Evaluation of a Commercial Enzymatic Method for the Determination of Total Serum Bile Acids

1980 ◽  
Vol 17 (6) ◽  
pp. 301-306 ◽  
Author(s):  
Per Tobiasson ◽  
Magnus Källberg
Keyword(s):  
Bile Acid ◽  
Bile Acids ◽  
Reference Range ◽  
Reference Value ◽  
Total Serum ◽  
Enzymatic Method ◽  
Normal Range ◽  
Serum Samples ◽  
Serum Bile Acids

An enzymatic method (Sterognost-3α® Flu, Nyegaard & Co, Oslo, Norway) for the determination of total serum bile acids has been evaluated, and reference values for fasting and postprandial total serum bile acids have been established. Precision was rather low in the lower normal range but satisfactory in the upper reference range and for elevated values. The upper reference value for fasting total serum bile acids was 5·0 μmol/l. Maximal postprandial elevations occurred at varying intervals after a test meal and the upper postprandial reference value was 12 μmol/l. Certain serum samples were also analysed by radioimmunological methods for conjugates of cholic and chenodeoxycholic acids. The values obtained with the enzymatic method correlated satisfactorily with those of the radioimmunoassay methods for most of the serum samples analysed. However, a number of samples with slightly elevated bile acid concentrations, as measured by the comparison radioimmunoassay methods, were found to have normal total serum bile acids when measured by the enzymatic method.

Effect of protein on the determination of total bile acids in serum.

1983 ◽  
Vol 29 (1) ◽  
pp. 171-175 ◽  
Author(s):  
N Q Hanson ◽  
E F Freier
Keyword(s):  
Bile Acid ◽  
Bile Acids ◽  
Hydroxysteroid Dehydrogenase ◽  
Inhibitory Effect ◽  
Normal Serum ◽  
Total Serum ◽  
Enzyme Preparations ◽  
Analytical Recovery ◽  
Fluorescence Light

Abstract We measured total serum bile acids on a fluorescence-light-scattering micro centrifugal analyzer by the direct enzymatic method with 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50) and with resazurin as a fluorogenic electron acceptor. We found that serum protein has an inhibitory effect on the measurement of bile acids, but this effect was eliminated by adding bovine serum albumin to the reaction mixture in a final protein concentration (12.2 g/L) that was high compared with that contributed by a normal serum specimen. The assay is a sensitive method that reaches equilibrium in 5 min. The method is microscale (5 microL of sample, 150 microL of working reagent), is easy to perform, and is accurate (analytical recovery = 104.1%) and precise (CV = 11.1 and 5.7% on specimens with bile acid concentrations of 7.6 and 35.4 mumol/L, respectively). Normal values are 1-12 and less than 9 mumol/L on nonfasting and fasting individuals, respectively. Pure 3 alpha-hydroxysteroid dehydrogenase must be used: we found several enzyme preparations that gave falsely high values for bile acid.

Enzyme inactivation in serum before determination of total bile acids.

1983 ◽  
Vol 29 (6) ◽  
pp. 1073-1075 ◽  
Author(s):  
N Q Hanson ◽  
E F Freier
Keyword(s):  
Bile Acid ◽  
Bile Acids ◽  
Final Concentration ◽  
Methanol Solution ◽  
Enzyme Inactivation ◽  
Total Serum ◽  
Sodium Pyruvate ◽  
Lactate Dehydrogenase Activity ◽  
Total Bile Acids

Abstract Endogenous NADH-generating enzymes must be inactivated before total serum bile acids can be measured accurately by the direct enzymic method. To do this, we pretreat the sera with NaOH, in a final concentration of 0.1 mol/L. Consequently, lactate dehydrogenase activity at least as high as 30 000 U/L is destroyed, obviating blank determinations. Values for bile acid in serum, so obtained, agree with values obtained after pretreatment with heat, an alkali-methanol solution, or sodium pyruvate, but our pretreatment has the advantages of ease, speed, economy, and negligible blank values.

Continuous-flow determination of primary bile acids, by bioluminescence, with use of nylon-immobilized bacterial enzymes.

1984 ◽  
Vol 30 (2) ◽  
pp. 206-210 ◽  
Author(s):  
A Roda ◽  
S Girotti ◽  
S Ghini ◽  
B Grigolo ◽  
G Carrea ◽  
...  
Keyword(s):  
Bile Acid ◽  
Bile Acids ◽  
Continuous Flow ◽  
High Performance ◽  
Immobilized Enzymes ◽  
Serum Samples ◽  
Light Emitting ◽  
Assay Tube ◽  
Bioluminescence Method

