Availability of intestinal microbial lysine for whole body
lysine homeostasis in human subjects
Cornelia C.
Metges1,2,
Antoine
E.
El-Khoury1,
Lidewij
Henneman3,
Klaus J.
Petzke2,
Ian
Grant3,
Shahinaze
Bedri1,
Paulo P.
Pereira1,
Alfred M.
Ajami4,
Malcolm F.
Fuller3, and
Vernon R.
Young1
1 Laboratory of Human
Nutrition, School of Science and Clinical Research Center,
Massachusetts Institute of Technology, Cambridge 02139;
4 MassTrace, Woburn, Massachusetts 01801;
2 Unit of Protein Metabolism, Department of
Biochemistry and Physiology of Nutrition, German Institute of Human
Nutrition, 14558 Bergholz-Rehbrücke, Germany; and
3 Rowett Research Institute, Bucksburn, Aberdeen
AB2 9SB, Scotland, United Kingdom
 |
ABSTRACT |
We have investigated
whether there is a net contribution of lysine synthesized de novo by
the gastrointestinal microflora to lysine homeostasis in six adults. On
two separate occasions an adequate diet was given for a total of 11 days, and a 24-h (12-h fast, 12-h fed) tracer protocol was performed on
the last day, in which lysine turnover, oxidation, and splanchnic
uptake were measured on the basis of intravenous and oral
administration of
L-[1-13C]lysine
and
L-[6,6-2H2]lysine,
respectively.
[15N2]urea
or
15NH4Cl
was ingested daily over the last 6 days to label microbial protein. In
addition, seven ileostomates were studied with
15NH4Cl.
[15N]lysine enrichment
in fecal and ileal microbial protein, as precursor for microbial lysine
absorption, and in plasma free lysine was measured by gas
chromatography-combustion-isotope ratio mass spectrometry. Differences
in plasma [13C]- and
[2H2]lysine
enrichments during the 12-h fed period were observed between the two
15N tracer studies, although the
reason is unclear, and possibly unrelated to the tracer form per se. In
the normal adults, after 15NH4Cl
and
[15N2]urea
intake, respectively, lysine derived from fecal microbial protein
accounted for 5 and 9% of the appearance rate of plasma lysine. With
ileal microbial lysine enrichment, the contribution of microbial lysine
to plasma lysine appearance was 44%. This amounts to a gross microbial
lysine contribution to whole body plasma lysine turnover of between 11 and 130 mg · kg
1 · day
1,
depending on the
[15N]lysine precursor
used. However, insofar as microbial amino acid synthesis is accompanied
by microbial breakdown of endogenous amino acids or their oxidation by
intestinal tissues, this may not reflect a net increase in lysine
absorption. Thus we cannot reliably estimate the quantitative
contribution of microbial lysine to host lysine homeostasis with the
present paradigm. However, the results confirm the significant presence
of lysine of microbial origin in the plasma free lysine pool.
lysine kinetics; amino acid requirement; tracer balance; lysine-15-nitrogen; gas chromatography-combustion-isotope ratio mass
spectrometry
 |
INTRODUCTION |
THE CURRENT INTERNATIONAL RECOMMENDATIONS for
indispensable amino acid requirements of adult humans (12) are derived
from nitrogen balance studies in men and women (22, 36). Earlier studies by us and others, using approaches based on
13C-labeled amino acid oxidation,
suggested that adult lysine requirements are considerably higher than
previously thought (23, 44, 45). Careful 24-h tracer studies have
provided general support for the minimal physiological requirement as
estimated in short-term experiments for phenylalanine, leucine, and
lysine, and possibly for other indispensable amino acids, and they have
established that the tracer-balance concept is valid (3, 11, 20).
However, there remains controversy about the quantitative values for
the amino acid requirements in adult humans, as reviewed by Waterlow (42).
Among the reasons suggested for the discrepancy between nitrogen
balance and tracer-derived amino acid requirement estimates is the
possibility that the metabolic requirement, i.e., the irreversible loss
of indispensable amino acids (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. On the basis of the interpretation of urinary 15N urea excretion after labeled
urea administration, it has been claimed that urea nitrogen can be
salvaged by urea hydrolysis in the colon and that this nitrogen can be
incorporated by the intestinal microflora into amino acids that are
subsequently absorbed by the host (18). Although it has been shown in
the pig that colonic absorption of amino acids is possible (30, 31),
experimental evidence in nonruminant animals does not indicate a
quantitatively important amino acid absorption from the colon (7, 17,
19). Nevertheless, results in the pig demonstrated that amino acids synthesized by the microflora can be absorbed supposedly in the small
intestine and used for host protein synthesis (25, 40). Tracer studies
in animals and humans have shown a transfer of nonspecific nitrogen
into dispensable and indispensable amino acids (1, 25, 32). For a
majority of amino acids, this may reflect nitrogen exchange or
reversible transamination. However, lysine does not undergo
transamination in mammalian tissue (6). Therefore, appearance of
15N-labeled lysine in body
proteins and plasma amino acids after administration of a
15N-labeled nitrogen source, such
as ammonium or urea, must reflect the de novo synthesis of lysine and
its subsequent absorption from the gastrointestinal tract. Comparative
experiments with germ-free and conventional rats (39) have confirmed
that lysine de novo synthesis is due to the activity of the indigenous
microflora in the gastrointestinal tract.
