Determination Of Total Bile Acids Research Articles

A highly sensitive electrically driven electrochemiluminescent assay for quantification of bile acids in human serum

A capillary electrically driven assay with electrochemiluminescent (ECL) detection for total bile acids in human serum was developed and fully validated. Quantification was performed by multiple reactions. First, the bile acids react with nicotinamide adenine dinucleotide (NAD(+)) under catalysis of 3α-hydroxysteroid dehydrogenase (3α-HSD), which is converted to 3-ketosteroid and concomitantly NAD(+) turns into reduced nicotinamide adenine dinucleotide (NADH). And then Ru(bpy)3(2+) is oxidized to be Ru(bpy)3(3+), which serves as an electron mediator, and reacts immediately with NADH coexisting in a carrier solution and is converted to Ru(bpy)3(2+*). NADH transfers an electron to form NAD(+) and the unstable excited-state species, Ru(bpy)3(2+*), which emits photons and gives out light when it decays to the ground state, Ru(bpy)3(2+). Consequently, the concentration of total bile acids could be determined by the electrochemiluminescent intensity. The assay was linear from 0.1 fmol L(-1) to 1000 fmol L(-1), with a detection limit of 0.02 fmol L(-1). The intra-day and inter-day precision had a coefficient of variation of less than 5.0%. The developed ECL assay had an acceptable correlation with an enzymatic cycling method commonly adopted in clinics for the determination of total bile acids (r = 0.7216). Based on the above-mentioned principle, we established a simple, accurate and highly sensitive approach for the determination of total bile acids. Furthermore, this assay has been applied successfully to the detection of total bile acids in human serum, indicating its practicality for bioanalysis.

Differential diagnosis of neonatal mild hypergalactosaemia detected by mass screening: clinical significance of portal vein imaging.

The aetiology of hypergalactosaemia in 100 neonates detected by screening using the Paigen method is discussed. Hypergalactosaemia was transient in 94 cases and persistent in 6. The aetiology among transient cases was unknown in 55, delayed closure of the ductus venosus in 19, heterozygous UDP-galactose 4-epimerase (GALE) deficiency in 16, and heterozygous galactose-1-phosphate uridyltransferase (GALT) deficiency in 6. The aetiology among persistent cases was hepatic haemangioendothelioma with portovenous shunting in 2, and patent ductus venosus with hypoplasia of the intrahepatic portal vein, citrin deficiency, homozygous GALE deficiency, and heterozygous GALE deficiency in one patient each. The abnormalities of the portal system were identified ultrasonographically at the initial consultation and measurements of the total bile acid and alpha-fetoprotein concentrations were helpful in resolving the differential diagnosis. The causes of hypergalactosaemia varied, but a major cause was portosystemic shunt. Evaluation of patients with hypergalactosaemia should not be limited to enzymatic analysis, but should also include hepatic imaging, especially ultrasonography. Additionally, determination of total bile acids and alpha-fetoprotein is helpful in identifying the aetiology of hypergalactosaemia in infants.

Mechanism of Trichloroethylene-Induced Elevation of Individual Serum Bile Acids: I. Correlation of Trichloroethylene Concentrations to Bile Acids in Rat Serum

The temporal relationship between trichloroethylene (TRI) and individual serum bile acids (SBA) has been investigated to gain insight into the mechanism of solvent-induced increases in SBA. Male Sprague-Dawley rats were treated with 1 mmol/kg TRI in corn oil, while control rats received only corn oil. Blood samples were collected from the abdominal aorta at 2, 4, 8, and 16 hr after dosing. Individual SBA were determined by high-performance liquid chromatography (HPLC). Liver and blood concentrations of TRI and one of its metabolites, trichloroethanol (TCEOH), were determined by gas chromatography. SBA levels reached their peak at 4 hr and returned to control levels by 16 hr. There was a relationship between SBA levels and TRI concentrations, which were also at their peak 4 hr after dosing. By 16 hr the levels were undetectable. However, peak blood concentrations of TCEOH were reached 8 hr after dosing, and remained high at 16 hr. Cholic acid and taurocholic acid showed the highest levels of bile acids. Some other bile acids were also elevated, including deoxycholic acid, taurodeoxycholic acid, ursodeoxycholic acid, chenodeoxycholic acid, and taurochenodeoxycholic acid. Determination of total bile acids in serum using an enzymatic/colorimetric method showed a similar pattern of response to that obtained with the HPLC analysis. The data are consistent with TRI having a rapid and specific effect on SBA levels by a mechanism other than liver cell damage.

