Amino acid use by the gastrointestinal tract of sheep given
lucerne forage
John C.
MacRae,
Les A.
Bruce,
David S.
Brown, and
A. Graham
Calder
Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB Scotland,
United Kingdom
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ABSTRACT |
Essential amino acid (EAA) utilization by
gastrointestinal tract (GIT) tissues has been investigated in sheep
given 800 and 1,200 g/day lucerne pellets. Animals prepared with
indwelling catheters into the aorta and the portal drained viscera plus
cannulas into the small intestine were infused with mixed
U-13C-labeled amino acid or
[1-13C]leucine tracers
into the jugular vein or directly into the small intestine. GIT
sequestration of EAA from arterial and luminal AA pools was determined
from tracer and tracee arterioportal concentration differences at both
levels of intake. Proportional tracer
13C-labeled EAA extraction of the
arterial supply, on first pass across the GIT during jugular infusion,
ranged from 0.063 for histidine to 0.126 for leucine. Recovery of
intestinally infused tracer
13C-EAA at the portal vein ranged
from 0.61 for histidine to 0.83 for valine. These data were independent
of intake. Calculated rates of tracee sequestration by GIT tissues
represented 0.45-0.65 of whole body EAA flux, except for
histidine, for which the values were much lower (0.25-0.32). With
the exception of phenylalanine, more than 0.8 of the EAA used by the
GIT was extracted from circulating blood, thus calling into question
the theory that GIT tissues make preferential use of EAA during
absorptive metabolism, restricting supply to peripheral tissues such as
skeletal muscle (growth) or mammary glands (lactation). Instead the GIT
seems to compete very successfully with these tissues for circulating
blood EAA.
stable isotopes
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INTRODUCTION |
EFFICIENCY OF UTILIZATION of absorbed essential amino
acids (EAA) by ruminants given fresh (24), conserved (5), or dried (20,
23) forages rarely exceeds 0.5-0.55. These values are lower than
the values of 0.7-0.75 reported in other species, such as pigs (4,
6). The reasons for these low efficiencies are not fully understood,
but one possibility is the disproportionate rates of protein synthesis
and accretion in the different tissues of the ruminant body. Cattle
muscle tissue represents 0.5 of total body protein mass, but protein
synthesis in these tissues represents only 0.16-0.22 of whole body
protein synthesis. In contrast, whereas the gastrointestinal tract
(GIT) represents only 0.05 of total protein mass, these tissues support
rates of protein synthesis that represent 0.32-0.45 of whole body
protein synthesis (17). Similarly, by combining daily rates of protein
synthesis (1) and accretion (25) in young weaned lambs, the same
discrepancies can be seen between the higher rates of synthesis and
lower rates of accretion in the GIT relative to muscle in ovines (18,
19).
Studies using a continuous infusion of
[3H]leucine (9) or a
flooding dose of
[3H]valine (1) to
measure rates of protein synthesis in different body tissues have
indicated that the GIT contributes 0.27-0.35 of whole body protein
synthesis in young growing lambs. These measurements were based on
specific activity of dissected tissues at slaughter and, although they
provided useful information, they were costly in terms of animals used.
Also, the measurements obtained related to only one moment in time
(i.e., the moment of slaughter). If the low efficiency of utilization
of EAA by the animal is related at least in part to the high
proportional sequestration of EAA by the GIT processes, it will be
important to develop methodology to determine rates of protein
synthesis in GIT tissues sequentially in the same animal as different
dietary treatments or perturbations of the GIT environment are imposed
on the animal.
The present study describes experiments in which either a single EAA
([1-13C]leucine) or
uniformly 13C-labeled mixed EAA
tracer (prepared from 13C-labeled
algae) was infused into the vascular circulation or the lumen of the
small intestine to determine the rates of EAA sequestration into GIT
tissues and secretions from arterial blood, which continuously perfuses
the GIT, and from the digesta, which generates AA and peptides for
absorption. To date, the methodology has been applied to only one
ration, but at two levels of intake.
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EXPERIMENTAL PROCEDURES |
Animals and diets.
Experiments were conducted on Suffolk × Finn Dorset
Wether lambs (30-40 kg) housed individually in metabolism crates
and given two levels of intake (800 and 1,200 g/day) of a lucerne
pellet [dry matter (DM) content 890 g/kg; N content 25 g/kg DM;
metabolizable energy (ME) content 10.5 MJ/kg DM] by means
of frequent (hourly) feeders for at least 3 wk before and throughout
the experimental period.
