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German Institute of Human Nutrition Potsdam-Rehbrücke, Department of Gastrointestinal Microbiology, 14558 Bergholz-Rehbrücke, Germany
1To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • polyamine formation • bacteroides • fusobacteria • gnotobiotic rats • pectin
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, McCormack and Johnson 1991
, McCormack et al. 1998
).
Moreover, polyamines are involved in the regulation of the
transcription of protooncogenes which play a role in mucosal cell
division (Patel and Wang 1997
). There is also increasing
evidence for the role of putrescine and spermidine in the promotion of
transformation of cells to malignancy (Seiler et al. 1998
). Considering these undesirable effects of polyamines,
knowledge about exogenous polyamines is of utmost importance. Exogenous
polyamines are ingested in large amounts with all types of foodstuffs
(Bardocz et al. 1993
, Romain et al. 1992
), and they are synthesized by the bacterial flora resident
in the lumen of the large bowel (Sarhan et al. 1989
,
Seidel and Scemama 1997
). Whereas the amounts of
polyamines available from food and their utilization in the body are
well-characterized (Bardocz et al. 1995
and 1998
,
Benamouzig et al. 1997
), the knowledge about the
microbial polyamine formation in the intestinal tract and the
contribution of this process to the body polyamine pool are
insufficient. This is in contrast to the well-accepted notion that
polyamines produced by the gut bacteria may be important for the host.
Osborne and Seidel (1989)
showed that the polyamine
concentration and the expression of bacterial lysine, ornithine and
arginine decarboxylase in luminal contents of rats increased in
response to colonic obstruction, resulting in increased proliferation
of the gut mucosa. The same rats treated with antibiotics did not
respond. The polyamines formed by intestinal bacteria undergo
enterohepatic circulation (Osborne and Seidel 1990
). In
contrast to human cells, bacteria can synthesize putrescine from either
ornithine or arginine, and cadaverine by decarboxyalation of lysine
(Tabor and Tabor 1985
). It is therefore not surprising
that the treatment of tumor-bearing patients or animals with
-difluoromethylornithine (the irreversible inhibitor of the
ornithine decarboxylase) is unsatisfactory. The antitumoral effect of
-difluoromethylornithine may be enhanced by the treatment with
antibiotics that are suitable for decontamination of the large bowel
(Hessels et al. 1989
, Sarhan et al. 1989
). Therefore, the inhibition of polyamine biosynthesis in
microorganisms is essential for an effective cancer therapy
(Bitonti and McCann 1987
). This requires detailed
information on the overall ability of microorganisms in the large bowel
to synthesize polyamines, on their metabolic behavior, and on the
factors that may influence these processes.
A previous study in our laboratory presented evidence that the
polyamine pattern found in the large intestine of germfree rats
differed markedly from that in conventional rats (Noack et al. 1996
). Moreover, the consumption of pectin- or
guar-containing diets altered the concentrations and the
composition of cecal polyamines in conventional rats but not in
germfree rats, demonstrating that bacterial polyamine synthesis was
stimulated by certain carbohydrates. Based on in vitro results obtained
with selected human intestinal bacteria (Noack et al. 1998
), we hypothesized that members of the genera
Bacteroides and Fusobacterium were mainly
responsible for the observed pectin-stimulated spermidine and
putrescine production in the large bowel of conventional rats.
