Simultaneous quantitation of fatty acids,sterols and bile acids in human stool by capillary gas-liquid chromatography

A simple method for the simultaneous gas-liquid chromatographic quantitation of fatty acids, sterols and bile acids from human fecal samples is described. The various compounds are directly converted into the n-butyl ester-trimethylsilyl ether derivatives, without prior isolation from the stool. Under these conditions, fecal bile acid derivatives are well resolved from each other and from those of fecal fatty acids and sterols without overlaps. The method was found to be reproducible and recoveries were similar to those obtained after exhaustive solvent extraction of fecal sterols, fatty acids and bile acids. Optimum derivatization conditions that allowed maximum recovery of fecal components with minimal destruction and application of the method for simultaneous bile acid, fatty acid and sterol analysis in human stool are described.     

General methods for the analysis of metabolic profiles of bile acids and related compounds in feces

A general method is described for the detailed qualitative and quantitative analysis of bile acids and related compounds from feces. The technique utilizes a novel combination of liquid-gel and liquid-solid extraction, lipophilic ion exchange chromatography, and capillary column gas-liquid chromatography coupled to mass spectrometry, which permits the detailed composition of bile acids in feces in terms of both the individual bile acids present and their mode of conjugation in the original fecal sample. The extraction, purification, and isolation procedures have been evaluated using fecal samples containing endogenous radioactive bile acid metabolites and from the addition of radiolabeled standards to fecal homogenates. The applicability of the general procedure is illustrated with examples from the analysis of bile acids and sterols in the feces collected from normal healthy subjects, patients with chronic diarrhea, and an adult female Sprague-Dawley rat. The flexibility of the method, and the general problems encountered in the extraction, purification, and isolation of bile acids and related classes of compounds from feces for subsequent analysis of gas-liquid chromatography are discussed in detail.     

Determination of underivatised sterols and bile acid trimethyl silyl ether methyl esters by gas chromatography-mass spectrometry-single ion monitoring in faeces

A method for quantification of total faecal sterols and bile acids (BAs) in human stool by using gas chromatography-mass spectrometry-single ion monitoring (GC-MS-SIM) is described. Cholesterol, coprostanol, coprostanone, cholestanol, iso-lithocholic acid (iso-LCA), lithocholic acid (LCA), iso-deoxycholic acid (iso-DCA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA), cholic acid (CA), and 12-oxo-deoxycholic acid (12-oxo-DCA) in faeces of 86 healthy subjects were determined. The sample preparation for sterol analysis requires hydrolysis and liquid extraction from matrix, but no derivatisation. The GC-flame ionisation detection (FID) and total ion current (TIC) in GC-MS were not sufficient for sterol and BA determination, whereas selectivity and specificity of the GC-MS-SIM ensured the analysis of sterols and BAs in faeces.     

The determination and excretion of individual human fecal bile acids

A fluorometric method, using resazurin, for the analysis of individual fecal bile acids separated by thin layer chromatography of crude fecal extracts is described. The method is precise and accurate. The assay was used to investigate the constancy of excretion of individual fecal bile acids in small random stool samples collected over three weeks in six humans. Relatively small but significant variations were found in each case, most were random but one was progressive.     

The steroids of 2000-year-old human coprolites.

Six samples of human coprolites, some more than 2,000 years old, were analyzed for fecal steroid composition. Despite this very lengthy period of storage, the fecal steroids of coprolites were remarkably similar to those of stool samples collected today. The sterol nucleus was clearly rather stable under the dry environmental conditions of the Nevada Caves. The steroid content (microgram/g dried weight) of coprolite was low in comparison to that of modern man. The bile acid/cholesterol and plant sterol/cholesterol ratios of the coprolite, however, were similar to these ratios of the stools of modern man. In the six coprolites, an average 73% of the neutral steroids was digitonin-precipitable. This precipitate was composed of cholesterol and three plant sterols (campesterol, stigmasterol, and beta-sitosterol) and their bacteria-modified products. A portion of the neutral steroids had been converted to products tentatively identified as epimers of these steroids. Individual bile acids were identified in the coprolite. The bile acid composition of the coprolite was similar to that of the stool of modern man.     

