Effects of some poorly digestible carbohydrates on bile acid bacterial transformations in the rat.

The effects of ingestion of poorly digestible carbohydrates on bacterial transformations of cholic acid and beta-muricholic acid were studied in rats fed on increasing levels of lactose, lactulose, amylomaize or potato starches. Each level was given for 3 weeks and, at the end of each dietary treatment, bile acid faecal composition was analysed and a group of six rats was killed every 4 h during 24 h to determine the amounts of fermented carbohydrate and fermentation characteristics (caecal pH, volatile fatty acids (VFA) and lactic acid concentrations). Fermentation of carbohydrates decreased caecal pH and enhanced caecal VFA and lactic acid concentrations. Irrespective of the poorly digestible carbohydrate, the variation of bacterial transformation always occurred in the same way: the bacterial transformation of beta-muricholic acid into hyodeoxycholic acid was the first to disappear, while omega-muricholic acid formation increased; second, cholic acid transformation decreased and finally all bile acid transformations were strongly affected. There was a significant correlation between bile acid transfer and the minimal caecal pH in vivo. This effect of pH was similar in vitro. To determine whether the levels of bacteria which transformed bile acids were modified, rats fed on the highest amounts of poorly digestible carbohydrates were introduced into isolators and carbohydrate feeding was stopped. Caecal pH recovered its initial value but bile acid transformations remained changed, suggesting that the intestinal microflora were modified by ingestion of fermentable carbohydrates.

Effective glucuronidation of 6 alpha-hydroxylated bile acids by human hepatic and renal microsomes.

The glucuronidation of bile acids is an established metabolic pathway in different human organs. The hepatic and renal UDP-glucuronyltransferase activities vary according to the bile acids concerned. Thus, hyodeoxycholic acid is clearly differentiated from other bile acids by its high rate of glucuronidation and elevated urinary excretion in man. To determine whether such in vivo observations are related to variations in bile acid structure, human hepatic and renal microsomes were prepared and time courses of bile acid glucuronidation measured with the bile acids possessing hydroxyl groups in different positions. Eleven [24-14C]bile acids were chosen or synthesized in respect of their specific combination of hydroxyl and oxo groups at the 3, 6, 7 and 12 positions and of their alpha or beta hydroxyl configurations. The results clearly demonstrate that bile acids with an hydroxyl group in the 6 alpha position underwent a high degree of glucuronidation. Apparent kinetic Km and Vmax values for UDP-glucuronyltransferase activities ranged over 78-66 microM and 1.8-3.3 nmol.min-1.mg-1 protein in the liver and over 190-19 microM and 0.5-9.2 nmol.min-1.mg-1 protein in the kidney. All the other bile acids tested, each of which possessed a 3 alpha-hydroxyl group and whose second or third hydroxyl was bound at the 6 beta, 7 or 12 positions, were glucuronidated to a degree far below that of the 6 alpha-hydroxylated bile acids. We conclude that an active and highly specific UDP-glucuronyltransferase activity for 6 alpha-hydroxylated bile acids exists in human liver and kidneys. Moreover, this activity results in the linkage of glucuronic acid to the 6 alpha-hydroxyl group and not to the usual 3 alpha-hydroxyl group of bile acids.

beta-Muricholic acid; potentiometric and cholesterol-dissolving properties.

Some physicochemical properties of beta-muricholic acid (3 alpha,6 beta,7 beta-trihydroxy-5 beta-cholanic acid), a major bile acid biosynthesized by rat liver, were determined and compared to those of ursodeoxycholic and chenodeoxycholic acids. From potentiometric studies, the following characteristics of beta-muricholic acid were shown: a low monomer solubility (13 microM), a high equilibrium precipitation pH (7.92 for 30 mM solution), an apparent critical micellar concentration of 4 mM, and a very low micellar capacity of the bile salt to dissolve the protonated bile acid. Sodium beta-muricholate solution (30 mM) poorly solubilized cholesterol, as indicated by a bile salt/cholesterol molar ratio of 1430, whereas saturation ratios obtained with chenodeoxycholate and ursoseoxycholate were 24 and 384, respectively. Sodium beta-muricholate (30 mM)/phosphatidylcholine/cholesterol mixtures contained non-micellar aggregates from very low cholesterol concentrations. At physiological phosphatidylcholine concentrations, sodium beta-muricholate (100 mM) dissolved cholesterol crystals via essentially lamellar liquid-crystal formation. These solubilizing properties might have important physiological relevance to the dissolution of cholesterol gallstones in man.

