United States Department of Agriculture/Agricultural Research Service Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030
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ABSTRACT |
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The objective of this study was to quantify the utilization of dietary and systemic phenylalanine for mucosal and hepatic constitutive protein synthesis in piglets. Seven female piglets (7.6 kg) bearing arterial, portal, peripheral venous, and gastric catheters were fed a high-protein diet and infused intragastrically with U-13C-labeled protein and intravenously with [2H(phenyl)5]phenylalanine ([2H5]phenylalanine) for 6 h. The isotopic enrichment of the two phenylalanine tracers was measured in arterial and portal blood, in mucosal and hepatic-free and protein-bound phenylalanine, and in very low-density apolipoprotein B-100, albumin, and fibrinogen. The relative isotopic enrichments of the tracers in mucosal-free (ratio of 2H5- to U-13C-labeled = 0.20 ± 0.05) and protein-bound (0.32 ± 0.08) phenylalanine differed significantly (P < 0.01). Although this suggests preferential use of arterial phenylalanine for mucosal protein synthesis, on a molar basis, 59 ± 6% of the mucosal protein was derived from dietary phenylalanine. There were significant differences (P < 0.025) between the relative labeling of the two tracers in arterial (ratio of 2H5- to U-13C-labeled = 1.25 ± 0.48) and portal (ratio of 2H5- to U-13C-labeled = 0.72 ± 0.18) phenylalanine. The mean ratio of the two tracers in all proteins of hepatic origin that were analyzed (0.69 ± 0.18) was similar to that of portal phenylalanine. We conclude that in the fed state portal phenylalanine is preferentially used for constitutive as well as secreted hepatic protein synthesis.
splanchnic metabolism; stable isotopes
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INTRODUCTION |
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PREVIOUS STUDIES OF THE labeling of rapidly synthesized mucosal (7, 8) or hepatic proteins (4, 6, 18, 20, 23) have provided evidence in favor of the preferential utilization of extracellular amino acids as protein synthetic precursors in both tissues. However, the intestinal mucosa and the liver receive amino acids and other nutrients from two extracellular sources. The question of whether there is preferential use of one of the two sources of extracellular amino acids has not been resolved, although results of studies with intragastric and intravenous amino acid tracers support the idea that portal amino acids may play a specific role in hepatic protein metabolism, at least in the synthesis of export proteins (3, 6, 23, 24).
In a previous study of mucosal and hepatic protein synthesis (23), we infused [1-13C]phenylalanine intragastrically and [2H]phenylalanine intravenously in fed piglets. On the basis of the relative isotopic enrichments of the two tracers in hepatic-free and very low-density apolipoprotein B-100 (apoB-100) phenylalanine, we concluded that portal amino acids were preferential sources for the synthesis of proteins secreted by the liver. However, this previous study did not address two important issues. First, the isotopic enrichment of the intragastric 13C-labeled tracer was too low to make adequate measurements of its incorporation into albumin. Second, because we were interested in quantifying the uptake of arterial phenylalanine by the portal-drained viscera (PDV) in both the fasted and fed states within a single infusion, we infused the 2H-labeled intravenous tracer for twice as long as the intragastric tracer. This precluded any calculations of the relative contributions of the two extracellular sources of phenylalanine to constitutive protein synthesis.
The present study was performed to address these issues. We used [U-13C]phenylalanine (given as part of an intragastric [U-13C]protein infusion) to increase the mass spectrometric sensitivity of our measurements and infused the enteral and intravenous tracers for the same period of time. We hypothesize that there is preferential utilization of arterial phenylalanine for mucosal protein synthesis and portal phenylalanine for hepatic protein synthesis.
