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German Institute of Human Nutrition, Department of Gastrointestinal Microbiology, 14558 Potsdam-Rehbrücke, Germany
1To whom correspondence should be addressed.
 |
ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-D-glucoside was a less effective inhibitor than
glucose, and 2-deoxy-D-glucose did not inhibit LA
conjugation at all. To analyze the concentration of carbohydrates in
intestinal contents, the LA-conjugating bacterial mixed culture and
human fecal microorganisms were introduced into germ-free rats.
Samples of feces and cecum and colon contents of both groups exhibited
in vitro LA-conjugating activity. Rats associated with human
intestinal microorganisms contained 5.7 ± 1.3 mmol glucose/L in
the cecal contents and 6.6 ± 1.0 mmol glucose/L in the colonic
contents. Rats associated with CLA-producing bacterial culture
contained 3.4 ± 1.3 mmol glucose/L in the cecal contents and 4.2
± 1.0 mmol glucose/L in the colonic contents. These values are
within a range that may explain the observed inhibition of LA
conjugation in vivo.
KEY WORDS: • conjugated linoleic acid • gnotobiotic rats • glucose • inhibition • intestinal microorganisms
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). A mixture of isomers of conjugated linoleic acid
(CLA) is produced that differ with respect to the position of the
double bonds (
79,
911 or
1012) and the
cis-trans stereochemistry (Ha et al. 1987
, Yurawecz et al. 1998
). Conjugated linoleic
acid is absorbed by ruminants and enriched in milk fat and body fat up
to 87 µmol CLA/g fat, depending on the feeding regimen
(Kelly et al. 1998
).
After anticarcinogenic activity of CLA was reported by Ha et al. (1987)
, several investigations established that CLA is an
effective anticarcinogen, inhibiting skin, mammary and forestomach
neoplasia in humans and rodents (Ha et al. 1990
,
Ip 1997
, Ip et al. 1999
, Liu and Belury 1997
). An antiatherogenic effect of CLA was postulated
(Nicolosi et al. 1997
), but questioned recently
(Munday et al. 1999
). Furthermore, report of a decrease
of body fat in mice has been published (Park et al. 1999
).
Although most CLA in the human diet seems to be of bacterial origin,
very little is known about the production of CLA by bacteria. In the
rumen, Butyrivibrio fibrisolvens is the only known organism
capable of CLA production (Kepler et al. 1966
).
Moreover, bacteria in the intestine of monogastric animals and humans
are capable of CLA production. The increase in CLA concentration in
various tissues of rats in response to feeding free LA was explained by
the activity of intestinal bacteria (Chin et al. 1994
).
However, the underlying formation of CLA seems to be restricted to the
feeding of free LA. In contrast to the situation in ruminants, the
consumption of LA esterified to glycerol by humans and rats did not
increase the amount of CLA in serum and various body tissues
(Chin et al. 1994
, Herbel et al. 1998
).
Recently, we demonstrated that gnotobiotic rats associated with a mixed
bacterial culture capable of in vitro CLA formation did not accumulate
CLA in various body tissues when fed a sunflower seed oil–fortified
diet (Kamlage et al. 1999
). This bacterial culture had
been enriched from a fecal sample of a human volunteer as described
recently (Kamlage et al. 1999
). CLA production of the
culture was highly oxygen sensitive and occurred only in the late
stationary growth phase (after 90 h). Only free, nonesterified LA
was a substrate of conjugation, and CLA was released into the medium
and not incorporated into cell lipids.
Fecal samples from these rats exhibited in vitro LA-conjugating activity in contrast to fecal samples from the germ-free control group. The aim of this study was to identify conditions that inhibit the LA-conjugation activity of the bacterial mixed culture in vitro. The results of our experiments led to a new hypothesis that may explain the observed absence of in vivo LA-conjugation activity.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Enrichment of the CLA-producing bacteria from a human fecal sample
was described recently (Kamlage et al. 1999
). The
culture was maintained in rubber-stoppered anoxic cultivation tubes
containing the following compounds (per L): 9 g tryptic peptone
from meat, 1 g proteose peptone, 3 g meat extract, 4 g
yeast extract, 3 g NaCl, 2 g Na2HPO4,
0.5 mL Tween 80, 0.1 g MgSO4 · 7 H2O, 5
mg FeSO4 · 7 H2O, 3.4 mg MnSO4
· 2 H2O, 0.25 g L-cysteine · HCl, 0.25 g L-cystine, 10 mg hemin and 1 mg resazurin. The gas phase
consisted of 80 vol% N2 and 20 vol% CO2, and
the pH was adjusted to 6.8–7.0. After autoclaving, 4 mmol/L of
filter-sterilized (0.2-µm sterile filter,
Sartorius, Göttingen, Germany) LA was added (Roth, Karlsruhe,
Germany). Cultures were shaken at 37°C on a rotary shaker (140
revolutions/min). After incubation for at least 90 h, samples were
subjected to CLA analysis (see below).