Effects of amylomaize starch on mineral metabolism in the adult rat: role of the microflora.

The effects of amylomaize starch on rat mineral metabolism were studied. To elucidate the role of bacterial fermentation, the effects of amylomaize starch were compared in germfree (GF) and conventional (CV) rats. A purified diet, sterilized by irradiation and containing either maize starch (M) or amylomaize starch (A), was fed to the rats. Feed intake was similar in all groups of rats except GF rats fed amylomaize starch ad libitum, whose feed intake was higher. One group of GF rats received amylomaize starch in restricted amounts (pair-fed rats). A balance study of energy, nitrogen and minerals (Ca, P, Mg, Na, K, Fe, Mn, Zn and Cu) was performed. Apparent absorption of Mg, P, Na, K and Zn (expressed as a percentage of the amount ingested) was lower in GF rats fed amylomaize starch than in GF rats fed maize starch. Since feed intake was higher in GF rats fed amylomaize starch ad libitum than in rats fed maize starch, mineral absorption (expressed in milligrams per day) was not reduced in rats fed ad libitum, whereas it was reduced in pair-fed rats. Mineral retention was similar in GF rats fed ad libitum, but was reduced in pair-fed rats. In CV rats fed amylomaize starch, cecal weight and cecal volatile fatty acids concentration were higher than those in CV rats fed maize starch. Apparent absorption of N and P was lower and that of Ca, Mg and Fe was higher in CV rats fed amylomaize starch than in rats fed maize starch.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucuronidation of bile acids in human liver, intestine and kidney. An in vitro study on hyodeoxycholic acid.

The activities of UDP-glucuronyl transferase(s) in homogenates and microsomal preparations of human liver, kidney and intestine were tested with hyodeoxycholic acid (HDC). The various kinetic parameters of the UDC-glucuronidation were determined from time course experiments. In both liver and kidney preparations, HDC underwent a very active metabolic transformation: liver Km = 78 microM, Vmax = 3.3 nmol . min-1 . mg-1 protein; kidney Km = 186 microM, Vmax = 9.9 nmol . min-1 . mg-1 protein. To our knowledge this is the first observation of both an extensive and comparable bile acid glucuronidation occurring in renal and hepatic tissues.

Metabolism of beta-muricholic acid in man.

Labeled beta-muricholic acid was obtained from germfree rats given [24-14C]-chenodeoxycholic acid. It was crystallized with the same unlabeled bile acid extracted from germfree rat pooled biles. Five patients fitted with a T-tube after cholecystectomy were given orally 100 mg of the bile acid. Metabolites of beta-muricholic acid in bile, urine and feces were studied. Glyco- and tauro-beta-muricholic acid were the only metabolites detected in bile. The urinary bile acid pattern was complex and included free, glyco- and sulfoconjugated beta-muricholic acid, but no glucuronide was observed. Analysis of fecal bile acid showed very few metabolites: the 3 beta-epimer was identified; the 6 beta- and 7 beta-hydroxyls were apparently not transformed by human intestinal microflora.

beta-Glucuronidase activities of intestinal bacteria determined both in vitro and in vivo in gnotobiotic rats.

The beta-glucuronidase activities of bacterial strains isolated from the rat intestinal tract were studied both in vitro in culture media and in vivo in the intestinal contents of gnotobiotic rats. Only 50 of 407 strains tested were found to be positive in vitro. They belonged to the three genera Clostridium, Peptostreptococcus, and Staphylococcus. The in vitro-negative strains were also negative in vivo. The beta-glucuronidase activities of the beta-glucuronidase activities of the positive strains were generally greater in vivo than in vitro. The highest in vivo activities were found in the intact bacterial cells and in the soluble fractions prepared from disrupted pellets. There was a discrepancy between the activities obtained from both conventional and gnotobiotic rats harboring selected positive strains, suggesting that the main beta-glucuronidase-positive strains have not yet been isolated from the intestines of conventional rats.

Increase in the ileal absorption rate of sodium taurocholate in germ-free or conventional rats given an amylomaize-starch diet.