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METHODS |
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Animals
The study was approved by the Baylor College of Medicine Animal Protocol Review Committee. Housing and care of the animals conformed to United States Department of Agriculture (USDA) guidelines. The study involved seven 28-day-old female crossbred (Large White × Duroc × Hampshire) piglets purchased from the Texas Department of Criminal Justice, Huntsville, TX. The pigs were received at the USDA/Agricultural Research Service (ARS) Children's Nutrition Research Center when they were 2 wk old and were fed a powdered milk replacer (Litter Life, Merrick, Union, WI) at a daily rate of 60 g/kg body wt.Study Design
The pigs were fed the diet in powdered form for 10 days. Surgery was performed on day 11 after an overnight fast. The surgery entailed the placement of catheters in the portal vein, in a carotid artery, in an external jugular vein, and in the stomach ~2 cm from the pyloric sphincter. An ultrasonic flow probe (either model 6S or 6R, Transonics, Ithaca, NY) was implanted around the common portal vein. The surgical procedures have been described in detail in previous publications (9, 21, 23) and were performed under isoflurane anesthesia using strict sterile techniques.After the surgery, the pigs were offered 25% of their preceding daily intake that night. This was followed by 50% intake on the first postoperative day, resuming full-feed intake on the second day after surgery. The 6-h tracer infusion protocol was carried out 5 days after surgery, by which time the animals had been growing at preoperative rates (200 to 250 g/day) for at least 2 days.
Infusion Protocol
The pigs were deprived of feed from 1800 to 0700. At 0700, baseline arterial and portal blood samples were taken and the pigs consumed a meal of liquid Litter Life that supplied 1/24th of the preceding daily intake. At the same time, an intragastric infusion of an aqueous suspension of U-13C-labeled Spirulina platensis (Martek, Malvern, MA) and an intravenous infusion of [2H5(phenyl)]phenylalanine ([2H5]phenylalanine) were started. Both tracers were infused at a rate of ~0.1 ml/min. The Spirulina infusion supplied ~15 µmol [U-13C]phenylalanine · kgImmediately after death, the abdomen was opened, and the proximal 2 m of small intestine and an aliquot (~5 g from a lateral lobe) of liver were removed, weighed, and frozen in liquid nitrogen and used for subsequent measurement of protein content, protein-bound phenylalanine isotopic enrichment, and amino acid composition. The remainder of the liver was removed and weighed.
Sample Preparation
Blood phenylalanine. The isotopic and concentration measurements of phenylalanine were made on whole blood. Samples (0.5 ml) for amino acid concentration measurements were mixed with an equal volume of an aqueous solution of methionine sulfone and centrifuged, at room temperature, through a 10-kDa cutoff filter. The filtrate was dried, and the amino acids were analyzed by reverse-phase HPLC of their phenylisothiocyanate derivatives (PicoTag, Waters, Woburn, MA). For isotopic analysis, 0.1 ml of a 10-kDa filtrate was mixed with 0.25 ml of HCl (0.1 mol/l) and applied to a 1-ml bed volume column of Dowex 50 W×8 (H+ form) at 4°C. The amino acids were eluted with 5 M NH4OH and dried under vacuum.
Plasma proteins. Plasma (700 µl) was carefully layered under 700 µl of a solution of NaCl (0.195 mol/l) and Na2EDTA (1 mmol/l) at pH 7.4 (final specific gravity 1.006 kg/l). The solution was centrifuged at 22°C for 3 h at 210,000 g in a 100.3 rotor in a Beckman (Palo Alto, CA) TL-100 ultracentrifuge. The very low-density lipoprotein fraction was removed by aspiration and apoB-100 was precipitated with isopropanol (10). The fibrin fraction of fibrinogen was isolated by mixing 0.1 ml of plasma with 40 µl of an aqueous thrombin solution (1 × 105 U/l) and 40 µl of CaCl2 (25 mmol/l). The fibrin was washed three times with saline (22). The total plasma protein in 10 µl of plasma was precipitated with TCA (0.6 mol/l), centrifuged, and washed repeatedly with TCA. Albumin was then extracted from the precipitate with 5 µl TCA (0.6 mol/l) and 1.0 ml of 100% ethanol (14). The supernatant was then dried. The dried protein samples were hydrolyzed in 1 ml of 6 M HCl at 110°C for 24 h and then analyzed using mass spectrometry.
