Phytate hydrolysis by germfree and conventional rats.

Phytic acid is naturally occurring compound that reduces intestinal absorption of many metals. Early work suggests that some dietary phytate may be hydrolyzed in the large intestines by bacteria, but more recently nutritionists have suggested that a mucosal enzyme is responsible. This paper reports a study intended to resolve this controversy. The hydrolysis of dietary phytic acid was measured in germfree and conventional rats fed either of two diets that differed in their calcium content. Negligible phytate hydrolysis occurred in the germfree rats, whereas 22 and 56% of the phytic acid was hydrolyzed by conventional rats fed high- and low-calcium diets, respectively. We concluded that bacteria were responsible for the hydrolysis of phytate in these diets and that any activity of endogenous enzyme was negligible.     

Serum zinc in aging germ-free and conventional rats.

Zinc nutritional status appears to decline with age in humans and rodents. Since germ-free rats outlive their conventional counterparts in better health, serum Zn levels were determined in male germ-free and conventional Lobund Wistar rats in samples originating from the Lobund Aging Study. Starting at 5 months of age, germ-free rats showed significantly higher serum Zn levels than did their conventional counterparts. In conventional rats sacrificed up to 30 months of age in apparently good health, serum Zn levels showed no effect of age, while a slight but significant increase with age was observed in the germ-free rats. In healthy germ-free adults (6-24 months of age), serum Zn concentrations were approximately 25% higher than those in comparable conventional animals. In conventional rats 18-30 months of age (average, 24.5 months), sacrificed because of an obvious moribund condition, serum Zn levels were significantly lower than those in rats of the same age range (average, 24.9 months) that were obviously healthy. Results suggest that the often observed higher absorptive capacity of the germ-free gut might have contributed to higher serum Zn levels, and that a decline in serum Zn concentration with age may be a consequence, rather than a causative factor, of declining health.     

Gastrointestinal nitric oxide generation in germ-free and conventional rats

Nitric oxide (NO) is a central mediator of various physiological events in the gastrointestinal tract. The influence of the intestinal microflora for NO production in the gut is unknown. Bacteria could contribute to this production either by stimulating the mucosa to produce NO, or they could generate NO themselves. Using germ-free and conventional rats, we measured gaseous NO directly in the gastrointestinal tract and from the luminal contents using a chemiluminescence technique. Mucosal NO production was studied by using an NO synthase (NOS) inhibitor, and to evaluate microbial contribution to the NO generation, nitrate was given to the animals. In conventional rats, luminal NO differed profoundly along the gastrointestinal tract with the greatest concentrations in the stomach [>4,000 parts per billion (ppb)] and cecum (approximately 200 ppb) and lower concentrations in the small intestine and colon (< or =20 ppb). Cecal NO correlated with the levels in incubated luminal contents. NOS inhibition lowered NO levels in the colon, without affecting NO in the stomach and in the cecum. Gastric NO increased greatly after a nitrate load, proving it to be a substrate for NO generation. In germ-free rats, NO was low (< or =30 ppb) throughout the gastrointestinal tract and absent in the incubated luminal contents. NO also remained low after a nitrate load. Our results demonstrate a pivotal role of the intestinal microflora in gastrointestinal NO generation. Distinctly compartmentalized qualitative and quantitative NO levels in conventional and germ-free rats reflect complex host microbial cross talks, possibly making NO a regulator of the intestinal eco system.     

Analysis of bile acids in conventional and germfree rats.

The well-known bile acid analysis technique used by us and others (Grundy, Ahrens, and Miettinen. 1965. J. Lipid Res. 6:397-410) does not allow for the detection of hyodeoxycholic acid, a product of quantitative importance in rodent feces. Using updated methodology, it was established that hyodeoxycholic acid and omega-muricholic acid, both apparent conversion products of beta-muricholic acid, occur in apppreciable amounts in intestinal contents and feces of conventional Wistar type Lobund rats. In conventional rats, these bile acids comprise about 50% of fecal bile acids; they are not found in intestinal contents or feces of germfree rats. Others have demonstrated that hyodeoxycholic acid if formed by combined action of gut flora and liver. A new method for the separation of conjugated and free bile acids in biological samples was developed. Results with this method confirmed the total conjugation of bile acids in the germfree rat, and the almost total deconjugation that takes place in the cecum of the conventional rat.     

Gastrointestinal absorption of estrone sulfate in germfree and conventional rats

Steroids are extensively excreted in the bile of rats. There was no significant difference in biliary excretion of steroid following administration of [3H]-estrone sulfate into the proximal small intestine (PSI) of conventional (CVL; 17.8 +/- 62%; mean +/- SD) or germfree (GF; 28.2 +/- 5.3) rats. A similar finding resulted from administration into the distal small intestine (DSI)-CVL, 22.3 +/- 11.8%; GF, 11.4 +/- 3.7%. However, when the drug was given into the caecum, excretion in the bile of CVL rats after 5 h was 59.1% whereas in GF rats it was only 1.7%. When estrone was injected into the PSI and DSI of CVL and GF rats, absorption (as judged by excretion in bile) was more rapid than that seen with estrone sulfate. Five hours after injection into the PSI, biliary excretion was, in CVL 88.2% and in GF 81.7% and after injection into the DSI excretion was, in CVL 84.7% and in GF 83.6%. Absorption of estrone from the caeca of GF rats was apparently reduced (49.0% and 25.3% excreted in the bile of CVL and GF rats respectively). There was no significant difference in bile flow rate between CVL and GF rats. These results give unequivocal evidence of intact absorption of estrone sulfate from the small intestine of the rat. The rate of absorption is however very much reduced compared to the non-sulphated steroid. Estrone sulfate is not absorbed intact in the caecum but is hydrolysed by the gut microflora prior to absorption.     

