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Deutsches Institut für Ernährungsforschung (German Institute of Human Nutrition), D-14558 Bergholz-Rehbrücke, Germany
2To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEstimation of microbial lysine...Site and precursor pool...Evidence for absorption of...Evidence for absorption of...Amino acid losses from...Nitrogen sources for microbial...Dietary effects on intestinal...Relevance for determining adult...REFERENCES |
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KEY WORDS: • intestinal bacteria • amino acid synthesis • tracer balance • large intestine • amino acid requirement
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEstimation of microbial lysine...Site and precursor pool...Evidence for absorption of...Evidence for absorption of...Amino acid losses from...Nitrogen sources for microbial...Dietary effects on intestinal...Relevance for determining adult...REFERENCES |
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) from the small to the large
intestine, but they are not uniformly distributed in either number,
species or metabolic activity (Table 1
) (Autenrieth 1998
, Goldin 1990
,
Hovgaard and Brondsted 1996
). Thus, for persons living
in Europe and North America, microbial counts increase along the
intestinal tract, with a bacterial concentration of
~102/mL at the proximal intestine and
105 to 109/mL in the region
of the distal ileum. The flora of the lower ileum is qualitatively
similar to that of feces, with the latter showing a bacterial count in
the range of 108 to
1012/mL. It has been shown that certain
Lactobacillus and Bifidobacteria strains and
commensal bacteria such as Escherichia coli can adhere to
the mucosal surface, in particular to the terminal ileum and cecum
(Alverdy et al. 1994
, Hendrickson et al. 1999
, Macfarlane and Cummings 1999
). For
pathogenic bacteria, adherence is thought to be a prerequisite for
invasion (Hendrickson et al. 1999
). Under normal
conditions, however, intestinal epithelial cells remain relatively free
of adherent bacteria (Hendrickson et al. 1999
), whereas
bacteria are attached to the mucus layer lining the intestinal walls
(Hume 1996
).
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, Mack et al. 1999
, Spitz et al. 1994
) and dietary factors
(Alverdy and Stern 1998
, Kim et al. 1998
,
Macfarlane and Cummings 1999
) but also can communicate
with the host cells to cause them to fulfill their needs as shown for
Bacteroides thetaiotaomicron (Gordon et al. 1997
, Hooper et al. 1998
). The
rhizobium-legume symbionts serve as an example for nonpathogenic
interactions in which the secretion of soluble factors by the bacteria
symbiont influences development and differentiation of the host.
Among the numerous biochemical reactions of which microorganisms
are capable (Bengmark 1998
, Goldin 1990
)
is the ability of amino acid production from nonspecific nitrogen
sources driven by energy generated from dietary and endogenous
fermentable carbohydrates (Matteuzzi et al. 1978
,
Reitzer and Magasanik 1996
, Sauer et al. 1975
). Various human and animal studies show that the
administration of nonspecific 15N (e.g., urea,
ammonium chloride) can be used to label microbial lysine and threonine,
which is subsequently termed the "15N labeling
paradigm." The appearance of 15N-labeled lysine
and threonine in body fluids or proteins presumably indicates their
absorption from microbial sources (Torrallardona et al. 1996a
) because these amino acids are not transaminated by
mammalian tissues.
Among the reasons suggested for the discrepancy between N balance
and tracer-derived amino acid requirement estimates is the
possibility that the metabolic requirement, i.e., the irreversible loss
of indispensable amino acids
(IAA)3
(of which oxidation is the major
component), is met not only by the diet but also by amino acids
synthesized de novo by the gastrointestinal microflora, which are then
absorbed. It is therefore crucial to better understand and quantify the
microbial biosynthesis of amino acids in the human gastrointestinal
tract and its potential role in providing IAA to meet human amino acid
requirement (Fuller and Garlick 1994
). Hence, the
objectives of this article are i) to compile the available
evidence on the contribution of microbial amino acids to the host’s
amino acid homeostasis, ii) discuss factors that may
influence estimates of microbial amino acid synthesis and
iii) to assess the importance of the available evidence of
microbial amino acid contribution in defining the adult requirement of
IAA.
