The use of rats associated with a human faecal flora as a model for studying the effects of diet on the human gut microflora

The activities of four bacterial biotransformation enzymes (β-glucosidase, β-glucuronidase, nitrate reductase and nitroreductase) were measured in the caecal contents of conventional flora rats or germ-free rats contaminated with a mixed, human faecal flora and compared with activities present in a fresh human stool preparation. Both the conventional flora rats and the rats inoculated with a human flora exhibited an enzyme profile generally similar to that of human faeces, although the conventional rat flora exhibited negligible nitrate reductase activity. The enzyme profile remained essentially unaltered in both human flora preparations following supplementation of the diet with pectin, whereas the conventional rat flora responded to this plant cell wall carbohydrate with a significant increase in nitrate reductase activity. The results demonstrate that enzymic activities of the human faecal microflora can be simulated in rats associated with a mixed population of human intestinal bacteria.     

The influence of the host on expression of intestinal microbial enzyme activities involved in metabolism of foreign compounds

The activities of four enzymes (β-glucosidase, β-glucuronidase, nitrate reductase and nitroreductase) in selected intestinal bacteria ( Escherichia coli, Clostridium sp., Streptococcus sp., Bacteroides sp. and Lactobacillus salivarius ) were measured after growth in vitro and in vivo . The five strains differed in their activites with Clostridium sp. being the most active for β-gjucosidase, β-glucuronidase and nitroreductase, and E. coli the most active producer of nitrate reductase. Enzyme activity in vivo tended to be higher than in vitro but there were instances where the comparative activities were reversed.     

Developmental changes to gut microflora metabolism in mice

W.E. BRENNAN-CRADDOCK, A.K. MALLETT, I.R. ROWLAND AND S. NEALE. 1992. Developmental changes in the activities of bacterial nitrate reductase, nitroreductase and β-glucuronidase and their response to fermentable dietary fibre, were investigated in caecal contents from suckling mice (2-week-old) and in mice aged 4–24 weeks fed either a purified fibre-free diet or that diet supplemented with 5% (w/w) pectin. There was no apparent age-related trend common to the three enzymes studied. Nitrate reductase activity in the mice fed the fibre-free diet did not markedly alter with age. Pectin administration, however, was associated with a significant increase in nitrate reductase activity, particularly in 4-week-old mice. Nitroreductase activity exhibited an overall upward trend in mice from 2 to 12 weeks and thereafter decreased. Caecal β-glucuronidase activity in mice increased sharply between 2 weeks and 4 weeks of age, thereafter not changing significantly until the 24th week. Pectin feeding had no consistent effect on activities either of nitroreductase or β-glucuronidase. The changes in enzyme activities with age were not related to the concentration of bacteria in the caecum, which was highest in the 2-week-old mice.
We conclude that the weaning is a period in which marked changes in caecal bacterial enzyme activities can occur.     

Histochemical changes in the fowl small intestine associated with enhanced absorption after feed restriction

Summary The morphology and histochemical enzyme pattern of the small intestine were investigated in chicks undergoing feed restriction. Corresponding intestinal sites were compared in both restricted birds and in control birds under normal feeding. Intestines from the restricted birds showed some atrophy, the villi being slightly shorter and thinner than normal after eight days restriction, and there was an increase in the activity of alkaline phosphatase, leucine naphthylamidase, acid phosphatase, -glucuronidase, non-specific esterase and succinic dehydrogenase in the absorptive cells.The significance of these findings has been discussed in relation to the enhanced absorptive capacity of the intestine during feed restriction and its similarity to other dietary stress factors that produce enhanced absorption. Possible mechanisms for the production of such mucosal changes have been considered. It was concluded that the enhanced absorption of nutrients in semi-starved animals is correlated with increased mucosal enzyme activities.     