Preparation of animals.
Each animal was prepared with jejunal and ileal T-shaped cannulas and
with indwelling catheters into the aorta and the portal and mesenteric
(n = 2) veins at least 6 wk before the
experiment. They were also prepared with indwelling jugular
catheters (for infusion of tracer) at least 24 h before
any infusion period in which this was required.
Venous and arterial catheters were prepared such that the portion of
the catheter to be inserted into the blood vessel was of silicone
rubber (Silastic; Dow Corning, Reading, Berkshire; 0.64 mm ID, 1.19 mm
OD for the venous catheter; 0.76 mm ID, 1.65 mm OD for the arterial
catheter). Silicone rubber catheters have been shown to be less
thrombogenic in sheep than medical grade polyvinyl chloride
(PVC). The portion that was outside the blood vessel was
of PVC (Dural Engineering, Dural, New South Wales, Australia; 0.8 mm
ID, 1.2 mm OD for the venous catheter; 2.0 mm ID, 3.0 mm OD for the
arterial catheter) to minimize the permeability of
O2 and
CO2 that is known to occur with
silicone rubber. The PVC portion of the catheter was fitted with an
outer sheath of large-diameter PVC to give rigidity and prevent kinking
of the catheters as they passed through the abdominal viscera. For each venous catheter the end to be inserted into the vein was prepared with
an atraumatic needle to facilitate its direct entry into the vessel.
All catheters and cannulas were inserted under general anesthesia
established by thiopentone sodium and maintained by a mixture of
nitrous oxide and halothane. Mesenteric and portal vein catheters were
established as described by Katz and Bergman (14), except that a direct
insertion technique (10) was used. During preparation of the portal
catheter, because the length of the silicone catheter eventually left
in the vessel was only ~2 cm, it was found to be advantageous to
secure the PVC portion of the catheter to the vein and pancreas,
proximal to the site of entry into the vessel, before sectioning the
silicone portion to its correct length and manipulating the tip back
into the vein.
The catheter for sampling of mesenteric blood was introduced at a point
where the capillary drainage from the small intestine first entered the
main mesenteric arcade, and the tip was inserted ~7 cm, to a point
before the junction with the ileocecal vein [see review by Seal
and Reynolds (27); Fig. 1A]. A
second catheter [for infusion of the downstream dilution marker
paraaminohippuric acid (PAH)] was inserted ~20-25 cm
upstream of the sampling catheter. Again the tip was ~7 cm inside the
vessel.
Once the catheters were established, simple T-shaped cannulas were
prepared in the jejunum (at the most proximal point where the capillary
drainage entered the mesenteric arcade) and at the terminal ileum
(400-500 mm from the ileocecal valve) (13). The venous catheters
were then exteriorized from the abdominal cavity at points lateral to
the longissimus dorsi on the right flank, and the cannulas
were exteriorized just below these, before the incision site was
closed.
An aortal catheter was inserted via the femoral artery of the left
hindlimb (12), and this catheter was fed subcutaneously up the leg to
an exteriorization site lateral to the longissimus dorsi on the left
side of the animal.
Stable mass isotopes.
Two separate tracers, both obtained from Martek (Columbia, MD), were
used in the experiments.
L-[1-13C]leucine
[99% molar percent excess (MPE)] was diluted to 15 mM with sterile
saline for infusion at ~5 µmol/min into the jugular vein or into
the jejunum (on separate occasions). A mixed
U-13C-labeled AA tracer was
prepared from lipid- and starch-extracted freeze-dried phototrophic
algal biomass that had been grown in an atmosphere of
[13C]CO2.
For infusion into the jugular vein, this material was acid hydrolyzed
to its constituent free AA, as described previously (15). For jejunal
infusion, the algal biomass was prepared as follows. Samples of algae
(1 g) were hydrolyzed under reflux in 200 ml of 1 M HCl in an
atmosphere of pure O2-free
N2 for 1.5 h. The resultant
solubilized 13C-AA mixture
consisted of 0.85-0.95 peptide/protein-bound AA. The EAA
composition (µmol/g original algae) of the totally hydrolyzed (jugular) and partially hydrolyzed (jejunal) tracer infusates is given
in Table 1. Comparison of the two infusates
indicates that the mild acid hydrolysis solubilized 0.56-0.72
(depending on individual EAA) of the total EAA in the algae.
