G. Parmentier

Trihydroxycoprostanic acid in the duodenal fluid of two children with intrahepatic bile duct anomalies

Abstract Using thin-layer chromatography, gas-liquid chromatography and mass spectrometry, trihydroxycoprostanic acid (3α, 7α, 12α-trihydroxy-5β-cholestan-26-oic acid) was identified in the duodenal fluid of 2 subjects with anomalies of the intrahepatic bile ducts. Sugject I was a case of intrahepatic cholestasis due to atresia of the interlobular bile ducts with familial incidence. The bile acid spectrum in the duodenal fluid of this patient was: 23% chenodeoxycholic acid, 58% cholic acid and 19% trihydroxycoprostanic acid. Subject 2 had a Zellweger-like syndrome with cholestasis and with scarely developed intrahepatic bile ductuli. The bile acid spectrum in the duodenal fluid of this patient was: 18% chenodeoxycholic acid, 37% cholic acid and 45% trihydroxycoprostanic acid. No trihydroxycoprostanic acid was found in seven healthy subjects, in three cases of cholestasis of infancy, or in ten cases of various disorders of the small intestine. Obviously, the excretion of trihydroxycoprostanic acid with the bile of Patients 1 and 2 was due to a reduced capacity of the hepatocytes to split off the 3 terminal carbon atoms of the side chain of trihydroxyprostanic acid. The cause of the impaired function of the hepatocytes remains to be established.

Synthesis of the specific monosulfates of cholic acid

The three isomeric cholic acid-monosulfates were synthetized and characterized. Cholic acid-3-sulfate was obtained by reacting cholic acid for 2 min with chlorosulfonic acid in pyridine and chromatography of the resulting bile salt mixture on Sephadex LH-20. The 7- and the 12-monosulfate were prepared by sulfation of the corresponding monohydroxy-diacetates followed by removal of the acetyl groups by alkaline hydrolysis and purification by chromatography on Sephadex LH-20. On TLC in n-butanol-acetic acid-water (10:1:1, v/v) the Rf values were 0.59 for cholic acid-3-sulfate, 0.52 for cholic acid-7-sulfate and 0.48 for cholic acid-12-sulfate. The time required for complete solvolysis at 37 degrees C in acid methanol-acetone (1:9) was 3 h for cholic acid-3-sulfate, 12 h for the 12-monosulfate and 18 h for the 7-monosulfate.

Synthesis and characteristics of the specific monosulfates of chenodeoxycholate, deoxycholate and their taurine or glycine conjugates

The isomeric monosulfates of chenodeoxycholate, deoxycholate, and their taurine or glycine conjugates, were synthesized and characterized. Reaction with chlorosulfonic acid in pyridine for 2 minutes mainly afforded the 3-monosulfates. To prepare the 7- or the 12-monosulfates, the 3-hydroxyl group was protected by carbethoxylation prior to sulfation of the 7- or 12-hydroxyl group for 24 h to 5 days; after sulfation, the protecting 3-carbethoxy function was removed by mild alkaline hydrolysis. The crude bile salt monosulfates were purified by chromatography on silica gel and on Sephadex LH-20 and were crystallized from methanolethanol-ethyl acetate. The results of elemental analysis demonstrated that the compounds were disodium dihydroxy bile salt monosulfates. Thin layer chromatography of the sulfates, and gas-liquid chromatography after oxidation and solvolysis, showed that the substances were pure and that the sulfate group was at the expected position.

Mechanism of biohydrogenation of cholesterol to coprostanol by Eubacterium ATCC 21408

