Daily delivery of dietary nitrogen to the periphery is stable
in rats adapted to increased protein intake
Céline
Morens,
Claire
Gaudichon,
Gilles
Fromentin,
Agnès
Marsset-Baglieri,
Ahmed
Bensaïd,
Christiane
Larue-Achagiotis,
Catherine
Luengo, and
Daniel
Tomé
Institut National de la Recherche Agronomique, Unité de
Physiologie de la Nutrition et du Comportement Alimentaire, Institut
National Agronomique de Paris-Grignon, F-75005 Paris, France
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ABSTRACT |
Dietary nitrogen was traced
in rats adapted to a 50% protein diet and given a meal containing
1.50 g 15N-labeled protein (HP-50 group). This group
was compared with rats usually consuming a 14% protein diet and fed a
meal containing either 0.42 g (AP-14 group) or 1.50 g (AP-50
group) of 15N-labeled protein. In the HP group, the muscle
nonprotein nitrogen pool was doubled when compared with the AP group.
The main adaptation was the enhancement of dietary nitrogen transferred
to urea (2.2 ± 0.5 vs. 1.3 ± 0.1 mmol N/100 g body wt in
the HP-50 and AP-50 groups, respectively). All amino acids reaching the
periphery except arginine and the branched-chain amino acids were
depressed. Consequently, dietary nitrogen incorporation into muscle
protein was paradoxically reduced in the HP-50 group, whereas more
dietary nitrogen was accumulated in the free nitrogen pool. These
results underline the important role played by splanchnic catabolism in adaptation to a high-protein diet, in contrast to muscle tissue. Digestive kinetics and splanchnic anabolism participate to a lesser extent in the regulation processes.
high-protein diet; rats; adaptation; nitrogen-15
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INTRODUCTION |
ADAPTATION TO A
PARTICULAR level of protein intake is a complex process involving
both short- and long-term regulation of the anabolic and catabolic
pathways that control nitrogen and amino acid (AA) homeostasis. Dietary
intake is a discontinuous process unevenly distributed throughout the
nycthemere. Both energy and macronutrient content vary between meals.
As a consequence, the body must be able to respond acutely to both
daily transitions between the fasted and fed states and changes in the
protein content of meals. A long-term adaptation to the habitual level
of protein intake also occurs. It is established that one of the
principal mechanisms of adaptation to an increase of this habitual
protein intake is the stimulation of splanchnic AA oxidation. The
activity of different gut and liver enzymes and transport systems
increases markedly after an adaptation period, thereby augmenting the
capacity for AA catabolism (7, 12, 20, 26, 29, 34, 35,
38).
However, although the modulatory role of liver catabolic pathways is
well established, less is known about the cooperative catabolic and
anabolic capacities of different splanchnic and peripheral tissues to
cope with an excess of dietary AA. The current view is that the
splanchnic area has a large but limited buffering capacity to respond
to a high-protein (HP) intake (8, 20, 27). Above the
limit, metabolic adaptive adjustments may be altered to an
accommodation status that can have deleterious effects. The regulation
of both splanchnic anabolism and peripheral AA and nitrogen metabolism
under these conditions is still not fully understood. There seems to be
a small increase of body protein synthesis (6, 14, 28,
32), which may in the fed state contribute to absorb the surplus
dietary AA, even if the mean daily protein turnover rate does not
appear to change (15). It may be possible to induce the
liver protein synthesis rate through an increase in dietary protein
intake (19). The results for the gut mucosa are more
controversial, depending on the species and the physiological state
(3, 9). Muscle protein synthesis does not seem to be
enhanced by HP intake (2) but, in contrast, may be
surprisingly reduced (40). Such observations likely depend on both the type of muscle studied and on the composition of the diet
in energy nutrients (46).
The objective of this work was to assess the effects of long-term
adaptation to a diet with an HP content (50% dry matter) on the
anabolic and catabolic capacities of different tissues closely involved
in the adaptation of AA and nitrogen metabolism, i.e., gut, liver,
kidney, and muscle, to an increase in protein intake. For this purpose,
rats were adapted for 15 days to either an HP diet (i.e., 50% protein)
or an adequate-protein (AP) diet (i.e., 14% protein). Protein and
nonprotein nitrogen were measured in the different tissues and the
plasma. The postprandial distribution and deamination of dietary AA
were studied in rats chronically consuming a HP diet (i.e., 50%
protein) after the ingestion of a meal containing 1.50 g of
15N-labeled protein and then compared with the response
obtained in rats adapted to an adequate protein diet (i.e., 14%
protein) after the ingestion of a meal containing either 0.42 or
1.50 g of 15N-labeled protein.
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MATERIALS AND METHODS |
Animals and Diets
Male Wistar rats (n = 112; Harlan), initially
weighing 196 ± 1 g, were housed in single stainless steel
wire cages in a room lit for 12 h daily (2030-0830) and
maintained at a temperature of ~23°C. The animals were divided into
the following three groups: two groups of rats (total n = 76) were adapted for 15 days to a control diet providing an adequate
intake of protein (AP group, 14% of total milk protein), whereas the
third group of 36 rats was fed for 15 days on an HP diet (HP group,
50% of total milk protein). The composition of the diets is given in
Table 1. Rats were fed according to a
schedule of three meals a day to adapt them to prompt consumption of
the diet (0830-0845: 3 g of food; 1330-1430 and
1830-1930: free access to food), as previously
described (27). On the morning of day 16 (0830-0845), 36 AP rats, i.e., rats adapted to an adequate protein
diet, ingested an experimental 3-g meal containing 14% of
15N-labeled milk protein (0.42 g protein). This group was
taken as the control (AP-14). The other 40 AP rats that were adapted to
an adequate protein diet ingested a 3-g meal containing 50% of
15N-labeled milk protein (1.50 g of protein). This group is
referred to as AP-50. The 36 rats of the HP group ingested a 3-g meal
containing 50% of 15N-labeled milk protein (1.50 g of
protein). This group was named HP-50. For more clarity, Fig.
