Henri Saxerholt

Development of five metabolic activities associated with the intestinal microflora of healthy infants

The establishment of a functionally active intestinal flora was followed in 17 healthy Swedish children from birth up to 6 months of age. Utilizing gas chromatography, spectrophotometry, and gel electrophoresis, feces were analyzed on certain biochemical markers that reflect the action of the intestinal flora in vivo. The establishment of the following five flora-related functions was investigated: production of short chain fatty acids (SCFAs), degradation of mucin, conversion of bilirubin to urobilinogen and of cholesterol to coprostanol, and inactivation of fecal tryptic activity (FTA). Production of SCFAs was the first function to be established, followed by bilirubin conversion and mucin degradation. No child showed conversion of cholesterol. The values of FTA were lower than in adults. This study indicates that the establishment of a functionally active flora is a slow process and that some functions are almost fully established before other functions have started to develop. Environmental factors, such as the diet, seem to be of importance. In general, the functions seem to develop slower in those children receiving breast milk exclusively than in those receiving formula supplements.

Influence of peroral antibiotics upon the biotransformatory activity of the intestinal microflora in healthy subjects

Abstract. The effects of ampicillin, clindamycin or metronidazole, given perorally for 6 days to eighteen healthy volunteers, upon the following intestinal microflora‐associated characteristics (MACs) were evaluated: breakdown of mucin, formation of coprostanol, hydrolysis of bilirubin conjugates, formation of urobilinogen, and of some short chain fatty acids (SCFAs), presence of β‐aspartylglycine and inactivation of trypsin. Clindamycin markedly influenced the expression of all characteristics, but trypsin and β‐aspartylglycine, resulting in a pattern very much alike what has been found in germ‐free animals. Ampicillin caused a significant reduction in total amount of SCFAs (P<0·05) and urobilinogen (P<0·05) present in the faecal samples. Metronidazole caused a significant reduction in the formation of coprostanol and the deconjugation of bilirubin (P<0·05). We conclude that orally given antibiotics may cause major alterations in several parameters reflecting the normal biotransformatory activity of the intestinal microflora, probably caused by severe disturbances in the intestinal ecosystem.

Intestinal microbial conversion of cholesterol to coprostanol in man

The intestinal microbial conversion of cholesterol to coprostanol has been measured in groups of healthy subjects before, during and after they received the antibiotics ampicillin, bacitracin, clindamycin, co‐trimoxazole, doxycycline, erythromycin, metronidazole, nalidixic acid, ofloxacin or vancomycin orally for 6 days. Before they received antibiotics, the subjects demonstrated two distinct patterns of cholesterol conversion. One pattern was characterised by extensive conversion of cholesterol, the other by little or no conversion. Intake of bacitracin, clindamycin, erythromycin, metronidazole and vancomycin significantly reduced the conversion to coprostanol. In the groups receiving ampicillin or doxycycline, marked reductions were found in most of the subjects. No alterations were found in the groups receiving co‐trimoxazole, nalidixic acid or ofloxacin. In 6 subjects no conversion of cholesterol to coprostanol was found up to 5 weeks after the end of the antibiotic intake. We conclude that orally given antibiotics may cause alterations in the intestinal conversion of cholesterol, reflecting changes in the anaerobiC., Gram‐positive component of the gut flora.

Influence of antibiotics on intestinal mucin in healthy subjects

To determine the effect on microbial breakdown of intestinal mucin healthy volunteers were treated orally with ten different antibiotics. The most pronounced effects were seen after administration of bacitracin, clindamycin or vancomycin: the electrophoretic mucin pattern in faeces changed from a normal conventional pattern to a specific pattern similar to that found in germ-free rats. Disturbed patterns were also observed in some of the subjects treated with ampicillin, doxycycline, erythromycin, metronidazole or nalidixic acid. In most cases the electrophoretic mucin pattern normalized within five weeks after the end of treatment. There were no effects on the microbial breakdown of intestinal mucin in the groups treated with ofloxacin or trimethoprim/sulfamethoxazole. Thus, administration of antimicrobial drugs in clinically recommended doses may cause long-term disturbances in one microflora-associated characteristic, the breakdown of intestinal mucin.

