495 results on '"Alan F. Hofmann"'
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2. Key discoveries in bile acid chemistry and biology and their clinical applications: history of the last eight decades
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Alan F. Hofmann and Lee R. Hagey
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bile acid transport ,enterohepatic circulation ,bile acid physical chemistry ,bile acid analysis ,bile acid metabolism ,Biochemistry ,QD415-436 - Abstract
During the last 80 years there have been extraordinary advances in our knowledge of the chemistry and biology of bile acids. We present here a brief history of the major achievements as we perceive them. Bernal, a physicist, determined the X-ray structure of cholesterol crystals, and his data together with the vast chemical studies of Wieland and Windaus enabled the correct structure of the steroid nucleus to be deduced. Today, C24 and C27 bile acids together with C27 bile alcohols constitute most of the bile acid “family”. Patterns of bile acid hydroxylation and conjugation are summarized. Bile acid measurement encompasses the techniques of GC, HPLC, and MS, as well as enzymatic, bioluminescent, and competitive binding methods. The enterohepatic circulation of bile acids results from vectorial transport of bile acids by the ileal enterocyte and hepatocyte; the key transporters have been cloned. Bile acids are amphipathic, self-associate in solution, and form mixed micelles with polar lipids, phosphatidylcholine in bile, and fatty acids in intestinal content during triglyceride digestion. The rise and decline of dissolution of cholesterol gallstones by the ingestion of 3,7-dihydroxy bile acids is chronicled. Scientists from throughout the world have contributed to these achievements.
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- 2014
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3. A simple and accurate HPLC method for fecal bile acid profile in healthy and cirrhotic subjects: validation by GC-MS and LC-MS[S]
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Genta Kakiyama, Akina Muto, Hajime Takei, Hiroshi Nittono, Tsuyoshi Murai, Takao Kurosawa, Alan F. Hofmann, William M. Pandak, and Jasmohan S. Bajaj
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extraction ,esterified bile acids ,bile acid 24-phenacyl ester ,derivatization ,liver cirrhosis ,high-performance liquid chromatography ,Biochemistry ,QD415-436 - Abstract
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.
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- 2014
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4. Bile salts of vertebrates: structural variation and possible evolutionary significance[S]
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Alan F. Hofmann, Lee R. Hagey, and Matthew D. Krasowski
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bile acids ,cholesterol ,enzymes ,metabolism ,molecular evolution ,phylogeny ,Biochemistry ,QD415-436 - Abstract
Biliary bile salt composition of 677 vertebrate species (103 fish, 130 reptiles, 271 birds, 173 mammals) was determined. Bile salts were of three types: C27 bile alcohols, C27 bile acids, or C24 bile acids, with default hydroxylation at C-3 and C-7. C27 bile alcohols dominated in early evolving fish and amphibians; C27 bile acids, in reptiles and early evolving birds. C24 bile acids were present in all vertebrate classes, often with C27 alcohols or with C27 acids, indicating two evolutionary pathways from C27 bile alcohols to C24 bile acids: a) a ‘direct’ pathway and b) an ‘indirect’ pathway with C27 bile acids as intermediates. Hydroxylation at C-12 occurred in all orders and at C-16 in snakes and birds. Minor hydroxylation sites were C-1, C-2, C-5, C-6, and C-15. Side chain hydroxylation in C27 bile salts occurred at C-22, C-24, C-25, and C-26, and in C24 bile acids, at C-23 (snakes, birds, and pinnipeds). Unexpected was the presence of C27 bile alcohols in four early evolving mammals. Bile salt composition showed significant variation between orders but not between families, genera, or species. Bile salt composition is a biochemical trait providing clues to evolutionary relationships, complementing anatomical and genetic analyses.
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- 2010
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5. A new, major C27 biliary bile acid in the Red-winged tinamou (Rhynchotus rufescens):(25R)-1β,3α,7α-trihydroxy-5β-cholestan-27-oic acid
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Lee R. Hagey, Genta Kakiyama, Akina Muto, Takashi Iida, Kumiko Mushiake, Takaaki Goto, Nariyasu Mano, Junichi Goto, Cleida A. Oliveira, and Alan F. Hofmann
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1β-hydroxylation ,C27 bile acids ,1β,3α,7α-trihydroxy-5β-cholestan-27-oic acid ,taurine conjugate ,LC-MS ,NMR ,Biochemistry ,QD415-436 - Abstract
The chemical structures of the three major bile acids present in the gallbladder bile of the Red-winged tinamou (Rhynchotus rufescens), an early evolving, ground-living bird related to ratites, were determined. Bile acids were isolated by preparative reversed-phase HPLC. Two of the compounds were identified as the taurine N-acylamidates of (25R)-3α,7α-dihydroxy-5β-cholestan-27-oic acid (constituting 22% of biliary bile acids) and (25R)-3α,7α,12α-trihydroxy-5β-cholestan-27-oic acid (constituting 51%). The remaining compound, constituting 21% of biliary bile acids, was an unknown C27 bile acid. Its structure was elucidated by LC/ESI-MS/MS and NMR and shown to be the taurine conjugate of (25R)-1β,3α,7α-trihydroxy-5β-cholestan-27-oic acid, a C27 trihydroxy bile acid not previously reported. Although C27 bile acids with a 1β-hydroxyl group have been identified as trace bile acids in the alligator, this is the first report of a major biliary C27 bile acid possessing a 1β-hydroxyl group.
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- 2009
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6. Evolution of the bile salt nuclear receptor FXR in vertebrates*s⃞
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Erica J. Reschly, Ni Ai, Sean Ekins, William J. Welsh, Lee R. Hagey, Alan F. Hofmann, and Matthew D. Krasowski
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fishes ,lampreys ,lithocholic acid ,molecular evolution ,molecular models ,nuclear hormone receptors ,Biochemistry ,QD415-436 - Abstract
Bile salts, the major end metabolites of cholesterol, vary significantly in structure across vertebrate species, suggesting that nuclear receptors binding these molecules may show adaptive evolutionary changes. We compared across species the bile salt specificity of the major transcriptional regulator of bile salt synthesis, the farnesoid X receptor (FXR). We found that FXRs have changed specificity for primary bile salts across species by altering the shape and size of the ligand binding pocket. In particular, the ligand binding pockets of sea lamprey (Petromyzon marinus) and zebrafish (Danio rerio) FXRs, as predicted by homology models, are flat and ideal for binding planar, evolutionarily early bile alcohols. In contrast, human FXR has a curved binding pocket best suited for the bent steroid ring configuration typical of evolutionarily more recent bile acids. We also found that the putative FXR from the sea squirt Ciona intestinalis, a chordate invertebrate, was completely insensitive to activation by bile salts but was activated by sulfated pregnane steroids, suggesting that the endogenous ligands of this receptor may be steroidal in nature. Our observations present an integrated picture of the coevolution of bile salt structure and of the binding pocket of their target nuclear receptor FXR.
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- 2008
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7. Isolation and chemical synthesis of a major, novel biliary bile acid in the common wombat (Vombatus ursinus): 15α-hydroxylithocholic acid
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Genta Kakiyama, Hideyuki Tamegai, Takashi Iida, Kuniko Mitamura, Shigeo Ikegawa, Takaaki Goto, Nariyasu Mano, Junichi Goto, Peter Holz, Lee R. Hagey, and Alan F. Hofmann
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bile acid synthesis ,detoxification of bile acids ,bile acid metabolism in marsupials ,15-hydroxylation ,lithocholic acid ,Biochemistry ,QD415-436 - Abstract
The major bile acids present in the gallbladder bile of the common Australian wombat (Vombatus ursinus) were isolated by preparative HPLC and identified by NMR as the taurine N-acylamidates of chenodeoxycholic acid (CDCA) and 15α-hydroxylithocholic acid (3α,15α-dihydroxy-5β-cholan-24-oic acid). Taurine-conjugated CDCA constituted 78% of biliary bile acids, and (taurine-conjugated) 15α-hydroxylithocholic acid constituted 11%. Proof of structure of the latter compound was obtained by its synthesis from CDCA via a Δ14 intermediate. The synthesis of its C-15 epimer, 15β-hydroxylithocholic acid (3α,15β-dihydroxy-5β-cholan-24-oic acid), is also reported. The taurine conjugate of 15α-hydroxylithocholic acid was synthesized and shown to have chromatographic and spectroscopic properties identical to those of the compound isolated from bile. It is likely that 15α-hydroxylithocholic acid is synthesized in the wombat hepatocyte by 15α-hydroxylation of lithocholic acid that was formed by bacterial 7α-dehydroxylation of CDCA in the distal intestine. Thus, the wombat appears to use 15α-hydroxylation as a novel detoxification mechanism for lithocholic acid.
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- 2007
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8. Two loci on chromosome 9 control bile acid composition: evidence that a strong candidate gene, Cyp8b1, is not the culprits⃞
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Ephraim Sehayek, Lee R. Hagey, Yee-Yan Fung, Elizabeth M. Duncan, Hannah J. Yu, Gösta Eggertsen, Ingemar Björkhem, Alan F. Hofmann, and Jan L. Breslow
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chenodeoxycholic acid ,sterol 12α-hydroxylase ,cholic acid ,Biochemistry ,QD415-436 - Abstract
An intercross between C57BL/6J and CASA/Rk mice was used to study the genetics of biliary bile acid composition. In parental strains, male C57BL/6J mice had significantly higher cholic acid (CA; 14%) and lower β-muricholic acid (βMC; 27%) than CASA/Rk mice, whereas females did not differ. However, quantitative trait locus analysis of F2 mice revealed no significant chromosome 9 loci in males but loci in females on chromosome 9 for percentage CA (%CA) at 72 centimorgan (cM) [logarithm of the odds (LOD) 5.89] and %βMC at 54 cM (LOD 4.09). Chromosome 9 congenic and subcongenic strains representing CASA/Rk intervals 38–73 cM (9KK) and 68–73 cM (9DKK) on the C57BL/6J background were made. In 9KK and 9DKK males, %CA was increased and %βMC was unchanged, whereas in 9KK but not 9DKK females, %CA was increased and %βMC was decreased. Sterol 12α-hydroxylase (Cyp8b1) channels bile acid precursors into CA and maps at chromosome 9 (73 cM). However, there was no significant difference in Cyp8b1 mRNA or enzymatic activity between parental mice, parental-congenic-subcongenic mice, or high-low biliary %CA F2 mice. In summary, two chromosome 9 loci control sexually dimorphic effects on biliary bile acid composition: a distal (68–73 cM) major determinant in males, and a more proximal (38–68 cM) major determinant in females. In this intercross, Cyp8b1, a strong candidate, does not appear to be responsible.