Although in uremic patients and human subjects consuming a low-protein
diet microbial lysine can be made available to the human host (15, 38),
there has been no previous attempt to quantify the significance of this
source of lysine in host tissue metabolism. The purpose of the present
investigation, therefore, was to quantify the contribution of microbial
lysine to whole body lysine turnover in healthy adult human subjects.
In a pilot study in pigs (25), we found that the degree of
[15N]lysine labeling
is dependent on the 15N source
([15N2]urea
or
15NH4Cl)
used. This article describes the appearance of
15N-labeled lysine in plasma after
oral administration of isonitrogenous amounts of
[15N2]urea
and15NH4Cl,
respectively, in normal young adults. In addition, lysine turnover,
lysine oxidation, and lysine splanchnic uptake were measured during a
24-h oral/intravenous tracer protocol. Because the intestinal site of
synthesis and subsequent absorption of microbial lysine is uncertain,
we studied, additionally, the microbial synthesis of lysine in
otherwise healthy subjects with ileostomies after administration of
15NH4Cl
by use of the same protocol. This enabled us to compare estimates of
the microbial lysine contribution to host tissue lysine homeostasis by
using fecal and ileal microbial protein as putative precursor pools for
the [15N]lysine absorption.
 |
MATERIALS AND METHODS |
Subjects and diets.
Six male subjects aged 21.7 ± 1.6 yr (weight 81.7 ± 13.3 kg;
height 1.82 ± 0.04 m) were recruited within the student population at the Massachusetts Institute of Technology (MIT) and the community of
the Boston-Cambridge area. The study was performed at the Clinical Research Center (CRC) of the MIT. The subjects were healthy according to medical history, physical examination, vital signs, blood
parameters, and urinalysis. They had no recent weight loss, unusual
dietary practice, or pharmacological treatment. Subjects who smoked and consumed more than five or more alcoholic drinks per week were excluded
from the study. Subjects were allowed to engage in their usual daily
activities but not to participate in competitive sports and perform
strenuous exercise. Body weight was monitored daily and did not change
throughout the experimental period. The purpose of the study and the
potential risks involved were fully explained to each of the subjects.
Written informed consent was obtained, and the subjects received
financial compensation for their participation in the study. The study
was approved by the MIT Committee on the Use of Humans as Experimental
Subjects and the MIT-CRC Advisory Committee.
A second group of seven otherwise healthy subjects were ileostomates
who were studied at the Human Nutrition Unit of the Rowett Research
Institute, Aberdeen, Scotland. The group comprised three men and four
women with terminal ileum ileostomies following ulcerative colitis or
cancer of the colon (1 subject). Their mean age, weight, and height
were 59.7 ± 12.3 yr, 71.6 ± 15.0 kg, and 1.68 ± 1.27 m,
respectively. Body weight did not change during the experimental period. Clinical examination and routine clinical chemistry parameters were in a normal range for their age. The intestinal resection was
performed
6 mo before the study. They did not take any medications and had no known diseases of the upper gastrointestinal tract. Each
subject gave informed written consent to participate in this study,
which was approved by the Joint Ethical Committee of Grampian Health
Board and University of Aberdeen.
All subjects received a protein (N) adequate, semi-synthetic, diet
based on a crystalline L-amino
acid mixture (Ajinomoto USA, Teaneck, NJ; 160 mg
N · kg
1 · day
1)
patterned essentially as in a hen's egg except for the moderate, but
presumably adequate, lysine and leucine content (total lysine and
leucine intake: 45 and 40 mg · kg
1 · day
1,
respectively; Table 1). To adjust for the
somewhat lower nitrogen contribution of leucine and lysine, glycine and
alanine were added. The level of energy intake was based on estimates
of resting energy expenditure and subjects' activity level. Average
diet composition for the MIT subjects is given in Table
2. The diet composition for the subjects
studied in Aberdeen was the same but adjusted for their different
energy requirements. No other foods or beverages were allowed except
tap water, decaffeinated tea or coffee with or without artificial
sweetener, and bouillon.
Total nitrogen intake for the MIT subjects was 167 mg · kg
1 · day
1,
due to the additional urea and ammonia tracer intake, supplied in
gelatine capsules, and a lysine supplement to account for the lysine
tracer given during the 24-h tracer protocol (see 24-h Tracer protocol). The ileostomates received a total
nitrogen intake of 163 mg · kg
1 · day
1.
The energy intake of the MIT subjects was 186 kJ · kg
1 · day
1,
whereas the ileostomates consumed 134 kJ · kg
1 · day
1
because of a lower level of physical activity. Two out of three meals
per day were consumed under the supervision of the dietary staff.
The MIT subjects received the experimental diet during each of two
randomly assigned 11-day periods with a break of 4-8 wk in
between, when subjects consumed their usual diet. During the two
periods, isonitrogenous amounts of either
[15N2]urea
or
15NH4Cl
(3.45 mg
15N · kg
1 · day
1;
2.1% of total N intake) were given. The
15N tracer was started on
day 5, in the morning. Therefore,
subjects received with each of the three daily meals one gelatine
capsule containing either
[15N2]urea
[580 mg/day; 99.1 atom percent (AP); Isotec, Miamisburg, OH] or
15NH4Cl
(1,000 mg/day; 99.8 AP; Isotec), respectively, during
days 5-10. The ileostomy subjects
were residents in the Human Nutrition Unit of the Rowett Research
Institute and consumed the same experimental diet for 10 days.
