Isolated fecal microorganisms capable of 7-alpha-dehydroxylating bile acids.

Strains of microorganisms capable of 7alpha-dehydroxylation of chenodeoxycholate were isolated from rat and human feces. All the strains were strictly anaerobic, non-motile, moderately themioresistant Gram-positive rods. They showed some saccharolytic properties with the production of both acid and gas. They were H(2)S-positive but indole-, skatole-, citrate-, catalase-, and oxidase-negative. The isolated strains capable of 7alpha-dehydroxylation of chenodeoxycholate were also able to oxidize the hydroxyl groups at C-3 and C-7 to keto groups. The following metabolites were isolated: 3-keto-7alpha-hydroxy-5beta-cholanoic acid, 3alpha-hydroxy-7-keto-5beta-cholanoic acid, 3alpha-hydroxy-5beta-cholanoic acid, and 3-keto-5beta-cholanoic acid. The isolated strains did not have the enzymes necessary for hydrolyzing conjugated bile acids. In mixed anaerobic cultures of fecal microorganisms, extensive reduction of the 3-keto group to the 3beta-hydroxyl group occurred. The microorganism(s) responsible for this reaction have as yet not been isolated.

Immunological studies in ulcerative colitis. IV. Origin of autoantibodies.

The incidence and height of antibody titers to colon, assayed by indirect hemagglutination with a heat stable colon extract from germ free rats, is significantly higher in sera from patients with ulcerative colitis than in those from healthy controls or from patients with amebic liver abscess or dysentery. While sera from ulcerative colitis patients and controls are indistinguishable in regard to incidence and height of antibody titers to Forsman antigen, Staphylococcus aureus S 209, Clostridium difficile, and several common strains of E. coli, they have elevated titers and increased incidence of antibodies to a heat stable antigen of E. coli O14. Patients with amebic dysentery have normal titers of such antibodies. Absorption of patients' sera with E. coli O14 antigen inhibits the colon directed hemagglutination reaction in approximately 30% of the cases tested. Likewise, the anti-E. coli O14 reaction can sometimes be inhibited with the colon extract. Other E. coli strains and other bacteria are inactive or have only weak inhibitory activity. Hemagglutination inhibition experiments show that germ free rat colon and E. coli O14 contain common structures, depicted by antibodies in the patients' sera. This pattern of reactivity closely resembles that seen in rats made autoimmune to colon by injection of newborn rabbit colon. E. coli O14 is known to carry a heterogenetic antigen present in lower concentration (or activity) in most Enterobacteriaceae. Hemagglutination inhibition experiments with rabbit antisera to E. coli O14 suggest that the antigen common for E. coli O14 and colon is related to this heterogenetic antigen. The findings imply that this antigen, which is constantly present in low concentrations in the human colon, may give rise to anticolon antibody formation in ulcerative colitis through breakage of tolerance. Since this antigen is present in healthy individuals as well, additional factors are required to explain the induction of anti-colon autoimmunity in ulcerative colitis.

Autoantibodies to colon in germfree rats monocontaminated with Clostridium difficile.

Germfree rats monocontaminated with the anaerobic microorganisms Clostridium difficile or another Clostridium species (strain G 62) produce auto-antibodies to colon antigen. The antigen can be extracted with phenol water from the feces of germfree rats. Antibodies, demonstrable by means of passive hemagglutination of antigen sensitized sheep erythrocytes appear after monocontamination for 35 days or longer. The indirect immunofluorescence techniques, applied to sections of germfree rat colon, gave positive mucosal staining. The staining was similar to that obtained with sera from patients with ulcerative colitis or from rats immunized with rabbit colon. No antibodies were found in the sera of germfree rats, germfree rats monocontaminated with various other bacteria, conventional rats of germfree origin, or conventional Sprague-Dawley rats. Although the anti-colon antibodies of the Clostridium infected rats reacted with the same feces extract as the antibodies of ulcerative colitis patients or of rabbit colon immunized rats, their specificity was different. While the latter cross-react with polysaccharide from E. coli O14, those from the Clostridium-infected exgermfree rats did not. Rats monocontaminated with Cl. difficile also developed antibodies to this organism, but no cross-reaction between Cl. difficile antigen and colon antigen could be demonstrated. This speaks against breakage of tolerance by cross-reacting bacterial antigen as the cause of autoimmunity in these rats. Other possible mechanisms for autoantibody production in this model are immunogenic alteration of gastrointestinal mucins by bacterial degradation, adjuvant effects of bacterial products, or both.

Metabolism of propachlor by the germfree rat.