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- 2006
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9. Identification of a novel bile acid in swans, tree ducks, and geese: 3α,7α,15α-trihydroxy-5β-cholan-24-oic acid
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Genta Kakiyama, Takashi Iida, Takaaki Goto, Nariyasu Mano, Junichi Goto, Toshio Nambara, Lee R. Hagey, Claudio D. Schteingart, and Alan F. Hofmann
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Anseriformes ,bile acid evolution ,bile acid conjugation ,Biochemistry ,QD415-436 - Abstract
By HPLC, a taurine-conjugated bile acid with a retention time different from that of taurocholate was found to be present in the bile of the black-necked swan, Cygnus melanocoryphus. The bile acid was isolated and its structure, established by 1H and 13C NMR and mass spectrometry, was that of the taurine N-acyl amidate of 3α,7α,15α-trihydroxy-5β-cholan-24-oic acid. The compound was shown to have chromatographic and spectroscopic properties that were identical to those of the taurine conjugate of authentic 3α,7α,15α-trihydroxy-5β-cholan-24-oic acid, previously synthesized by us from ursodeoxycholic acid. By HPLC, the taurine conjugate of 3α,7α,15α-trihydroxy-5β-cholan-24-oic acid was found to be present in 6 of 6 species in the subfamily Dendrocygninae (tree ducks) and in 10 of 13 species in the subfamily Anserinae (swans and geese) but not in other subfamilies in the Anatidae family. It was also not present in species from the other two families of the order Anseriformes. 3α,7α,15α-Trihydroxy-5β-cholan-24-oic acid is a new primary bile acid that is present in the biliary bile acids of swans, tree ducks, and geese and may be termed 15α-hydroxy-chenodeoxycholic acid.
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- 2006
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10. A phylogenetic survey of biliary lipids in vertebrates1
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Antonio Moschetta, Fang Xu, Lee R. Hagey, Gerard P. van Berge-Henegouwen, Karel J. van Erpecum, Jos F. Brouwers, Jonathan C. Cohen, Molly Bierman, Helen H. Hobbs, Joseph H. Steinbach, and Alan F. Hofmann
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bile salts ,cholesterol ,gas chromatography ,mass spectrometry ,phosphatidylcholine ,sphingomyelin ,Biochemistry ,QD415-436 - Abstract
Biliary lipids (bile salts, phospholipids, cholesterol, plant sterols) were determined in 89 vertebrate species (cartilaginous and bony fish, reptiles, birds, and mammals), and individual phospholipid classes were measured in 35 species. All samples contained conjugated bile salts (C27 bile alcohol sulfates and/or N-acyl amidates of C27 and/or C24 bile acids). Phospholipids were generally absent in the bile of cartilaginous fish and reptiles and were present in low amounts relative to bile salts in bony fish and most birds. In mammals, the phospholipid-bile salt ratio varied widely. The bile from species with low biliary phospholipid-bile salt ratios often contained a high proportion of sphingomyelin, confirmed by HPLC-MS. In species with a high phospholipid-bile salt ratio, the predominant biliary phospholipid was phosphatidylcholine (PC). The phospholipid-bile salt ratio correlated weakly with the calculated weighted hydrophobic index value. Cholesterol was present in the bile of virtually all species, with plant sterols uniformly being present in only trace amounts. The cholesterol-bile salt ratio tended to be higher in mammals than in nonmammals, but bile of all species was unsaturated.Thus, most nonmammalian vertebrates have relatively low levels of biliary phospholipid and cholesterol, suggesting that cholesterol is eliminated predominantly as bile salts. Mammals have a higher phospholipid and cholesterol to bile salt ratio, with the dominant phospholipid being PC.
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- 2005
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11. Chemical synthesis of (22E)-3α,6β,7β-trihydroxy-5β-chol-22-en-24-oic acid and its taurine and glycine conjugates
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Genta Kakiyama, Takashi Iida, Atsushi Yoshimoto, Takaaki Goto, Nariyasu Mano, Junichi Goto, Toshio Nambara, Lee R. Hagey, and Alan F. Hofmann
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unsaturated bile acid ,tauro-Δ22-β-muricholic acid ,Δ22-β-muricholyltaurine ,glyco-Δ22-β-muricholic acid ,Δ22-β-muricholylglycine ,Biochemistry ,QD415-436 - Abstract
A method for the synthesis of Δ22-β-muricholic acid (Δ22-β-MCA), (22E)-3α,6β,7β-trihydroxy-5β-chol-22-en-24-oic acid, and its taurine and glycine conjugates (Δ22-β-muricholyltaurine and Δ22-β-muricholylglycine) is described. The key intermediate, 3α,6β,7β-triformyloxy-23,24-dinor-5β-cholan-22-al, was prepared from β-muricholic acid (β-MCA) via the 24-nor-22-ene and 24-nor-22,23-diol derivatives. Wittig reaction of the aldehyde with (carbomethoxymethylene) triphenylphosphorane and subsequent hydrolysis gave (unconjugated) Δ22-β-MCA. Condensation reaction of the unconjugated acid with taurine or glycine methyl ester using diethylphosphorocyanide yielded the naturally occurring taurine or glycine conjugate (N-acylamidate) of Δ22-β-MCA.These synthetic reference compounds are now available for investigation of the metabolism of β-MCA by bacterial and hepatic enzymes in the rat and should also be useful as substrates for reductive deuteration or tritiation to give the 22,23-2H or 3H-β-MCA.
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- 2004
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12. An N-acyl glycyltaurine conjugate of deoxycholic acid in the biliary bile acids of the rabbit
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Lee R. Hagey, Claudio D. Schteingart, Steven S. Rossi, H-T. Ton-Nu, and Alan F. Hofmann
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bile acid metabolism ,lithocholic acid ,chenodeoxycholic acid metabolism ,enterohepatic circulation ,bile acid conjugation ,Biochemistry ,QD415-436 - Abstract
The biliary bile acid composition of the adult and neonatal domestic rabbit, as well as that of the adult brush rabbit, was characterized. In adult domestic rabbits, the dominant bile acid present was deoxycholic acid (88“% of total bile acids), a secondary bile acid formed by the bacterial 7-dehydroxylation of cholic acid. Although most of the bile acids present were conjugated with glycine, two exceptions were observed. About 3”% of deoxycholic acid was conjugated, in N-acyl linkage, with glycyl-taurine. Chenodeoxycholic acid, which composed
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- 1998
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13. Efficient extraction of bile acid conjugates with tetraheptylammonium chloride, a liquid ion exchanger
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Alan F. Hofmann
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extraction ,bile acid conjugates ,tetraheptylammonium chloride ,liquid ion exchanger ,alkaloid glucosiduronates ,alkyl sulfates ,Biochemistry ,QD415-436 - Abstract
Ethyl acetate or chloroform solutions of tetraheptylammonium chloride, an oil-soluble quaternary amine, quantitatively extract polar, anionic lipids such as steroid or bile salt conjugates from aqueous solution by a process of anion exchange.
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- 1967
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14. Rapid preparation of tritium-labeled bile acids by enolic exchange on basic alumina containing tritiated water
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Alan F. Hofmann, Patricia A. Szczepanik, and Peter D. Klein
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exchange labeling bile acid epimers ,thin-layer chromatography isotope fractionation ,Biochemistry ,QD415-436 - Abstract
When a 3-keto bile acid methyl ester was chromatographed on basic alumina inactivated with tritiated water, the enolic hydrogen atoms at C-2 and C-4 exchanged with tritium atoms. The 3H-labeled keto ester was reduced with borohydride, and the resultant mixture of 3α- and 3β-hydroxy epimers was resolved by preparative thin-layer chromatography to yield a pure 2,4-3H-labeled bile acid ester. Lithocholic, chenodeoxycholic, deoxycholic, and cholic acids having a specific activity of 1-10 μc/μmole were prepared from their 3-keto derivatives. The tritium label remained intact during alkaline saponification in vitro and enterohepatic cycling in vivo in human subjects
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- 1968
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15. Use of 3H-labeled triether, a nonabsorbable oil-phase marker, to estimate fat absorption in rats with cholestyramine-induced steatorrhea
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Reginald G.H. Morgan and Alan F. Hofmann
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glycerol triether ,ether lipid metabolism ,3H-labeled glycerol triether ,1-hexadecyl-2,3-didodecyl glycerol ,1-hexadecoxy-2,3-didodecoxypropane ,Biochemistry ,QD415-436 - Abstract
A tritium-labeled glycerol triether was tested as a nonabsorbable oil-phase marker in studies of fat absorption in normal rats and in rats with steatorrhea induced by various doses of cholestyramine. Animals were fed a test meal containing 3H-labeled triether and 14C-labeled trilinolein. Fat absorption was estimated in the following three ways: (a) by isotope ratios (the change in 3H/14C in the test meal and in feces); (b) by isotope recovery (the total fecal excretion of 14C radioactivity); and (c) by chemical recovery (the total fecal fat excretion). Absorption calculated from isotope ratios agreed well with that calculated from isotope recovery over a range of fat absorption of 50-100%, thus validating the use of this lipid marker under these conditions of fat malabsorption. Absorption calculated from chemical recovery was consistently poorer than that calculated from isotope ratios or isotope recovery, thus suggesting that cholestyramine increased the excretion of nondietary (endogenous) fat. Triether may be of value for studying the absorption of compounds present predominantly in the oil phase during digestion and may have significant advantages over other proposed lipid markers.
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- 1970
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16. Synthesis and metabolism of glycerol-3H triether, a nonabsorbable oil-phase marker for lipid absorption studies
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Reginald G.H. Morgan and Alan F. Hofmann
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14C-labeled glycerol triether ,1-hexadecyl-2,3-didodecyl glycerol ,1-hexadecoxy-2,3-didodecoxypropane ,glycerol triether synthesis ,glycerol triether metabolism ,ether lipid metabolism ,Biochemistry ,QD415-436 - Abstract
A saturated mixed-chain glycerol triether, 1-hexadecyl-2,3-didodecyl glycerol (1-hexadecoxy-2,3-didodecoxypropane), was synthesized with 3H at positions 9 and 10 or 14C at position 1 of the hexadecyl moiety. In acute feeding experiments in rats, less than 0.2% of the triether was absorbed, based on lymph and fecal recoveries. Radioactivity was present exclusively as triether in feces, indicating that it was not degraded by digestive or bacterial enzymes. Chronic feeding experiments in rats confirmed the nonabsorbability of the triether and further indicated that it was nontoxic, did not influence the absorption of dietary fat, and mixed intimately with the fat present in colonic contents and feces. The triether that was absorbed was deposited as triether in adipose tissue, liver, and spleen. When administered intraperitoneally to mice, the triether was stored in the tissues and was not metabolized. When the triether was partitioned between an oil phase of triolein or fatty acid and monoglyceride, and an aqueous micellar phase, the triether remained exclusively in the oil phase. The triether appears to be an ideal nonabsorbable oil-phase marker for use in lipid absorption studies.
