Enzymatic methods may underestimate the total serum bile acid concentration

Area of Science:

• Biochemistry
• Analytical Chemistry
• Clinical Chemistry

Background:

• Enzymatic assays using bacterial 3α-hydroxysteroid dehydrogenase are standard for serum total bile acid (BA) quantification.
• These assays are generally considered precise and robust despite their non-specific nature.

Purpose of the Study:

• To evaluate the impact of varying bile acid (BA) spectrums on the reliability of enzymatic BA quantification.
• To compare enzymatic assay results with a more specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method.

Main Methods:

• Standard solutions of 23 human and murine bile acids (BAs) were analyzed using a commercial enzymatic assay.
• Total BA concentrations in human and rat cholestatic serum samples were measured via enzymatic assay and LC-MS/MS.

Main Results:

• Individual BAs exhibited significant variability in response to the enzymatic assay, with relative signal intensities ranging from 100% (glycocholic acid) to 20% (α-muricholic acid).
• The enzymatic assay markedly underestimated total BA concentrations in both human and rat cholestatic sera compared to LC-MS/MS.
• Assay performance was highly dependent on the specific BA spectrum present in the sample.

Conclusions:

• The reliability of enzymatic assays for total bile acids is significantly influenced by the BA spectrum.
• Marked underestimation of total BA concentrations can occur, particularly with atypical BA profiles, such as those found in rodents.
• Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is recommended for accurate BA quantification in samples with abnormal BA spectrums.