Bengt E. Gustafsson

The establishment of some microflora associated biochemical characteristics in feces from children during the first years of life.

ABSTRACT. This report presents a new approach to the study of the colonization of the digestive tract after birth. We have examined the development of four microflora associated characteristics, MACs, defined as the recording of any anatomical structure, biochemical or physiological function in the macroorganism, which has been influenced by the microflora. These MACs may create a basis for later investigations into the impact of diarrheal diseases and antibiotic therapy. The following biochemical characteristics were studied in feces from children of 0‐61 months of age: conversion of cholesterol to coprostanol and bilirubin to urobilins, inactivation of trypsin and degradation of mucin. These results indicate establishment of microbes capable of converting bilirubin to urobilins within the second year of life. The mucin degrading and cholesterol converting microbes are established in most of the children during the same period. Tryptic activity was found to be absent in meconium, present in feces from all children up to 21 months of age, and absent in 6 out of 15 children in the age group 46‐61 months. The study indicates that the establishment of the MACs in the digestive tract is a remarkably long drawn out process

On serum-cholesterol levels and neutral fecal sterols in germ-free rats; bile acids and steroids 59.

Abstract On a semisynthetic diet without significant amounts of cholesterol, germ-free rats had significantly higher serum-cholesterol values than control rats, the mean value for germ-free animals being 110 mg.% and for controls 77 mg.%. On a diet with completely steroid-free synthetic fat, germ-free animals excreted daily 2.6 mg. of neutral sterols in feces. At least 82% of these consisted of cholesterol.

Ultrastructure of the enlarged cecum in germfree rats

SummaryThe cecum of germfree rats, as studied by light microscopy and scanning and transmission electron microscopy, differs in many respects from the cecum of conventional rats. Epithelial cells in germfree rats are taller and have larger nuclei and longer microvilli than similar cells in conventional rats. The cecal mucosa of germfree rats shows a larger variability in the arrangement of the crypts of Lieberkühn than does the mucosa of conventional rats. Some crypts are funnel-shaped and connected close to the mucosal surface with adjacent similar crypts to form long valleys. Less wide crypts open on elevated regions of the mucosal surface. The lamina propria of germfree animals is devoid of plasma cells but rich in mast cells. Germfree animals show hypertrophy of the tunica muscularis externa.In conventional rats the cecal lumen contains a large variety of morphologically different bacteria. However, the lumen of the crypts of Lieberkühn contains only one type of elongated bacteria, which are present in large amounts. This finding suggests that symbiotic relations may be of particular importance in the crypts of Lieberkühn in the cecum.

Rat intestinal 6-bromo-2-naphthyl glycosidase and disaccharidase activities: I. Enzymic properties and distribution in the digestive tract of conventional and germ-free animals

Abstract The properties and suitable conditions of assay of three 6-bromo-2-naphthyl glycosidase (α-glucosidase, β-glucosidase, and β-galactosidase) activities in rat small-intestinal mucosal extracts have been investigated. An activity unit has been defined which is comparable with the unit for disaccharidase activity. The 6-bromo-2-naphthyl-glycosidase activities and corresponding disaccharidase (maltase, invertase, lactase, and cellobiase) activities have been measured in homogenates of different parts of the intestinal tract of conventional and germ-free rats, in order to obtain information about the origin of the enzymes. The small-intestinal activities are exerted by enzymes produced by the animal itself. Part of the 6-bromo-2-naphthyl-α-glucosidase and a small fraction of the maltase may originate from the pancreas; the other activities are formed in the small-intestinal mucosa. In the large intestine of conventional rats the enzymes arriving with the small-intestinal content (secreted from the pancreas or released from desquamated small-intestinal mucosal cells) are mixed with bacterial enzymes, which have higher pH optima and different specificity. In the germ-free rats, in which these bacterial enzymes are absent, some of the enzymes produced by the small intestine or pancreas are accumulated in large amounts in the cecal content. These enzymes are destroyed by the intestinal bacteria in the conventional rats.

