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Sources in Humans
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.
KEY WORDS: intestinal bacteria amino acid synthesis tracer balance large intestine
amino acid requirement
The human intestinal tract is colonized by 400 species of tion of 102/mL at the proximal intestine and 105 to 109/mL
bacteria (Gordon et al. 1997) from the small to the large in the region of the distal ileum. The flora of the lower ileum
intestine, but they are not uniformly distributed in either is qualitatively similar to that of feces, with the latter showing
number, species or metabolic activity (Table 1) (Autenrieth a bacterial count in the range of 108 to 1012/mL. It has been
1998, Goldin 1990, Hovgaard and Brondsted 1996). Thus, for shown that certain Lactobacillus and Bifidobacteria strains and
persons living in Europe and North America, microbial counts commensal bacteria such as Escherichia coli can adhere to the
increase along the intestinal tract, with a bacterial concentra- mucosal surface, in particular to the terminal ileum and cecum
(Alverdy et al. 1994, Hendrickson et al. 1999, Macfarlane and
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 thought to be a prerequisite for invasion (Hendrickson et al.
symposium was sponsored by the National Dairy Council; International Dairy 1999). Under normal conditions, however, intestinal epithe-
Federation; United Kingdom Dairy Association; Dairy Farmers of Canada; Davisco
Foods International, Inc.; New Zealand Milk; CAMPINA MELKUNIE, Zaltbommel,
lial cells remain relatively free of adherent bacteria (Hendrick-
The Netherlands; Land OLakes; and CERIN. Published as a supplement to The son et al. 1999), whereas bacteria are attached to the mucus
Journal of Nutrition. Guest editors for this publication were Gregory D. Miller, layer lining the intestinal walls (Hume 1996).
National Dairy Council, Rosemont, IL, and Daniel Tome, Institut National
Agronomique, Paris, France. The human host has developed a subtle balance between its
2
To whom correspondence should be addressed. resident microflora and the innate mucosal defense systems. It has
1857S
1858S SUPPLEMENT
TABLE 1
Microbial counts along the human gastrointestinal tract
Bacterial
Location Length Transit pH counts Abundant species
m h g1
been shown that microbial activity is responsive to metabolic and al. 1980) that microbial lysine can be incorporated into host
pathogen challenges (Hendrickson et al. 1999, Mack et al. 1999, body protein, but no attempt had been made to quantify this
Spitz et al. 1994) and dietary factors (Alverdy and Stern 1998, contribution. Utilization of microbially derived IAA has been
Kim et al. 1998, Macfarlane and Cummings 1999) but also can confirmed in pigs and rats fed protein-free or low protein diets
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
and lysine contribution ranged from 8 to 17% and from 5 to Site and precursor pool of microbial amino acid absorption
21%, respectively. These estimates correspond to a microbial
contribution of lysine and threonine to the plasma flux ranging The scientific literature has been largely concerned with
from 11.7 to 67.9 and from 21 to 44.7 mg kg1 d1 in the impact of colonic fermentation products affected by the
normal adults, respectively. intake of prebiotics and probiotics (Macfarlane and Cum-
For comparative purposes, Table 3 presents a summary of mings, 1999) in regard to colon cancer protection and sys-
the available data on microbial lysine appearance in the host temic immunity. A possible role of the ileal microflora for host
body protein or amino acid pools in rats, pigs and humans. In amino acid nutriture has not been considered so far. In con-
applying the 15N labeling paradigm, between 1 and 20% of trast to the available evidence that quantitative important
circulating plasma lysine, urinary lysine and body protein amino acid absorption occurs only in the small intestine, the
lysine of the host is derived from intestinal microbial sources possibility of amino acid absorption from the large intestine
(Table 3). There is a trend toward higher numbers when the should not generally be excluded for reasons indicated later. In
ileal microbial protein was used as precursor, whereas it ap- addition, it is still an open question in which form microbial
pears that the calculated contribution to body protein lysine is amino acids are absorbed (i.e., free amino acids or peptides).