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Estimation of microbial lysine contribution to lysine homeostasis of the host using 15N labeling paradigm |
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TOPABSTRACTINTRODUCTIONEstimation of microbial lysine...Site and precursor pool...Evidence for absorption of...Evidence for absorption of...Amino acid losses from...Nitrogen sources for microbial...Dietary effects on intestinal...Relevance for determining adult...REFERENCES |
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,
Tanaka et al. 1980
) that microbial lysine can be
incorporated into host body protein, but no attempt had been made to
quantify this contribution. Utilization of microbially derived IAA has
been confirmed in pigs and rats fed protein-free or low protein
diets (Torrallardona et al. 1994
and 1996a
), whereas it
was reported that absorption of microbial lysine in the rat was
exclusively due to coprophagy (Torrallardona et al. 1996b
). In a study in protein-energy–malnourished infants, the
appearance of urinary 15N lysine after the oral
intake of 15N urea was observed (Yeboah et al. 1996
). In recent studies in adult humans on nitrogen
(protein)-adequate diets, we observed a significant contribution of
microbially derived lysine and threonine to the free plasma lysine and
threonine pool (Metges et al. 1999a
and 1999b
).
In the following, the experimental design used and the main outcomes of
our studies are summarized, and results are compared with similar
studies in animals and men. Healthy young adults and a group of
otherwise healthy subjects with ileostomies were adapted to an adequate
diet based on a crystalline amino acid mixture for four days
(Metges et al. 1999a
). On two different occasions and
after taking appropriate baseline samples between days 5 and 11,
isonitrogenous amounts of
15NH4Cl or
15N2 urea, respectively,
were added daily to the diet of the normal subjects to label
microbially synthesized amino acids. The ileostomy subjects were
studied with 15NH4Cl only.
The comparison between
15NH4Cl and
15N2 urea was made because
a pilot study in minipigs suggested a different metabolic and microbial
fate of those two sources of nonspecific nitrogen (Metges et al. 1996
). The study of the two groups of subjects, one with an
intact gastrointestinal tract and one without a large intestine
(ileostomates), enabled us to compare estimates of microbial lysine and
threonine contribution by using ileal and fecal microbial protein as
putative precursor for lysine and threonine absorption. The use of gas
chromatography–combustion isotope ratio mass spectrometry allowed us
to measure 15N enrichment of circulating free
plasma amino acids, which would have not been possible using
preparative ion exchange (Torrallardona et al. 1996a
,
Yeboah et al. 1996
). The fractional appearance of
microbial lysine (or threonine) in the circulating plasma was
calculated as the ratio of plasma free 15N lysine
(or 15N threonine) to the presumable precursor,
i.e., fecal or ileal microbial protein-bound lysine (or threonine),
respectively (Table 2
). In an attempt to quantify the microbial lysine and threonine
contribution to the host homeostasis, we multiplied this ratio by the
plasma lysine and threonine turnover, respectively, measured via
intravenous 13C lysine infusion in the same
subjects or taken from the literature (Zhao et al. 1986
)
(Table 2)
. Microbial threonine and lysine contribution ranged from 8 to
17% and from 5 to 21%, respectively. These estimates correspond to a
microbial contribution of lysine and threonine to the plasma flux
ranging from 11.7 to 67.9 and from 21 to 44.7 mg ·
kg-1 · d-1 in normal
adults, respectively.
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presents a summary of the available data on microbial lysine appearance
in the host body protein or amino acid pools in rats, pigs and humans.
In applying the 15N labeling paradigm, between 1
and 20% of circulating plasma lysine, urinary lysine and body protein
lysine of the host is derived from intestinal microbial sources (Table 3)
. There is a trend toward higher numbers when the ileal microbial
protein was used as precursor, whereas it appears that the calculated
contribution to body protein lysine is lower.