Experiments with growing cells.
To investigate the substrate inhibition of LA conjugation, concentrated anaerobic stock solutions of various mono-, di-, tri- and polysaccharides, aminosugars, sugar alcohols and pyruvate were sterilized by autoclaving (meso-erythritol, glycerol, meso-inositol, D-sorbitol, inulin, starch from potatoes) or filter-sterilized (N-acetyl-D-glucosamine, D-arabinose, D-fructose, D-galactose, D-glucose, D-glucosamine, D-mannose, D-ribose, D-xylose, D-cellobiose, D-lactose, D-lactulose, D-maltose, D-saccharose, D-trehalose, D-melezitose, D-raffinose, sodium fumarate, D/L-sodium lactate, sodium pyruvate). Substrates were added to the medium described above to a final concentration of 33 mmol/L (monosaccharides, aminosugars, sugar alcohols and pyruvate), 16.5 mmol/L (disaccharides, fumarate and D/L-lactate), 11 mmol/L (trisaccharides) and 1 g/L (polysaccharides), respectively. For each substrate, four parallel samples were inoculated with the CLA-producing mixed culture and shaken at 37°C on a rotary shaker. After incubation for 90 h, samples were subjected to CLA analysis (see below). Cultures without substrate served as controls.
The concentration-dependent inhibition of LA conjugation by glucose
and the effects of the glucose analogs
methyl--D-glucoside and 2-deoxy- D-glucose
(Sigma, Deisenhofen, Germany) were investigated by inoculation of media
containing increasing concentrations of filter-sterilized
substrates from 1 to 20 mmol/L with the mixed bacterial culture.
Animal experiment.
Specifications of the germ-free rat strain
AVN-Ipcv-Wistar-Rehbrücke were given recently (Kamlage et al. 1999
). The protocol for the animal experiment was approved
by the Ministry of Nutrition, Agriculture and Forestry, Brandenburg,
Germany.
Germ-free male rats (n = 12; 5 wk old), weighing 117 ± 7.5 g, were divided randomly into two groups. They were fed an irradiated pelleted diet (25 kGy) consisting of a commercial rat breeding diet (type 1311, Altromin, Lage, Germany) with the following composition (per kg): crude protein (225 g), crude fat (50 g), crude fiber (45 g), ash (65 g), moisture (135 g) and nitrogen free extract (480 g). The diet was supplemented with 60 g of sunflower seed oil (Brölio, Hamm, Germany), 20 mg BHT as an antioxidant and 7.5 g CaCO3 per kilogram diet, resulting in 110 g fat and 12 g calcium per kilogram diet. The diet was stored at room temperature. Rats were maintained in plastic film isolators and housed in polycarbonate cages (2 rats/cage) on irradiated wood chips at 22 ± 2°C, 55 ± 5% relative humidity with a 12-h light:dark cycle (0700–1900 h). The rats were weighed once per week. They had free access to diet and autoclaved distilled water. Coprophagy was not prevented. Diet samples were taken at the end of the experiment from the isolators and analyzed for total fat, LA and CLA (see below). The six rats of the first group were each inoculated intragastrically with 0.5 mL of a suspension of 2 g human feces in 2 mL of the medium described above. The six rats of the second group were inoculated intragastrically with 0.5 mL of the CLA-producing mixed bacterial culture described above.
Fresh fecal samples were taken directly from the anus of every rat twice per week and analyzed for bacterial cell counts and LA-conjugating activity (see below). A period of 4 wk was required to establish stable cell counts in the gnotobiotic rats and to verify LA-conjugating activity. After 4 wk, the rats were killed by CO2 inhalation. Cecal and colonic contents were removed under sterile conditions and analyzed for LA-conjugating activity. Remaining cecal and colonic contents were stored frozen at -20°C and analyzed for glucose and total reducing carbohydrates (see below).
Determination of LA-conjugating activity, LA and CLA.
Two hundred microliters each of the anoxic suspensions prepared from
feces or from cecum or colon contents (see above) were transferred to
two rubber-stoppered anoxic cultivation tubes filled with 5 mL of
the liquid medium described. The gas phase was 80 vol% N2
and 20 vol% CO2. Filter-sterilized LA was added to a
final concentration of 4 mmol/L and the cultures were shaken at 37°C.
Media without inoculum served as controls. LA and CLA was analyzed by
HPLC after lipid extraction as described recently (Kamlage et al. 1999
).
Quantification of glucose and total reducing carbohydrates.
