Integrative physiology of splanchnic glutamine and ammonium metabolism

Dawei Yang1, Jeffrey W. Hazey2, France David1, Jittendra Singh1, Ryan Rivchum1, Jason M. Streem1, Mitchell L. Halperin3, and Henri Brunengraber1

Departments of 1 Nutrition and 2 Surgery, Case Western Reserve University, Cleveland, Ohio 44106; and 3 Renal Division, Department of Medicine, St Michael's Hospital, University of Toronto, Toronto, Ontario M5B 1A6, Canada


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The substrates for hepatic ureagenesis are equimolar amounts of ammonium and aspartate. The study design mimics conditions in which the liver receives more NH+4 than aspartate precursors (very low-protein diet). Fasted dogs, fitted acutely with transhepatic catheters, were infused with a tracer amount of 15NH4Cl. From arteriovenous differences, the major NH+4 precursor for hepatic ureagenesis was via deamidation of glutamine in the portal drainage system (rather than in the liver), because there was a 1:1 stoichiometry between glutamine disappearance and NH+4 appearance, and the amide (but not the amine) nitrogen of glutamine supplied the 15N added to the portal venous NH+4 pool. The liver extracted all this NH+4 from glutamine deamidation plus an additional amount in a single pass, suggesting that there was an activator of hepatic ureagenesis. The other major source of nitrogen extracted by the liver was [14N]alanine. Because alanine was not produced in the portal venous system, we speculate that it was derived ultimately from proteins in peripheral tissues.

gluconeogenesis; intestinal tract; liver; mass spectrometry; proteins; urea; zonation


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

UREA SYNTHESIS is the main route of nitrogen disposal in virtually all terrestrial mammals (for a review, see Ref. 28). Most of this nitrogen in urea results from the complete oxidation of proteins (8, 16, 28). Because of a steady state and a relatively constant protein intake, urea nitrogen excretion during a given day is largely equivalent to the protein intake of that day; however, in fact it is derived only in part from ingested proteins, the remainder being supplied by body proteins that were catabolized on that day (and resynthesized from the amino acids of dietary origin). Humans who consume proteins at 1.0-1.5 g · kg-1 · day-1 excrete urea at a relatively constant rate in the 24-h cycle (4). There is good evidence that an amount of urea equal to ~20% of the usual daily urea production rate is hydrolyzed to NH+4 and HCO-3 in the lumen of the gastrointestinal (GI) tract by urease derived from local microoorganisms in normal subjects (7, 8, 12, 14, 15, 21, 28).

Proteins are essential components of the body's lean body mass. Although they are macronutrients that are ingested and oxidized daily, their primary function is not to provide energy but rather to be made into structural and contractile elements, enzymes, transporters, hormones, and defense elements such as antibodies. In contrast to carbohydrates and lipids, amino acids contain nitrogen (and sulfur), which must be disposed of. Because true gluconeogenesis (GNG, i.e., the conversion of amino acids to glucose1) and ureagenesis share common intermediates until late in this metabolic pathway (argininosuccinate), Jungas et al. (16) and Halperin and Rolleston (10) have emphasized that GNG and ureagenesis are in fact components of the same metabolic process (Fig 1). Moreover, the two nitrogen (N) atoms that are incorporated into the urea molecule are in two different forms, i.e., one via free NH+4 and the other via aspartate (Fig. 1). Nevertheless, under conditions of low-protein intake and continuous splanchnic urea hydrolysis (i.e., a process that generates NH+4 but does not supply aspartate), it is not clear what the source of aspartate will be in the liver. If one-half of the NH+4 load could be converted to aspartate by glutamate dehydrogenase and aspartate-aminotransferase reactions, one would expect to find that the rise in urea appearance would be equal to the rate of supply of NH+4 to the liver, and that both nitrogens would almost equally be 15N enriched with the same molar percent enrichment (MPE) as NH+4. In contrast, if NH+4 were extracted by the liver but not converted to aspartate at a sufficient rate, one would expect to find a higher rate of appearance of urea, because a net supply of aspartate by proteolysis would be obligated, and this aspartate would not be 15N enriched. If, indeed, proteolysis is needed in this setting, it could help to explain the protein catabolism seen in patients consuming a low-protein diet (22, 27).