Twenty germ-free and twenty conventional male Fischer 344 rats, 3 months old, were fed ad lib. diets based either on normal or on amylomaize starch for 1 month. The absorption rate of sodium taurocholate, 25 mmol/l, was determined in vivo in jejunal or ileal segments. Each determination included five rats. Jejunal absorption rate was not modified either by the amylomaize-based diet or by the digestive microflora. Ileal absorption rate was slightly higher in germ-free than in conventional rats. Ileal absorption rate was largely increased by the amylomaize-based diet. This increase was slightly larger in germ-free rats than in conventional rats. It is suggested that dietary amylomaize starch has an action on the active absorption process of bile acid.

Dietary fiber and cholesterol and bile acid metabolism in axenic (germfree) and holoxenic (conventional) rats. III. Effect of non-sterilized pectin.

Axenic (germfree) and holoxenic (conventional) rats were given a pectin-containing diet (5%) or a pectin-free diet. The diet was not sterilized and the axenic state was maintained by the addition of antibiotics. Pectin modified little or not at all the quantity of bile acids in the small intestine, fecal bile acid excretion and fecal elimination of the sum of cholesterol and bile acids. It similarly decreased the concentration of liver cholesterol and the amount of bile acids in the cecum and large intestine of both germfree and conventional rats. Pectin decreased the absorption coefficient of dietary cholesterol and its plasma concentration and increased fecal cholesterol elimination in conventional rats but not in germfree animals. The data imply that some of the actions of pectin depend on the presence of the intestinal flora but the exact nature of this relationship is unclear.

Absence of transformation of beta-muricholic acid by human microflora implanted in the digestive tracts of germfree male rats.

Germfree rats biosynthetize cholic and beta-muricholic acids. The latter does not exist in humans. Germfree rats were given human fecal suspensions. These rats degraded cholic acid into deoxycholic acid but failed to metabolize beta-muricholic acid.

Effects of Maillard's reaction products on apparent mineral absorption in different parts of the digestive tract. The role of microflora.

1.5 and 3% of Maillard's reaction products (MRP), prepared by heating glycine and glucose, were incorporated ino a semi-synthetic diet. The diet was sterilized by irradiation. MRP caused a change in apparent absorption of calcium and magnesium in germfree (GF) and conventional (CV) rats. The absorption of these minerals was reduced in the small intestine of CV and GF, perhaps due to the action of MRP on enterocyte metabolism. This effect was compensated for only in CV rats by increased absorption of these minerals in the cecum and the colon. The presence of microflora and the increase in cecal volume in CV rats suggested that MRP fermentation increased mineral absorption in the cecum and colon. However, although total apparent absorption of magnesium and phosphorus was higher in CV rats ingesting MRP, the urinary excretion of these minerals was also higher and their retention was not better. The effect of MRP in GF rats was not compensated for in the cecum, and mineral retention was significantly reduced in GF rats ingesting MRP.

Effects of microflora on the dimensions of enterocyte microvilli in the rat.

The length and diameter of enterocyte microvilli at mid-villus position were measured on electron-micrographs. The duodenum, jejunum and ileum of axenic (germfree) and holoxenic (conventional) inbred rats fed the same diet have been studied. The microvilli were significantly shorter in all these intestinal regions when the microflora was present. The decrease in microvillus length (due to the presence of microflora), expressed as a percentage of the length in axenic rat, was 5% in the duodenum, 9% in the jejunum and 18% in the ileum. This was not true of microvillus diameter where only the values found for the ileum of axenic rat were significantly lower, the others not differing among themselves. In conclusion, the presence of a total microflora led to a decrease in the length of enterocyte microvilli. These results have been discussed in relation with studies on renewal and enzyme activity in the intestinal epithelium.

Effect of wheat bran upon gastro-intestinal transit in germ-free and conventional rats.

Germ-free and conventional rats were fed a semi-synthetic diet including 10% wheat bran for 1 month. They were compared to similar rats fed the same diet without bran. Transit of polyethylene glycol (PEG) 4000 was studied in giving the marker either in saline or in the diet. The presence of a bacterial flora was found to accelerate small intestine transit and total transit. The ingestion of solid food slowed gastric emptying but had little effect on small intestine transit. Wheat bran slowed gastric emptying of PEG in most of the experimental groups. It had no significant effect on small intestine transit except in germ-free rats having received the marker in solid food. It accelerated total transit in germ-free and conventional rats. The results suggest that bran mainly exerts its action in the hindgut. The role of microflora in this action of bran is discussed.