Three major metabolites of propachlor were isolated from the excreta of germfree rats given 14C-labeled propachlor orally. In contrast, 11 urinary metabolites, six of which were 2-methylsulfonylacetanilides not present in excreta of germfree rats, were isolated from control rats given 14C-labeled propachlor orally. Enterohepatic circulation and microbial metabolism in the intestine were necessary for production of the methylsulfonyl-containing and other metabolites of propachlor in the conventional rat.

Improved intramuscular blood flow and normalized metabolism in lateral epicondylitis after botulinum toxin treatment.

Lateral epicondylitis is a common cause of elbow pain, and decreased microcirculation in extensor carpi radialis brevis (ECRB) has recently been suggested to contribute to the symptoms. The purpose of this pilot study was to investigate the treatment response after injection of botulinum toxin type A. Ten patients with unilateral epicondylitis and decreased intramuscular blood flow in ECRB participated. Handgrip, 2-pinch grip and muscle strength during radial deviation and dorsal extension of the wrist were recorded. Perceived pain during contraction was evaluated with the Visual Analogue Scale (VAS) and function in daily activities was assessed using the Disability of Arm, Shoulder and Hand instrument (DASH) and the Canadian Occupational Performance Measure instrument (COPM). Intramuscular blood flow was recorded by laser Doppler flowmetry, and microdialysis was used to analyze muscle metabolism. The difference in intramuscular blood flow between the control and the affected side had decreased 3 and 12 months after treatment (P=0.03). Lactate concentration at the 12-month follow-up had decreased (P=0.02); perceived pain was reduced and function in daily activities had improved. Injection of botulinum toxin is an alternative treatment for epicondylitis. Symptom relief may be due to enhanced microcirculation causing an aerobic metabolism.

Soya--a dietary source of the non-steroidal oestrogen equol in man and animals.

The dietary origin of the weak oestrogen equol (7-hydroxy-3-(4'-hydroxyphenyl)-chroman) present in human urine has been investigated using gas chromatography-mass spectrometry. Feeding experiments with different food constituents and monitoring the urinary excretion of equol revealed that soya food yields more than 0.1 mg urinary equol/g flour ingested. From this source the glucoside of daidzein (4',7-dihydroxyisoflavone) has been isolated and identified as a precursor of equol. Both equol and daidzein were characterized as monoglucuronide conjugates in human urine and the concentration of urinary equol exceeded the concentrations of the classical oestrogens by 100- to 1000-fold after ingestion of a single meal containing soya protein. The potential biological significance of this result is discussed.

Effects of feeding chenodeoxycholic acid on metabolism of cholesterol and bile acids in germ-free rats.

The aim of this investigation was to study the influence of chenodeoxycholic acid administration on cholesterol and bile acid synthesis in germ-free rats. Seven rats were fed a basal diet and 2 groups of 4 rats received the same diet supplemented with 0.4 and 1% chenodeoxycholic acid, respectively. After 6 weeks, feces were collected in one 3- and one 4-day pool for analysis of cholesterol and bile acids. When the sampling period was finished, the rats were killed and the liver microsomal fractions isolated. The activities of HMG CoA reductase and cholesterol 7 alpha-hydroxylase were determined, the 7 alpha-hydroxylase by a mass fragmentographic method. The 2 dominating bile acids in the untreated rats were cholic acid and beta-muricholic acid. During treatment with chenodeoxycholic acid, 60--70% of this bile acid was converted into alpha- and beta-muricholic acid, indicating a high activity of the 6 beta-hydroxylase. The excretion of cholic acid was almost completely inhibited and the 7 alpha-hydroxylase activity was decreased ca 75% in the rats fed 1% chenodeoxycholic acid. The activity of the hepatic HMG CoA reductase as unchanged. The fecal excretion of cholesterol increased 2--3 times. An accumulation of cholesterol was seen in the rats treated with 1% chenodeoxycholic acid, which was probably a result of the decreased catabolism of cholesterol to bile acids.

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.

Influence of intestinal microflora on the tryptic activity during lactation in rats.

On comparing germ-free and conventional rats, inactivation of the tryptic activity was found to take place in the caecum of conventional adult rats only. A microbial intestinal inactivation of the tryptic activity was established in suckling conventional rats within 10 days after birth. At 3 weeks of age, suckling germ-free rats were found to have less faecal tryptic activity than their early-weaned littermates.

The physiological importance of the colonic microflora.

