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Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1
3To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • pigs • protein synthesis • visceral organs • diet composition
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In pigs, it has
been established that about 75% of endogenously secreted gut protein
is reabsorbed, while the remainder passes the terminal ileum and is
thus considered a protein loss to the pig (Souffrant et al. 1993
).
These endogenous gut nitrogen losses
(ENL)5
are generally much higher and more variable than previously estimated
using the conventional methods of feeding a protein-free diet or
mathematical regression to zero protein intake (de Lange et al. 1989
,
Jansman et al. 1995
, Nyachoti et al. 1997b
, Schulze et al. 1994
).
Moreover, it has been estimated that >50% of ENL in growing pigs
originates from the small intestine (Low 1985
).
Studies on endogenous gut protein have, thus far, concentrated on ENL
at the distal ileum under various experimental conditions but not its
possible influence on protein synthesis in visceral organs. Because of
the significant implications of gut protein synthesis on amino acid and
energy requirements of the animal (Moughan 1995
,
Nyachoti et al. 1997a
), it is important to understand
how this is influenced when pigs are fed diets that stimulate high ENL.
Visceral organs have high protein turnover rates relative to other body
tissues (Lobley et al. 1980
). In order to study
the relationship between ENL and protein synthesis rates (PSR) in
visceral organs, a method is required for estimating PSR with high
turnover rates. Conventional methods, based on a continuous infusion of
labeled amino acids, underestimate PSR in visceral organs because they
do not reflect accurately the PSR that turn over rapidly or that are
exported from the tissues (Southorn et al. 1992
).
Furthermore, conventional methods present problems with selecting the
amino acid precursor pool. A procedure involving injection of a
flooding-dose of labeled amino acids (Garlick et al. 1980
) has been used to estimate PSR in individual rat
tissues within 10 min, thus allowing measurements to be made in tissues
such as the liver and small intestines which have high protein turnover
rates. Use of the large-dose method to measure PSR in large animals
is not feasible without changes largely because of cost. Southorn et al. (1992)
have demonstrated the use of this method to estimate
intestinal and liver PSR in sheep by decreasing the specific
radioactivity to one-tenth of the level used by Garlick et al. (1980)
and by increasing the incorporation time from 10 to 20, 40 or 60
min. Applying this adapted method to estimate PSR in the visceral
organs of the pig should provide an insight into the significance of
ENL in pig nutrition.
The current study aimed at using the phenylalanine flooding-dose
technique as outlined for large animals by Southorn et al. (1992)
to
estimate protein synthesis in the visceral organs of growing pigs fed
diets designed to induce either low or high ENL.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Growing Yorkshire barrows with an average initial body weight of 18 kg were obtained from the University of Guelph Arkell Swine Research farm for use in the present study. They were housed individually in metabolic crates with smooth, transparent side walls and tender-foot floors in a temperature-controlled room (20–22°C) and allowed to adapt to their new environment and diets for 14 to 16 d before undergoing surgery and a further 1 wk between surgery and the actual study.
Two diets based on either casein-cornstarch (CC) or
barley-canola meal (BCM) known to induce low [7 g/kg dry matter
intake (DMI)] and high (18 g/kg DMI) ENL, respectively
(Nyachoti et al. 1997b
, Table 1
) were used in this experiment. The diets were formulated to meet or
exceed NRC (1988)
requirements for vitamins and minerals and to contain
a similar digestible energy to protein ratio. Pigs were given their
daily feed allowance in two equal amounts (at 0800 and 2000h) and
intake was restricted to 2.6 times maintenance energy requirements
(NRC 1988
). During the last 3 d before the start of
infusion, the feeding schedule was changed to three-hourly feedings
so as to maintain steady-state conditions (Lobley et al. 1992
).