Abstract The mechanism of biohydrogenation of cholesterol to coprostanol was studied by incubating Eubacterium ATTC 21408 with [3α- 3 H, 4- 14 C]cholesterol or [4β- 3 H, 4- 14 C]cholesterol in a brain-thioglycollate medium under anaerobic conditions. Conversion of [3α- 3 H, 4- 14 C]cholesterol into coprostanol occurred with loss of 65 % of tritium. However, part of the tritium might have been lost not during but after reduction of cholesterol to coprostanol. That this might have been the case was demonstrated by the observation that incubation of Eubacterium 21408 with preformed [3α- 3 H, 4- 14 C]coprostanol resulted in a loss of 40% of the tritium. Hence, Eubacterium 21408 carries out reversible oxidation-reduction reactions at the C-3 hydroxyl group and neither loss nor retention of tritium during conversion of [3α- 3 H]-cholesterol to corpostanol warrants conclusions as to the mechanisms of the biohydrogenating pathway. 1. 2. Coprostanol produced by incubating Eubacterium 21408 with [4β- 3 H, 4- 14 C]-cholesterol had retained 81 % of the tritium originally present in cholesterol. However, more than 90 % of the tritium in cholesterol had been transferred to the C-6 position in coprostanol, indicating that the conversion of cholesterol into coprostanol involved isomerisation of the 5,6-double bond to a 4,5-double bond and an intramolecular shift of the major amount of tritium from C-4 to C-6. These data support the hypothesis that the major pathway for biohydrogenation of cholesterol by Eubacterium 21408 involves the intermediate formation of 4-cholesten-3-one followed by reduction of the latter to corprostanol.

Bile acids in peroxisomal disorders

Abstract. We examined serum bile acids in patients with different peroxisomal disorders. Patients with Zellweger syndrome (n= 23), infantile form of Refsum disease (n= 6) and neonatal adrenoleukodystrophy (n= 4) consistently had increased levels of bile acid precursors. Patients with X‐linked adrenoleukodystrophy, (n= 5) classical Refsum disease (n= 3), hyperpipecolic acidaemia (n= 4) and rhizomelic chondrodysplasia punctata (n= 9) did not have increased bile acid precursor levels. Total serum bile acids (41 μg ml‐1) and the percentage of bile acid precursors (80%) were highest in typical Zellweger patients who died young. Long‐living Zellweger patients, neonatal adrenoleukodystrophy patients and infantile Refsum disease patients had, on average, less cholestasis and a lower percentage of bile acid precursors. We also observed that total serum bile acids and the percentage of bile acid precursors decreased with age in longliving Zellweger patients. Screening for bile acid precursors, combined with very long chain fatty acids analysis is, in our experience, an easy and reliable firstline approach to the detection of peroxisomal disorders.

Age-related differences in plasmalogen content of erythrocytes from patients with the cerebro-hepato-renal (Zellweger) syndrome: implications for postnatal detection of the disease

Phosphatidylethanolamine plasmalogen levels were determined in erythrocytes from controls and 13 patients with the cerebro-hepato-renal (Zellweger) syndrome. It was found that in Zellweger patients 20 weeks of age or younger, erythrocyte phosphatidylethanolamine plasmalogen levels were lowered whereas in older patients (except in one) normal levels were found. The results obtained suggest a close relationship between the age of the patients at sampling and the phosphatidylethanolamine plasmalogen levels in their erythrocytes. A possible explanation for these findings and the implications for the postnatal detection of Zellweger syndrome are discussed.

Influence of microbial bile salt desulfation upon the fecal excretion of bile salts in gnotobiotic rats

The fecal excretion of intraperitoneally injected 24-14C-labeled taurocholate (TCA), taurolithocholate (TLCA) and the respective 3-sulfate esters (TCA-3-S; TLCA-3-S), were compared in germfree (GF) rats, conventional (CV) rats, and in gnotobiotic rats associated with Clostridium Cl-8 or this same strain Cl-8 plus the bile desulfating Clostridium S1, respectively. TCA and TLCA were about two times more rapidly excreted by CV animals than by GF animals; the time required for 50% excretion of total label injected (t 1/2) was 6.6 days vs 14.9 for TCA, and 4.4 vs 8.9 for TLCA. In GF and in CV animals, TCA-3-S and TLCA-3-S were excreted more rapidly than their nonsulfated analogues; the t 1/2 values of TCA-3-S and TCA were 2.7 days vs 14.9 in GF rats, and 3.1 vs 6.6 days in CV animals. The t 1/2 values of TLCA-3-S and TLCA were 2.7 days vs 8.9 in GF rats, and 1.5 vs 4.4 days in CV rats. In gnotobiotic rats associated with Clostridium strains S1 + Cl-8, fecal bile salts were nearly 100% deconjugated and desulfated and the 50% excretion times of TCA-3-S and TLCA-3-S approximated to those of TCA and TLCA in GF animals. T 1/2 of TCA-3-S in gnotobiotic S1 + Cl-8 animals was 12.2 days vs 14.9 for TCA in GF animals. In gnotobiotic S1 + Cl-8 animals the t 1/2 of TLCA and TLCA-3-S was 12.5 and 11.0 days, respectively. These results illustrate clearly the important effect the intestinal microflora has upon the metabolic half-life of bile salts. Moreover, they demonstrate that desulfation of bile salts by the intestinal microflora takes place in intestinal segments from where a certain degree of reabsorption is still possible, and thus point to the fact that microbial desulfation is an important variable in the overall elimination of bile salts.