Beginning on the morning of day 5, the
1,000-mg amount of
15NH4Cl
was given in three gelatine capsules per day, and this continued until
the end of day 10. Hence, 3.9 mg
15N · kg
1 · day
1
(2.5% of total N intake) were ingested. This subject group did not
receive
[15N2]urea.
24-h Tracer protocol.
At 1600 on day 10, the
subjects consumed their last meal in the CRC and were then admitted as
inpatients to the MIT Medical Department. Two small catheters were
placed into veins of the nondominant arm by use of aseptic sterile
procedures. One catheter was introduced into an antecubital vein for
tracer infusion, and one catheter was placed into a superficial dorsal
vein of the hand for blood collection (10). Beginning at 1800 on
day 10 of the diet, oral doses (1/24
of daily dose) of the respective 15N tracer
([15N2]urea
or
15NH4Cl)
were given hourly. Hourly portions of
15N tracers were weighed
individually and dissolved in ~25 ml of tap water immediately before
ingestion. Simultaneuosly, a 24-h infusion protocol was performed in
which an
L-[1-13C]lysine · HCl
tracer (99 AP; MassTrace, Woburn, MA) was administered intravenously
and
L-[6,6-2H2]lysine · HCl
(99 AP; MassTrace) was given by mouth in hourly intervals to assess
lysine kinetics. Because the analytic data of two of the six subjects
were questionable, they were not included in data evaluation. The
primed, constant intravenous infusion of
[13C]lysine was
administered at a known rate of 3.5 µmol · kg
1 · h
1
(prime 5.25 µmol · kg
1 · h
1
). The bicarbonate pool was primed (0.6 µmol/kg) with
13C sodium bicarbonate (99 AP;
Cambridge Isotope Laboratories, Andover, MA). The oral tracer
[2H2]lysine
was given at a rate of 2.5 µmol · kg
1 · h
1
(prime 3.75 µmol/kg). All tracers were tested to be sterile and pyrogen free by an independent laboratory. The intravenous tracers were
prepared in physiological saline under sterile conditions and were
infused with a screw-driven pump at a rate of 8 ml/h (model 22; Harvard
Apparatus, Millis, MA). The oral tracer was dissolved in distilled
water and given in hourly doses of 8 ml.
The feeding regimen on the tracer day
11 consisted of 10 equal hourly meals (each supplying
1/10 of total 24-h intake), which were started at 0600. Because the
subjects remained in bed during the 24-h tracer protocol period, energy
but not nitrogen supply was decreased to ~83% of that for the prior
10 days. The 24-h tracer protocol was not performed in the ileostomates.
Breath 13CO2
background enrichment.
To correct the breath
13CO2
baseline for food-derived changes, a tracer-free study was performed in
three additional young MIT subjects (20.7 ± 0.9 yr; 61.6 ± 15.0 kg; 1.65 ± 0.1 m). They received the standard
experimental diet plus unlabeled ammonium chloride, as described in
Subjects and diets, and 30-min breath samples were taken. To
account for the lysine intake by the tracers, unlabeled lysine was
added as hourly oral doses. The mean value for each 30-min interval of
the 24-h protocol was used to correct the
13C enrichment in breath during
[13C]lysine tracer infusion.
Sample collection.
Urine and feces from MIT subjects were collected over 24 h starting on
day 3 of the dietary period and
continued throughout the remainder of the experimental period
(day 3 0800 to day
11 1800). The subjects were instructed to collect feces
directly in clean plastic bags and then to freeze the sample promptly
at
20°C. During the 24-h tracer protocol, urine was
collected at consecutive 3-h intervals. Fasted blood samples were drawn
into heparinized tubes from an antecubital arm vein by venipuncture on
days 3 or
4 (baseline samples for
[15N]lysine
enrichments) and day 8. Throughout the
24-h tracer protocol, hourly blood samples were taken via the catheter
in the hand vein. Ten minutes before blood sampling, the hand was
placed into a customized warming box (65°C air temperature) to
obtain arterialized blood.
Breath samples were taken every 30 min for measurement of breath
13CO2.
Between 360 and 720 min, no breath samples were obtained so that
subjects could maintain the normal sleeping pattern. Instead, blood
samples (2 ml) were used to liberate
13CO2
from blood bicarbonate, as previously described (10). Total carbon
dioxide production (
CO2)
was determined by indirect calorimetry, also as described previously
(10).
Starting on day 5, 24-h ileostomy
fluid was collected continuously and pooled over 24-h periods. Subjects
emptied their bags whenever fluid was present and froze the fluid
immediately. One fasted blood sample was obtained in the morning of
day 10. Because no baseline samples of
ileal fluids and plasma were taken, total urinary nitrogen was used as
a baseline for calculation of 15N
enrichments (atom percent excess, APE).
Sample analysis.
Ileal digesta and feces were pooled daily, and the microbial fraction
was obtained by differential centrifugation as recently described (39).
The microbial pellet was precipitated, and the protein was hydrolyzed
and purified by filtration. Nitrogen content in ileal digesta was
determined by a micro-Kjeldahl method. Lysine content in ileal
microbial protein was determined after acid hydrolysis by ion exchange
chromatography (33). Plasma was separated in a refrigerated centrifuge
at 1,000 g and stored frozen at
20°C until analysis. The isolation of free amino acids was
performed as described by Metges and Petzke (24).