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Fig. 1.   Stoichiometry of gluconeogenesis (GNG) and ureagenesis. In this schematic representation, we emphasize that one-half of the nitrogen from amino acid catabolism must be converted to NH+4 and the other one-half to aspartate to support both glucose and urea synthesis. This represents a single pathway, with common intermediates up to its late stages. In the experimental design, a supply of NH+4 without aspartate was provided.

The question we addressed in the studies to be reported is, "What is the physiological response to a supply of nitrogen to the liver that differs markedly from the 1:1 stoichiometry for the nitrogen precursors (NH+4 and aspartate) illustrated in Fig. 1?" To address this question, we took advantage of a new sensitive technique we developed to measure the concentration and 15N enrichment of NH+4 in biological fluids (30). This technique also measures the 15N enrichment of L-[5-15N]glutamine after isolation and treatment with glutaminase and the average 15N enrichment of urea nitrogen after isolation and treatment with urease. The study was conducted in anesthetized dogs infused with tracer amounts of 15NH4Cl. The data to be presented show that 33% of the glutamine delivered to the portal venous drainage bed was deamidated, and this provided the liver with a large NH+4 load. A near-equal quantity of nitrogen in the form of [14N]alanine was extracted from the portal venous blood by the liver, but it was not derived from the intestinal tract as judged by the absence of an arterial-portal vein concentration difference for alanine.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. All animal experiments were approved by the Animal Care and Use Committee of Case Western Reserve University. After three orientation experiments, seven mongrel dogs (24 h fasted, 20-24 kg) were anesthetized and ventilated with 50% O2 + 0.75-1.75% halothane + 49% N2O. The temperature of the dog, monitored via a rectal thermometer, was kept at 37-37.5°C by use of heating pads irrigated with circulated warm water. Catheters were placed in the femoral artery, the superficial front paw vein, and the urinary bladder. After laparotomy, a catheter was inserted into the portal vein and positioned so that its tip extended to a point between the confluent of the pancreaticoduodenal vein and the liver hylum. The abdomen was closed with metal clips. Under X-ray control, a catheter was channeled from a femoral vein (contralateral to the femoral artery catheter) to a renal vein. At the end of the experiment, after euthanasia of the dog with intravenous pentobarbital, the abdomen was reopened and the positions of the portal and hepatic vein catheters were verified.

Protocol. To measure hepatic plasma flow, indocyanine green (0.10 mg · m-2 · min-1) was infused from -0.5 to 3 h. Dogs were infused with tracer amounts of 15NH4Cl (99%) for 3 h (0.7 µmol · min-1 · kg-1 as a 310 mM solution). Blood was collected every 30 min from the femoral artery, portal vein, and hepatic vein for assays of the concentration and enrichment of substrates (see Analytical procedures).

Analytical procedures. The hepatic plasma flow was calculated from the concentrations of indocyanine green measured spectrophotometrically at 805 nm in samples of arterial and hepatic vein plasma. To measure oxygen uptake, blood PO2, PCO2, hemoglobin, and pH were measured to calculate the O2 content in the arterial, portal venous, and hepatic venous blood (18). The techniques to measure the 15N enrichments of NH+4, glutamine-amide, and urea nitrogen were recently described (30). Briefly, to measure the 15N enrichment of plasma NH+4, chilled blood was centrifuged and the plasma was treated with AG-50-X8-Na+,K+ resin at neutral pH. After the resin was rinsed with water, NH+4 was eluted with 4 M NaCl. The eluate was treated with formaldehyde and NaOH to form hexamethylenetetramine (HMT). The latter was extracted and its 15N enrichment assayed by gas chromatography-mass spectrometry (GC-MS). The enrichment of HMT is four times that of plasma NH+4, because the HMT molecule contains 4 nitrogen atoms derived from NH+4. To measure the concentration of NH+4, a second aliquot of plasma was spiked with an internal standard of 15NH4Cl and treated as above. Measurements conducted on whole blood and plasma showed that the concentration of NH+4 in whole blood was 1.5 times that of the corresponding plasma. The 15N enrichment of NH+4 was the same in whole blood and in plasma (30).