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Surgical procedures, preparation of infusate and infusion procedures
used in the current study were described previously (Nyachoti et al. 1998
). Six animals were assigned at random to one of the
two diet treatments. Starting 1.5 h after feeding, each pig was
infused with a solution of unlabeled phenylalanine (150 mmol ·
L-1) in water containing 230 MBq · L-1
L-[ring 2, 6-3H] phenylalanine (American
Radiolabeled Chemicals, St. Louis, MO) for 12 min at a rate of 10 mL/kg
body weight to give a dose of about 2.3 MBq/kg body weight. This dose
level was almost twice that used by Southorn et al. (1992)
and
one-eighth that used in the original study by Garlick et al. (1980)
. Southorn et al. (1992)
found significant differences in
specific radioactivity in plasma and intracellular free pools and only
a 57% flooding level in the liver. Increasing the amounts of
radioactivity infused in this study was done so as to minimize such
differences and increase the accuracy of measurements in all tissues
studied. After 20 min, timed from the end of infusion, pigs were killed
by a lethal injection of sodium pentobarbitone via the infusion
catheter. The start of infusion was sequentially delayed to allow a 3-h
interval between animals. Blood samples (5 mL) were drawn 10 min before
the start of infusion, at 3-min intervals during infusion, and at 5-min
intervals after infusion until slaughter. Samples of the liver,
pancreas, duodenum (taken as the first 1 m of the small
intestine), jejunum, ileum (taken as the last 1 m of the small
intestine), colon, cecum and skeletal muscle were quickly excised
immediately after death and chilled with ice-cold irrigation saline
to minimize postmortem metabolism (Southorn et al. 1992
). Each sample, except for muscle, was then blotted with an
absorbent paper, weighed and then wrapped in aluminum foil before rapid
freezing in liquid nitrogen. Measurements of PSR in muscle were done to
allow comparisons with other studies. The time from dissecting to
chilling of each sample was recorded accurately and considered in the
calculation of protein synthesis. Sampled organs were weighed to allow
calculation of total protein synthesis. The whole sampling procedure
was accomplished in <5 min following death. The experimental protocol
was approved by the Animal Care Committee at the University of Guelph,
and pigs were cared for according to the guidelines of the Canadian
Council on Animal Care.
Sample preparation and analyses.
Blood samples were centrifuged at 1500 x g for 15
min and the recovered plasma subdivided into three batches before being
stored at -20°C until required for analysis. Plasma insulin
concentration was determined using a radioimmunoassay (Coat-A-count;
Diagnostic Products, Los Angeles, CA) while glucose concentration was
measured using the method of Trinder (1969)
(Sigma Diagnostic Procedure
No. 315). Plasma glucose and insulin concentrations were determined in
the present study so as to further assess the impact of a flooding dose
of phenylalanine on pigs (Nyachoti et al. 1998
).
Tissue samples (1 g) were homogenized in 5 mL of 20 g/L of perchloric acid with an Ultra-Turax T25 tissue disrupter (Janke & Kunkel, IKA Labortechnik, Staufen, Germany) and then centrifuged at 1500 x g for 15 min. The supernatant was recovered and kept frozen at -20°C until required for further processing. The precipitate was washed twice with 8 mL of 20 g/L of perchloric acid, resuspended in 8 mL of 1 mol · L-1 sodium hydroxide and then left to stand in a water bath set at 37°C for 1.5 h to solubilize the proteins. The solubilized protein was recovered by adding 4 mL of 200 g/L of cold perchloric acid and letting the mixture to stand on ice for 20 min. Precipitated protein was recovered by centrifugation at 2000 x g for 15 min followed by two washings with 8 mL of 20 g/L of perchloric acid.
The phenylalanine content in the precipitated protein pellet was
determined following hydrolysis in 10 mL of 6 mol · L-1
of hydrochloric acid in sealed, nitrogen-flushed tubes at 110°C for
24 h. The hydrolyzed samples were left to cool for 30 min and then
thoroughly mixed before transferred into 125-mL Erlenmeyer flasks. Each
tube was rinsed twice with deionized water and the washing added to
their respective hydrolysates. The samples were then diluted to ~50
mL using deionized water and mixed thoroughly by swirling before
filtering about 4 mL of each sample through 0.22 µm filters with low
protein binding ability (Millipore, Mississauga, Ontario, Canada). One
mL of the filtered hydrolysate samples and all of the supernatant
samples were cleaned through 2.5 mL of a cation exchange resin (AG
50W-X8; Bio-Rad laboratories Ltd., Mississauga, Ontario, Canada) to
remove salts and other contaminants that interfere with derivatization
of amino acids with phenylisothiocynate (Sève et al. 1986
, Southorn et al. 1992
). Phenylalanine
concentration in the clean hydrolysate, supernatant and plasma samples
was determined according to Bidlingmeyer et al. (1984)
. Plasma and
supernatant samples were analyzed in duplicate while hydrolysates were
analyzed in triplicate. Thirty-five µL of each sample were
injected for amino acid separation using a 3.9 mm x 30 cm
Pico.Tag reverse-phase column (Waters, Mississauga, Ontario,
Canada) maintained at 48°C. The run time was shortened from 90 to 30
min and the gradients modified to allow a clear separation of the
phenylalanine peak. The phenylalanine peak was collected over a time
window starting and ending at least 1 min before and after the elution
time of phenylalanine using a Waters Fraction Collector (Waters,
Milford, MA). The level of radioactivity in the collected fractions was
determined by liquid scintillation counting on a liquid scintillation
system (Model LS 6000; Beckman Instruments, Fullerton, CA) after adding
10 mL of Biodegradable Counting Scintillant (Amersham Canada, Oakville,
Ontario, Canada). Tissue protein content was measured according to
Smith et al. (1985)
by the calorimetric reaction with bicinchoninic
acid (Sigma Chemicals, St. Louis, MO).
