Criteria and Significance of Dietary Protein

Sources in Humans

Contribution of Microbial Amino Acids to Amino Acid

Homeostasis of the Host
Cornelia C. Metges
Deutsches Institut fur Ernahrungsforschung (German Institute of Human Nutrition), D-14558 Bergholz-
Rehbrucke, Germany

ABSTRACT Among the reasons suggested for the discrepancy between N balance and tracer-derived indispensable
amino acid (IAA) requirement estimates is the possibility that the metabolic requirement is met not only by the diet but
also by IAA 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. Here, the available evidence on the contribution
of microbial amino acids to the hosts amino acid homeostasis, applying the 15N labeling paradigm, is summarized.

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Between 1 and 20% of circulating plasma lysine, urinary lysine and body protein lysine of the host, respectively, is
derived from intestinal microbial sources and corresponds to a gross microbial lysine contribution of 11 68 mg kg1
d1 in adult humans with an adequate protein intake when fecal or ileal microbial lysine enrichment is used as
precursor. Factors affecting estimates of net microbial IAA contribution are discussed. It appears that the small intestine
is responsible for a large part of microbial lysine uptake, although some absorption from the large intestine cannot be
excluded. Nonoxidative lysine losses from the human gastrointestinal tract, which were found to be between 3.9 to 8.5
mg kg1 d1, are necessary to estimate the net contribution of microbial IAA. It is reasonable to assume that microbial
amino acid synthesis in the human gastrointestinal tract utilizes a mixture of various nitrogen sources, i.e., endogenous
amino acids, urea and ammonia. Microbes in the small intestine may rely more on endogenous amino acids. Deprivation
of nutrients, the intake of certain dietary nonstarch oligosaccharides, lipids, as well as protein intake level and source
and level of consumption of certain amino acids can affect the composition and metabolic activity of the intestinal
microflora and thus its fermentation products potentially available to the host. In conclusion, with the use of the 15N
labeling paradigm, a significant contribution of microbial lysine to the host lysine homeostasis is found. However, to
assess the net contribution of microbial IAA and its importance in defining the adult IAA requirement, this is not the
ultimately successful experimental strategy because the interpretation of results is complicated by the nitrogen recycling
in the gut, the uncertainty of the precursor pool of absorption and the limited data on nonoxidative IAA losses from the
human gastrointestinal tract. J. Nutr. 130: 1857S1864S, 2000.

KEY WORDS: intestinal bacteria amino acid synthesis tracer balance large intestine
amino acid requirement

The human intestinal tract is colonized by 400 species of
bacteria (Gordon et al. 1997) 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 concentra-
1 Cummings 1999). For pathogenic bacteria, adherence is
Presented at the symposium Criteria and Significance of Dietary Protein
Sources in Humans, held in San Francisco, CA, on October 4, 1999. The
symposium was sponsored by the National Dairy Council; International Dairy
Federation; United Kingdom Dairy Association; Dairy Farmers of Canada; Davisco
Foods International, Inc.; New Zealand Milk; CAMPINA MELKUNIE, Zaltbommel,
The Netherlands; Land OLakes; and CERIN. Published as a supplement to The
Journal of Nutrition. Guest editors for this publication were Gregory D. Miller,
National Dairy Council, Rosemont, IL, and Daniel Tome, Institut National
Agronomique, Paris, France.
2
To whom correspondence should be addressed.
tion 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
thought to be a prerequisite for invasion (Hendrickson et al.
1999). Under normal conditions, however, intestinal epithe-
lial cells remain relatively free of adherent bacteria (Hendrick-
son et al. 1999), whereas bacteria are attached to the mucus
layer lining the intestinal walls (Hume 1996).
The human host has developed a subtle balance between its
resident microflora and the innate mucosal defense systems. It has

0022-3166/00 $3.00 2000 American Society for Nutritional Sciences.

1857S
1858S
Microbial counts along the human gastrointestinal tract
Location Length Transit
m h
Oral cavity 160 s
Stomach 0.6 15
Duodenum 0.2 12
Jejunum 1.5
Ileum 2 26
Cecum/colon 1.8 870 5.57
1 Adapted from Autenrieth 1998 and Hovgaard and Brondsted 1996.
been shown that microbial activity is responsive to metabolic and
pathogen challenges (Hendrickson et al. 1999, 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 micro-
organisms 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 irrevers-
ible 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 micro-
flora, 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 hosts amino acid homeostasis, ii) discuss
factors that may influence estimates of microbial amino acid
synthesis and iii) to assess the importance of the available evi-
dence of microbial amino acid contribution in defining the adult
requirement of IAA.
Estimation of microbial lysine contribution to lysine
homeostasis of the host using 15N labeling paradigm
It was observed earlier in uremic patients and in subjects
consuming low protein diets (Giordano et al. 1968, Tanaka et
3
Abbreviation used: IAA, indispensable amino acid.
SUPPLEMENT
TABLE 1
Bacterial
pH counts Abundant species
g1
15 0104 Lactobacilli
57 0103 Lactobacilli, Bacteroides
67 105 Enterobacteria, Bacteroides
67.5 1069 Bifidobacteria, Enterococci
101012 Enterococci, Enterobacteria, Clostridia,
Bacteroides
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
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(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-energymalnourished infants, the appearance of uri-
nary 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 appropri-
ate 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
15
N2 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 contri-
bution 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
13
C lysine infusion in the same subjects or taken from the
literature (Zhao et al. 1986) (Table 2). Microbial threonine
 AMINO ACID HOMEOSTASIS OF THE HOST 1859S