Tissue Proteins and Amino Acids
The mucosa from the intestinal segment was isolated by freeze-thaw disruption of the mucosal structure (19). Weighed aliquots (~5 g) of mucosa and liver were homogenized (Ultra Turrax, Tekmar, Germany) with water (1:1 wt/wt) at 4°C. One milliliter of the homogenate was then treated with 1 ml of perchloric acid (1 mol/l) and centrifuged (15,000 g for 10 min) in a Microfuge. The supernatant was removed and brought to pH 4-6 with KOH (5 mol/l). After removal of the potassium perchlorate, the amino acid fraction was isolated by cation exchange chromatography as described for the blood amino acid fraction. The protein precipitate was redissolved in 5 ml of NaOH (0.3 mol/l), and 1 ml of precipitate was used for determination of protein by the Biuret method. The remaining protein was reprecipitated with 0.15-0.2 ml of perchloric acid (11.7 mol/l), washed with two changes of ice-cold ethanol, and suspended in 2 ml water. An aliquot of the homogenate was mixed with an equal volume of HCl (10.8 mol/l) and hydrolyzed at 110°C for 24 h in a sealed tube. A known aliquot of the hydrolysate was dried, redissolved in HCl (0.1 mol/l), and used for amino acid analysis. A second aliquot was purified by ion exchange chromatography and used for mass spectrometry.Mass Spectrometry
Mass spectrometric analysis of the amino acids was carried out with the n-propyl ester heptafluorobutyramide derivative (13). Gas chromatography was performed on a 5890 series II gas chromatograph (Hewlett Packard, Palo Alto, CA), using a DB5 fused silica column (J & W Scientific, Folson, CA). Mass spectrometric analysis was by methane-negative chemical ionization using a 5989B (Hewlett Packard) quadruple mass spectrometer. Ions with a mass-to-charge ratio of 383-392 were monitored. All runs were performed at least in triplicate. The crude ion spectra were converted to tracer-to-tracee ratios using, as baseline, the ion spectrum of phenylalanine purified from samples taken immediately before the tracer infusions commenced.Calculations
Portal mass balance (in µmol · kg
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(1) |
Portal tracer balance (in µmol
tracer · kg1 · h
1)
was calculated as follows
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(2) |
Fractional portal balance (in percent input) was calculated as follows
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(3) |
Mucosal protein tracer incorporation (in µmol of labeled
phenylalanine · kg1 · h
1)
was calculated as follows
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(4) |
Hepatic protein tracer incorporation (in µmol of labeled
phenylalanine · kg1 · h
1)
was calculated as follows
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(5) |
Total phenylalanine incorporation into mucosal protein (in
µmol · kg1 · h
1)
was calculated next. Because the
[U-13C]phenylalanine
infusion rate and the arterial flux of
[2H5]phenylalanine
to the PDV are known, the incorporation of the two tracers into mucosal
protein can be expressed as a proportion of the respective tracer
inputs. On the assumption that both the tracer and tracee phenylalanine
are metabolized identically, then the total incorporation of unlabeled
(tracee) phenylalanine into mucosal protein can be calculated as
follows for the enteral tracer
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(6) |
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(7) |
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(8) |
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(9) |
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(10) |
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(11) |
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(12) |
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(13) |
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(14) |
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(15a) |
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(15b) |
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(15c) |
Because we did not obtain samples of hepatic venous blood, we were unable to make direct measurements of the hepatic balance of phenylalanine. We therefore adopted an indirect approach to the calculation of the uptake of amino acids by the liver. The reasoning is outlined below.
For a nutritionally essential amino acid, the only sources of the intracellular free pool are transport from the blood and entry from tissue proteolysis. When the primary pool of tracer is the blood (as in the present experiment), then the isotopic enrichment of the tissue-free amino acid is lower than that of the blood amino acid, and the degree of isotopic dilution is a function of the relative rates of transport from the blood and proteolysis. Thus, if the rate of proteolysis and the isotopic enrichment of the amino acid in the blood and tissue-free pools are known, then the rate of uptake of the labeled amino acid from the blood can be calculated from
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(16) |
Note that in this calculation we have assumed that 75% of total hepatic blood flow is portal and that the portal and arterial amino acids contribute to the hepatic-free pool in proportion to their respective flow rates. However, because the tracer-to-tracee ratios of arterial and portal phenylalanine differed by only 20%, the assumption with regard to the distribution of hepatic blood flow between the arterial and portal inputs in fact has a negligible effect on the final value for hepatic tracer amino acid uptake.
