Metabolic Profiling of Bile Acids in Human and Mouse Blood by LC-MS/MS in Combination with Phospholipid-Depletion Solid-Phase Extraction (original) (raw)
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Diagnostics, 2020
Bile acids (BA) play a pivotal role in cholesterol metabolism. Their blood concentration has also been proposed as new prognostic and diagnostic indicator of hepatobiliary, intestinal, and cardiovascular disease. Liquid chromatography tandem mass spectrometry (LC–MS/MS) currently represents the gold standard for analysis of BA profile in biological samples. We report here development and validation of a LC–MS/MS technique for simultaneously quantifying 15 BA species in serum samples. We also established a reference range for adult healthy subjects (n = 130) and performed a preliminary evaluation of in vitro and in vivo interference. The method displayed good linearity, with high regression coefficients (>0.99) over a range of 5 ng/mL (lower limit of quantification, LLOQ) and 5000 ng/mL for all analytes tested. The accuracies were between 85–115%. Both intra- and inter-assay imprecision was <10%. The recoveries ranged between 92–110%. Each of the tested BA species (assessed on ...
Journal of Chromatography B, 2012
The role of bile acids in cell metabolism, membrane biology and cell signaling is increasingly recognized, thus making necessary a robust and versatile technique to extract, separate and quantify a large concentration range of these numerous molecular species. HPLC-MS/MS analysis provides the highest sensitivity to detect and identify bile acids. However, due to their large chemical diversity, extraction methods are critical and quite difficult to optimize, as shown by a survey of the literature. This paper compares the performances of four bile acid extraction protocols applied to either liquid (serum, urine, bile) or solid (stool) samples. Acetonitrile was found to be the best solvent for deproteinizing liquid samples and NaOH the best one for stool extraction. These optimized extraction procedures allowed us to quantitate as much as 27 distinct bile acids including sulfated species in a unique 30 min HPLC run, including both hydrophilic and hydrophobic species with a high efficiency. Tandem MS provided a non ambiguous identification of each metabolite with a good sensitivity (LOQ below 20 nmol/l except for THDCA and TLCA). After validation, these methods, successfully applied to a group of 39 control patients, detected 14 different species in serum in the range of 30-800 nmol/l, 11 species in urine in the range of 20-200 nmol/l and 25 species in stool in the range of 0.4-2000 nmol/g. The clinical interest of this method has been then validated on cholestatic patients. The proposed protocols seem suitable for profiling bile acids in routine analysis.
Journal of Chromatography B, 2009
Beside their role as lipid solubilizers, bile acids (BAs) are increasingly appreciated as signaling factors. As ligands of G-protein coupled receptors and nuclear hormone receptors BAs control their own metabolism and act on lipid and energy metabolism. To study BA function in detail, it is necessary to use methods for their quantification covering the structural diversity of this group. Here we present a simple, sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the analysis of bile acid profiles in human plasma/serum. Protein precipitation was performed in the presence of stable-isotope labeled internal standards. In contrast to previous LC-MS/MS methods, we used a reversed-phase C18 column with 1.8 m particles and a gradient elution at basic pH. This allows base line separation of 18 bile acid species (free and conjugated) within 6.5 min run time and a high sensitivity in negative ion mode with limits of detection below 10 nmol/L. Quantification was achieved by standard addition and calibration lines were linear in the tested range up to 28 mol/L. Validation was performed according to FDA guidelines and overall imprecision was below 11% CV for all species. The developed LC-MS/MS method for bile acid quantification is characterized by simple sample preparation, baseline separation of isobaric species, a short analysis time and provides a valuable tool for both, routine diagnostics and the evaluation of BAs as diagnostic biomarkers in large clinical studies.
Journal of Chromatography B, 2008
We report a sensitive, generic method for quantitative profiling of bile acids and other endogenous metabolites in small quantities of various biological fluids and tissues. The method is based on a straightforward sample preparation, separation by reversed-phase high performance liquid-chromatography mass spectrometry (HPLC-MS) and electrospray ionisation in the negative ionisation mode (ESI−). Detection is performed in full scan using the linear ion trap Fourier transform mass spectrometer (LTQ-FTMS) generating data for many (endogenous) metabolites, not only bile acids. A validation of the method in urine, plasma and liver was performed for 17 bile acids including their taurine, sulfate and glycine conjugates. The method is linear in the 0.01-1 M range. The accuracy in human plasma ranges from 74 to 113%, in human urine 77 to 104% and in mouse liver 79 to 140%. The precision ranges from 2 to 20% for pooled samples even in studies with large number of samples (n > 250). The method was successfully applied to a multi-compartmental APOE*3-Leiden mouse study, the main goal of which was to analyze the effect of increasing dietary cholesterol concentrations on hepatic cholesterol homeostasis and bile acid synthesis. Serum and liver samples from different treatment groups were profiled with the new method. Statistically significant differences between the diet groups were observed regarding total as well as individual bile acid concentrations.