1 summarizes the experimental schedule. The composition of the experimental meals is described in Table 1. The
study was carried out in accordance with the recommendations of the
French Committee for Animal Care. As previously described (27), the 15N-labeled milk was produced at the
Institut National Agronomique experimental farm in Grignon with the
help of Dr. P. Schmidely (Department of Animal Sciences, INA-PG,
Grignon, France), and the isotopic enrichment of isolated milk protein
was 0.4535 atom percent excess.

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Fig. 1.
Diagram of the experimental protocol. Rats were adapted for 15 days
(D) to either an adequate-protein (AP) diet (14% protein) or a
high-protein (HP) diet (50% protein). On the 16th day, rats received
an experimental meal containing either 14 or 50% protein. Rats were
killed at times varying from 0 to 5 h after the meal.
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Experimental Protocol
The rats were killed at 0, 1, 2, 3, 4, and 5 h after the
15N-labeled meal. The average number of animals at each
time point was six. They were injected with 13.6 mg/100 g body wt of
pentobarbital sodium for anesthesia and 5,000 IU heparin (Laboratoires
Leo, Saint-Quentin en Yvelines, France) to permit blood sampling.
Sampling Procedures
Blood was removed from the peritoneal cavity after rupture of
both the abdominal aorta and vena cava. Plasma was stored at
80°C
until analysis. Urine was collected from the bladder and from an
absorbent paper, which had been placed under the cage from the
beginning of the experiment. Stomach, gut and cecum digesta, liver,
small intestine and colon mucosa, one kidney, and one gastrocnemius muscle were sampled. The small intestine was divided into three equal
parts that were everted and scraped as well as the colon to collect the
mucosa. All samples were weighed and stored at
80°C until analysis.
Analytical Methods
Urea was measured in blood and urine using the urease/glutamate
dehydrogenase reaction (HYCEL kit, Le Rheu, France). Urinary urea was
extracted by cation exchange chromatography on Dowex resin (Dowex
AG50X8; Bio-Rad, Ivry sur Seine, France) as previously described
(18) and was stored at 4°C until isotopic determination. Plasma was deproteinized with 1 mol/l HCl and then neutralized with 0.1 mol/l NaH2PO4. Plasma urea was then extracted
using the same procedure.
For AA analysis, plasma was first deproteinized with
sulfosalicylic acid (50 mg/ml). The supernatant was dried and
resuspended in a lithium citrate buffer (pH 2.2) for analysis. Plasma
AA concentrations were determined using HPLC with a postcolumn
ninhydrin derivatization system (Amino System 3000; Bio-Tek
Instruments, St. Quentin en Yvelines, France).
Tissue samples were crushed in 4 vol of 0.9% NaCl. TCA (10% final
concentration) was used to precipitate the protein. After centrifugation, the supernatant (containing free AA and small peptides)
was frozen and dried, and the pellet (containing protein) was
washed one time in 0.9% NaCl and then frozen and lyophilized. The
soluble fraction was designated as the nonprotein fraction, and the
insoluble fraction was designated as the protein fraction.
Isotopic Determinations
Before the isotopic determination of 15N
enrichment, resins were eluted with 2.5 mol/l KHSO4. An
isotopic ratio-mass spectrometer (Optima; Fisons Instruments,
Manchester, UK) coupled to an elemental analyzer (NA 1500 series 2;
Fisons Instruments) was used to measure 15N enrichment in
urinary and blood urea as well as in protein and nonprotein fractions
of tissues and gastrointestinal digesta, as previously described
(27). Total nitrogen in the protein and nonprotein
fractions of tissues (i.e., gut and colon mucosa, liver, muscle,
kidney) was measured using the elemental nitrogen analyzer.
Calculations and Statistics
All results were expressed in quantities per 100 g of body
weight, since the rats in the three groups did not have exactly the
same weight at the end of the adaptation period.
Body muscle weight was evaluated assuming that it represented 45% of
total body mass, as previously described (27). The gastrocnemius was taken to be representative of the whole muscular mass.
Dietary nitrogen in samples.
The dietary nitrogen present in samples (Ndiet, mmol) was
calculated as follows
where Ntot is the amount of total nitrogen in the
sample, and APEs and APEm are the
15N enrichment excess of the sample and the meal, respectively.
The dietary nitrogen present in the urea body pool
(Ndiet-urea, mmol) was calculated according to the formula
where BW is the body weight, Curea is the
concentration of urea in the plasma, and APEureas is the
15N enrichment excess of the plasma urea sample.
Sixty-seven and ninety-two percent were the mean percentage of body
water in the rat and the mean percentage of water in plasma,
respectively (36). The total transfer of dietary
nitrogen to urea was calculated as the sum of the dietary nitrogen
excreted in urinary urea and the dietary nitrogen present in the body
urea pool.
Intestinal flux of dietary nitrogen absorption.
The intestinal flux of dietary nitrogen absorption
(QA) was calculated from the amounts of dietary
nitrogen that disappeared from the stomach and intestinal lumen within
1 h
where NdietSt,
NdietIt, and
NdietCt are the amount of dietary
nitrogen recovered at time point t from the stomach, small
intestine, and cecum, respectively, and Ningest is the
amount of ingested nitrogen.
Protein fractional synthesis rate in gut mucosa.
Protein fractional synthesis rates in the gut mucosa (FSR, %/day) were
calculated assuming that the luminal AA were the precursor pool for
protein synthesis (39), according to the formula
where APEmucosa is the 15N enrichment
excess of the mucosa sample, and t is the incorporation time
expressed in hours.
Daily incorporation of dietary nitrogen.
The daily incorporation of dietary nitrogen into different body pools
was extrapolated from experimental data for the AP-14 and HP-50 groups,
assuming that the morning meal was representative of the other meals
during the day, using the formula
where Nincorp is the amount of dietary nitrogen
daily transferred to the pool (tissue or deamination),
Ndaily is the amount of nitrogen consumed daily (480 and
1,600 mg for AP and HP rats, respectively), and Ningest is
the nitrogen content of the experimental meal (64 and 240 mg for AP-14
and HP-50 rats, respectively).
The results are expressed as means ± SE. Differences between
groups were tested using ANOVA (Proc GLM, SAS version 6.11). P < 0.05 was considered to be significant.
