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The isolation and further characterization of the bilirubin tetrapyrroles in bile-containing human duodenal juice and dog gall-bladder bile.

ABSTRACT: Bilirubin and its conjugates were extracted from either dog gall-bladder bile or bile-containing human duodenal juice into chloroform containing 10mm-tetraheptylammonium chloride. The intact bilirubin tetrapyrroles were then separated by t.l.c. Structural elucidation was made after coupling of the individual pigments with diazonium salts. Four azopigments were detected: azopigment alpha(o) or dipyrrolic azobilirubin; azopigment delta or dipyrrolic azobilirubin monoglucuronide; azopigment alpha(3) or dipyrrolic azobilirubin monoglucoside; and, from dog gall-bladder bile, azopigment alpha(2). The last conjugate required further verification of its structure. After methanolysis, it was shown by combined g.l.c.-mass spectrometry to contain xylose in a 1:1 molar ratio with the azopigments of bilirubin. Human bile contained 86% bilirubin diglucuronide, 7% bilirubin monoglucuronide monoglucoside diester, 4% bilirubin monoglucuronide and 3% bilirubin. Dog gall-bladder bile had a considerably different composition; it contained 47% bilirubin diglucuronide, 40% bilirubin monoglucuronide monoglucoside diester, 8% bilirubin monoglucuronide, 4% bilirubin diglucoside, 1-2% bilirubin and traces of conjugates containing xylose. The total bilirubin content and proportions of the conjugates did not change in bile that was frozen and stored at -20 degrees C under N(2), whereas in the chloroform/tetraheptylammonium chloride extract, similarly stored, total pigment was slowly lost and the diglucuronide conjugate converted into the monoglucuronide.

SUBMITTER: Gordon ER

PROVIDER: S-EPMC1183615 | biostudies-other | 1977 Oct

REPOSITORIES: biostudies-other

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Project description:BACKGROUND: Endoscopic sphincterotomy has been shown to inhibit stone formation in the gall bladder of experimental animals. AIMS: To investigate the alterations in bile composition and gall bladder motility after endoscopic sphincterotomy. PATIENTS: A study was performed of gall bladder bile composition and gall bladder motility in patients with gallstone disease ((n = 20; age 40-60 years, median age 55 years: seven men), with gall bladder calculi (n = 12) and with diseased gall bladder (chronic inflammation) without gall bladder calculi (n = 8)), who had received endoscopic sphincterotomy for common bile duct stones. Age and sex matched disease controls comprised 20 patients with gallstone disease but without stones and an intact sphincter of Oddi (with gall bladder calculi (n = 10) and diseased gall bladder without gall bladder calculi (n = 10)). METHODS: Gall bladder motility was assessed by ultrasound. Duodenal bile collected by nasoduodenal tube after stimulation of gall bladder by intravenous ceruletid infusion was analysed for cholesterol, phospholipid, and bile acid concentrations, cholesterol saturation index, and nucleation time. RESULTS: There was a significant reduction in mean (SEM) fasting volume (12.5 (1.7) ml v 26.4 (2.5) ml; p < 0.001) and mean (SEM) residual volume (4.34 (0.9) ml v 14.7 (0.98) ml; p < 0.001), and increase in mean (SEM) ejection fraction (65.7 (4.2)% v 43.6 (5.52)%; p < 0.001) and mean (SEM) rate constant of gall bladder emptying (-0.031/min v -0.020/min; p < 0.01) in patients who had been subjected to endoscopic sphincterotomy. Median nucleation time was significantly longer (17 days v 6 days; p < 0.006) in treated patients. There was a reduction in total mean (SEM) lipid concentrations (6.73 (0.32) g/dl v 7.72 (0.84) g/dl; p < 0.05), cholesterol (5.6 (1.5) mmol/l v 10.3 (2.23) mmol/l; p < 0.001) and CSI (0.72 (0.15) v 1.32 (0.31); p < 0.001). There was no significant change in mean (SEM) phospholipid (25.6 (3.5) mmol/l v 23.4 (6.28) mmol/l) and bile acid (93.7 (7.31) mmol/l v 105.07 (16.6) mmol/l) concentrations. CONCLUSIONS: After endoscopic sphincterotomy there was enhanced contractility of the gall bladder, accompanied by a prolongation of nucleation time and reduction in cholesterol saturation index.

Project description:A method is presented that allows the isolation of eight different phenylazo derivatives of bile bilirubin. In step I of the isolation procedure, three bilirubin fractions (bilirubin fractions 1, 2 and 3) from human hepatic bile are separated by reverse-phase partition chromatography on silicone-treated Celite with the use of a solvent system prepared from butan-1-ol and 5mm-phosphate buffer, pH6.0. Azo coupling is then performed with diazotized aniline. The three azo pigment mixtures are subjected to step II, in which the above chromatography system is used again. With each azo pigment mixture this step brings about the separation of a non-polar and a polar azo pigment fraction (azo 1A and azo 1B, azo 2A and azo 2B, and azo 3A and azo 3B from bilirubin fractions 1, 2 and 3 respectively). Approximately equal amounts of non-polar and polar pigments are obtained from bilirubin fractions 1 and 2, whereas bilirubin fraction 3 yields azo 3B almost exclusively. In step IIIA the non-polar azo pigment fractions are fractionated further by adsorption chromatography on anhydrous sodium sulphate with the use of chloroform followed by a gradient of ethyl acetate in chloroform. Three azo pigments are thus obtained from both azo 2A (azo 2A(1), azo 2A(2) and azo 2A(3)) and azo 3A (azo 3A(1), azo 3A(2) and azo 3A(3)). The 2A pigments occur in approximately the following proportions: azo 2A(1), 90%; azo 2A(2), 10%; azo 2A(3), traces. The pigments are purified by crystallization, except for the A(3) pigments, which are probably degradation products arising from the corresponding A(2) pigments. In step IIIB the polar azo pigment fractions are subjected to reverse-phase partition chromatography on silicone-treated Celite with the use of a solvent system prepared from octan-1-ol-di-isopropyl ether-ethyl acetate-methanol-0.2m-acetic acid (1:2:2:3:4, by vol.). Azo pigment fractions 2B and 3B each yield six azo pigments (azo 2B(1) to azo 2B(6) and azo 3B(1) to azo 3B(6) respectively) together with small amounts of products of hydrolysis (azo 2A(B) and azo 3A(B)). Only one azo B pigment is obtained from bilirubin fraction 1, and this azo pigment is probably of the B(2) type. The yields of the azo 3B pigments suggest that these pigments are present in approximately the following proportions: azo 3B(1), 0-0.4%; azo 3B(2), traces; azo 3B(3), traces; azo 3B(4), 10%; azo 3B(5), 50%; azo 3B(6), 40%. Azo pigments 2B(1) to 2B(6) are estimated to occur in similar proportions. Since pairs of correspondingly numbered azo pigments from bilirubin fractions 1, 2 and 3 do not separate on rechromatography together (e.g. azo 2A(1) co-chromatographs with azo 3A(1), and azo 2B(6) co-chromatographs with azo 3B(6)), it is concluded that such pigments are chemically identical. The structures of the isolated phenylazo derivatives are discussed in an accompanying paper (Kuenzle 1970c).

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The isolation and further characterization of the bilirubin tetrapyrroles in bile-containing human duodenal juice and dog gall-bladder bile.

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