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.     

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.     

Cytokine responsiveness in germfree and conventional NMRI mice

We have investigated the proliferative response of thymocytes from different mouse strains to cytokines in vitro. Interleukin 2 (IL-2), IL-4 and IL-7 induced proliferation of thymocytes from NMRI/KI (a locally bred NMRI mouse strain), NMRI/H ('traditional' NMRI mice), C3H/HeJ and C3H/HeN mice. NMRI/KI thymocytes showed the most prominent proliferation in response to IL-1 alpha and IL-1 beta. IL-3, IL-5, IL-6, IL-8, IL-10, tumour necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma), inhibin and lipopolysaccharide (LPS) induced no thymocyte proliferation. Germfree NMRI/KI mouse thymocytes showed a significantly lower proliferation in response to IL-1 alpha and IL-1 beta than conventional mice. Rat tissues, previously shown to contain lymphocyte activating factors (LAFs), were also tested. Skin, tongue, esophagus, proventricular stomach, testis and placenta were all positive in the LAF assay utilizing NMRI/KI thymocytes, whereas none of the tissue extracts could induce proliferation in NMRI/H thymocytes. The higher cytokine responsiveness in conventional mice compared with germfree might suggest that exposure to microflora induces a higher state of activation of the immune system. The LAF assay, utilizing NMRI/KI thymocytes, is a highly sensitive IL-1 bioassay with a detection level of 1 pg/ml for IL-1 beta and 2 pg/ml for IL-1 alpha. The specificity of the assay is increased by utilizing NMRI/H mice to exclude the presence of IL-2, IL-4 and IL-7.     

Husbandry of the "nude" mouse in conventional and germfree environments.

The "nude" mouse is a unique tool for immunologic studies. Its relatively short life span dictates the application of rigid environmental controls to increase longevity if the mouse is to assume the role of a practical experimental animal. In this paper we discussed the husbandry procedures employed to raise "nude" mice in our facilities under conventional, defined flora, and germfree conditions. Conventional and defined flora mice were raised on laminar flow stay-clean rocks, and germfree "nudes" were housed in self-contained germfree isolators. The major cause of morbidity and mortality among conventional and defined flora "nude" mice was fulminating hepatitis. We presented evidence that the etiologic agent of the disease was mouse hepatitis virus (MHV). Germfree "nude" mice were completely free from viral and bacterial diseases.     

Buoyant density analysis of myeloid colony-forming cells in germfree and conventional mice.

Granulocyte-macrophage colony-forming cells (CFUc), in the bone marrow of germfree and conventioal CBA mice, were compared quantitatively and qualitatively. Cells were separated on the basis of their buoyant density by equilibrium centrifugation in continuous albumin density gradients. CFUc in the density subpopulations were detected by culture in agar containing three different types of colony stimulating factor (CSF). The sources of the CSF were post-endotoxin mouse serum (CSFES), mouse lung conditioned medium (CSFMLCM) and human urine (CSFHU). Mice were removed from the germfree environment and the buoyant density status of their CFUc was examined 1, 4 and 8 weeks later. No difference was found between germfree and conventional mice in the number of nucleated cells per femur or in their modal density. Neither was the number of CFUc per femur different. The cell cycle status of CFUc, as determined by the thymidine suicide technique was not significantly different. Functional heterogeneity was found among the density subpopulations for both groups of mice. This depended on the type of CSF. The density distribution of CFUc was significantly different in germfree mice. There were proportionately more low density CFUc. The mean modal density of CFUc under CSFES stimulation was less by 0.0045 g/cm3 in germfree mice. The removal of mice from the germfree environment resulted in a shift of the distribution to higher densities. The trend was towards the conventional situation. The significance of the buoyant density status of CFUc is discussed.     

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.     

Differences in survival among germfree mice following transfer to a conventional colony.

Sex and strain differences in survival were studied in 7-9 month old germfree mice following transfer to a conventional colony. One outbred and three inbred strains were observed. All outbred CD-1 mice survived transfer and in 4 months increased their weight by 50%. The majority of inbred mice survived 7 months after transfer. Sex differences in survival were evident throughout the experimental period and were most marked 7 months after transfer. An unexpected new finding was the viability of the male sex in germfree mice after transfer. Possible explanations are considered.     

Trypsin and chymotrypsin activity of the intestinal content in germfree, monoassociated and conventional rabbits.

Trypsin (T) and chymotrypsin (CHT) activities in luminal contents of the ileum, caecum and sigmoideum were followed in conventional (6 animals), monoassociated (5) and germfree (5) rabbits by pH-stat automatic titration using p-toluenesulphonyl-L-arginine methylester and acetyl-L-tyrosine ethylester as substrates. In conventional rabbits with complete microbial flora an aborally increasing decline of both proteolytic activities of luminal contents was determined (ileum T 198.2 - CHT 100.0; signmoideum T 10m.2 - CHT 68.8 mrg/g of intestinal content). Monoassociated animals represent a group different from both germfree and conventional animals. Trypsin and chymotrypsin of intestinal contents were not significantly altered by the presence of megacaecum in germfree rabbits (ileum T 219.2 - CHT 160.2; sigmoideum T 208.8 - CHT 110.8 mug/g of intestinal content). Chymotrypsin in the intestinal contents appears more labile and more affected by microbial flora than trypsin.     

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.     

Modulation of lipolysis by endotoxin and indomethacin in fat cells of germfree and conventional dogs.

Germfree fat cells released significantly less FFA and glycerol under basal conditions (i.e. in the absence of hormonal stimulation) than conventional cells. The lipolytic response to norepinephrine stimulation (0.2 μg/ml) was not different in the two cell populations.$\text{E. coli}$ endotoxin increased basal lipolysis and norepinephrine stimulated (0.2 μg/ml) FFA release in adipocytes from conventional dogs, while having no consistent influence on lipolysis of adipocytes from germfree dogs. The endotoxin effect was not dose dependent (0.2–2.0 μg/0.5 ml cell suspension).Indomethacin (5.0 μg/ml) significantly increased basal FFA and glycerol release from cells of germfree origin, and FFA efflux from cells of conventional dogs. Endotoxin obviated the influence of indomethacin on basal lipolysis of germfree cells.Endotoxin by itself did not alter cAMP concentrations in adipocytes from germfree dogs. The combination of indomethacin and endotoxin, however, significantly increased intracellular cyclic nucleotide concentrations.Compared to conventional fat cells, germfree fat cells are characterized by significantly reduced basal lipolysis, lack of a consistent lipolytic response to endotoxin stimulation and dissociation of the lipolytic response and cAMP levels by the combined influence of endotoxin and indomethacin.