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- 1970
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17. Experimental cholelithiasis in the rabbit induced by cholestanol feeding: effect of neomycin treatment on bile composition and gallstone formation
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Alan F. Hofmann, Victor Bokkenheuser, Robert L. Hirsch, and Erwin H. Mosbach
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5α-cholestan-3β-01 metabolism ,rabbit ,3α,12α-dihydroxy-5α-cholanoic acid ,3α,7α,12α-dihydroxy-5α-cholanoic acid ,experimental cholelithiasis ,gallstones ,Biochemistry ,QD415-436 - Abstract
Fed cholestanol is converted by the rabbit to 5α-bile acids which coprecipitate with the normally occurring 5β-bile acids to form gallstones composed of calcium and sodium glycoallodeoxycholate and glycodeoxycholate. The present study shows that oral administration of large doses of neomycin prevents gallstone formation in the cholestanol-fed rabbit and reduces the elevated concentration of allodeoxycholic acid in bile, with a reciprocal increase in allocholic acid concentration. The reduction in the concentration of allodeoxycholic acid and in the incidence of gallstones is proportional to the dose of neomycin; at a concentration of allodeoxycholic acid below about 20% of total bile acids, gallstone formation does not occur. Neomycin probably exerts its action by modifying the anerobic intestinal flora which dehydroxylate allocholic acid to allodeoxycholic acid; if so, this suggests that both hepatic and bacterial transformations are essential steps in the pathogenesis of cholestanol-induced cholelithiasis.The bile of rabbits on a normal diet contains allodeoxycholic acid (5% of total bile acids). A similar decrease in allodeoxycholic acid concentration and reciprocal increase in allocholic acid concentration is observed when neomycin is administered to rabbits on a normal diet.
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- 1968
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18. Separation of 1- and 2-monoglycerides by thin-layer adsorption chromatography on hydroxyl-apatite
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Alan F. Hofmann
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Biochemistry ,QD415-436 - Published
- 1962
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19. One-step quantitative extraction of medium-chain and long-chain fatty acids from aqueous samples
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Manley Cohen, R.G.H. Morgan, and Alan F. Hofmann
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medium-chain triglycerides ,free fatty acid ,titration ,octanoic acid ,12-hydroxystearic acid ,feces ,Biochemistry ,QD415-436 - Abstract
Medium-chain (C6 and longer) fatty acids, as well as 12-hydroxystearic and long-chain fatty acids, can be quantitatively extracted into toluene and titrated in the toluene phase with tetrabutylammonium hydroxide. The method may be useful in determinations of fecal and serum fatty acids and of the products of lipolysis.
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- 1969
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20. Thin-layer adsorption chromatography of free and conjugated bile acids on silicic acid
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Alan F. Hofmann
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Biochemistry ,QD415-436 - Published
- 1962
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21. Obeticholic acid improves hepatic bile acid excretion in patients with primary biliary cholangitis
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Alan F. Hofmann, Mary Erickson, Kristoffer Kjærgaard, Susanne Keiding, David Shapiro, Michael Sørensen, Anna C. Schacht, Ole Lajord Munk, Jacob Horsager, and Kim Frisch
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0301 basic medicine ,INDOCYANINE GREEN ,Receptors, Cytoplasmic and Nuclear ,Chronic liver disease ,Gastroenterology ,chemistry.chemical_compound ,0302 clinical medicine ,CIRRHOSIS ,GENE-EXPRESSION ,Bile acid ,Liver Cirrhosis, Biliary ,Ursodeoxycholic Acid ,Intrahepatic cholestasis ,Obeticholic acid ,Middle Aged ,G protein-coupled bile acid receptor ,Ursodeoxycholic acid ,Treatment Outcome ,FXR ,HEPATOBILIARY SECRETION ,CHOLYLSARCOSINE ,Alkaline phosphatase ,Female ,030211 gastroenterology & hepatology ,FARNESOID-X-RECEPTOR ,medicine.drug ,medicine.medical_specialty ,medicine.drug_class ,Bile acid-binding proteins ,Molecular imaging ,Chenodeoxycholic Acid ,digestive system ,Bile Acids and Salts ,Excretion ,03 medical and health sciences ,Farnesoid X receptor ,Double-Blind Method ,Gastrointestinal Agents ,Internal medicine ,medicine ,Humans ,KINETICS ,Aged ,Hepatology ,business.industry ,Biological Transport ,Alkaline Phosphatase ,medicine.disease ,URSODEOXYCHOLIC ACID ,SALT EXPORT PUMP ,Bile Ducts, Intrahepatic ,030104 developmental biology ,chemistry ,Positron-Emission Tomography ,Liver cirrhosis ,Hepatocytes ,business - Abstract
Background & Aims: Obeticholic acid (OCA) is an agonist of the nuclear bile acid receptor farnesoid X receptor, which regulates hepatic bile acid metabolism. We tested whether OCA treatment would influence hepatic transport of conjugated bile acids in patients with primary biliary cholangitis (PBC) who responded inadequately to treatment with ursodeoxycholic acid (UDCA). Methods: Eight UDCA-treated patients with PBC with alkaline phosphatase ≥1.5 times the upper limit of normal range participated in a double-blind, placebo-controlled study. While continuing on UDCA, the patients were randomised to two 3-month crossover treatment periods with placebo and OCA, in random order, separated by a 1-month washout period without study treatment. After each of the two treatment periods, we determined rate constants for transport of conjugated bile acids between blood, hepatocytes, biliary canaliculi, and bile ducts by positron emission tomography of the liver using the conjugated bile acid tracer [N-methyl- 11C]cholylsarcosine ( 11C-CSar). The hepatic blood perfusion was measured using infusion of indocyanine green and Fick's principle. Results: Compared with placebo, OCA increased hepatic blood perfusion by a median of 11% (p = 0.045), the unidirectional uptake clearance of 11C-CSar from blood into hepatocytes by a median of 11% (p = 0.01), and the rate constant for secretion of 11C-CSar from hepatocytes into biliary canaliculi by a median of 73% (p = 0.03). This resulted in an OCA-induced decrease in the hepatocyte residence time of 11C-CSar by a median of 30% (p = 0.01), from group median 11 min to 8 min. Conclusions: This study of UDCA-treated patients with PBC showed that, compared with placebo, OCA increased the hepatic transport of the conjugated bile acid tracer 11C-CSar, and thus endogenous conjugated bile acids, from hepatocytes into biliary canaliculi. As a result, OCA reduced the time hepatocytes are exposed to potentially cytotoxic bile acids. Lay summary: Primary biliary cholangitis is a chronic liver disease in which the small bile ducts are progressively destroyed. We tested whether the treatment with obeticholic acid (OCA) would improve liver excretion of bile acids compared with placebo in 8 patients with primary biliary cholangitis. A special scanning technique (PET scan) showed that OCA increased the transport of bile acids from blood to bile. OCA thereby reduced the time that potentially toxic bile acids reside in the liver by approximately one-third.
- Published
- 2021
22. Reply to: 'Obeticholic acid and hepatic bile acids: Excellent study faulty conclusion'
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Kim Frisch, Susanne Keiding, and Alan F. Hofmann
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Bile Acids and Salts ,chemistry.chemical_compound ,medicine.medical_specialty ,Hepatology ,chemistry ,Liver ,Internal medicine ,medicine ,Obeticholic acid ,Chenodeoxycholic Acid ,Hepatic bile ,Gastroenterology - Published
- 2021
23. Two Major Bile Acids in the Hornbills, (24R,25S)-3α,7α,24-Trihydroxy-5β-cholestan-27-oyl Taurine and Its 12α-Hydroxy Derivative
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Takashi Iida, Kuniko Mitamura, Tetsuya Saito, Kaoru Omura, Shigeo Ikegawa, Lee R. Hagey, Alan F. Hofmann, Hiroaki Ogata, Rika Satoh, Biao Zhou, and Satoshi Kurabuchi
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0301 basic medicine ,Taurine ,Buceros ,medicine.drug_class ,Stereochemistry ,Hornbill ,Crystallography, X-Ray ,Biochemistry ,Bile Acids and Salts ,Birds ,03 medical and health sciences ,chemistry.chemical_compound ,Penelopides panini ,medicine ,Animals ,Chromatography, High Pressure Liquid ,Molecular Structure ,Bile acid ,biology ,Organic Chemistry ,Diastereomer ,Gallbladder ,Stereoisomerism ,Cell Biology ,biology.organism_classification ,Great hornbill ,030104 developmental biology ,chemistry ,Derivative (chemistry) - Abstract
Two major bile acids were isolated from the gallbladder bile of two hornbill species from the Bucerotidae family of the avian order Bucerotiformes Buceros bicornis (great hornbill) and Penelopides panini (Visayan tarictic hornbill). Their structures were determined to be 3α,7α,24-dihydroxy-5β-cholestan-27-oic acid and its 12α-hydroxy derivative, 3α,7α,12α,24-tetrahydroxy-5β-cholestan-27-oic acid (varanic acid, VA), both present in bile as their corresponding taurine amidates. The four diastereomers of varanic acid were synthesized and their assigned structures were confirmed by X-ray crystallographic analysis. VA and its 12-deoxy derivative were found to have a (24R,25S)-configuration. 13 additional hornbill species were also analyzed by HPLC and showed similar bile acid patterns to B. bicornis and P. panini. The previous stereochemical assignment for (24R,25S)-VA isolated from the bile of varanid lizards and the Gila monster should now be revised to the (24S,25S)-configuration.
- Published
- 2016
24. Chemical Synthesis of Uncommon Natural Bile Acids: The 9α-Hydroxy Derivatives of Chenodeoxycholic and Lithocholic Acids
- Author
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Kyoko Iida, Takashi Iida, Alan F. Hofmann, Kaoru Omura, Kazunari Namegawa, and Naoya Nakane
- Subjects
0301 basic medicine ,Biological Products ,Lithocholic acid ,Chemistry ,Decarbonylation ,Sodium chlorite ,Deoxycholic acid ,Markovnikov's rule ,Molecular Conformation ,Cholic acid ,General Chemistry ,General Medicine ,Chenodeoxycholic Acid ,Chemical synthesis ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,Chenodeoxycholic acid ,Drug Discovery ,Organic chemistry ,Lithocholic Acid - Abstract
The chemical synthesis of the 9α-hydroxy derivatives of chenodeoxycholic and lithocholic acids is reported. For initiating the synthesis of the 9α-hydroxy derivative of chenodeoxycholic acid, cholic acid was used; for the synthesis of the 9α-hydroxy derivative of lithocholic acid, deoxycholic acid was used. The principal reactions involved were (1) decarbonylation of conjugated 12-oxo-Δ(9(11))-derivatives using in situ generated monochloroalane (AlH2Cl) prepared from LiAlH4 and AlCl3, (2) epoxidation of the deoxygenated Δ(9(11))-enes using m-chloroperbenzoic acid catalyzed by 4,4'-thiobis-(6-tert-butyl-3-methylphenol), (3) subsequent Markovnikov 9α-hydroxylation of the Δ(9(11))-enes with AlH2Cl, and (4) selective oxidation of the primary hydroxyl group at C-24 in the resulting 3α,9α,24-triol and 3α,7α,9α,24-tetrol to the corresponding C-24 carboxylic acids using sodium chlorite (NaClO2) in the presence of a catalytic amount of 2,2,6,6-tetramethylpiperidine 1-oxyl free radical (TEMPO) and sodium hypochlorite (NaOCl). The (1)H- and (13)C-NMR spectra are reported. The 3α,7α,9α-trihydroxy-5β-cholan-24-oic acid has been reported to be present in the bile of the Asian bear, and its 7-deoxy derivative is likely to be a bacterial metabolite. These bile acids are now available as authentic reference standards, permitting their identification in vertebrate bile acids.