The influence of incubation pH on the activity of rat and human gut flora enzymes

The activities of three bacterial biotransformation enzymes (β-glucuronidase, β-glucosidase, nitrate reductase) were determined in suspensions of rat caecal contents or human faeces over the pH range 6–8. All three enzymes were influenced by pH, as exemplified by β-glucosidase activity which diminished as pH increased. In other instances the rat and human flora showed distinct profiles, with nitrate reductase activity undetectable in human faeces below pH 6–6, whereas the rat caecal flora displayed optimal reduction of nitrate around neutrality. The most pronounced host-species difference was found with β-glucuronidase, which showed maximal activity at pH 6–0 in human faecal bacteria, while the rat caecal flora expressed greatest activity at pH 8–0. All three enzyme activities were associated with that fraction of rat caecal or human faecal material sedimented by centrifugation at 5000 g for 15 min, with little or no metabolism occurring in the 11000 g supernatant fluid. The results demonstrate that pH has a pronounced effect on the enzymic activity of bacterial preparations from rat and human sources.     

Low β-glucuronidase enzyme activity and mutations in the human β-glucuronidase gene in mild mucopolysaccharidosis type VII, pseudodeficiency and a heterozygote

Deficiency of β-glucuronidase is the cause of the human lysosomal storage disorder mucopolysaccharidosis type VII (MPS VII). The wide interfamilial variation in the presentation of this disorder complicates clinical diagnosis. Since greatly reduced β-glucuronidase enzyme activity may also be found in healthy individuals (pseudodeficiency), diagnosis based on the biochemical phenotype is also difficult. This is illustrated by the patients studied here, who had extremely mild symptoms confined to the spine, or tachycardia, or upper respiratory infection, and who had low β-glucuronidase activity, and excessive granulation of granulocytes and monocytes on routine blood smears. Low enzyme activity was caused by mutations in the β-glucuronidase gene in all cases. One patient was homozygous for the previously described D152N allele. Family information and ³⁵SO₄-uptake studies clearly demonstrated that he was pseudodeficient, with symptoms unrelated to his low β-glucuronidase activity. Two patients of another family were compound heterozygotes for a C38G and a Y626H allele, and were probably extremely mild MPS VII patients. The low β-glucuronidase activity in another mild MPS VII patient was due to reduced biosynthesis of stable mRNA from one allele, and a W446X mutation on the second. Extremely low β-glucuronidase enzyme activity was also found in the serum of a carrier of a 1801ΔT allele, possibly as a consequence of a dominant-negative effect. A combination of investigations is necessary in order to differentiate between mild disease and pseudodeficiency in individuals with enzyme activities close to the threshold. Received: 31 May 1997 / Accepted: 26 Augsut 1997     

Effects of dietary fructan on cecal enzyme activities in rats

In this study, we have attempted to determine the effects of dietary fructose polymers (fructan), high molecular-weight β-(2,6)-linked levan, and low-molecular-weight β-(2,1)-linked inulin, on two intestinal enzymes (β-glucuronidase and β-glucosidase). As a preliminary experiment, when intestinal microflora were cultured in anaerobic media harboring levan or its oligosaccharides, bacterial cell growth was observed in the levanoligosaccharide-supplemented media, but not in the levan-supplemented media, indicating that levan’s size is important for the utilization by intestinal bacteria of levan as an energy source. In our animal study, the intake of a levan-rich diet was determined to significantly attenuate the activity of the harmful enzyme β-glucuronidase, but did not affect the activity of β-glucosidase.     

Hydrolysis of iodothyronine glucuronides by obligately anaerobic bacteria isolated from human faecal flora

Abstract 35 bacterial strains isolated from the human faecal flora were screened for hydrolysis of the glucuronides of 3,3',5-triiodothyronine and 3,3'-diiodothyronine. Two Gram-positive obligately anaerobic strains possessed glucuronidase activity. These strains probably belong to the genus Eubacterium , but ethanol was produced in high concentrations during glucose fermentation, which makes final classification difficult. Considering the number of bacteria in the intestinal flora (> 10⁸/ml) and the biliary excretion of iodothyronine conjugates, the strains must be able to hydrolyse a major part of the total daily intestinal supply of these iodothyronine metabolites. The study extends previous observations with faecal suspensions of human and rat origin [24]. The relevance of bacterial β-glucuronidase activity for a possible enterohepatic circulation of iodothyronines is discussed.     