Because 15N enrichment in lysine
in the plasma samples was extremely low, a highly sensitive methodology
was used. [15N]lysine
and the other amino acids of plasma and microbial protein were
derivatized to form
N-pivaloyl-i-propyl esters and
measured by gas chromatography-combustion-isotope ratio mass
spectrometry (GC-C-IRMS). Briefly, amino acid esters are separated on a
capillary column (Ultra-2, Hewlett-Packard, Waldbronn, Germany) by gas
chromatography (HP 5890, Hewlett-Packard). The effluent is directed
on-line into a combustion interface. Amino acids are combusted in an
oxidation furnace at 980°C to
CO2,
N2,
NOx, and
H2O. Combustion gases are reduced
in a reduction oven at 600°C,
CO2 and
H2O are eliminated, and
N2 is directed into the ion source
of an isotope ratio mass spectrometer (Finnigan delta S, Finnigan MAT,
Bremen, Germany). Details on this methodology have been reported
elsewhere (24, 25). Samples were measured in duplicate.
Urinary 15N baseline enrichment
was measured by gas IRMS (Europa Scientific, Crewe, UK) (39).
Plasma free [13C]- and
[2H2]lysine
enrichment was measured in
trifluoracetyl-n-propanol ester by
GC-MS. Plasma (100 µl) was subjected to cation exchange (Bio-Rad AG
50W-X8, 100-200 mesh, H+
form). After evaporation of the eluate, 0.5 ml of freshly prepared propanol-HCl (1:5 acetyl chloride-propanol, vol/vol) was added, and the
mixture was heated at 90°C for 30 min. Propyl esters were dried
under N2. Then 100 µl
acetonitrile and 100 µl trifluoroacetic anhydride were added, and the
mixture was heated at 60°C for 30 min. After evaporation of excess
reagent under N2 at room
temperature, the residue was redissolved in 0.5 ml ethyl acetate and
transferred to autosampler microvials. All reagents were purchased from
Sigma-Aldrich (St. Louis, MO) and were of analytical grade.
Samples (1.5 µl) were injected splitless into a Varian (Walnut Creek,
CA) Saturn 2000 quadrupole ion trap GC-MS equipped with a model 3400 GC
and then onto a DB-WAX capillary column (0.25 mm × 30 m, 0.25 µm film thickness; J&W Scientific, Folsom, CA). Helium was the
carrier gas, set to a head pressure of 55 kPa and adjusted so as to
deliver 1 ml/min at 250°C. The injector temperature was programmed
to rise from 140 to 190°C at 15°C/min, whereas the oven
temperature was programmed to rise from 180 to 250°C at
35°C/min after a 2-min isothermal phase. Sample retention times under these conditions averaged 6 min.
Isotopic enrichments were monitored in EI/MS/MS mode under
default settings to accommodate a filament emission of 90 µA, with a
multiplier gain of autotune + 200 V and an automatic gain control target of 5,000. For the prescan, the parent ions at or above mass-to-charge ratio (m/z) 380 (C13H18N2O4F6)
were isolated in an 8-amu parent mass window
(m/z 377-384). During the
subsequent analytic scan, the same parent window was isolated and
subjected to nonresonant excitation so as to produce a predominant
(product) ion centered at m/z 380. These were sampled using a narrow rF scan, and the ion current ratios
at m/z 381/380 and 382/380,
corresponding to the two lysine tracer/tracee ratios of interest, were
computed postrun from the narrow-range product ion scan data. Plasma
samples were analyzed in duplicate.
For purposes of calibration, a training data set was created by
compiling the mass spectral response of graded mixtures of [13C]- and
[2H2]lysine,
together with unlabeled lysine, over a 0- to 10-mole fractional range
for each tracer. Multilinear regression was then used to generate a
prediction equation, correlating the two ion pair ratios obtained
spectrometrically on standards as the explanatory variable for
tracer-to-tracee (tracer/tracee) mole ratios. Thus, for
[13C]lysine in the
presence of
[2H2]lysine
plus tracee, the regression relationship between tracer/tracee mole
ratio (MR1) was described by MR1 = 1.070 × RAE1
0.261 × RAE2; and for
[2H2]lysine
in the presence of
[13C]lysine plus
tracee, the tracer/tracee mole ratio (MR2) relationship was described
by MR2 =
0.261 × RAE1 + 1.070 × RAE2.
In these equations, the paired ion current ratios RAE1 and RAE2
represent the baseline corrected relative abundances at
m/z 381/380 and 382/380, respectively.
The equation was then applied in determining ion pair mole ratios from
the corresponding spectral information afforded by the plasma samples,
and the resulting mole ratios were converted algebraically into tracer
mole fractional excess (MFE) for each tracer, after subtraction of the
corresponding baseline plasma values for each set of samples. Analysis
of replicate standards (n = 5) under
these conditions showed a coefficient of variation ranging from 2 to
7%, the latter values being those for the detection of 0.5 MFE of
either lysine isotopolog in the presence of a 5 MFE of the other. Also,
by way of validation, the accuracy of estimates for this methodological
approach was found to fall within 5% of expected values on average,
based on analysis of sham mixtures prepared gravimetrically and with
known tracer(s)-to-tracee composition.
Evaluation of primary data.
Lysine oxidation (Lys Ox) was computed for each half-hour interval
during the 24-h tracer protocol, except during the first 2 h of the
study, when it was taken to be equal to that measured at the end of the
2-h period to reduce any possible effect of the lysine and bicarbonate
prime
where
13C plasma lysine enrichment is
the average of the two enrichments determining the specific half-hour
interval. Mean lysine oxidation for the fasted and fed states was
calculated during 480-720 min and 1,020-1,320 min of the
tracer period, respectively.
and
# = APE/100
and ## = recovery of
13CO2
[as reported by El-Khoury et al. (9); fasted state: 76.6%; fed
state: 85.1%].
