The Journal of Nutrition Vol. 128 No. 9 September 1998, pp. 1517-1524

Dietary Amino Acids Are the Preferential Source of Hepatic Protein Synthesis in Piglets^1,2,3,4

Barbara Stoll, Douglas G. Burrin, Joseph Henry, Hung Yu, Farook Jahoor, and Peter J. Reeds⁵

USDA/ARS Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX

	ABSTRACT

Abstract Introduction Methods Results Discussion References

To investigate the utilization of dietary amino acids for hepatic protein synthesis, seven female pigs ( 28 d old, 7.5 kg) were implanted with catheters in a carotid artery, the jugular and portal veins, and the stomach. A portal flow probe was also implanted. The pigs were fed a high protein diet once hourly and infused intragastrically with [U-¹³C]algal protein for 6 h. Amino acid labeling was measured in arterial and portal blood, in the hepatic free and protein-bound pools and in apolipoprotein B-100 (apoB-100), albumin and fibrinogen. The isotopic enrichments of apoB-100-bound [U-¹³C]threonine, leucine, lysine and phenylalanine were 33, 100, 194 and 230% higher than those of their respective hepatic free amino acid pools (P < 0.01). Using the labeling of apoB-100 to estimate that of the protein synthetic precursor, the fractional rate of hepatic protein synthesis was 42 ± 2%/d. Between 5 and 8% of the dietary tracer amino acids was used for hepatic protein synthesis. In contrast to the small intestinal mucosa, in which the majority of the metabolized amino acids were apparently catabolized, protein synthesis utilized from 48% (threonine) to 90% (lysine) of the hepatic uptake of tracer amino acids. It appears that hepatic protein synthesis consumes nutritionally significant quantities of dietary essential amino acids in first pass and that extracellular, especially portal, essential amino acids are channeled to hepatic protein synthesis in the fed state.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

The fractional rates of protein synthesis in the liver and gut are considerably higher than those of other tissues; together the two tissues account for 25% of whole-body protein synthesis. Because the liver receives extracellular amino acids from two sources (arterial and portal) and also has a substantial rate of proteolysis (Mortimore et al. 1988), the kinetics of the hepatic amino acid exchange are complex and their regulation is poorly understood. We (Berthold et al. 1995, Jahoor et al. 1994, Reeds et al. 1992) and others (Cayol et al. 1996, Lichtenstein et al. 1990, Parhofer et al. 1990) have used the steady-state labeling of VLDL apolipoprotein B-100 (apoB-100), a rapidly turning over protein of hepatic origin, to estimate the plateau isotopic enrichment of the hepatic protein synthetic precursor pool. The results of these studies suggest that extracellular (rather than mixed intracellular) amino acids are the major contributors to hepatic protein synthesis. Other results (Berthold et al. 1995, Cayol et al. 1996) imply that dietary (i.e., portal) amino acids may be preferentially utilized for hepatic protein synthesis. However, with the exception of a recent study with labeled phenylalanine (Stoll et al. 1997), there is little direct information to substantiate these implications and specifically no data on the relationship between the labeling of apoB-100 and that of the hepatic free amino acid pools.

There is now a considerable literature that suggests that between 20 and 50% of essential amino acids absorbed from the intestinal lumen are metabolized in first pass by the tissues of the splanchnic bed (Biolo et al. 1992, Hoerr et al. 1991 and 1993, Matthews et al. 1993). On the basis of measurements of the portal balances of either enterally administered [1-¹³C]phenylalanine (Stoll et al. 1997) or systemically administered [¹³C/¹⁵N]leucine (Yu et al. 1990 and 1992), it appears that the metabolism of dietary amino acids by the mucosal cells accounts for the majority of their total splanchnic metabolism. This paper reports the second part of an enteral tracer infusion study described by Stoll et al. (1998). In this portion of the study, we were concerned with two questions. First, what is the contribution of hepatic protein metabolism to the first-pass splanchnic utilization of dietary amino acids? Second, does uptake from the blood make a disproportionate contribution to the hepatic protein synthetic precursor pool?