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RESULTS |
Daily Food Intake, Weight Gain, and Biochemical Characteristics in
the Fasted State
Growth and food intake were measured every day for 15 days in rats
fed AP or HP diets (Fig. 2). During the
first 2 days of the adaptation period, rats in the HP group lost weight
(
6.9 ± 0.6 g/day). Thereafter, the weight gain was 5.5 ± 0.2 g/day for the rats in the AP group and 5.2 ± 0.1 g/day for
those in the HP group and did not differ significantly. The 15-day
cumulative weight gain in rats consuming the 50% protein diet was
significantly lower than that seen among rats consuming the 14%
protein diet (54.9 ± 2.2 vs. 67.6 ± 2.2, P < 0.001). This was because of diminished food intake during the first
3 days. The food efficiency ratio calculated over the 15-day adaptation
period (cumulative weight gain/cumulative food intake) was
significantly higher among rats in the AP group than among those in the
HP group (0.020 ± 0.0004 and 0.017 ± 0.001 g/kJ,
respectively, P < 0.0001).

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Fig. 2.
Daily food intake (A) and cumulative weight
gain (B) of rats during adaptation to an AP
(n = 76) or HP (n = 40) diet for 15 days. Data are means ± SE. *P < 0.05, AP vs.
HP.
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After the 15-day adaptation period, the weights, protein, and
nonprotein nitrogen contents of different tissues (Table
2), as well as fasted plasma urea and AA
levels (Table 3), were measured in rats
adapted to AP or HP diets. The contribution of proximal and median
intestinal mucosa, colon mucosa, liver, and kidneys to body mass was
significantly heavier (P < 0.05) in animals given the
HP diet for 2 wk compared with those given the AP diet. Similar differences were obtained when the total mass of each organ was taken
into account (data not shown). For instance, the liver weight was
increased from 9.3 ± 0.2 g in the AP group to 11.2 ± 0.2 g in the HP group. The total amount of protein nitrogen was
significantly higher in the proximal and median intestinal mucosa in
the HP group. The total nonprotein nitrogen content was higher in the liver (+93 mmol/100 g body wt) and to a greater extent in the muscle of
animals in the HP group. The differences between groups were similar
whether or not nitrogen was reported to the body weight. Fifteen days
of adaptation to the HP diet resulted in striking changes to certain
biochemical plasma characteristics. In the fasted state, plasma urea
concentrations were more than double among HP rats compared with AP
rats, whereas plasma protein concentrations did not differ
significantly. Plasma AA concentrations measured in the fasted state
were also markedly influenced by the level of protein in the adaptation
diet. All AA, except the branched-chain type (BCAA) and Arg, were
depressed, and particularly the essential AA His, Met, and Thr and the
nonessential AA Gly, Ser, and Gln (P < 0.05). The most
spectacular reduction was found for Met, the concentration of which was
nine times lower in the HP than in the AP group (4.2 ± 0.9 vs.
36.1 ± 4.8 µmol/l, respectively). In contrast, concentrations
of BCAA were increased by 70% in HP rats.
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Table 2.
Organ weights and total protein and nonprotein nitrogen of tissues
collected from rats adapted for 2 wk to either a 14% protein or 50%
protein diet
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Nitrogen Kinetics and Distribution in the Tissues After the
Ingestion of a 15N-Labeled Protein Meal
After adaptation for 15 days to an AP or an HP diet, the rats
received a low (0.42 g) or a high (1.50 g) 15N-labeled
protein meal. The digestion kinetics and the postprandial incorporation
of dietary nitrogen in the protein and nonprotein fractions of gut
mucosa, liver, plasma protein, kidney, and muscles were measured during
the first 5 h after ingestion of the meal.
Digestive kinetics.
The digestive kinetics responded differently to an acute or chronic
increase in protein intake. The gastric emptying rate, expressed as a
percentage of nitrogen ingested, was moderately affected by adaptation
to the HP diet (AP-50 vs. HP-50 groups; Fig.
3A). When compared with the
AP-50 group, a slight delay in emptying rate was observed during the
first 2 h in the AP-14 group, but, after 3 h, an inversion
was seen, with slower gastric emptying of protein in the AP-50 group.
More dietary nitrogen was recovered in the intestinal lumen of rats in
the HP-50 group than in the AP-50 group (Fig. 3B). As a
consequence, the protein content of the meal (14 or 50%) had an effect
on the hourly flux of dietary nitrogen absorption by the small
intestine (Fig. 3C). In the control group (AP-14), the
maximum flux was delayed compared with that seen in the AP-50 and HP-50
groups (2 vs. 1 h). Moreover, the maximal flux of dietary nitrogen
absorption was reduced by the adaptation period, since the peak value
was lowered from 52 to 42% of ingested nitrogen in the AP-50 and HP-50
groups, respectively.

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Fig. 3.
Gastric emptying (A), dietary nitrogen in the
gut lumen (B), and intestinal absorption flux (C)
in rats adapted to an HP or AP diet and then ingesting either a 50 or
14% 15N-labeled protein meal (HP-50, n = 36; AP-50, n = 40; and AP-14, n = 36).
Data are means ± SE. Values with the same letter are not
significantly different, P < 0.05. Note that the scale
in B is different from A and C.
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Incorporation of dietary nitrogen.
The incorporation of dietary nitrogen in the protein and nonprotein
fractions of some tissues is shown in Fig.
4. In the gut mucosa, incorporation did
not differ between AP-50 and HP-50 rats (Fig. 4A). It
increased significantly during the postprandial period and finally
reached 93 ± 8 µmol/100 g body wt in the HP-50 group and
104 ± 11 µmol/100 g body wt in the AP-50 group. There was
almost no difference in the amount of dietary nitrogen recovered in the
nonprotein fraction in the two groups, except at 1 and 5 h where
values were slightly but significantly higher in the HP-50 group. No
significant differences were found in the colon in either the protein
or the nonprotein fractions (data not shown). Protein FSR (%/day) for
the mucosa were calculated in the proximal, median, and distal small
intestine and in the colon (Table 4). The
15-day adaptation period had no effect (AP-50 vs. HP-50 data), and FSR
was highest in the proximal segment and lowest in the colon.

