Quantitative Bile Acids Analysis
The Duke Proteomics and Metabolomics Shared Resource utilizes the Biocrates Bile Acids Kit to analyze endogenous bile acids, including 19 conjugated and unconjugated, primary and secondary bile acids. Bile acids have many biological functions, including biomarkers for liver function, signaling molecules with hormone-like functions and biomarkers for gut microbiota. The Biocrates Bile Acids Kit utilizes Acquity UPLC coupled to a Xevo TQ-S triple quadrupole mass spectrometry by Waters Corporation to perform quantitative multiplexed analysis of up to 16 human or 19 murine bile acids in plasma samples. Bile Acids profiled with the kit are shown in Table 1 below. Serum samples also can be analyzed with the bile acid kit; other sample matrices may be compatible but will need to be considered on a case-by-case basis. In a typical plasma or serum sample from a healthy donor, 10 bile acids are present in amounts greater than the limit of quantitation. Composite variables and indices calculated using the individual bile acid quantity measures have also been shown to be descriptive of certain clinical conditions. More details on this approach with references can be found at Biocrates Scientific Background information. Pricing is performed on a per sample basis, plus the cost of consumables from Biocrates. For more details see our pricing page or contact Lisa St. John-Williams or Will Thompson.
Much like the Biocrates p180 kit, a 96 well plate is used for analysis, which includes calibration standards and isotopically labeled internal standards along with quality control samples and blanks. Up to 80 experimental samples can be analyzed on one plate. Only 10 uL of sample is required in each well per analysis. A typical chromatogram for separation of bile acid standards from the standard curve is shown in the figure below.
Negative electrospray ionization source conditions allow for the bile acids to be more readily detected by MS/MS instrumentation. The typical concentration range detected with the Biocrates Bile Acids Kit is 0.05 – 10 uM, although this range can vary depending on the specific bile acid to be analyzed. Quantitation of bile acids is performed using the calibration curve generated as part of the standard analysis.
The table below details abbreviations used in the chromatogram shown above along with the full bile acid name and type. Primary bile acids originate from direct synthesis by liver cells. Before secretion, primary bile acids are conjugated with either glycine or taurine within the liver to form the conjugated bile acids, commonly known as bile salts. In healthy humans, bile, overall, is about 70% glyco-conjugated and 20% tauro-conjugated. Once the bile salts are secreted into the intestine, gut bacteria partially dehydroxylates and removes the glycine and taurine conjugation to produce secondary bile acids.
The figure below summarizes the biochemical pathways of bile acid production and reveals relationships between different classes of bile acids. Bile acid abbreviations correspond to those detailed in the above table. All bile acids are produced from cholesterol and two key intermediates, 7-alpha-hydroxycholesterol and 4-cholesten-7a-ol-3-one, which are the rate determining steps in the pathway.
Figure used with permission of the author. doi: 10.1371/journal.pone.0025482