On the basis of previous work, we hypothesized that the contribution of the systemic amino acid pool to hepatic protein synthesis would be high, but that the contribution of the extracellular supply would differ among essential amino acids.

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

Animals.  The study was approved by the Baylor College of Medicine Animal Protocol Review Committee. Housing and care of the animals conformed to USDA guidelines. The study involved seven 28-d-old female crossbred piglets purchased either from the Department of Animal Science, Texas A & M University, College Station, TX or from the Texas Department of Criminal Justice (TDCJ), Huntsville, TX. The two groups of animals were of similar genetic background (Large White × Duroc × Hampshire). The pigs were received at the CNRC when they were 2 wk old and fed powdered milk replacer (Litterlife, Merrick, Union , WI) at a rate of 60 g/kg body weight. The proximate composition (g/kg dry matter) of Litterlife is 500 carbohydrate, 100 fat and 250 whey-predominant protein. The calculated energy density is 18 MJ gross energy/kg dry matter. The amino acid composition is given in Stoll et al. (1998).

Study design  This is described in detail by Stoll et al. (1998). The pigs were fed the diet in powdered form for 10 d. Food was removed overnight before surgery (Ebner et al. 1994, Reeds et al. 1996, Stoll et al. 1997); after the surgery, the pigs were offered 25% of their preceding daily intake that night, followed by 50% intake for the first postoperative day, resuming full feed intake on d 2 after surgery. The 6-h tracer infusion protocol was carried out 5 d after surgery, by which time the animals had been growing at preoperative rates (200-250 g/d) for at least 2 d.

Infusion protocol.  The pigs were deprived of feed from 1800 to 0700 h, baseline arterial and portal blood samples were taken and the pigs consumed a meal of liquid Litterlife that supplied one twenty-fourth of the preceding daily intake. Immediately thereafter, an aqueous suspension of [U-¹³C]Spirulina platensis (Martek Corporation, Malvern, MA) was infused into the gastric catheter at a rate of ~0.1 mL/min. The spirulina supplied ~60 mg crude protein/(kg·h). Its amino acid composition and the tracer:tracee ratios of threonine, lysine, leucine and phenylalanine are given in Stoll et al. (1998). Throughout the infusion, the pigs consumed hourly meals of Litterlife. Each meal supplied one twenty-fourth of their preceding daily intake [i.e., 600 mg protein/(kg·h)]. Arterial and portal blood samples (3 mL) were taken at hourly intervals until 5 h of tracer infusion, and then at 15-min intervals until the animals were killed with an arterial injection of sodium pentobarbital (50 mg/kg body weight) and sodium phenytoin (5 mg/kg) (Beutanasia-D; Schering-Plough Animal Health, Kenilworth, NJ). Blood samples were collected in EDTA tubes and were frozen immediately in liquid nitrogen.

Immediately 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 and frozen in liquid nitrogen. The remainder of the liver was removed, weighed and frozen for subsequent measurement of protein content and amino acid composition.

Sample preparation  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 VLDL fraction was removed by aspiration and apoB-100 was precipitated with isopropanol (Egusa et al. 1983). 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 (Stein et al. 1992). The total plasma protein in 10 µL of plasma was precipitated with trichloracetic acid (0.6 mol/L), centrifuged and washed repeatedly with trichloracetic acid. Albumin was then extracted from the precipitate with 5 µL trichloracetic acid (0.6 mol/L) and 1.0 mL of 100% ethanol (Korner and Debro 1956). The supernatant was then dried.

Hepatic protein and amino acids. A 5-g aliquot of liver was homogenized (Ultra Turrax, Tekmar, Germany) with water (1:1 wt/wt) at 4°C. One milliliter of the homogenate was mixed with 1 mL of perchloric acid (1 mol/L). The homogenate was centrifuged (15000 × g for 10 min) in a microfuge. The supernatant was 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 on a 1-mL bed volume column of Dowex AG50X8 (Biorad, Richmond, CA). The protein precipitate was redissolved in 5 mL of NaOH (0.3 mol/L) and 1 mL was taken for determination of protein by the biuret method (Hubscher et al. 1965). 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. A known 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 under nitrogen. The hydrolysate was dried, redissolved in water, redried and resuspended in HCl (0.1 mol/lL). The amino acid fraction was isolated by cation exchange chromatography. One portion of the dried fraction was taken for negative chemical ionization gas chromatography-mass spectrometry as described previously (Jahoor et al. 1994, Stoll et al. 1997 and 1998). A second portion was taken for amino acid analysis by reverse-phase HPLC of their phenylisothiocyanate derivatives (Picotag system, Waters, Woburn, MA). Amino acid concentrations were assessed against standards that had been incubated in HCl (5.4 mol/L) overnight.

