Abstract
Bile acids (BAs) are a group of chemically related steroids recognized as regulatory molecules whose profiles can change in different physio-pathological situations. We have developed a sensitive, fast, and reproducible ultraperformance liquid chromatography/multiple reaction monitoring/mass spectrometry method to determine the tissue and sera BA profiles in different species (human, rat, and mouse) by quantifying 31 major and minor BA species in a single 21-min run. The method has been validated according to FDA guidelines, and it generally provides good results in terms of intra- and interday precision (less than 8.6% and 16.0%, respectively), accuracy (relative error measurement between -11.9% and 8.6%), and linearity (R(2) > 0.996 and dynamic ranges between two and four orders of magnitude), with limits of quantification between 2.5 and 20 nM. The new analytical approach was applied to determine BA concentrations in human, rat, and mouse serum and in liver tissue. Our comparative study confirmed and extended previous reports, showing marked interspecies differences in circulating and hepatic BA composition. The targeted analysis revealed the presence of unexpected minoritary BAs, such as tauro-alpha-Muricholic acid in human serum, thus allowing us to obtain a thorough profiling of human samples. Its great sensitivity, low sample requirements (25 µl of serum, 5 mg of tissue), and comprehensive capacity to profile a considerable number of BAs make the present method a good choice to study BA metabolism in physiological and pathological situations, particularly in toxicological studies.
Highlights
Bile acids (BAs) are a group of chemically related steroids recognized as regulatory molecules whose profiles can change in different physio-pathological situations
An ultraperformance liquid chromatography (UPLC)-multiple reaction monitoring (MRM)-mass spectrometry (MS) method for targeted BA profiling in different biological matrices has been developed in our laboratory
In the glycine-conjugated species, an m/z value of 74 was selected as the common product ion for all the glycine conjugates, and m/z values of 432.3, 448.3, 464.3, and 458.3 were selected as the precursor ions for mono, di, and tri-hydroxylated and glycodehydrocholic acid (GDHCA) bile acids, respectively
Summary
Bile acids (BAs) are a group of chemically related steroids recognized as regulatory molecules whose profiles can change in different physio-pathological situations. We have developed a sensitive, fast, and reproducible ultraperformance liquid chromatography/multiple reaction monitoring/mass spectrometry method to determine the tissue and sera BA profiles in different species (human, rat, and mouse) by quantifying 31 major and minor BA species in a single 21-min run. Targeted profiling of circulating and hepatic bile acids in human, mouse, and rat using a UPLC-MRMMS-validated method. PXR or VDR) and the G-protein coupled receptor TGR5 and can activate several cell signaling pathways (i.e., JNK, ERK, or AKT) [5,6,7,8] By activating these receptors and signaling cascades, BAs regulate their own homeostasis and fatty acid, lipoprotein, glucose, and energy metabolism [5, 7, 9]. The primary signaling function of BAs seems to be the regulation of metabolic flux in the liver and the gastrointestinal tract during the feed/fast cycle; they are involved in the control of cell proliferation and inflammatory processes [5, 9]
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