Cecum and colon content samples of ~0.2 g were exactly weighed, diluted fivefold with distilled water and sonified for 10 min. Samples were centrifuged (20,000 x g, 15 min, room temperature) and analyzed in duplicate for glucose and for total reducing carbohydrates.
For glucose determination, 0.5 mL ethanol was added to 200
µL of the supernatants and the samples were
centrifuged (20,000 x g, 15 min, room
temperature). The supernatants were dried in a rotoevaporator at 4°C
(Jouan, Saint-Herblain, France), and 200 µL
distilled water was added to each tube. Glucose was determined
enzymatically as described (Kunst et al. 1984
). Controls
received water instead of sample or glucose-6-phosphate dehydrogenase.
For determination of total reducing sugars (Southgate 1991
), supernatants were diluted 100-fold with distilled water.
To a 0.5-mL sample, 0.5 mL water, 1.0 mL of a solution containing
5.3 g Na2CO3/L and 0.65 g KCN/L, and
1.0 mL of a solution containing 0.5 g
K3Fe(CN)6/L were added and the samples were
incubated in a boiling water bath for 15 min. After cooling, 5.0 mL of
a solution containing 1.5 g
Fe(NH4)(SO4)2/L and 0.025 mol
H2SO4/L was added and the absorption was
determined at 700 nm. Calibration was done with a solution containing
glucose, fructose, maltose and cellobiose, each at a concentration of
1.25 mmol/L. Controls received water instead of sample or
Fe(NH4)(SO4)2.
Determination of LA and CLA in diet samples.
Diet samples (~0.2 g) were exactly weighed, ground at room
temperature to a powder and the lipids were extracted with 10 mL of
petroleum ether. The lipids were saponified in duplicate and analyzed
as described (Kamlage et al. 1999
).
Statistical analysis.
Results are expressed as means ± SD. The effects of
glucose and methyl--D-glucoside were evaluated using the
nonparametric Kruskal-Wallis test on ranks and the two-sided
U-test. P-values 
0.02 were considered to be
significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Without any substrate added, concentrations of 0.2–1.4 mmol CLA/L were produced from 4 mmol LA/L by the bacterial mixed culture. In the presence of N-acetyl-D-glucosamine, D-arabinose, D-cellobiose, D-fructose, fumarate, D-galactose, D-glucosamine, D-glucose, glycerol, inulin, D-lactose, D-lactulose, D-maltose, D-mannose, D-melezitose, sodium pyruvate, D-raffinose, D-ribose, saccharose, D-sorbitol, starch, D-trehalose and D-xylose, the CLA concentrations in the cultures did not exceed the CLA concentrations in the uninoculated controls. The growth of the bacteria was not inhibited by the substrates as evident from microscopic examinations.
In contrast, meso-inositol, meso-erythritol and D/L-lactate did not inhibit CLA production of the culture. This indicated that a simple osmotic effect cannot explain the inhibition of LA conjugation by the various carbohydrates. It is not known whether the growth of the LA-conjugating species in the mixed bacterial culture was specifically repressed or whether the enzymatic CLA-producing activity was inhibited.
To investigate the inhibitory effect of glucose in more detail, media
with increasing concentrations of glucose were inoculated with the
CLA-forming culture and after incubation for 90 h, analyzed
for CLA (Fig. 1A
). In the presence of >2 mmol glucose/L, a significant (P
< 0.01) inhibition of CLA production became evident. The CLA
concentrations found in cultures with >5 mmol glucose/L were in the
range of those measured in uninoculated controls, indicating that
essentially no LA conjugation had occurred in these cultures. The
glucose concentration that inhibited LA conjugation by 50% was
calculated to be 1.5 mmol glucose/L.
|
-D-glucoside was a weaker inhibitor of LA
conjugation than glucose (Fig. 1B
). In contrast, 2-deoxy-
D-glucose in concentrations of up to 20 mmol/L did not
inhibit LA conjugation (data not shown), supporting the view that the
observed inhibition of LA conjugation was not caused by a simple
osmotic effect. Animal experiment.
The total fat concentration of the diet was 83.2 ± 2.9 g/kg, containing 1500 ± 160 µmol LA/g fat and 7.6 ± 1.2 µmol CLA/g fat. The gain in body weight did not differ significantly between the groups (data not shown).
The activities of LA conjugation in samples obtained from rats
associated with human fecal microorganisms and from rats associated
with the LA-conjugating mixed bacterial culture are given in
Table 1
. The LA-conjugating activities in fecal samples were stable
throughout the experiment. These data indicated that the microorganisms
capable of in vitro LA conjugation had successfully colonized the
rats’ intestinal tract.
|
. Although the glucose
concentrations varied considerably, it is clear that the concentrations
of glucose and total reducing sugars were within a range that inhibited
completely CLA synthesis in vitro. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Herbel et al. 1998
,
Kamlage et al. 1999
). Thus, for humans, the sole source
of CLA is the diet. Nevertheless, intestinal microorganisms exhibit in
vitro LA-conjugating activity. Here, we present evidence for the
inhibition of bacterial CLA synthesis by glucose and other
carbohydrates. The concentrations of glucose and total reducing
carbohydrates found in cecum and colon contents of gnotobiotic rats
were sufficiently high to explain the observed inhibition of CLA
synthesis in vivo.