The aim of the present study was to examine this hypothesis by performing feeding experiments with germfree rats colonized with selected species of Bacteroides and Fusobacterium. We also wanted to find out whether the pectin-dependent increase in polyamines resulted from an increase in bacterial cell numbers or from a stimulation of bacterial polyamine synthesis. The results obtained provide a more specific view on the factors that govern bacterial polyamine synthesis in the large bowel and its impact on the large bowel.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Germfree male Albino Wistar rats (n = 18) (Germfree Animal Unit of the German Institute of Human Nutrition, Potsdam-Rehbrücke, Germany; initial body weight 130 ± 20 g) were randomly assigned to one of three treatment groups of six rats: group 1, monoassociated + fiber-free diet; group 2, diassociated + fiber-free diet; and group 3, diassociated + pectin-containing diet. The groups consuming the fiber-free diet (mono- or diassociated, respectively) were included to study the effect of coculturing on both bacterial growth and polyamine production in the cecum of the gnotobiotic rats. The two diassociated groups (fiber-free or pectin-containing diet, respectively) were compared to examine the effect of pectin. The rats were housed individually in wire-bottomed cages arranged in sterile isolators equipped with a sterile water supply. Separate isolators were used for each treatment group. The room was regulated to a 12-h light 12-h dark schedule and maintained at 22 ± 2°C. The experiment included two steps: (i) germfree rats were mono- or diassociated with microorganisms of the human intestinal flora and (ii) feeding of purified diets without or with pectin.
Experimental step 1.
In the morning of d 1 and 2, 1 mL of a bacterial suspension of Bacteroides thetaiotaomicron containing 3.16 x 1013 cfu/L (colony-forming units/L) was applied by intragastric intubation to each rat of each treatment group. In the morning of d 4 and 5 the rats of two treatment groups were colonized additionally with 1 mL of a suspension of Fusobacterium varium containing 4.2 x 1013 cfu/L. During the period of microbial colonization, the rats of all treatment groups were fed nonpurified diet, Altromin 1310 (Altromin GmbH, Lage, Germany) sterilized by gamma radiation.2
Experimental step 2.
On d 8, the Altromin diet was replaced by purified diets. The rat group
monoassociated with B. thetaiotaomicron, and one of the
groups diassociated with B. thetaiotaomicron + F. varium
received a polyamine-deficient, fiber-free basal diet. The second
group diassociated with B. thetaiotaomicron + F. varium
were fed basal diet supplemented with 10% pectin (Copenhagen Pectin,
Lille Skensved, Denmark) as soluble dietary fiber. The nutrient
composition, the preparation and the polyamine contents of the diets
were described previously (Noack et al. 1998
). For
sterilization, all diets were subjected to 25 kGy of gamma radiation.
Rats were adapted to the diet for 7 d, and thereafter fed the
respective diet for 10 d. On d 3, 8, 10, 15 and 25 of the
experimental period, fecal samples of all rats were taken for
enumeration of bacterial counts and for polyamine analysis. The rats
were given free access to the diets and water. Food intake was
determined daily and calculated as gram dry food · rat-1
· d-1. Each rat was weighed at the beginning and at the
end of the study. All treatments and diets were formally approved by
the Animal Welfare Committee of Brandenburg.
Microorganisms for colonization of the gut.
Bacteroides thetaiotaomicron (B. thetaiotaomicron, type
strain No. 2079; German Collection of Microorganisms and Cell Cultures,
Braunschweig, Germany) were grown in Wilkins-Chalgren broth.
Fusobacterium varium (F. varium, type
strain No. 8501; American Type Culture Collection, Rockville, MD) were
cultivated in Wilkins-Chalgren broth supplemented with
Na2HPO4 (2.0 g/L medium) and cysteine
hydrochloride (250 µg/L medium). Both organisms were cultivated
anaerobically for 24 h at 37°C using the Hungate technique
(Hungate 1969
). All chemicals and media were purchased
from Merck (Darmstadt, Germany) and from OXOID (Unipath GmbH, Wesel,
Germany).
Collection and chemical analysis of samples.
Rats were killed on d 25 of the experimental period by ether anesthesia
and decapitation at 0800 h. Both the terminal end of the ileum and
the proximal end of the colon were tied; thereafter the cecum was
removed, immediately weighed and the cecum content was collected.