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Table 1.
Amino acid concentrations of totally hydrolyzed algal preparations
infused into the jugular vein and partially hydrolyzed
preparations infused into the jejunal cannula
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Tracer infusions and blood and digesta sampling.
The 13C-EAA derived from 1 g of
original algal biomass (see Table 1 for AA composition) was made up to
~160 ml in sterile saline and infused continuously (20 ml/h) into the
jugular vein or into the jejunum over an 8-h period (i.e., one site of
infusion per experiment with at least 10-day intervals between
infusions to allow for dilution of enrichment in circulating EAA to
near-background values). Where
[1-13C]leucine tracer
was used it was dissolved in a similar quantity of saline and infused
at ~5 µmol/min. Over the last 7 h of each infusion, PAH was infused
at a rate of 4 mmol/h (in 20 ml saline containing 4,750 IU heparin)
into the mesenteric vein to allow determination of portal blood flow by
downstream dilution. The heparin infusion allowed continuous
withdrawal, by peristaltic pump, of aortal, portal, and mesenteric
blood (~8 ml/h into ice-cold syringes) over the last 5 h of the
tracer infusion.
During the second half of the jejunal infusions, ileal digesta samples
were collected at regular intervals for the measurement of
13C enrichment in individual EAA
to determine the extent of tracer absorption, i.e., as nonrecovery of
infused tracer. The quantities of
13C-EAA reaching the ileum were
calculated from a knowledge of the flow of EAA determined using the
ruthenium-103
phenanthroline/51Cr-labeled EDTA
dual-phase marker procedure as described previously (20).
Analyses.
During the
L-[13C]leucine
infusion, the hourly blood samples were immediately analyzed for pH,
PO2, and
PCO2 with
use of an ABL3 blood gas analyzer (Radiometer, Denmark). Duplicate
samples of whole blood (1 ml) were also introduced into evacuated tubes containing 1 ml of a frozen mixture (9:1) of lactic acid and silicone antiform. These were then stored at
20°C for subsequent
measurement of blood
[13C]CO2
enrichment.
All blood samples taken from both
L-[13C]leucine
and U-13C-labeled mixed EAA tracer
infusions were prepared for gas chromatography-mass spectrometry (GCMS)
and PAH analysis as follows. For GCMS analyses, ~4 ml of whole blood
(accurately weighed to 2 decimal places) were hemolyzed by addition of
an equal volume (weighed) of a solution containing
L-norleucine (0.1 mmol),
dithioerythritol (1 mmol), and 2-ketohexanoic acid (0.02 mmol). The
mixed solution was subdivided into four 2-ml aliquots,
which were stored separately in 5-ml plastic tubes at
20°C.
Samples of blood were obtained before each tracer infusion and prepared
similarly to provide natural abundance/background samples for GCMS
analysis.
Samples of the ileal digesta (0.5-0.7 g) were hydrolyzed in 2 ml
of 6 N HCl under reflux for 18 h at 110°C. The supernatant was
rotary evaporated to dryness and washed twice with distilled water
before being redissolved in 3 ml of water and poured onto a 2-ml column
of AG50W (H+) resin. The resin
was washed with water (10 ml), and the EAA were diluted with 4 M
NH4OH (8 ml) followed by water (4 ml). The NH4OH-water eluant was
freeze-dried and redissolved in 2 ml of distilled water for analysis.
For PAH determination, ~1 g of whole blood (weighed) was
deproteinized with ~10 ml of 12% trichloroacetic acid (weighed) and centrifuged at 1,500 g for 10 min. The
supernatant was refrigerated at <4°C overnight before analysis.
Chemical analysis.
Portal vein blood flow rates were determined from the concentration of
PAH in the respective hourly samples of blood (15). Leucine
concentration and enrichment in samples taken during the L-[13C]leucine
infusion (7) and enrichments and concentrations of all EAA in samples
taken during the U-13C-AA infusion
(15) were determined as described previously.
