳态中相互联系的作用的深刻见解。有人提出，尿液总BA曲线可以作为宿主-肠道微生物BA共代谢功能状态的理想足迹。总之，这项工作为人类C24 BA代谢组学分析提供了强大的工具，弥合了过去时代的GC-MS技术与生物医学研究中目前流行的LC-MS技术之间的差距。本方法在临床，转化研究中的进一步应用，