Influence of an estrone-desulfating intestinal flora on the enterohepatic circulation of estrone-sulfate in rats

The fecal and urinary excretion of orally administered [4-14C]estrone-3-sulfate was studied in germfree (GF) rats, conventional (CV) rats and gnotobiotic rats selectively associated with estrone-desulfating and/or cecal-volume reducing microorganisms. The time required to excrete 50% of the total label recovered (t 1/2) was 22 h in CV rats vs 32 h in GF rats. Gnotobiotic rats selectively associated with a cecal volume-reducing flora (CRF rats) excreted the label even faster (t 1/2 = 13 h) than CV rats. Association of GF rats as well as CRF rats with estrone-desulfating microorganisms (termed S1 + S2 + R9 rats and CRF + S1 + S2 + R9 rats, respectively) led to a slower excretion of labeled products (t 1/2 = 38 h in S1 + S2 + R9 rats and t 1/2 = 27 h in CFR + S1 + S2 + R9 rats). Intestinal microbial desulfation also increased the relative part of the urinary excretion from 4% in GF rats to 8% in S1 + S2 + R9 rats and from 3% in CRF rats to 9% in CFR + S1 + S2 + R9 rats. We conclude that intestinal microbial desulfation enhances the enterohepatic circulation of orally administered estrone-3-sulfate.

A further study of the bile acids in infants with coprostanic acidemia

The structure of the bile acids in serum of infants with coprostanic acidemia was further investigated. The identity of 3 alpha-hydroxy-5 beta-cholestan-26-oic acid and 3 beta-hydroxy-5-cholesten-26-oic acid was confirmed. The biosynthesis of the 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-C29 dicarboxylic bile acid does not start from beta-sitosterol.

Synthesis and 29-14C-labeling of 3α,7α,12α-trihydroxy-27-carboxymethyl-5β-cholestan-26-oic acid. A bile acid occurring in peroxisomal diseases

The synthesis and 14C-labeling of 3 alpha, 7 alpha, 12 alpha-trihydroxy-27-carboxymethyl-5 beta-cholestan-26-oic acid by two different approaches is described. One of them involves chain elongation of cholic acid via Wittig-Horner condensation of its formylated 24-aldehyde with tetraethyl phosphonoglutarate. The resulting cholestenoate, on deprotection and hydrogenation, affords the unusual C29 bile acid in good yield. An alternative procedure consists in a malonic ester synthesis starting from the formylated 24-alcohol which, after conversion into a mesylate, is reacted with sodium salt of 2-carboethoxy-gamma-butyrolactone. Alkaline hydrolysis, decarboxylation, esterification with diazomethane and selective tosylation of the newly introduced primary hydroxyl function give a C28 precursor, which is easily chain-elongated into a labeled or unlabeled C29 bile acid by reaction with cyanide and hydrolysis. Due to the easy lactonization of some of the C28 intermediates, the latter method provides a better way for introducing a C-29 label than the sequence usually employed for carboxyl labeling of bile acids and consisting in a decarboxylative halogenation of the parent acid followed by substitution of the norhalogenide with [14C]cyanide and hydrolysis. The structure of the synthesized acid or its dimethyl ester is confirmed by 13C nuclear magnetic resonance spectroscopy and mass spectrometry, and is also shown by gas liquid chromatography to be identified with an authentic sample of biosynthetic C29 dioic bile acid extracted from body fluids of Zellweger patients.