Endocrine Abstracts

Background: Bile acids are cholesterol metabolites that influence glucose and energy homeostasis via receptors including FXR and TGR5. Translational endocrinology needs robust, isomer-resolving bile acid quantification in clinically relevant matrices.

Aim: To develop and validate a high-resolution HPLC–ESI–MS assay for major bile acids and key precursors, and to demonstrate utility across human and mouse samples.

Methods: An HPLC–ESI–Orbitrap method (negative ion mode) was optimised to quantify 17 bile acids (unconjugated, glycine- and taurine-conjugated) plus two precursors (dihydroxycholestanoic acid and trihydroxycholestanoic acid) using three deuterated internal standards. Sensitivity, linearity and within-/between-day precision and accuracy were evaluated using pooled plasma spiked at four QC levels. The assay was applied to healthy human plasma and faeces and to mouse plasma and tissues. For biological validation, Swiss mice received cephalothin (2 mg/mL) and neomycin (2 mg/mL) in drinking water for 17 days (n = 10/group).

Results: Limits of detection (LOD) were 0.05–0.30 nmol/mL and lower limits of quantification 0.1–2.0 nmol/mL, with chromatographic separation of key isomer groups. Precision/accuracy met predefined criteria for 15 analytes across the analytical range. We quantified 11 bile acids in human plasma and faeces; human plasma profiles were dominated by glycochenodeoxycholic acid, chenodeoxycholic acid and cholic acid (~65% of total quantified bile acids). Cross-species profiling showed expected differences: in mouse plasma, taurine conjugates comprised >95% of total bile acids, with taurocholic acid and tauro-β-muricholic acid representing >80%. Antibiotic treatment decreased most unconjugated bile acids (often below LOD) and increased most tauro-conjugated bile acids up to 10-fold in colon and caecal content.

Conclusion: This validated platform enables sensitive bile acid and precursor profiling in human plasma/faeces with complementary mouse data, supporting endocrine–metabolic phenotyping and biomarker work involving dihydroxycholestanoic acid and trihydroxycholestanoic acid in disorders of cholesterol–bile acid metabolism.