Sulfated Bile Acids Research Articles

Synthesis of bile acid monosulphates by the isolated perfused rat kidney.

Perfusion of an isolated rat kidney with labelled bile acids, in a protein-free medium, resulted in the urinary excretion of the labelled bile acid, 3% being converted into polar metabolities in 1h. These metabolities were neither glycine nor taurine conjugates, nor bile acid glucuronides, and on solovolysis yielded the free bile acid. On t.l.c. the metabolite of [24-14C]lithocholic acid had the mobility of lithocholate 3-sulphate. The principal metabolite of [24-14C]chenodeoxycholic acid had the mobility of chenodeoxycholate 7-sulphate; trace amounts appeared as chenodeoxycholate 3-sulphate. [35S]sulphate was incorporated in chenodeoxycholic acid by the kidney, resulting in a similar pattern of sulphation. No disulphate salt of chenodeoxycholic acid was detected. These findings lend support to the hypothesis that renal synthesis may account for some of the bile acid sulphates present in urine in the cholestatic syndrome in man.

Preparation of the 3-monosulphates of cholic acid, chenodeoxycholic acid and deoxycholic acid

1. The 3-sulphates of cholic, chenodeoxycholic and deoxycholic acids were prepared as crystalline disodium salts. 2. The method described shows that it is possible to prepare specific sulphate esters of polyhydroxy bile acids and to remove protecting acyl groups without removing the sulphate. 3. A study of bile acid sulphate solvolysis showed that none of the usual methods give the original bile acid in major yield in a single step. 4. An understanding of the preparation, properties and methods of solvolysis of bile acid sulphates is basic for investigations of cholestasis and liver disease.

Bile Acids in Bile during Long-term Chenodeoxycholic Acid Treatment

Relative concentrations of conjugated and sulfated bile acids in duodenal bile were measured in 5 patients before and during treatment with 0.50-0.75 g of chenodeoxycholic acid per day for 3-4 months. Lithocholic acid constituted 0.8-3.3% (mean 1.8%) of total conjugated and sulfated bile acids before and 0-5.4% (mean 2.6%) during treatment. Lithocholic acid was the predominant bile acid in the sulfate fraction in three patients and chenodeoxycholic acid in two. Sulfated bile acids constituted less than 1% of total bile acids and did not increase during treatment. Ursodeoxycholic acid, the other major metabolite of chenodeoxycholic acid, was found in higher amounts during therapy. The unsaturated bile acid 3beta-hydroxy-delta5-cholenic acid, which was found exclusively as its sulfate ester, showed a slight fall. The average G/T conjugation ratio rose from 2.2 to 4.5.

Renal Synthesis of Bile Acid Sulphates: Evidence from Man and the Isolated Perfused Rat Kidney

Renal Synthesis of Bile Acid Sulphates: Evidence from Man and the Isolated Perfused Rat Kidney

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-β-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 μM (range 1.1–16.4) including 0.7 μM 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 μM, 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.

Chenodeoxycholic Acid Therapy for Gallstones: Effectiveness, Toxicity and Influence on Bile Acid Metabolism

Chenodeoxycholic acid (1 g daily) was administered to 10 patients with gallstones and three patients with biliary stricture and recurrent cholangitis. Four gallstone patients showed diminution or disappearance of stones including one patient whose stone was in the common bile duct. The patients with recurrent cholangitis showed marked improvement in symptoms during treatment. Serum bile acid levels were significantly elevated in 8 gallstone patients during treatment. Liver biopsy in eight gallstone patients during treatment revealed minor changes in five. Lithocholic acid and bile acid sulphates were found in only small amounts in the bile of patients during treatment. No significant trend in biliary lipid composition during treatment was observed. There was no overall trend in the group of patients whose stones disappeared or diminished. Changes in biliary bile acid composition and in bile acid pool sizes were variable following treatment and could not be correlated with the clinical results of treatment. A further trial of chenodeoxycholic acid is recommended in patients with stones in the biliary tree and recurrent cholangitis who are not amenable to surgical treatment.

Sulfated and Nonsulfated Bile Acids in Urine, Serum, and Bile of Patients with Hepatobiliary Diseases

