Determination of serum bile acids by glass capillary gas-liquid chromatography.

Bile acids were extracted from serum samples by chromatography on Amberlite XAD-2 and, after alkaline or enzymic hydrolysis, purified by chromatography on aluminium oxide. The quantitation was carried out by gas-liquid chromatography with an OV-101 glass capillary column using their methyl ester trimethylsilyl derivatives. The mean total amount of cholic, chenodeoxycholic and deoxycholic acids in a group of healthy fasting women was 2.14 mumol/l, in a group of fasting pregnant women at 8-12 weeks of gestation 1.13 mumol/l and at 38-41 weeks of gestation 2.10 mumol/l. In patients with cholestasis of pregnancy the total bile acid levels varied from 6 to 86 mumol/l.     

Determination of bile acids in serum by capillary gas-liquid chromatography.

A glass capillary column and an appropriate relatively simple procedure for sample preparation have been developed for determination of serum bile acids. Sample preparation involved extraction with Amberlite XAD-2, solvolysis of sulfates, enzymatic hydrolysis with cholylglycine hydrolase, methylation and silylation. Because of complete chromatographic separation of bile acid trimethylsilylether derivatives from cholesterol on the capillary column, an additional step for elimination of cholesterol could be omitted. Trimethylsilylether derivatives were separated on a 20 meter x 0.3 mm i.d. glass capillary column covered with a crystal layer of barium carbonate and coated with polyethyleneglycol 20,000 as liquid phase according to Grob, K. and Grob, G. (1976) J. Chromatogr.125, 471--485, and Grob, K., Grob, G. and Grob, Jr., K., (1977) Chromatographia 10, 181--187. Overall recovery of the major human conjugated bile acids ranged from 86 to 89%. Reproducibility of bile acid determination was satisfactory in both normal and pathological serum with elevated bile acid concentrations (coefficient of variation 7.6 to 10.0%). The mean concentrations of cholic, deoxycholic, chenodeoxycholic and lithocholic acid in the serum of healthy subjects were 0.9, 1.0, 1.7 and 0.2 mumol/l in males, and 1.0, 0.8, 1.4 and 0.2 mumol/l in females.     

Serum cholesterol determination by gas-liquid chromatography

A gas-liquid chromatographic (GLC) method adapted from Schmit and Mater is described for the measurement of total and free cholesterol in serum. The method has been found to be simple, sensitive and precise. The results have been compared with the colorimetric method of Carr and Drekter for total cholesterol and the method of Sperry and Webb for free cholesterol. For total cholesterol lower values were obtained by the GLC method both in sera from patients without signs of liver disease and in sera with high total bilirubin. There was a tendency for somewhat higher values for free cholesterol by the GLC method than by the colorimetric method. This difference was not significant. The present study has demonstrated that the GLC method for total and free cholesterol determination is more specific than colorimetric methods. Gas-liquid chromatography should be given consideration as a reference method for the estimation of free cholesterol and total cholesterol.     

Direct quantitation of cholestanol in plasma by gas-liquid chromatography.

A gas-liquid chromatographic (GLC) method for quantitation of cholestanol in plasma was developed using SP-1000 without the need for argentation thin-layer chromatography and silylation. Cholestanol was resolved well before cholesterol and the phytosterols. This method was sensitive, specific for cholestanol, and reproducible.     

The measurement of sulphated and non-sulphated bile acids in serum using gas-liquid chromatography.

A method is described to assay sulphated and non-sulphated bile acids in serum using gas-liquid chromatography. Previously described techniques have been substantially modified to allow analysis of free and conjugated salts of the four major bile acids with particular care to ensure quantitative recoveries of lithocholic acid, its conjugates and sulphate esters. Losses of lithocholic acid inherent in some methods have been reduced by avoidance of column chromatography with alumina and extraction of lipid contaminants into heptane. Assay of the proportion of serum bile acids present as sulphate esters is achieved by the routine use of column chromatography to separate sulphated bile acids from non-sulphated bile acids followed by solvolysis of the sulphated bile acids before deconjugation. Careful selection of the conditions of strong alkaline hydrolysis ensures deconjugation of all bile salt conjugates including lithocholic conjugates which are not completely hydrolysed in weaker alkaline solutions. The trifluoroacetate derivatives of the methyl esters of the bile acids are chromatographed using 5-beta-cholanic acid as an internal standard with clear separation of the four major bile acids from the internal standard. In 10 fasting control subjects the mean serum total bile acid concentration was 5.3 muM (RANGE 1.1-16.4) including 0.7 mum sulphated bile acid (range 0-1.8). In 10 patients with acute viral hepatitis the total bile acid concentration was elevated in some but normal in others (mean 44.9 muM, range 2.7-80.3). The percentage of the total bile acid sulphated was not significantly different in the hepatitis patients compared to controls (controls 13%, range 0-35; hepatitis 23%, range 0-52). Lithocholic acid made up 13% of the total bile acid in controls (0-32%) and 18% in hepatitis patients (0-53%). Most of this lithocholic acid was sulphated (controls 81%, range 30-100; hepatitis 67%, range 37-100). Unconjugated bile acids were demonstrated in the serum of a few patients with acute viral hepatitis but in no control subjects.     