Fluorimetric enzymatic flow-injection determination of bile acids in human serum

A fast, simple and convenient method for the individual and total determination of bile acids based on the enzymatic oxidation of the analytes and the fluorimetric measurement of a reaction product (the reduced form of the coenzyme, NADH) is proposed. The method allows the determination of the analytes (taurocholic, chenocholic, glycocholic and chenodesoxycholic acid) over the range of 0.3–10.0 μg ml−1, with an r.s.d. smaller than 3.0% in each case. It was applied to the determination of total bile acids in human serum. The simplicity of the method makes it useful for screening clinical samples in order to avoid implementing chromatographic processes on samples with analyte overall contents clearly below pathological limits.

The clinical value of faecal bile acid determination in patients with chronic diarrhoea of unknown origin.

To evaluate the diagnostic value of faecal total bile acid determination in adult patients with chronic diarrhoea, output and concentration of 3 alpha-hydroxy-bile acids were determined in 28 patients. The patients had normal results of primary biochemical, radiologic, and endoscopic examinations. Stools were sampled for 3 days while on a standardized diet. The patients were observed until final diagnosis was obtained; Crohn's disease was diagnosed in 5 cases, osmotic diarrhoea in 4, bacterial overgrowth in 3, laxative abuse in 2, other specific causes in 3, and persisting lack of specific diagnosis in 11. No difference in output or concentration of faecal bile acids was found between the groups. Six patients with high bile acid outputs benefited from treatment with cholestyramine. Accordingly, determination of total bile acids in stools cannot be recommended as a primary test in patients with chronic diarrhoea.

Enzymatic fluorimetric determination of the individual bile acids in blood serum

An enzymatic fluorimetric method is described for the determination of total bile acids (cholic acid and deoxycholic acid), primary bile acids (cholic and chen acids and individual bile acids in serum without prior separation of the acids. Total and primary bile acids are determined by equilibrium procedures by conver of the 3α- and 7α-hydroxy bile acids to 3-oxo and 7-oxo bile acids by α-NAD +, in the presence of 3α- and 7α-hydroxysteroid dehydrogenase (HSD), respectively, and measurement of the generated NADH fluorimetrically. Chenodeoxycholic acid is determined with 7α-HSD in the presence of cholic and deoxycholic acids by a differential kinetic procedure, and cholic and deoxycholic acids are calculated by difference. Interferents are removed by treatment of serum with Sachrom rein. Only 1.00 ml of serum is required. Low cost, simplicity and reliability are the main features of the method. The recovery of bile acids added to serum averaged 103% (range 83–122%). The method is suitable for routine use in small clinical laboratories.

Effect of protein on determination of total bile acids in serum is minimized by preheating the sample.

Journal Article Effect of protein on determination of total bile acids in serum is minimized by preheating the sample. Get access K Linnet K Linnet Search for other works by this author on: Oxford Academic Google Scholar Clinical Chemistry, Volume 29, Issue 10, 1 October 1983, Pages 1871–1872, https://doi.org/10.1093/clinchem/29.10.1871a Published: 01 October 1983

Enzyme inactivation in serum before determination of total bile acids.

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.

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

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.

Determination of total bile acids in serum. A comparison of a radioimmunoassay with an enzymatic-fluorimetric method

A solid phase radioimmunoassay kit method for total conjugated bile acids has been compared to an enzymatic fluorimetric method for total serum bile acids. The methods were compared with respect to: precision, cross-reactivity (molar equivalence) of different bile salts, recovery of different bile salts from serum, the reference range for a healthy population, linearity, coefficient of correlation, diagnostic effectiveness, cost and ease of assay. Both assays seemed equally capable of predicting the presence or absence of liver disease. Radioimmunoassay had little advantage over the enzymatic-fluorimetric method. Its relative ease was far outweighed by its greater cost and poorer analytical performance.