Furthermore, the entire cecum tissue was cleaned with ice-cold
isotonic NaCl, blotted dry and weighed. The difference between the
weight of the total cecum (cecum content + tissue) and the cecum tissue
represents the total cecum content. The cecal pH was measured with a
microprocessor pH meter 537 A (Wissenschaftlich-Technische
Werkstätten GmbH, Weilheim, Germany). For polyamine analysis, the
samples were frozen in liquid nitrogen, lyophilized and stored at 4°C
in a desiccator until analysis (Noack 1998
). The
lyophilized material was also used for determination of total nitrogen
by the Kjeldahl method and for analyzing the contents of undigested
pectin (Blumenkranz and Asboe-Hansen 1973
,
Kunerth and Youngs 1984
). The analysis of
short-chain fatty acids
(SCFA)3
was performed with freshly collected cecum content. Samples were
prepared as described by Pomare et al. (1985)
. Briefly,
about 150–300 mg cecum content was diluted fivefold, homogenized and
centrifuged at 22,000 x g for 5 min at 4°C. The
supernatant (200 µL) was mixed with 23.6 µL 2-ethylbutyric acid (12
mol/L, internal standard), 280 µL HClO4 (0.36 mol/L), 270
µL NaOH (1 mol/L), frozen at -20°C and lyophilized. The dry
residue was mixed with 100 µL formic acid and 400 µL acetone,
thereafter centrifuged at 22,000 x g for 15 min.
One microliter of the supernatant was injected into a Hewlett Packard
5890, Serie II gas chromatograph fitted with a 25-m Carbowax 20 M
capillary column (i.d. 0.32 mm; Hewlett Packard-GmbH, Waldbronn,
Germany) and a flame ionization detector. The column temperature was
125°C and helium was the carrier gas. The flow rate was 12 mL/min.
The splitting rate was set at 1:10. All samples were analyzed in
duplicate.
Microbial analysis.
Feces or cecum contents were freshly collected in sterile tubes placed in ice and immediately transferred into an anaerobic chamber (MK3 anaerobic workstation; dW Scientific, West Yorkshire, England). Feces or cecum content (~0.5 g) was diluted 100-fold with prereduced Sörensen buffer (pH 6.8, 0.033 mol/L)4 by weighing. Following homogenization, the specimens were subjected to a series of 10-fold dilutions (up to 10-10) in prereduced Sörensen buffer. Numbers of bacteroides and total anaerobic counts were determined by plating 0.05 mL of each dilution in duplicate on Columbia agar plates supplemented with 5% sheep blood (BioMerieux, Nürtingen, Germany). Fusobacteria were differentiated from bacteroides by plating the dilutions described above on Brilliant Green agar plates.5 The inoculated media were incubated anaerobically at 37°C for 3 d and the colonies subsequently enumerated. The viable counts are expressed as log10 of colony-forming unit (cfu)/g pectin-free dry weight of feces or log10 cfu/total pectin-free dry cecum content.
Statistical analysis.
Data are expressed as means ± SEM. Germfree
rats (n = 18) were randomly assigned to one of
three treatments (n = 6): (i) fiber-free diet +
bacterial monoassociation, (ii) fiber-free diet + diassociation or
(iii) pectin diet + diassociation. The effect of the different
treatments on the cecal polyamine concentrations, total-N, dry
mass, pH, and SCFA concentrations was to be analyzed. Differences
between treatment groups for putrescine and spermine were analyzed by
Student’s t test for independent samples. Differences
among treatment groups for spermidine, total-N, dry mass, pH and
SCFA concentrations were established by one-way ANOVA
(SPSS® software for WINDOWSTM, Version 6.1.2.; SPSS, Chicago, IL, 1995). To compare mean values where appropriate, the least
significance difference (LSD) test (SPSS 1995) was used.
To analyze the effect of treatment on microbial counts in cecum of
mono- and disassociated ex-germfree rats, logarithmic data
transformation to log10 cfu were applied. Differences among
treatments for total counts were analyzed by one-way ANOVA. The LSD
test (SPSS 1995) was used to confirm differences among
treatment groups. Differences among treatments for fusobacterial counts
were analyzed by Student’s t test for independent
samples. The effect of time on the polyamine contents in feces in the
different treatment groups during the entire experimental period was
analyzed by two-way ANOVA for treatment and time. In every case
interactions between main effects treatment x time were observed
(P < 0.001). Therefore, differences among
colonization times in each treatment group were analyzed by one-way
ANOVA for days after microbial association. Significance of differences
was established at P 
0.05.