Previously unrecognized virus from submaxillary glands of gnotobiotic and conventional rats

Ashe, Warren K. (National Institute of Dental Research, Bethesda, Md.), Henry W. Scherp, and Robert J. Fitzgerald. Previously unrecognized virus from submaxillary glands of gnotobiotic and conventional rats. J. Bacteriol. 90:1719-1729. 1965.-A serially transmissible cytopathic agent was isolated from histologically normal submaxillary glands, but not from various other tissues or specimens, from 74 of 97 gnotobiotic and conventional rats. Triturates of the glands or subsequent culture supernatant fluids induced specific cytopathic effects (CPE) in monolayer cultures of primary rabbit kidney cells (14 passages), a line of human skin cells (8 passages), and HeLa cells (17 passages). Transfer of supernatant fluids containing infected cells enhanced transmissibility. Neutralization of the CPE was demonstrated with sera of gnotobiotic and conventional rats and with homologous rabbit antiserum. A cold hemagglutinin specific for rabbit erythrocytes is associated with, but separable from, the infectious particle. Cultures of the agent induced no discernible effect on inoculation by various routes into suckling, weanling, or adult conventional mice, rats, and hamsters. Weanling and adult rabbits were also unaffected. Cultures for bacteria in gland extracts and infected cell supernatant fluids were uniformly negative. Negative cultures on PPLO media and negative arginine deiminase tests indicated that this agent is not a Mycoplasma. The data indicate that it is a virus whose biological and physical properties distinguish it from the known cytomegaloviruses. Because it has been found so far only in rat submaxillary glands, this agent is designated provisionally as RSMG virus.     

Strain differences in faecal tryptic activity of germ-free and conventional rats

Regardless of diet (semi-synthetic or lab chow) or strain (AGUS or SD), germ-free rats have tryptic activity in their faeces, whereas conventional rats never do. The activity in faeces from germ-free AGUS rats was significantly higher than from SD rats.     

Pre-epithelial mucus layer in the colon of conventional and germ-free rats

Summary The pre-epithelial mucus layer (PML) and epithelial mucins were studied by mucin histochemistry in 10m-thick celloidinstabilized cryostat sections in the proximal and distal colon of conventional and germ-free rats aged 120 and 350 days. No continuous PML was found in the proximal colon. A continuous mucus blanket, of fairly homogenous thickness, was observed in the distal colon, where the PML-thickness was 40±24 m at 120 days of age and 44±22 at 350 days of age in conventional rats, and 25±17m (120 days) and 22±10 (350 days) in germ-free rats. The stainability of the PML by periodic acid-Schiff and Alcian Blue at pH 2.5 and 1.0 was stronger in conventional rats than in germ-free rats, indicating higher concentrations of mucosubstances and of acid and sulphated mucins, respectively. The PML of the conventional rat distal colon showed a stratified structure of up to eight sublayers. In the distal colon of germ-free rats, the whole gut wall thickness was reduced 47% compared to the conventional rat (germ-free: 185±73m, conventional: 350±115m). No stratification of the PML was observed. The presence of intestinal microflora obviously had a strong influence on the thickness, compactness, mucin content, mucin composition and structure of the pre-epithelial mucus layer.     

Urinary profiles of volatile and acid metabolites in germfree and conventional rats

Qualitative and quantitative differences in the urinary excretion of volatile and acidic metabolites in germfree and conventional rats were examined by capillary gas chromatography and gas chromatography/mass spectrometry. A number of carbonyl compounds, including several short-chain aliphatic ketones and acetophenone, were higher in the conventional urines, while many heterocyclic compounds (furan derivatives, benzothiazole and others) were lower. Both qualitative and quantitative differences were observed in the urinary excretion of acidic metabolites. Three meta-hydroxy phenolic acids appeared only in the conventional rat urines, while levels of many other aromatic and aliphatic acids were also higher.     

Glycosphingolipid patterns of the gastrointestinal tract and feces of germ-free and conventional rats

Acid and non-acid glycosphingolipids of stomach, small and large intestine, and stimulated feces of germ-free and conventional rats of the same stain have been isolated and characterized. The glycosphingolipid patterns of the intestinal organs were chemically and immunologically very similar between the two groups of rats and relatively unaffected by the presence of an intestinal microbial flora. The major exception was the presence of hematoside with N-glycoloylneuraminic acid (NeuGc) (NeuGc alpha 2----3Gal beta 1----4Glc beta 1----1Cer) in the stomach of conventional rats not found in the stomach of germ-free animals. Glycosphingolipids of stimulated feces of germ-free animals were derived from epithelial cells mainly of the small intestine and showed no signs of degradation. Glycosphingolipids of feces of conventional rats completely retained the pattern of blood group A-, B-, and H-active glycolipids as found in sterile feces but contained less of hematoside and more of lactosylceramide. This effect was probably due to degradation by bacteria, as demonstrated in vitro with the production of lactosylceramide after treatment of the isolated acid glycolipids of sterile feces with neuraminidase from Clostridium perfringens. The amount of total non-acid glycosphingolipids per dry weight was similar for stomach, was 50% higher for small intestine, and 300% higher for large intestine of germ-free animals compared to conventional animals. Due to the presence of large amounts of mucins the dry sterile feces contained 12% less non-acid glycolipids than conventional feces. However, calculated per rat per day the germ-free animal excreted more of non-acid glycosphingolipids (1.8 and 1.2 mg, respectively).