Calculations and statistical analysis.
PSR were calculated by the method of Garlick et al. (1983)
using the
following equation:
 |
where Ks is the fractional rate of protein synthesis in percentage of the tissue protein pool synthesized per day; SRAh is the specific radioactivity of bound phenylalanine in the protein hydrolysate from the tissue; SRAf is the phenylalanine-specific radioactivity in the precursor pool used for calculation; and t is the incorporation time in days of 3H-phenylalanine into protein (includes the time taken to sample each tissue). Ksvalues were calculated in four different ways; assuming t to be the time from the start or end of infusion to chilling of tissue samples and the amino acid precursor pool to be either the plasma-free or intracellular free pool. The SRA in plasma-free phenylalanine was averaged over the various sampling times from 3 min after the start of infusion to the slaughter time. The level of flooding achieved in different tissues was calculated by expressing the SRA in the intracellular free-pool phenylalanine of each tissue as a percentage of SRA in plasma-free phenylalanine. Flooding level was also assessed by expressing the phenylalanine SRA in plasma as a percentage of phenylalanine SRA in the infusate. The absolute rates of tissues protein synthesis in grams of protein per day were calculated by multiplying the Ks by the total protein content present in each tissue on the day of the experiment.
Data were analyzed by the General Linear Models procedure of SAS
(Statistical Analysis System; SAS Institute, Cary, NC). Plasma insulin,
glucose and phenylalanine levels were compared at various sampling
times using repeated measure ANOVA. Treatment means for protein
synthesis and level of specific radioactivity in the plasma or
intracellular precursor pool were compared by Student’s t
test (Steel and Torrie 1980
). Variation is presented as
the SEM. Means were declared significantly different at a
probability level of P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), the amount of each diet offered differed. Feed intake
was 667 and 782 g/d dry matter for the CC- and BCM-fed pigs,
respectively.
At most sampling times, plasma glucose concentrations were higher
(P < 0.05) in the CC-fed pigs than in the
BCM-fed pigs (Fig. 1A
). However, within dietary treatments there was no change
(P > 0.10) in plasma glucose concentration during and
after infusion, and no interaction between diet and time was observed
(P > 0.10). Over the entire study period, it averaged
5.78 ± 0.18 and 5.19 ± 0.25 mmol ·
L-1 of plasma for the CC- and BCM-fed pigs,
respectively. At most sampling times, the CC-fed pigs had higher
(P < 0.01) plasma insulin levels than the BCM-fed
pigs (Fig. 1B
). Within each dietary treatment group, plasma
insulin levels declined (P < 0.05) from baseline
values within 3 min after the start of infusion and then remained
constant thereafter. There was no interaction between diet and time in
plasma insulin levels (P > 0.10).
|
). It then dropped quickly before leveling out at about 1585 and 1790
µmol · L-1 for the casein- and BCM-fed
pigs, respectively. The pattern was very similar for the two dietary
treatments although levels were higher (P < 0.05) at
some sampling times in the BCM-fed pigs than in the CC-fed pigs.
The SRA of plasma-free phenylalanine in plasma rose to a plateau
value within 3 min of starting the infusion and did not change
(P > 0.10) thereafter (Fig. 3
). There were no differences (P > 0.10) in
plasma-free phenylalanine SRA between the CC- and BCM-fed pigs
at any sampling time. The level of flooding achieved in the
intracellular-free phenylalanine pool in the studied organs is
presented in Table 2
. The levels of flooding in all tissues did not differ (P
> 0.10) in the two dietary groups. In both groups, the lowest
flooding level (~68%) was observed in the pancreas while the highest
level (~83%) was observed in the liver and ileum. The
flooding levels calculated by expressing mean phenylalanine SRA in
plasma as a percentage of phenylalanine SRA in the infusate was 72.4
± 1.3% and 74.9 ± 1.4%, for the CC- and BCM-fed pigs,
respectively. Again these levels did not differ (P
> 0.10).
