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

The synthesis of 12α-hydroxylated bile acids (12HBAs)
and
non-12α-hydroxylated bile acids (non-12HBAs) occurs via classical
and alternative pathways, respectively. The composition of these BAs
is a crucial index for pathophysiologic assessment. However, accurately
differentiating 12HBAs and non-12HBAs is highly challenging due to
the limited standard substances. Here, we innovatively introduce 12α-hydroxysteroid
dehydrogenase (12α-HSDH) as an enzymatic probe synthesized by
heterologous expression in Escherichia coli, which can specifically and efficiently convert 12HBAs in vitro
under mild conditions. Coupled to the conversion rate determined by
liquid chromatography-high resolution mass spectrometry (LC–HRMS),
this enzymatic probe allows for the straightforward distinguishing
of 210 12HBAs and 312 non-12HBAs from complex biological matrices,
resulting in a BAs profile with a well-defined hydroxyl feature at
the C12 site. Notably, this enzyme-driven LC-HRMS approach can be
extended to any molecule with explicit knowledge of enzymatic transformation.
We demonstrate the practicality of this BAs profile in terms of both
revealing cross-species BAs heterogeneity and monitoring the alterations
of 12HBAs and non-12HBAs under asthma disease. We envisage that this
work will provide a novel pattern to recognize the shift of BA metabolism
from classical to alternative synthesis pathways in different pathophysiological
states, thereby offering valuable insights into the management of
related diseases.