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Fig. 4.
Dietary nitrogen incorporation in protein and nonprotein
fractions of gut mucosa (A), liver (B), and
muscle (C) [mmol dietary N/100 g body wt (BW)] in rats
adapted to an HP or AP diet and then ingesting either a 50 or 14%
15N-labeled protein meal (HP-50, n = 36;
AP-50, n = 40; and AP-14, n = 36). Data
are means ± SE. *P < 0.05, AP-50 vs. HP-50,
protein (P) fraction. P < 0.05, AP-50 vs. HP-50,
nonprotein (NP) fraction.
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Table 4.
FSR (%/day) of gut mucosa, calculated 1 h after meals in rats
adapted or not to an HP or AP diet and ingesting a 50%
15N-labeled protein meal
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In the liver, the incorporation of dietary nitrogen in both the protein
and nonprotein fractions (Fig. 4B) was higher in HP-50 rats
during the first 3 h (at 2 h, for instance: protein nitrogen, 179 ± 37 vs. 107 ± 12 µmol/100 g body wt in HP-50 and
AP-50 groups, respectively; and nonprotein nitrogen: 46 ± 4 vs.
28 ± 4 µmol/100 g body wt in HP-50 and AP-50 groups,
respectively, P < 0.05). In muscle, marked differences
were observed in the dietary nitrogen kinetics of both protein and
nonprotein fractions (Fig. 4C). During the first 4 h
after the meal, more dietary nitrogen was recovered in the nonprotein
fraction of HP-50 rats than in that of AP-50 rats (at 2 h:
374 ± 40 vs. 187 ± 35 µmol/100 g body wt in HP-50 and
AP-50 groups, respectively, P < 0.05). In contrast,
more dietary nitrogen was incorporated in the muscle protein of AP-50
animals. However, because of the high variability of results in the
AP-50 group, the differences were only significant at 3 h
(868 ± 152 vs. 306 ± 136 µmol/100 g body wt in AP-50 and
HP-50 groups, respectively, P < 0.05).
Finally, Table 5 shows the overall
distribution of dietary nitrogen in the different samples collected
5 h after the meals in the three groups of rats. Nonnegligible
amounts of dietary nitrogen still remained in the gastrointestinal
tract (stomach and gut), particularly in the AP-50 group (9.6% of
ingested nitrogen). In the other groups, nonabsorbed nitrogen
represented 3.5 and 5.0% of ingested nitrogen. More dietary nitrogen
was incorporated in plasma proteins when rats were given HP food for
the first time (AP-50 group) than after a 15-day adaptation period.
Five times more dietary nitrogen was seen in the kidney protein
fraction of rats in the HP-50 group.
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Table 5.
Dietary nitrogen distribution in body nitrogen pools 5 h after
ingestion of a meal containing 14 or 50% 15N-labeled total
milk protein by rats adapted to either an AP or HP diet
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Plasma AA Kinetics and Dietary and Total Nitrogen Transfer to Urea
The maximum variations in plasma AA concentrations, observed after
the 50% protein meal, were attenuated after HP adaptation, since the
postprandial concentration of total AA increased by 31% in AP-50 rats
and by 18% in HP-50 rats (data not shown).
Transfers of dietary nitrogen to body and urinary urea differed
considerably in rats receiving the HP diet for the first time (AP-50)
and after a 15-day adaptation period (HP-50) (Fig.
5). In the AP-50 group, the transfer of
dietary nitrogen to body urea regularly increased to reach a maximum of
0.571 ± 0.108 mmol/100 g body wt at 5 h, whereas a peak of
0.647 ± 0.127 mmol/100 g body wt was observed at 3 h among
HP-50 rats (Fig. 5A). Dietary nitrogen excretion in urinary
urea (Fig. 5B) was greatly enhanced by adaptation to the HP
diet; for instance, 5 h after the meals the amount of dietary
nitrogen recovered in urinary urea reached 1.679 ± 0.266 mmol/100
g body wt in HP-50 rats and only 0.723 ± 0.121 mmol/100 g body wt
in rats belonging to the AP-50 group (P < 0.001). This amount reached only 0.089 ± 0.017 mmol/100 g body wt in the AP-14 group. The kinetics also differed, since a plateau was achieved in the
HP-50 group after 3 h, whereas a sustained rise was observed in
the AP-50 group.

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Fig. 5.
Transfer kinetics of dietary nitrogen to body urea
(A) and to urinary urea (B) in rats adapted to an
AP or HP diet and then ingesting either a 50 or 14%
15N-labeled protein meal (HP-50, n = 36; AP-50, n = 40; and AP-14, n = 36). Data are means ± SE. Values with the same letter are not
significantly different, P < 0.05.
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Cumulative total and dietary nitrogen transfer to urinary plus body
urea, i.e., total deamination, was evaluated in the three groups during
the postprandial period (Fig. 6). The
production of total urea over the entire study period was greatly
enhanced in the HP-50 group compared with the AP-50 group (6.02 ± 0.99 vs. 3.36 ± 0.36 mmol N/100 g body wt in the HP-50 and AP-50
groups, respectively, 5 h after the meal). In these two groups,
dietary nitrogen represented an important part of the total nitrogen
transferred to urea (between 25 and 40% in the HP-50 group and 19 and
42% in the AP-50 group), although it never represented >23% in the AP-14 group. Finally, 5 h after the meal, the total deamination, calculated as the sum of dietary nitrogen transferred to urinary and
body urea, was 1.7 times higher in the HP-50 group than in the AP-50
group (2.19 ± 0.48 vs. 1.29 ± 0.22 mmol/100 g body wt, respectively, P < 0.001) and only reached 0.13 ± 0.03 mmol/100 g body wt in the AP-14 group.

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Fig. 6.
Cumulative dietary and total nitrogen transfer to urea in
rats adapted to an AP or HP diet and then ingesting either a 50 or 14%
15N-labeled protein meal (HP-50, n = 36 ;
AP-50, n = 40; and AP-14, n = 36).
a,b,cTotal nitrogen: values with the same letter do not
differ significantly, P < 0.05. d,e,fDietary nitrogen: values with the same letter do not
differ significantly, P < 0.05.
