Analytical evaluation of three enzymatic assays for measuring total bile acids in plasma using a fully-automated clinical chemistry platform

Abstract

BackgroundAlthough the clinical significance of measuring bile acids concentration in plasma or serum has been recognized for long in patients with hepatobiliary disease and/or bile acid malabsorption, the reference separation techniques are expensive and mostly unsuitable for early diagnosis and for measuring large volumes of samples. Therefore, this study was aimed to evaluate the analytical performance of three commercial enzymatic techniques for measuring total bile acids in plasma using a fully-automated clinical chemistry platform.MethodsThree commercial enzymatic assays (from Diazyme, Randox and Sentinel) were adapted for use on a Cobas Roche c501. We performed imprecision and linearity studies, and we compared results with those obtained using a reference liquid chromatography-mass spectrometry (LC-MS) technique on an identical set of lithium-heparin plasma samples.ResultsTotal imprecision was optimal, always equal or lower than 3%. All assays had optimal linearity between 3–138 μmol/L. The comparison studies showed good correlation with LC-MS data (Spearman’s correlation coefficients always >0.92), but all plasma samples values were significantly underestimated using the commercial enzymatic assays (-44% for Diazyme, -16% for Randox and -12% for Sentinel). The agreement at the 10 and 40 μmol/L diagnostic thresholds of total bile acids in plasma ranged between 86–92%. This discrepancy was found to be mainly attributable to a heterogeneous composition in terms of bile acids content of the three assay calibrators.ConclusionsThis study suggests that the analytical performance of the three commercial enzymatic assays is excellent, thus confirming that automation of this important test by means of enzymatic assessment may be feasible, practical, reliable and supposedly cheap. Nevertheless, the underestimation of values compared to the reference LC-MS also suggests that the local definition and validation of reference ranges according to the combination between the specific enzymatic assay and the different clinical chemistry platforms may be advisable.

The association of serum total bile acid with non-alcoholic fatty liver disease in Chinese adults: a cross sectional study

“…An automatic biochemistry analyzer (Hitachi 7360; Hitachi Ltd., Tokyo, Japan) and commercially available kits were used to measure serum biochemistries according to accompanying manuals. Specifically, serum BAs were measured using enzymatic assay [24].…”

“…Currently, no convenient/applicable serum BA composition assessment techniques are available in non-academic clinics. So far, penetration of "classical" laboratory methods, including high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS), into the clinics is minimal due to the need of expensive equipment and time-consuming protocols [24]. Because of that, we had to resort to the conventional enzymatic assessment of blood TBA in this study, which is widely used in clinical settings…”

Enzymatic methods may underestimate the total serum bile acid concentration

“…Taken together, although enzymatic assays for measurement of total BA concentration are quite simple and straightforward, with good analytical performance [9], results depend greatly on the BA spectrum in the analyzed sample as well as on the composition of the calibrator. https://doi.org/10.1371/journal.pone.0236372.g003…”

“…Commercially available calibrators typically contain strongly reacting BAs (GCA, CDCA, or TDCA) [9]. Therefore, the BA concentration in samples with an "atypical" spectrum of BAs will more-or-less be underestimated.…”

Bile Acids Quantification by Liquid Chromatography–Tandem Mass Spectrometry: Method Validation, Reference Range, and Interference Study

“…Nonetheless, the measurement of BA in biological fluids is not an easy enterprise. Conventional methods consist of enzymatic techniques based on the use of dehydrogenases acting on hydroxyl groups in BA structures, and thus allowing quantification of total BA [13]. Their use is of limited value when comparisons are made between populations in which BA differ not necessarily in total concentration, but rather in relative percentages.…”