Rapid Detection of Small Molecule Metabolites in Serum of Hepatocellular Carcinoma Patients Using Ultrafast Liquid Chromatography-Ion Trap-Time of Flight Tandem Mass Spectrometry.

A method was developed for analyzing broad spectrum small molecule metabolites in the serum of hepatocellular carcinoma (HCC) patients based on ultrafast liquid chromatography-ion trap-time of flight tandem mass spectrometry (UFLC-IT-TOF MS). Serum samples were collected from 80 HCC patients and healthy persons. After pretreatment process for protein precipitation, the supernatant was analyzed with the UFLC-IT-TOF MS to obtain information on the metabonomics of small molecules. The eight compounds of glycocholic acid, choline glycerophosphate, acetyl-L-phenylalanine, oleamide, tetradecanamide, acetylcarnitine, lysolecithin and glycochenodeoxycholic acid in the HCC group were identified with significant differences from those in the health group (P <0.01). By using multidimensional analysis of variation coefficient and principal component analysis for the repeatability and 48 h stability, the method was demonstrated to have good repeatability, excellent precision, and high stability, which can satisfy the metabonomics research requirement. The high throughput and practical usability of the method further shows perspective for metabonomic analysis of large-batch serum samples.

Absence of glycochenodeoxycholic acid (GCDCA) in human bile is an indication of cholestasis: a 1H MRS study.

The utility of (1)H MR spectroscopy in detecting chronic cholestasis has been investigated. The amide proton region of the (1)H MR spectrum of human bile plays a major role in differentiating cholestatic (Ch) patterns from the normal ones. Bile obtained from normal bile ducts contains both taurine and glycine conjugates of bile acids--cholic acid (CA), chenodeoxycholic acid (CDCA), and deoxycholic acid (DCA). Absence of a glycine-conjugated bile acid glycochenodeoxycholic acid (GCDCA) has been observed in bile samples obtained from primary sclerosing cholangitis (PSC) patients. A total of 32 patients with various hepatobiliary diseases were included in the study. Twenty-one patients had PSC and 11 had normal cholangiograms. One PSC patient was excluded from the study because of a bad spectrum. Seventeen out of the 20 PSC patients showed an absence of GCDCA in their (1)H MR spectrum of bile. Six of the 11 reference patients with normal cholangiogram also showed spectra similar to those of PSC, indicating the possibility of cholestasis. DQF-COSY and TOCSY experiments performed on bile samples from PSC patients also revealed absence of phosphatidylcholine (PC) in some of the bile samples, suggesting possible damage to the cholangiocytes by the toxic bile. These observations suggest that analysis of human bile by (1)H MRS could be of value in the diagnosis of chronic Ch liver disorders.

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.

Changes in intragastric bile acid composition in patients receiving cimetidine postoperatively.

Enterogastric reflux has been implicated as a possible etiologic mechanism in gastritis both after partial gastrectomy and in those with an intact pylorus. We studied the effects of cimetidine on bile acid concentration and composition by high-performance liquid chromatography. The gastric aspirates collected for this study came from 27 prospectively randomized patients receiving intravenous cimetidine (200 mg every 6 hours) and 25 patients given a placebo. Total bile acid concentration of aspirates was determined spectrophotometrically. Marked differences were noted in conjugated bile acids. Glycochenodeoxycholic acid, a toxic dihydroxy bile acid, was decreased after cimetidine compared with results from the placebo. The ratio of less toxic trihydroxylated to more toxic dihydroxylated bile acids was significantly increased. Enterogastric reflux itself seemed unaltered by cimetidine; likewise, the concentration of total bile acids in the cimetidine group was similar to that among patients receiving placebo. These changes in bile salt composition with cimetidine may help explain its salutary effects in gastritis, over and above its ability to reduce gastric hydrogen ion secretion.

Effects of biliary bile acid composition on biliary cholesterol saturation in gallstone patients treated with chenodeoxycholic acid and/or ursodeoxycholic acid.

Chenodeoxycholic acid (cheno) and ursodeoxycholic acid (urso) dissolve cholesterol gallstones in humans. In the present study conjugation of biliary bile acids with glycine and taurine and their effects on biliary cholesterol saturation were investigated during treatment with cheno, urso, and cheno-urso. Ten patients were included in this study, and every patient served as his own control. Each of the treatment periods lasted for 3 mo. During treatment with cheno or urso, daily doses of 11.9-15.6 mg/kg were administered, while during treatment with cheno-urso each bile acid was administered at one-half the dose. In the control period biliary bile acids consisted of 31.8 +/- 2.8% glycocheno, 10.9 +/- 1.2% taurocheno, 1.0 +/- 0.1% glycourso, and 0.3 +/- 0.1% taurourso. During the three treatment periods dihydroxy bile acids in bile and glycine conjugation of these dihydroxy bile acids increased significantly (P < 0.05). During treatment with urso the amounts of glycourso in bile were positively correlated to the dose of urso administered (P < 0.05). No correlation existed between urso dose and the amounts of taurourso in bile. Biliary cholesterol was 9.0 +/- 1.0 mol% in the control period and decreased during treatment with cheno, urso, and chenourso to 5.2 +/- 0.5, 3.7 +/- 0.3, and 3.8 +/- 0.3 mol%, respectively. Cholesterol saturation index corrected for the biliary content of glycourso and taurourso was 1.2 +/- 0.1 in the control period and decreased during treatment with cheno, urso, and cheno-urso to 0.8 +/- 0.1, 1.0 +/- 0.1 and 0.7 +/- 0.1, respectively. Thus urso treatment led to the lowest biliary content of cholesterol, but cheno-urso treatment led to significantly lower cholesterol saturation indices than urso treatment (P < 0.05).

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.

A rapid, sensitive method for accurate analysis of individual bile acids in biological fluids by high performance thin-layer chromatography and densitometry.

1. A rapid new micromethod for quantitative analysis of individual bile acids in duodenal juice by high performance thin-layer chromatography (HPTLC) and densitometry is described and evaluated by comparison with standard TLC and spectrophotometry. 2. Advantages of HPTLC over TLC include more rapid separation, better resolution and more sensitive detection (5 - 10 fold), without the need for prior extraction. Densitometry provides simple, direct and rapid quantitation. 3. The method is accurate and reliable over a range of bile acid concentrations. In the 0.5 mM range, recovery was greater than 89%, and coefficients of variation for within-day analysis were 2 - 12% and for between-day analysis were 6 - 18% for the individual bile acids. Twenty analyses can be performed by one worker in a single day. 4. We conclude that the method offers several advantages over most currently described techniques, is suitable for routine use and is deserving of wider application.