Distribution of acid phosphatase and β-glucuronidase in the hypoplastic cerebellum of jaundiced Gunn rats

Summary Activities of acid phosphatase and -glucuronidase in the cerebella of young jaundiced (j/j) and non-jaundiced (j/+; control) Gunn rats were studied with the enzyme histochemical method. The cerebellum of j/+ rats showed high acid phosphatase activities in Purkinje cells and neurons in the cerebellar nuclei. In j/j rats, a number of neurons were lost and numerous microglialike cells with a high acid phosphatase activity appeared in the hypoplastic cerebellum. Although -glucuronidase activity was rarely detected in the control cerebellum, a high enzyme activity was observed associated with microglialike cells in j/j rats. The present results provide a cytological basis for the reported differential increase in the activities of these lysosomal enzymes in the j/j rat cerebellum.     

Enzymes of orithine metabolism in adult developing rat intestine

The levels of 11 enzymes, most of them involved in the metabolism of orithine, were measured in whole upper intestine, or in duodenum, small intestine and colon of adult rats. The developmental formations in small intestine of arginase, orithine aminotransferase, and orithine transcarbamylase were compared with those in liver. Changes with age (late gestation to adult) of the intestinal activities of pyrroline-5-carboxylate reductase, proline oxidase and glutamyl transpeptidase are also described.The results suggests that the proximal part of the intestine is well endowed with enzymes involved in the conversion of ornithine to proline as well as to citrulline. Fetal intestine is rich in proline oxidase and pyrroline-5-carboxylate reductase. The peak levels of ornithine aminotraferase found in intestine in the first 3 postnatal weeks were higher than seen in any other rat tissue.Some of the properties of arginase, ornithine aminotransferase and pyrroline-5-carboxylate reductase in small intestine were compared with those in liver. Isozymes of arginase in small intestine differed from those in liver; the kinetic properties of ornithine aminotransferase were similar in the two tissues. In intestine of 14-day-old rats, the orithine aminotransferase reaction was reversible, forming ornithine from pyrroline-5-carboxylate. The intestinal pyrroline-5-carboxylate reductase was cold-labile as was the hepatic enzyme in rat.     

Enzymes of ornithine metabolism in adult and developing rat intestine.

The levels of 11 enzymes, most of them involved in the metabolism of ornithine, were measured in whole upper intestine, or in duodenum, small intestine and colon of adult rats. The developmental formations in small intestine of arginase, ornithine aminotransferase, and ornithine transcarbamylase were compared with those in liver. Changes with age (late gestation of adult) of the intestinal activities of pyrroline-5-carboxylate reductase, proline oxidase and glutamyl transpeptidase are also described. The results suggest that the proximal part of the intestine is well endowed with enzymes involved in the conversion of ornithine to proline as well as to citrulline. Fetal intestine is rich in proline oxidase and pyrroline-5-carboxylate reductase. The peak levels of ornithine aminotransferase found in intestine in the first 3 postnatal weeks were higher than seen in any other rat tissue. Some of the properties of arginase, ornithine aminotransferase and pyrroline-5-carboxylate reductase in small intestine were compared with those in liver. Isozymes of arginase in small intestine differed from those in liver; the kinetic properties of ornithine aminotransferase were similar in the two tissues. In intestine of 14-day-old rats, the ornithine aminotransferase reaction was reversible, forming ornithine from pyrroline-5-carboxylate. The intestinal pyrroline-5-carboxylate reductase was cold-labile as was the hepatic enzyme in rat.     

