H. Eyssen

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.

Interdigestive myoelectric complex in germ-free rats

The effect of intestinal bacteria on the interdigestive myoelectric complex (IDMEC) was studied by recordings of the electrical activity of the proximal and distal small bowel in fasting germ-free (n=6), gnotobiotic (n=7), and conventional (n=6) rats in vivo. Germ-free and gnotobiotic rats were operated and studied in germ-free or gnotobiotic conditions. The IDMEC period (mean±sd) in the proximal and distal small bowel was significantly longer in germ-free rats (20±2 min and 102±14 min) as compared to conventional rats (13±1 min and 58±5 min). Association of germ-free rats with a limited flora (gnotobiotic rats) decreased the IDMEC period significantly (15± 1.5 min and 75±14 min). The migration velocity of the IDMEC was inversely related to the IDMEC period. These observations confirm previous data suggesting that the interdigestive motor complex has a role in the homeostasis of the bacterial content of the small bowel.

Infantile Refsum's disease: Biochemical findings suggesting multiple peroxisomal dysfunction

Infantile Refsums disease was diagnosed in three male patients, presenting with facial dysmorphia, retinitis pigmentosa, neurosensory hearing loss, hepatomegaly, osteopenia and delayed growth and psychomotor development.An elevated plasma phytanic acid concentration and a deficient phytanic acid oxidase activity in fibroblasts were found with an accumulation of very long chain fatty acids in plasma and fibroblasts. There were elevated pipecolic acid levels in plasma, urine and CSF, and abnormal bile acid metabolites in plasma.Deficient activity of acylCoA: dihydroxyacetone phosphate acyl transferase was found in thrombocytes and fibroblasts of these patients as well as an impairedde novo plasmalogen biosynthesis in fibroblasts. These biochemical abnormalities, previously described in the Zellweger syndrome, suggest multiple peroxisomal dysfunction in our patients.

IIeal dysfunction and bacterial overgrowth in patients with Crohn's disease

Abstract. The intestinal bile acid metabolism was studied in sixty‐one patients with non‐operated Crohns disease (twenty‐seven ileitis and thirty‐four ileocolitis patients) by means of the 14C‐glycocholate breath test with marker‐corrected faecal analysis before and after a short course of antibiotics. The results of the combined breath and faecal analysis were compared with the data of other tests detecting bacterial overgrowth and ileal dysfunction. Fifteen of the sixty‐one patients (25%) presented with a 14C excretion pattern consistent with bacterial overgrowth of the small bowel. Repetition of the combined breath and faecal analysis after antibiotic treatment revealed that concurrent ileal dysfunction was present in at least six of these fifteen patients. In twenty other patients elevated marker‐corrected 14C faecal excretion indicated ileal dysfunction. Thus, the overall incidence of ileal dysfunction amounted to 26/61 (44%). The sensitivity of the bile acid breath test with marker‐corrected stool analysis was comparable to that of aerobic and anaerobic jejunal cultures in twenty non‐selected patients for the detection of bacterial overgrowth, and to that of chemical bile acid measurement in stools for the detection of ileal dysfunction. The bile acid breath test with faecal analysis was more sensitive than measurement of glycine‐taurine ratio in bile (twenty patients) and the Schilling test.

Non-infectious intracisternal A-type particles in a sarcoma-positive, leukemia-negative mouse cell line transformed by murine sarcoma virus (MSV)

Cell lines derived from C3H mouse embryos are described. Untransformed MO-cells were found susceptible to transformation by Kirsten MSV. Two types of transformed cell lines were isolated. MO-P-cells produced infectious MSV and contained extracellular C-type particles. MO4-cells did not release infectious focus forming virus; however, upon superinfection with murine leukemia viruses (MLV), infectious MSV was released. Not superinfected MO4-cells contained multiple intracisternal A-type particles. In this respect these cells differ from the MSV genome-carrying NP-cells (1) which do not produce particles nor viral antigen, and from S+L−-cells (2) which release extracellular, non-infectious C-type particles.

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.

Formation and regeneration of Penicillium chrysogenum protoplasts

Abstract1.High yields of protoplasts from Penicillium chrysogenum Wisc. 49,2105 were obtained by using a combined enzyme system containing either Cellulase from Oxoporus plus an extract of Helix pomatia gut juice, or cellulase plus an enzyme preparation from the culture filtrate of Streptomyces graminofaciens ATCC 12,705. When 15 h old mycelium was incubated with one of these enzyme systems in 0.55 M NaCl at pH 5.6 nearly all of the mycelium was transformed into protoplasts within 4–5 h.2.The process of protoplast regeneration was studied both on solid and in liquid media. Approximately 50% of the protoplasts regenerated within 8–10 h. Addition of yeast extract to the medium accelerated the speed of regeneration. Microscopically, 3 patterns were seen by which protoplasts could regenerate to normal mycelium.

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.

The deficient degradation of synthetic 2- and 3-methyl-branched fatty acids in fibroblasts from patients with peroxisomal disorders

SummaryThe oxidation of pristanic and phytanic acids by human skin fibroblasts was compared to that of their synthetic analogues, 2-methylpalmitic and 3-methylmargaric acids. The synthetic compounds and natural substrates were degraded at comparable rates in control and X-linked adrenoleukodystrophy fibroblasts. The α-decarboxylation of 3-methylmargaric acid, similarly to that of phytanic acid, was affected in Refsum disease and Zellweger syndrome, but not in X-linked adrenoleukodystrophy. The β-oxidation of 2-methylpalmitic acid, similarly to that of pristanic acid, was deficient in fibroblasts derived from patients suffering from Zellweger syndrome, confirming the importance of peroxisomes in the breakdown of 2-methyl-branched fatty acids. No deficiency was observed in fibroblasts from X-linked adrenoleukodystrophy patients. The 1-14C-labelled 2- and 3-methyl-branched fatty acids, which are easier to synthesize that the natural analogues, are therefore valuable tools for the diagnosis of human peroxisomal disorders.