lower. 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
body (valine, histidine) show rather comparable numbers, and the glutamate provides nitrogen for the synthesis of most
whereas glutamic acid and alanine, which are known for of the other amino acids. Lysine biosynthesis proceeds from
extensive nitrogen exchange, differ by a factor of 2 between aspartate via the intermediate diaminopimelic acid, required
normal subjects and ileostomates, thereby indicating an up- by many bacteria for the biosynthesis of cell wall (Morrison
take of 15N-labeled nitrogen by the large intestine (Table 2). and Mackie 1996). Hence, if 15NH4Cl is given orally,15N-
However, this does not shed light onto the identity of the labeled ammonia might be quite rapidly incorporated into
substances absorbed. microbial amino acids (Metges et al. 1999a, Fig. 4). Further-
On the other hand, several reports indicate that amino acid more, the 15N ammonia can reach the liver directly via the
absorption from the large intestine is negligible in nonrumi- portal vein or transferred through the intestinal wall where it
nant animals (Darragh et al. 1994, Hume et al. 1993). How- is introduced into dispensable (mainly arginine, glutamine)
ever, Fuller and Reeds (1998) recently summarized data on N and also some IAA via transamination as well as being trans-
balance measurements in pigs when protein or amino acids ferred to be incorporated into urea. In this context, it is
were infused into the large intestine and found that the whole interesting to note that in our study in healthy adult subjects,
body N balance was always slightly improved. This suggests only 38.7% of the 15N ingested as 15NH4Cl was excreted as
that there is protein digestion and that there might be absorp- urinary urea, whereas the isonitrogenous dose of 15N urea
tion of amino acids. However, the identity of the substances resulted in a significantly higher fractional excretion of 56.7%
absorbed may still be in question because no nutritional ben- (Metges et al. 1999b).
efit was seen when lysine was infused into the cecum, although The 15N from nonspecific nitrogen sources is also returned
the latter finding does not exclude the possibility of peptide to the intestinal tract as 15N-labeled amino acids and as 15N
absorption as mentioned earlier. urea in endogenous secretions (pancreatic, biliary and muco-
In conclusion, it appears as if the small intestine is respon- sal) (Fuller and Reeds 1998). Intravenous infusion of 15N-
sible for a large part of microbial lysine uptake, although some labeled amino acids is followed by labeled plasma amino acids
Nunes 1992). This suggests the following possibilities: 1) in indigenous gut microflora, and thereby possibly the microbial
the pig (as a human model), the upper digestive tract repre- protein synthesis might be affected.
sents the main site of urea secretion and urea reaches the colon Deprivation of nutrients such as glucose can induce the
mainly by the digesta flow from the small to the large intestine; formation of adhesive organelles to enable bacteria to gain
2) given that the major part of urea hydrolysis takes place access to nutrient sources within or adjacent to host cells
juxtamucosal, ammonia derived from urea breakdown might (reviewed by Alverdy and Stern 1998). It was shown that
never appear in the perfusate because it is directly absorbed or gram-negative bacteria establish glycocalyx-coated microcolo-
fixed as amino acid nitrogen and absorbed; and 3) urease nies on epithelial cells during periods of luminal nutrient
activity in the small intestine might be lower than that in the deprivation (reviewed by Alverdy and Stern 1998), and it
large intestine. could be speculated that feeding purified or chemically defined
Interestingly, in contrast to the dogma that ureagenesis is diets such as used in a number of experiments investigating
restricted to the liver, in a study with jejunal enterocytes of microbial lysine utilization with low contents of fermentable
postweaning pigs, urea synthesis from glutamine, ornithine, carbohydrates (e.g., Giordano et al. 1968, Metges et al.,
aspartate, arginine and NH4Cl was observed (Wu 1995), 1999a) might have changed the microbial environment.
which points to the possibility of a tightly connected nitrogen Chemically defined liquid diets fed to rats for 1 week resulted
recycling between juxtamucosal ureolytic bacteria and the in overgrowth of coliform strains and altered bacterial trans-
enterocyte. location from the gut (Alverdy et al. 1990). It was also
Furthermore, a comparison of the intake of isonitrogenous observed that in humans, overall microbial cell counts de-
amounts of 15N2 urea and 15NHCl in healthy human subjects creased by 10 20% when purified diets were ingested (Blaut,
(Metges et al. 1999b) showed that the degree of 15N labeling M., personal communication). On the other hand, overall
in the microbial amino acids was, as for plasma free amino dietary restriction (60% of ad libitum food intake) had little
acids, higher with 15NH4Cl than with [15N2]urea. However, effect on the fecal microflora of female Fischer 344 rats (Hen-
Relevance for determining adult IAA requirement not allow an estimation of the net microbial contribution
because of the various uncertainties, as discussed.
Using the 15N labeling paradigm, it could be shown that
microbial lysine is used for lysine homeostasis in normal hu- LITERATURE CITED
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