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Site and precursor pool of microbial amino acid absorption |
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TOPABSTRACTINTRODUCTIONEstimation of microbial lysine...Site and precursor pool...Evidence for absorption of...Evidence for absorption of...Amino acid losses from...Nitrogen sources for microbial...Dietary effects on intestinal...Relevance for determining adult...REFERENCES |
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) in regard to colon cancer protection and systemic
immunity. A possible role of the ileal microflora for host amino acid
nutriture has not been considered so far. In contrast to the available
evidence that quantitative important amino acid absorption occurs only
in the small intestine, the possibility of amino acid absorption from
the large intestine should not generally be excluded for reasons
indicated later. In addition, it is still an open question in which
form microbial amino acids are absorbed (i.e., free amino acids or
peptides). Because use of the 15N labeling
paradigm requires knowledge of the true precursor enrichment for
absorption, it is crucial to address these concerns.
Intestinal absorption of amino acids has been shown to be maximal in
the mid-lower jejunum, and human studies with ileal tubes show that
at the ileum level, dietary nitrogen is still recovered, suggesting a
role of the ileum for complete uptake of dietary amino acids
(Gaudichon et al. 1999
). This conclusion is supported by
the observation that peptide transporters were upregulated in the
distal regions of intestine by a high protein diet (Erickson et al. 1995
). There is evidence that enterocytes high on the
villus are mainly responsible for absorption. In studies of the
injection of mRNA from rat small intestine into Xenopus
laevis oocytes, expression of three types of lysine transport has
been identified (Munck and Munck 1994
). The peptide
transporter PEPT1 has been localized to the apical microvillus plasma
membrane of the absorptive epithelial cells of the rat small intestine
and shown to be responsive to nutritional condition (Erickson et al. 1995
, Ogihara et al. 1999
). PEPT1 appears to
be exclusively expressed in small intestinal tissues, but no PEPT1 mRNA
could be detected in large intestinal tissues of various species
(Chen et al. 1999
, Doring et al. 1998
).
However, weak signals of PEPT2 specific fragments have been identified
in rabbit colon (Doring et al. 1998
), although the
importance of this finding for human colon epithelial cells remains to
be seen.
M cells, an epithelial cell phenotype that occurs only over organized
mucosal lymphoid follicles, deliver samples of foreign material
(antigens and microorganisms) via transepithelial transport from the
lumen to organized lymphoid tissues within the mucosa of the small and
large intestines (Sansonetti and Phalipon 1999
). In
healthy animals, it is likely that spontaneous bacterial translocation
occurs at a low rate but that bacteria are killed by the host immune
defense. Hence, it would be theoretically possible that
15N-labeled bacterial material enters the
circulating plasma via this pathway as indicated for
14C-labeled E. coli in mice
(Gianotti et al. 1995
).
Absorption of microbial protein–derived amino acids would require that
microbial protein breakdown occurs at the ileum. There is evidence for
high proteolytic activity in human ileal effluents due to small
intestinal peptidases but also due to bacterial proteases
(Macfarlane et al. 1988
and 1989
). For some bacteria,
peptides derived from microbial protein breakdown and dead and lysed
bacteria cells may not be immediately reincorporated into microbial
protein but instead released into the surrounding medium (Cotta and Russell 1996
). It is interesting to note that
peptide-bound amino acids contributed to ~50% to the portal
plasma amino acid pool in the rat (Seal and Parker 1991
). However, the extent to which these peptides may be
derived from microbial protein is not known.
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Evidence for absorption of microbial amino acids from small intestine |
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TOPABSTRACTINTRODUCTIONEstimation of microbial lysine...Site and precursor pool...Evidence for absorption of...Evidence for absorption of...Amino acid losses from...Nitrogen sources for microbial...Dietary effects on intestinal...Relevance for determining adult...REFERENCES |
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). No enrichment has been found in the small intestine with
the exception of the ileum, whereas a substantial enrichment in the
cecum, followed by an increase toward the distal colon comparable to
the enrichments in feces, has been observed (Torrallardona et al. 1994
). Based on the comparison of enrichments of both
isotopes in the digesta and the carcass, it was suggested that
microbial lysine has been absorbed in the small intestine. The same
authors studied the site of microbial lysine absorption by returning
15N-labeled digesta into the ileum of unlabeled
pigs. They found that at least 75% of total microbial lysine
absorption occurred in the small intestine (Torrallardona et al. 1996b
).