A simple and accurate HPLC method for fecal bile acid profile in healthy and cirrhotic subjects: validation by GC-MS and LC-MS

We have developed a simple and accurate HPLC method for measurement of fecal bile acids using phenacyl derivatives of unconjugated bile acids, and applied it to the measurement of fecal bile acids in cirrhotic patients. The HPLC method has the following steps: 1) lyophilization of the stool sample; 2) reconstitution in buffer and enzymatic deconjugation using cholylglycine hydrolase/sulfatase; 3) incubation with 0.1 N NaOH in 50% isopropanol at 60°C to hydrolyze esterified bile acids; 4) extraction of bile acids from particulate material using 0.1 N NaOH; 5) isolation of deconjugated bile acids by solid phase extraction; 6) formation of phenacyl esters by derivatization using phenacyl bromide; and 7) HPLC separation measuring eluted peaks at 254 nm. The method was validated by showing that results obtained by HPLC agreed with those obtained by LC-MS/MS and GC-MS. We then applied the method to measuring total fecal bile acid (concentration) and bile acid profile in samples from 38 patients with cirrhosis (17 early, 21 advanced) and 10 healthy subjects. Bile acid concentrations were significantly lower in patients with advanced cirrhosis, suggesting impaired bile acid synthesis.     

Capillary gas chromatographic analysis of serum bile acids as the n-butyl ester–trimethylsilyl ether derivatives

Gas chromatographic separations of n-butyl ester-trimethylsilyl ether derivatives of several common bile acids were compared with those of the corresponding methyl ester-trimethylsilyl ether derivatives on a CP-Sil-5 CB capillary column. Both types of derivatives were similarly resolved from each other. However, the n-butyl ester-trimethylsilyl ether derivatives of the bile acids showed longer retention times than the corresponding methyl ester-trimethylsilyl ethers and unlike the methyl ester-trimethylsilyl ether derivatives, were completely resolved from and eluted later than the trimethylsilyl ethers of common plasma sterols including sitosterol. A simplified method of plasma work-up for quantitation of bile acids and application of the above method in quantification of plasma bile acids in humans is described.     

Effect of bile acid deconjugation on the fecal excretion of steroids

The effect of microbiological deconjugation of bile acids on total bile acid and neutral sterol fecal excretion by adult male rats has been studied. A screening method utilizing mice allowed selection of a Clostridium perfringens type A strain, which accelerated cholesterol catabolism in mice. When this species of bacteria was associated with germfree rats, the fecal bile acids were excreted as free bile acids (deconjugated), however the quantities of bile acids excreted were not increased compared with those of germfree rats. Conventional rats excrete twice as much bile acids (all deconjugated) as do the germfree and C. perfringens-associated rats. It is, therefore, unlikely that the microbiological deconjugation of bile acids is responsible for the increased fecal excretion of bile acids seen in conventional rats. The C. perfringens-associated rats excreted identical kinds and quantities of fecal neutral sterols as did the germfree rats.     

Alteration of biliary ursodeoxycholic acid in guinea pig during early stages of cholestyramine feeding

The effects of cholestyramine feeding on biliary ursodeoxycholic acid, fecal excretion of bile acids and neutral sterols on cholesterol 7α-hydroxylase and hepatic HMG-CoA reductase were examined in the guinea pig. In the bile there was a 57% decrease in the concentration of ursodeoxycholic acid while an increase was observed in the concentration of chenodeoxycholic acid. Cholestyramine feeding for ten days resulted in a decrease in plasma cholesterol levels and an increase in both hepatic HMG-CoA reductase and cholesterol 7α-hydroxylase activities. The fecal excretion of both bile acids and neutral sterols was significantly increased.     

Gas chromatography-mass spectrometry of isobutyl ester trimethylsilyl ether derivatives of bile acids and application to the study of bile sterol and bile acid biosynthesis in rat liver epithelial cell lines