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RESULTS |
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The mean body weight of the pigs was 7.62 ± 0.31 kg. The mass of mucosa isolated from the proximal 2 m of the jejunum was 27.9 ± 4.1 g (3.66 ± 0.35 g/kg), and the protein-bound phenylalanine in this sample was 892 ± 122 µmol (117 ± 10 µmol/kg). It is important to note that the mucosal mass, and hence the phenylalanine content of the protein of the proximal 2 m of the jejunum, was 36% (P < 0.01) higher than in the previous groups of pigs that we have studied (23). The mean liver weight of the animals was 261 ± 15 g (34.3 ± 1.3 g/kg), and the protein-bound phenylalanine was 10,700 ± 1,900 µmol (1,400 ± 150 µmol/kg).
The time courses of labeling of the two tracers in arterial and portal
blood and in apoB-100 are shown in Figs. 1
(intragastric tracer) and 2 (intravenous
tracer). The data show that isotopic steady state in all three pools
was achieved by ~3 h of infusion. Data on the mass and tracer
balances at steady state are summarized in Table
1. On average, the portal phenylalanine
mass balance (59.6 µmol · kg1 · h
1)
was only 40% of phenylalanine intake, and the portal balance of the
intragastric tracer (7 µmol · kg
1 · h
1)
accounted for 46.7% of the infusion. Both values were also
significantly less than the values reported previously (50 and 66%,
respectively; Refs. 23, 24). The difference between the fractional mass and enteral tracer balances was significant
(P < 0.05) and suggested a
continuing utilization of arterial phenylalanine by the PDV. This was
confirmed by direct measurements of the balance of the intravenous
tracer across the PDV, which showed that 6.4% of the arterial tracer
flux of the intravenous
[2H5]phenylalanine
tracer was removed across this tissue bed.
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Whole body phenylalanine kinetics calculated with the two tracers are
summarized in Table 2. All the values for
phenylalanine entry rate were higher than our previous estimates in fed
piglets, and the difference between the apparent phenylalanine flux
measured with the two tracers suggested that first-pass utilization of the intragastric tracer by the tissues of the splanchnic bed accounted for 63% of dose. Of this, 83% (54% of dose) represented first-pass intestinal tracer metabolism. The hepatic extraction of the tracer was
9.9% of dose, a value similar to our previous estimates (10.9% of
dose; Ref. 23). Entry of phenylalanine from hepatic proteolysis, calculated from the difference between lines 2 and 3 of Table 2, was 34 ± 7 µmol · kg1 · h
1.
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The labeling of mucosal phenylalanine is shown in Table
3. The relative contributions of the two
tracers to the mucosal-free phenylalanine pool
(intravenous-to-intragastric ratio of 0.20) were significantly
(P < 0.05) different from their
contributions to protein labeling (intravenous-to-intragastric ratio of
0.32), a result that suggests preferential utilization of the arterial phenylalanine for protein synthesis. The apparent fractional rates of
mucosal protein synthesis calculated with the two tracers were also
different (P < 0.01).
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In Table 4, we have used the data on
mucosal protein labeling (Table 3) and the portal balances of the two
tracers (Table 1) to calculate the absolute quantities of dietary and
arterial phenylalanine incorporated into mucosal protein. These
calculations suggest that, despite the channeling of arterial
phenylalanine into mucosal protein synthesis, 59% of the mucosal
protein-bound phenylalanine was derived from the diet. The estimated
fractional rate of mucosal protein synthesis (70 ± 8% per day)
calculated from these data was significantly
(P < 0.05) lower than either of the
estimates based on the relative labeling of the protein-bound and free
pools of phenylalanine.