Lysine turnover (rate of appearance, Lys
Ra) was calculated as follows
where
i is the tracer infusion rate
(µmol · kg
1 · 30 min
1),
Ei is the enrichment in AP of the
administered isotope
([13C]- or
[2H2]lysine),
and Ep is the enrichment in plasma
at isotopic steady state during fasting (480-720 min) and feeding
(1,020-1,320 min).
Lysine splanchnic uptake (Lys Spl Upt) was computed for the fasted and
fed states from plasma enrichments of orally and intravenously administered lysine
(E[2H2]Lys
and
E[13C]Lys) at isotopic steady state, normalized for infusion rate (ir) of tracers,
as follows
This
fraction represents the fraction of lysine taken up by the gut and the
liver during its first pass.
The contribution of microbial
[15N]lysine (Contr
Lysmicr) to total lysine
appearance in the peripheral circulation was estimated from the
fraction of lysine that was of microbial origin and the lysine
turnover. Thus the former was calculated from the ratio of the mean
enrichment of
[15N]lysine in plasma
(Ep [15N]Lys)
for the fast and fed states to the enrichment of
[15N]lysine in either
fecal or ileal microbial lysine
(Em [15N]Lys). These values were then multiplied by the appropriate lysine turnover (Lys Ra) value, derived from the
intravenously administered
[1-13C]lysine tracer.
Thus
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Statistics.
All results were expressed as means ± SD. The hypotheses to be
tested were 1) that
[13C]lysine kinetics
after intake of isonitrogenous amounts of
15NH4Cl
and
[15N2]urea,
respectively, would not differ from each other,
2) microbial lysine contribution to
host metabolism would not differ when
15NH4Cl
and
[15N2]urea,
respectively, were given as 15N
tracers to the MIT subjects, and 3)
that 15N enrichment of ileal
microbial lysine (in ileostomates) would be lower than fecal microbial
lysine (in MIT subjects) after the intake of
15NH4Cl.
Hence we used a two-sided, paired
t-test to evaluate group means for
hypotheses 1 and
2 and a one-sided, unpaired
t-test to compare group means of
ileostomates and MIT subjects (hypothesis 3). To compare the fasted and fed states during the
24-h tracer protocol in the MIT subjects, the mean values were compared
using a two-sided, paired t-test. A
P value of <0.05 was considered to
be significant. Nonsignificantly different means were labeled NS.
 |
RESULTS |
[15N]lysine labeling.
The plasma lysine 15N enrichments
in the MIT subjects on days 3 and
4, before the intake of the two
15N tracers, did not differ from
each other, and they corresponded to the
15N natural abundance
([15N2]urea
period: 0.3671 ± 0.0003;
15NH4Cl
period: 0.3668 ± 0.0004 15N
AP). Because a baseline plasma sample was not available for the
ileostomates, urinary total nitrogen was measured. The result (0.3658 ± 0.0003 15N
AP) was similar to plasma lysine of the MIT subjects.
The plasma [15N]lysine
enrichment (APE) after 6 days of
15NH4Cl
tracer intake was threefold greater than after
[15N2]urea
administration in MIT subjects in the fasted state (Table 3; Fig. 1).
This difference between the two
15N tracers was already apparent
on day 8 ([15N2]urea:
0.0028 ± 0.0011;
15NH4Cl:
0.0067 ± 0.0014; NS). Mean enrichments on day
8 after
15NH4Cl
and
[15N2]urea,
respectively, were significantly different from zero (one-sided
t-test;
P < 0.01;
P < 0.05).
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Table 3.
15N enrichment of plasma lysine and microbial protein
(ileal and fecal) lysine, respectively, in MIT subjects and
ileostomates during administration of
[15N2]urea and
15NH4Cl
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Fig. 1.
Daily pattern of plasma lysine 15N
enrichment on days 10-11
throughout 24-h hourly oral intake of 0.14 mg
15N · kg 1 · h 1,
either as
15NH4Cl
( ) or
[15N2]urea
( ) after 6 days of intake of the respective
15N tracer. Values are means ± SD for 6 subjects. Results are expressed as difference in enrichment
[15N atom percent excess
(APE)] from initial value on experimental days
3 and 4 (considered
zero enrichment baseline). Feeding began with small meals at 0600 (720 min) and was continued until 1500 (1,260 min), providing in total
(tracer included) 45 mg dietary
lysine · kg 1 · day 1.
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During the 24-h tracer protocol on days
10 to 11, the mean
plateau enrichments of plasma
[15N]lysine after
15NH4Cl
intake differed slightly between the fasted and fed states (0.0176 ± 0.0087 vs. 0.0118 ± 0.0034 APE; NS; Fig. 1; Table 3). After
[15N2]urea
consumption, fasted and fed state plasma
[15N]lysine
enrichments were not different (0.0056 ± 0.0035 vs. 0.0059 ± 0.0053 APE; NS; Fig. 1; Table 3).
Comparison of fasted-state plateau enrichments (during the 24-h tracer
protocol), after
[15N2]urea
and
15NH4Cl
tracer intake, respectively, showed significantly higher values with
the
15NH4Cl
tracer, although variability (25-100%) was high
(P < 0.05; Table 3). During the fed
state, values after
15NH4Cl
tracer intake also tended to be higher than after
[15N2]urea,
but this difference did not reach statistical significance.