An automated method for the determination of total bile acids in serum

An automated modification of the earlier described enzymatic methods [10, 12] for the determination of total bile acids in serum is presented. The procedure involves two main steps, the inactivation of unspecific NADH generating enzyme activity in serum, followed by a transformation of the bile acids by a 3α-hydroxysteroid: NAD-oxidoreductase (3α-HSD) to the corresponding 3-keto compounds. Using an LKB reaction rate analyser, the NAD-containing 3 a-HSD preparation (Sterognost-3α) serves as starting reagent, and the NADH formed is measured as the increase in absorbance at 340 nm. The recovery of bile acid standards in 50 g/l albumin averaged 94% and in sera 91%. Accuracy was evaluated by comparison of the results on fourteen sera to gas liquid chromatography analysis; the means agreed within 2.2% (r = 0.99). A commercial control serum agreed within 1.5% to the recommended value. Precision was estimated on three pooled sera and found to be essentially the same for within run and day to day series, the stand...

An automated method for the determination of total bile acids in serum

An automated method for the determination of total bile acids in serum

Cholestasis induced by sodium taurolithocholate in isolated hamster liver.

The mechanism of cholestasis (decreased bile flow) induced by taurolithocholate in the isolated perfused hamster liver was investigated. Taurocholate was infused to maintain bile acid output, and sulfobromophthalein (BSP) was administered to establish a BSP transport maximum in bile. The effects of taurolithocholate on bile flow and on the biliary secretion of BSP and bile acid anions were determined.A significant dose-response correlation was found between taurolithocholate and the degree of cholestasis. No significant hepatic morphologic alterations were observed. At low doses, cholestasis was reversible. A multiple regression equation was developed to validate the steroid dehydrogenase determination of total bile acids in bile that contained BSP. During cholestasis, output of bile acid was maintained by a significantly increased concentration of bile acid. Hepatic removal rate and transport maximum of BSP were significantly decreased, whereas BSP concentration, conjugation, and hepatic content were unaffected. The concentrating capacity for BSP in bile appeared to be the rate-limiting factor in BSP transport. Individual bile acids were determined by gas-liquid chromatography. Of the injected taurolithocholate, 40-50% was recovered in bile as lithocholic acid, 30% was converted to chenodeoxycholic acid, and only traces of lithocholic acid were detected in the perfusate after 4 hr. Cholic and chenodeoxycholic acids comprised 75-89%, and lithocholic acid comprised 11-25% of bile acids in bile after taurolithocholate injection; only traces of deoxycholic acid were seen. Small amounts of taurolithocholate sulfate were detected in bile by thinlayer chromatography. The outputs of sodium and potassium in bile were significantly diminished during cholestasis.A substantial fraction (75%) of basal bile flow in the isolated hamster liver was estimated to be independent of bile acid secretion. Cholestasis occurred after taurolithocholate, whereas bile acid secretion was maintained. The results indicate that the most likely mechanism for acute cholestasis induced by taurolithocholate in isolated hamster liver was interference with the bile acid-independent fraction of canalicular or ductular bile flow or both.

Gas-liquid chromatographic determination of human fecal bile acids

A method for the determination of total bile acids in human feces that is suitable for routine application is described and discussed. Bile acids are extracted from freeze-dried feces with acetic acid and toluene, in the presence of the internal standard 23-nordeoxycholic acid. After saponification of the extract, bile acids and the internal standard are methylated and converted by mild chromic acid oxidation into their ketonic derivatives. The resultant mixture of a few stable compounds can be separated and measured quantitatively by gas-liquid chromatography on a methylsiloxane polymer. A reference bile acid mixture including the internal standard is also taken through the entire procedure with each series of samples. It has been demonstrated that, in spite of the omission of the usual purification steps, the method is specific for bile acids.

Spectrofluorometric Determination of Total Bile Acids in Bile

Spectrofluorometric Determination of Total Bile Acids in Bile