- Published
- 2016
25. Bile Microinfarcts in Cholestasis Are Initiated by Rupture of the Apical Hepatocyte Membrane and Cause Shunting of Bile to Sinusoidal Blood
- Author
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Aya A. Abbas, Alan F. Hofmann, Reham Hassan, Ute Hofmann, Selahaddin Sezgin, Peter L.M. Jansen, Patricio Godoy, Nachiket Vartak, Frank Lammert, Steven Dooley, Jan G. Hengstler, Dirk Drasdo, Kai Markus Schneider, Christian Trautwein, Yasser A. Ahmed, Michael Spiteller, Raymond Reif, Lars Kuepfer, Verena Keitel, Georgia Guenther, Ahmed Ghallab, A Zaza, Department of Forensic Medicine and Veterinary Toxicology [Qena], Faculty of Veterinary Medicine [Qena], South Valley University [Qena]-South Valley University [Qena], Dr. Margarete Fischer-Bosch Institute for Clinical Pharmacology [Stuttgart], Technische Universität Dortmund [Dortmund] (TU), Leibniz Research Centre for Working Environment and Human Factors [Dortmund] (IFADO), Rheinisch-Westfälische Technische Hochschule Aachen (RWTH), South Valley University [Qena], Heinrich Heine Universität Düsseldorf = Heinrich Heine University [Düsseldorf], Computational Systems Biology [Leverkusen], Bayer Technology Services [Leverkusen], Universität Mannheim [Mannheim], Saarland University [Saarbrücken], Modelling and Analysis for Medical and Biological Applications (MAMBA), Inria de Paris, Institut National de Recherche en Informatique et en Automatique (Inria)-Institut National de Recherche en Informatique et en Automatique (Inria)-Laboratoire Jacques-Louis Lions (LJLL (UMR_7598)), Université Paris Diderot - Paris 7 (UPD7)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), University of California [San Diego] (UC San Diego), University of California, Maastricht University [Maastricht], RS: FSE MaCSBio, Maastricht Centre for Systems Biology, RS: FHML MaCSBio, RS: FPN MaCSBio, Heinrich-Heine-Universität Düsseldorf [Düsseldorf], Rheinisch-Westfälische Technische Hochschule Aachen University (RWTH), Universität Mannheim, Universitätsklinikum RWTH Aachen - University Hospital Aachen [Aachen, Germany] (UKA), and University of California (UC)
- Subjects
0301 basic medicine ,Male ,Pathology ,medicine.medical_specialty ,Necrosis ,ATP Binding Cassette Transporter, Subfamily B ,mice ,injury ,[SDV]Life Sciences [q-bio] ,Bone canaliculus ,digestive system ,Nephropathy ,Bile Acids and Salts ,03 medical and health sciences ,0302 clinical medicine ,Cholestasis ,INFLAMMATION ,medicine ,Animals ,Mice, Knockout ,Kidney ,Hepatology ,cholemic nephropathy ,RAT HEPATOCYTES ,Chemistry ,Bile Canaliculi ,Optical Imaging ,[SDV.MHEP.HEG]Life Sciences [q-bio]/Human health and pathology/Hépatology and Gastroenterology ,medicine.disease ,ACIDS ,[INFO.INFO-MO]Computer Science [cs]/Modeling and Simulation ,TRANSPORT ,3. Good health ,Mice, Inbred C57BL ,Disease Models, Animal ,030104 developmental biology ,medicine.anatomical_structure ,Biliary tract ,Hepatocyte ,[INFO.INFO-TI]Computer Science [cs]/Image Processing [eess.IV] ,Hepatocytes ,030211 gastroenterology & hepatology ,medicine.symptom ,Immunostaining - Abstract
International audience; Bile duct ligation (BDL) is an experimental procedure that mimics obstructive cholestatic disease. One of the early consequences of BDL in rodents is the appearance of so-called bile infarcts that correspond to Charcot-Gombault necrosis in human cholestasis. The mechanisms causing bile infarcts and their pathophysiological relevance are unclear. Therefore, intravital two photon-based imaging of BDL mice was performed with fluorescent bile salts (BS) and non-BS organic anion analogues. Key findings were followed up by matrix-assisted laser desorption ionization imaging, clinical chemistry, immunostaining, and gene expression analyses. In the acute phase, 1-3 days after BDL, BS concentrations in bile increased and single-cell bile microinfarcts occurred in dispersed hepatocytes throughout the liver caused by the rupture of the apical hepatocyte membrane. This rupture occurred after loss of mitochondrial membrane potential, followed by entry of bile, cell death, and a "domino effect" of further death events of neighboring hepatocytes. Bile infarcts provided a trans-epithelial shunt between bile canaliculi and sinusoids by which bile constituents leaked into blood. In the chronic phase, ≥21 days after BDL, uptake of BS tracers at the sinusoidal hepatocyte membrane was reduced. This contributes to elevated concentrations of BS in blood and decreased concentrations in the biliary tract. Conclusion: Bile microinfarcts occur in the acute phase after BDL in a limited number of dispersed hepatocytes followed by larger infarcts involving neighboring hepatocytes, and they allow leakage of bile from the BS-overloaded biliary tract into blood, thereby protecting the liver from BS toxicity; in the chronic phase after BDL, reduced sinusoidal BS uptake is a dominant protective factor, and the kidney contributes to the elimination of BS until cholemic nephropathy sets in. (Hepatology 2019;69:666-683). SEE EDITORIAL ON PAGE 473 R ecently, the concept of an ascending patho-physiology of cholestatic liver disease has been proposed, whereby lesions start in large or small bile ducts followed by the involvement of upstream structures, such as the bile canalicular network and liver parenchyma. (1) Our knowledge of the responses of the biliary tree to cholestasis stems mainly from studies in rodents after bile duct ligation
- Published
- 2018
26. Guts and gall: bile acids in regulation of intestinal epithelial function in health and disease
- Author
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Alan F. Hofmann, Péter Hegyi, József Maléth, Stephen J. Keely, and Julian R.F. Walters
- Subjects
0301 basic medicine ,Gastrointestinal Diseases ,Physiology ,Disease ,Biology ,Microbiology ,Bile Acids and Salts ,03 medical and health sciences ,0302 clinical medicine ,Accessory organs ,Physiology (medical) ,Gall ,Animals ,Humans ,Intestinal Mucosa ,Molecular Biology ,chemistry.chemical_classification ,Gastrointestinal tract ,Epithelial Cells ,General Medicine ,11 Medical And Health Sciences ,06 Biological Sciences ,Intestines ,030104 developmental biology ,Enzyme ,chemistry ,030211 gastroenterology & hepatology ,Medical science ,Function (biology) - Abstract
Epithelial cells line the entire surface of the gastrointestinal tract and its accessory organs where they primarily function in transporting digestive enzymes, nutrients, electrolytes, and fluid to and from the luminal contents. At the same time, epithelial cells are responsible for forming a physical and biochemical barrier that prevents the entry into the body of harmful agents, such as bacteria and their toxins. Dysregulation of epithelial transport and barrier function is associated with the pathogenesis of a number of conditions throughout the intestine, such as inflammatory bowel disease, chronic diarrhea, pancreatitis, reflux esophagitis, and cancer. Driven by discovery of specific receptors on intestinal epithelial cells, new insights into mechanisms that control their synthesis and enterohepatic circulation, and a growing appreciation of their roles as bioactive bacterial metabolites, bile acids are currently receiving a great deal of interest as critical regulators of epithelial function in health and disease. This review aims to summarize recent advances in this field and to highlight how bile acids are now emerging as exciting new targets for disease intervention.
- Published
- 2018
27. Bile Acids and the Microbiome in the Cow: Lack of Deoxycholic Acid Hydroxylation
- Author
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Zhouji Chen, Thomas H. Herdt, N. K. Ames, Alan F. Hofmann, and Lee R. Hagey
- Subjects
0301 basic medicine ,Taurine ,Microbiota ,Organic Chemistry ,Deoxycholic acid ,Glycine ,Cell Biology ,Hydroxylation ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,chemistry ,Liver ,Animals ,Bile ,Cattle ,Female ,Microbiome ,Carbon Radioisotopes ,Deoxycholic Acid - Published
- 2018
28. Causal Role of Bile Acids in Irritable Bowel Syndrome–Constipation
- Author
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Alan F. Hofmann
- Subjects
medicine.medical_specialty ,Constipation ,Hepatology ,business.industry ,Gastroenterology ,MEDLINE ,medicine.disease ,Bile Acids and Salts ,Irritable Bowel Syndrome ,Internal medicine ,medicine ,Bile ,Humans ,medicine.symptom ,business ,Biomarkers ,Irritable bowel syndrome - Published
- 2019
29. Focused metabolomics using liquid chromatography/electrospray ionization tandem mass spectrometry for analysis of urinary conjugated cholesterol metabolites from patients with Niemann–Pick disease type C and 3β-hydroxysteroid dehydrogenase deficiency
- Author
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Masami Togawa, Hiroshi Nittono, Nariyasu Mano, Junichi Goto, Kousaku Ohno, Takashi Iida, Hiroaki Yamaguchi, Masamitsu Maekawa, Alan F. Hofmann, and Miki Shimada
- Subjects
Male ,Spectrometry, Mass, Electrospray Ionization ,Urinary system ,Clinical Biochemistry ,Urine ,Conjugated system ,Young Adult ,chemistry.chemical_compound ,Metabolomics ,Tandem Mass Spectrometry ,medicine ,Humans ,Niemann–Pick disease, type C ,Chromatography ,Adrenal Hyperplasia, Congenital ,Cholesterol ,Infant ,Niemann-Pick Disease, Type C ,General Medicine ,medicine.disease ,Liquid chromatography electrospray ionization tandem mass spectrometry ,chemistry ,Biochemistry ,Glycine ,Female ,Biomarkers ,Chromatography, Liquid - Abstract
Background Various conjugated cholesterol metabolites are excreted in urine of the patients with metabolic abnormalities and hepatobiliary diseases. We aimed to examine the usefulness of precursor ion scan and neutral loss scan for the characterization of conjugated cholesterol metabolites in urine. Methods A mixture of authentic standards of conjugated cholesterol metabolites was used for investigating the performance of the present method. The urine of patients with Niemann–Pick diseases type C and 3β-hydroxysteroid dehydrogenase deficiency were analysed by precursor ion scan of m/z 97, 74, and 124. Results A precursor ion scan of m/z 97 was effective for identifying conjugates with ester sulphates on hydroxyl groups whereas ester sulphates on phenolic alcohols were signalled by a neutral loss scan of 80 Da. Monosaccharide-conjugated cholesterol metabolites were signalled by a precursor ion scan of m/z 113. Although precursor ion scan of m/z 74 and 124 was effective for finding glycine- and taurine-conjugated metabolites, high intensity of product ions ( m/z 74 and 124) disturbed measurement of other multiply conjugated metabolites. The urine samples contained many conjugated cholesterol metabolites, and there were several disease-specific intense peaks. We found several unknown intense peaks with three known peaks in urine of the Niemann–Pick type C patient. In the patient with 3β-hydroxysteroid dehydrogenase deficiency, intense peaks that were tentatively identified as 5-cholenoic acid sulphates and their glycine and taurine conjugates were present. Conclusion The method should lead to the discovery of new urinary biomarkers for these disturbances of cholesterol catabolism and transport.