Large amounts of bile acid sulfate were found in the urine of patients with hepatobiliary diseases. In patients with acute hepatitis, daily excretion of bile acid into urine was 68.24 plus or minus 51.80 mumoles per day, and the percentage of sulfated bile acid was 83.4 plus or minus 16.7%. In patients with chronic hepatitis and cirrhosis, a slight increase of urinary bile acid was observed (2.89 plus or minus 2.69 and 5.27 plus or minus 4.28 mumoles per day, respectively), and the percentage of sulfated bile acid was 73.9 plus or minus 28.6 and 44.6 plus or minus 30.4%, respectively. In patients with obstructive jaundice, a moderate increase of urinary bile acid was found (32.62 plus or minus 18.35 mumoles per day), and the percentage of sulfated bile acid was 58.3 plus or minus 22.6%. In patients with hepatobiliary diseases, the elevation of both levels of sulfated and nonsulfated bile acids in serum was observed. The percentage of sulfated bile acid was 9% in normal serum, and varied from zero to 82.8% in pathological sera. A remarkable increase of sulfated bile acid was found in patients with obstructive juandice and acute hepatitis, while a slight elevation was found in patients with chronic hepatitis and cirrhosis. Sulfated bile acid in bile was nonexistent or below 0.5% of total bile acid. According to these findings, the increased bile acid in serum of patients with hepatobiliary diseases might be more easily excreted into the urine as sulfated bile acid.

Measurement of sulfated and nonsulfated bile acids in human serum and urine

Amberlite XAD-2 was used to extract bile acids from urine or diluted serum of patients with hepatobiliary diseases. Columns containing Sephadex LH-20 were then used to separate the sulfated and nonsulfated bile acids. Thin-layer chromatography of the sulfated bile acid fraction obtained from urine revealed several spots with R(F) values different from those of the taurine or glycine conjugates. According to thin-layer chromatographic mobilities, gas-liquid chromatographic analyses, infrared spectra, and elementary analysis of the sulfated material, one of these sulfated bile acids was identified as glycochenodeoxycholic acid monosulfate, and the others were presumed to be taurochenodeoxycholic acid sulfate and glycocholic acid sulfate. A large amount of bile acid sulfate was found in urine of patients with hepatobiliary diseases. They accounted for 35.5-93.3% of total urinary bile acids and consisted of both di- and trihydroxycholanoic acids, with chenodeoxycholic acid as the major acid. Sulfated bile acids were also found in serum, and accounted for 1.8-21.2% of the total bile acids. Only dihydroxycholanoic acids (mainly chenodeoxycholic) were identified.

Sulfated bile acid in urine of patients with hepatobiliary diseases.

AbstractThe presence of sulfated bile acid in urine of patients with hepatobiliary diseases was recognized by using an Amberlite XAD‐2 column for extraction of bile acid and a Sephadex LH‐20 column for separation of sulfated (sulfate of either taurine or glycine conjugate) and nonsulfated bile acid (taurine and glycine conjugate). Sulfated and nonsulfated bile acid, obtained after a Sephadex LH‐20 column of urinary extract of the patient with acute hepatitis, was identified by thin layer chromatography. Sulfated bile acid showed a spot with different Rf value from that of taurine‐conjugated, glycine‐conjugated and free bile acid, and solvolysis of sulfated bile acid resulted in a compound with the same Rf value as glycodihydroxycholanoic acid. A large amount of bile acid sulfate was found in urine of patients with hepatobiliary diseases. The sulfated bile acid in these urine samples occupied from 57.1 to 93.3% of total bile acid, and consisted of both di‐and trihydroxycholanoic acid (major part, chenodeoxycholic acid). As no solvolysis was carried out in previous works, bile acid sulfate in urine, as described in this paper, was not determined at all.

Sulfated and nonsulfated bile acid in human serum.

AbstractThe presence of sulfated bile acid in serum of healthy persons and patients with hepatobiliary diseases was recognized by using an Amberlite XAD‐2 column for extraction of bile acid and utilizing a Sephadex LH‐20 column for separation of sulfated bile acid (sulfate of either taurine or glycine conjugate), and nonsulfated bile acid (taurine and glycine conjugate. The values of nonsulfate determined in this study were almost similar to the level of total serum bile acid reported by previous workers. A small amount of sulfated bile acid was found in normal serum, and the percentage of sulfated to total bile acid was ca. 10%. In patients with hepatobiliary diseases, both levels of sulfated and nonsulfated bile acid in serum rose, but sulfate did not increase in parallel to the level of nonsulfate. A remarkable increase of serum‐sulfated bile acid could be observed in patients with cholestasis, while a slight elevation was noted in patients with chronic hepatocellar insufficiency. The percentage of sulfated to total bile acid was from 1.8 to 21.2% in patients with hepatobiliary diseases. Sulfated bile acid in serum consisted of only dihydroxycholanoic acid, deoxycholic and chenodeoxycholic acid. As no solvolysis was not carried out in previous works, bile acid sulfate in serum as described in this study was not determined at all.