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Table 5 summarizes the data on hepatic and
plasma protein labeling. As we have observed previously, the
steady-state isotopic enrichment of apoB-100-bound phenylalanine was
approximately threefold higher than that of the bulked free
phenylalanine pool. Furthermore, the ratios of the isotopic enrichments
of the two tracers in arterial and portal phenylalanine were
significantly different. The ratios of the tracers in apoB-100, hepatic
constitutive protein, as well as in plasma albumin and fibrinogen were
similar to one another and were not significantly different from the
ratio of the two tracers in portal phenylalanine. The fractional rates
of synthesis of the various proteins calculated with the two tracers
were essentially the same when the calculation used the isotopic
enrichment of apoB-100 phenylalanine as the denominator in the
calculation.
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In Table 6, we used the data on hepatic
protein synthesis and the isotopic enrichments of extracellular and
intracellular phenylalanine to calculate the uptake of phenylalanine in
first pass by the liver. The estimated rate of phenylalanine entry from hepatic proteolysis (27 ± 5 µmol · kg1 · h
1)
was similar to that calculated from blood phenylalanine kinetics and
shown in Table 2 (34 ± 7 µmol · kg
1 · h
1).
The calculated first-pass uptake of phenylalanine (11.6 µmol · kg
1 · h
1)
was 18% of the portal balance. Of the phenylalanine removed by the
liver in first pass, we estimate that 64% was incorporated into
hepatic constitutive protein.
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DISCUSSION |
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Protein synthesis in the gastrointestinal tract and the liver makes a disproportionate contribution to whole body protein turnover. Exact quantification of the rates of protein synthesis in these two organs has proved difficult because of inexact knowledge of the sources of amino acids used to support protein synthesis and the problems that are posed by the metabolic compartmentation of the free amino acid pools of both tissues. This compartmentation has been revealed by a variety of approaches, including direct measurements of aminoacyl-tRNA labeling in the liver (2, 25), by measurements of the steady-state isotopic enrichments of rapidly turning over proteins of hepatic (6, 18, 20) and mucosal (7, 8) origin, and by the results of other dual-tracer studies (1, 3, 17). The aim of this study was to quantify the degree of first-pass utilization of enteral phenylalanine within the intestine and liver and to measure the relative contributions of dietary (or portal) and arterial phenylalanine to constitutive protein synthesis in the liver and the proximal jejunal mucosa.
First-Pass Metabolism of Enteral Amino Acids
There is now extensive literature on the first-pass splanchnic extraction of enteral amino acids, especially in humans (e.g., Refs. 5, 11, 12, 16). These studies revealed variations among amino acids with values ranging from 50% for phenylalanine (5) to <25% for leucine (12, 16). Our previous measurements with simultaneous intragastric (1-13C-labeled) and intravenous (2H5-labeled) phenylalanine tracers indicated that, in fed piglets, first-pass splanchnic metabolism of the enteral tracer accounted for 45% of dose, and of this 76% occurred in the intestine. These data were similar to earlier measurements in adult dogs (26, 27). In the present work, performed with a similar diet but using a U-13C-labeled protein as the source of enteral tracer phenylalanine, splanchnic first-pass tracer phenylalanine metabolism was 63% of dose, a value that is 40% higher than that in our previous report. This reflected an apparently higher first-pass utilization (53% of dose) of phenylalanine by the intestine.Although it might be argued that the difference in apparent intestinal first-pass metabolism might have resulted from incomplete digestion of the U-13C-labeled protein tracer, we think this is unlikely because the portal balance of unlabeled phenylalanine (40% of intake) was also significantly lower, in relation to intake, than in our previous study (23). We believe that the difference between this study and our past results reflects differences in the husbandry of the animals used in the two studies. In our previous work, animals were purchased from a research facility (Texas A & M Univ., College Station, TX) where they had been held under relatively sterile conditions. The present animals were purchased from the Texas Dept. of Criminal Justice and had been housed outside. Hence, they had the opportunity to root and had probably ingested soil and fibrous material. This, we believe, underlies the fact that the pigs obtained from the Texas Dept. of Criminal Justice had a significantly (36%; P < 0.01) greater mucosal mass than that of the pigs obtained from the research facility. It seems likely, therefore, that the increased first-pass metabolism of the enteral tracer and dietary phenylalanine resulted from a higher rate of enteral amino acid metabolism. This, in turn, implies that environmental factors that affect mucosal mass, including increased exposure to potential pathogens, have a measurable and nutritionally significant effect on the systemic availability of dietary amino acids.