- Published
- 2015
30. Chemical Synthesis of Rare Natural Bile Acids: 11α-Hydroxy Derivatives of Lithocholic and Chenodeoxycholic Acids
- Author
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Shoujiro Ogawa, Alan F. Hofmann, Takashi Iida, Kyoko Iida, Kazunari Namegawa, and Kaoru Omura
- Subjects
0301 basic medicine ,Biological Products ,Bile acid ,medicine.drug_class ,Organic Chemistry ,Deoxycholic acid ,Cholic acid ,Molecular Conformation ,Stereoisomerism ,Cell Biology ,Chenodeoxycholic Acid ,Biochemistry ,Chemical synthesis ,Medicinal chemistry ,Catalysis ,Bile Acids and Salts ,03 medical and health sciences ,chemistry.chemical_compound ,Hydrolysis ,030104 developmental biology ,chemistry ,Hexamethylphosphoramide ,medicine ,Lithocholic Acid ,Tetrahydrofuran - Abstract
A method for the preparation of 11α-hydroxy derivatives of lithocholic and chenodeoxycholic acids, recently discovered to be natural bile acids, is described. The principal reactions involved were (1) elimination of the 12α-mesyloxy group of the methyl esters of 3α-acetate-12α-mesylate and 3α,7α-diacetate-12α-mesylate derivatives of deoxycholic acid and cholic acid with potassium acetate/hexamethylphosphoramide; (2) simultaneous reduction/hydrolysis of the resulting △11 -3α-acetoxy and △11 -3α,7α-diacetoxy methyl esters with lithium aluminum hydride; (3) stereoselective 11α-hydroxylation of the △11 -3α,24-diol and △11 -3α,7α,24-triol intermediates with B2 H6 /tetrahydrofuran (THF); and (4) selective oxidation at C-24 of the resulting 3α,11α,24-triol and 3α,7α,11α,24-tetrol to the corresponding C-24 carboxylic acids with NaClO2 catalyzed by 2,2,6,6-tetramethylpiperidine 1-oxyl free radical (TEMPO) and NaClO. In summary, 3α,11α-dihydroxy-5β-cholan-24-oic acid and 3α,7α,11α-trihydroxy-5β-cholan-24-oic acid have been synthesized and their nuclear magnetic resonance (NMR) spectra characterized. These compounds are now available as reference standards to be used in biliary bile acid analysis.
- Published
- 2017
31. Seventy Years of Polyethylene Glycols in Gastroenterology: The Journey of PEG 4000 and 3350 From Nonabsorbable Marker to Colonoscopy Preparation to Osmotic Laxative
- Author
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John S. Fordtran and Alan F. Hofmann
- Subjects
0301 basic medicine ,medicine.medical_specialty ,Osmosis ,medicine.medical_treatment ,Laxative ,Colonoscopy ,Gastroenterology ,History, 21st Century ,Polyethylene Glycols ,03 medical and health sciences ,0302 clinical medicine ,Drug Stability ,Internal medicine ,PEG ratio ,Medicine ,Humans ,Therapeutic Irrigation ,Hepatology ,medicine.diagnostic_test ,business.industry ,Extramural ,History, 19th Century ,History, 20th Century ,030104 developmental biology ,Solubility ,Laxatives ,030211 gastroenterology & hepatology ,business - Published
- 2017
32. N-Methyltaurine N-acyl amidated bile acids and deoxycholic acid in the bile of angelfish (Pomacanthidae): A novel bile acid profile in Perciform fish
- Author
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Ayumi Ohsaki, Takashi Iida, Kuniko Mitamura, Hiroaki Ogata, Alan F. Hofmann, Kiyoshi Asahina, Rika Satoh, Tetsuya Saito, Shigeo Ikegawa, and Lee R. Hagey
- Subjects
Taurine ,medicine.drug_class ,Clinical Biochemistry ,Molecular Conformation ,Biology ,Biochemistry ,Bile Acids and Salts ,chemistry.chemical_compound ,Endocrinology ,Species Specificity ,Bile acid conjugation ,medicine ,Animals ,Bile ,Pygoplites diacanthus ,Molecular Biology ,Pharmacology ,Bile acid ,Organic Chemistry ,Deoxycholic acid ,Cholic acid ,Stereoisomerism ,biology.organism_classification ,Perciformes ,chemistry ,Anaerobic bacteria ,CYP8B1 ,Lipid digestion ,Deoxycholic Acid - Abstract
Two novel N-acyl amidated bile acids, N-methyltaurine conjugated cholic acid and N-methyltaurine conjugated deoxycholic acid, were found to be major biliary bile acids in two species of angelfish the regal (Pygoplites diacanthus) and the blue-girdled (Pomacanthus navarchus) angelfish. The identification was based on their having MS and NMR spectra identical to those of synthetic standards. A survey of biliary bile acids of 10 additional species of angelfish found 7 with N-methyltaurine conjugation. In all 12 species, conjugated deoxycholic acid (known to be formed by bacterial 7-dehydroxylation of cholic acid) was a major bile acid. In all previous studies of biliary bile acids in fish, deoxycholic acid has been present in only trace proportions. In addition, bile acid conjugation with N-methyltaurine has not been detected previously in any known vertebrate. N-methyltaurine conjugated bile acids are resistant to bacterial deconjugation and dehydroxylation, and such resistance to bacterial enzymes should aid in the maintenance of high concentrations of bile acids during lipid digestion. Our findings suggest that these species of angelfish have a novel microbiome in their intestine containing anaerobic bacteria, and describe the presence of N-methyltaurine conjugated bile acids that are resistant to bacterial attack.
- Published
- 2014
33. LC/ESI-MS/MS analysis of urinary 3β-sulfooxy-7β-N-acetylglucosaminyl-5-cholen-24-oic acid and its amides: New biomarkers for the detection of Niemann–Pick type C disease
- Author
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Hiroaki Yamaguchi, Yasushi Misawa, Miki Shimada, Nariyasu Mano, Masami Togawa, Junichi Goto, Takashi Iida, Masamitsu Maekawa, Genta Kakiyama, Kousaku Ohno, Hiroshi Nittono, Ayako Sotoura, and Alan F. Hofmann
- Subjects
Adult ,Male ,Spectrometry, Mass, Electrospray Ionization ,Taurine ,Adolescent ,medicine.drug_class ,Urinary system ,Clinical Biochemistry ,Urine ,Biochemistry ,Matrix (chemical analysis) ,Young Adult ,chemistry.chemical_compound ,Endocrinology ,Tandem Mass Spectrometry ,Liquid chromatography–mass spectrometry ,medicine ,Humans ,Molecular Biology ,Pharmacology ,Chromatography ,Bile acid ,Organic Chemistry ,Selected reaction monitoring ,Infant ,Niemann-Pick Disease, Type C ,Saponins ,chemistry ,Case-Control Studies ,Glycine ,Female ,Biomarkers - Abstract
We developed a sensitive, reliable, and accurate LC/ESI-MS/MS method for measurement of 3β-sulfooxy-7β-N-acetylglucosaminyl-5-cholen-24-oic acid and its glycine and taurine amides in urine. This atypical C24 bile acid has been reported previously to be present in the urine of patients with Niemann-Pick Type C (NPC) disease. In the method, targeted analytes are concentrated at the front edge of a trapping column, Shim-pack MAYI-C8, which permits elimination of contaminating molecules in the urinary matrix. The trapped analytes are then eluted, separated on a YMC-Pack Pro C18, and quantified with MS/MS using selected reaction monitoring. The method could detect (as amount injected) 2pg of nonamidated 3β-sulfooxy-7β-N-acetylglucosaminyl-5-cholen-24-oic acid, 2pg of its glycine-amide, and 0.6pg of its taurine-amide, and is linear up to 300pg. The method was then used to measure the three analytes in the urine of NPC patients (N=2), 3β-hydroxysteroid dehydrogenase deficiency patients (N=2), and healthy volunteers (N=8). Measurable concentrations of all three analytes were present in all subjects. The urinary concentration of the sum of all three analytes was four hundred times greater in the 3month NPC patient and 40times greater in the adult patient than that of healthy volunteers. The markedly elevated urinary concentration of 3β-sulfooxy-7β-N-acetylglucosaminyl-5-cholen-24-oic acid and its amides in NPC patients suggests that these compounds may be valuable biomarkers for detection of NPC disease.
- Published
- 2013
34. Ursodeoxycholic acid attenuates colonic epithelial secretory function
- Author
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Michael Scharl, Orlaith B. Kelly, Magdalena S. Mroz, Stephen J. Keely, Padraic G. Fallon, Alan F. Hofmann, Carolina Colliva, Frank E. Murray, Aldo Roda, John F. Gilmer, Roberto Pellicciari, and Joseph B. J. Ward
- Subjects
medicine.medical_specialty ,Forskolin ,Lithocholic acid ,Bile acid ,Physiology ,medicine.drug_class ,Bile acid malabsorption ,Biology ,medicine.disease ,Ursodeoxycholic acid ,chemistry.chemical_compound ,Endocrinology ,chemistry ,In vivo ,Internal medicine ,Chenodeoxycholic acid ,medicine ,Secretion ,medicine.drug - Abstract
Dihydroxy bile acids, such as chenodeoxycholic acid (CDCA), are well known to promote colonic fluid and electrolyte secretion, thereby causing diarrhoea associated with bile acid malabsorption. However, CDCA is rapidly metabolised by colonic bacteria to ursodeoxycholic acid (UDCA), the effects of which on epithelial transport are poorly characterised. Here, we investigated the role of UDCA in the regulation of colonic epithelial secretion. Cl(-) secretion was measured across voltage-clamped monolayers of T84 cells and muscle-stripped sections of mouse or human colon. Cell surface biotinylation was used to assess abundance/surface expression of transport proteins. Acute (15 min) treatment of T84 cells with bilateral UDCA attenuated Cl(-) secretory responses to the Ca(2+) and cAMP-dependent secretagogues carbachol (CCh) and forskolin (FSK) to 14.0 ± 3.8 and 40.2 ± 7.4% of controls, respectively (n = 18, P < 0.001). Investigation of the molecular targets involved revealed that UDCA acts by inhibiting Na(+)/K(+)-ATPase activity and basolateral K(+) channel currents, without altering their cell surface expression. In contrast, intraperitoneal administration of UDCA (25 mg kg(-1)) to mice enhanced agonist-induced colonic secretory responses, an effect we hypothesised to be due to bacterial metabolism of UDCA to lithocholic acid (LCA). Accordingly, LCA (50-200 μm) enhanced agonist-induced secretory responses in vitro and a metabolically stable UDCA analogue, 6α-methyl-UDCA, exerted anti-secretory actions in vitro and in vivo. In conclusion, UDCA exerts direct anti-secretory actions on colonic epithelial cells and metabolically stable derivatives of the bile acid may offer a new approach for treating intestinal diseases associated with diarrhoea.