TABLE 2
Appearance of various microbial indispensible and nonindispensable amino acids in the plasma pool
measured after oral administration of 15NH4Cl and 15N2 urea1
15N tracer

15NH4Cl 15N2 urea

Amino acid Ileal2 Fecal3 Fecal3

% mg kg1 d1 % mg kg1 d1 % mg kg1 d1

Lysine 21 67.9 9 28.9 5 11.7

Threonine4 17 44.7 14 36.8 8 21.0
Valine 80 88 14
Histidine 52 49 20
Proline 41 73 24
Glutamic acid 72 138 15
Alanine 63 111 25
Glycine 93 182 41

1 Summarized from Metges et al. 1999a, 1999b.

2 Ileal effluent of ileostomy subjects.

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3 Normal subjects.
4 Threonine plasma flux taken from Zhao et al. 1986.

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 kg1 d1 in
normal adults, respectively.
For comparative purposes, Table 3 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 ap-
pears that the calculated contribution to body protein lysine is
lower.
Site and precursor pool of microbial amino acid absorption
The scientific literature has been largely concerned with
the impact of colonic fermentation products affected by the
intake of prebiotics and probiotics (Macfarlane and Cum-
mings, 1999) in regard to colon cancer protection and sys-
temic immunity. A possible role of the ileal microflora for host
amino acid nutriture has not been considered so far. In con-
trast 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

TABLE 3
Contribution of microbial lysine to host plasma free, urinary and body protein lysine pool: Comparison of data
from rats, pigs and human subjects measured via oral administration of nonspecific 15N

Dietary Microbial
Species intake 15N tracer1 lysine Host lysine Contribution Reference

d1 APE 15N %

Normal adult2 11/1862 15NH4Cl Fecal Plasma 7.53 Metges et al. 1999a
15N2 urea Fecal Plasma 4.63
Ileostomy patient 11/1342 15NH4Cl Ileal Plasma 21.1
Malnourished infants 0.54.61 15N2 urea Urinary urea Urine 0.9 Yeboah et al. 1996
Normal adult Not given Lactose-[15N]ureide Fecal Urine 7.9 Gibson et al. 1998
Young pigs 21 15NH4Cl Ileal Carcass 3.0 Torrallardona et al. 1994
Cecal Carcass 0.9
Minipigs (ileorectal 3.5 15NH4Cl Ileal Plasma albumin 15.3 Metges et al. 1996
anastomosis)
15N2 urea Ileal Plasma albumin 8.2
Young rats 0 15NH4Cl Fecal Carcass 2.8 Torrallardona et al. 1996a

1 g protein kg1 d1.