Plasma [15N]lysine
enrichment of ileostomates on day 10 was somewhat but not significantly lower than that in MIT subjects
(Table 3). However, it should be noted that the ileostomates had
consumed the
15NH4Cl
tracer for only 5 days and had not received tracer during the night
before the final blood sample was drawn for analysis.
The 15N enrichment of microbial
lysine from feces and ileal fluid increased steadily after initiation
of the tracer (Fig. 2), and after 3 days
following the start of 15N intake
an apparent plateau was reached (mean slope 0.02; NS; Fig. 2). The
[15N]lysine enrichment
with
15NH4Cl
as tracer was higher than that with
[15N2]urea,
but the difference did not reach statistical significance (Table 3). In
comparison, the enrichment of ileal microbial lysine was significantly
lower than that for fecal microbial lysine in MIT subjects after
administration of
15NH4Cl
(P < 0.001; Fig. 2; Table 3). At the
end of the experimental period, after
15NH4Cl
and
[15N2]urea
intake, respectively, fecal microbial lysine was on average 15 and 25 times more enriched than plasma lysine (Table 3). In contrast, ileal
microbial lysine enrichment was 4.5 times higher than plasma lysine
enrichment in the ileostomates after
15NH4Cl
administration.

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|
Fig. 2.
Microbial fecal
(15NH4Cl:
;
[15N2]urea:
) and ileal
(15NH4Cl:
) lysine enrichments throughout experimental time frame. Daily
15N intake started on
day 5. Results are expressed as
difference in enrichment (15N APE)
from initial value on experimental days
3 and 4 (considered
zero enrichment baseline). Samples were not obtained on
day 11. Note that on some days
(days 5, 7, and
9), fecal samples of only 4 MIT
subjects could be obtained and that ileostomates ingested 13% more
15N · kg 1 · day 1
than MIT subjects because of their lower body weight.
|
|
Daily total nitrogen output via ileal fluid (621 ± 310 g/day) was
1.51 ± 0.45 (SD) g on day 9. The
lysine content in ileal microbial protein was 4.75 g/16 g N. Hence,
assuming that, as in the pig, 50% of total ileal nitrogen was of
microbial origin (43) and that the lysine concentration in endogenous
protein is similar to mixed body protein (~8%) (27), ~0.22 g
lysine of microbial origin and ~0.38 g lysine of endogenous origin
were lost daily under the current experimental conditions. This
corresponds to an approximate ileal total loss of 8.5 mg
lysine · kg
1 · day
1.
The 15N ratio of plasma free
lysine to microbial lysine did not differ between the two
15N tracers ingested (Table
4). However, if the plasma lysine
enrichment of the MIT subjects given the
15NH4Cl
tracer was related to ileal microbial lysine enrichment, the ratio was
higher (Table 4; fasted state: P < 0.01; fed state: P < 0.001).
Computed for the fasted state only, in ileostomates, when ileal
microbial lysine enrichment is used as a precursor, the apparent
fractional contribution of microbial lysine to plasma lysine was lower
than in MIT subjects (0.21 vs. 0.44; Table 4).
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|
Table 4.
Ratio of 15N enrichment in plasma free lysine to microbial
protein lysine after [15N2]urea and
15NH4Cl intake, calculated either from fecal or
ileal microbial lysine enrichment
|
|
Lysine kinetics.
As expected, the course of
13CO2
background enrichment in three subjects in the tracer-free study after
consumption of the experimental diet for 10 days changed only minimally
with the experimental diet (typically
0.0008 APE fasted state;
0.0003 APE fed state). The course of
13CO2
enrichment and
13CO2
production throughout the 24-h tracer protocol for the
15NH4Cl
and
[15N2]urea
intakes was apparently different, although this did not reach
statistical significance at any given interval (Fig.
3, A and
B).



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Fig. 3.
Pattern of change during a 24-h primed continuous intravenous infusion
of [1-13C]lysine
(infusion rate 3.5 µmol · kg 1 · h 1).
Values are 13C enrichment of
expired carbon dioxide (A), µmol
13CO2
expired (B), and lysine oxidation
(C) for each 30-min interval
throughout 24-h tracer protocol (means ± SD of 4 subjects). Feeding
began with small meals at 0600 (720 min) and was continued until 1500 (1,260 min), providing in total (tracer included) 45 mg dietary
lysine · kg 1 · day 1.
, After
15NH4Cl
intake; , after
[15N2]urea
intake.
|
|
The lysine oxidation rates, however, were significantly higher with
15NH4Cl
tracer intake for the intervals 540-720 min (fasted state) and
1,110-1,200 min (fed state), respectively
(P < 0.05; Fig. 3C). Total lysine oxidation measured
over 24 h was 34.2 mg · kg
1 · day
1
during
15NH4Cl
tracer intake and 19.2 when
[15N2]urea
was given (Table 5). Lysine turnover,
estimated with the intravenous
[13C]lysine tracer,
was higher when the
15NH4Cl
tracer was given than it was with
[15N2]urea,
but this was only significant for the fed state (Table 5). When lysine
turnover was estimated with the orally administered [2H2]lysine,
the same difference between the
15N tracers was seen in the fed
but not the fasted state. Lysine turnover estimated with the oral
tracer was higher (Table 5). Lysine splanchnic uptake was estimated not
to differ between 15N
tracers.
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|
Table 5.
