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Effect of saccharin ingestion on stool composition in relation to caecal enlargement and increased stool hydration.

In an attempt to determine why sodium saccharin feeding causes caecal enlargement (Anderson & Kirkland, Fd Cosmet. Toxicol. 1980, 18, 353) and increased stool hydration (Anderson, Fd Cosmet. Toxicol. 1979, 17, 195), stools from rats fed diets containing 0, 1, 3, 5 or 7.5% sodium saccharin were analysed. Saccharin ingestion resulted in a small increase in stool ash but no change in lipid or non-saccharin nitrogen concentrations (mg/g dry stool). Saccharin treatment also resulted in a dose-dependent increase in the stool content of carbohydrate soluble in 1 N-NaOH. The results have led to the hypothesis that saccharin feeding results in a dose-dependent increase in the stools in the content of hygroscopic polysaccharides which may be derived from the diet or synthesized by an intestinal microorganism(s). The polysaccharide in conjunction with the high stool saccharin content causes caecal enlargement and increased stool hydration. A possible relationship between this effect of saccharin and urinary-tract toxicity is suggested.

Animals↗

Gas chromatographic profiling of phenolic acids in urine of patients with cirrhosis of the liver.

Phenolic acids are analysed within the profile of the organic acids in urine of patients with cirrhosis. For the following constituents an increased urinary excretion is observed: 4-hydroxyphenylpyruvic acid, 4-hydroxyphenyllactic acid, 4-hydroxyphenylacetic acid, 4-hydroxybenzoic acid, 4-hydroxyhippuric acid, vanillic acid, homovanillic acid, 4-hydroxymandelic acid, 4-hydroxy-3-methoxyphenylpropionic acid and p-cresol. The phenols are metabolites of tyrosine and are produced in the liver, in extrahepatic tissues and by intestinal microorganisms. They are suggested as biochemical control parameters for the metabolizing function of the liver, for the effect of therapy and for the existence of portal-systemic venous collaterals.

Chromatography, Gas↗

Structural identification of two metabolites of catechins and their kinetics in human urine and blood after tea ingestion.

Tea is a popular beverage consumed worldwide. The metabolic fate of its major constituents, catechins, however, is not well-known. In this study, two catechin metabolites were detected in the urine and plasma of human volunteers after ingestion of green tea. These metabolites were identified by LC/ESI-MS and NMR as (-)-5-(3',4', 5'-trihydroxyphenyl)-gamma-valerolactone (M4) and (-)-5-(3', 4'-dihydroxyphenyl)-gamma-valerolactone (M6). The renal excretion of M4 and M6 had a 3 h lag time and peaked 7.5-13.5 h after ingestion of a single dose of green tea, while (-)-epigallocatechin (EGC) and (-)-epicatechin peaked at 2 h. M4 and M6 were two major tea metabolites with urinary cumulative excretions as high as 8-25 times the levels of EGC and (-)-epicatechin in some of our subjects, and accounted for 6-39% of the amounts of ingested EGC and (-)-epicatechin. Both the metabolites appeared to be produced by intestinal microorganisms, with EGC and (-)-epicatechin as the precursors of M4 and M6, respectively. Repeated ingestion of green tea produced a slight accumulative effect of the metabolites. They were also detected in the plasma, exhibiting kinetics similar to those of the urinary metabolites, and in the feces. Study on these metabolites may help us further understand the cancer chemopreventive actions and other beneficial effects of tea.

Adult↗

On the effects of Fusarium toxin-contaminated wheat and the feed intake level on the metabolism and carry over of zearalenone in dairy cows.

The aim was to investigate the effect of feeding Fusarium toxin-contaminated wheat to dairy cows on the metabolism and carry over of zearalenone (ZON) and its metabolites at different feed intakes. Fourteen dairy cows equipped with rumen and duodenal fistulae were used. The experiment consisted of a control period in which the uncontaminated wheat was fed and a mycotoxin period in which the Fusarium toxin-contaminated wheat (8.21 mg deoxynivalenol (DON) and 91 microg ZON kg(-1) dry matter (DM)) was replaced by the control wheat (0.25 mg DON kg(-1) and 51 microg ZON kg(-1) DM). The wheat portion of the concentrate fed daily amounted to 55% on a DM basis. The ration was completed with maize and grass silage (50:50), whereby the maize silage contained 62 microg ZON kg(-1) DM. Feed intakes were adjusted to the current performance of the individual cows. The ZON metabolites alpha-zearalenol (alpha-ZOL) and beta-zearalenol (beta-ZOL) were recovered at the duodenum beside the parent toxin ZON. The recovery of ingested ZON as ZON plus alpha-ZOL plus beta-ZOL at the duodenum ranged between 19 and 247%. The portion of ZON (ranging from 29 to 99%) of the ZON plus alpha-ZOL plus beta-ZOL flow at the duodenum increased significantly with increasing ZON feed intake, whereas the portion of beta-ZOL (up to 57%) decreased significantly. In contrast, portions of ZON in faeces (32-100%), alpha-ZOL (up to 39%) and beta-ZOL (up to 43%) of ZON plus alpha-ZOL plus beta-ZOL were independent of ZON intake. It seems that a lower retention time of the feed and the toxins in the rumen as an effect of the increased feed intake may limit the ruminal metabolization of ZON. The relatively steady recovery of ingested ZON as ZON, alpha-ZOL and beta-ZOL in faeces at the different levels of ZON intake would suggest a further reduction of ZON by intestinal microorganisms. Furthermore, ZON and its metabolites in the milk were lower than the detection limits at daily ZON and DM intakes between 75 and 1125 microg and 5.6 and 20.5 kg day(-1), respectively, and milk yields (fat corrected milk, FCM) between 10 and 42 kg day(-1).