Our investigations in pigs with end-to-end ileorectal anastomosis and
patients with ileostomy indicate that microbial amino acids can be
synthesized in the small intestine, and their appearance in the free
plasma pool indicates their absorption from that site (Metges et al. 1996
and 1999a
). However, ileostomy as well as ileorectal
anastomosis is prone to secondary colonization and possibly alterations
of digesta transit rate in the gut. Hence, although it might be not
completely comparable to the microbial situation in an intact
gastrointestinal tract, it demonstrates the principal possibility of
microbial lysine and threonine absorption from the small intestine.
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Evidence for absorption of microbial amino acids from large intestine |
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TOPABSTRACTINTRODUCTIONEstimation of microbial lysine...Site and precursor pool...Evidence for absorption of...Evidence for absorption of...Amino acid losses from...Nitrogen sources for microbial...Dietary effects on intestinal...Relevance for determining adult...REFERENCES |
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). However, the mere
appearance does not imply the absorption of intact microbial amino
acids. Also, the finding that 30–100% of free lysine, threonine,
serine, histidine and arginine from an enzymatic casein hydrolysate
solution disappeared from the cecum of pigs (Olszewski and Buraczewski 1978
) has to be viewed with caution because the
surgical isolation of the cecum and the continued antibiotic lavages
generate rather artificial conditions that may not be comparable to
normal conditions.
Using 15N-labeled rectum content, the appearance
of 15N lysine and other 15N
amino acids were detected in the colic branch of the ileocolic vein
3 h after infusion into the cecum (Niiyama et al. 1979
). In the same study, it has been shown that the enrichment
of amino acids in the digesta differed largely between the free and the
protein-bound amino acid pool (Table 4
). This observation emphasizes the importance of knowing the true
precursor pool for microbial amino acid absorption.
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suggests a
role of the colon in the absorption of nitrogen (Metges et al. 1999a
and 1999b
). Amino acids undergoing a moderate nitrogen
exchange in the body (valine, histidine) show rather comparable
numbers, whereas glutamic acid and alanine, which are known for
extensive nitrogen exchange, differ by a factor of 2 between normal
subjects and ileostomates, thereby indicating an uptake of
15N-labeled nitrogen by the large intestine
(Table 2)
. However, this does not shed light onto the identity of the
substances absorbed.
On the other hand, several reports indicate that amino acid absorption
from the large intestine is negligible in nonruminant animals
(Darragh et al. 1994, Hume et al. 1993
).
However, Fuller and Reeds (1998
) recently summarized
data on N balance measurements in pigs when protein or amino acids were
infused into the large intestine and found that the whole body N
balance was always slightly improved. This suggests that there is
protein digestion and that there might be absorption of amino acids.
However, the identity of the substances absorbed may still be in
question because no nutritional benefit was seen when lysine was
infused into the cecum, although the latter finding does not exclude
the possibility of peptide absorption as mentioned earlier.
In conclusion, it appears as if the small intestine is responsible for a large part of microbial lysine uptake, although some absorption from the large intestine cannot be excluded as based on new information on peptide transporters in colon tissue.
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Amino acid losses from gastrointestinal tract |
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TOPABSTRACTINTRODUCTIONEstimation of microbial lysine...Site and precursor pool...Evidence for absorption of...Evidence for absorption of...Amino acid losses from...Nitrogen sources for microbial...Dietary effects on intestinal...Relevance for determining adult...REFERENCES |
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.