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RESULTS |
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The intake of food and energy as well as the weight gain of rats did not differ among the treatment groups. The food intake was 17.5 ± 0.25, 17.4 ± 0.27 and 17.5 ± 0.21g/d, resulting in an intake of energy of 289.3 ± 4.05, 287.7 ± 4.45 and 289.6 ± 3.48kJ/d for rats fed pectin-free diet + monoassociation, pectin-free diet + diassociation, and pectin-containing diet + disassociation, respectively. Values for the pectin treatment group were calculated on the basis of pectin-free dry mass of the diet. The body weight gain of the rats was 4.8 ± 0.16, 4.1 ± 0.10 and 4.1 ± 0.17g/d for the respective treatment groups.
Total cecum tissue and content, dry mass, total nitrogen and pH.
Total cecum tissue weight did not differ among the groups. In the mono-
and diassociated rats fed the fiber-free diet, total cecum content,
dry mass, total nitrogen and cecum pH were not different (Table 1
). However, consumption of the pectin diet by diassociated rats led to
60% greater total cecum content and the dry mass as compared to the
diassociated pectin-free treatment group (P < 0.001). Pectin also influenced the total nitrogen content which was
significantly lower (P < 0.001) in the cecum of the
pectin group than in that of the respective pectin-free group.
Furthermore, the diassociated rats fed the pectin diet had a lower
cecal pH (P < 0.002) than the diassociated rats fed
the pectin-free diet. The cecal content of undigested pectin in the
rats fed the pectin diet was 37.0 ± 1.5 g/100 g dry mass.
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In all three treatment groups, the microbial counts in the cecum of
mono- and diassociated rats were equal to or exceeded
1012 cfu (Table 2
). The total microbial counts in cecal contents of the diassociated
treatment groups (pectin-free and pectin group) were significantly
higher (P < 0.001) than in the cecum of the
monoassociated pectin-free group. It appears that the type of
carbohydrate in the diet (starch in the pectin-free diet or pectin)
did not affect the colonization of B. thetaiotaomicron and
F. varium in the cecum of diassociated rats as the total
microbial counts, and the F. varium counts in the
diassociated rats were similar, no matter whether they were fed the
pectin-free or pectin-containing diet. On the average the
proportion of F. varium was 12.4 ± 2.5% of the total
microbial counts in the cecum content, indicating that B.
thetaiotaomicron was the numerically dominant organism in the
microbial community.
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Spermidine was the predominant polyamine in cecal contents of
ex-germfree rats monoassociated with B. thetaiotaomicron
and fed the pectin-free diet (Table 3
). Putrescine and spermine were found in equal but low concentrations.
Diassociation of germfree rats with B. thetaiotaomicron +
F. varium in contrast led to an alteration of the cecal
polyamine pattern, independent of the consumed diet. Not only the
spermidine concentration was higher in the diassociated than in the
monoassociated rats, but also putrescine increased so much that it
became the predominant polyamine. The diassociated rats consuming the
pectin diet had higher cecal concentrations of all polyamines than
those fed the pectin-free diet (P < 0.01).
Cadaverine was not detected in any case.
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SCFA concentrations in cecum contents were affected by the status of
microbial colonization and the carbohydrate supplemented to the diet.
Thus, in the cecal contents of rats monoassociated with B.
thetaoitaomicron and fed the pectin-free diet, acetate and
propionate were the only SCFA detected (Table 4
). The concentration of total SCFA was significantly lower than that of
the diassociated treatment groups (P < 0.0001). In
diassociated rats consuming the pectin-free diet, the cecal
concentration of acetate was 70% higher than in the monoassociated
rats, and butyrate was formed in the former but not in the latter
group. The concentration of propionate was not altered in response to
microbial colonization. The proportions of acetate, propionate and
butyrate in relation to total SCFA were 75, 21 and 4%, respectively.