Calculations  Portal amino acid flux.

(1)

(2)

in which t/T is the tracer:tracee ratio(mol [U-¹³C]amino acid/mol [U-¹²C]amino acid.

Portal amino acid balance.

(3)

(4)

in which Conc. is concentration (µmol/L).

(5)

Whole-body amino acid flux. Whole-body (i.e., extra-intestinal) amino acid flux was calculated with the conventional two-pool model (Reeds 1992), except that the portal tracer balance (Equation 4) was used as the tracer input. Thus:

(6)

The flux of individual amino acids was then converted into protein equivalents with the mass contribution of each amino acid to whole-body protein (Davis et al. 1993).

The entry of each amino acid from body protein degradation, conventionally calculated as amino acid flux minus amino acid intake (see for example, Berthold et al. 1995), was calculated in this paper as follows:

(7)

i.e., Equation 6  Equation 3.

Once again this was converted to body protein equivalents as above.

Tracer incorporation into hepatic constitutive protein [µmol/(kg·h)] =

(8)

in which 1/6 accounts for the 6-h incorporation period.

Fractional rate of hepatic protein synthesis (%/d).

(9)

Calculations were made with either the labeling of the hepatic free amino acid or the steady-state labeling of apoB-100 as the denominator (see Results).

Fractional rate of plasma protein synthesis. For the calculation of fibrinogen and albumin synthesis, it was assumed that the steady-state tracer:tracee ratio of a given amino acid in arterial apoB-100 defined the steady-state labeling of the albumin and fibrinogen precursor pool, and that label was incorporated in a monoexponential fashion. Thus fractional synthesis rate (h¹) was calculated from

(10)

in which k is the fractional rate of synthesis and t the time of infusion (h). In the results section, the fractional rate of protein synthesis is presented as %/h, i.e., 100 × Equation 10.

For both proteins, we used measurements of the tracer:tracee ratio in the product protein from 3 to 6 h of infusion in the numerator, with the mean tracer:tracee ratio of apoB-100 over 4 to 6 h as the denominator.

Hepatic tracer amino acid uptake. Because we did not obtain samples of hepatic venous blood, we were unable to make direct measurements of the hepatic balance of amino acids. We therefore adopted an indirect approach to the calculation of the uptake of amino acids by the liver. The reasoning was as follows.

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 this 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 the following: Although we know that under the circumstances of this experiment there was net protein deposition in the liver, the rate of hepatic protein deposition (~3%/d) is much less than the rate of hepatic protein turnover (>40%/d). Thus, the measured rate of hepatic protein synthesis is a close approximation to that of hepatic proteolysis. The rate of uptake of the tracer amino acid was calculated with Equation 11:

Hepatic tracer uptake [µmol/(kg·h)]

(11)

in which the rate of tracer incorporation was that calculated with Equation 8, and incorporation and uptake were expressed as µmol/(kg·h).

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:tracee ratio of the arterial amino acid was >80% of that of the portal, 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.

Total splanchnic utilization of tracer amino acid. Intestinal metabolism + hepatic uptake, i.e., Equation 5 + Equation 11.

Statistics.  All values are shown as the mean ± the between-animal SD. Differences between amino acids in the relationships of the tracer:tracee ratios of the various pools (e.g., apoB-100-bound vs. hepatic free) and of the protein synthetic rates were assessed by one-way ANOVA, with amino acid as the independent variable, followed by a post-hoc t test with the appropriate Bonferroni adjustment for four comparisons. A value of P (two-tailed) <0.05 was taken as indicating significance.