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Ks (%/d) after 20 or 32 min of
incorporation were affected by dietary treatment only in the liver
(P < 0.01) and colon (P
< 0.05) (Table 3
). Within the two dietary treatments, the pancreas and muscle had the
highest and lowest Ks, respectively,
compared to other tissues. Only Ks in the
pancreas calculated using SRA in the intracellular-free
phenylalanine pool were higher (P < 0.05) than those calculated using plasma-free phenylalanine SRA for
both treatments. In all other tissues, intracellular-free pool
based values were only numerically higher than those based on the
plasma-free pool. As expected, Kswere lower based on 32 min than 20 min of
incorporation (Table 3
). However, the trends in treatment
effects were the same as those observed based on a 20-min incorporation
period.
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). No differences were observed in the other visceral tissues although
synthesis rates in the CC-fed pigs were numerically higher in most
tissues compared to the BCM-fed pigs (Table 4
). However, a
trend (P < 0.10) toward a higher protein content
in jejunum of CC- than BCM-fed pigs was observed when calculated
assuming plasma as precursor pool after 20 min of incorporation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In the current study,
plasma glucose concentrations (Fig. 1A
) were within the
range of values (5.48 to 6.03 mmol · L-1)
reported for plasma glucose concentration in growing pigs
(Antinmo et al. 1978
, Le Floc’h et al. 1995
, Nyachoti et al. 1998
). Furthermore, within
each treatment, these values did not change significantly during and
after the infusion period. This supports earlier findings indicating no
significant change in plasma glucose concentration following a flooding
dose of phenylalanine in pigs (Nyachoti et al. 1998
) and
sheep (Southorn et al. 1992
).
For both dietary treatments, circulating plasma insulin concentrations
dropped slightly but significantly within 3 min of the start of
infusion and then remained unchanged thereafter. This is in agreement
with previous observations (Nyachoti et al. 1998
). The
overall plasma insulin concentrations observed in the CC group were
similar to literature values (84.4 to 89.0 pmol
L-1) from studies where pigs were fed similar
diets (Le Floc’h et al. 1995
, Nyachoti et al. 1998
). The higher level of plasma insulin (and plasma glucose)
in the CC-fed pigs compared to the BCM-fed pigs could be
expected as the CC diet was much more digestible than the BCM diet
(Nyachoti et al. 1997b
). Apparently when a
flooding-dose of phenylalanine is infused over a relatively long
period (12 min) as opposed to giving a bolus injection (usually lasting 10–15 s; Lobley et al. 1992
, McNurlan et al. 1979
) plasma
insulin levels remain essentially unchanged. The minor changes in
plasma glucose and insulin levels indicate that the flooding-dose
procedure as used in the current study did not have a significant
impact on the metabolic status of the pigs and is thus unlikely to have
influenced observed Ks and PSR.
The purpose of administering a large dose of phenylalanine is to
rapidly bring the SRA of phenylalanine in all amino-acid precursor
pools to the same level so as to eliminate the uncertainty regarding
the precursor pool to use in determining
Ks values (Davis et al. 1989
, Garlick et al. 1980
, McNurlan et al. 1979
). Specific radioactivity of phenylalanine in plasma
rose to maximum level within 3 min of starting the infusion, and this
value did not change significantly over the course of the experiment
for both dietary treatments. This suggests that flooding was indeed
achieved in the present study (Fig. 3)
. Another way of assessing the
adequacy of the flooding method is to relate SRA in the various
intracellular-free phenylalanine pools to SRA in the
plasma-free phenylalanine pool. Theoretically, these ratios should
approach unity. However, there will never be unity because of the
continuous dilution of partly labeled plasma phenylalanine with
unlabeled amino acids derived from protein degradation in the
intracellular free amino-acid pool (Davis et al. 1989
).
The flooding levels observed in the liver and jejunum (Table 2
) agree closely with findings of earlier studies with the flooding
dose procedure in rats (Garlick et al. 1980
,
McNurlan et al. 1979
). In sheep, Southorn et al. (1992)
observed a substantially lower flooding level (57 to 67%) in the liver
than in the current study: an observation they attributed to potential
differences in hepatic structure among species or contamination of
samples with highly labeled blood. The flooding level in the duodenum
was lower in the current study than that observed (92%) in sheep
(Southorn et al. 1992
).
It has been suggested that it is more difficult to flood skeletal
muscle tissues because of the larger free amino acid pool and the
relatively low permeability of skeletal muscle cell membranes
(Davis et al. 1989
). Furthermore, skeletal muscle
tissues have lower PSR compared to visceral organs (Lobley 1988
). This could explain why the flooding level in the
skeletal muscle of about 80% seen in the current study is slightly
lower than the literature values. High flooding in the muscle (91 to
100%) relative to SRA in the infusate has been observed in 4-kg
piglets given a bolus injection of 3H-phenylalanine and
killed 10 min later (Sève et al. 1986
). The
differences in the amount of radioactivity in infusates, mode of
administration (injection vs. 12 min infusion) and length of
incorporation time (10 min vs. 32 min) may explain part of the
differences in flooding levels observed in the two studies. Also,
differences in protein metabolism, due to differences in body weight
and genotype of the pigs used in the two studies, may have contributed
to differences in flooding levels.
