Profiling urinary bile acids by targeted liquid chromatography-tandem mass spectrometry

Abstract

Bile acids (BA) are C24 steroids synthesized from cholesterol in the liver. Apart from emulsification of fatty food components, they function as endocrine signaling molecules. As such, bile acids bear great potential as future biomarkers in diagnosis and monitoring of metabolic diseases. However, hardly any data exist on BA in urine. Therefore, the present study aimed at developing and implementing a new method for the quantification of urinary bile acids using targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS). The targeted approach included 18 BA: 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. Furthermore, ursodeoxycholic acid (UDCA) and five BA in their sulfated forms (LCA-S, GLCA-S, TLCA-S, GCDCA-S, GDCA-S) were analyzed. Another goal consisted in presenting first reference values of urinary bile acids during childhood and to investigate their excretion patterns in obese children and adolescents. Finally, potential correlations between urinary bile acids and nutrition were examined. The method required 2 mL of urine and sample preparation consisting of protein precipitation and solid phase extraction. Stable isotopes of BA were included as internal standards (IS). The method was successfully validated and then applied to samples of 80 healthy children as well as 237 obese children of various age groups. The results were presented in three different units ([nmol/L], [nmol BA/mmol Crea] and [nmol/d]). Regardless of the unit, sulfated bile acids (GCDCA-S, GLCA-S, GDCA-S, TLCA-S) dominated in both study groups, CA and GCA were the two dominant non-sulfated BA. Lower bile acid sulfation and amidation in obese children may point to obesity-related limitations in hepatic metabolic capacity. Glycine-amidation appeared to be less prone to obesity-related impairments. Urinary concentration of GCDCA-S decreased with higher carbohydrate intake, while it increased with higher fat or protein intake, respectively.