Animal Feed↗

Comment on the carcinogenic potential of di(2-ethylhexyl) phthalate.

Analysis of the carcinogen bioassay of di(2-ethylhexyl) phthalate (DEHP) has shown that the designated maximum tolerated dose was exceeded in the low- and high-dose groups of male rats, in the high-dose group of female rats, and in the low- and high-dose groups of female mice. Significant differences in tumor incidence among small populations of laboratory animals within the testing facility further confounded interpretation of the bioassay. Critical data on food consumption, nutritional status, clinical signs, clinical pathology, and intestinal microorganisms are lacking. This review concludes that because of major deficiencies in the available data, the studies cannot be interpreted as showing a carcinogenic effect due to DEHP alone. Epigenetic mechanisms to explain the biologic effects are examined.

Animals↗

RESISTANCE OF THE MOUSE'S INTESTINAL TRACT TO EXPERIMENTAL SALMONELLA INFECTION. II. FACTORS RESPONSIBLE FOR ITS LOSS FOLLOWING STREPTOMYCIN TREATMENT.

Determinations of pH, Eh, and concentrations of acetic, butyric and lactic acids were made on the content of cecum and transverse colon of groups of mice killed 1, 3, and 5 days after oral administration of 50 mg streptomycin. Control observations on untreated mice are reported in the preceding communication. Heat-killed supenatants of suspensions of bowel content were tested in vitro for their ability to inhibit multiplication of our standard streptomycin-resistant strain of Salmonella enteritidis during aerobic and anaerobic incubation. Also tested in like fashion were series of cultures in broth buffered at various pH levels and containing acetic, butyric, and lactic acids in varying concentrations. In colon content of mice on the 1st day after streptomycin treatment, the pH had risen and the concentrations of the fatty acids fallen, a combination of effects which adequately accounts for its inability to inhibit multiplication of Salmonella in vitro and in vivo. By the 3rd day after streptomycin treatment, pH and fatty acid concentrations had returned to normal levels. The susceptibility of mice to oral challenge on the 3rd day was explained by the finding that lactic acid had accumulated in colon content to levels which, in broth, effectively counteracted the activity of inhibitory concentrations of the fatty acids. Other cocarboxylic acids also antagonized the inhibitory activity of the fatty acids; glucose did not.

Acetates↗

MUCUS IN INTESTINAL CONTENTS OF GERMFREE RATS.

The fecal excretion of total nitrogen and of total hexosamines has been determined in germfree and conventional rats. Germfree rats excreted more hexosamines than the conventional rats, while no difference in the nitrogen excretion was found. Infection of the germfree rats with a normal flora resulted in a temporarily increased excretion of hexosamines and nitrogen over a period of 2 to 3 days after which they reached the level of the conventional animals. The contents of the germfree cecum contained 65 to 137 mg of hexosamines and 57 to 127 mg of nitrogen as compared to 1.2 to 5.3 and 7.4 to 23 mg in conventional animals. The high figures for hexosamines were due to an increase in the total amount of contents in the cecum and to a fivefold increase in the concentration of hexosamine-containing material. Studies on the distribution of hexosamine-containing cecal contents between sediment and supernatant after centrifugation at 20,000 g for 2 hours demonstrated that 5 to 10 per cent of the hexosamines occurred in the sediment in the germfree rats, while 75 to 85 per cent was found in this fraction in the conventional rats. The soluble part of the cecal contents in germfree as well as in the conventional rats contained 70 per cent of hexosamines in molecules with a molecular weight above approximatively 100,000 as found by gel filtration experiments on sephadex gels. The higher weight of the germfree cecal wall was reflected in a high total amount of nitrogen and hexosamines. Isolated strains of bacteria capable of reducing the cecal size in vivo did not show any capacity to degrade the mucus in vitro in a test system, where a full intestinal flora was highly active.

Animals↗

THE DEVELOPMENT OF THE BACTERIAL FLORA IN THE GASTROINTESTINAL TRACT OF MICE.