Intestinal absorption, excretion, and biotransformation of hyodeoxycholic acid in man.

Five patients fitted with a biliary T-tube after cholecystectomy were given orally a tracer dose of [14C]hyodeoxycholic acid and 500 mg of the same unlabeled acid. Intestinal absorption and biotransformation, liver metabolism, bile secretion, fecal and urinary excretions of this acid or of its metabolites were studied. Hyodeoxycholic acid was well absorbed by the human intestine. It was not subjected to intestinal transformations and, particularly, did not produce a significant amount of lithocholic acid, which does not support the existence of intestinal bacterial 6 alpha-dehydroxylases. The percentage of hyodeoxycholic acid and of its metabolites recovered in bile varied from 11.5 to 31%. Two major metabolites were isolated from bile: glycohyodeoxycholic acid and hyodeoxycholic acid glucuronide. Analysis of urinary bile acids showed that a large proportion (30-84%) of the administered hyodeoxycholic acid was excreted by the kidney as a glucuronide. The large extent of both glucuronidation and urinary excretion of hyodeoxycholic acid is a unique example of bile acid metabolism and excretion in man.

[Characterization of the antigen in circulating immune complexes and application to the detection of streptococcal antigens in human infectious endocarditis]. / Caractérisation de l'antigène des complexes immuns circulants et application à la mise en évidence d'antigènes streptococciques lors des endocardites infectieuses humaines.

Immune complexes from patients with subacute endocarditis were used to develop a methodology for characterization of the antigen involved in human circulating immune complexes. This model was chosen because it permits isolation of the causative agent of the streptococcal infection thought to contain the antigen present in the immune complex. A comparison was made between two methods for characterization of bacterial antigen bound to antibody. In the first, animals were immunized with purified immune complexes and the production of antistreptococcal antibodies was investigated. In the second method, a sandwich ELISA was developed, implying the double specificity of the immune complex (human immunoglobulin and streptococcal antigen). The latter method gave the best results, detecting antigen in the immune complexes of 8 out of 10 patients' sera. The application of this method to other immunopathological situations is discussed.

Effect of microflora and lactose on the absorption of calcium, phosphorus and magnesium in the hindgut of the rat.

For 4 weeks, 3-month old germfree (GF) and conventional (CV) rats were given a semi-synthetic diet sterilized by irradiation with or without 10% of lactose. During the 5th week, 0.2% of titanium oxide (TiO2) was added to the diet and the rats were killed at regular intervals throughout the light/dark cycle. The patterns of TiO2 and 45Ca excretion were similar, indicating that TiO2 was a good marker of unabsorbed calcium transit. The apparent absorption coefficient of calcium, magnesium and phosphorus was determined in the ileum, caecum, large intestine and faeces by the mineral/TiO2 ratio. The effects of microflora and lactose varied with the mineral and the digestive tract level studied. --In the small intestine, microflora had no effect on the apparent absorption of calcium and magnesium but did have an unfavorable influence on phosphorus absorption. Lactose increased calcium and magnesium absorption, and this increase was similar in GF and CV rats, but lactose had a favorable effect on phosphorus absorption only in CV rats. --In the caecum, microflora had an unfavorable effect on the apparent absorption of calcium and magnesium and a favorable effect on phosphorus absorption. The ingestion of lactose reduced calcium and magnesium absorption in the caecum of GF rats and phosphorus absorption in the caecum of CV animals. --In the colon, mineral absorption was not significant in either CV or GF rats receiving the lactose-free diets. Lactose ingestion caused the absorption of calcium, magnesium and phosphorus to rise significantly only in GF rats. This absorption contributed to the stronger effect of lactose on total calcium and phosphorus absorption in GF rats.

[Biotin deficiency in the germ-free rat and propionic acidemia]. / Carence en biotine chez le rat axénique et acidémie propionique.

Biotin deficiency was induced in germ-free rats using three experimental protocols. The results showed the important role of biotin during gestation and suckling. The earlier the deprivation, the earlier the deficiency and the severer the symptoms. In this vitamin deficiency, symptoms occurred that were not observed in the control rats, such as the formation of an intestinal volvulus in rats ingesting an L-isoleucine-supplemented diet. The main biochemical anomalies characteristic of propionic acidemia (ketoacidosis and increased urinary elimination of propionic acid) due to propionyl CoA carboxylase deficiency in man were not observed in the rats that were deprived of biotin for 200 days even after a dietary load of L-isoleucine. Only a small urinary excretion of propionyl-glycine and tiglylglycine was observed. We observed a drop in enzymatic propionyl CoA carboxylase activity in the liver that was proportional to the severity of the vitamin deficiency.