In contrast to the results on intestinal metabolism, the estimates of first-pass hepatic utilization of dietary phenylalanine between the two studies were similar. In our previous study, the difference between total splanchnic first-pass extraction and intestinal extraction was 10.9% of dose, whereas the present results suggest a value of 9.9% of dose. Furthermore, calculations of hepatic proteolysis in the previous and the present study were similar.
Sources for Mucosal and Hepatic Protein Synthesis
In previous studies, we presented evidence (based on comparisons of the relative labeling of intragastric and intravenous tracer phenylalanine in arterial and portal blood and in apoB-100 and fibrinogen) that suggested preferential use of portal phenylalanine for the production of hepatic secretory proteins. These data were similar to the results of other investigations of albumin synthesis in humans (6) and fibrinogen synthesis in dogs (3). Because of metabolic zonation in the liver, the results could have reflected compartmentation of secretory protein synthesis, i.e., preferential synthesis of secreted proteins in periportal hepatocytes. Therefore, a primary objective of the present study was to extend the data to the synthesis of hepatic constitutive proteins.The present results confirm our earlier observation that the steady-state isotopic enrichment of apoB-100 considerably exceeded that of the hepatic-free phenylalanine, suggesting channeling of extracellular phenylalanine to hepatic protein synthesis. In addition, on the basis of the relative isotopic enrichments of the two tracers in the various protein-bound pools of phenylalanine, we conclude that hepatic proteins, both secretory and constitutive, were derived almost exclusively from portal phenylalanine. The tracer incorporation results in the mucosa also confirmed earlier observations of simultaneous utilization of both arterial and enteral phenylalanine for mucosal protein synthesis (1, 17) and suggested that there was also channeling of arterial phenylalanine toward protein synthesis. Even so, when the molar rates of phenylalanine delivery from the diet and the arterial circulation were taken into account, 59% of the mucosal protein-bound phenylalanine was of direct dietary origin.
In the liver, firm calculations of the fractional rate of protein synthesis can, we believe, be made by using apoB-100 labeling to define that of the hepatic protein synthetic precursor pool. However, because of uncertain knowledge of the true isotopic enrichment of the mucosal protein synthetic precursor pool, the mucosal protein labeling cannot be used to calculate accurate estimates of the rate of mucosal protein synthesis. Indeed, because of the channeling of arterial phenylalanine, the two tracers gave significantly different estimates of the mucosal protein synthesis rate. In the present work, we were able to express the mucosal tracer incorporation data as a proportion of the unidirectional uptake of the enteral (tracer infusion rate) and arterial (portal [2H5]phenylalanine balance) tracers. This allowed the calculation of the absolute rate of mucosal protein synthesis, i.e., moles unlabeled phenylalanine incorporated per unit time. This, in turn, allows an estimate of the fractional rate of mucosal protein synthesis without knowledge of the isotopic enrichment of the protein synthetic precursor pool. It was notable therefore that the value so derived was significantly less than the values that were based on conventional precursor-product calculations. This observation implies that the isotopic enrichment of the mucosal protein synthetic precursor pool of phenylalanine was significantly higher than that of the mixed acid, soluble-free pool. This conclusion is supported by the recent demonstration in fed piglets that showed that during a [2H]leucine infusion the steady-state isotopic enrichment of prolactase exceeds that of the mucosal-free leucine pool (8).
It appears, therefore, that in both the liver and the intestinal mucosa extracellular amino acids are channeled to protein synthesis without mixing freely with the intracellular pool of phenylalanine. The reverse is also implied, i.e., that phenylalanine derived from proteolysis is recycled to only a limited extent within the cell.
The cellular mechanisms that underlie these kinetic observations remain obscure. It may be important that both tissues are specialized with regard to the regulated extraction from and the release of amino acids to the extracellular compartment, and it is possible that inward and outward amino acid transport proceeds at different sites. Such a mechanism is easy to envision in the enterocyte because the arterial and lumenal contributions to the free pool arise from transport at two functionally distinct surfaces (the brush-border and basolateral membranes) and there is no reason to believe that amino acids transported across the basolateral membrane necessarily suffer the same metabolic fate as newly transported dietary amino acids. Indeed, there is evidence that shows metabolic channeling of other dietary amino acids in the mucosa (19).