To measure the 15N enrichment of the amide nitrogen of glutamine and the average enrichment of the two nitrogens of urea, aliquots of the water eluate of the resin were treated with glutaminase and urease, respectively. The NH+4 released by the action of these enzymes was converted to HMT, and the 15N enrichment of the latter was assayed by GC-MS. To measure the 15N enrichment of the amine nitrogen of glutamine, glutamate derived from the treatment of glutamine with glutaminase was converted to a tert-butyldimethylsilyl derivative and assayed by GC-MS.

Statistical analyses. Data are presented as means ± SE. Statistical differences were evaluated by the two-tailed t-test.


    RESULTS
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INTRODUCTION
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RESULTS
DISCUSSION
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Acid-base data. The acid-base effects of the infusion of the tracer of NH4Cl are shown in Table 1. The plasma [HCO-3] was close to 20 mM before the infusion, a normal value for this parameter in the dog (11). There was a small and not statistically significant decline in the plasma [HCO-3] to 19 mM at the end of the 3-h experimental period. There was no significant change in arterial blood lactate level.

                              
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Table 1.   Acid-base values in arterial blood

Levels of nitrogenous metabolites in arterial blood and their 15N enrichment. The concentration of NH+4 in arterial blood was 35 ± 4 µM during the tracer infusion and did not vary significantly over the 3-h time of observation (Fig. 2A, Table 2). The NH+4 infused was 15N enriched to help define the sources of nitrogen incorporated into urea. The profile for the 15N enrichment of NH+4 in the artery, hepatic vein, and portal vein blood during the tracer infusion is shown in Fig 2B. The 15N enrichment of NH+4 was virtually stable after 1 h in both of the venous sampling sites, whereas a plateau was not quite achieved in the arterial compartment.


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Fig. 2.   Plasma concentration (A) and 15N enrichment of NH+4 (molar percent enrichment, MPE, B) in femoral artery (ART), portal vein (PV), and hepatic vein (HV) of dogs infused for 3 h with tracer 15NH+4. Data are presented as means ± SE (n = 7).


                              
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Table 2.   Concentrations of NH+4, glutamine, and alanine across liver

The other major circulating nitrogenous metabolite that was enriched with 15N was glutamine. There was a progressive rise in the MPE of the amide nitrogen of glutamine in arterial blood with time during the tracer infusion (Fig 3). In contrast, there was no detectable 15N enrichment of the amine nitrogen of glutamine. The 15N enrichment of the amide nitrogen of glutamine in the hepatic vein was 70% that of the weighted enrichment of glutamine entering the liver.


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Fig. 3.   15N enrichment of glutamine amide in plasma of the same dogs as in Fig. 2.

Urinary excretion. The urinary excretion of nitrogen (urea + NH+4) was 10.5 µmol N · min-1 · kg-1 (Table 3), and this rate remained constant over the 3-h period of observation (Fig. 4). More than 96% of the nitrogen excreted was in the form of urea (Table 3). Because plasma concentrations of urea remained between 3.5 and 3.7 mM (Fig. 4), the excretion of urea is equivalent to its net production.

                              
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Table 3.   Organ balances and urinary excretion of substrates



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Fig. 4.   Profiles of plasma urea concentration () and urea excretion ().

Portal drainage system. Three sets of data provide evidence concerning the metabolic source of NH+4 added to the portal venous blood. First, NH+4 was produced because its concentration was close to 100 µM higher in portal venous than in arterial blood (Table 2). Second, the source of this NH+4 could be glutamine, because the concentration of glutamine was >= 100 µM lower in portal venous than in arterial blood (Table 2). Third, the use of 15N enrichment suggested a precursor (glutamine)-product (NH+4) relationship. In more detail, because the concentration of NH+4 was fourfold higher in the portal venous than in the arterial blood (Table 2), one would anticipate the MPE of 15NH+4 in this drainage system to fall to 25% if the source of NH+4 was an 14N precursor (e.g., urea). In fact, the MPE only fell by close to 50% in the portal vein (Fig. 2B). These data are best explained by having a major precursor of NH+4 that was predominantly 15N enriched. It is of interest that the amide nitrogen of glutamine was 15N enriched (Fig. 3) with an MPE of ~40% of that of arterial NH+4 by the end of the experimental period (compare Figs. 3 and 2B). Moreover, as mentioned above, the amine nitrogen of glutamine was not enriched with 15N.