The present concept of the intestinal microflora has been reviewed with stressing the fact that it represents an entity with a weight comparable to one of the larger organs of the body. It is composed of about 500 different strains, many of which as yet have not been isolated because 95% of them are anaerobic and fastidious in their growth requirements. The intimate relationship of the flora to the surface epithelium of the intestines and the Lieberkühn's crypts are brought forward. The production of germfree animals is reviewed as an accounting of the most obvious differences between the germfree animal and its conventional counterparts, as far as gastroenterology is concerned. This includes the protective function of the normal intestinal flora, the caecum enlargement in germfree rodents, the influence of the flora on the metabolism of bilirubin, intestinal mucin, and pancreatic enzymes. Differences found in germfree animals due to the absence of the microbial flora related to the metabolism of fatty acids, bile acids, cholesterol and steroid hormones are also reviewed. The germfree animal has even proven to be an important research tool in such fields as carcinogenesis and studies of Crohn's disease and ulcerative colitis. There is need of a wider use of the germfree animal as a baseline in studies on the interference of the intestinal microbial flora with factors and conditions of great physiological and clinical importance.

Decreased intramuscular blood flow in patients with lateral epicondylitis.

The purpose of this pilot study was to investigate intramuscular microcirculation in extensor carpi radialis brevis (ECRB) in patients with lateral epicondylitis. Ten patients with unilateral epicondylitis, mean duration of symptoms of 39 (12-96) months participated. The diagnosis was based on clinical examination and none was under treatment for the last 6 months. Isometric handgrip strength, 2-pinch grip strength and muscle strength during radial deviation and dorsal extension were determined. Functional perceived pain was evaluated by a modified behaviour rating scale and perceived pain during contraction by visual analogue scale. Intramuscular and skin blood flow was recorded by a laser-Doppler flowmetry system technique (LDF) during stable temperature condition. Intramuscular blood flow was significantly lower in the affected side, 22.7+/-9.8 perfusion units (PU), as compared with 35.2+/-11.9 PU in the control side (P=0.01). There was no difference in skin blood flow or temperature between the affected and the control side. A positive correlation was found between the duration of symptoms and the difference in intramuscular blood flow between the affected and the control arm (r=0.65, P=0.06). The present data indicate that decreased microcirculation and anaerobic metabolism in ECRB may contribute to the lateral epicondylitis symptoms.

Impaired enteric degradation of pancreatic endopeptidases in antibiotic-treated rats.

Conventional Sprague-Dawley rats were fed by gastric tube for 5 days with either benzylpenicillin, ampicillin, doxycycline, or clindamycin. In contrast to the pretreatment period fibrinolytic activity and active and immunoreactive trypsin and immunoreactive elastase were present in fecal extracts after 4 days of antibiotic administration. This is consistent with findings in germfree rats and represents an alteration in the intestinal microflora. The germfree characteristics persisted until a suspension of cecal contents from normal rats was administered by enema on the 26th day. In fecal extracts from the clindamycin-treated rats considerable amounts of active and immunoreactive enzymes were, however, still found 10 days after the enema. A possible explanation is that clindamycin or a metabolite remains in the intestinal tract for a long time. The pathophysiological significance of this finding is unknown.

Development of germfree animal characteristics in conventional rats in antibiotics.

Conventional (CONV) rats were fed by stomach tube for five days with either benzylpenicillin, ampicillin, tetracykline, oxitetracykline, neomycin, bacitracin + neomycin, kanamycin or colistin. On the 2nd-3rd day all the animals developed one or several of the following symptoms or characteristics typical for germfree (GF) rats: no coprostanol formation, no stercobilin production, a GF pattern after gel electrophoresis of fecal supernatant and proteolytic activity in the feces. Under the same conditions succinylsulfathiazole or metronidazole had much less pronounced effects than the antibiotics. When clofibrate, acetylsalicylic acid or ferrous sulphate were administered the effects were none or negligible. The GF characteristics persisted for several weeks after the end of the administration of the drugs. In some instances this was the case up to 7 weeks, when the animals were contaminated by anal route with a suspension of the cecum contents from intact CONV animals. On the 2nd day after this treatment the GF characteristics had disappeared.

Microbial conversion of bilirubin to urobilins in vitro and in vivo.

No urobilins are formed from bilirubin in germ-free rats. To isolate and investigate the strains of intestinal microorganisms responsible for this transformation, a suitable test medium was adopted. The strength of the medium and a rather high initial pH were found to be of importance. In this medium, suspensions of rat faeces and a single strain, Cl. ramosum (G62), converted bilirubin to urobilins. Cultivations of Cl. ramosum (G62) together with E. coli significantly enhanced the conversion, whereas addition of 4 other bacterial strains was without the influence. The highest in vitro formation of the urobilins was about 10% of the bilirubin present. When the 6 strains investigated in vitro were established in EXG rats, the in vivo conversion of bilirubin to urobilins was found to be about 15%, compared to 70% in CONV rats.

Intestinal water-soluble mucins in germfree, exgermfree and conventional animals.