Mechanism of Cholestasis: 6. Bile acids in human livers with or without biliary obstruction

The bile acid pattern was determined in 12 surgical biopsy specimens, and in four necropsy specimens of human liver. In normal livers, cholic acid is usually the predominant one. Only traces of lithocholic acid were found. In severe biliary obstruction, cholic acid rose conspicuously, whereas chenodeoxycholic acid rose only when cholestasis was protracted. In the patient with the most prolonged jaundice, chenodeoxycholic acid exceeded cholic acid. Lithocholic acid and bile acid sulfate esters comprised only a minor fraction of the total bile acids in cholestasis. The sum of deoxycholic acid and chenodeoxycholic acid parallels the degree of feathery degeneration as an expression of cholestatic liver cell injury. It is assumed that the rise of cholic acid, which does not appreciably interact with the microsomal biotransformation system in contrast to the dihydroxylated bile acids, represents a transient protective mechanism in human extrahepatic biliary obstruction.

A Versatile Method for Analysis of Bile Acids in Plasma

Abstract Plasma or serum is diluted ten times with 0.1 M NaOH. Bile acids are extracted and purfied by passing the sample through a column of Amberlite XAD-2. The resin adsorbs the bile acids, which are then eluted with ethanol. After addition of sodium hydroxide and hydrolysis, the free bile acids are extracted by Amberlite XAD-2 and are then methylated and analyzed by gas chromatography. The degree of purification obtained in the first extraction step is sufficient to permit an analysis of conjugated bile acids or bile acid sulfates by thin-layer or Sephadex LH-20 chromatography.

Bile acid sulfates. II. Formation, metabolism, and excretion of lithocholic acid sulfates in the rat

Sulfate esterification has been shown previously to be a prominent feature of lithocholate metabolism in man. These studies were undertaken to ascertain whether this metabolic pathway is also present in rats, and to investigate the physiological significance of bile acid sulfate formation. Lithocholic acid-24-(14)C was administered to bile fistula rats, and sulfated metabolites were identified in bile by chromatographic and appropriate degradative procedures. They constituted only a small fraction (2-9%) of the total metabolites but a more significant fraction (about 20%) of the secreted monohydroxy bile acids, most of the lithocholate having been hydroxylated by the rat liver. When sulfated glycolithocholate was administered orally, it was absorbed from the intestine without loss of the sulfate, presumably by active transport, and secreted intact into the bile. In comparison with non-sulfated lithocholate, an unusually large fraction (24%) of the sulfated bile acid was excreted in the urine, and fecal excretion took place more rapidly. Both the amino acid and sulfate moieties were extensively removed prior to excretion in the feces. Hydroxylation of bile acid sulfates or sulfation of polyhydroxylated bile acids did not occur to any great extent, if at all.

Bile acid sulfates. I. Synthesis of lithocholic acid sulfates and their identification in human bile

Sulfate esters of lithocholic, glycolithocholic, and taurolithocholic acids were synthesized using sulfur trioxide in pyridine; they were purified by crystallization from methanol or ethanol as the diammonium salts, and their chemical compositions, infared spectra, and chromatographic behavior were determined. Strong alkaline hydrolysis of these sulfates, as commonly performed during quantitative and qualitative analyses of conjugated bile salts, was found to result in a number of degradation products, presumably through disruption of the C-O bond of the hydroxyl group and conversion of the original steroid to isolithocholate and other (possibly olefinic) compounds. After oral administration of lithocholate-(14)C to three patients with cholelithiasis, radioactive metabolites having the chromatographic properties of sulfated lithocholates were isolated from bile and, confirming a preliminary report (1), were identified as sulfated glycolithocholate and taurolithocholate by their characteristic chromatographic mobilities during a series of specific hydrolytic procedures and by crystallizing them to constant specific activities with the synthetic sulfates. The fraction of endogenous lithocholate present in bile as the sulfate was calculated for two patients by isotope dilution and was shown to be 41% and 75% of the total. Sulfation can be expected to affect the physiological and pharmacological properties of lithocholates and may, therefore, influence the toxic properties of these compounds.

The formation of bile acid sulfates: a new pathway of bile acid metabolism in humans.