In the diassociated rats consuming the pectin diet, the concentrations
of all SCFA increased in comparison with the rats consuming the
pectin-free diet. The relative increases in propionate, butyrate
and total SCFA were 94, 99 and 34%, respectively.
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Microbial counts in feces were determined in all treatment groups throughout the experiment (25 d) to monitor the bacterial colonization of the gut. High and stable numbers were observed for total counts with 12.10 ± 0.14, 12.37 ± 0.11 and 12.54 ± 0.08 Log10 cfu/g dry mass for rats fed pectin-free diet + monoassociation, pectin-free diet + diassociation, and pectin-containing diet + diassociation, respectively. Total fecal counts were different only between diassociated rats consuming the pectin diet and monoassociated rats consuming the pectin-free diet (P < 0.02). Furthermore, the fecal numbers of fusobacteria in both of the diassociated treatment groups were also stable over the study period: 11.56 ± 0.09 Log10 cfu/g dry mass for the diassociated rats fed the pectin-free diet and 11.85 ± 0.20 Log10 cfu/g dry mass for the rats fed the pectin-containing diet. The proportion of fusobacteria was on the average 18.0% of the total bacterial counts in feces of these treatment groups.
Development of polyamine concentrations in feces.
The polyamine concentrations and patterns in feces were similar
in the three treatment groups within the first 3 d after
monoassociation of the rats with bacteroides, and spermidine was the
dominant polyamine (Table 5
). In the monoassociated rats fed the pectin-free diet, however, the
spermidine concentration decreased steadily from d 8 on after the
association. On d 25 only 43% of the original spermidine concentration
was still present. Nevertheless, throughout the entire experiment, the
fecal spermidine concentration was higher than that of any other
polyamine. The concentrations of putrescine and spermine did not alter
during the experimental period in this treatment group (rats
monoassociated with Bacteroides and fed the pectin-free
diet). In the diassociated rats consuming either pectin-free or
pectin diet, the fecal putrescine concentrations increased on d 8,
reached their maximal values on d 15 or d 10 and decreased thereafter
again. However, in no case were the putrescine concentrations
determined at the end of the experimental period so low as at its
beginning. In contrast, the spermidine concentration decreased on d 8
or 10 and reached a minimum on d 25 corresponding to 35 or 43% of the
original concentrations (pectin-free or pectin diet, respectively). In
both diassociated treatment groups, the spermine concentrations were
maximal at the beginning of the study, decreasing to only 30–40% of
the original concentrations on d 10, and thereafter spermine remaining
constant until the end of the experiment.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
In the present study, the polyamine pattern in cecal contents and
feces changed completely when germfree rats consuming a
polyamine-deficient fiber-free diet were colonized with B.
thetaiotaomicron. Spermidine now became the predominant polyamine
just as in conventional rats. The high microbial counts and the
bacterial propionate formation in the cecum of monoassociated rats
indicate that the associated bacteria were metabolically active and
would therefore have the ability to synthesize spermidine de novo just
like Bacteroides does in vitro (Noack et al. 1998
). As expected, the additional colonization of rats
associated with Bacteroides and fed a pectin-free or
pectin-containing diet with F. varium resulted in
elevated total microbial counts. Although the proportion of
fusobacteria was only 12.4% of the total bacteria present in cecal
contents of the diassociated treatment groups, putrescine became the
predominant polyamine in response to the presence of
Fusobacterium. The fusobacteria-associated intestinal
putrescine formation observed in this study is in accordance with
previous experiments which demonstrated that conventional rats fed a
pectin-supplemented diet, have higher cecal putrescine
concentrations and higher counts of bacteroides-fusobacteria than
the control rats fed a pectin-free diet (Noack et al. 1998
). That the total counts of cecal bacteria were higher in
the diassociated than in the monoassociated rats cannot only be
attributed to the fusobacteria being additionally present but also to
an increase in the Bacteroides cell counts by about log 1.0.