24-h Lysine kinetics in MIT subjects receiving
15NH4Cl and
[15N2]urea, respectively, in small oral
doses during continuous intravenous infusion of
[1-13C]lysine and hourly oral administration of
[6,6-2H2]lysine
|
|
If the ratio of plasma
[15N]lysine to
microbial [15N]lysine
is used as a direct measure of the fraction of circulating lysine that
is derived from the intestinal microflora, then the estimate of the
contribution of fecal microbial lysine to lysine turnover, measured by
[1-13C]lysine per day
with the
15NH4Cl
data, was 28.9 ± 29.7 mg · kg
1 · day
1,
and with
[15N2]urea
it was 11.7 ± 7.5 mg · kg
1 · day
1
(NS; 4 subjects). However, when the microbial contribution was calculated on the basis of ileal microbial lysine enrichment with the
15NH4Cl
data, it was 129.6 ± 85.6 mg · kg
1 · day
1
for the MIT subjects. If a similar lysine turnover in ileostomates is
assumed, a possible 67.9 ± 13.7 mg · kg
1 · day
1
lysine was derived from microbial sources. The quantitative, nutritional meaning of the estimates will be presented in
DISCUSSION.
 |
DISCUSSION |
Although the present data confirm the less extensive, earlier
investigations by others on body
[15N]lysine labeling
after administration of
15N-labeled nitrogen precursors
(15, 38), a major question posed at the outset of this investigation
was the quantitative extent to which microbially synthesized lysine was
made available for metabolism in host tissues and its net contribution
to the lysine economy of the individual subject. This question turns
out to be far more difficult to answer from the present data than we had anticipated. After methodological and design aspects are addressed, the various reasons for this will be discussed.
We have measured plasma and microbial
[15N]lysine without
consideration of its enantiomeric form. A study investigating
D-amino acid contents in
cultures of several bacteria revealed that
D-lysine was not detectable,
whereas, for example, D-alanine
and D-aspartic acid ranged
between 8 and 80% and 2 and 60% of total amino acids, respectively
(5). In serum of adult rats ingesting standard food, no
D-lysine was detected (37).
Also, to the best of our knowledge,
D-lysine in human plasma has not
been reported in the literature, although other plasma amino acids
(serine, alanine, aspartic acid, phenylalanine, tyrosine) have been
shown to be present in D form
(2, 28). Therefore, we do not consider that a significant contribution
of D-lysine is likely here.
Nevertheless, if this were not the case, then we would have
overestimated the availability of microbially derived lysine for host utilization.
The reason for including ileostomates in this study is that, although
microbial activity is associated principally with the large intestine,
it is by no means confined solely to that part of the gastrointestinal
tract (8, 13), and the flora of the lower ileum is qualitatively
similar to that of feces (8). Given that the major sites of amino acid
absorption are in the small intestine, it was important to determine
the 15N enrichment in the
microbial protein of digesta as close as possible to those sites and
where there was no functional large intestine to complicate
interpretation of the results. Nevertheless, we recognize that ileal
fluid of ileostomates may not be the same as the contents of an intact
ileum (4, 16, 29, 41); however, our assessment, for the present
purpose, was that conventional ileostomy resembles the environment in
the intact ileum more than it does the colon, and this gave us the
rationale for comparing and contrasting the data from the two groups of
subjects studied here.
To help readers understand our results and the discussion that follows,
we present a scheme in Fig. 4 showing that
15N from either
15NH4Cl
or
[15N2]urea,
after breakdown by microbial urease, is incorporated either directly or
indirectly into microbial lysine (and other microbial amino acids) in
the small intestine. This depicts the complexity of the pathways that
lysine might take within the intestine and between the intestine and
host tissues, which introduces difficulties in readily interpreting the
quantitative significance of our findings.
The 15N from nonspecific nitrogen
sources is returned to the intestinal tract in the
15N-labeled amino acids in
endogenous secretions (pancreatic, biliary, mucosal) and as
15N urea (Fig. 4). Intravenous
infusion of 15N-labeled amino
acids is followed by labeled plasma amino acids appearing rapidly in
the jejunal fluids and pancreatic secretions (14, 21). The quantity of
endogenous protein that is recycled in the intestine makes it a
potentially significant source of nitrogen for microbial growth. In
pigs, 90% of all endogenous nitrogen secreted into the gut was found
to be reabsorbed (19). Although these experiments do not explain the
involvement of the enteric flora in this process, it is probable that
microbial proteolytic activity is involved because mucins and some
other digestive secretions are resistant to mammalian digestive enzymes.
The differences we observed between ileal and fecal microbial protein
in [15N]lysine
enrichment are in accord with investigations in the pig (40) showing
that, after the pigs were fed 15NH4Cl for 10 days, [15N]lysine enrichment increased
dramatically from the ileum to the caecum. Also, the difference in
fecal microbial and plasma lysine 15N enrichment seen in this study
between
15NH4Cl
and
[15N2]urea
confirms earlier results in the pig (25) and is presumably due, in
part, to the greater retention of
15N label from ammonium chloride
(24a). However, because of high interindividual variation the
difference did not always reach statistical significance. If allowance
is made for the different 15N
retention, then the microbial enrichments would not appear to be
different, although an additional reason for the different enrichments
might be differences in ammonia nitrogen and urea nitrogen metabolism
by the microflora (35, 26).