The liver produced 10.5 µmol urea N · min-1 · kg-1, and the GI tract produced 3.4 ± 0.14 µmol NH+4 · min-1 · kg-1, largely from the amide nitrogen of glutamine (3.2 ± 0.21 µmol · min-1 · kg-1, Table 3). Hence glutamine metabolism in the GI tract could supply close to one-third of the nitrogen converted to urea in the liver, or almost two-thirds of the free NH+4 needed (i.e., one-half of the 10.5 µmol urea N · min-1 · kg-1; see Table 3). We could not identify the fate of the amine nitrogen of glutamine because there was no significant appearance of alanine (Table 2) or glutamate in portal venous blood (not shown).

The O2 uptake by the gut was 15.2 ± 4.5 µmol · min-1 · kg -1, a value that was four- to fivefold higher than the uptake of glutamine. This amount of O2 would be needed to completely oxidize glutamine (Eq. 1) if glutamine were the exclusive fuel for the entire portal drainage system.
glutamine + 5 O<SUB>2</SUB> → 5 CO<SUB>2</SUB> + 3 H<SUB>2</SUB>O + 2 NH<SUB>3</SUB> (1)

Events in the liver. The amount of NH+4 extracted by the liver was close to 4 µmol · min-1 · kg-1 (Table 3). The other major source of nitrogen extracted by the liver was alanine (3 µmol · min-1 · kg-1), most of which was derived from a systemic source because there was no significant difference in alanine concentrations between the artery and portal vein (Table 2). Thus NH+4 and alanine supplied close to 75% of the total nitrogen converted to urea. The concentration of NH+4 was very low in the hepatic vein (16 µM, Table 1), reflecting the complete extraction of the NH+4 derived from glutamine and more than one-half of the arterial NH+4 delivered to the liver (Table 2).

The 15N enrichment of arterial urea N increased linearly by 0.3%/h throughout the experiment (Fig. 5). Note that this is an average enrichment of the two N of urea, because the assay required the conversion of urea N to NH+4 by urease before the making of HMT.


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Fig. 5.   Average labeling of plasma urea nitrogen in the same dogs as in Fig. 2.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The principal findings in this study were that the major sources of NH+4 extracted by the liver of fasted dogs appeared to be the amide nitrogen of glutamine after its deamidation in the portal drainage system rather than in the liver. The other major source of nitrogen extracted by the liver was from alanine, whose ultimate source was the systemic rather than the portal drainage system. The 15N portion of the protocol strongly suggested that the 15N incorporated into urea was derived primarily from 15NH+4 produced from the amide nitrogen of glutamine and that the alanine nitrogen did not mix appreciably with the 15NH+4 pool.

NH+4 is a critically important, yet toxic, metabolic intermediate in the degradation of amino acids (Fig. 1). Therefore, its concentration in plasma and cells must be maintained at very low levels. Accordingly, the metabolism of NH+4 is difficult to monitor in vivo with tracer techniques, because 1) multiple samples are needed in view of its rapid turnover, and 2) current techniques for measuring the 15N enrichment of plasma NH+4 require fairly large volumes of blood. For these reasons, we developed a technique that is capable of measuring the concentration and a low 15N enrichment of NH+4 on smaller blood samples (30). With this technique, the metabolism of NH+4 and glutamine can be followed in organs in vivo. When combined with the traditional arteriovenous measurements of metabolites, along with an index of blood flow rate to specific organs, valuable new insights about this metabolism in vivo can be obtained.