The derivatization of bile acids into trimethylsilyl ether isobutyl ester (IBTMS) and of neutral sterols into trimethylsilyl ether (TMS) allowed the separation on an OV-1 capillary gas chromatography column of 15 bile steroids as follows: cholesterol, 7 alpha-hydroxycholesterol, 6 beta-hydroxycholesterol, 6 alpha-hydroxycholesterol, 7 beta-hydroxycholesterol, lithocholate, deoxycholate, 25-hydroxycholesterol, chenodeoxycholate, cholate, murocholate, hyodeoxycholate, ursodeoxycholate, hyocholate, and beta-muricholate. Fragmentation data of the coupled gas chromatographic-mass spectrometric (GC-MS) analysis of these nine bile acids as IBTMS derivatives under electron impact and chemical ionizations (methane, isobutane, and ammonia) are given. The ammonia chemical ionization appears to be the best mode for compound identification and quantitation due to fragmentations into high mass ions. The comparison of methylene units of the five sterols as TMS derivatives and of each type of methyl, TMS, or isobutyl ester of the nine bile acids as TMS ethers showed that isobutyl esterification increased dramatically the retention time of the bile acids, allowing their separation after the neutral sterols. Different methods of GC-MS analysis were applied to the study of bile steroid secretion in long-term rat liver epithelial cell lines, either serum-supplemented cell lines or serum-free cell lines, growing in serum-free medium since the primary explanation or after adaptation of serum-supplemented lines to this medium. It is demonstrated for the first time that liver epithelial cell lines maintain the metabolic pathway leading from synthesized cholesterol to dioxygenated sterols and the two normal main primary bile acids of the liver, chenodeoxycholic acid and cholic acid, up to 32-47% of the in vivo daily rate, and in addition the production of alpha-muricholic acid, the bile acid marker of murine liver.     

Cholesterol gallstones in alloxan-diabetic mice

Normal and alloxan-diabetic male mice (Crj-ICR) were fed a diet containing 0.5% cholesterol for 5 and 10 weeks, and gallbladder bile was analyzed for cholesterol, phospholipids and bile acids, feces for sterols and bile acids, and plasma and liver for cholesterol, phospholipids, and triglycerides. Normal mice developed no gallstones but the diabetic mice developed cholesterol gallstones with an incidence of 70% by 5 weeks and 80% by 10 weeks after feeding of the cholesterol diet. Diabetic mice fed the ordinary diet also developed stones (23%) by 10 weeks. In the diabetic mice, the gallbladder was enlarged about threefold, and biliary lipid concentration, diet intake, and fecal excretion of sterols and bile acids increased but body weight decreased. Cholic acid and beta-muricholic acid comprised over 40% each of the total biliary bile acids in normal mice, but cholic acid increased to about 80% and beta-muricholic acid decreased to a few percent in the diabetic mice. Fecal excretion of bile acids increased after cholesterol feeding in both normal and diabetic mice, but the increased bile acid in the normal animals was beta-muricholic acid and that in the diabetic mice was deoxycholic acid. The mice that developed gallstones showed a marked increase in biliary cholesterol value and decreases in gallbladder bile and bile acid concentration, but no difference in biliary and fecal bile acid composition, bile acid synthesis, fecal sterols, or plasma and liver lipid levels. Cholesterol absorption was increased in the diabetic mice when examined by plasma 14C/3H ratio and fecal 14C-labeled sterol excretion after a single oral administration of [14C]cholesterol and a simultaneous intravenous injection of [3H]cholesterol. These data led to the conclusion that cholesterol gallstones developed in alloxan-diabetic mice fed excess cholesterol, due to the hyperphagia and the enhancement of cholesterol absorption caused by increases in the synthesis and secretion of cholic acid.     

Sensitive detection of specific repair endonucleases: radial diffusion assay utilizing differential alkaline denaturation of supercoiled and nicked PM2 DNA in agarose gels

An improved method for separation and quantitation of sulfated neutral and acidic steroids in human feces was developed. The procedure consists of separation of sulfated steroids on Sephadex LH-20 and hydrolysis by cholylglycine hydrolase followed by quantitation and identification of the trimethylsilylether derivatives by gas-liquid chromatography and gas-liquid chromatography-mass spectroscopy. Using this procedure, we detected no sulfated bile acids in human feces. However, sulfated cholesterol was detected in the sulfated bile acid fraction obtained from human fecal extracts. Analysis showed that cholesterol sulfate comprised 12.3, 11.2, and 31.0% of the total neutral sterol fraction in the three fecal samples. Using our procedures, cholesterol sulfate and bile acid sulfates in a biological mixture can be quantitated and identified when they are present.     

Importance of jejunum for bile acid absorption in pigeons: effect of jejunal resection on plasma cholesterol and cholesterol excretion

Previous studies from our laboratory suggested that the jejunum could be the major site of bile acid absorption in the pigeon. To confirm this observation directly, the effects of jejunal resection on the fecal excretion of bile acids and neutral sterols and on the level of plasma cholesterol were examined in the pigeon. This surgical procedure specifically increased (p less than 0.05) the excretion of bile acids, with no change in the excretion of neutral sterols. The total fecal steroid excretion in the pigeons with jejunal resection was significantly (P less than 0.01) higher than that in the control group. Jejunal resection also decreased the plasma cholesterol level in the pigeon. These results confirm our earlier suggestion that the jejunum is the major site of bile acid absorption in pigeons, unlike that in mammals, in which the ileum is the major site.     