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.

Azo Reduction of Salicyl-Azo-Sulphapyridine in Germ-free and Conventional Rats

Abstract1. Salicyl-azo-sulphapyridine was recovered mainly as azo-cleavage products in conventional rats after oral as well as after intraperitoneal administration of the drug.2. After administration of salicyl-azo-sulphapyridine to germ-free rats, the recovery of azo-cleavage products was 1·7% of the dose after oral and 10% of the dose after intraperitoneal dosage.3. It is concluded that salicyl-azo-sulphapyridine is reductively cleaved at the azo linkage by the action of the intestinal microflora and that azo-reductases in the tissues play a minor role.

Effect of Some Biologically Active Amines on the Cecum Wall of Germfree Rats.

Summary The cecum wall of germfree and conventional rats was compared with respect to content of and sensitivity to biologically active amines. The concentration of nor-adrenaline, 1-adrenaline, dopamine, serotonin, acetylcholine and histamine was found to be of the same order of magnitude in germfree and conventional rats. Strips of the cecum wall from conventional rats exhibited regular spontaneous muscle contractions whereas in germfree rats such an activity was never seen. The cecum wall of the germfree rats was generally less sensitive to acetylcholine, serotonin, 1-adrenaline and histamine. This was most pronounced for acetylcholine. In both kinds of cecum wall, the type of response to the amines administered was similar except for serotonin.

Inorganic sulphate, sulphite and sulphide as sulphur donors in the biosynthesis of sulphur amino acids in germ-free and conventional rats.

Abstract 1. 1. Incorporation of 35S from 35S-labelled sodium sulphate, sulphite and sulphide into the sulphur-containing amino acids cysteine/cystine and methionine was investigated in young germ-free and conventional rats. The animals were killed 3 h after intraperitoneal injection of the labelled compound. 2. 2. After injection of [35S]sulphate or [35S]sulphite both cysteine and methionine were labelled slightly in conventional rats, whereas no labelling of sulphur amino acids was detectable in germ-free animals. 3. 3. After injection of [35S]sulphide, cysteine was labelled to approximately the same extent in both conventional and germ-free rats. Labelling of methionine in conventional animals was much smaller and in germ-free rats not detectable. 4. 4. These findings prove that inorganic sulphur at the sulphide level of oxidation can be used in rat tissues for the synthesis of cysteine by a mechanism not involving methionine as the ultimate sulphur donor, most likely by enzymic sulphhydrylation of serine. The complete lack of incorporation of 35C from labelled sulphate and sulphite into cysteine in germ-free animals indicates that these compounds are not reduced to sulphide in noteworthy amounts in rat tissues. The intestinal microorganisms are thus responsible for the utilization of sulphate and sulphite in the sulphur amino acid synthesis observed in contentional rats.

Comparison of Bile Acids in Intestinal Contents of Germfree and Conventional Rats.

Summary Two days after administration of cholic acid-24-C14 to germfree and conventional rats the intestinal contents were separated by centrifugation at 25,000 X g. 1-The labeled bile acids throughout the intestinal tract of germfree rats were recovered in the supernatant. 2. In the conventional rats almost all labeled bile acids in the small intestine were recovered in the supernatant whereas 27-44% of the labeled bile acids in cecum were found in the sediment. 3. The labeled bile acids in the sediment after centrifugation of cecal content had a higher per cent of deoxycholic and 3-hydroxy-12-keto-cholanic acid than those present in the supernatant.

The metabolism of pentachloromethylthiobenzene in germ-free and conventional rats

1. Both germfree and conventional rats excreted over 80% of oral doses of pentachloromethylthio[14C]benzene in the faeces.2. The faeces from germ-free rats contained mainly N-acetyl-S-(methylthiotetrachlorophenyl)cysteine.3. The faeces from conventional rats contained bis-(methylthio)tetrachlorobenzene and non-extractable residues in about equal amounts.