GCMS determines only fragment ions that contain all the carbon atoms of
the AA (15). Only the m + 0 and
m + n
ions, where n is the number of carbon
atoms in the molecule, were monitored, and thus MPE cannot be
calculated. Rather, values represent the relative enrichment (RE) of
(m + n)/ [(m + 0) + (m + n)], with the calculation based on
(Rs
Ro)/ (1 + Rs
Ro) (8), where Rs and
Ro are the ratios
of the (m + n)/(m + 0) ions in an enriched and natural abundance sample,
respectively. However, because the algal protein AA contained >99%
of the molecules in the (m + n) form, i.e., all carbon atoms
fully labeled, for the EAA reported here, RE will approximate to MPE.
Calculations.
Whole body flux (WBF) rates for individual EAA were calculated from the
RE of the arterial blood collected from 5-8 h of jugular vein
infusion of tracer, with corrections to accommodate infusion of
nontracer quantities of AA.
where
REa is the enrichment of free EAA in arterial blood and Inf
is rate of infusion (µmol/min) of the
U-13C-EAA or the
[1-13C]leucine.
The fractional sequestration of each arterial EAA into the total GIT
proteins was determined during the jugular infusion of the respective
tracers from the equation
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(1)
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where
[A] and [P] are concentrations (µM) of EAA in
arterial and portal whole blood, respectively, and the subscripts a and p are arterial and portal, respectively.
Quantities of EAA (mmol/day) sequestered into GIT proteins from
arterial sources (ARTseq) were
then calculated from the equation
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(2)
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where
PBF is portal blood flow (liters/min).
The calculations of fractional sequestration of each EAA into small
intestine tissue protein during absorption of digesta-derived EAA were
made on the assumption that the
13C-tracer that disappeared from
the intestine [i.e., infusate minus that measured at the ileum;
depending on EAA, this recovery of jejunal infusate at the ileum ranged
from 4.5% (histidine) to 10% (threonine); mean value for all EAA = 7 ± 1.4% of the infused dose] mixed with the liberated AA
peptides that were absorbed from the intestine. On this basis, the
fraction of the tracer that was absorbed but not detected at the portal
vein was assumed to be sequestered into GIT proteins. Recovery of
U-13C-EAA or
[1-13C]leucine at the
portal drained viscera (PDV) was determined from the net portal flux of
13C-EAA, with correction made for
the amount of recirculated arterial 13C-EAA sequestered into proteins
on passage across the GIT (i.e., before addition of the jejunal infused
13C-AA), using the equation
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(3)
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where
I is the rate of 13C-EAA infused
into the jejunum and Iil is
nonabsorbed infusate detected in the ileum (µmol/min).
Quantities of EAA sequestered into small intestine tissue proteins
(mmol/day) were then calculated from the equation
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(4)
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where
LUM is luminal, App is apparent, and Abs is absorption.
App · Abs · AA (mmol/day) is the
disappearance of AA from the small intestine, reported
separately (17) and Il · endog · AA (mmol/day) is the calculated endogenous AA leaving the ileum, determined by linear extrapolation of the relationship between ileal AA
flow vs. duodenal AA flow (determined when these sheep were given 800- and 1,200-g intake of the ration) to zero duodenal flow as described
previously (28).
Statistics.
Primary measurements, including the WBF and
S1 and
S2 extraction
data, are presented as means ± SE of individual estimates. Where,
as in the case of the arterial 13C
extraction data
(S1) and the
recovery of jugular 13C-labeled
infusate at the PDV
(S2), the data
from sheep given the 800- and 1,200-g intake levels were not
significantly different, as assessed using Student's
t-test, they have been combined across intakes for presentation. Derived data on rates of EAA sequestration by
GIT tissues obtained from Eq. 2 and
Eq. 4 are presented with standard
errors that have been calculated as the summated variance of the
calculated means, i.e., derived from the individual variances of the
component data.
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RESULTS |
The WBF of individual EAA, the calculated equivalent whole body protein
fluxes based on these EAA fluxes, and the EAA composition of body
tissues (25) for sheep given the 800- and 1,200-g lucerne pellet
rations are shown in Fig. 1. On average,
the WBF values at the higher intake levels were 1.62 ± 0.067 relative to those at the lower level. Equivalent protein fluxes (g/day)
calculated from the leucine, lysine, threonine, valine, and isoleucine
data were similar (216 ± 5.3 g protein/day on the 800 g intake; 334 ± 8.7 g protein/day on the 1,200 g intake).

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Fig. 1.