In vitro regulation of 3-hydroxy-3-methylglutarylcoenzyme a reductase and acylcoenzyme a: Cholesterol acyltransferase activities by phoshorylation-dephosphorylation in rabbit intestine

The regulation of 3-hydroxy-3-methylglutarylcoenzyme A reductase and acylcoenzyme A: cholesterol acyltransferase activities by phosphorylation-dephosphorylation in rabbit intestine was studied in vitro. Preparing intestinal microsomes in the presence of 50 mM NaF caused a 64% decrease in the reductase activity. It had no effect on acyl-CoA: cholesterol acyltransferase activity. Microsomes that were prepared in NaF were incubated with intestinal cytosol, a partially purified phosphatase from cytosol, and Escherichia coli alkaline phosphatase. All three preparations increased 3-hydroxy-3-methylglutaryl-CoA reductase by two- or three-fold suggesting dephosphorylation and ‘reactivation’ of enzyme activity. Cytosol caused a 78% increase in acyl-CoA: cholesterol acyltransferase activity, but neither the partially purified phosphatase nor the E. coli alkaline phosphatase affected the acyltransferase activity. Microsomes incubated with increasing concentrations of MgCl₂ and ATP decreased both the activities of 3-hydroxy-3-methylglutaryl-CoA reductase and acylcoenzyme A: cholesterol acyltransferase in a step-wise fashion. Whereas this inhibitory effect was specific for reductase, the effect on acyl-CoA: cholesterol acyltransferase activity was secondary to the presence of ATP in the assay mixture. The 8500×g supernatant of intestinal whole homogenate from isolated intestinal cells or scraped mucosa was incubated with MgCl₂, ATP and NaF. In microsomes prepared from this supernatant, the activity of 3-hydroxy-3-methylglutaryl-CoA reductase was significantly decreased. Again, no change was observed in the acyltransferase activity. The rate of cholesterol esterification in isolated intestinal cells was not affected by 0.1 mM cAMP or 50 mM NaF. We conclude that under conditions which regulate 3-hydroxy-3-methylglutaryl-CoA reductase activity in rabbit intestine by phosphorylation-dephosphorylation, no regulation of acyl-CoA: cholesterol acyltransferase activity is observed.     

The effects of transgalactosylated oligosaccharides on gut flora metabolism in rats associated with a human faecal microflora

The effect on various caecal bacteria and their metabolic activities of feeding diet containing transgalactosylated oligosaccharides (TOS) with or without Bifidobacterium breve (administered in the drinking water) was investigated in rats colonized with a human faecal microflora. TOS (5% w/w in diet) or TOS plus B. breve, given for 4 weeks, induced increases in caecal concentration of total anaerobic bacteria, lactobacilli and bifidobacteria, and decreases in numbers of enterobacteria. Caecal pH was significantly reduced by feeding TOS, as were the activities of β-glucuronidase and nitrate reductase. In contrast, β-glucosidase activity was increased in TOS-fed rats.
Dietary TOS was also associated with decreased conversion, by caecal contents, of the dietary carcinogen 2-amino-3-methyl-3H-imidazo[4, 5- f ] quinoline (IQ) to its genotoxic 7-hydroxy derivative.     

In vitro regulation of 3-hydroxy-3-methylglutarylcoenzyme A reductase and acylcoenzyme A: cholesterol acyltransferase activities by phosphorylation-dephosphorylation in rabbit intestine

