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

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

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

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

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.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

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

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.
WBF (mmol/day) = [(0.99/RE<SUB>a</SUB>) − 1] × Inf × 1.44
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
<IT>S</IT><SUB>1</SUB> = <FR><NU>([A] × RE) − ([P] × RE<SUB>p</SUB>)</NU><DE>([A] × RE<SUB>a</SUB>)</DE></FR> (1)
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
ART<SUB>seq</SUB> = [A] × [PBF] × <IT>S</IT><SUB>1</SUB> × 1.44 (2)
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
<IT>S</IT><SUB>2</SUB> = <FR><NU>[([P]RE<SUB>p</SUB> − [A]RE<SUB>a</SUB>) + ([A]RE<SUB>a</SUB> × <IT>S</IT><SUB>1</SUB>)] × PBF</NU><DE>I − I<SUB>il</SUB></DE></FR> (3)
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
LUM<SUB>seq</SUB> = (App · Abs · AA + Il · endog · AA) × (1 − <IT>S</IT><SUB>2</SUB>) (4)
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.

    RESULTS
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References

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).

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

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

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

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.

    DISCUSSION
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Abstract
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Procedures
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Discussion
References

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.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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AJP Gastroint Liver Physiol 273(6):G1200-G1207
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