Whole body fluxes (mmol/day) of individual essential amino acids (EAA)
in sheep given 800 (A) and 1,200 g/day (B)
lucerne pellets and infused with mixed
U-13C-labeled amino acids (AA) or
L-[13C]leucine
tracers. Protein equivalent fluxes have been calculated from the EAA
composition of different body tissues, scaled relative to their
approximate proportional contributions to whole body flux (WBF), i.e.,
gastrointestinal tract (GIT) 0.4; carcass 0.2; skin 0.2; liver 0.15;
wool 0.05 (see Refs. 1 and 25 and present data).
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The proportional extraction of tracer
13C-AA during passage across the
GIT measured during jugular infusions of
U-13C-EAA or
[1-13C]leucine tracer are given
in Table 2. These values are means across
both intakes. Values of threonine, isoleucine, lysine, and leucine were
all similar (range 0.11-0.14), but those of valine (0.08),
phenylalanine (0.085), and histidine (0.063) were lower (P < 0.05). Extraction of
[13C]leucine was
identical whether infused alone as
[1-13C]leucine or as part of the
U-13C-labeled algal mixed EAA
tracer. Where [1-13C]leucine
was infused, the arteriovenous difference of
[13C]CO2
across the GIT accounted for 6.7 ± 0.76%
(n = 10) of the infused dose or 16.8 ± 2.55% of the
[13C]leucine
sequestered by the GIT.
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Table 2.
Proportional extraction of arterial 13C-labeled EAA by PDV
tissues during mixed [U-13C]leucine or
L-[13C]leucine tracer infusions into the
jugular vein
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The recoveries of 13C-EAA at the
portal vein after intestinal infusion of the mixed
U-13C-labeled peptide tracer and
the single
[1-13C]leucine tracer
are given in Table 3. Again, there was no
difference between recoveries at the two levels of intake or between
leucine given as part of the
U-13C-peptide mixture or as the
single free AA. For five of the EAA, recoveries were in the range
0.76-0.83, but recoveries of phenylalanine (0.65) and histidine
(0.61) were lower (P < 0.05). Where
[1-13C]leucine was
infused, the arteriovenous difference of
[13C]CO2
across the GIT accounted for a further 4.2 ± 0.51%
(n = 12) of the
13C infused as leucine tracer, or
~21% of the sequestered
[13C]leucine taken up
by the GIT. However, 0.75 ± 0.038 of this
[13C]CO2
can be accounted for as oxidation of recycled
[13C]leucine extracted
from the arterial EAA pool during the course of the intestinal infusion
(assuming the same rates of EAA extraction and oxidation determined
during the jugular infusion), and so the
[13C]CO2
generated during absorptive metabolism of the luminally derived tracer
EAA accounted for only ~5% of the tracer leucine sequestered during
absorptive metabolism.
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Table 3.
Proportional recovery of 13C-EAA at the portal vein during
mixed [U-13C]leucine or
L-[13C]leucine tracer infusions into the
jejunum
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Data presented in Table 2, when combined with the rates of EAA
traversing the GIT (i.e., arterial blood EAA concentration × portal blood flow rates), give estimates of individual EAA
sequestration by GIT tissue from the blood EAA pool (see
Eq. 2). Calculation of the equivalent
data for rates of EAA sequestration from digesta EAA during absorption
is more complicated. Here the reciprocals of the data from Table 3 were
combined with the rates of true absorption of EAA (see
Eq. 4). The true absorption of EAA
was calculated from a knowledge of the apparent absorption of EAA from
the small intestine and an estimate of the flow of endogenous ileal EAA
derived from the intercept of the relationship between ileal EAA flow
(y-axis) and duodenal EAA flow
(x-axis), as intake of ration was
increased from 800 to 1,200 g, as described previously (28). Data used
to derive the true absorptions and the mean values at both levels of
intake are given in Table 4.
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Table 4.
Rates of duodenal and ileal flow, apparent absorption from small
intestine, calculated endogenous ileal flow, and true absorption of
EAA in sheep given 800 and 1,200 g/day grass pellets
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Estimates of daily rates of EAA sequestration from both arterial and
luminal sources in sheep given both levels of intake are given in Fig.
2. With the exception of phenylalanine, the majority (range
0.75-0.87; mean 0.82; SE 0.011) of this sequestration was from the
arterial blood pool.
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DISCUSSION |
Technical considerations.
