Determination Of Individual Bile Acids Research Articles

Quantification of Bile Acids in Cerebrospinal Fluid: Results of an Observational Trial.

(1) Background: Bile acids, known as aids in intestinal fat digestion and as messenger molecules in serum, can be detected in cerebrospinal fluid (CSF), although the blood-brain barrier is generally an insurmountable obstacle for bile acids. The exact mechanisms of the occurrence, as well as possible functions of bile acids in the central nervous system, are not precisely understood. (2) Methods: We conducted a single-center observational trial. The concentrations of 15 individual bile acids were determined using an in-house LC-MS/MS method in 54 patients with various acute and severe disorders of the central nervous system. We analyzed CSF from ventricular drainage taken within 24 h after placement, and blood samples were drawn at the same time for the presence and quantifiability of 15 individual bile acids. (3) Results: At a median time of 19.75 h after a cerebral insult, the concentration of bile acids in the CSF was minute and almost negligible. The CSF concentrations of total bile acids (TBAs) were significantly lower compared to the serum concentrations (serum 0.37 µmol/L [0.24, 0.89] vs. 0.14 µmol/L [0.05, 0.43]; p = 0.033). The ratio of serum-to-CSF bile acid levels calculated from the respective total concentrations were 3.10 [0.94, 14.64] for total bile acids, 3.05 for taurocholic acid, 14.30 [1.11, 27.13] for glycocholic acid, 0.0 for chenodeoxycholic acid, 2.19 for taurochenodeoxycholic acid, 1.91 [0.68, 8.64] for glycochenodeoxycholic acid and 0.77 [0.0, 13.79] for deoxycholic acid; other bile acids were not detected in the CSF. The ratio of CSF-to-serum S100 concentration was 0.01 [0.0, 0.02]. Serum total and conjugated (but not unconjugated) bilirubin levels and serum TBA levels were significantly correlated (total bilirubin p = 0.031 [0.023, 0.579]; conjugated bilirubin p = 0.001 [0.193, 0.683]; unconjugated p = 0.387 [-0.181, 0.426]). No correlations were found between bile acid concentrations and age, delirium, intraventricular blood volume, or outcome measured on a modified Rankin scale. (4) Conclusions: The determination of individual bile acids is feasible using the current LC-MS/MS method. The results suggest an intact blood-brain barrier in the patients studied. However, bile acids were detected in the CSF, which could have been achieved by active transport across the blood-brain barrier.

Determination of individual bile acids in acute respiratory distress syndrome reveals a specific pattern of primary and secondary bile acids and a shift to the acidic pathway as an adaptive response to the critical condition.

Cholestasis and elevated serum bile1 acid levels are common in critically ill patients. This study aims to define the specific pattern of bile acids associated with acute respiratory distress syndrome (ARDS) and the changes in pattern over time. Prospective observational study. Serum samples of 70 ARDS patients were analyzed for primary bile acids (cholic acid, chenodeoxycholic acid) and secondary bile acids (deoxycholic acid, litocholic acid, and ursodeoxycholic acid) as well as their glycine and taurine glycation products. Primary bile acid levels increased from day zero to day five by almost 50% (p<0.05). This change bases on a statistically significant increase in all primary bile acids between day 0 and day 5 (cholic acid [CA] p=0.001, taurocholic acid [TCA] p=0.004, glycocholic acid [GCA] p<0.001, chenodeoxycholic acid [CDCA] p=0.036, taurochenodeoxycholic acid [TCDCA] p<0.001, glycochenodeoxycholic acid [GCDCA] p<0.001). Secondary bile acids showed predominantly decreased levels on day 0 compared to the control group and remained stable throughout the study period; the differences between day zero and day five were not statistically significant. Non-survivors exhibited significantly higher levels of TCDCA on day 5 (p<0.05) than survivors. This value was also independently associated with survival in a logistic regression model with an odds ratio of 2.24 (95% CI 0.53-9.46). The individual bile acid profile of this ARDS patient cohort is unique compared to other disease states.The combination of changes in individual bile acids reflects a shift toward the acidic pathway of bile acid synthesis. Our results support the concept of ARDS-specific plasma levels of bile acids in a specific pattern as an adaptive response mechanism.