- Published
- 2013
35. A novel varanic acid epimer – (24R,25S)-3α,7α,12α,24-tetrahydroxy-5β-cholestan-27-oic acid – is a major biliary bile acid in two varanid lizards and the Gila monster
- Author
-
Alan F. Hofmann, Rika Satoh, Mizuho Une, Takashi Iida, Kuniko Mitamura, Shoujiro Ogawa, Lee R. Hagey, Narimi Kato, and Shigeo Ikegawa
- Subjects
Taurine ,Heloderma ,medicine.drug_class ,Clinical Biochemistry ,Biochemistry ,Bile Acids and Salts ,chemistry.chemical_compound ,Endocrinology ,biology.animal ,medicine ,Animals ,Gila monster ,Biliary Tract ,Molecular Biology ,Pharmacology ,biology ,Bile acid ,Cholesterol ,Lizard ,Organic Chemistry ,Lizards ,Stereoisomerism ,Varanus olivaceus ,biology.organism_classification ,chemistry ,Epimer ,Cholestanols - Abstract
A key intermediate in the biosynthetic pathway by which C(24) bile acids are formed from cholesterol has long been considered to be varanic acid, (24ξ,25ξ)-3α,7α,12α-24-tetrahydroxy-5β-cholestan-27-oic acid. The (24R,25R)-epimer of this tetrahydroxy bile acid, in the form of its taurine N-acyl amidate, was thought to be the major biliary bile acid in lizards of the family Varanidae. We report here that a major biliary bile acid of three lizard species - the Komodo dragon (Varanus komodoensis), Gray's monitor (Varanus olivaceus), and the Gila monster (Heloderma suspectum) - is a novel epimer of varanic acid. The epimer was shown to be (24R,25S)-3α,7α,12α,24-tetrahydroxy-5β-cholestan-27-oic acid (present in bile as its taurine conjugate). The structure was established by mass spectroscopy and by (1)H and (13)C nuclear magnetic spectroscopy, as well as by synthesis of the compound.
- Published
- 2012
36. Synthesis of multiply deuterated 3- and 21-monosulfates of allo-tetrahydrocorticosteroids as internal standards for mass spectrometry
- Author
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Rika Satoh, Takashi Iida, Rina Matsumoto, Alan F. Hofmann, Shoujiro Ogawa, Kanta Sato, Sachi Fujioka, Shigeo Ikegawa, Masataka Nakajima, Takayuki Mabuchi, Kaori Nagae, and Kuniko Mitamura
- Subjects
Pharmacology ,Analyte ,Chromatography ,Sulfates ,Chemistry ,Electrospray ionization ,Organic Chemistry ,Clinical Biochemistry ,Chemistry Techniques, Synthetic ,Reference Standards ,Mass spectrometry ,Biochemistry ,Mass Spectrometry ,chemistry.chemical_compound ,Endocrinology ,Deuterium ,Adrenal Cortex Hormones ,Liquid chromatography–mass spectrometry ,Molecule ,Hydrogen–deuterium exchange ,Methylene ,Molecular Biology - Abstract
The accurate analysis of trace components in complex biological matrices requires the use of reliable internal standards. For liquid chromatography/mass spectrometry analysis, the stable isotope-labeled analogues of the analyte molecules are the most appropriate internal standards. In this paper the synthesis of the 3- and 21-monosulfates of allo-tetrahydrocorticosteroids labeled with four or five deuterium atoms is described. The principal reactions used were (1) hydrogen-deuterium exchange reaction of active methylene groups adjacent to 3- and 11-oxo group of 17,20;20,21-bismethylenedioxy derivatives of 5α-3-ketosteroids and/or 5α-11-ketosteroids with NaOD in CH(3)OD followed by reduction with NaBD(4), (2) epimerization of the 3β-hydroxy group into a 3α configuration, (3) sulfation of hydroxy groups at C-3 or C-21 in the resulting substrates with sulfur trioxide-trimethylamine complex, and (4) removal of 17,20;20,21-bismethylenedioxy groups with hydrogen fluoride in ethanol. Isotopic purity was found to be satisfactory by MS, and NMR properties of the new compounds were tabulated. The labeled compounds can be used as internal standards in liquid chromatography/mass spectrometry assays for clinical and biochemical studies.
- Published
- 2012
37. Detection of Δ4-3-oxo-steroid 5β-reductase deficiency by LC–ESI-MS/MS measurement of urinary bile acids
- Author
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Alan F. Hofmann, Takayuki Hoshina, Jun Mori, Tsuyoshi Murai, Takao Kurosawa, Tatsuki Mizuochi, Akina Muto, Atsushi Unno, Hiroshi Nittono, Shigeo Ikegawa, Shoujiro Ogawa, Takashi Iida, Akira Ohtake, Akihiko Kimura, Hajime Takei, and Lee R. Hagey
- Subjects
Spectrometry, Mass, Electrospray Ionization ,medicine.medical_treatment ,Clinical Biochemistry ,Urine ,Tandem mass spectrometry ,Sensitivity and Specificity ,Biochemistry ,Gas Chromatography-Mass Spectrometry ,Analytical Chemistry ,Steroid ,Bile Acids and Salts ,chemistry.chemical_compound ,Tandem Mass Spectrometry ,medicine ,Humans ,chemistry.chemical_classification ,Creatinine ,Chromatography ,Cholesterol ,Selected reaction monitoring ,Infant ,Reproducibility of Results ,Cell Biology ,General Medicine ,Metabolic pathway ,Enzyme ,chemistry ,Child, Preschool ,Oxidoreductases ,Chromatography, Liquid - Abstract
The synthesis of bile salts from cholesterol is a complex biochemical pathway involving at least 16 enzymes. Most inborn errors of bile acid biosynthesis result in excessive formation of intermediates and/or their metabolites that accumulate in blood and are excreted in part in urine. Early detection is important as oral therapy with bile acids results in improvement. In the past, these intermediates in bile acid biosynthesis have been detected in neonatal blood or urine by screening with FAB-MS followed by detailed characterization using GC-MS. Both methods have proved difficult to automate, and currently most laboratories screen candidate samples using LC-MS/MS. Here, we describe a new, simple and sensitive analytical method for the identification and characterization of 39 conjugated and unconjugated bile acids, including Δ(4)-3-oxo- and Δ(4,6)-3-oxo-bile acids (markers for Δ(4)-3-oxo-steroid 5β-reductase deficiency), using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). In this procedure a concentrated, desalted urinary sample (diluted with ethanol) is injected directly into the LC-ESI-MS/MS, operated with ESI and in the negative ion mode; quantification is obtained by selected reaction monitoring (SRM). To evaluate the performance of our new method, we compared it to a validated method using GC-MS, in the analysis of urine from two patients with genetically confirmed Δ(4)-3-oxo-steroid 5β-reductase deficiency as well as a third patient with an elevated concentration of abnormal conjugated and unconjugated Δ(4)-3-oxo-bile acids. The Δ(4)-3-oxo-bile acids concentration recovered in three patients with 5β-reductase deficiency were 48.8, 58.9, and 49.4 μmol/mmol creatinine, respectively by LC-ESI-MS/MS.
- Published
- 2012
38. Hepatobiliary Secretion Kinetics of Conjugated Bile Acids Measured in Pigs by 11C-Cholylsarcosine PET
- Author
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Michael Sørensen, Susanne Keiding, Alan F. Hofmann, Nikolaj Worm Ørntoft, Kim Frisch, Peter Ott, Aage Kristian Olsen Alstrup, Frank Viborg Mortensen, Kasper Jarlhelt Andersen, and Ole Lajord Munk
- Subjects
medicine.medical_specialty ,medicine.drug_class ,Swine ,digestive system ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Internal medicine ,medicine ,Animals ,Radiology, Nuclear Medicine and imaging ,Secretion ,Carbon Radioisotopes ,Biliary Tract ,Bile acid ,business.industry ,Cholic acid ,Cholic Acids ,Sarcosine ,Venous blood ,Bile Salt Export Pump ,Kinetics ,medicine.anatomical_structure ,Endocrinology ,chemistry ,Common hepatic duct ,Liver ,Biliary tract ,030220 oncology & carcinogenesis ,Positron-Emission Tomography ,030211 gastroenterology & hepatology ,Female ,business ,Perfusion - Abstract
The aim was to develop a method for quantification of hepatobiliary uptake and secretion of conjugated bile acids using PET and the (11)C-labeled conjugated bile acid analog [N-methyl-(11)C]cholylsarcosine ((11)C-CSar).METHODS: Six pigs (13 experiments) underwent dynamic (11)C-CSar PET of the liver with measurements of hepatic blood perfusion and (11)C-CSar concentrations in arterial, portal and hepatic venous blood. In three pigs (seven experiments) bile was collected from a catheter in the common hepatic duct (CHD). PET data were analyzed by a two-tissue compartmental model with calculation of rate constants for transport of (11)C-CSar between blood, hepatocytes and intra- and extra-hepatic bile ducts. PET results were validated against invasive blood and bile measurements.RESULTS: Directly measured secretion rate of (11)C-CSar into bile was equal to removal rate from blood at steady state. Hepatocytes accordingly did not accumulate bile acids but simply facilitated transport of bile acids from blood into bile against a measured concentration gradient of 4,000 from blood to bile. In experiments with catheter in CHD, rate constant for secretion of (11)C-CSar from hepatocytes into bile was 25% of that in experiments without a catheter (P < 0.05) which we interpreted as mild cholestasis caused by the catheter. The catheter caused an increased backflux of (11)C-CSar from hepatocytes to blood and hepatic blood flow was 25% higher than in experiment without catheter. The capacity of the overall transport of (11)C-CSar from blood into bile, as quantified by intrinsic clearance, was significantly lower in experiments with catheter than in pigs without catheter (P < 0.001). PET and blood measurements correlated significantly (P < 0.05).CONCLUSION: In vivo kinetics of hepatobiliary secretion of conjugated bile acids can now be determined by dynamic (11)C-CSar PET.
- Published
- 2015
39. Novel, major 2α- and 2β-hydroxy bile alcohols and bile acids in the bile of Arapaima gigas, a large South American river fish
- Author
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Kuniko Mitamura, Shoutaro Sekiguchi, Kaoru Omura, Kazunari Namegawa, Rika Sato, Satoshi Kurabuchi, Naoya Nakane, Hiroaki Ogata, Jan Raines, Alan F. Hofmann, Shigeo Ikegawa, Lee R. Hagey, Takashi Iida, and Tetsuya Saito
- Subjects
0301 basic medicine ,Taurine ,medicine.drug_class ,Clinical Biochemistry ,ved/biology.organism_classification_rank.species ,Salt (chemistry) ,DEPT ,Biochemistry ,Hydroxylation ,Bile Acids and Salts ,03 medical and health sciences ,chemistry.chemical_compound ,Endocrinology ,medicine ,Animals ,Molecular Biology ,Pharmacology ,chemistry.chemical_classification ,Chromatography ,Bile acid ,ved/biology ,Organic Chemistry ,Cholic acid ,Fishes ,030104 developmental biology ,chemistry ,Arapaima gigas ,Cholestanols - Abstract
Bile alcohols and bile acids from gallbladder bile of the Arapaima gigas, a large South American freshwater fish, were isolated by reversed-phase high-performance liquid chromatography. The structures of the major isolated compounds were determined by electrospray-tandem mass spectrometry and nuclear magnetic resonance using (1)H- and (13)C-NMR spectra. The novel bile salts identified were six variants of 2-hydroxy bile acids and bile alcohols in the 5α- and 5β-series, with 29% of all compounds having hydroxylation at C-2. Three C27 bile alcohols were present (as ester sulfates): (24ξ,25ξ)-5α-cholestan-2α,3α,7α,12α,24,26-hexol; (25ξ)-5β-cholestan-2β,3α,7α,12α,26,27-hexol, and (25ξ)-5α-cholestan-2α,3α,7α,12α,26,27-hexol. A single C27 bile acid was identified: (25ξ)-2α,3α,7α,12α-tetrahydroxy-5α-cholestan-26-oic acid, present as its taurine conjugate. Two novel C24 bile acids were identified: the 2α-hydroxy derivative of allochenodeoxycholic acid and the 2β-hydroxy derivative of cholic acid, both occurring as taurine conjugates. These studies extend previous work in establishing the natural occurrence of novel 2α- and 2β-hydroxy-C24 and C27 bile acids as well as C27 bile alcohols in both the normal (5β) as well as the (5α) "allo" A/B-ring juncture. The bile salt profile of A. gigas appears to be unique among vertebrates.