This study is thought to be the first attempt to determine rates of EAA
sequestration into GIT tissues and associated secretions using a
trans-organ approach coupled with isotope kinetic measurements that can
apportion unidirectional fluxes of EAA from the arterial blood pool and
from the digesta EAA during absorptive metabolism. Using this method,
it is possible to make measurements repeatedly on live animals for as
long as catheter patency allows and with the restriction on the
proportion of the total blood volume that can be withdrawn without
causing stress to the animal. Previously, most estimates of protein
synthesis by GIT tissues were determined in acute experiments in which
animals were administered with tracer, usually into the bloodstream,
and then killed so that GIT tissues could be sampled for tracer
incorporation (1, 9). In this methodology, the fractional synthetic
rate of the tissues is measured and the values are transposed to
absolute (g/day) rates of synthesis on the basis of the protein content
of the tissue at slaughter. Such approaches have several disadvantages
relative to the present procedure. First, they are by definition
one-off (spot) measurements and so cannot be used to follow changes in
GIT metabolism caused by nutritional, physiological, or environmental
perturbations. Second, they measure only the tracer incorporation into
GIT tissue protein and take no account of the requirements for
digestive enzyme and mucin secretions, which could be considerable.
Third, they employ only one route of tracer administration (usually
intravenous), even though it is known that small intestine tissues draw
on both digesta-derived and blood EAA as substrates for protein
synthesis.
In these initial technique developments, in which a multiple tracer
approach was used to monitor a range of EAA simultaneously, it was
considered that perhaps
13C-labeled free EAA would not be
the most suitable tracer for determining rates of sequestration of
luminally derived EAA during absorptive metabolism, where presumably
the tracee is being produced in situ from degradation of digesta
protein. In an attempt to provide a more relevant tracer,
13C-labeled algal protein was
treated with only mild acid hydrolysis to produce the infusate, in
which more than 80% of AA was peptide bound (Table 1). However,
comparison of data on the recoveries of
[13C]leucine at the
portal vein during infusion of
13C-labeled free leucine or the
mixed 13C tracer, where 0.89 of
the leucine was in peptide form, did not seem to substantiate this
concern in that values from the two tracers were very similar (Table
3).
This observation may have significance in terms of the recent
controversy regarding the possibility that a proportion of the AA
absorbed from the GIT is in peptide form (30, 31). Although recent
studies from this group have reported the presence of a functional
peptide transporter in brush-border membrane vesicles prepared from
sheep intestines (3), the present
[13C]leucine data
(Table 3) would seem to indicate that any peptide-bound [13C]leucine taken up
in this way is probably hydrolyzed by peptidases within the enterocyte
before release into venous drainage. Otherwise, recovery of the
peptide-bound
[13C]leucine from the
mixed U-13C-labeled jejunal tracer
infusate as free
[13C]leucine at the
portal vein would have been lower than corresponding values for the
recovery of the
[1-13C]leucine jejunal
infusate. Recoveries of
[13C]phenylalanine and
histidine at the portal vein were lower than for the other tracers
(Table 3). Whether this may indicate some minor transfer of these
tracers in non-free AA (nondetected) form is unclear. It has been
suggested that both of these AA are used in peptide form by other
tissues (2). However, further experimentation using peptide-bound and
free forms of each tracer would be required to confirm whether they are
transferred to the liver and beyond as peptides.
Jugular infusions of the mixed
13C-EAA tracer used fully
hydrolyzed algae, but again the rate of arterial sequestration of
[13C]leucine
determined from this tracer was very similar to the data obtained from
L-[13C]leucine
alone (see Table 2). These preliminary observations open up the
possibility of using a single EAA as a representative tracer in two
different isotopic forms, thus allowing the measurement of the arterial
and luminal sequestration rates simultaneously in one single
experimental period, rather than separately during two experimental
periods, as was carried out in these experiments. Convenient dual
markers with respect to sensitivity on GCMS assays would be
[13C]leucine and
D3-leucine or
[13C]phenylalanine and
D5-phenylalanine. With these
markers, measurements could be made frequently and sequentially while
imposing different types of nutritional, physiological, and
environmental challenges on the GIT.
Whole body flux.