Determination of Bile Acids in Piglet Bile by Solid Phase Extraction and Liquid Chromatography-Electrospray Tandem Mass Spectrometry.

An LC/MS/MS-based method was developed for the determination of individual bile acids (BA) and their conjugates in porcine bile samples. The C18-based solid-phase extraction (SPE) procedure was optimized so that all 19 target BA and their glycine and taurine conjugates were collected with high recoveries for standards (89.1-100.2%). Following this, all 19 compounds were separated and quantified in a single 12 min chromatographic run. The method was validated in terms of linearity, sensitivity, accuracy, precision, and recovery. An LOD in the low ppb range with measured precisions in the range of 0.5-9.3% was achieved. The recoveries for all of the 19 analytes in bile samples were all >80%. The validated method was successfully applied to the profiling of BA and their conjugates in the bile from piglets treated with exogenous glucagon-like peptide-2 (GLP-2) in a preclinical model of neonatal parenteral nutrition-associated liver disease (PNALD). The method developed is rapid and could be easily implemented for routine analysis of BA and their conjugates in other biofluids or tissues.

Determination of intralumenal individual bile acids by HPLC with charged aerosol detection

An isocratic HPLC charged aerosol detector (CAD) method was developed, validated, and applied for the determination of individual bile acids in human gastric and duodenal aspirates. The method requires a low volume of aspirates (50-100 microl) and minimal sample pretreatment. A Hypersil BDS RP-C(18) column (250 x 4.6 mm, 5 microm particle size) was equilibrated with a mobile phase composed of methanol-[ammoniun formate 20 mM, formic acid 0.5%, triethylamine 0.2% (pH 3)] 67:33 v/v. Its flow rate was 1 ml/min. The elution times for taurocholate, glycocholate, taurochenodeoxycholate, ursodeoxycholate, glycochenodeoxycholate, cholate, and glycodeoxycholate were approximately 9.9, 16.2, 18.2, 21.3, 31.6, 34.5, and 38.5 min, respectively. Calibration curves in the mobile phase were constructed in the concentration range of 0.5-500 microM. Limits of detection and quantification were in the range of 0.07-0.60 microM and 0.20-1.80 microM, respectively. This method was applied first, in gastric aspirates collected in the fasted state, in which bile acid presence is minimal and, second, in duodenal aspirates collected in the fed state, in which a large number of potentially interfering compounds exists. Intra-day relative standard deviation in fasted gastric aspirates and in fed duodenal aspirates was less than 2.2% and 6.0%, respectively.

High performance liquid chromatographic determination of individual bile acids in serum for automatic diagnosis of various liver and biliary diseases.

A reversed phase high performance liquid chromatographic method for the high sensitivity determination of individual bile acids in serum using a C18 column with a ternary solvent system combined with fluorometric techniques using immobilized enzymes is described. A computer-assisted diagnosis system using pattern recognition was developed to assist the clinical diagnosis of various liver and biliary diseases. A total consistency rate of 95% can be reached using this system.

Determination of individual bile acids in serum by high performance liquid chromatography