The regulation of 3-hydroxy-3-methylglutarylcoenzyme A reductase and acylcoenzyme A:cholesterol acyltransferase activities by phosphorylation-dephosphorylation in rabbit intestine was studied in vitro. Preparing intestinal microsomes in the presence of 50 mM NaF caused a 64% decrease in the reductase activity. It had no effect on acyl-CoA:cholesterol acyltransferase activity. Microsomes that were prepared in NaF were incubated with intestinal cytosol, a partially purified phosphatase from cytosol, and Escherichia coli alkaline phosphatase. All three preparations increased 3-hydroxy-3-methylglutaryl-CoA reductase by two- or three-fold suggesting dephosphorylation and 'reactivation' of enzyme activity. Cytosol caused a 78% increase in acyl-CoA:cholesterol acyltransferase activity, but neither the partially purified phosphatase nor the E. coli alkaline phosphatase affected the acyltransferase activity. Microsomes incubated with increasing concentrations of MgCl2 and ATP decreased both the activities of 3-hydroxy-3-methylglutaryl-CoA reductase and acylcoenzyme A:cholesterol acyltransferase in a step-wise fashion. Whereas this inhibitory effect was specific for reductase, the effect on acyl-CoA:cholesterol acyltransferase activity was secondary to the presence of ATP in the assay mixture. The 8500 X g supernatant of intestinal whole homogenate from isolated intestinal cells or scraped mucosa was incubated with MgCl2, ATP and NaF. In microsomes prepared from this supernatant, the activity of 3-hydroxy-3-methylglutaryl-CoA reductase was significantly decreased. Again, no change was observed in the acyltransferase activity. The rate of cholesterol esterification in isolated intestinal cells was not affected by 0.1 mM cAMP or 50 mM NaF. We conclude that under conditions which regulate 3-hydroxy-3-methylglutaryl-CoA reductase activity in rabbit intestine by phosphorylation-dephosphorylation, no regulation of acyl-CoA:cholesterol acyltransferase activity is observed.     

A note on lactobacilli and β-glucuronidase activity in the intestinal contents of mice

A comparison was made of caecal β-glucuronidase activity in the caecal contents of mice with or without lactobacilli as members of the normal microflora. Male mice that did not have lactobacilli as intestinal inhabitants had approximately 52% more caecal β-glucuronidase activity than their female counterparts. Colonization of male mice by lactobacilli reduced the β-glucuronidase activity to that of female mice.     

A possible mechanism for the changes in hepatic and intestinal alkaline phosphatase activities in bile-duct-ligated rats or guinea pigs

The effects of bile-duct ligation on hepatic and intestinal (jejunum) alkaline phosphatase activities were studied using rats and guinea pigs. In ligated rats, the enzyme activity was increased 4.1-fold in the liver after 24 h and 2.8-fold in the intestine after 12 h. In guinea pigs, the hepatic and intestinal enzyme activities were increased 2.3-fold and 1.5-fold after 100 and 24 h, respectively. The intestinal activity was induced sooner after ligation than hepatic activity. The induction of alkaline phosphatase was inhibited by prior treatment of animals with amanitin, an inhibitor of RNA polymerase activity. This result indicates that the induction is associated with de novo enzyme synthesis. The content of cyclic AMP in liver and intestine increased immediately after ligation. The increase in alkaline phosphatase activities was also inhibited by pretreatment with chlorpromazine, an inhibitor of adenylate cyclase activity. Hence, cellular cyclic AMP may be implicated in playing a role in the induction of alkaline phosphatase by bile-duct ligation.     

Dietary Modification of the Activation State of Intestinal 3-Hydroxy-3-methylglutaryl Coenzyme A (HMG-CoA) Reductase

To ascertain whether the phosphorylation-dephosphorylation reaction is actually involved in the in vivo regulation of intestinal 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, dietary modulation of the activation state of the enzyme was studied in isolated epithelial cells of rats. Substitution of a sucrose-enriched semipurified diet for the commercial non-purified diet caused a significant increase in jejunal activity with a concomitant decrease in ileal activity. Jejunal activity increased without influencing the activation state whereas at the early stage of dietary manipulation, there was a rapid decrease in apparent activity compared to total activity in the ileum, hence the reduction of the activation state. These observations favor the view that the phosphorylation (inactivation) reaction is responsible for the regulation of intestinal HMG-CoA reductase in vivo. In contrast, dietary fat-dependent stimulation of jejunal reductase activity was mainly attributable to an increase in enzyme protein rather than in the level of the activation. The results suggest a complex controlling feature of the cholesterol synthesis in the intestine.     