Other routes of lysine loss have been considered to be minor and thus
were not included in the calculation of lysine balance. Lysine
oxidation, in contrast to leucine oxidation, is different when given
orally and intravenously (El-Khoury et al. 1998
), and
the intravenous 13C lysine tracer results in an
underestimation of lysine oxidation, suggesting that first-pass
splanchnic and possibly microbial oxidation contributes to the whole
body lysine oxidation rate.
Assuming that there is no quantitative important absorption of amino
acids from the large intestine, it is thought that IAA entering the
cecum through the ileocecal sphincter are lost to the body.
Cummings and Macfarlane (1997)
summarize that on mixed
European diets, total ileal N is 2–3 g/d, whereas fecal N is 2–4 g/d,
suggesting that the colon is in approximate N balance. Daily
irreversible lysine losses at the terminal ileum have been estimated
from the ileal effluent of subjects on protein-free diets to be 4.6
and 3.9 mg · kg-1after a 2-d intake of an
antibiotic (Fuller et al. 1994
). This suggests that
under these experimental conditions, the losses are mainly of
endogenous origin. When receiving a mixed diet providing ~1 g protein
· kg-1 · d-1 in the
form of a crystalline amino acid mixture, a daily loss of 8.5 mg ·
kg-1 has been estimated (Metges et al. 1999a
), and a comparative value can be calculated for threonine
(Metges et al. 1999b
).
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Nitrogen sources for microbial amino acid synthesis |
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TOPABSTRACTINTRODUCTIONEstimation of microbial lysine...Site and precursor pool...Evidence for absorption of...Evidence for absorption of...Amino acid losses from...Nitrogen sources for microbial...Dietary effects on intestinal...Relevance for determining adult...REFERENCES |
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) but only
if there is sufficient ATP to drive microbial protein synthesis. As
known from rumen bacteria, acetate and CO2 and,
to a lesser degree, propionate derived from microbial carbohydrate
fermentation, form an active precursor pool of carbon for amino acid
synthesis (Sauer et al. 1975
). Ammonia is assimilated to
form glutamate and glutamine (Reitzer and Magasanik 1996
), and the glutamate provides nitrogen for the synthesis of
most of the other amino acids. Lysine biosynthesis proceeds from
aspartate via the intermediate diaminopimelic acid, required by many
bacteria for the biosynthesis of cell wall (Morrison and Mackie 1996
). Hence, if
15NH4Cl is given
orally,15N-labeled ammonia might be quite rapidly
incorporated into microbial amino acids (Metges et al. 1999a
, Fig. 4). Furthermore, the 15N
ammonia can reach the liver directly via the portal vein or transferred
through the intestinal wall where it is introduced into dispensable
(mainly arginine, glutamine) and also some IAA via transamination as
well as being transferred to be incorporated into urea. In this
context, it is interesting to note that in our study in healthy adult
subjects, only 38.7% of the 15N ingested as
15NH4Cl was excreted as
urinary urea, whereas the isonitrogenous dose of
15N urea resulted in a significantly higher
fractional excretion of 56.7% (Metges et al. 1999b
).
The 15N from nonspecific nitrogen sources is also
returned to the intestinal tract as 15N-labeled
amino acids and as 15N urea in endogenous
secretions (pancreatic, biliary and mucosal) (Fuller and Reeds 1998
). Intravenous infusion of
15N-labeled amino acids is followed by labeled
plasma amino acids appearing in the gastrojejunal fluids within
~3 h after onset of the infusion (Gaudichon et al. 1994
). Leterme et al. (1996)
showed in pigs that
the tracer appears in pancreatic secretions within 50 min of the
consumption of a 15N-labeled diet. This finding
is confirmed by our observations in the pig showing substantial
15N enrichment of amino acids (i.e., lysine,
alanine, glycine, leucine, isoleucine and glutamic acid) in duodenal
and jejunal proteins after a 10-d administration of
15NH4Cl (Metges et al. 1996
, C. C. Metges, unpublished data). The quantity of
endogenous protein that is recycled in the intestine makes it a
potentially significant source of nitrogen for microbial growth.