	RESULTS

Abstract Introduction Methods Results Discussion References

The mean body weight of the pigs was 7.53 ± 0.96 kg, liver weight was 264 ± 32 g and liver weight:body weight was 33.8 ± 1.9 g/kg. The concentration (µmol/g protein) of threonine, leucine, lysine and phenylalanine in hepatic protein was 441 ± 44, 821 ± 86, 626 ± 43 and 298 ± 41, respectively.

As judged by the fact that the slope of the line relating the amino acid tracer:tracee ratios with time was not significantly different from zero, arterial and portal free and apoB-100-bound amino acids had attained isotopic steady state by 3 h of infusion (data not shown). The mean values for the tracer:tracee ratios of arterial, portal, apoB-100, hepatic free and protein-bound amino acids and estimates of whole-body amino acid and protein turnover are shown in Table 1. The arterial and portal data (Table 1) have been reported previously (Stoll et al. 1998) and are shown here to facilitate the comparisons with the other amino acid pools. The estimates of whole-body protein degradation were between 8.3 and 10.4 g protein/(kg·d) with no significant differences among the amino acids.

The relationships between the tracer:tracee ratios of the amino acids in the various sampled pools are summarized in Table 2. The tracer:tracee ratios of threonine, leucine and phenylalanine in apoB-100 were close to those in arterial blood (ratio not significantly different from one). However, the tracer:tracee ratio of apoB-100 lysine was 19% (P < 0.05) higher than that of arterial lysine. Because the portal free amino acids were all more isotopically enriched than the arterial amino acids, the ratio of the isotopic enrichments of apoB-100 amino acid:portal amino acid was lower than the apoB-100:arterial ratio, although it was still close to unity (0.82-0.93). The tracer:tracee ratio of apoB-100 lysine was not significantly different from that of portal lysine (labeling ratio 1.02 ± 0.09).

With all four amino acids, the steady-state tracer:tracee ratios of the apoB-100-bound amino acids were significantly higher (P < 0.001) than those of their respective hepatic free amino acid pools. There were also highly significant differences (P < 0.01) among the amino acids. ApoB-100 threonine was 33% more highly labeled than hepatic free threonine, whereas apoB-100 [U-¹³C]lysine and phenylalanine were three times more enriched than their respective free amino acid pools.

The labeling of hepatic protein is also shown in Table 2. The ratio of hepatic protein-bound:apoB-100-bound labeling varied little among amino acids (between amino acid CV 4.2%), and when apoB-100 labeling was used to define the isotopic enrichment of the protein synthetic precursor pool, the fractional rate of hepatic constitutive protein synthesis was 42 ± 2%/d. The ratio of hepatic protein-bound amino acid:hepatic free amino acid was widely variable among the different amino acids. As a result, calculations of liver protein synthesis, based on hepatic free amino acid labeling, ranged from 74%/d (threonine) to 227%/d (phenylalanine).

Table 3 summarizes the data on the portal flow and the absolute incorporation of the tracer amino acids into the constitutive proteins of the liver. The portal tracer flux (i.e., portal concentration × tracer:tracee ratio × portal blood flow) contributed between 12% (lysine) and 59% (threonine) of the whole-body flux. Although the incorporation of the tracer amino acids into hepatic protein expressed as a proportion of the portal tracer amino acid flux varied among the amino acids, when amino acid incorporation into hepatic protein was expressed in terms of the portal balance (i.e., the net input of tracer from the intragastric infusion into the portal vein), there was much less between-amino acid variation. Nevertheless, a significantly higher proportion of the portal balance of threonine (11.8%) was incorporated into hepatic protein than that of the other amino acids (9.8-10.1%). Total tracer incorporation into hepatic protein [1.0-3.0 µmol/(kg·h)] was similar in magnitude to our previous estimates of tracer incorporation into mucosal protein [1.2-3.5 µmol/(kg·h); Stoll et al. 1998].