There are many reports of whole body protein synthesis rates in many
different species, including rats, mice, sheep, cows, and humans. When
measured at the maintenance level of intake and reported relative to
the metabolic body size of the animal [i.e., live weight
(LW0.75)],
these values are very similar for most studies [15-16 g
protein synthesized/kg
LW0.75 per
day (11)]. In the present study the 800 g lucerne pellet ration
provided ~1.15 of the metabolizable energy required to maintain these
animals and the animals were in nitrogen equilibrium (N retention = 0.2 ± 0.19 g N/day). Their calculated whole body protein flux rate,
determined from the WBF of those EAA giving similar values (Fig. 1),
which of course does not account for any oxidation of the EAA, was
equivalent to 15.0 g protein/kg LW0.75
per day.
It is not clear why protein equivalent values determined using
phenylalanine and histidine tracers do not conform with other values.
However, it is becoming clear that at least some body tissues obtain a
proportion of their phenylalanine substrate for protein synthesis from
non-free AA (peptide/protein) (2), which might account for the lower
fluxes of free phenylalanine relative to the other EAA. The higher
rates for histidine are more difficult to understand unless these
relate to the substantial requirements for histidine, for hemoglobin,
and, to a lesser extent, neurotransmitter synthesis. In a recent study
from this group (16) it was found that the whole body protein flux of
histidine calculated from plasma histidine enrichments was similar to
values calculated using other EAA fluxes but that the value calculated
from whole blood histidine enrichments was twice these values.
GIT metabolism.
GIT protein turnover is probably a more significant proportion of whole
body protein turnover in ruminants than in animals with a single
stomach because of the extensively modified forestomach region in
ruminants and also because of the nature of the rations they consume
(i.e., more fibrous and of lower digestibility than those usually
consumed by monogastric animals). Indeed, a previous report of rates of
GIT tissue protein synthesis in young (8-wk-old) lambs indicated a
major increase in GIT use of
[13C]valine when the
animals were weaned from suckling to solid food; rates of protein
synthesis in the GIT increased from 42 to 77 g/day, which represents an
increase from 12 to 27% of whole body synthesis (1).
Total protein deposition in those lambs was ~40 g/day (1), a value
similar to that determined for the higher level of intake (1,200 g/day)
in the present study (N retention 6.6 ± 0.74 g/day). In previous
experiments from this laboratory it was found that <3% of the total
extra protein retained by growing lambs is deposited in GIT tissues
(25) and so the GIT must be considered to be in near equilibrium with
respect to the tissue protein pool, i.e., the rates of protein
synthesis and degradation are very similar. Where this is the case in
tissues such as skeletal muscle, it can be assumed that the rate of EAA
sequestration from arterial blood for tissue protein synthesis will be
matched by the rate of release of EAA from protein degradation to
venous blood. However, with the GIT this concept is complicated by
several factors, namely, 1)
substrate for tissue synthesis is from both arterial and, in the case
of the small intestine, luminal sources,
2) a proportion of GIT tissue
protein turnover involves desquamation of cellular materials that pass
down the tract and are potentially available for subsequent redigestion
and resorption, 3) there are many
glandular and epithelial secretory mechanisms that provide digestive
enzymes and mucins into the tract, and again these are available for
subsequent redigestion/absorption, and
4) it is possible that digestive
secretions and desquamated cellular debris released from one section of
the GIT (e.g., the forestomach) can be subsequently redigested and reabsorbed in a more distal region (e.g., the small intestine).
This latter phenomenon seems to be borne out by additional data
obtained from these sheep. When EAA absorption from the small intestine
was compared with net fluxes of EAA at the mesenteric (small intestine
only) and portal (whole GIT) drained viscera, recovery of absorbed EAA
was higher at the mesenteric than the portal drained viscera, even
though all mesenteric drainage enters the portal vein. It was argued
that this could be accounted for if part of the degradation component
of the protein turnover process in regions of the GIT anterior to, or
distal to, the small intestine was via the luminal route, thus creating
a negative arteriovenous difference across these regions, which
subsequently "diluted" the positive arteriovenous difference
created by EAA absorption across the small intestine.
Unfortunately, net flux measurements across the GIT do not give any
indication of the rates of EAA use by the GIT tissues and associated
secretory organs, and so the present tracer procedures were developed
in an attempt to quantify the unidirectional fluxes of both arterial
(across the whole tract) and luminal (presumably only across the small
intestine during absorption) EAA.
It is thought that data given in Fig. 2
represent the first reports of the relative rates of EAA sequestration
for protein synthesis from blood and luminal routes and for a number of
EAA simultaneously. As can be seen from Fig.