Effect of amylomaize starch on cholesterol and bile acid metabolisms in germfree (axenic) and conventional (holoxenic) rats.

Germfree and conventional rats were given a semi-synthetic diet containing either normal cornstarch or an amylomaize starch. The experimental groups thus formed were compared to assess the effects of these two types of starch and to determine if digestive tract microflora was involved in these effects. The presence of amylomaize starch decreased body growth in germfree and conventional rats, increasing food intake in the former and decreasing it in the latter. In conventionals, amylomaize starch decreased the apparent digestibility of the ration only slightly, while in germfrees it diminished apparent digestibility considerably. The cecal weight of germfree animals was not modified by amylomaize starch but that of conventional rats was increased fourfold. In both types of rat, amylomaize starch largely decreased the plasma concentration of cholesterol, largely increased the total amount of bile acids in the small intestine but slightly modified the fecal elimination of cholesterol and bile acids. It augmented the cholesterol concentration in the liver of germfrees and decreased it in conventionals while, on the contrary, it diminished the total amount of bile acids in the hind gut in the former and augmented it in the latter. This starch did not change bile acid deconjugation in conventional rats but considerably decreased other bacterial transformations of cholesterol and bile acids. Digestive tract microflora was undoubtedly involved in the action of amylomaize starch on cecal weight, ration digestibility, food intake, hepatic cholesterol concentration, the amount of bile acid in the hind gut and obviously in the transformation of cholesterol and bile acids. It did not play a role in the other effects of this starch: the strong decrease in the concentration of plasma cholesterol was the direct effect of amylomaize starch on rat metabolism.

Microbial flora in the digestive tract and action of lactose on mineral metabolism.

Germ-free (GF) and conventional (CV) adult rats were given for 4 weeks a semi-synthetic diet containing 10 p. 100 lactose (L) or no lactose (LO). The axenic state had an unfavourable effect on sodium and potassium absorption. On the contrary, it increased the absorption and retention of calcium, phosphorus and magnesium. The favourable effect of lactose on magnesium metabolism was not different in GF and CV rats. On the contrary, lactose increased the absorption and retention of phosphorus and calcium more in GF than in CV animals. It augmented the amount of calcium present in a soluble, ultrafiltrable form in the caecum of only the GF rats. These results are discussed and compared to previous data on younger rats. They lead to the hypothesis that the voluminous caecum of the GF rat is the site of a calcium absorption which is accentuated by presence of lactose in the diet.

Dietary fiber and cholesterol and bile acid metabolisms in axenic (germfree) and holoxenic (conventional) rats. I. -- Effect of wheat bran.

Axenic (GF) and holoxenic (CV) rats were given a semi-synthetic diet containing no fiber (0) or 10 p. 100 bran (B). The GFO, GFB, CVO and CVB groups were compared to assess the action of the flora and the bran. The bran diet did not change body growth, food intake or cecal water content. It caused only a slight decrease in the digestive utilization of the diet in the holoxenic rats and a decline in cecal weight in the axenic rats. The bran diet modified unabsorbed cholesterol transit in both types of rats, and slightly altered dietary cholesterol absorption which was a little lower in axenics than in holoxenics. In the former (GF), the bran-containing diet dit not change either the plasma or the hepatic cholesterol concentration. In the latter (CV), it increased plasma cholesterol which was lower in CVO rats than in the other three groups and decreased hepatic cholesterol which was thus lower in the CVB lot than in the others. The bran diet reduced fecal cholesterol elimination in axenic and holoxenic rats. This decrease was a little higher in the latter. The digestive tract microbial flora was thus implicated in the effect of bran on those characteristics of cholesterol metabolism. The bran did not change fecal bile acid composition in the axenics. In the holoxenics, it contributed to make this composition uniform because without bran, individual compositions varied. The microbial flora thus changed many aspects of bran action on cholesterol and bile acid metabolisms. But total cholesterol and bile acid elimination was only slightly affected by those two factors.