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DISCUSSION |
The purpose of this work was to evaluate the adaptation of
nitrogen and AA metabolism to an HP diet (50% protein) in rats. Our
findings pointed out that the principal adaptation occurred in the
organs of the splanchnic area through the dramatic enhancement of
catabolic pathways. The pattern of dietary AA reaching the peripheral
zone was significantly modified, with a large increase in the transfer
of BCAA and a reduction in the other essential and nonessential AA.
Paradoxically, the chronic consumption of a HP diet resulted in a
twofold increase in the nonprotein nitrogen pool in the muscle and in a
lower incorporation of dietary nitrogen in muscle protein. In contrast
to catabolic activation, the anabolic pathway was either moderately
enhanced or remained unchanged, depending on the tissue.
Activation of Splanchnic Catabolism
The principal adaptive regulation pathways responding to an
increase of the habitual protein content of the diet were seen in the
splanchnic tissues and subsequently in the kidneys, with dramatic
stimulation of both AA deamination and urea production. The liver is
known to play a central role in postprandial dietary AA metabolism,
since it modulates the inflow of AA from the intestine and controls AA
supply to peripheral tissues. One of the most remarkable changes
induced by adaptation to a HP diet is an increase in AA oxidation,
especially in the fed state (15). Although we did not
specifically study AA oxidation, the production of urea that represents
80-85% of the nitrogen excreted in urine is highly correlated
with AA catabolism. We observed a net increase in plasma uremia, which
almost doubled when the HP group was compared with the AP group, and in
total urea excretion over the experimental period. Similarly, the
surplus nitrogen provided by dietary AA was mainly directed toward
deamination when animals were adapted to a HP intake. This shows the
stimulation of hepatic AA-metabolizing enzymes such as alanine and
aspartate aminotransferases or glutamate dehydrogenase that was
demonstrated in rats adapted to a 51% protein diet (8).
Rémésy et al. (34) reported an increase in
mitochondrial glutamine hydrolysis in hepatocytes isolated from animals
fed a 70% casein diet, this being an important step in urea
production. However, because the gut mucosa metabolizes >90% of the
luminal Glu and Gln (33a, 43a), the liver is likely to be less
important in the catabolism of those AA. The lower baseline
concentration of Gln in animals fed the HP diet could also be explained
by its high level of utilization in kidneys, since this AA is the major precursor of urinary ammonia (5), and by a diminution in
de novo synthesis from BCAA in muscle (25). This probably
also leads to a decrease of plasma Ala (45). As a result,
with the remarkable exception of BCAA and Arg, plasma concentrations of numerous AA were diminished in the rats of the HP group, whereas the
protein intake of those animals was almost three times that of animals
in the AP group. The increase in Arg concentration must reflect the
intestinal and hepatic activation of the urea cycle, and it has clearly
been shown that the expression of branched-chain aminotransferase was
not regulated in the liver but rather in the kidneys, muscle, and heart
(43). All of these adaptive processes result in the
remarkable stability of plasma AA concentrations in the postprandial state.
Is the Muscle Nitrogen Metabolism Regulated?
Our study showed that the dietary nitrogen content in the
nonprotein fraction of muscle was considerably increased, whereas less
dietary nitrogen (even though it was not significant) was incorporated
in the protein pool. Several studies have supported the idea that HP
consumption does not enhance muscle protein synthesis (2, 23,
40). For instance, Masanés et al. (24)
recently reported a decreased FSR in the muscles of Zucker rats fed a
36% protein diet vs. rats receiving a 9% protein diet. This
observation agrees with the assumption made by Abumrad et al.
(1), who suggested that, when skeletal muscle is exposed
to excessive amounts of BCAA, most of what is incorporated is not used
for protein synthesis but sequestrated in a free AA pool, which may be
mobilized later. If the apparent increased availability of AA in the
intracellular pool is in fact mainly the result of an increase in BCAA,
as indicated by the plasma AA concentrations, this suggests that muscle
weakly reacts to modifications of the AA pattern, since the oxidation of BCAA in the muscle does not seem to be enhanced. Because the availability of other AA is depressed, protein synthesis must be slowed
through the paradoxical reduction in substrate availability. We should
note the fact that, in our study, the isocaloric exchange between diets
was performed on carbohydrates. It can be argued that the daily insulin
secretion in HP rats was lower than that of AP rats, as supported by
the work of Takahashi et al. (41). However, although
insulin is known to exert an anabolic effect on protein, several
authors have suggested that this effect is stronger in splanchnic
proteins, especially albumin, than in peripheral proteins (10,
42). It also can be argued that, because an HP-diet consumption
results in an energy loss through enhanced thermogenesis
(16) and because protein synthesis is energy demanding, muscle protein synthesis is diminished when protein is isocalorically exchanged with carbohydrates. Very few data are available to support or
refute this hypothesis because, in rat studies, isocaloric exchanges
are mostly performed on carbohydrates because of the low amount of fat
in the standard diets. However, Taillandier et al. (40),
who made an exchange between fat and protein, also reported a
diminished FSR in the tibialis. Moreover, the limited energy
availability of HP diets resulting from the cost of AA absorption and
deamination and protein synthesis is highly controversial (4, 13,
17). In our view, muscle has a very low capacity of adaptation
to a modification of the peripheral AA pattern. Because the surplus
BCAA is not reduced by activated oxidation, they accumulate in the free
pool and are proportionally less incorporated into protein. This would
also be favored by low insulin secretion.
Minor Regulatory Pathways
Intestinal regulation.