Quantitative information comes from experiments in growing pigs in
which digesta from the upper intestinal tract was transferred between
one pig fed 15N and another that was not
(Krawielitzki et al. 1990
). This study suggests that
90% of all endogenous nitrogen secreted into the gut is reabsorbed,
although the experiments do not throw any light on the involvement of
the enteric flora in this process. However, because mucus glycoproteins
and some other digestive secretions are resistant to mammalian
digestive enzymes, microbial proteolytic activity is involved
(Macfarlane et al. 1988
, Quigley and Kelly 1995
).
Approximately 60–70% of newly synthesized urea is excreted in the
urine, whereas the remainder is degraded by microbial urease. It has
been claimed that urea nitrogen can be salvaged in the colon and that
this nitrogen can be incorporated by the intestinal microflora into
amino acids that are subsequently absorbed by the host (Jackson 1993
, 1995
). Bacterial growth on sources of
nitrogen other than ammonia is also possible but slower than with
ammonia because the rate of ammonia generation from these sources
(i.e., amino acids or urea) appears to be a growth-limiting factor
(Reitzer and Magasanik 1996
). In rats, urease activity
in small intestinal contents (units/g collected content) was 15%
compared with that found in the large intestine (Kim et al. 1998
). As shown for the human colon and the bovine rumen,
ureolytic bacteria seem to be mainly located close to the intestinal
walls (Hume 1996
), which might explain why there was
only low enrichment in cecal luminal ammonia when
15N urea was infused intravenously into human
subjects (Wrong et al. 1985
). When urea is intravenously
infused into pigs, urea concentrations in the jejunal and in the
colonic perfusate increase significantly. In contrast, ammonia
concentration measured in the same animals was not significantly
changed (Malmlof and Simoes Nunes 1992
). This suggests
the following possibilities: 1) in the pig (as a human
model), the upper digestive tract represents the main site of urea
secretion and urea reaches the colon mainly by the digesta flow from
the small to the large intestine; 2) given that the major
part of urea hydrolysis takes place juxtamucosal, ammonia derived from
urea breakdown might never appear in the perfusate because it is
directly absorbed or fixed as amino acid nitrogen and absorbed; and
3) urease activity in the small intestine might be lower
than that in the large intestine.
Interestingly, in contrast to the dogma that ureagenesis is restricted
to the liver, in a study with jejunal enterocytes of postweaning pigs,
urea synthesis from glutamine, ornithine, aspartate, arginine and
NH4Cl was observed (Wu 1995
),
which points to the possibility of a tightly connected nitrogen
recycling between juxtamucosal ureolytic bacteria and the enterocyte.
Furthermore, a comparison of the intake of isonitrogenous amounts of
15N2 urea and
15NHCl in healthy human subjects (Metges et al. 1999b
) showed that the degree of
15N labeling in the microbial amino acids was, as
for plasma free amino acids, higher with
15NH4Cl than with
[15N2]urea. However, the
differences were far smaller than those for plasma amino acids: the
15N enrichments of microbial amino acids were
only approximately twice as high with ammonium chloride as with urea,
whereas free dispensable amino acids and leucine and valine in plasma
were between 10 and 20 times higher after
15NH4Cl than after
[15N2]urea. In contrast,
the other IAA that we measured, lysine, threonine and histidine, were
more equally labeled with 15N from the two
sources, although the enrichment was still higher with
15NH4Cl (Metges et al. 1999a
and 1999b
). This indicates that an oral dose of urea
is a relatively more effective source of N for microbial amino acid
synthesis than it is as a source of N for the synthesis of tissue
endogenous amino acids.
Thus, taken together, it is reasonable to assume that microbial amino acid synthesis in the human gastrointestinal tract uses a mixture of various nitrogen sources, i.e., ammonia derived from amino acids and urea. Because urea hydrolysis is dependent on the availability of microbial urease activity, which is apparently lower in the small intestine than in the colon, microbes in the small intestine may rely more on endogenous amino acids and ammonia from gastrointestinal secretions.
