Enzyme-Driven LC-HRMS Approach for Specific Recognition of 12α-Hydroxy Bile Acids

Area of Science:

• Biochemistry
• Metabolomics
• Enzymology

Background:

• Bile acids (BAs) are crucial for pathophysiologic assessment, with 12α-hydroxylated bile acids (12HBAs) and non-12α-hydroxylated bile acids (non-12HBAs) synthesized via distinct pathways.
• Differentiating 12HBAs and non-12HBAs is challenging due to limited standard substances.

Purpose of the Study:

• To develop a novel enzymatic method for accurately distinguishing 12HBAs and non-12HBAs in complex biological samples.
• To establish a new approach for analyzing bile acid profiles and understanding metabolic shifts.

Main Methods:

• Heterologous expression of 12α-hydroxysteroid dehydrogenase (12α-HSDH) in *Escherichia coli* as an enzymatic probe.
• Utilizing liquid chromatography-high resolution mass spectrometry (LC-HRMS) coupled with the enzymatic conversion rate to profile bile acids.
• Demonstrating the method's applicability across species and in disease models like asthma.

Main Results:

• The 12α-HSDH probe specifically and efficiently converts 12HBAs under mild conditions.
• The enzyme-driven LC-HRMS approach successfully distinguished 210 12HBAs and 312 non-12HBAs from complex matrices.
• The method revealed cross-species bile acid heterogeneity and alterations in asthma.

Conclusions:

• This enzyme-driven LC-HRMS approach provides a straightforward method for profiling bile acids based on C12 hydroxylation.
• The technique offers a novel pattern to recognize shifts between classical and alternative bile acid synthesis pathways.
• This work provides valuable insights into bile acid metabolism and its implications for disease management.