One way in which animals adapt to dietary change is by regulating
the rates of intestinal nutrient absorption. Our study demonstrates that adaptation to a HP diet occurred also at the digestive level by
changing the kinetics of nitrogen absorption kinetics. In fact, after 2 wk of adaptation, gastric evacuation was slightly delayed during the
first 2 h after ingestion when compared with the rate in
nonadapted rats receiving a HP meal. This result contradicts that of
Shi et al. (37), but in this study the experimental meal
consisted of soluble peptides, and the results were based on follow-up
of the liquid phase. We also observed that dietary nitrogen remained
longer in the lumen of rats adapted to the HP diet but not when HP food
was presented for the first time, a finding consistent with an early
observation by Peraino et al. (33). It has also been shown
that intestinal enzymes and peptide and AA transporters adapt to diets
containing different levels of protein (12, 20); this
result is surprising and contradicts that of Karasov et al.
(21). However, HP rations have been reported to modify
different AA transport systems to varying degrees (12, 21, 22,
44).
Although luminal events appear to be involved, at least slightly,
in adaptation to HP diets, the intestinal mucosa is not affected by a
chronic increase in protein intake. Indeed, incorporation of the tracer
into mucosal protein was very similar whether the HP diet was given for
the first time or after 2 wk of adaptation. The FSR can be calculated
assuming that luminal AA were the direct precursors of protein
synthesis in the mucosa, an assumption that is sustained in the light
of recent reports in the literature (3, 39). We reported
FSR values ranging from 38 to 66%/day in the proximal intestine and
from 10 to 23%/day in the distal portion; these values are consistent
with those published elsewhere (30, 31). The mucosal FSR
is insensitive to chronic consumption of a HP diet, thus confirming the
results published by Masanés et al. (24), who showed
that the 36% protein diet had no effect on the FSR in the small intestine.
Hepatic anabolism.
In the liver, the stimulation of dietary nitrogen incorporation in
constitutive hepatic protein was higher in the HP-50 group than in the
AP-50 group during the first 3 h of the postprandial period, as if
the protein synthesis system was "ready" to respond to the large
influx of dietary AA after the meal. This can result from both a
stimulated synthesis rate, as shown by several authors (11,
19), and an increased amount of available dietary nitrogen for
synthesis. Indeed, the amount of dietary nitrogen recovered in the
hepatic nonprotein fraction was found to be higher. Last, our study
throws new light on the possible role of plasma protein, especially
albumin, in peripheral AA homeostasis. Indeed, we were surprised to
observe a higher level of dietary nitrogen incorporation in the plasma
protein when rats were given the HP meal for the first time than when
they had chronically ingested an HP diet. Plasma protein could thus be
interpreted as a pool capable of reacting quickly to an AA surplus when
the catabolic capacities of the liver are overloaded, as was the case
in the AP-50 group.
Regulation of Dietary Nitrogen Metabolism: A Synthesis
Because our study allowed us to determine the immediate
distribution of dietary nitrogen after a single meal, we intended to
extrapolate our results to identify the daily regulations of dietary
and endogenous nitrogen metabolism when the chronic ingestion of
protein is increased three times. For this purpose, the single 15N-labeled meal was considered to be representative of the
daily protein ingestion; the protein ingested with the experimental meal is 13.7 and 12.3% of the daily protein ingestion in the HP and
the AP group, respectively. Figure 7
shows how dietary nitrogen is distributed through the different
compartments. The most spectacular increase was that of dietary AA
nitrogen transferred to urea, which was enhanced 11-fold, whereas the
transfer of endogenous nitrogen to urea was only increased by 2.7-fold.
An interesting finding of this work was that the splanchnic zone tended
to maintain the transfer of dietary AA to the periphery at a constant
level. As a direct consequence, the daily incorporation of dietary
nitrogen in the protein fraction of muscles was almost identical
whether the rats ingested 3 or 10 g of protein/day. Although it
increased nitrogen intake threefold, splanchnic protein incorporated
two times as much dietary nitrogen resulting principally from the liver
(2 times), whereas muscles incorporated the same amount whether rats
ingested either 3 or 10 g of protein/day.

View larger version (30K):
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|
Fig. 7.
Daily dietary nitrogen distribution among several body
nitrogen pools in rats given an AP or HP diet (indicated by underline).
Calculations were made for an average rat weighting 300 g.
Endogenous nitrogen deamination was calculated assuming that the daily
nitrogen balance is nil and that growth represents 0.3 g of
protein/day. Results are expressed in mg equivalent protein (N × 6.25). Data are means ± SE. *P < 0.05, AP-14 vs.
HP-50.
|
|
In conclusion, our study, although confirming the predominant role of
liver in the adaptation processes to a HP diet, throws new light on the
acute and midterm mechanisms that ensure a relatively constant amount
of dietary nitrogen reaching the periphery. The enhancement of
catabolic and (to a lesser extent) anabolic systems in the liver
induces a reduction of the availability of all peripheral plasma AA
except BCAA and Arg, which results in the absence of hyperaminoacidemia
during the postprandial phase. Our study suggests that a habitual HP
intake could both dramatically increase the nonprotein nitrogen pool
and, in the context of a diluting effect, reduce the incorporation of
dietary nitrogen in muscle protein.
 |
ACKNOWLEDGEMENTS |
Jean François Huneau is acknowledged for assistance and very
helpful scientific discussion. Sophie Daré is thanked for the contribution in amino acid analyses.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: C. Gaudichon, INRA UPNCA, INA-PG, 16 Rue Claude Bernard, 75231 Paris, Cedex 05, France (E-mail: gaudicho{at}inapg.inra.fr).
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. Section 1734 solely to indicate this fact.
Received 21 November 2000; accepted in final form 10 May 2001.
 |
REFERENCES |
1.
Abumrad, NN,
Williams P,
Frexes-Steed M,
Geer R,
Flakoll P,
Cersosimo E,
Brown LL,
Melki I,
Bulus N,
Hourani H,
Hubbard M,
and
Ghishan F.
Inter-organ metabolism of amino acids in vivo.
Diabetes Metab Rev
5:
213-226,
1989[ISI][Medline].
2.
Almurshed, KS,
and
Grunewald KK.
Dietary protein does not affect overloaded skeletal muscle in rats.
J Nutr
130:
1743-1748,
2000[Abstract/Free Full Text].
3.
Bouteloup-Demange, C,
Boirie Y,
Dechelotte P,
Gachon P,
and
Beaufrere B.
Gut mucosal protein synthesis in fed and fasted humans.