With regard to compartmentation of the free amino acid pool of the liver, Vidrich et al. (25) developed a statistical model for amino acid metabolic compartmentation in the liver in which they argued for the idea of specific activation of amino acids located on the inner face of the amino acid transporters. The implication of this model is that, in the fed state, in which inward transport exceeds outward transport, there is preferential use of newly transported amino acids for protein synthesis. The model and our results also imply that amino acids derived from hepatic proteolysis are specifically transported out of the hepatocyte. Interestingly, Vidrich et al. (25) also found that as rats approached the fasted state (a condition in which the rates of inward and outward transport are closer and hepatic proteolysis is increased), the equilibration between intracellular and aminoacyl-tRNA-bound valine labeling became more complete. This observation has been confirmed recently with regard to both leucine and phenylalanine in fasted pigs (2). In this context, it might be of significance that lysosomal proteolysis is particularly important in the liver and that the lysosome represents a compartment that is physically separated from the cytosolic amino acid pool. That being so, it is possible that release of amino acids and/or small peptides from the lysosome is predisposed toward outward transport rather than reentry into the aminoacyl-tRNA pool.
The other hepatic protein metabolic phenomenon that demands explanation is the evidence from the work of ourselves (23) and others (3, 4, 6) that portal, rather than arterial, amino acids are used preferentially for protein synthesis in the liver of fed animals and humans. These previous works concentrated on secretory protein synthesis, and it seemed to us entirely possible that the differences reflected functional rather than kinetic zonation. However, the present results suggest that the same compartmentation also applies to hepatic resident protein synthesis, in which case an argument based on functional zonation is more difficult to sustain.
There seems to us to be three possible explanations. First, the contribution of arterial blood flow to total hepatic blood flow could be much lower than is generally presumed. This seems unlikely largely because the hemoglobin oxygen saturation in the portal blood of fed piglets is low and a low hepatic arterial flow would compromise the energy status of the hepatocytes.
Second, although it is generally held that mixing of capillary blood derived from the portal and arterial circulations occurs within a very short distance of their respective entry into the acinar unit, hepatocytes may possibly show some degree of functional polarization, in which different amino acid transporters are exposed to the arterial and portal inputs. Although we know of no evidence for this proposition, it is noteworthy that the ratio of the two tracers in the hepatic-free amino acid pool was similar to the ratio found in portal blood, an observation that indicates preferential transport of amino acids derived from this input.
The third possibility is that the rate of protein synthesis in the periportal hepatocytes is considerably higher than in the perivenous cells, so that the apparent preferential utilization of portal amino acids reflects merely the fact that protein synthesis in periportal hepatocytes dominates the overall protein metabolic activity of the liver. There is some autoradiographic evidence to support this proposition (15).
However, irrespective of the underlying mechanism, the present data extend the growing body of evidence that suggests that 1) intestinal amino acid metabolism quantitatively dominates overall splanchnic metabolism and 2) dietary amino acids play a role as direct protein synthetic precursors for mucosal and hepatic protein synthesis.
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ACKNOWLEDGEMENTS |
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We are grateful to Leslie Loddeke for her sound editorial advice.
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FOOTNOTES |
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This work is a publication of the USDA/ARS Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine and Texas Children's Hospital, Houston, TX.
The contents of this publication do not necessarily reflect the views or policies of the USDA; mention of trade names, commercial products, or organizations does not imply endorsement from the U.S. Government.
This work was supported in part by National Heart, Lung, and Blood Institute Grant RO1-HD-33920 (D. G. Burrin) and by federal funds from the USDA/ARS Cooperative Agreement 58-6250-6-001. B. Stoll was supported in part by the Alexander von Humboldt-Stiftung fund.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: P. J. Reeds, USDA/ARS Children's Nutrition Research Center, Dept. of Pediatrics, Baylor College of Medicine, 1100 Bates, Houston, TX 77030.
Received 13 May 1998; accepted in final form 18 September 1998.
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