- Published
- 2015
40. Two Inborn Errors of Bile Acid Biosynthesis: The Need for Recognition and Treatment by Primary Bile Acid Replacement
- Author
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Alan F. Hofmann
- Subjects
0301 basic medicine ,Bile acid biosynthesis ,Primary (chemistry) ,Bile acid ,business.industry ,medicine.drug_class ,Gastroenterology ,Metabolism ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Biochemistry ,Pediatrics, Perinatology and Child Health ,Medicine ,030211 gastroenterology & hepatology ,business - Published
- 2017
41. Identification of S-acyl glutathione conjugates of bile acids in human bile by means of LC/ESI-MS
- Author
-
Alan F. Hofmann, Shigeo Ikegawa, Toshiaki Shimizu, Naohiro Hori, Kuniko Mitamura, Hiroshi Nittono, Kyoichi Takaori, Mitsuyoshi Suzuki, Hajime Takikawa, and Takashi Iida
- Subjects
Adult ,Spectrometry, Mass, Electrospray Ionization ,Lithocholic acid ,Electrospray ionization ,Clinical Biochemistry ,Biochemistry ,Bile duct cancer ,Bile Acids and Salts ,chemistry.chemical_compound ,Endocrinology ,Biotransformation ,Liquid chromatography–mass spectrometry ,medicine ,Animals ,Bile ,Humans ,Molecular Biology ,Aged ,Pharmacology ,Chromatography ,Organic Chemistry ,Infant ,Glutathione ,Middle Aged ,medicine.disease ,Ursodeoxycholic acid ,Orders of magnitude (mass) ,Rats ,chemistry ,Chromatography, Liquid ,medicine.drug - Abstract
Previous work from this laboratory has reported the biotransformation of bile acids (BA) into the thioester-linked glutathione (GSH) conjugates via the intermediary metabolites formed by BA:CoA ligase and shown that such GSH conjugates are excreted into the bile in healthy rats as well as rats dosed with lithocholic acid or ursodeoxycholic acid. To examine whether such novel BA–GSH conjugates are present in human bile, we determined the concentration of the GSH conjugates of the five BA that predominate in human bile. Bile was obtained from three infants (age 4, 10, and 13 months) and the BA–GSH conjugates quantified by means of liquid chromatography (LC)/electrospray ionization (ESI)-linear ion trap mass spectrometry (MS) in negative-ion scan mode, monitoring characteristic transitions of the analytes. By LC/ESI-MS, only primary BA were present in biliary BA, indicating that the dehydroxylating flora had not yet developed. GSH conjugates of chenodeoxycholic and lithocholic acid were present in concentrations ranging from 27 to 1120 pmol/ml, several orders of magnitude less than those of natural BA N-acylamidates. GSH conjugates were not present, however, in the ductal bile obtained from 10 adults (nine choledocholithiasis, one bile duct cancer). Our results indicate that BA–GSH conjugates are formed and excreted in human bile, at least in infants, although this novel mode of conjugation is a very minor pathway.
- Published
- 2011
42. Chemical synthesis of the (25R)- and (25S)-epimers of 3α,7α,12α-trihydroxy-5α-cholestan-27-oic acid as well as their corresponding glycine and taurine conjugates
- Author
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Kuniko Mitamura, Shigeo Ikegawa, Shoujiro Ogawa, Takashi Iida, Matthew D. Krasowski, Alan F. Hofmann, and Lee R. Hagey
- Subjects
chemistry.chemical_classification ,Taurine ,Magnetic Resonance Spectroscopy ,Decarboxylation ,Organic Chemistry ,Cholic acid ,Stereoisomerism ,Cholic Acid ,Cell Biology ,Biochemistry ,Chemical synthesis ,Bile Acids and Salts ,chemistry.chemical_compound ,Malonate ,Dicarboxylic acid ,chemistry ,Glycine ,Organic chemistry ,Ethyl chloroformate ,Molecular Biology ,Cholestanols - Abstract
The (25R)- and (25S)-epimers of C(27) 3α,7α,12α-trihydroxy-5α-cholestan-27-oic acid as well as their corresponding N-acylamidate conjugates with glycine or taurine were prepared starting from cholic acid in 14 steps. The principal reactions involved were (1) reduction of a key intermediary C(24)allo-cholic acid performate with NaBH(4)/triethylamine/ethyl chloroformate, (2) iodination of the resulting 3,7,12-triformyloxy-5α-cholan-24-ol with I(2)/triphenylphosphine; (3) nucleophilic substitution of the iodo derivative with diethylmethyl malonate/NaH; and (4) hydrolysis of the resulting 3,7,12-triformyloxy-25-methyl-26,27-diethyl ester with KOH, followed by decarboxylation of the geminal dicarboxylic acid with LiCl. N-Acylamidation of the resulting (25R)/(25S)-3α,7α,12α-trihydroxy-5α-cholestan-27-oic acid mixture with glycine or taurine afforded the corresponding epimeric mixtures of the glycine and taurine conjugates. The (25R)- and (25S)-epimers of the three variants of unconjugated and conjugated 3α,7α,12α-trihydroxy-5α-cholestan-27-oic acid were efficiently separated by HPLC on a reversed-phase C(18) column and their structural characteristics, particularly the chiral center at C-25, delineated using (1)H and (13)C NMR. These synthetic compounds should be useful as authentic reference standards for establishing their presence in bile as well as being useful in studies on the biosynthesis of allo-bile acids from cholesterol.
- Published
- 2011
43. Characterization of non-enzymatic acylation of amino or thiol groups of bionucleophiles by the acyl-adenylate or acyl-CoA thioester of cholic acid
- Author
-
Eriko Aoyama, Takashi Iida, Shigeo Ikegawa, Alan F. Hofmann, Kuniko Mitamura, and Toshihiro Sakai
- Subjects
chemistry.chemical_classification ,Taurine ,Stereochemistry ,Acylation ,Cholic acid ,Esters ,Cholic Acid ,Glutathione ,Thioester ,Biochemistry ,Adenosine Monophosphate ,Analytical Chemistry ,Amino acid ,chemistry.chemical_compound ,Transacylation ,chemistry ,Thiol ,Organic chemistry ,Coenzyme A - Abstract
Acyl-adenylates and acyl-CoA thioesters of bile acids (BAs) are highly electrophilic acyl-linked metabolites which can undergo transacylation reactions with amino and thiol groups of nucleophilic groups on acceptor molecules such as amino acids, peptides, and proteins. Here, non-enzymatic acylation at pH 7.4 of glycine, taurine, glutathione (GSH), and N-acetylcysteine (NAC) by cholyl-adenylate (CA-AMP) was compared with that mediated by cholyl-CoA thioester (CA-CoA) using a 1:1 mixture of stable isotopically labeled CA-AMP and unlabeled CA-CoA. The transacylation products of these substrates were analyzed by liquid chromatography/electrospray ionization linear ion-trap mass spectrometry in negative-ion detection mode. CA-AMP was more reactive than CA-CoA with the amino group of glycine or taurine than with the thiol group of GSH or NAC. In contrast, CA-CoA was more reactive than CA-AMP with the thiol group of GSH or NAC and was far less reactive with the amino group of glycine or taurine. These differences in the reactivity of CA-AMP as compared with that of CA-CoA towards amino and thiol groups may be attributed to the electrophilicity of the carbonyl carbon of these acyl-linked cholic acid metabolites and the nucleophilicity of the amino and thiol group in the bionucleophiles that were studied.
- Published
- 2011
44. Synthesis of the 3-sulfates of N-acetylcysteine conjugated bile acids (BA-NACs) and their transient formation from BA-NACs and subsequent hydrolysis by a rat liver cytosolic fraction as shown by liquid chromatography/electrospray ionization-mass spectrometry
- Author
-
Kuniko Mitamura, Risa Nakai, Tateaki Wakamiya, Toshihiro Sakai, Alan F. Hofmann, Takashi Iida, and Shigeo Ikegawa
- Subjects
Spectrometry, Mass, Electrospray Ionization ,Electrospray ,Magnetic Resonance Spectroscopy ,medicine.drug_class ,Electrospray ionization ,Biochemistry ,Chemical synthesis ,Analytical Chemistry ,Bile Acids and Salts ,Hydrolysis ,Cytosol ,Sulfation ,Limit of Detection ,medicine ,Animals ,Chromatography ,Bile acid ,Sulfates ,Chemistry ,Selected reaction monitoring ,Nuclear magnetic resonance spectroscopy ,Acetylcysteine ,Rats ,Liver ,Chromatography, Liquid - Abstract
Previous work from this laboratory has reported the chemical synthesis of N-acetylcysteine (NAC) conjugates of natural bile acids (BAs) and shown that such novel conjugates can be formed in vivo in rats to which NAC has been administered. The subsequent fate of such novel conjugates is not known. One possible biotransformation is sulfation, a major pathway for BAs N-acylamidates in patients with cholestatic liver disease. Here, we report the chemical synthesis of the 3-sulfates of the S-acyl NAC conjugates of five natural BAs (cholic, chenodeoxycholic, deoxycholic, ursodeoxycholic, and lithocholic). We also measured the sulfation of N-acetylcysteine-natural bile acid (BA-NAC) conjugates when they were incubated with a rat liver cytosolic fraction. The chemical structures of the BA-NAC 3-sulfates were confirmed by proton nuclear magnetic resonance, as well as by means of electrospray ionization-linear ion trap mass spectrometry with negative-ion detection. Upon collision-induced dissociation of singly and doubly charged deprotonated molecules, structurally informative product ions were observed. Using a triple-stage quadrupole instrument, selected reaction monitoring analyses by monitoring characteristic transition ions allowed the achievement of a highly sensitive and specific assay. When BA-NACs were incubated with a rat liver cytosolic fraction to which 3'-phosphoadenosine 5'-phosphosulfate was added, sulfation occurred, but the dominant reaction was hydrolysis of the S-acyl linkage to form the unconjugated BAs. Subsequent sulfation occurred at C-3 on the unconjugated BAs that had been formed from the BA-NACs. Such sulfation was proportional to the hydrophobicity of the unconjugated bile acid. Thus, NAC conjugates of BAs as well as their C-3 sulfates if formed in vivo are rapidly hydrolyzed by cytosolic enzymes.