The Journal of Lipid Research, 2012
Bile acids (BAs) are major components of bile formed from cholesterol through various enzymatic reactions in hepatocytes. Before being excreted into bile canaliculi, primary BAs synthesized in the liver (i.e., cholic acid [CA] and chenodeoxycholic acid [CDCA] in humans and alphamuricholic acid [ ␣ MCA] and beta-muricholic acid [  MCA] in rodents) (1, 2) are mainly conjugated with taurine or glycine amino acids through the terminal side-chain carboxylic group present in the BA structure. Once in the intestine, primary BAs are deconjugated and converted into secondary BAs by microbiota. Then, most BAs are reabsorbed back to the liver, conjugated by hepatocytes, and re-excreted into bile to complete enterohepatic circulation (3). In the past, BAs were considered to be mere detergents required for the solubilization and absorption of dietary fats. However, BAs are now recognized as regulatory molecules capable of activating specifi c receptors. BAs are Abstract Bile acids (BAs) are a group of chemically related steroids recognized as regulatory molecules whose profi les 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 profi les 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 quantifi cation 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 confi rmed 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 profi ling of human samples. Its great sensitivity, low sample requirements (25 µl of serum, 5 mg of tissue), and comprehensive capacity to profi le 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.-García-Cañaveras, J. C., M. T. Donato, J. V. Castell, and A. Lahoz. Targeted profi ling of circulating and hepatic bile acids in human, mouse, and rat using a UPLC-MRM-MS-validated method.
Analytical and Bioanalytical Chemistry, 2014
An ultra high performance liquid chromatography tandem mass spectrometry method (UHPLC-MS/MS) was developed for the determination of 33 target and 28 unknown bile acids (BAs) in biological samples. Sixty-one BAs could be measured in 20 min using only a small amount of sample and with a simple sample preparation. The method proved to be very sensitive (limit of detection 5-350 pg/mL, lower limit of quantitation 0.1-2.6 ng/ mL), linear (R 2 >0.99) and reproducible (typically CV <15 % in biological matrixes). The method was used to analyze human adipose tissue, plasma, and serum (from same subjects) and mouse serum, gall bladder, small intestine, and colon samples (from same animals). Cholic acid, ursodeoxycholic acid, and chenodeoxycholic acid, deoxycholic acid, and their conjugates (mainly glycine, but also taurine conjugates) were the main metabolites in human samples, and cholic acid, glycine cholic acid, and several taurine conjugates in mouse samples. Using the method, 28 unknown BAs could also be detected. UHPLC-MS/ MS spectra, accurate mass, and tissue distribution suggested that nine of the unknown bile acids were taurine conjugates, 13 were glycine conjugates, and six were intact BAs, respectively. To our knowledge, this was the first time BAs were detected in adipose tissue. Results showed that 17 targeted BAs were found at ng/g level in human adipose tissue. Our findings give a novel insight of the endogenous role of BAs in adipose tissue and their role as biomarkers (e.g., in metabolic diseases).
Analytical chemistry, 2015
Bile acids are important end products of cholesterol metabolism. While they have been identified as key factors in lipid emulsification and absorption due to their detergent properties, bile acids have also been shown to act as signaling molecules and intermediates between the host and the gut microbiota. To further the investigation of bile acid functions in humans, an advanced platform for high throughput analysis is essential. Herein, we describe the development and application of a 15 min UPLC procedure for the separation of bile acid species from human biofluid samples requiring minimal sample preparation. High resolution time-of-flight mass spectrometry was applied for profiling applications, elucidating rich bile acid profiles in both normal and disease state plasma. In parallel, a second mode of detection was developed utilizing tandem mass spectrometry for sensitive and quantitative targeted analysis of 145 bile acid (BA) species including primary, secondary, and tertiary b...
Annals of Clinical Biochemistry: International Journal of Laboratory Medicine, 2010
Background WEQAS, one of the largest EQA (External Quality Assessment) providers in the UK, offers a bile acid EQA scheme, with linear serum pools containing cholic acid, deoxycholic acid and chenodeoxycholic acid, reflecting levels observed in obstructive cholestasis. Total bile acids are currently measured routinely by non-specific enzymatic methods. Traceability of results to the SI unit utilizing reference target values is the preferred method of comparison of returned results where available, ensuring the transfer of accuracy from definitive methods to routine methods. Methods Target values have been assigned to EQA material utilizing isotope dilution gas chromatography mass spectrometry (ID-GCMS). The methodology was based on published routine methods and adapted for use as a ID-GCMS target method. The total bile acid target value was reported as the sum of the three major bile acids measured: cholic acid, deoxycholic acid and chenodeoxycholic acid. Results The produced target...
Analysis of Bile Acids Profile in Human Serum by Ultrafiltration Clean-up and LC-MS/MS
Chromatographia, 2012
Bile acids (BAs) are useful biomarkers for the diagnosis of many diseases. The pathologies related to bile acid synthesis are often expressed in the first years of life and may lead to serious liver injury. Here we present a sensitive and rapid method for the analysis of the main 14 bile acids in human serum by liquid chromatography-tandem mass spectrometry. The chromatographic separation is performed using a core-shell column which provides improved separation, highly desirable considering the small structural differences among the analytes. All isomeric BAs of interest were resolved in less than 9 min. Sample pretreatment consisted in ultrafiltration of serum after addition of methanol by means of centrifugal filter devices. The calculated LOQs ranged between 2 and 5 ng mL -1 with linearity of the calibration curves in the 5-5,000 ng mL -1 range for all the BAs. The extraction recoveries for all the analytes were higher than 80 %. Intraday and inter-day coefficients of variation were all below 15 %. The method proposed has been validated and has been applied for the analysis of serum of pediatric patients. This simple procedure allowed minimal consumption of serum sample (about 100 lL) and a rapid assay, easily implementable in routine analysis.