Selective culture media, and equipment for anaerobic incubation of large numbers of specimens, have been developed to facilitate the quantitative enumeration of the various aerobic and anaerobic bacterial species present in the gastrointestinal tract. The evolution of this flora has been followed in young mice from several colonies by cultivating homogenates of the different parts of the gastrointestinal tract at daily intervals from the time of birth to the time of weaning. It has been found that the lactobacilli and anaerobic streptococci become established immediately after birth and persist in large numbers, not only in the large intestine but also in the stomach and in the small intestine. In contrast, the anaerobic bacilli of the bacteroides group become established only after the 16th day; they multiply only in the large intestine but persist in this organ in very large numbers. Other bacterial species become established at different periods of time after birth, exhibit characteristic anatomic localizations, and greatly fluctuate in numbers. In general, the populations of enterobacilli and enterococci decrease precipitously after having reached a maximum level shortly after the beginning of colonization.

Animals↗

INDIGENOUS, NORMAL, AND AUTOCHTHONOUS FLORA OF THE GASTROINTESTINAL TRACT.

The bacterial flora of the gastrointestinal tract differs qualitatively and quantitatively from one colony of mice to another. Certain components of this flora, however, are always present in large and approximately constant numbers in healthy adult mice, irrespective of the colony from which the animals are derived. Lactobacilli and anaerobic streptococci are extremely numerous in the stomach, the small intestine, and the large intestine. In contrast, organisms of the bacteroides group proliferate only in the large intestine. These three bacterial species persist at approximately constant levels in their characteristic localization throughout the life span of healthy animals. They are closely associated with the walls of the digestive organs, and are probably concentrated in the mucous layer. A few experiments carried out with rats and young swine indicate that lactobacilli are also present in large numbers in the stomach of these animal species. It is suggested that some of the components of the gastrointestinal flora have become symbiotic with their hosts in the course of evolutionary development and thus constitute a true autochthonous flora. The other components of the indigenous flora are acquired early in life either through accidental contact or because they are ubiquitous in the environment. The "normal" flora is that which is always present in the environment of the animal colony under consideration.

Animals↗

Simultaneous determination of carbohydrates and products of carbohydrate metabolism in fermentation mixtures by HPLC.

An improved procedure for separating and quantitating carbohydrates, alcohols, and organic acids in fermentation mixtures metabolized by intestinal microflora is described. The high-pressure liquid chromatographic method is efficient, reproducible, and sensitive. A column packed with cation-exchange resin in the hydrogen form, eluted isocratically with 0.028 M H2SO4 at 40 degrees C separates the compounds of interest. The eluate is monitored with ultraviolet and refractive index detectors in series. On-line acquistion and storage of detector output by a computer allows post-analysis data manipulation and quantitation. Using this method, the metabolic profiles for the fermentation of glucose, fructose, lactose, and sucrose by several intestinal microorganisms are characterized and compared.

Carbohydrate Metabolism↗

Inactivation of Clostridium difficile cytotoxin by the neutrophil myeloperoxidase system.

The cytotoxin of Clostridium difficile was examined for sensitivity to oxidant secretory products of neutrophils. Exposure to myeloperoxidase, H2O2, and a halide resulted in loss of toxin activity measured by tissue-culture cytotoxicity. The peroxide requirement was provided by reagent H2O2, a peroxide-generating enzyme (glucose oxidase), or a peroxide-producing intestinal microorganism, Lactobacillus acidophilus. Human neutrophils stimulated by phorbol myristate acetate caused similar toxin inactivation. In both the cell-free and the neutrophil systems, inactivation of toxin required halides and was abrogated by azide, cyanide, or catalase. Neutrophils from patients with lack of myeloperoxidase or failure to produce H2O2 were impaired in toxin inactivation unless myeloperoxidase or H2O2, respectively, was added. The reducing agent 2-mercaptoethanol enhanced toxin activity. These data suggest a similarity between C difficile cytotoxin and the classic thiol-activated cytolysins. Moreover, they raise the possibility that neutrophils are involved in oxidative detoxification of microbial products.

Bacterial Proteins↗

Some nutritional effects of folate-binding protein in bovine milk on the bioavailability of folate to rats.

The excretions of folate compounds into both the urine and bile were investigated in rats after the administration of pteroylglutamic acid (PteGlu) with or without the folate-binding protein (FBP) prepared from bovine milk. When the sample solution, containing either free or bound [3H]PteGlu (i.e., bound to the FBP from milk), was delivered to rats intragastrically via oral intubation, the amounts of [3H]PteGlu excreted into the feces did not change. On the other hand, the urinary excretion of 3H-labeled folate compounds, especially [3H]5-methyltetrahydrofolic acid (5-CH3-H4PteGlu), after the administration of bound [3H]PteGlu was significantly lower (P less than 0.01) than that after the administration of free [3H]PteGlu. The urinary excretion of [3H]5-CH3-H4PteGlu was directly proportional to the initial amount of free [3H]PteGlu administered. The similar effect of FBP was also observed when the biliary excretion of 3H-labeled folate compounds was investigated in situ. Furthermore, the incorporation of [3H]PteGlu into folate-requiring intestinal microorganisms was considerably reduced when it was bound to FBP. These results suggest that milk FBP has some nutritional effects on the bioavailability of folate in vivo.

Animals↗