At least in the pig, the upper digestive tract (stomach and small
intestine) represents the main site of urea secretion (Malmlof and Simoes Nunes 1992
, Mosenthin et al. 1992
).
The possibility of urea diffusion through the colon wall might be
difficult to detect, assuming that the major part of urea hydrolysis
and utilization for microbial amino acid synthesis takes place
juxtamucosal. In any case, it appears that urea as a nitrogen source
for microbial protein synthesis is of greater significance in the colon
than in the small intestine, although there also is an ample supply of
amino acids from small intestinal secretions, bacterial protein and
undigested food protein. Chacko and Cummings (1988)
report that total nitrogen at the human terminal ileum (2–3 g ·
d-1) consists of 10–15% urea/ammonia/nitrate
and free amino acids, 48–51% protein and 34–42% peptides.
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Dietary effects on intestinal microbial IAA synthesis |
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TOPABSTRACTINTRODUCTIONEstimation of microbial lysine...Site and precursor pool...Evidence for absorption of...Evidence for absorption of...Amino acid losses from...Nitrogen sources for microbial...Dietary effects on intestinal...Relevance for determining adult...REFERENCES |
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Deprivation of nutrients such as glucose can induce the formation of
adhesive organelles to enable bacteria to gain access to nutrient
sources within or adjacent to host cells (reviewed by Alverdy and Stern 1998
). It was shown that gram-negative bacteria
establish glycocalyx-coated microcolonies on epithelial cells
during periods of luminal nutrient deprivation (reviewed by
Alverdy and Stern 1998
), and it could be speculated that
feeding purified or chemically defined diets such as used in a number
of experiments investigating microbial lysine utilization with low
contents of fermentable carbohydrates (e.g., Giordano et al. 1968
, Metges et al., 1999a
) might have changed
the microbial environment. Chemically defined liquid diets fed to rats
for 1 week resulted in overgrowth of coliform strains and altered
bacterial translocation from the gut (Alverdy et al. 1990
). It was also observed that in humans, overall microbial
cell counts decreased by ~10–20% when purified diets were ingested
(Blaut, M., personal communication). On the other hand, overall dietary
restriction (60% of ad libitum food intake) had little effect on the
fecal microflora of female Fischer 344 rats (Henderson et al. 1998
). However, short-term starvation induced a 7500-fold
increase in E. coli bacteria adherent to cecal epithelium in
mice (Hendrickson et al. 1999
).
Dietary fat content could also have an impact on microflora
because reports indicate bactericidal effects of various fatty acids
and monoglycerides on gram-positive bacteria (Petschow et al. 1998
, Sprong et al. 1999
). Feeding rats
diets containing milk, yogurt, lactose or cellulose considerably
decreased urease activity and ammonia production (Kim et al. 1998
), which was thought to be due to the growth of nonurease,
nonammonia producers, such as lactobacilli for the milk product–based
diets.
Numerous studies have shown that the ingestion of certain nonstarch
oligosaccharides can affect the composition of the microflora and
subsequently of their fermentation products (e.g., Hylla et al. 1998
, Macfarlane and Cummings 1999
). However,
this goes beyond the scope of the present review, and the reader is
referred to the relevant literature.
Using the 15N labeling paradigm in a recent study
in minipigs, our preliminary results indicate that microbial lysine
isolated from ileal chyme is apparently more 15N
labeled when pigs were adapted to a low lysine but otherwise adequate
diet (Backes et al., unpublished data). It has been shown in growing
pigs that there is an adaptation to lysine amino acid deficiency in
that lysine concentration in whole body protein decreases whereas other
amino acids are more concentrated (Batterham et al. 1990
). Amino acid compositions of endogenous nitrogen
secretions are dependent on the protein status of the animal (de Lange et al. 1989
). Hence, it is possible that lysine content
or other nitrogenous compounds of endogenous secretions in
lysine-deficient animals are lower than in animals adequately
supplied with lysine, which could lead to changes in the intestinal
microenvironment.