The stoichiometry involved in hepatic NH+4 metabolism played a central role in our thinking (Fig. 1). An equal number of NH+4 and aspartate molecules must be provided within the liver for the combined synthesis of new glucose molecules plus urea, because hepatic true GNG and ureagenesis are different ways of naming the same metabolic pathway (10, 16). We reasoned that a relatively large amount of NH+4 but a small exogenous amount of aspartate would be provided to the liver in patients who eat a low-protein diet. This could create a metabolic problem if aspartate could not be made quickly enough in the liver from exogenous NH+4 plus fumarate (Fig. 1) and/or oxaloacetate formed via pyruvate carboxylase. With use of the above points as linchpins for our approach, there were unexpected findings, challenges to existing hypotheses for the control of glutamine metabolism, and a novel mechanism was suggested as a partial explanation for the protein catabolic state found in patients with end-stage renal disease. In quantitative terms, there could be a large supply of NH+4 delivered to the liver in patients with poor renal function if a large amount of urea were to enter the colon [because of the high urea concentration in body fluids and a urea transport system in the intestinal mucosa (14)], where bacterial urease would convert it to NH+4 + HCO-3 (or NH3 + CO2). After absorption, NH+4 is delivered via the portal vein to the liver. Moreover, with a portal blood flow of close to 1,000 l/day in adult human subjects and the daily hydrolysis of 100 mmol of urea (20% of production) in their colon (8, 28), the [NH+4] in the portal vein would rise by an average of close to 200 µM. This should lead to a supply of NH+4 that greatly exceeds exogenous aspartate when a low-protein diet is consumed.

Portal drainage system. There was a surprising finding at the outset in the portal drainage system, because we did not expect to find a much higher [NH+4] in the portal venous than in the arterial blood in fasted dogs unless its source was [14N]urea that was hydrolyzed in the colon by bacterial urease. Three lines of evidence suggested that the source of the extra portal vein NH+4 was the amide (15N) nitrogen of glutamine (Fig 6). First, the decline in the concentration of glutamine was similar to the rise in the [NH+4] when portal venous and arterial blood are compared (Table 2). Second, the concentration of NH+4 in the portal drainage system increased fourfold (Table 2), whereas its MPE only halved (Fig. 2B), implying that most of the NH+4 produced was derived from a pool with a 15N MPE similar to 15NH+4. This in effect suggests that the amide (or 15N nitrogen) and not the amino (or 14N nitrogen) of glutamine or the nitrogens in urea (which are mostly 14N) was the source of this 15NH+4. Third, given the stoichiometry of glutamine oxidation to CO2 + NH3, there could only be complete oxidation of this fuel if glutamine were the only fuel oxidized by the entire portal venous drainage bed (Eq. 1); this is an unlikely possibility (29).


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Fig. 6.   Compartmentation of 15N enrichment of urea precursors labeled from exogenous 15NH+4 in liver, with cytosolic compartment (left) and mitochondrial compartment (right of vertical double lines). In mitochondrial compartment, 15NH+4 is incorporated into aspartate, carbamoyl-phosphate, and then citrulline. The [15N]aspartate does not appear to equilibrate with [14N]aspartate in the cytosol. The other important mitochondrial reaction converts pyruvate to malate via pyruvate carboxylase. The exit of citrulline is linked to the entry of ornithine, and the exit of malate occurs via the dicarboxylate carrier in exchange for inorganic phosphate (Pi). Transamination reactions in cytosol generate [14N]aspartate needed for synthesis of argininosuccinate. The precursor of malate, oxaloacetate (OAA), could be formed via pyruvate metabolism or from fumarate via cytosolic steps as illustrated. alpha KG, alpha -ketoglutarate; PEP, phosphoenolpyruvate; GDH, glutamate dehydrogenase.

The site for glutamine deamidation in fasted dogs was in the portal drainage bed, and this differs from the scheme proposed by Haüssinger et al. (13). On the basis of data gathered in isolated rat livers perfused with NH4Cl or glutamine, Haüssinger inferred that the hydrolysis of glutamine in vivo occurs in periportal hepatocytes (17) because they contain high activities of glutaminase and carbamoyl phosphate synthetase (CPS). The fate of the amine nitrogen of glutamine was not established in the present study because there was no detectable release of glutamate into the portal vein, and the major transamination product, alanine (Table 3), did not appear in significant amounts in the portal venous blood. One possible explanation is that glutamate was transformed into compounds such as ornithine or citrulline in the intestinal tract. These compounds are poorly extracted by the liver and could be converted to arginine in the proximal convoluted tubule of the kidney (for a review, see Ref. 1). This allows arginine to avoid immediate hepatic hydrolysis and be delivered to other organs, where it can serve as the substrate for protein synthesis and for the production of the critically important messenger nitric oxide (24).