2 kJ kg1.
3 Mean of fasted and fed state; not significantly different between 15NH4Cl and 15N2 urea tracers.
1860S SUPPLEMENT
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 conclu-
sion 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 ab-
sorption. 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 ex-
pressed 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 for-
eign material (antigens and microorganisms) via transepithe-
lial transport from the lumen to organized lymphoid tissues
within the mucosa of the small and large intestines (San-
sonetti 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 ma-
terial enters the circulating plasma via this pathway as indi-
cated 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 hu-
man 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.
Evidence for absorption of microbial amino acids from
small intestine
15
N and 14C lysine enrichment of microbial protein after
the ingestion of 15NH4Cl and 14C polyglucose changes
throughout the gastrointestinal tract of pigs (Torrallardona et
al. 1994). No enrichment has been found in the small intes-
tine 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
TABLE 4
Appearance of cecal microbial amino acids in the colic
branch of the ileocolic vein of a pig 3 hours after
administration of labeled microbes into the cecum1
Precursor pool used for calculation of
absorption
Amino acid Free amino acids Microbial protein
Lysine 25 4
Threonine 2 3
Phenylalanine 0.2 3
Histidine 0.4 19
Leucine 0.07 3
Alanine 0.2 2
Glycine 0.03 0.6
Ammonia 0 0
1 Calculated from data of Niiyama et al. 1979.
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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 anas-
tomosis 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 ab-
sorption from that site (Metges et al. 1996 and 1999a). How-
ever, ileostomy as well as ileorectal anastomosis is prone to
secondary colonization and possibly alterations of digesta tran-
sit rate in the gut. Hence, although it might be not completely
comparable to the microbial situation in an intact gastroin-
testinal tract, it demonstrates the principal possibility of mi-
crobial lysine and threonine absorption from the small intes-
tine.
Evidence for absorption of microbial amino acids from large
intestine
After cecal administration of 15N-labeled yeast, 15N label
appeared in the body protein of infants (Heine et al. 1987).
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.
The comparison of the appearances of microbial amino
acids in the free plasma pool amino acids after ingestion of the
15
NH4Cl tracer when ileal and fecal microbial enrichments
are being used (Table 2) 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
 AMINO ACID HOMEOSTASIS OF THE HOST
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 up-
take 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 nonrumi-
nant animals (Darragh et al. 1994, Hume et al. 1993). How-
ever, 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 absorp-
tion of amino acids. However, the identity of the substances
absorbed may still be in question because no nutritional ben-
efit 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 respon-
sible 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.
Amino acid losses from gastrointestinal tract
Oxidation is the major component of metabolic disposal of
IAA and is related to the level of dietary IAA supply, as shown
in various stable isotope studies by Young and Borgonha
(2000). 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 absorp-
tion 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 23 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 kg1after a 2-d intake
of an antibiotic (Fuller et al. 1994). This suggests that under
these experimental conditions, the losses are mainly of endog-
enous origin. When receiving a mixed diet providing 1 g
protein kg1 d1 in the form of a crystalline amino acid also possible but slower than with ammonia because the rate of
mixture, a daily loss of 8.5 mg kg1 has been estimated
(Metges et al. 1999a), and a comparative value can be calcu-
lated for threonine (Metges et al. 1999b).
Nitrogen sources for microbial amino acid synthesis
Ammonia is the preferred source of nitrogen for the growth
of enteric bacteria (Reitzer and Magasanik 1996) 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 fer-
mentation, 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),
1861S
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). Further-
more, 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 trans-
ferred 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 muco-
sal) (Fuller and Reeds 1998). Intravenous infusion of 15N-
labeled amino acids is followed by labeled plasma amino acids
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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, ala-
nine, glycine, leucine, isoleucine and glutamic acid) in duo-
denal and jejunal proteins after a 10-d administration of
15
NH4Cl (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 nitro-
gen 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 diges-
tive 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 ex-
creted 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 incorpo-
rated by the intestinal microflora into amino acids that are
subsequently absorbed by the host (Jackson 1993, 1995). Bac-
terial growth on sources of nitrogen other than ammonia is
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 enrich-
ment 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
1862S SUPPLEMENT

Nunes 1992). This suggests the following possibilities: 1) in
the pig (as a human model), the upper digestive tract repre-
sents 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,
indigenous gut microflora, and thereby possibly the microbial
protein synthesis might be affected.
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 microcolo-
nies 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 trans-
location from the gut (Alverdy et al. 1990). It was also
observed that in humans, overall microbial cell counts de-
creased 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 (Hen-

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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
15
NH4Cl 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 mi-
crobial 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 ni-
trogen at the human terminal ileum (23 g d1) consists of
10 15% urea/ammonia/nitrate and free amino acids, 48 51%
protein and 34 42% peptides.
Dietary effects on intestinal microbial IAA synthesis
Reports on the effect of dietary factors on the intestinal
microflora have been mainly focused on pathogenic microbial
species. However, this does not mean that these factors could
not influence the composition or density of the nonpathogenic
derson et al. 1998). However, short-term starvation induced a
7500-fold increase in E. coli bacteria adherent to cecal epithe-
lium 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 grow-
ing 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 endog-
enous 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 oligo-
saccharides, lipids, milk products as well as protein intake and the
level of consumption of certain amino acids can affect the com-
position or metabolic activity of the intestinal microflora and thus
its fermentation products potentially available to the host.
 AMINO ACID HOMEOSTASIS OF THE HOST
Relevance for determining adult IAA requirement
Using the 15N labeling paradigm, it could be shown that
microbial lysine is used for lysine homeostasis in normal hu-
man subjects. However, to define the net contribution of
microbial IAA to meet the human IAA requirement, the 15N
labeling paradigm is not the ultimately successful experimental
strategy because interpretation of results is complicated by the
nitrogen recycling into and from the gut. To quantify the net
contribution of microbial amino acids to meet the metabolic
demand for IAA would require that the material used to
produce microbial IAA would be otherwise lost or of no
further value for the body. Hence, to the extent that the
growth of the microbes that give rise to the labeled lysine is
supported by the degradation of endogenous protein, then the
appearance of microbially derived lysine in plasma can be seen
as part of the normal mechanism by which endogenous nitro-
gen and amino acids are recycled rather than microbial amino
acids serving as a net source of amino acids, which are addi-
tional to those supplied in the diet (Metges et al. 1999a).
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 constit-
uent proteins and amino acids in that spatial domain, then the
15
N enrichment of lysine being absorbed from the gastrointes-
tinal 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 difficul-
ties 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 secre-
tions 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 d1) of highly uniformly labeled non-
starch polysaccharide (with the disadvantage of severely af-
fecting 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 possi-
ble 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
1863S
not allow an estimation of the net microbial contribution
because of the various uncertainties, as discussed.
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