Determination of fetal bile acids in biological fluids from neonates by gas chromatography-negative ion chemical ionization mass spectrometry

A method has been developed for microanalysis of fetal bile acids in biological fluids from neonates by capillary gas chromatography-mass spectrometry using negative-ion chemical ionization of pentafluorobenzyl ester-dimethylethylsilyl ether derivatives of bile acids. Calibration curves for the bile acid derivatives are useful over the range 0.1–100 pg and the detection limit for bile acids was 1 fg (S/N=5) using isobutane as a reagent gas. Recoveries of the bile acids and their glycine and taurine conjugates from bile acid-free serum and dried blood discs ranged from 92 to 101% and from 93 to 108%, respectively, of the added amounts of their standard samples. The analysis of bile acids on a dried blood disc, meconium and urine from infants, exhibited significant hydroxylation at the 1β-, 2β-, 4β and 6α-positions of the usual bile acids, cholic and chenodeoxycholic acids, for the urinary or fecal excretion of bile acids in the fetal and neonatal periods. The present method was applied clinically to analyze bile acids on a dried blood disc from neonatal patients with congenital biliary atresia and hyper-bile-acidemia.     

Capillary gas-liquid chromatography of glycine-conjugated bile acids without prior hydrolysis

A method is described for the gas-liquid chromatographic (GLC) analysis of intact glycine conjugates of the major bile acids present in human plasma. It is, therefore, now possible to analyze glycine-conjugated and unconjugated bile acids together on a single GLC column without the necessity for a hydrolytic step. A large number of derivatives of bile acid glycine conjugates were examined, but only acetate- and silyl ether-derivatives of carboxylic acid methyl esters were found initially to be suitable. It was not possible to make acetates consistently, and trimethylsilyl ethers did not allow resolution of the glycine conjugates of cholic and chenodeoxycholic acids. Dimethylethylsilyl ether methyl ester derivatives were subsequently found to give the best results. Chromatographic conditions for successful analysis of these derivatives were examined and it was found to be necessary to use wall-coated capillary columns of thin film thickness (0.12 micron) and very high carrier gas flow rates (ca. 20 ml/min hydrogen). Using acetonitrile and Bond Elut extraction, fractionation on Sep-Pak SIL cartridges, and derivatization as dimethylethylsilyl ether methyl esters, the capillary gas-liquid chromatography of intact glycine-conjugated bile acids from human plasma was demonstrated for the first time.     

Cholesterol metabolism during ketoconazole treatment in man

Ketoconazole, an antifungal antibiotic, inhibits cholesterol synthesis by blocking demethylation of lanosterol. Effects of this inhibition were studied on serum cholesterol, lipoproteins and cholesterol precursors, biliary lipid composition, and fecal steroid elimination in five patients with prostate cancer treated with large doses of ketoconazole. The serum level of total cholesterol fell by 27%, that of LDL cholesterol by 41% and that of LDL apoB by 32% with ketoconazole alone; the fall in the total cholesterol level of a patient treated with ketoconazole-cholestyramine was 65%. Serum contents of free lanosterol and dihydrolanosterol increased up to 250 times, yet the total concentrations remained less than 2 mg/dl. Of the other cholesterol precursor sterols only those with delta 8-double bond increased several times, indicating that in addition to 14 alpha-demethylation, ketoconazole also interfered with metabolism of later intermediary sterols to some extent. Compared with serum sterols, lanosterols were enriched in biliary and fecal sterols up to 10-20 times. Fecal lanosterol output increased from 12 to 247 mg/day, and comprised over 20% fecal steroids of endogenous origin. Bile acid synthesis was significantly decreased, the proportion of chenodeoxycholic acid being markedly reduced in both biliary and fecal bile acids. Cholesterol absorption appeared to decrease yet fecal neutral sterol output and cholesterol synthesis were unchanged and the overall sterol synthesis was increased. It thus appears that ketoconazole inhibits cholesterol elimination as bile acids. However, by blocking 14 alpha-demethylation, it results in effective drainage of sterol nucleus as lanosterols into bile and feces, which, in turn, is associated with a marked reduction in low density lipoprotein (LDL) cholesterol level probably through activation of hepatic LDL apoB receptors.     