Abstract We describe a continuous-flow bioluminescence method for measuring primary bile acid in serum, with nylon as the solid support. 7 alpha-Hydroxysteroid dehydrogenase (EC 1.1.1. 159), a bacterial luciferase (EC 1.14.14.3), and NAD+:FMN oxido-reductase (EC 1.6.8.1) are covalently co-immobilized on a nylon coil (1 m X 1.0 mm i.d.). The assay is highly specific for 7 alpha-hydroxy bile acids. Other bile acids and steroids do not interfere. The continuous-flow light-emitting system, in which the reactor (nylon coil) is placed in front of a photomultiplier tube inside a luminometer, is versatile and simple. The flow is air-segmented, and serum samples (5-50 microL) can be injected directly. Concentration and response are linearly related from 10 to 2500 pmol per assay tube. The precision of the method is satisfactory (CV 5-10%), both inter- and intra-assay. We validated the technique by comparing results with those by RIA, enzyme immunoassay, and "high-performance" liquid chromatography. More than 20 samples an hour can be analyzed, with no carryover. The nylon-immobilized enzymes are stable for more than two months, and greater than 500 samples can be analyzed with use of a few milligrams of enzymes. Normal values for bile acid content of serum ranged from 1 to 2.5 mumol/L, in agreement with those obtained by other methods.

scholarly journals Hypercortisolism in patients with cholestasis is associated with disease severity

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Verena Theiler-Schwetz ◽  
Hansjörg Schlager ◽  
Barbara Obermayer-Pietsch ◽  
Tatjana Stojakovic ◽  
Günter Fauler ◽  
...  
Keyword(s):  
Bile Acid ◽  
Adrenal Gland ◽  
Bile Acids ◽  
Disease Severity ◽  
Total Serum ◽  
Acth Stimulation ◽  
Serum Bile Acids ◽  
Baseline Cortisol ◽  
Duct Ligation ◽  
Gland Function

Abstract Background Cholestasis might lead to an impairment of adrenal function as suggested by in vitro and in vivo data as well as by clinical findings. Bile acid and adrenal steroid metabolism not only share the receptors farnesoid X receptor (FXR) and the G protein-coupled bile acid receptor 1 (TGR5), but supraphysiological bile acid levels were found to stimulate steroidogenesis independent of FXR and TGR5. Our previous experimental findings revealed that mice fed bile acids or subjected to common bile duct ligation develop hypercortisolemia. We thus aimed to assess adrenal gland function in patients with cholestasis. Methods Adrenal gland function was assessed in 36 patients with cholestasis and in 32 patients without cholestasis by measuring total serum cortisol, adrenocorticotropic hormone (ACTH), as well as the increase of cortisol 20 and 30 min after administration of 1 µg of ACTH. Bile acid levels and bile acid pool composition were determined by high-resolution mass spectrometry. Results Patients with cholestasis per definition had markedly elevated levels of alkaline phosphatase (AP), bilirubin and serum bile acids. Baseline cortisol and maximum cortisol after ACTH stimulation were significantly higher in patients with cholestasis compared to controls. Increase of cortisol after ACTH stimulation and ACTH did not differ. In the cholestasis group, baseline cortisol correlated with bilirubin but not with AP, total serum bile acids and levels of conjugated and unconjugated bile acid species. Patients with duration of cholestasis < 6 months (n = 30) had significantly higher baseline cortisol levels than those with long standing cholestasis (> 6 months), together with higher bilirubin levels. Conclusions We find no evidence of adrenal insufficiency in non-cirrhotic patients with cholestasis. In contrast, patients with cholestasis show hypercortisolism associated with disease severity as mirrored by levels of bilirubin. Lack of ACTH increase in cholestasis suggests a direct effect of cholestasis on adrenals and not on the pituitary gland. Further studies are needed to elucidate the mechanism of cortisol elevation in patients with cholestasis and its clinical significance.

Continuous-flow enzymatic determination of total serum cholesterol and method standardization with CDC-calibrated pooled sera.

1980 ◽  
Vol 26 (7) ◽  
pp. 896-902
Author(s):  
M A MacAulay ◽  
C L Jacklyn ◽  
J M Mathers ◽  
V A Storm
Keyword(s):  
Serum Cholesterol ◽  
Continuous Flow ◽  
Cholesterol Content ◽  
Comparison Method ◽  
Total Serum ◽  
Total Serum Cholesterol ◽  
Enzymatic Method ◽  
Factors Affecting ◽  
Assigned Value

Abstract We compared Boehringer Mannheim's enzymatic kit for the continuous-flow (AutoAnalyzer II) determination of serum cholesterol with Technicon's N-24a extraction method. Results for patients' samples analyzed by the enzymatic method were higher than those by the comparison method. To evaluate accuracy in the cholesterol determinations, we enrolled the enzymatic method into the Center for Disease Control's (CDC) Lipid Standardization program. We calibrated the method by use of a pooled sera for which cholesterol content was assigned by CDC after analysis by their reference Abell-Kendall procedure. We discuss the difficulties with available calibration material and limitations in the application of some commercial control materials to the enzymatic cholesterol method. The continuous-flow variables, Michaelis-Menton constants, percent ester-hydrolase activity, and other factors affecting the performance of the enzyme-linked cholesterol method are evaluated. We believe pooled sera with an assigned value for cholesterol content is the best calibrator material.

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