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Studies on bile acids: The microquantitative separation of cellular bile acids by gas-liquid chromatography.

ABSTRACT: 1. A method is described for the quantitative isolation of bile acids from cellular material. Homogenates of rat liver are freeze-dried and extracted exhaustively with 95% (v/v) ethanol containing 0.1% (v/v) of aq. ammonia (sp.gr. 0.88) and purified by anion-exchange chromatography on Amberlyst A-26. 2. The extracted bile acid conjugates are subjected to either of two hydrolytic procedures, one involving chemical and the other enzymic agents. A unique feature in this study is the introduction of an enzyme, a clostridial peptide-bond hydrolase, for the rapid cleavage of bile acid conjugates, replacing the classical drastic chemical hydrolysis with strong alkali. 3. After hydrolysis, free bile acids are methylated and converted into their trifluoroacetates for final determination by gas-liquid chromatography on a triple component column, FS-1265-SE30-NGS. 4. For the purpose of identification of peaks, bile acid methyl esters are converted into their trimethylsilyl ethers by allowing the methyl esters to react with a new and potent silyl donor, bis(trimethylsilyl)acetamide. 5. The technique affords us a means of studying the metabolism of bile acids at the cellular and subcellular levels in tissues.

SUBMITTER: Okishio T

PROVIDER: S-EPMC1270311 | biostudies-other | 1967 Mar

REPOSITORIES: biostudies-other

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Project description:Bile acids (BAs) are cholesterol metabolites with important biological functions. They undergo extensive host-gut microbial co-metabolisms during the enterohepatic circulation, creating a vast structural diversity and resulting in great challenges to separate and detect them. Based on the bioanalytical reports in the past decade, this work developed three chromatographic gradient methods to separate a total of 48 BA standards on an ethylene-bridged hybrid (BEH) C18 column and high-strength silica (HSS) T3 column and accordingly unraveled the factors affecting the separation and detection of them by liquid chromatography coupled with mass spectrometry (LC-MS). It was shown that both the acidity and ammonium levels in mobile phases reduced the electrospray ionization (ESI) of BAs as anions of [M-H]-, especially for those unconjugated ones without 12-hydroxylation. It was also found that the retention of taurine conjugates on the BEH C18 column was sensitive to the strength of formic acid and ammonium in mobile phases. By using the volatile buffers with an equivalent ammonium level as mobile phases, we comprehensively demonstrated the effects of the elution pH value on the retention behaviors of BAs on both the BEH C18 column and HSS T3 column. Based on the retention data acquired on a C18 column, we presented the ionization constants (pK a) of various BAs with the widest coverage beyond those of previous reports. When we made attempts to establish the structure-retention relationships (SRRs) of BAs, the lack of discriminative structural descriptors for BA stereoisomers emerged as the bottleneck problem. The methods and results presented in this work are especially useful for the development of reliable, sensitive, high-throughput, and robust LC-MS bioanalytical protocols for the quantitative metabolomic studies. Graphical Abstract Nonlinear curve fitting of capacity factors and elution pH value for the separation of common unconjugated bile acids.

Project description:We developed a highly sensitive method for quantifying 21 bile acids (BAs) in the rat liver by capillary liquid chromatography tandem mass spectrometry (cLC/MS/MS) with one-pot extraction. High recovery rates were obtained for the one-pot methods with either methanol (MeOH) extraction or MeOH/acetonitrile (ACN) (1:1, v/v) mixture extraction; the results obtained for the MeOH/ACN mixture solution were better than the results obtained for MeOH. Thus, we determined that the one-pot method with MeOH/ACN was the most suitable method for the efficient extraction of BAs in the liver. Targeted BAs were well separated by cLC with gradient elution using ammonium acetate (NH4OAc)-MeOH mobile phases. Method validation proved that the intra-day and inter-day accuracies and precisions were primarily less than ±20 and 20% relative standard deviation, respectively. Also, the limit of detection (LOD) and the limit of quantitation (LOQ) were 0.9-10 and 2.3-27 ng/g liver, which proves the high sensitivity of the method. Finally, we quantitated 21 BA concentrations in the liver samples of normal and nonalcoholic steatohepatitis (NASH) rats, both of which were derived from stroke-prone spontaneously hypertensive five (SHRSP5) /Dmcr rat. The hepatic BA profiles were found to be substantially different between the normal and NASH groups; the two groups were clearly separated along the first component axis in the score plots of the principal component analysis. In particular, 10 BAs (β-muricholic acid (MCA), glyco (G-) cholic acid (CA), G-chenodeoxycholic acid (CDCA), tauro (T-) CA, T-CDCA, T-ursodeoxycholic acid (UDCA), T-lithocholic acid (LCA), T-hiodeoxycholic acid (HDCA), T-α-MCA, and T-β-MCA) were significantly different between the two groups using Welch's t-test with the false discovery rate correction method, demonstrating BA disruption in the NASH model rat. In conclusion, this method was able to quantify 21 BAs in the rat liver and will evaluate the hepatic BA pathophysiology of rat disease models.

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Studies on bile acids: The microquantitative separation of cellular bile acids by gas-liquid chromatography.

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