Fibrinogen and albumin synthesis rates are summarized in Table 4. The values were similar in general to those determined in previous studies with tracer phenylalanine in piglets (Stoll et al. 1997). The estimated rate of synthesis of both proteins determined with apoB-100 threonine was slightly but significantly (P < 0.025) lower than when apoB-100 leucine, lysine or phenylalanine was used to calculate albumin and fibrinogen synthesis.

In Table 5, we summarize the intestinal and hepatic utilization of the enterally administered tracer amino acids. As already reported (Stoll et al. 1998), the intestinal mucosa utilized 35-37% of the enteral dose of leucine, lysine and phenylalanine and a significantly higher (P < 0.05) proportion of the threonine tracer (Table 5). The same appeared to apply to the hepatic uptake of the portal tracer. The liver extracted 13-18% of the net portal input of leucine, lysine and phenylalanine but 33% of the net portal input of threonine. Intestinal metabolism accounted for the majority (76-84%) of the total splanchnic extraction of the respective amino acids, but mucosal protein incorporation accounted for a minor portion of the intestinal utilization. However, in the liver (Table 5), the sum of constitutive and plasma protein incorporation accounted for 48, 73, 90 and 80% of the hepatic uptake of threonine, leucine, lysine and phenylalanine, respectively. As a result, mucosal and hepatic protein synthesis consumed similar proportions of the ingested tracer amino acids (5.4-8.5%).

	DISCUSSION

Abstract Introduction Methods Results Discussion References

Previous studies of splanchnic and intestinal metabolism of amino acids have been conducted in adult human beings (Biolo et al. 1992, Hoerr et al. 1992 and 1993, Matthews et al. 1993) and adult dogs (Yu et al. 1990 and 1992) and have used free amino acids as tracers. This study extended this work to the growing animal and investigated the first-pass splanchnic metabolism of multiple amino acids provided as a digestible protein source labeled uniformly with ¹³C. This enabled us to study the metabolism of a number of amino acids simultaneously. We elected to examine only four amino acids: threonine, because it is a major component of the intestinal mucins; lysine, because the chemical score of the diet suggested that lysine was the first limiting amino acid; and leucine and phenylalanine, as representatives of two nonlimiting essential amino acids whose sites of catabolism are generally presumed to be different. The major objectives of the work were to quantify the relative contributions of intestinal and hepatic metabolism to the first-pass utilization of enteral amino acids and to determine the contribution to first-pass metabolism of protein synthesis in the mucosa and liver. In our previous paper (Stoll et al. 1998), which concentrated on the intestinal tissues, we found that although about one third of the dietary amino acids were removed in first pass by the intestinal cells, incorporation into mucosal protein represented a minor fate of the enteral [U-¹³C]amino acids. We also found that ~30% of the protein nitrogen intake appeared as ammonia and alanine in the portal blood. On the basis of these results, we concluded that there was nutritionally significant first-pass catabolism of dietary amino acids by the mucosa.

Utilization of amino acids for hepatic protein synthesis.  The intestinal mucosa and the liver are unique among mammalian tissues in that they receive two extracellular sources of amino acids, i.e., dietary and arterial amino acids in the mucosa, and portal and arterial amino acids in the liver. In addition, in both tissues, the entry of amino acids derived from intracellular proteolysis makes a substantial contribution to the total free amino acid pool. Previous studies based on intravenous infusions of isotopically labeled amino acids have shown that there is a substantial isotopic dilution in the free amino acid pools of both the mucosa and the liver (Waterlow et al. 1978); this phenomenon greatly complicates the calculation of the true rate of tissue protein synthesis. The present results show that even when the primary site of administration of tracer to the liver is the portal circulation, there is still a considerable isotopic dilution in the hepatic free pool. This observation emphasizes the key role of proteolysis in maintaining the intracellular amino acid pool.

There is much evidence to suggest that the isotopic enrichment of the tissue free amino acids does not define exactly that of the protein synthetic precursor pool. Because the determination of the isotopic enrichment of the amino acyl tRNA is technically very difficult, as witnessed by the limited literature on hepatic amino acyl tRNA labeling (Airhart et al. 1974, Bauman et al. 1994, Vidrich et al. 1977), there has been considerable interest in developing alternative approaches to the problem. One approach is to quantify the labeling kinetics of proteins that have very rapid rates of turnover. The VLDL pool of apoB-100 is rapidly processed to lipoprotein particles of higher specific gravity, so that from a kinetic perspective, VLDL apoB-100 has a very high rate of turnover (55-75%/h in piglets; Stoll et al. 1997). As a result, VLDL-apoB-100 can be brought rapidly to an isotopic steady state in which its labeling directly defines that of the hepatic amino acid pool from which it derives.