Water-soluble intestinal mucins were investigated in germfree (GF), exgermfree (EXG) and conventional (CONV) rats and in GF and CONV mice. After agar gel electrophoresis, all GF animals had similar specific band patterns demonstrated by PAS and Toluidine Blue. These patterns, never seen in CONV animals, disappeared in GF animals infected either with intestinal contents from CONV rats or mono-infected with a mucin converting microorganism, labelled Peptostreptococcus N. The intestinal microflora seem to have a profound influence on the water-soluble mucins, and specific microorganisms appear to be involved in the conversion of these substances. Any CONV animal with a GF mucin pattern in the faeces must be considered to have a disturbance of the normal intestinal microflora.

Influence of cholesterol feeding on liver microsomal metabolism of steroids and bile acids in conventional and germ-free rats.

The present investigation has aimed at defining the factor responsible for the differences in microsomal metabolism of steroids between germ-free and conventional rats. Cholesterol, cholic acid, taurocholic acid, and chenodeoxycholic acid were fed to conventional and germ-free male rats and the effects on liver microsomal metabolism of 4-[4-14C]androstene-3,17-dione, 5alpha-[4-14C]androstane-3alpha,17beta-diol, [4-14C]-cholesterol, 7alpha-hydroxy-4-[6beta-3H]cholesten-3-one, and [24-14C]lithocholic acid were studied. The most consistent effects were found with dietary cholesterol that stimulated the activities of several of the hydroxylases active on 4-androstene-3,17-dione and 5alpha-androstane-3alpha,17beta-diol and that decreased the 5alpha reduction of 4-androstene-3,17-dione, increased the 7alpha hydroxylation of cholesterol, decreased the 12alpha hydroxylation of 7alpha-hydroxy-4-cholesten-3-one, and increased by 6beta hydroxylation of lithocholic acid. These effects of cholesterol feeding on the microsomal metabolism of steroids in conventional rats made the pattern of microsomal enzyme activities resemble that characteristic of germ-free rats. Cholesterol feeding led to a pronounced increase in the intestinal concentration of beta-muricholic acid in conventional rats. Furthermore, cholesterol feeding to conventional animals led to an intestinal ratio of chenodeoxycholic acid (including its metabolites alpha- and beta-muricholic acid and hyodeoxycholic acid) to cholic acid (including deoxycholic acid) that was almost identical to that in germ-free rats. Conventionalization of germ-free rats for a period of up to 56 days led only to a partial normalization of the liver microsomal metabolism of 5alpha-[4-14C]androstane-3alpha, 17beta-diol and 7alpha-hydroxy-4-[6beta-3H]cholesten-3-one and of the liver microsomal concentration of cytochrome P-450. The concentration of cholesterol was higher in both total liver homogenate and liver microsomal fraction of germ-free rats than in corresponding preparations from conventional rats. In conclusion, it is suggested that cholesterol is one of the factors responsible for the different microsomal metabolism of steroids in germ-free and conventional rats. It is also suggested that cholesterol may play a role as regulator of microsomal enzyme activities.

Detection of bilirubin conjugates in faeces of germfree rats.

The presence of bilirubin conjugates in faeces from germfree (GF) and conventional (CONV) rats was tested after using affinity chromatography and Porapak Q chromatography as clean-up procedures. The bilirubin conjugates were detected as their ethyl anthranilate azopigments after separation by thin layer chromatography (tlc). Azopigments prepared from bile of GF and CONV rats served as a reference material for the tlc analysis. After tlc of the faecal azopigment preparations it was concluded that GF rat faeces contains bilirubin conjugates, while faeces from CONV rats is devoid of conjugated bilirubin. The findings suggest that the main bilirubin conjugates present in faeces from GF rats are of the glucuronic acid type.

Clindamycin-induced alterations in intestinal microflora-associated characteristics in rats.

Conventional Sprague-Dawley rats were treated with clindamycin, 40 mg/kg/day and 0.04 mg/kg/day, for 5 days. At a dose of 40 mg/kg/day, microflora-associated characteristics (MACs), such as shape, color, and consistency of feces, proteolytic activity, electrophoretic pattern, and cholesterol and bilirubin metabolism were transformed into values like those found in germfree rats: germfree animal characteristics (GACs). The effect on the proteolytic activity lasted longest. It did not disappear until one or two enemas with cecal contents from intact conventional rats were administered. At a dose of 0.04 mg/kg/day, effects on the proteolytic activity and cholesterol metabolism were seen. With the exception of one rat, the effect on proteolytic activity did not disappear until one or two enemas were given. The results indicate that clindamycin, even in very small daily doses, has a profound and long-lasting influence on many intestinal MACs in rats.