A high performance liquid chromatographic (HPLC) method for analysis of 4 free and 8 conjugated bile acids in submicromolar quantities in serum is described using precolumn derivatization with 4-bromomethyl-7-methoxycoumarin (BMC) and fluorescence detection. Bile acids were extracted from serum with 0.4 M sodium bicarbonate, adsorbed onto a Sep-Pak C18 cartridge and eluted with methanol. The extract was derivatized with BMC in acetonitrile using 18-crown-6 crown ether as catalyst and the BMC labelled glycine conjugates and free bile acids were analysed using acetonitrile + methanol + water gradient elution and detection at 320/385 nm. Using a novel and simple approach, taurine conjugates were isolated by extracting the dried, derivatized material with water, in contrast to previous methods which required column chromatography cleanup to isolate the taurine conjugates prior to derivatization. The isolated taurine conjugates were then hydrolysed enzymatically, extracted, derivatized and analysed as free-bile acids. Recoveries of individual bile acids varied from 83-96% for free and glycine conjugates and 72-83% for taurine conjugates. Coefficients of variation were in the range of 5.1-12.5%. In addition to the simpler and shorter procedure for taurine conjugates, this method has increased sensitivity over most other procedures and improved HPLC separation for the various bile acids and conjugates with equivalent recovery and reproducibility compared with other published methods.

Direct enzymic fluorimetric method for the determination of individual bile acids in bile

We describe a new, simple, fluorimetric enzymic method for the determination of individual bile acids in bile. Bile is diluted 4000-fold with water and 3α- and 7α-hydroxy bile acids are determined by equilibrium methods and chenodeoxycholic acid is determined by a differential kinetic method, without any prior separation and preparatory step. The equilibrium methods are based on the reaction of 3α- or 7α-hydroxy bile acids with β-NAD + in the presence of the enzyme 3α- or 7α-hydroxysteroid dehydrogenase (HSD). The kinetic method is based upon the reaction of chenodeoxycholic acid with β-Nad + in the presence of the enzyme 7α-HSD. All measurements are monitored fluorimetrically. Cholic and deoxycholic acid are calculated by difference. Recovery experiments gave satisfactory results. Gallbladder bile from patients was analysed for the three major bile acids. The proposed method is suitable for clinical use.

A highly sensitive determination of individual serum bile acids using high-performance liquid chromatography with immobilized enzyme

We have developed a highly sensitive method for the simultaneous determination of individual bile acids in serum using high-performance liquid chromatography (HPLC) combined with immobilized 3α-hydroxysteroid dehydrogenase. Both the HPLC column and the immobilized-enzyme column are suitable for use with alkaline solutions necessary in working with this enzyme system. With this method we were able to determine simultaneously fifteen different serum bile acids.

Separation and determination of bile acids by high-performance liquid chromatography using immobilized 3α-hydroxysteroid dehydrogenase and an electrochemical detector

A high-performance liquid chromatographic method using an immobilized 3α-hydroxysteroid dehydrogenase column and an electrochemical detector was developed for the determination of individual bile acids in serum and bile. Bile acids in the eluate from a Radial-Pak A column reacted with NAD in the enzyme column to generate NADH, which was monitored by a voltammetric detector after mixing with phenazine methosulphate solution. Each bile acid was measurable at the 20 pmole level at the highest sensitivity of the detector. The mean recoveries and reproducibilities of bile acids were 86.7-104.6% [coefficient of variation (C.V.) = 0.3-8.7%] within-assay and 83.8-103.4% (C.V. = 1.6-7.0%) between-assay.

Spectrofluorimetric determination of individual bile acids in biological fluids: Peripheral plasma

A micromethod is reported for the isolation and quantitative determination of individual conjugated bile acids in peripheral plasma of human subjects and experimental animals. A mild saponification procedure, extraction, thin-layer Chromatographie separation, acetylation, rechromatography and spectrofluorimetry are involved in the procedure. Results of control experiments using bile acid isotopes as internal standard and those of a simultaneous gas-chromatographic estimation of the metabolites proved the method to be suitable for clinical and experimental purposes.

Determination of individual bile acids in biological fluids by thin-layer chromatography and fluorimetry.

A method is described for the separation and determination of individual bile acids in serum and in intestinal contents, employing thin-layer chromatography and fluorimetry. The mean recovery from intestinal juice was 94% and from serum 82%. Normal values for the individual bile acids of serum and examples of clinical applications of the method are given.