This increase was accompanied by a further elevation of the cecal
spermidine. It can be generally stated that the polyamine pattern
observed in cecal contents reflected the presence of the bacteria in
the cecum and their ability to synthesize a particular polyamine. The
amount of polyamines synthesized by the bacteria in the cecum was also
influenced by the content of dietary pectin. Pectin caused a further
elevation of all polyamine concentrations including that of spermine.
Pectin, which is not hydrolyzed by small intestinal enzymes, may serve
as a carbon source for a variety of saccharolytic bacteria including
B. thetaiotaomicron, which are capable of utilizing pectin
as a substrate (McCarthy et al. 1985
, Salyers and Leedle 1983
). The fact that the cell numbers in both
diassociated treatment groups were similar and the cecal contents of
SCFA were higher (P < 0.001) and the cecal pH was
lower (P < 0.002) in rats fed the pectin diet than in
rats fed the pectin-free diet suggests that the bacterial metabolic
activity is stimulated by pectin. In agreement with this notion, only
75% of the polymeric pectin fed to rats diassociated with
Bacteroides and Fusobacterium was recovered from
the cecum, indicating that 25% had been fermented.
The elevated spermine content found in the cecum of the diassociated
rats fed the pectin diet cannot be of bacterial origin as bacteria are
incapable of spermine synthesis (Tabor and Tabor 1985
).
As intestinal tissues contain high amounts of spermine (Sessa et al. 1995
), the high cecal spermine may be explained by an
increased cell proliferation of cecal tissue mediated through the
bacterial production of SCFA (Lupton and Kurtz 1993
,
Zhang and Lupton 1994
) and possibly through bacterial
polyamine formation.
Bacteroides species are primarily saccharolytic
(Bryant 1974
). In contrast, fusobacteria derive their
energy from the fermentation of amino acids (Roger et al. 1998
). The concomitant association of rats with two bacterial
species having complementary metabolic abilities and substrate
requirements may be responsible for the observed stimulation of the
metabolic activity of the bacterial community.
We used this simple model of bacterial mono- and diassociated
ex-germfree rats to examine the question whether microbial
polyamine formation in the cecum as stimulated by pectin has any
quantitative importance for the host. For this purpose we calculated:
(i) the amount of the polyamines potentially formed by the bacteria in
the cecum based on the in vitro polyamine formation rates of the
bacterial species present (Noack et al. 1998
) and
compared it with the actually found cecal polyamine contents
(Table 6
). The cecal contents of polyamines produced in vivo by the bacteria
were about two times higher than the calculated contents.
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). (ii) Bacteroides thetaiotaomicron and
F. varium synthesize in vitro 1µmol spermidine and 15.13
µmol putrescine/g bacterial dry mass, respectively. The proportion of
fusobacteria in cecum content of diassociated rats was 12.4% of the
total bacterial mass. The average daily dietary polyamine intake of rats fed the pectin-free or the pectin-containing diet (calculated from the polyamine content of the diet and the daily food consumption) was 0.05 µmol putrescine, 0.824 µmol spermidine and 0.368 µmol spermine. The amounts of polyamines synthesized in the cecum of the diassociated rats consuming the pectin diet were 84-fold higher for putrescine and fourfold higher for spermidine than the amounts consumed daily with the diet. Our data suggest that bacteria are able to synthesize high amounts of polyamines in vivo and that this synthesis is stimulated by polysaccharides such as pectin.