Previous studies using intravenous infusions of amino acids in humans (Berthold et al. 1995, Cayol et al. 1996, Reeds et al. 1992) and piglets (Jahoor et al. 1994, Stoll et al. 1997) have shown that, despite the expectation of substantial isotopic dilution in the hepatic free pool, the isotopic enrichment of apoB-100-bound amino acids was within 20% of that of the plasma free amino acids. This result favors the idea of preferential incorporation of extracellular amino acids into the apoB-100 precursor pool and the present results confirm this idea. Furthermore, the results support the conclusions drawn from studies with intragastric tracer amino acids (Berthold et al. 1995, Cayol et al. 1996), that in the fed state, there may be preferential utilization of portal amino acids for apoB-100 synthesis.

As far as we can ascertain, with the exception of our recent study with enterally administered [1-¹³C]phenylalanine (Stoll et al. 1998), there have been no investigations of the relationship between the labeling of hepatic free amino acids and those incorporated in apoB-100. The present results confirmed earlier work (see for example Airhart et al. 1974, Garlick et al. 1975, McNurlan et al. 1979) and showed a substantial isotopic dilution of amino acids in the hepatic free pool. However, although there was variation among the amino acids, for all four, the steady-state isotopic enrichment of apoB-100 exceeded that of the hepatic free amino acid. This result confirms earlier measurements of valyl tRNA labeling (Airhart et al. 1974, Vidrich et al. 1977) and provides direct evidence for substantial compartmentalization in the hepatic free amino acid pools in vivo.

It should be emphasized that this conclusion applies strictly to the synthesis of apoB-100. However, we would argue that the same phenomenon applies to hepatic protein synthesis in general because it was quite clear that the free amino acid pool could not have defined the labeling of the precursors of hepatic constitutive proteins. This was because measurements of the rate of hepatic protein synthesis, based on measurements of free amino acid isotopic enrichments, varied over at least a threefold range, which is an irrational result. In fact, as we have shown in previous work with albumin (Jahoor et al. 1994), the only basis on which the results for the incorporation of different amino acids into hepatic protein could be reconciled with one another was to use the apoB-100 labeling data in the calculation.

It should also be emphasized that this conclusion applies specifically to the fed state. In a recent study of the labeling of hepatic leucyl and phenylalanyl tRNA in fasted adult mini pigs (Baumann et al. 1994), substantial isotopic dilution in the hepatic free amino acid pool was found, but there was complete equilibration between the hepatic free and amino acyl tRNA pools. However, it is noteworthy that in the study of valyl tRNA labeling in fed and postabsorptive rats (Vidrich et al. 1977), isotopic equilibration between the free and amino acyl tRNA-bound amino acids became more complete as the animals approached the fasted state. In this context, it should also be pointed out that the pool defined as "free amino acids" is a crude acid extract, in which any structural compartmentalization will have been destroyed by the isolation procedure. It is well established that lysosomal protein degradation is a major pathway for the catabolism of resident hepatic proteins (Mortimore et al. 1988), and amino acids contained within the lysosomes represent a pool that is confined, at least temporarily, to a defined subcellular structure. As such, lysosomal amino acids are physically separated from pools destined for protein synthesis. The results published by Vidrich et al. (1977) in addition to the present results imply that amino acids derived from hepatic proteolysis, and hence contained in the lysosomal compartment, play little or no role as precursors for hepatic protein synthesis in the fed state. In the fasted state, however, because of the change in the relative rates of inward and outward amino acid transport on the one hand and in hepatic protein turnover on the other, it appears that the lysosomal and protein synthetic pools are more effectively mixed. However, whatever the explanation at the mechanistic level, the results underscore the extreme importance of the accurate definition of the labeling of the protein synthetic precursor pool in studies of hepatic protein synthesis.

