ESPE2021 ePoster Category 2 Fat, metabolism and obesity (59 abstracts)
Exploring Urinary Bile Acids as Potential Markers of Metabolism: Reference Values in Children by Targeted LC-MS/MS
Marcel Schauermann 1 , Rong Wang 1 , Michaela F. Hartmann 1 , Thomas Remer 2 , Yifan Hua 2 , Michael Shmoish 3 , Abdullah Bereket 4 , Malgorzata Wasniewska 5 , Ze’ev Hochberg 6 , Aneta Gawlik 7 & Stefan A. Wudy 1
1Steroid Research and Mass Spectrometry Unit, Division of Pediatric Endocrinology & Diabetology, Center of Child and Adolescent Medicine, Justus-Liebig-University, Gießen, Germany; 2DONALD Study Center, Department of Nutritional Epidemiology, Institute of Nutrition and Food Science, University of Bonn, Dortmund, Germany; 3Bioinformatics Knowledge Unit, Lorry I. Lokey Interdisciplinary Center for Life Sciences and Engineering, Technion-Israel Institute of Technology, Haifa, Israel; 4Department of Pediatric Endocrinology, Marmara University Faculty of Medicine, Istanbul, Turkey; 5Department of Pediatric, Gynecological, Microbiological and Biomedical Sciences, University of Messina, Via Consolare Valeria, Messina, Italy; 6The Ruth and Bruce Rappaport Faculty of Medicine, Technion - Israel Institute of Technology, Haifa, Israel; 7Department of Pediatrics and Pediatric Endocrinology, School of Medicine in Katowice, Medical University of Silesia, Upper Silesia Children’s Care Health Centre, Katowice, Poland
Bile acids (BA) are C24 steroids synthesized from cholesterol in liver. Originally, they were mainly considered to function as emulsifiers supporting resorption of lipophilic food compounds and excretion of metabolic products. Recently, their additional biological and endocrine functions in the regulation of metabolism have aroused interest of researchers. In contrast to BA in blood, it is surprising that hardly any data exist on BA in the most accessible human biofluid urine. Therefore, a targeted liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed and validated to analyze 18 BA simultaneously. The method contains the measurement of the primary BA cholic acid (CA) and chenodeoxycholic acid (CDCA) and the secondary BA deoxycholic acid (DCA) and lithocholic acid (LCA) as well as glycine and taurine conjugates of these four BA (GCA, TCA, GCDCA, TCDCA, GDCA, TDCA, GLCA, TLCA). Furthermore, ursodeoxycholic acid (UDCA) and five BA in their sulfated forms (LCA-S, GLCA-S, TLCA-S, GCDCA-S, GDCA-S) were analyzed. The method required 2 mL of urine and sample preparation consisting of protein precipitation and solid phase extraction. The method achieved good linearity (R2 > 0.99) and recovery (90.49% - 113.99%). LOQ was determined as 6.25 ng/ml for each compound (except for GCDCA: 18.75 ng/ml). Intra-day/inter-day precision and accuracy ranged from 0.42% to 11.47% and 85.75% to 110.99%, respectively. No significant matrix effect was observed. The method was successfully applied to samples of 80 healthy children of various age groups. CA (median: 55.2 ng/ml) and GCA (48.9 ng/ml) were the two dominant non-sulfated BA. However, sulfated BA showed much higher concentrations, with GCDCA-S (337.5 ng/ml) showing the highest levels among all BA, followed by GLCA-S (197.4 ng/ml) and GDCA-S (183.2 ng/ml). In total, 86.5% of quantified BA were sulfated. The total concentrations of glycine amidated BA measured were higher than taurine amidated and non-amidated ones. No obvious trends between urinary BA and age or sex, respectively were observed. We established a method for profiling concentrations of urinary BA in humans. Our data showed that urinary BA are mostly present in their sulfated form in children’s urine, indicating hepatic sulfation to be the major metabolic pathway for urinary BA excretion in humans. The study provides important information on the ontogenesis and metabolism of human BA. Our reference data for urinary BA from healthy children lay the foundation for further investigations into their potential role as metabolic and hepatic biomarkers.
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