Measurement of cholesterol in serum by gas chromatography/mass spectrometry at moderate mass resolution, with a nonendogenous cholesterol isomer as internal standard

We describe a gas chromatography/mass spectrometry method for the quantitative analysis of cholesterol in serum. A structural isomer of cholesterol, 7,(5 alpha)-cholesten-3 beta-ol, is used as an internal standard, its primary advantage being its lesser cost relative to that of a stable-isotope-labeled analog. Analysis of the National Bureau of Standards Certified Reference Serum (SRM 909) was used to validate the method. The results show this method to be highly accurate (bias = -0.6%) and precise (CV = 1.6% between-run, 1.2% within-run). The performance of this method is, therefore, sufficiently good to allow its use as a reference method for determinations of cholesterol in serum.     

Preparation of fecal samples for assay of volatile fatty acids by gas-liquid chromatography and high-performance liquid chromatography

We describe a procedure for preparing fecal samples for determination of volatile fatty acids (VFAs) by gas-liquid chromatography (GLC) and "high-performance" liquid chromatography (HPLC). The simple, one-step procedure involves only ultrafiltration through a membrane with a molecular-mass cutoff of 3000 Da. As revealed by the GLC chromatograms, ultrafiltration appears to be as effective as steam distillation in sample clean-up. It also enables higher, more reproducible analytical recoveries of long-chain VFAs. The VFA content of the filtrate can also be measured by HPLC. Use of the ion-exclusion mechanism completely resolves isobutyric acid and butyric acid on a cation-exchange column. The mean (+/- SD) percentage distribution values of VFAs (measured by GLC) from five healthy subjects were 56.0 +/- 3.5 (acetic acid), 17.0 +/- 5.3 (propionic acid), 2.9 +/- 1.5 (isobutyric acid), 18.8 +/- 5.8 (butyric acid), 2.3 +/- 1.2 (isovaleric acid), and 2.9 +/- 0.8 (valeric acid).     

Effect of pectin on serum cholesterol,fecal bile acids and biliary lipids in normolipidemic and hyperlipidemic individuals

Pectin, 40–50 g/day for two weeks administered to nine normolipidemic and hyperlipidemic patients, had no effect on serum triglycerides but did cause a significant decrease in the serum total and unesterified cholesterol of hypercholesterolemic subjects in particular. This was associated with increased excretion of fecal bile acids and total steroids and increased concentration of plasma methyl sterols. Thus, the serum cholesterol reduction by pectin appears to be caused by increased cholesterol elimination into stools as bile acids which is then balanced by enhanced cholesterol synthesis. The composition of biliary bile acids and lipids was not changed and secondary bile acids and sterols decreased inconsistently in feces. The measurement of fecal dry weight suggested that the bulk of the pectin was degraded by bacteria during passage through the intestine. Consequently fecal mass and dry weight were not consistently increased, suggesting that pectin may not be an ideal fibre for increasing fecal bulk in functional colonie disorders.     

A new method for simultaneous determination of bile acids in human bile without hydrolysis

A method for simultaneous determination of major bile acids in human bile without prior hydrolysis is described. The unconjugated, glycine- and taurine-conjugated bile acids are separated into groups by ion-exchange chromatography on a newly developed lipophilic gel, piperidinohydroxypropyl Sephadex LH-20 (PHP-LH-20). Subsequently, resolution of each group into cholate, ursodeoxycholate, chenodeoxycholate, deoxycholate and lithocholate is attained into two stages by high-performance liquid chromatography on a micron-Bondapak C18 column. First, 0.3% ammonium carbonate/acetonitrile (9 : 4, v/v) is used for separation of the latter three as a mobile phase. Cholate and ursodeoxycholate are separated in 0.3% ammonium carbonate/acetonitrile (11 : 4, v/v). The present method is applicable to quantitation of free and conjugated bile acids in human bile with satisfactory accuracy and precision.