Tissue distribution of the splanchnic utilization of dietary amino acids.  A major objective of these studies was to partition the utilization of dietary amino acids between the portal-drained viscera and the liver. With the exception of the work of Yu et al. (1990 and 1992) in the dog, there is little information on the distribution of splanchnic amino metabolism between these two components in monogastric species, although there is a larger literature on ruminants (see for example, Bergman and Pell 1986, Lobley et al. 1996 and 1997).

Yu et al. (1990) made direct measurements of both portal and hepatic balance of leucine and concluded that the portal drained viscera dominated splanchnic amino acid metabolism. Unfortunately, in this work, we were unable to make direct measurements of the hepatic tracer balance, and inferred the uptake by the liver from the rate of protein turnover and the isotopic dilution in the respective amino acids. Nevertheless, these results were remarkably similar to those obtained by Yu et al. (1990) and suggested that intestinal metabolism accounted for 76% (leucine) to 84% (threonine) of the total splanchnic tracer metabolism.

From a nutritional perspective, it is extremely important to partition this substantial metabolism between protein synthesis and complete catabolism. If the first-pass metabolism reflects incorporation into tissue or plasma proteins, then, apart from the energy expended in synthesizing the protein, the amino acids will eventually recycle into the free amino acid pool. Essential amino acid catabolism, however, represents a real loss from the body.

In our previous paper (Stoll et al. 1998), we concluded on the basis of both isotope incorporation into mucosal protein and portal ammonia and amino acid-nitrogen balance, that amino acid catabolism was a major part of the mucosal first-pass utilization of dietary amino acids. The present results suggest that essentially the reverse is true for the liver and that hepatic protein synthesis accounted for the majority of the metabolism of the portal amino acids taken by the liver. Indeed, in the case of lysine, hepatic protein synthesis utilized 90% of the total hepatic lysine uptake. Although at face value this is a surprising result, it is entirely compatible with the fact that the plateau isotopic enrichment of lysine in VLDL apoB-100 was equal to that of portal lysine, i.e., that the pool of lysine incorporated into hepatic protein was of virtually exclusive and immediate extracellular origin.

There are nutritional implications of these conclusions. First, despite the extremely well-documented presence of the catabolic enzymes for all of the essential amino acids in the liver, the results imply that although the liver is critical to nitrogen metabolism, hepatic metabolism may be less important in the immediate carbon catabolism of dietary amino acids than has hitherto been assumed. This would be compatible with the recent proposition of Basile-Filho et al. (1997) that oral tracers appear to give quantitatively more accurate estimates of phenylalanine carbon balance than intravenous tracers. Second, if amino acid oxidation is the major energy source for the intestinal mucosa, then the first-pass catabolism of dietary essential amino acids may be a fixed source of nutritional inefficiency that will vary primarily with the mass of the intestinal tissues. In other words, other dietary or environmental factors that alter mucosal mass will alter the systemic availability of dietary protein. However, it should be emphasized that at present our conclusion is based on indirect evidence and requires direct substantiation by appropriate studies with carbon-labeled essential amino acids. Finally, the results that we present here and in our previous paper (Stoll et al. 1998) imply that about a third of the intake of lysine, leucine and phenylalanine and more than half of the intake of threonine are metabolized between the site of absorption from the intestinal lumen and appearance in the peripheral circulation. We estimate that the animals in this study were depositing ~40% of their intake of 100 g protein. Because these results suggest that no more than 50% of the dietary protein was available for protein deposition, they also imply that the nutritional efficiency (~80%) of peripheral protein deposition is much higher than has hitherto been assumed, and that much closer and systematic attention should be paid to the role of splanchnic, especially intestinal, amino acid metabolism in models of amino acid requirements and the subsequent development of nutritional recommendations.

	FOOTNOTES

Manuscript received 2 February 1998. Initial reviews completed 6 March 1998. Revision accepted 26 April 1998.

	ACKNOWLEDGMENT

We are extremely grateful to Leslie Loddeke for her skilled editorial advice.

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