Am J Physiol Endocrinol Metab
274:
E541-E546,
1998[Abstract/Free Full Text].
4.
Bradfield, RB,
and
Jourdan MH.
Relative importance of specific dynamic action in weight-reduction diets.
Lancet
2:
640-643,
1973[ISI][Medline].
5.
Brosnan, JT.
The 1986 Borden award lecture. The role of the kidney in amino acid metabolism and nutrition.
Can J Physiol Pharmacol
65:
2355-2362,
1987[ISI][Medline].
6.
Cayol, M,
Boirie Y,
Rambourdin F,
Prugnaud J,
Gachon P,
Beaufrere B,
and
Obled C.
Influence of protein intake on whole body and splanchnic leucine kinetics in humans.
Am J Physiol Endocrinol Metab
272:
E584-E591,
1997[Abstract/Free Full Text].
7.
Chu, SH,
and
Hegsted DM.
Adaptive response of lysine and threonine degrading enzymes in adult rats.
J Nutr
106:
1089-1096,
1976[ISI][Medline].
8.
Colombo, JP,
Cervantes H,
Kokorovic M,
Pfister U,
and
Perritaz R.
Effect of different protein diets on the distribution of amino acids in plasma, liver and brain in the rat.
Ann Nutr Metab
36:
23-33,
1992[ISI][Medline].
9.
Davis, TA,
Burrin DG,
Fiorotto ML,
and
Nguyen HV.
Protein synthesis in skeletal muscle and jejunum is more responsive to feeding in 7-than in 26-day-old pigs.
Am J Physiol Endocrinol Metab
270:
E802-E809,
1996[Abstract/Free Full Text].
10.
De Feo, P,
Horber FF,
and
Haymond MW.
Meal stimulation of albumin synthesis: a significant contributor to whole body protein synthesis in humans.
Am J Physiol Endocrinol Metab
263:
E794-E799,
1992[Abstract/Free Full Text].
11.
Eisenstein, RS,
and
Harper AE.
Relationship between protein intake and hepatic protein synthesis in rats.
J Nutr
121:
1581-1590,
1991[ISI][Medline].
12.
Erickson, RH,
Gum JR, Jr,
Lindstrom MM,
McKean D,
and
Kim YS.
Regional expression and dietary regulation of rat small intestinal peptide and amino acid transporter mRNAs.
Biochem Biophys Res Commun
216:
249-257,
1995[ISI][Medline].
13.
Garlick, PJ.
Protein synthesis and energy expenditure in relation to feeding.
Int J Vitam Nutr Res
56:
197-200,
1986[ISI][Medline].
14.
Garlick, PJ,
McNurlan MA,
and
Ballmer PE.
Influence of dietary protein intake on whole-body protein turnover in humans.
Diabetes Care
14:
1189-1198,
1991[Abstract].
15.
Garlick, PJ,
McNurlan MA,
and
Patlak CS.
Adaptation of protein metabolism in relation to limits to high dietary protein intake.
Eur J Clin Nutr
53, Suppl1:
S34-S43,
1999[ISI][Medline].
16.
Garrow, JS.
The contribution of protein synthesis to thermogenesis in man.
Int J Obes
9:
97-101,
1985[ISI][Medline].
17.
Garrow, JS,
and
Hawes SF.
The role of amino acid oxidation in causing specific dynamic action' in man.
Br J Nutr
27:
211-219,
1972[ISI][Medline].
18.
Gaudichon, C,
Mahe S,
Benamouzig R,
Luengo C,
Fouillet H,
Dare S,
Van Oycke M,
Ferriere F,
Rautureau J,
and
Tome D.
Net postprandial utilization of [15N]-labeled milk protein nitrogen is influenced by diet composition in humans.
J Nutr
129:
890-895,
1999[Abstract/Free Full Text].
19.
Hayase, K,
Koie M,
and
Yokogoshi H.
The quantity of dietary protein affects brain protein synthesis rate in aged rats.
J Nutr
128:
1533-1536,
1998[Abstract/Free Full Text].
20.
Jean, C,
Rome S,
Mathé V,
Huneau JF,
Aattouri N,
Fromentin G,
Larue-Achagiottis C,
and
Tomé D.
Food intake and metabolic adaptation in rats fed a high protein diet.
J Nutr
131:
91-98,
2001[Abstract/Free Full Text].
21.
Karasov, WH,
Pond RSd,
Solberg DH,
and
Diamond JM.
Regulation of proline and glucose transport in mouse intestine by dietary substrate levels.
Proc Natl Acad Sci USA
80:
7674-7677,
1983[Abstract].
22.
Karasov, WH,
Solberg DH,
and
Diamond JM.
Dependence of intestinal amino acid uptake on dietary protein or amino acid levels.
Am J Physiol Gastrointest Liver Physiol
252:
G614-G625,
1987[Abstract/Free Full Text].
23.
Laurent, BC,
Moldawer LL,
Young VR,
Bistrian BR,
and
Blackburn GL.
Whole-body leucine and muscle protein kinetics in rats fed varying protein intakes.
Am J Physiol Endocrinol Metab
246:
E444-E451,
1984[Abstract/Free Full Text].
24.
Masanes, R,
Fernandez-Lopez JA,
Alemany M,
Remesar X,
and
Rafecas I.
Effect of dietary protein content on tissue protein synthesis rates in Zucker lean rats.
Nutr Res
19:
1017-1026,
1999[ISI].
25.
Matthews, DE,
and
Campbell RG.
The effect of dietary protein intake on glutamine and glutamate nitrogen metabolism in humans.
Am J Clin Nutr
55:
963-970,
1992[Abstract].
26.
McCarthy, DM,
Nicholson JA,
and
Kim YS.
Intestinal enzyme adaptation to normal diets of different composition.
Am J Physiol Gastrointest Liver Physiol
239:
G445-G451,
1980[Abstract/Free Full Text].
27.
Morens, C,
Gaudichon C,
Metges CC,
Fromentin G,
Baglieri A,
Even PC,
Huneau JF,
and
Tome D.
A high-protein meal exceeds anabolic and catabolic capacities in rats adapted to a normal protein diet.