Using the 15N enrichment of fecal
microbial lysine with
15NH4Cl
as the label, we estimated an uptake of ~29
mg · kg
1 · day
1;
with
[15N2]urea,
the estimate was 12 mg · kg
1 · day
1,
although this was not statistically different. It is obvious, however,
that the digesta undergo many changes during passage from the duodenum
to the rectum, and it is unlikely that the
[15N]lysine in
microbial protein from feces is truly representative of that occurring
at sites of amino acid absorption in the intestine. It was for that
reason that we sampled ileal digesta from ileostomates as being closer
to the sites of absorption. The corresponding estimate of microbial
lysine absorption for the ileostomates (when the same rate of lysine
turnover is assumed as in the MIT subjects) is ~68
mg · kg
1 · day
1.
If the 15N enrichment of lysine in
ileal digesta were assumed to represent the
15N enrichment of absorbed lysine
in the MIT subjects given
15NH4Cl,
their uptake of microbial lysine would be calculated to be ~130
mg · kg
1 · day
1,
a value that exceeds by ~10-fold both the apparent dietary
requirement and, therefore, the irreversible loss of body lysine (12).
In considering the plausibility of these different estimates of
microbial lysine uptake, one must consider that they refer to the gross
uptake of amino acid synthesized by the microflora and do not
necessarily represent a net increase of that magnitude in the total
lysine available for metabolism. To the extent that 1) the growth of the microbes that
give rise to the labeled lysine is supported by the degradation of
endogenous protein (Fig. 4) and 2)
luminal amino acids of endogenous origin are oxidized by intestinal
tissues, then the microbially derived lysine in plasma can be seen as
part of the normal mechanism by which endogenous nitrogen and amino
acids are recycled, rather than microbial amino acids serving as a net
source of amino acids, which are additional to those supplied in the
diet. Support for this view comes from previous
[1-13C]leucine
oxidation studies (10, 11), where there is an excellent concordance
between both predicted and measured protein oxidation when estimated
from leucine oxidation and N excretion values, which would not have
been the case if there had been a significant net uptake of leucine
from the gut microflora. There seems little reason to expect, if there
were a significant entry of microbially derived lysine into body
tissues, that this would not also apply to the other nutritionally
indispensable amino acids, including leucine.
Although the true 15N enrichment
of the labeled lysine that is actually absorbed might be closer to that
of the ileal digesta protein than to that of the feces, it could quite
conceivably be higher (30). Furthermore, if
15N-labeled substrates entering
the gut lumen 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.
The loss of lysine at the terminal ileum was estimated in the present
study to be ~8.5
mg · kg
1 · day
1.
Presumably there is also loss of endogenous lysine due to bacterial and
tissue intestinal oxidative metabolism (Fig. 4), as indicated by the
measured first-pass lysine extraction by splanchnic tissues (and
microflora) of ~25% of the dietary lysine (Table 5). It could be,
therefore, that any entry of lysine of microbial origin helps to
counteract the endogenous lysine losses, rather than adding in a net
way to the total exogenous (diet) input of lysine.
The lysine oxidation rates measured in this study are considerably
lower than the ~45
mg · kg
1 · day
1
level of lysine intake, indicating that we have significantly underestimated the true whole body lysine oxidation rate. This is in
accord with our previous oral/intravenous
[1-13C]lysine tracer
studies (9). Lysine turnover rates were in the same range as reported
earlier and were somewhat higher for the orally adminstered lysine
tracer (9). It was surprising, however, that there were differences
between various measures of lysine kinetics between the two different
studies in which either
[15N2]urea
or
15NH4Cl
was used as a tracer (Table 5). There are a number of possible explanations. First, it could be due to the
15N tracers per se. However, this
seems unlikely, because the amount of nitrogen supplied with the two
15N tracers was equal. If account
is taken of the 1.6-fold higher 15N retention with
15NH4Cl
compared with
[15N2]urea
(24a), and it is assumed that all of the additional nitrogen retained
was used to produce microbial lysine, this would amount to a difference
between the two tracers of only 3 mg · kg
1 · day
1.
This does not explain the observed difference in lysine kinetics between the two tracers (Table 5). Second, although administration of
NH4Cl causes acidosis and
increases protein breakdown and amino acid oxidation (34), this occurs
at doses that are severalfold higher than the total 1 g/day dose used
in this study. Third, and on the basis of the foregoing comments, the
observed differences would appear to be due to experimental and
biological factors that cannot be identified at this time.
In conclusion, to reliably estimate the quantitative contribution made
by lysine (or other indispensable amino acids) of bacterial origin to
host metabolism, use of the present paradigm seems doubtful. Although
our data do suggest a potentially significant nutritional uptake into
body tissues of amino acids of microbial origin, the quantification of
this uptake, and its net contribution to the amino acid economy,
require a better understanding of nitrogen and carbon transactions of
amino acids and related compounds between the intestinal tissues and
the microflora within the gastrointestinal tract.
 |
ACKNOWLEDGEMENTS |
We are grateful to the staff of the MIT Clinical Research Center
and the Unit Protein Metabolism of the German Institute of Nutrition
for skillful technical assistance.
 |
FOOTNOTES |
This research was supported by National Institutes of Health Grants
DK-42101, P-30-DK-40561, and RR-88, and a grant from the Deutsche
Forschungsgemeinschaft, Bonn, Germany.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for correspondence and reprint requests: C. C. Metges,
German Institute of Human Nutrition, Arthur-Scheunert-Allee
114-116, 14558 Bergholz-Rehbrücke, Germany (E-mail:
metges{at}www.dife.de).
Received 18 November 1998; accepted in final form 24 May 1999.
 |
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