Targeted quantitative analysis of urinary bile acids by liquid chromatography-tandem mass spectrometry: Method development and application to healthy and obese children

Abstract

Bile acids (BA) are C24 steroids synthesized from cholesterol in liver. Hardly any data exist on BA in the most accessible human biofluid urine. As bile acids bear great potential as future biomarkers in diagnosis and monitoring of metabolic diseases, we 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). A second goal consisted in creating first reference values of urinary bile acids during childhood and to investigate their excretion patterns in obese children and adolescents.

Our 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).

Our method is capable of simultaneously determining 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.

After successful validation (intra-day precision 1.02 % - 11.07 %; inter-day precision 0.42–11.47 %.; intra-day accuracy 85.75 % - 108.90 %; inter-day accuracy 86.76 % - 110.99 %; no significant matrix effect; recovery 90.49 % - 113.99 %)., the method was applied to samples of 80 healthy children as well as 237 obese children of various age groups.

Sulfated BA showed the highest concentrations, with GCDCA-S (nmol/L, medians, controls vs. obese 588.4 vs. 360.2) being the most abundant among all BA, followed by GLCA-S (353.9 vs. 344.8) and GDCA-S (319,3 vs. 323.9). CA (135.1 vs. 174.6) and GCA (100.2 vs. 92.4) were the two dominant non-sulfated BA.

In conclusion, we developed a LC-MS/MS method for the simultaneous determination of 18 urinary bile acids in children and adolescents. We created reference values and investigated obese children. Sulfated bile acids dominated in both study groups. Lower bile acid sulfation and amidation in obese children point to limitations in their hepatic metabolic capacity.