In a previous study we showed that ENL at the distal ileum were larger
in BCM-fed pigs than in the CC-fed pigs (Nyachoti et al. 1997b
). Based on this prior observation, we hypothesized that
feeding a diet that increases ENL will also induce a high rate of
protein synthesis in gut tissues and that this will represent an
important energy loss to the animal (Nyachoti et al. 1997a
). The current data, however, do not support this
hypothesis (Tables 3
and 4)
. Of the tissues studied, significant
differences in Ks were observed only
in the liver and colon (Table 3)
. The absolute amount of protein
synthesized per day was significantly different only in the liver and
pancreas, but these differences were not evident when the synthesis
rates were corrected for the metabolic body weight
(kg0.75) of the pigs (data not shown). The
availability of nutrients in the CC diet was significantly higher than
in the BCM diet as shown in our previous study (Nyachoti et al. 1997b
). According to Armentano (1994)
and Volman et al. (1998)
, the liver is a major site for amino acid metabolism and,
therefore, the high protein turnover rates observed in the livers of
CC-fed pigs were likely due to their adaptation to metabolize the
relatively large amount of available nutrients in the CC diet.
Differences in endogenous nitrogen flow observed in our previous
experiment may have been due to the differences in the efficiency of
endogenous nitrogen reabsorption rather than due to differences in
rates of protein synthesis in the gut tissues. A similar proposition
has been made by Grala (1998)
. For example, among the factors that
influence the flow of ENL at the terminal ileum are the content and
type of dietary fiber (Boisen and Moughan 1996
,
Nyachoti et al. 1997a
). Dietary fiber increases
endogenous nitrogen not only through its abrasiveness, which increases
the sloughing off of the gut mucosa, but also through increasing
digesta viscosity, which in turn hinders adequate interaction between
endogenous protein and digestive enzymes (Chesson 1993
).
This results in reduced recycling of endogenous gut protein, thus
increasing ENL. It is important to note, however, that in the current
study we looked at the overall protein turnover rates as opposed to
turnover rates of specific proteins. It may as well be that the
turnover rates of certain types of proteins (e.g., mucoproteins) were
affected to a different extent (Lien et al. 1997
).
Based on phenylalanine SRA in the plasma-free pool, the rates of
muscle protein synthesis after 32 min of incorporation for the CC-
(19.4%) and BCM- (17.9%) fed pigs are in close agreement with results
(16.4 to 18.2%) in 10-d suckled piglets (Sève et al. 1986
). However, the current results and those of Sève et al. (1986)
indicate that protein synthesis in the muscle of growing
pigs is much higher than the rate of 7.6% per day obtained in the
gastrocnemius muscle of 20–30 kg pigs (Edmunds et al. 1978
, Simon et al. 1978
) or in the leg muscle of
70-kg pigs (Garlick et al. 1976
). It is important to
note, however, that both Edmunds et al. (1978)
and Garlick et al. (1976)
used the constant infusion of tracer amino acids: a technique
that is known to underestimate the rates of protein turnover in various
tissues (Lobley 1988
).
In conclusion, diet composition affected the rates of protein synthesis only in the liver and colon of growing pigs. This means that feeding pigs a BCM diet that induces higher ENL than a CC diet does not necessarily increase PSR in the small intestine from which >50% of ENL originates. It appears that the observed differences in ENL when feeding the CC and BCM diets are due to differences in reabsorption and not secretion of endogenous proteins.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Financial support was provided by Finnfeeds International, Marlborough, U.K., and the Natural Sciences and Engineering Research Council of Canada.
4 Current address: Department of Animal Sciences, University of Illinois, 1207 West Gregory Drive, Urbana, IL 61801.
5 Abbreviations used: BCM, barley-canola meal diet; CC, casein-cornstarch diet; CP, crude protein; DE, digestible
energy; DMI, dry matter intake; EBW, empty body weight; ENL, endogenous gut nitrogen losses; Ks, fractional rates of protein synthesis; PSR, protein synthesis rate; SRA, specific radioactivity.
Manuscript received May 19, 1999. Initial review completed July 18, 1999. Revision accepted November 3, 1999.
|
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