Deconjugation of bilirubin conjugates and urobilin formation by conventionalized germ-free rats

The amounts of conjugated bilirubin and urobilins/urobilinogen were determined semiquantitatively in faeces of germ-free (GF) rats during GF condition and after conventionalization by oral administration of faeces suspension from conventional (CONV) rats. The amount of bilirubin conjugates, detected as their ethyl anthranilate azopigments, decreased rapidly 1 day after conventionalization. Thin-layer chromatography analysis of the corresponding faecal azopigment preparations showed that some azopigments started to disappear a few days after the conventionalization, indicating that their corresponding bilirubin conjugates were deconjugated by the bacteria in the intestine. On day 21 after conventionalization, only two azopigments were detected, namely the unconjugated and glucuronic acid conjugated dipyrroles of bilirubin, respectively, thus indicating the presence of only one bilirubin conjugate, the monoglucuronide. After 69 days no azopigments could be detected, indicating the total absence of conjugated bilirubin in these faeces samples. No urobilins were detected in faeces of the rats during their GF state, but these metabolites appeared in faeces one day after conventionalization and increased during a few days to a CONV level.

Intestinal deconjugation of bilirubin in germfree and conventional rats

The content of conjugated bilirubin (CB) was about the same in small intestinal contents from germfree (GF) and conventional (CONV) rats. Caecal contents from GF rats contained considerably more CB than from CONV rats. The results indicated that the caecum is the major site of microbial deconjugation of CB in the CONV rat. Separation of azopigments prepared from small intestinal contents of GF and CONV rats revealed similar patterns. Similar separation patterns were also observed with azopigments prepared from caecal contents and faeces of GF rats. Some chemical properties of CB prepared from GF rat faeces were studied.

HPLC separation and quantification of bilirubin and its glucuronide conjugates in faeces and intestinal contents of germ-free rats

We describe an accurate reverse-phase high-performance liquid chromatography (HPLC) method for the separation and quantification of unconjugated bilirubin (UCB) and its monoglucuronide (BMG) and diglucuronide (BDG) conjugates in faeces and intestinal contents from germ-free (GF) rats. We demonstrated that female GF rats excreted predominantly BMG and that the percentage of this conjugate was at most 71.7% of the total bilirubin excreted with the faeces. The highest percentages for BDG and the UCB were 27.9% and 6.0%, respectively. The bile pigment composition in duodenal contents was 59.8% BDG and 40.2% BMG (median percentage) and was 47.7% BDG, 50.1% BMG and 2.2% UCB in ileal contents. Deconjugation of BDG to BMG was profound in caecal contents with 26.0% BDG, 67.4% BMG and 6.6% UCB. Endogenous (mammalian) beta-glucuronidase activity was present in intestinal contents throughout the entire length of the intestine and in faeces of the GF rats. The results indicated that it is very likely that endogenous beta-glucuronidase plays a role in the deconjugation of bilirubin glucuronides as well as of other glucuronides in the intestine of the GF rat.

Methods for determination of conjugated bilirubin in rat faeces

Conjugated bilirubin was prepared from the faeces of germ-free (GF) rats by three different preparative methods. The bilirubin conjugate preparations were coupled with diazotized ethyl anthranilate and the formed ethyl anthranilate azopigments were quantified spectrophotometrically and separated by thin-layer chromatography (tlc). The most polar azopigment was purified by tlc and subjected to ammonolysis followed by tlc of the released saccaride. As a result of this procedure, only glucuronic acid was detected as the conjugating saccaride thus indicating that the most polar azopigment prepared from GF rat faeces was the delta ethyl anthranilate azopigment. Reference azopigments were prepared from GF rat small intestinal contents and subjected to separation by tlc. The azopigment pattern was very similar to the pattern obtained with the faecal azopigment preparations and a maximum of ten separated azopigment spots were detected. The findings indicated that, in addition to bilirubin glucuronides, other bilirubin conjugates with unknown structure are excreted with the faeces of GF rats. One of the preparative methods used for the preparation of conjugated bilirubin from GF rat faeces was tested on faeces from conventional (CONV) rats. From these preparations, no ethyl anthranilate azopigments were formed, thus indicating that faeces from CONV rats is devoid of conjugated bilirubin.

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.

Enteral nutrition and the function of the intestinal microflora in healthy adults

An oral feeding formula was given to 5 healthy volunteers for 8 days. Faecal samples were collected before, during and after the feeding period. The effect of enteral nutrition (EN) on the following seven intestinal microflora-associated characteristics (MACs) was studied: formation of urobilinogen, coprostanol and deoxycholic acid, degradation of mucin and beta-aspartylglycine, faecal tryptic activity, and production of short-chain fatty acids (SCFAs). None of the microbial functions studied were lost during the study. The urobilinogen level increased during EN (P < 0.05) but it seems reasonable to assume that this was a concentration effect due to a decrease in stool mass. The concentration of SCFAs decreased during EN (P < 0.05) and this reflects the absence of dietary fibre in the feed used.