Feeding diets containing 20% protein compared with 5% to septic and
control guinea pigs resulted in increased bacterial translocation
measured by the instillation of 14C-labeled
E. coli, with nonsignificantly higher counts in the liver of
control animals (Nelson et al. 1996
). In addition, mice
fed glutamine-enriched diets had a lower degree of translocation to
the tissues (liver, spleen and lymph nodes) (Gianotti et al. 1995
).
These results indicate that the intake of certain dietary oligosaccharides, lipids, milk products as well as protein intake and the level of consumption of certain amino acids can affect the composition or metabolic activity of the intestinal microflora and thus its fermentation products potentially available to the host.
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Relevance for determining adult IAA requirement |
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TOPABSTRACTINTRODUCTIONEstimation of microbial lysine...Site and precursor pool...Evidence for absorption of...Evidence for absorption of...Amino acid losses from...Nitrogen sources for microbial...Dietary effects on intestinal...Relevance for determining adult...REFERENCES |
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). Furthermore, if
15N-labeled substrates entering the gut lumen
(whether plasma-derived amino acids, endogenous proteins or urea)
were used preferentially by microbes in juxtaposition to the intestinal
wall with turnover and release of their constituent proteins and amino
acids in that spatial domain, then the 15N
enrichment of lysine being absorbed from the gastrointestinal tract
would not be accurately reflected by either the ileal or fecal
microbial protein-bound lysine.
In a pig, experiment 14C polyglucose was used as
a tracer to label microbial lysine (Torrallardona et al. 1994
), and it might appear that this could have solved the
problems related to nitrogen cycling. The authors estimated the
microbial lysine contribution to body lysine based on cecal enrichment
to be 0.2% compared with ~1% estimated using the
15N labeling paradigm in the same animal.
However, the discussed difficulties in interpreting the results using a
carbon-labeled tracer do persist, although to a lesser degree, for
very similar reasons. First, the difficulty in selecting the true
precursor pool (i.e., small or large intestine, luminal or juxtaposal,
free or protein/peptide-bound amino acids) would remain. Second, it
cannot be excluded that carbon derived from carbohydrates that are not
readily accessible by the enzymes of the host might return into the
gastrointestinal tract as IAA via endogenous secretions and
sluffed-off cells (Simon et al. 1986
) and thus can
be reincorporated into microbial protein. Simon et al. (1986
) estimated for a 34-kg pig that in 12 h, 0.79 g
leucine was secreted into the upper gastrointestinal tract derived from
the plasma pool. The possibility of using
13C-labeled polyglucose instead of
14C in humans would require a comparably large
amount (50–100 g · d-1) of highly uniformly
labeled nonstarch polysaccharide (with the disadvantage of severely
affecting the normal gastrointestinal transit rate) to be able to
detect 13C-labeled lysine in the plasma pool.
When introducing a potential microbial IAA contribution to the input side of the tracer balance equation, a potentially irreversible loss of IAA from the human gastrointestinal tract must also be considered. A microbial contribution to the input side only would overestimate the dietary supply if a potential gastrointestinal loss is not accounted for. It is therefore possible that the microbial contribution and the gastrointestinal losses cancel themselves out.
In conclusion, we and others have shown that there is a significant presence of microbially derived lysine in the body protein and plasma pool of humans and nonruminant animals. However, the current use of the 15N labeling paradigm does not allow an estimation of the net microbial contribution because of the various uncertainties, as discussed.
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FOOTNOTES |
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3 Abbreviation used: IAA, indispensable amino acid.
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TOPABSTRACTINTRODUCTIONEstimation of microbial lysine...Site and precursor pool...Evidence for absorption of...Evidence for absorption of...Amino acid losses from...Nitrogen sources for microbial...Dietary effects on intestinal...Relevance for determining adult...REFERENCES |
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