Bile acids are normally conjugated at the C24 carboxyl group with the amino acids taurine and glycine prior to their excretion into the bile of man and most higher species. In previous studies on biliary metabolites of C'4-labeled lithocholic acid in humans,' the expected taurine and glycine conjugates were observed, but approximately half of the labeled material occurred as more polar metabolites. In this report, data are presented which identify these compounds as the 3a-sulfate esters of glycolithocholic and taurolithocholic acids. The formation of these sulfate esters delineates a new pathway of bile acid metabolism in man, and has important implications for the clinical significance of lithocholic acid, an endogenous steroid with extensive biological toxicity. Methods.-Lithocholic acid-24-C'4 (50 tc, New England Nuclear Corp.) was given orally to two patients with functioning gallbladders, 36 hours prior to cholecystectomy for cholelithiasis. At operation, bile was obtained from the gallbladder. Thin-layer chromatography was performed using Silica Gel H (E. Merck A. G., Darmstadt, Germany) and the following phase systems: Butanol 1 :2 butanol 50, acetic acid 5, water 5 (pH 1); Butanol 3: butanol 50, 0.01 M Tris buffer 9.25, propionic acid 0.75 (pH 3.0); Propionic acid:' propionic acid 15, isoamyl acetate 20, water 5, n-propanol 10; S154: trimethylpentane 25, ethyl acetate 25, acetic acid 0.25. Radioactive spots on chromatoplates were detected with a Vanguard glass plate scanner (Vanguard Instrument Co., LaGrange, Ill.). Sulfate esters of glycolithocholic and taurolithocholic acids were prepared using pyridine-sulfur trioxide5 and crystallized as the ammonium salts. Details of the synthesis and properties of the compounds will be described separately. Solvolysis was performed using a modification of the method of Burstein and Lieberman.6 The steroid sulfate was dissolved in ethanol, acidified to pH 1 or less with 2 N HCI, and diluted with 9 volumes of acetone. The solvolysis mixture was then incubated at room temperature for one to three days. Some esterification occurred, and the resulting ethyl esters were hydrolyzed by refluxing for two hours in 5 per cent methanolic KOH. Results.-In the previous study, labeled biliary metabolites of orally administered lithocholic acid-24-C14 were shown to include glycolithocholic acid, taurolithocholic acid, and two polar compounds, I and II.' In this study, essentially similar results were obtained in both patients; four corresponding labeled biliary metabolites were observed on thin-layer chromatography using the Butanol 1 system. When bile was chromatographed with the Butanol 3 system, the labeled metabolites separated into two areas, A and B (Fig. 1). The compounds in area A, with the same mobility as glycolithocholic and taurolithocholic acid standards, were eluted and rechromatographed with the Butanol 1 system. Two radioactive spots, with mobilities corresponding to glycolithocholic acid and taurolithocholic acid, were identified. The

Excretion of bile acids in normal rabbits.

ARTICLESExcretion of bile acids in normal rabbitsJA Gregg, and Poley JRJA Gregg, and Poley JRPublished Online:01 Nov 1966https://doi.org/10.1152/ajplegacy.1966.211.5.1147MoreSectionsPDF (1 MB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInEmailWeChat Previous Back to Top Next Download PDF FiguresReferencesRelatedInformationCited ByRobust and Comprehensive Targeted Metabolomics Method for Quantification of 50 Different Primary, Secondary, and Sulfated Bile Acids in Multiple Biological Species (Human, Monkey, Rabbit, Dog, and Rat) and Matrices (Plasma and Urine) Using Liquid Chromatography High Resolution Mass Spectrometry (LC-HRMS) Analysis7 April 2021 | Journal of the American Society for Mass Spectrometry, Vol. 32, No. 8MODULATION OF BILE ACIDS INDUCED BY PARAQUAT IN RABBITSDrug Metabolism and Drug Interactions, Vol. 11, No. 4Cholic Acid Synthesis from 26-Hydroxycholesterol and 3-Hydroxy-5-cholestenoic Acid in the RabbitJournal of Biological Chemistry, Vol. 264, No. 7The bile acid composition of rabbit and cat gall-bladder bileJournal of Steroid Biochemistry, Vol. 8, No. 10Sterol and bile acid metabolism during development. 1. Studies on the gallbladder and intestinal bile acids of newborn and fetal rabbitSteroids, Vol. 29, No. 1Bile acids XLVII. 12α-hydroxylation of precursors of allo bile acids by rabbit liver microsomesBiochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, Vol. 409, No. 2Bile salts and bile formation in rats, rabbits and guinea pigsComparative Biochemistry and Physiology Part A: Physiology, Vol. 50, No. 3Bile composition in the pregnant rabbitThe American Journal of Digestive Diseases, Vol. 17, No. 1Effects of rose bengal on bile secretion in the rabbit: inhibition of a bile salt-independent fractionGut, Vol. 11, No. 2Bacterial 7-dehydroxylation of cholic acid and allocholic acidJournal of Lipid Research, Vol. 10, No. 4Bile acid composition of bile from germ-free rabbitsBiochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, Vol. 176, No. 1Experimental cholelithiasis in the rabbit induced by cholestanol feeding: effect of neomycin treatment on bile composition and gallstone formationJournal of Lipid Research, Vol. 9, No. 2Bile salt evolutionJournal of Lipid Research, Vol. 8, No. 6 More from this issue > Volume 211Issue 5November 1966Pages 1147-1151 Copyright & PermissionsCopyright © 1966 by American Physiological Societyhttps://doi.org/10.1152/ajplegacy.1966.211.5.1147PubMed5924035History Published online 1 November 1966 Published in print 1 November 1966 Metrics