Gas-liquid chromatographic determination of human fecal bile acids

A method for the determination of total bile acids in human feces that is suitable for routine application is described and discussed. Bile acids are extracted from freeze-dried feces with acetic acid and toluene, in the presence of the internal standard 23-nordeoxycholic acid. After saponification of the extract, bile acids and the internal standard are methylated and converted by mild chromic acid oxidation into their ketonic derivatives. The resultant mixture of a few stable compounds can be separated and measured quantitatively by gas-liquid chromatography on a methylsiloxane polymer. A reference bile acid mixture including the internal standard is also taken through the entire procedure with each series of samples. It has been demonstrated that, in spite of the omission of the usual purification steps, the method is specific for bile acids.     

Fecal neutral steroids and bile acids from germfree rats

The amount and composition of fecal neutral sterols and bile acids excreted by adult male germfree and conventional rats have been determined. The amounts of neutral sterols excreted were 12.8 (germfree) and 19.5 (conventional) mg/kg of body wt per day. The germfree rats excreted cholesterol and lathosterol (methostenol was not assayed); the conventional rats excreted coprostanol and coprostanone in addition. The amounts of bile acids excreted were 11.3 (germfree) and 21.4 (conventional) mg/kg of body wt per day. The bile acids excreted by the rats were tentatively identified as tauro--muricholate, tauro-alpha-muricholate, and tauro-cholate, besides an unidentified component. The conventional rats excreted the corresponding unconjugated acids as well as many other unconjugated bile acids. No significant correlation was found between the amount of coprosterols and the total amount of neutral sterols excreted by the conventional rats. This suggests that bacterial reduction of cholesterol is not an important mechanism of increasing neutral sterol excretion of conventional rats as compared to germfree rats. Evidence is presented that suggests that this difference in neutral sterol excretion is due to changes in intestinal secretion and sloughing between the two types of animal. The factors reponsible for the differences in bile acid excretion have not been identified.     

Comparison among fecal secondary bile acid levels, fecal microbiota and Clostridium scindens cell numbers in Japanese

Bile acid 7alpha-dehydroxylation by intestinal bacteria, which converts cholic acid and chenodeoxycholic acid to deoxycholic acid (DCA) and lithocholic acid (LCA), respectively, is an important function in the human intestine. Clostridium scindens is one of the most important bacterial species for bile acid 7alpha-dehydroxylation because C. scindens has high levels of bile acid 7alpha-dehydroxylating activity. We quantified C. scindens and secondary bile acids, DCA and LCA, in fecal samples from 40 healthy Japanese and investigated their correlation. Moreover, we used terminal restriction fragment length polymorphism (T-RFLP) analysis to investigate the effect of fecal microbiota on secondary bile acid levels. There was no correlation between C. scindens and secondary bile acid in fecal samples. On the other hand, T-RFLP analysis demonstrated that fecal microbiota associated with high levels of DCA were different from those associated with low levels of DCA, and furthermore that fecal microbiota in the elderly (over 72 years) were significantly different from those in younger adults (under 55 years). These results suggest that intestinal microbiota have a stronger effect on DCA level than does the number of C. scindens cells.     

l-Alanine-α-Ketobutyrate Aminotransferase of Pseudomonas sp.

Modified fungal product 4-O-methylascochlorin (MAC) is an experimental agent affecting lipid and carbohydrate metabolism in mammals. The hypocholesterolemic properties of MAC were studied using rats fed on a standard laboratory diet. Because of the insolubility in water, reproducibility of the hypocholesterolemic activity had usually been poor for rats fed ad libitum. The difficulty was overcome by controlled reverse-phase feeding; MAC significantly lowered serum total cholesterol (s-TC) in rats only when given by gastric intubation soon after diet intake.

MAC increased fecal excretion of neutral and acidic sterols and also increased biliary flow accompanying increments in biliary cholesterol, bile acids and phospholipids. A much larger increase in neutral sterols was characteristic for MAC. However, intestinal absorption of cholesterol and cholic acid was unaffected by MAC. Three mechanisms therefore seemed to be working in hypocholesterolemic activity: (a) withdrawal of hepatic cholesterol into bile, (b) a larger fecal loss of sterols following increment of biliary sterols and (c) enhanced bile acid synthesis compensating the larger fecal loss. A negative sterol balance often leads to an increase in hepatic cholesterogenesis. However, cholesterogenesis, as judged from incorporation of the precursors, was unchanged by MAC.