- Published
- 2011
45. Identification of bile acid S-acyl glutathione conjugates in rat bile by liquid chromatography/electrospray ionization–linear ion trap mass spectrometry
- Author
-
Shigeo Ikegawa, Kuniko Mitamura, Alan F. Hofmann, Naohiro Hori, and Takashi Iida
- Subjects
Male ,Spectrometry, Mass, Electrospray Ionization ,Lithocholic acid ,medicine.drug_class ,Electrospray ionization ,Clinical Biochemistry ,Molecular Conformation ,Mass spectrometry ,Biochemistry ,Bile Acids and Salts ,chemistry.chemical_compound ,Endocrinology ,Liquid chromatography–mass spectrometry ,Bile acid conjugation ,medicine ,Animals ,Bile ,Rats, Wistar ,Molecular Biology ,Pharmacology ,Chromatography ,Bile acid ,Organic Chemistry ,Stereoisomerism ,Glutathione ,Ursodeoxycholic acid ,Rats ,chemistry ,Chromatography, Liquid ,medicine.drug - Abstract
Acyl-adenylates and acyl-CoA thioesters of bile acids (BAs) are reactive acyl-linked metabolites that have been shown to acylate the thiol group of glutathione (GSH); the reaction is catalyzed by glutathione S -transferase (GST) and the product is a thioester-linked BA-GSH conjugate. Such GSH conjugates are present in bile in lithocholic acid and ursodeoxycholic acid dosed-rats. To determine whether such novel BA-GSH conjugates are present in the bile of normal rats, we first synthesized the GSH conjugates of the major and minor biliary BAs of the rat and defined their MS and proton NMR properties. We then analyzed the BA-GSH composition in the bile of anesthetized biliary fistula rats by means of liquid chromatographic separation and electrospray ionization–linear ion trap mass spectrometric detection in negative- and positive-ion scan modes, monitoring characteristic transitions of the analytes. GSH conjugates of cholic, ω-muricholic, hyodeoxycholic, deoxycholic, 12-oxolithocholic, and lithocholic acids were present with concentrations in the range of 1.4–2.8 nmol/ml, some four orders of magnitude less than those of natural BA N -acyl amidates. Our results indicate that BA-GSH conjugates are formed and excreted in bile in the healthy rat, although this novel mode of BA conjugation is a very minor pathway.
- Published
- 2011
46. Complex Evolution of Bile Salts in Birds
- Author
-
Alan F. Hofmann, Nicolas Vidal, Matthew D. Krasowski, and Lee R. Hagey
- Subjects
Gut microflora ,Herbivore ,Taxon ,Ecology ,Molecular evolution ,Zoology ,Animal Science and Zoology ,Omnivore ,Biology ,digestive system ,Lipid digestion ,Article ,Ecology, Evolution, Behavior and Systematics - Abstract
Bile salts are the major end-metabolites of cholesterol and are important in lipid digestion and shaping of the gut microflora. There have been limited studies of bile-salt variation in birds. The purpose of our study was to determine bile-salt variation among birds and relate this variation to current avian phylogenies and hypotheses on the evolution of bile salt pathways. We determined the biliary bile-salt composition of 405 phylogenetically diverse bird species, including 7 paleognath species. Bile salt profiles were generally stable within bird families. Complex bile-salt profiles were more common in omnivores and herbivores than in carnivores. The structural variation of bile salts in birds is extensive and comparable to that seen in surveys of bile salts in reptiles and mammals. Birds produce many of the bile salts found throughout nonavian vertebrates and some previously uncharacterized bile salts. One difference between birds and other vertebrates is extensive hydroxylation of carbon-16 of bile salts in bird species. Comparison of our data set of bird bile salts with that of other vertebrates, especially reptiles, allowed us to infer evolutionary changes in the bile salt synthetic pathway.
- Published
- 2010
47. Plasma Bile Acid Concentrations in Humans: Suggestions for Presentation in Tabular Form
- Author
-
Hanns-Ulrich Marschall and Alan F. Hofmann
- Subjects
0301 basic medicine ,medicine.medical_specialty ,Hepatology ,Bile acid ,medicine.drug_class ,business.industry ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Endocrinology ,Internal medicine ,medicine ,030211 gastroenterology & hepatology ,Presentation (obstetrics) ,business - Published
- 2018
48. Hepatobiliary Disposition of 3α,6α,7α,12α-Tetrahydroxy-Cholanoyl Taurine: A Substrate for Multiple Canalicular Transporters
- Author
-
Alan F. Hofmann, Paiboon Jungsuwadee, Vandana Megaraj, Takashi Iida, and Mary Vore
- Subjects
Pharmacology ,medicine.medical_specialty ,Taurine ,Bile acid ,medicine.drug_class ,Multidrug resistance-associated protein 2 ,Cholic acid ,Pharmaceutical Science ,Taurocholic acid ,medicine.disease ,Excretion ,chemistry.chemical_compound ,Endocrinology ,chemistry ,Cholestasis ,Internal medicine ,medicine ,Enzyme kinetics - Abstract
Tetrahydroxy bile acids become major biliary bile acids in Bsep(−/−) mice and Fxr(−/−) mice fed cholic acid; we characterized disposition of these novel bile acids that also occur in patients with cholestasis. We investigated mouse Mrp2 (mMrp2) and P-glycoprotein [(P-gp) mMdr1a]-mediated transport of a tetrahydroxy bile acid, 6α-OH-taurocholic acid (6α-OH-TC), and its biliary excretion in wild-type and Mrp2(−/−) mice in the presence or absence of N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918), a P-gp and breast cancer resistance protein inhibitor. 6α-OH-TC was rapidly excreted into bile of wild-type mice (78% recovery); coinfusion of GF120918 had no significant effect. In Mrp2(−/−) mice, biliary excretion was decreased (52% recovery) and coinfusion of GF120918 further decreased these values (34% recovery). In wild-type, but not Mrp2(−/−), mice, 6α-OH-TC increased bile flow 2.5-fold. Membrane vesicle transport studies of 6α-OH-TC (0.05–0.75 mM) yielded saturation kinetics with a higher apparent affinity for mMrp2 (Km = 0.13 mM) than for mMdr1a (Km = 0.33 mM); mBsep transported 6α-OH-TC with positive cooperativity (Hill slope = 2.1). Human multidrug resistance-associated protein (MRP) 2 and P-gp also transported 6α-OH-TC but with positive cooperativity (Hill slope = 3.6 and 1.6, respectively). After intraileal administration, the time course of 6α-OH-TC biliary recovery was similar to that of coinfused taurocholate, implying that 6α-OH-TC can undergo enterohepatic cycling. Thus, Mrp2 plays a key role in 6α-OH-TC biliary excretion, whereas P-glycoprotein plays a secondary role; Bsep likely mediates excretion of 6α-OH-TC in the absence of Mrp2 and P-gp. In Bsep(−/−) mice, efficient synthesis of tetrahydroxy bile acids that are Mrp2 and P-gp substrates can explain the noncholestatic phenotype.
- Published
- 2010
49. Major Biliary Bile Acids of the Medaka (Oryzias latipes): 25R- and 25S-Epimers of 3α,7α,12α-Trihydroxy-5β-cholestanoic Acid
- Author
-
Mizuho Une, Takashi lida, Takaaki Goto, Kiyoshi Asahina, Hideyuki Tamegai, Alan F. Hofmann, Matthew D. Krasowski, Junichi Goto, Kumiko Mushiake, Nariyasu Mano, Lee R. Hagey, and Shoujiro Ogawa
- Subjects
Taurine ,Chromatography ,Molecular Structure ,Bile acid ,biology ,medicine.drug_class ,Oryzias ,Cholic acid ,Diastereomer ,Carbon-13 NMR ,biology.organism_classification ,Article ,Bile Acids and Salts ,chemistry.chemical_compound ,chemistry ,Biochemistry ,medicine ,Animals ,Bile ,Animal Science and Zoology ,Cholestanols ,CYP8B1 ,Phylogeny - Abstract
The biliary bile salts of the medaka, the Japanese rice fish (Oryzias latipes) were isolated and identified. Only bile acids were present, and all were N-acylamidated with taurine. Three bile acids, constituting 98% of total bile acids, were isolated by chromatography and their structure inferred from their properties compared to those of synthetic standards when analyzed by liquid chromatographytandem mass spectrometry. The dominant bile acid was the 25R-epimer (82%) of 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestan-27-oic acid. The 25S-epimer was also present (11%), as was cholic acid (5%). Complete (1)H and (13)C NMR signal assignments of the C-25 epimers were made by using a combination of several 1D- and 2D-NMR techniques. The (1)H and (13)C NMR chemical shifts and spectral patterns of the hydrogen and carbon atoms, being close to the asymmetric centered at C-25, provided confirmatory evidence in that they distinguished the two epimeric diastereomers. The medaka is the first fish species identified as having C(27) biliary bile acids as dominant among its major bile salts.
- Published
- 2010
50. Bile salts of vertebrates: structural variation and possible evolutionary significance[S]
- Author
-
Lee R. Hagey, Matthew D. Krasowski, and Alan F. Hofmann
- Subjects
enzymes ,QD415-436 ,Biology ,phylogeny ,digestive system ,Biochemistry ,Bile Acids and Salts ,Evolution, Molecular ,Hydroxylation ,Structural variation ,chemistry.chemical_compound ,Endocrinology ,Phylogenetics ,Molecular evolution ,biology.animal ,Animals ,Humans ,chemistry.chemical_classification ,bile acids ,Cholesterol ,molecular evolution ,Thematic Review ,Vertebrate ,cholesterol ,Cell Biology ,Metabolism ,Enzyme ,chemistry ,Vertebrates ,metabolism ,Cholestanols - Abstract
Biliary bile salt composition of 677 vertebrate species (103 fish, 130 reptiles, 271 birds, 173 mammals) was determined. Bile salts were of three types: C(27) bile alcohols, C(27) bile acids, or C(24) bile acids, with default hydroxylation at C-3 and C-7. C(27) bile alcohols dominated in early evolving fish and amphibians; C(27) bile acids, in reptiles and early evolving birds. C(24) bile acids were present in all vertebrate classes, often with C(27) alcohols or with C(27) acids, indicating two evolutionary pathways from C(27) bile alcohols to C(24) bile acids: a) a 'direct' pathway and b) an 'indirect' pathway with C(27) bile acids as intermediates. Hydroxylation at C-12 occurred in all orders and at C-16 in snakes and birds. Minor hydroxylation sites were C-1, C-2, C-5, C-6, and C-15. Side chain hydroxylation in C(27) bile salts occurred at C-22, C-24, C-25, and C-26, and in C(24) bile acids, at C-23 (snakes, birds, and pinnipeds). Unexpected was the presence of C(27) bile alcohols in four early evolving mammals. Bile salt composition showed significant variation between orders but not between families, genera, or species. Bile salt composition is a biochemical trait providing clues to evolutionary relationships, complementing anatomical and genetic analyses.
- Published
- 2010
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