In our study, the intestinal concentrations of the individual
polyamines declined from cecum to feces when determined at the end of
the experimental period. Since the microbial counts were constantly
high in feces and cecal contents throughout the study, the data could
be explained by an absorption of polyamines in cecum and colon. The
decrease in polyamines (25–72% of putrescine, 44–74% of spermidine)
from cecum to feces was observed in all treatment groups. We speculate
that the rats had an increased demand for polyamines because
polyamine-deficient diets were consumed. Previous studies suggested
that dietary polyamines are essential for meeting the body’s need for
polyamines (Bardocz 1993
). We therefore propose that the
polyamines synthesized by the bacteria resident in the cecum may be
used in a similar way. In particular putrescine, which was shown in
this study to be produced by fusobacteria, may be of physiological
importance since it can be utilized as energy substrate in the gut when
required (Bardozc et al. 1998
). Intraluminal putrescine
acts as a growth factor for the gut (Ginty et al. 1998
,
Loser et al. 1999
) and is responsible for the healing of
injured intestinal mucosa (Otani et al. 1998
). The
pectin-stimulated polyamine formation by intestinal microorganisms
observed in our study may explain the improved postoperative
adaptability and intestinal functions following massive bowel
resections in humans and animals upon ingestion of
pectin-containing diets (Roth et al. 1995
). In
situations where cell proliferation is required, bacterial polyamine
formation is beneficial for the host organism. In other situations,
bacterial polyamines may also be harmful. The role of bacterial
polyamines in the gut for tumor growth has been discussed extensively
(Patel and Wang 1997
, Seiler et al. 1990
). In light of these undesirable effects of microbial
polyamines and the stimulation of bacterial polyamine formation by
pectin reported here, foodstuffs containing high amounts of
indigestible polysaccharides such as pectin may be an additional risk
for cancer patients. In agreement with this notion, pectin may enhance
tumorigenesis (Jacobs and Lupton 1986
).
Previous in vitro experiments have shown that gram-positive rods
such as bifidobacteria and eubacteria are not able to synthesize
polyamines when grown in polyamine-free medium (Noack et al. 1998
); results for eubacteria not published. Owing to the
inability of these bacteria to synthesize polyamines, the
supplementation of the diet with indigestible oligosaccharides,
particularly fructo-oligosaccharides, which cause bifidobacteria to
become numerically dominant in the gut (Bouhnik et al. 1999
, Kleessen et al. 1997
, Kruse et al. 1999
), offers the theoretical possibility to reduce the
bacterial formation of polyamines in the large intestine when
desirable.
In summary, the colonic microbiota are able to produce large amounts of polyamines in vivo. Indigestible polysaccharides such as pectin enhance polyamine synthesis by certain microbes such as B. thetaiotaomicron which are capable of fermenting pectin. Our model study also indicates that, simultaneously, polyamine formation by other bacterial species in the gut is stimulated by the supply of suitable metabolizable substrates.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: cfu, colony-forming
units; LSD, least significant difference; SCFA, short-chain fatty
acids.
4 Sörensen buffer used for microbial
analysis (0.033 mol/L). All substances were purchased from Merck
(Darmstadt, Germany) and from Difco (Augsburg, Germany). The
composition was as followes (g/L or µL/L): agar, 1.0;
KH2PO4, 4.5; NaHPO4 ·
2H2O, 6.0; cysteine-HCl, 0.25; thioglycolic acid, 400.
The final pH was adjusted to 6.8. This solution (4.5 mL) was filled in
tubes and autoclaved (20 min, 121°C).
5 Brilliant green agar used for enumeration of
Fusobacterium varium and differentiation from
Bacteroides thetaiotaomicron. All substances were
purchased from Merck, Sigma (Deisenhofen, Germany) and from BioMerieux
(Nürtingen, Germany). Substances were solved in distilled
H2O under stirring in the following order (g/L or µL/L):
Columbia Agar, 42.5; Tween 80, approximately; 200; Brilliant Green,
0.008 (the mixture was cooked for 1min); cysteine-HCl, 0.5 (the
mixture was cooked again for 2 min). Thereafter, the medium is ready
for use. The medium must not be autoclaved.
Manuscript received September 14, 1999. Initial review completed November 15, 1999. Revision accepted January 4, 2000.
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REFERENCES |
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|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2. Bardocz S., Duguid T. J., Brown D. S., Grant G., Pusztai A., White A., Ralph A. The importance of dietary polyamines in cell regeneration and growth. Brit. J. Nutr. 1995;73:819-828[Medline]
3.
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