One of the most surprising findings of our study were the relatively
high concentrations of glucose and reducing sugars in intestinal
contents. These may result from the bacterial degradation of
nondigestible carbohydrates such as resistant starch, and are
intermediates in the ensuing fermentation to butyrate, propionate,
acetate, formate, lactic acid and CO2. In rumen
fluid from cows consuming a forage-based diet, considerably lower
concentrations of glucose (0.55 mmol/L) and reducing sugars (0.64
mmol/L) were reported (Piwonka et al. 1994
) compared
with the results for cecum and colon contents in this study. The
concentrations found in rumen fluid are in a range that in our
experiments would inhibit LA-conjugation activity only partially.
In the rumen, CLA is synthesized from LA esterified to glycerol, e.g.,
from sunflower seed oil (Kelly et al. 1998
). This is in
contrast to the situation in the cecum and colon of monogastrics
(Chin et al. 1994
, Kamlage et al. 1999
).
This difference may be explained by differences in the composition of
the microbial communities of the rumen on the one hand and the cecum
and colon on the other hand. For instance, B. fibrisolvens,
a ruminal bacterium capable of CLA synthesis (Kepler et al. 1966
), is not a member of the bacterial culture studied in this
investigation (Kamlage et al. 1999
).
The exact mechanism of the glucose-mediated inhibition of LA
conjugation is still unknown. Theoretically, glucose may have either
inhibited the growth of the LA-conjugating bacteria in the mixed
culture or the expression or activity of the LA-conjugating enzyme
system. LA-conjugation activity of the bacterial mixed culture is
dependent on the growth phase and occurs only in the late stationary
phase (Kamlage et al. 1999
). It is not yet known whether
the metabolic conditions of the stationary growth phase in vitro are
similar to the conditions in vivo when the bacteria grow inside the
cecum or colon. We speculate that the restriction of LA-conjugating
activity to the late stationary growth phase and the observed
inhibition by glucose and other substrates were caused by the same
mechanism. It is conceivable that the absence of glucose and other
carbohydrates or stationary growth phase conditions signaled
unfavorable growth conditions to the cells.
The experiments with glucose analogs might give a first hint to the
inhibition mechanism. 2-Deoxy-D-glucose is taken up and
phosphorylated, but not metabolized any further by most bacteria. This
glucose analog did not inhibit LA conjugation, indicating that it is
not glucose itself, but a metabolite of glucose degradation that
mediates the observed effect. Methyl--D-glucoside may be
transported but, depending on the organism studied, it may be
phosphorylated (Vadeboncoeur and Trahan 1982
) or
nonphosphorylated (Brocklehurst et al. 1977
). The weak
inhibitory effect of methyl--D-glucoside on LA
conjugation seems to contradict the results observed with
2-deoxy-D-glucose. One possible explanation is a slow
degradation of methyl--D-glucoside, releasing small
amounts of glucose (Brocklehurst et al. 1977
).
Nothing is known about the possible physiologic advantage of the
inhibition of LA conjugation by glucose for the microorganisms.
Moreover, the benefit of CLA synthesis itself is obscure. CLA was
proposed to be an intermediate in the biohydrogenation of LA to stearic
acid carried out by the concerted activity of rumen microorganisms
(Kelly et al. 1998
).
|
ACKNOWLEDGMENTS |
|---|
Manuscript received December 16, 1999. Revision accepted March 28, 2000.
|
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ha Y. L., Grimm N. K., Pariza M. W. Anticarcinogens from fried ground beef: heat altered derivatives of linoleic acid. Carcinogenesis 1987;8:1881-1887
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Ha Y. L., Storkson J., Pariza M. W. Inhibition of benzo(a)pyrene-induced mouse forestomach neoplasia by conjugated dienoic derivatives of linoleic acid. Cancer Res 1990;50:1097-1101
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Ip C. Review of the effects of trans fatty acids, oleic acid, n-3 polyunsaturated fatty acids, and conjugated linoleic acid on mammary carcinogenesis in animals. Am. J. Clin. Nutr. 1997;66(suppl):1523S-1529S
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Kepler C. R., Hirons K. P., McNeill J. J., Tove S. B. Intermediates and products of the biohydrogenation of linoleic acid by Butyrivibrio fibrisolvens. J. Biol. Chem. 1966;241:1350-1354
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