Bandaru S. Reddy

Intestinal disaccharidase activities in the growing germfree and conventional rats.

Abstract Disaccharidase (maltase, invertase, trehalase, lactase, and cellobiase) activities have been studied in homogenates of small intestine of the germfree and conventional rats from birth through 120 days of age. The pH optimum values for germfree and conventional rats were comparable. In adult germfree rats, the pattern of distribution of disaccharidase activities in different sections of small intestine was similar to that of conventional rats. Lactase and cellobiase activities were substantial at birth, reached maximum at 7 days of age, and then decreased gradually to a plateau at 30 days of age in both groups. Maltase activity was low at birth, whereas no invertase and trehalase activities could be detected up to 7 days of age, and these activities developed rapidly during the third week of life in both groups. Differences in activity of these disaccharidases between germfree and conventional rats were not apparent until after weaning (21 days) when the germfree animal showed a higher activity than the conventional rat. When the germfree rats were conventionalized by the introduction of cecal contents from the conventional rats, the disaccharidase activities were reduced to normal conventional levels. These results indicate that intestinal disaccharidases are synthesized by the animal itself, and that the intestinal microorganisms do not contribute to any major extent to their production.

Efficiency of digestion in germ-free and conventional rabbits.

1. Germ-free (GF) and conventional (CV) rabbits wearing collars to prevent coprophagy were fed an autoclaved diet with added cellulose. Their faecal excretion was analysed to determine nutrient digestibility. 2. Clearly distinguishable hard faeces were excreted by the GF rabbit only if the diet contained at least 15% cellulose. Unlike CV rabbits, the GF rabbits did not consume their soft faeces even when permitted to do so. Soft faeces made up a larger proportion of the total output of GF than of CV rabbits. Food intake and total dry-matter excretion per kg body-weight were similar in both groups. 3. Although digestibility of dry matter was similar in the two groups, in the GF rabbits there was a higher digestibility of crude fat and true protein and a lower digestibility of crude fibre and nitrogen-free extract. GF rabbits excreted a higher percentage of ingested calcium and phosphorus in the urine than did CV rabbits. 4. The results suggest that intestinal microbes, even without the enhancing effect of coprophagy, aid in the digestion of carbohydrate by rabbits. The greater faecal excretion of crude fat and true protein by CV rabbits could result from poorer digestion and absorption, but could also represent nutrients synthesized by microbes from simpler materials. The reingestion of faecal crude fat and true protein might therefore improve the quality of the total nutrient intake. The results suggest ways of assuring an adequate dietary intake by GF rabbits in the absence of contributions from an intestinal microflora.

Studies on the mechanism of calcium and magnesium absorption in germfree rats.

Abstract The effect of intestinal microflora on the activities of brush border Ca 2+ -ATPase, Mg 2+ -ATPase, and alkaline phosphatase, and mucosal calcium-binding protein activity in the small intestine was studied to elucidate whether the increased absorption of calcium and magnesium in the germfree rats is mediated through an increase in the activities of above enzymes. The pH optimum values of Ca 2+ -ATPase, Mg 2+ -ATPase, and alkaline phosphatase for germfree and conventional rats were comparable. The brush border protein concentration in both germfree and conventional rats was maximal in the duodenum and decreased distally. Germfree rats showed an increase of brush border protein content in the jejunum and ileum. Germfree status resulted in a significant increase of brush border Ca 2+ -ATPase, Mg 2+ -ATPase, and alkaline phosphatase activities in the small intestine. The calcium-binding protein activity of the mucosal preparations of small intestine was increased under the germfree conditions. These results indicate that the enhanced levels of intestinal brush border Ca 2+ -ATPase, Mg 2+ -ATPase and alkaline phosphatase together with mucosal calcium-binding protein might be responsible for increased calcium and magnesium absorption observed in germfree rats.

Effect of Protein-Calorie Restriction on Brain Amino Acid Pool in Neonatal Rats

Summary Newborn rats fed restricted calories during the first 2 days of life showed a depression of brain weight, and the levels of glutamic acid, glutamine, alanine, serine, and GAB A compared to those allowed to suckle their mothers normally. Also it was found that the levels of valine, leucine, isoleucine, histidine, arginine, cystine, ornithine, urea, and ammonia were increased in newborn rats maintained on restricted calories. It is concluded that calorie malnutrition, even for a short period, during the neonatal development is associated with alteration in the brain amino acid pool.

Metabolic enzymes in liver and kidney of the germfree rat

Abstract The effect of intestinal microflora on the activities of NADP-dependent dehydrogenases in liver and kidneys, and on fatty acid synthetase, ATP-citrate lyse, cytochrome oxidase and succinate dehydrogenase activities in liver of rats were studied. Germfree rats showed a decrease in liver succinate dehydrogenase activity, and no effect on liver cytochrome oxidase activity. The absence of an intestinal microflora resulted in a significant decrease of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities, and a substantial increase of ATP-citrate lyase and fatty acid synthetase activities in the liver. Liver malate dehydrogenase activity was comparable in both germfree and conventional animals. Germfree status had no effect on the activities of NADP-dependent dehydrogenases in the kidney. It is concluded that the absence of an actively metabolizing microflora was associated with quantitative shifts in the activity of hepatic enzymes involved in the metabolism of carbohydrates and fats.

Protein Metabolism in Germfree Rats Fed Chemically Defined, Water-Soluble Diet and Semisynthetic Diet

The importance of germfree animals and chemically defined, water-soluble diets developed at Lobund Laboratory in nutritional and immunological studies was discussed in detail by Pleasants (1). Nutritional adequacy of this diet has been demonstrated by the fact that it supported reproduction of germfree mice into fifth generation and the growth of germfree rats from birth through maturity (2). Although these results indicate that the diet is at least qualitatively adequate for germfree rats and mice, it is of considerable importance to determine if the animal is physiologically “normal” in a chemical environment so limited and so defined. So, as a further test of nutritional adequacy and physiological normality, germfree and conventional rats fed this diet were compared to similar rats fed a semisynthetic diet based on casein-starch using morphology, serum amino nitrogen, pancreatic enzyme levels, liver protein, nitrogen balance and protein efficiency ratio (PER) as criteria. In addition, these studies would indicate to what extent the intestinal microflora could influence the protein metabolism of the host.

Sudden Death in Germfree Mice Reared Through Successive Generations on Chemically Defined Liquid Diet

A diet for germfree animals which consists of chemically defined, low-molecular-weight nutrients, and which can be prepared in liquid form for sterilization by filtration, brings the experimental animal one step farther toward complete control of nutritional and antigenic variables. Such a diet, consisting of purified amino acids, sugars, ethyl linoleate, vitamins and minerals, all dissolved in a single water solution, has been fed to germfree rats (1) and to germfree mice (2,3). The fat-soluble portion of the diet had been solubilized in the water solution by use of Polysor-bate 80, These preliminary experiments had implicated the nonionic detergent as a cause of persistent diarrhea in some mice. Changes in the lymph nodes of germfree mice fed the detergent diet also resembled those observed by Mori and Kato (4) in conventional rats fed polysorbate. For these reasons the detergent was eliminated and the fat-soluble portion of the diet was prepared and fed separately from the water-soluble portion, according to a procedure reported at the 1966 meeting of this Association. Diet prepared in this way has now been subjected to long-term testing of its nutritional adequacy.