J Nutr
130:
2312-2321,
2000[Abstract/Free Full Text].
28.
Motil, KJ,
Matthews DE,
Bier DM,
Burke JF,
Munro HN,
and
Young VR.
Whole-body leucine and lysine metabolism: response to dietary protein intake in young men.
Am J Physiol Endocrinol Metab
240:
E712-E721,
1981[Abstract/Free Full Text].
29.
Moundras, C,
Remesy C,
and
Demigne C.
Dietary protein paradox: decrease of amino acid availability induced by high-protein diets.
Am J Physiol Gastrointest Liver Physiol
264:
G1057-G1065,
1993[Abstract/Free Full Text].
30.
Nakshabendi, IM,
Obeidat W,
Russell RI,
Downie S,
Smith K,
and
Rennie MJ.
Gut mucosal protein synthesis measured using intravenous and intragastric delivery of stable tracer amino acids.
Am J Physiol Endocrinol Metab
269:
E996-E999,
1995[Abstract/Free Full Text].
31.
O'Keefe, SJ,
Haymond MW,
Bennet WM,
Oswald B,
Nelson DK,
and
Shorter RG.
Long-acting somatostatin analogue therapy and protein metabolism in patients with jejunostomies.
Gastroenterology
107:
379-388,
1994[ISI][Medline].
32.
Pacy, PJ,
Price GM,
Halliday D,
Quevedo MR,
and
Millward DJ.
Nitrogen homeostasis in man: the diurnal responses of protein synthesis and degradation and amino acid oxidation to diets with increasing protein intakes.
Clin Sci (Colch)
86:
103-116,
1994[ISI][Medline].
33.
Peraino, C,
Rogers QR,
Yoshida M,
Chen ML,
and
Harper AE.
Dietary factors affecting the rate of disappearance of casein from the gastrointestinal tract.
Can J Biochem Physiol
37:
1475-1491,
1959[ISI].
33a.
Reeds, PJ,
Burin DJ,
Jahoor F,
Wykes L,
Henry J,
and
Frazer EM.
Enteral glutamate is almost completely metabolized in first pass by the gastrointestinal tract in infant pigs.
Am J Physiol Endocrinol Metab
270:
E413-E418,
1996[Abstract/Free Full Text].
34.
Remesy, C,
Morand C,
Demigne C,
and
Fafournoux P.
Control of hepatic utilization of glutamine by transport processes or cellular metabolism in rats fed a high protein diet.
J Nutr
118:
569-578,
1988[ISI][Medline].
35.
Schimke, RT.
Adaptive characteristics of urea cycle enzymes in the rat.
J Biol Chem
237:
459-468,
1962[Free Full Text].
36.
Sharp, PE,
and
La Regina MC.
The Laboratory Rat. Boca Raton, FL: CRC, 1998.
37.
Shi, G,
Leray V,
Scarpignato C,
Bentouimou N,
Bruley des Varannes S,
Cherbut C,
and
Galmiche JP.
Specific adaptation of gastric emptying to diets with differing protein content in the rat: is endogenous cholecystokinin implicated?
Gut
41:
612-618,
1997[Abstract/Free Full Text].
38.
Shiraga, T,
Miyamoto K,
Tanaka H,
Yamamoto H,
Taketani Y,
Morita K,
Tamai I,
Tsuji A,
and
Takeda E.
Cellular and molecular mechanisms of dietary regulation on rat intestinal H+/peptide transporter PepT1.
Gastroenterology
116:
354-362,
1999[ISI][Medline].
39.
Stoll, B,
Burrin DG,
Henry JF,
Jahoor F,
and
Reeds PJ.
Dietary and systemic phenylalanine utilization for mucosal and hepatic constitutive protein synthesis in pigs.
Am J Physiol Gastrointest Liver Physiol
276:
G49-G57,
1999[Abstract/Free Full Text].
40.
Taillandier, D,
Guezennec CY,
Patureau-Mirand P,
Bigard X,
Arnal M,
and
Attaix D.
A high protein diet does not improve protein synthesis in the nonweight-bearing rat tibialis anterior muscle.
J Nutr
126:
266-272,
1996[ISI][Medline].
41.
Takahashi, RF,
Curi R,
and
Carpinelli AR.
Insulin secretion to glucose stimulus in pancreatic islets isolated from rats fed unbalanced diets.
Physiol Behav
50:
787-791,
1991[ISI][Medline].
42.
Tessari, P,
Zanetti M,
Barazzoni R,
Vettore M,
and
Michielan F.
Mechanisms of postprandial protein accretion in human skeletal muscle. Insight from leucine and phenylalanine forearm kinetics.
J Clin Invest
98:
1361-1372,
1996[Abstract/Free Full Text].
43.
Torres, N,
Lopez G,
De Santiago S,
Hutson SM,
and
Tovar AR.
Dietary protein level regulates expression of the mitochondrial branched-chain aminotransferase in rats.
J Nutr
128:
1368-1375,
1998[Abstract/Free Full Text].
43a.
Windmueller, HG,
and
Spaeth AE.
Intestinal metabolism of glutamine and glutamate from the lumen as compared to glutamine from blood.
Arch Biochem Biophys
171:
662-672,
1975[ISI][Medline].
44.
Wolffram, S,
and
Scharrer E.
Effect of feeding a high protein diet on amino acid uptake into rat intestinal brush border membrane vesicles.
Pflügers Arch
400:
34-39,
1984[ISI][Medline].
45.
Yang, RD,
Matthews DE,
Bier DM,
Wen ZM,
and
Young VR.
Response of alanine metabolism in humans to manipulation of dietary protein and energy intakes.
Am J Physiol Endocrinol Metab
250:
E39-E46,
1986[Abstract/Free Full Text].
46.
Yoshizawa, F,
Nagasawa T,
Nishizawa N,
and
Funabiki R.
Protein synthesis and degradation change rapidly in response to food intake in muscle of food-deprived mice.
J Nutr
127:
1156-1159,
1997[Abstract/Free Full Text].
Am J Physiol Endocrinol Metab 281(4):E826-E836
0193-1849/01 $5.00
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