Single-step analysis of individual conjugated bile acids in human bile using 1H NMR spectroscopy.

1H and 13C NMR spectra of intact human bile were assigned using one-dimensional (1H and 13C) and two-dimensional (1H-1H and 1H-13C) experiments. Individual conjugated bile acids--glycocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, and taurochenodeoxycholic acid--were identified. The bile acids were quantified accurately and individually in a single step by using distinct and characteristic amide signals. Making use of 13C NMR, the study also suggests a way to analyze unconjugated bile acids separately, if present. Chemical shift assignments and rapid single-step analysis of individual conjugated bile acids from intact bile presented herein may have immense utility in the study of bile acid metabolism and deeper understanding of hepatobiliary diseases.

Steroid metabolism along the gastrointestinal tract of the cannulated pig.

Steroid metabolism along the gastrointestinal tract of the cannulated pig was studied. Thi was achieved by fitting simple gut cannulas in the terminal ileum, caecum and mid-colon of three Landrace x large white boars, which enabled convenient collection of digesta and faecal samples at defined time points. Biochemical analyses showed that the neutral steroid profile of the pig is similar to that of man, dominated by cholesterol and its bacterial metabolite coprostanol. In contrast, pigs consuming a normal diet excrete appreciably lower quantities of neutral sterols in faeces. The major primary bile acids detected were the glycine and taurine amidates of hyocholic and chenodeoxycholic acids, which were rapidly converted to the free bile acids and subsequently dehydroxylated to hyodeoxycholic and lithocholic acids respectively, in the terminal ileum and caecum. Bacterial deconjugation and 7 alpha-dehyrdoxylation are virtually complete in the caecum with negligible further metabolism in the colon and faeces. On a wet weight basis the concentration of both neutral and acid steroids was shown to increase aborally. Inclusion of dietary fibre in the form of cellulose (Solka floc) and guar gum reduced steroid concentration considerably at all sites of the large intestine, which is consistent with their stool bulking effects. In conclusion, this study shows that intestinal steroid metabolism in the pig is similar to that in man despite slightly different bile acid profiles and, therefore, the multicannulated pig may serve as a useful model of man in chemoprevention studies of colorectal cancer.

The use of negative ion thermospray liquid chromatography/tandem mass spectrometry for the determination of bile acids and their glycine conjugates.

A study was carried out on the negative ion thermospray liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry of a group of bile acids and their glycine conjugates. The liquid chromatographic/tandem mass spectrometric experiments were performed using low-energy collisions on a triple-quadrupole mass spectrometer. It was found that relatively high collision energies and collision gas pressures were required to produce fragmentation and that some unusual fragments were produced.

Colorimetric determination of glycine conjugates of bile acids.

A simple assay was developed for the determination of glycine-conjugated bile acids. Samples containing the glycine-conjugated bile acids in the range from 15 to 250 nmol were acidified with HCl, and extracted with ethyl acetate containing ethanol (50 ml/l). An aliquot of the organic phase was evaporated to dryness, and the dried residue was treated to develop the color by the addition of acetic anhydride, pyridine and a trace amount of phosphoric acid. the absorbance was measured at 429 or 456 nm after the reaction at 50 degrees C for 2 h. Color development did not occur with unconjugated and taurine-conjugated bile acid. Beer's law was obeyed from 12.5 to 200 nmol in a cuvette. The recovery of the conjugates from the rabbit gall bladder bile and liver homogenate was satisfactory. This method requires no hydrolysis step and is applicable to the determination of glycine-conjugated bile acids in bile, duodenal aspirate and liver homogenate.

High performance liquid-chromatographic analysis of individual bile acids: free, glycine- and taurine-conjugated bile acids.

High performance liquid-chromatographic analyses of individual bile acids (cholic acid, chenodeoxycholic acid, deoxycholic acid, and lithocholic acid), free and conjugated with glycine and taurine, are described. The analyses of free and glycine-conjugated bile acids are based on esterification of carboxyl group of bile acids with O-(p-nitrobenzyl)-N, N'-diisopropylisourea (PNBDI). Moreover, ursodeoxycholic acid, hyocholic acid, hyodeoxycholic acid and 3beta-hydroxy-5-cholenoic acid also are able to analyse by this method. These bile acids in biological sample were extracted by an Amberlite XAD-2 column, and separated by DEAE-Sepharose CL-6B into free, glycine- and taurine-conjugated bile acids. After the separation, free and glycine-conjugated bile acids were esterified with PNBDI directly. Because taurine-conjugated bile acids are unable to be esterified with PNBDI, these bile acids were hydrolyzed by NaOH in order to make free bile acids, and then they were esterified. Because the p-nitrobenzyl ester of bile acids has characteristic ultraviolet absorption, these compounds were separated to individual bile acids by high performance liquid-chromatography, and detected by an UV-detector. An analysis of individual bile acids in human bile was demonstrated.