Influence of bile salts on β-glucuronidase activity of intestinal bacteria

T. FUJISAWA AND M. MORI. 1996. The β-glucuronidase activity of intact cells of Escherichia coli and Clostridium perfringens was increased in the presence of bile salts. In contrast, bile salts had inhibitory effects on the activity of β-glucuronidase extracted from the lysed cells. These results suggest that the permeability of the bacterial cells is increased by the presence of bile salts, and that bile salts may significantly enhance bacterial β-glucuronidase activity in the intestinal tract.     

Transported Enzymes in Sciatic Nerve and Sensory Ganglia of Rats Exposed to Maternal Antibodies Against Nerve Growth Factor

Abstract: The accumulations by axoplasmic transport of selected enzyme activities proximal and distal to a ligature placed on the sciatic nerve were monitored in rats exposed in utero to maternal antibodies to nerve growth factor (NGF) and in control rats. Littermates of the animals exposed to anti-NGF were shown elsewhere to have had a 70% reduction in the number of sensory neurons in dorsal root ganglia and a 90% reduction in number of neurons in superior cervical (sympathetic) ganglion. The accumulation of F^--sensitive acid phosphatase activity was depressed 75% both proximal and distal to the tie. Accumulation of F^--resistant acid phosphatase activity was depressed nearly 50% proximal to the tie. Distal accumulation of this activity did not occur in either group of rats. Accumulation of acetylcholinesterase activity was not affected. Proximal accumulation of glutamic dehydrogenase activity was depressed 30%. Distal accumulation of the activities of β-glucuronidase and hexokinase was depressed 50%. In the lumbar dorsal root ganglia, dry weight was reduced 40%, and the activities of peroxide-sensitive, F^--resistant acid phosphatase and of the mitochondrial enzymes hexokinase, glutamic dehydrogenase, glutamic-oxalacetic transaminase, and NAD-dependent isocitric dehydrogenase were all reduced a little more, 45–50% per ganglion. However, the activities of the lysosomal enzymes, F^--sensitive acid phosphatase and β-glucuronidase, of the peroxide-resistant, F^--resistant acid phosphatase, and of the mitochondrial enzyme glutaminase were all reduced about 60% per ganglion. The results of these measurements were interpreted to suggest that much, and perhaps all, of the F^--sensitive acid phosphatase activity in motion in peripheral nerve in rat is confined to sensory axons.     

Characterization of the new β-glucuronidase from <Emphasis Type="Italic">Streptococcus equi</Emphasis> subsp. <Emphasis Type="Italic">zooepidemicus</Emphasis>

Recently, a new gene encoding β-glucuronidase from Streptococcus equi subsp. zooepidemicus (SEZ) was identified and expressed in Escherichia coli. In this paper, the characterization of the enzyme is described. Specific enzyme activity was 120,000 U/mg purified protein at 37°C and pH = 7.0. The temperature and pH value, at which the enzyme has the highest specific activity, were determined and were found to be approximately 52°C and 5.6, respectively. The mutant strain SEZ glcHis was designed for the efficient isolation of β-glucuronidase from S. equi subsp. zooepidemicus. It was observed that the specific activity of β-glucuronidase in the cytoplasmic extract of a mutated strain was about 45% lower than in the cytoplasmic extract of a wild-type strain. The specific activity of purified β-glucuronidase from SEZ glcHis was four times as low as β-glucuronidase purified from E. coli. Comparing the specific activity of purified streptococcal β-glucuronidase from E. coli with E. coli β-glucuronidase (the enzyme with the highest specific activity was supplied by Sigma), the former is 1.8 higher than the latter.