3, total sequestration (vascular and
luminal) of the individual EAA was in most cases proportional to the
relative molar concentrations of these in GIT tissue protein
[reported previously from comparative slaughter studies
(25)]. However, individual rates, expressed as a proportion of
total body flux (see Fig. 2), were higher than previous reports in
which single AA, e.g., valine (1) and leucine (9), were used as tracers
in terminal experiments. Animals used in those previous
experiments were younger and smaller than those used in the present
study; therefore, it may be that physiological age is a factor relating
to the much higher proportional contribution of GIT flux to WBF in the
present experiment (valine 0.62 and 0.67; leucine 0.43 and 0.48; Fig.
2) relative to those reported for young weaned lambs using the flood
dose [3H]valine
procedure (0.27 of WBF) (1) or an
L-[4,5-3H]leucine
continuous infusion procedure (0.17-0.34; Ref. 9). On the other
hand, the need for EAA for digestive secretions, including mucins, plus
the use of luminally derived AA, neither of which would have been
measured in the previous studies, could have contributed to the
differences. Further work will be needed to examine this aspect,
perhaps by incorporating a terminal flood dose procedure into an
experimental design to directly compare flux rate determined by
arteriovenous difference and tissue incorporation.

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Fig. 2.
Rates (mmol/day) of AA sequestration with GIT tissues and associated
secretions from arterial (open portion of bars) and luminal (stippled
portion of bars) precursors in sheep given 800 (A) and 1,200 g/day (B) lucerne pellets. Values in parentheses indicate
arterial sequestration as a proportion of total GIT sequestration.
Error bars relate to arterial or luminal means as separate
measurements.
|
|

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Fig. 3.
Relationship between proportional rates of EAA sequestration into GIT
proteins (from arterial plus luminal sources) and proportional EAA
composition of GIT tissue protein in similar sheep.
|
|
In the meantime, it would appear from the present study that there is a
need to reconsider the concept of GIT protein metabolism relation to
whole body metabolism. Previous considerations of whether use of EAA by
GIT tissue may, at least in part, be causal to the low efficiency of
utilization of absorbed AA in ruminants (19, 22) have centered on their
reported preferential use during absorptive metabolism (29). Data in
Fig. 2 seem to indicate that although some EAA is sequestered from
digesta protein during absorptive metabolism, a much greater
contribution comes from circulating blood EAA. This suggests that the
GIT is in open competition with other peripheral tissues, e.g.,
skeletal muscle in growth and the mammary gland in lactation. If so,
then presumably the proportional extraction of EAA by GIT tissues
reflects the high rates of protein metabolism required to maintain the
cellular turnover and secretory functions of the digestive machinery.
Further investigation is required to determine whether this then raises the possibility of altering the potential for other tissues (e.g., skeletal muscle and/or the mammary gland) to receive additional EAA as a result of reducing the demands of the GIT, either by nutritional manipulation or even by alterations in the environmental conditions of the GIT. It would appear that when the GIT environment is
changed (increased) by parasitic infection, rates of protein synthesis
in other tissues are reduced (18). Similarly, when nonpathogenic
microbes are eliminated from the tract with the use of antimicrobial
agents and the mitotic index of intestinal tissue is reduced (22),
whole body nitrogen retention increases (19, 22).
Using the methods developed in this study, it should be possible to
follow changes in EAA sequestration into GIT processes within
individual animals and so obtain a better understanding of the
consequences of GIT metabolism on the rest of animal protein metabolism. However, care will need to be taken to select appropriate tracer EAA, particularly where comparisons are to be made across experiments. Comparison of WBFs (Fig. 1) and the proportions of these used for GIT sequestration (Fig. 2) for the different EAA tracers
indicate that the branched chain AA, plus threonine and lysine, all
seem to give reasonably similar data, but phenylalanine and
histidine may need to be considered separately.
 |
ACKNOWLEDGEMENTS |
This study was funded by the Scottish Agriculture and Fisheries
Department as part of their core budget to the Rowett Research Institute.
 |
FOOTNOTES |
Address for reprint requests: J. C. MacRae, Rowett Research Institute,
Bucksburn, Aberdeen AB21 9SB, Scotland, UK.
Received 17 January 1996; accepted in final form 15 October 1996.
 |
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