The deamidation of glutamine in the intestinal tract seems to be a paradox if one focuses only on NH+4 rather than glutamine (5, 6, 26). A derivative of glutamate, N-acetyl glutamate, is the physiological activator of CPS (23), the first enzymatic reaction that removes toxic NH+4 in the process of converting it into the nontoxic nitrogenous end-product, urea. Therefore, if some of this intestinally derived glutamate had reached the hepatocytes, it could have led to a high level of N-acetyl glutamate without requiring the production of glutamate by hepatocytes.

Events in the liver. The liver removed even more than all the extra NH+4 (derived from glutamine amide) delivered to it in portal venous blood in a single pass (the hepatic vein [NH+4] was significantly lower than the [NH+4] in arterial blood, Table 2). Second, only one-half of the nitrogen in newly synthesized urea appeared to be 15N enriched. Our basis for this impression is derived from the fractional synthetic rate (FSR) of urea, which was calculated in two different ways. First, we compared the output of urinary urea with the urea body pool size (calculated from plasma concentration and a volume of distribution of 2/3 of body weight). This yielded an FSR of 10-12%/h, which is similar to published values in humans, where the pool size is close to 200 mmol (5 mM × 40 liters) and the excretion of urea is 480 mmol/day or 20 mmol/h (8, 28). Second, after finding very low 15N enrichment of plasma aspartate (<0.2%), as had also been found in humans infused with tracer 15NH4Cl (25), we assumed that urea was labeled only from free 15NH+4. Accordingly, we divided the linear rate of labeling of plasma urea nitrogen (Fig. 5) by one-half the 15N enrichment of NH+4 entering the liver, and the result was an FSR of 10%. The similarity between the stoichiometric and the isotopic calculations of the FSR for urea supports the assumption made in the latter calculation. Under the conditions of tracer infusion, it was not possible to detect doubly labeled [15N2]urea by GC-MS of the intact molecule. If both urea N atoms were equally labeled at 0.9% at 3 h (Fig. 5), the molar fraction of doubly labeled urea would be <0.008%, which would not have been detected.

The fact that only one of the urea nitrogens seemed to be labeled was not anticipated, because NH+4 in the portal compartment was 15N enriched, and it is the most abundant source of the 15N in urea. Moreover, to be incorporated into urea, 15NH+4 had to enter the mitochondrial compartment and gain access to CPS (Fig. 6). Unless there was a surprising streaming, one would anticipate that this 15NH+4 would label the mitochondrial glutamate pool via glutamate dehydrogenase. Moreover, intramitochondrial aspartate should also have 15N labeling. Therefore, to explain why mitochondrial [15N]glutamate did not label aspartate used for the synthesis of urea, we favor the following scheme. The pathway of aspartate metabolism for the synthesis of argininosuccinate is located primarily in the cytosolic compartment (Fig. 6). The major source of its nitrogen was alanine (Table 3), which would undergo transamination with alpha -ketoglutarate (alpha KG) in this compartment. The glutamate so formed would not contain 15N and would undergo a second cytosolic transamination reaction, with [14N]aspartate being one of the products. There are two possible sources for cytosolic oxaloacetate: fumarate, which is a product of argininosuccinate lyase, and pyruvate via intramitochondrial pyruvate carboxylase, with the transfer of the carbon skeleton of oxaloacetate to the cytosol in the form of malate via the dicarboxylate carrier (reviewed in Ref. 3). The latter process would also transfer the reducing equivalents that are needed for GNG from amino acid-derived pyruvate to the cytosol. When cytosolic fumarate is the source of oxaloacetate, flux through GNG must be very small. This would be likely if the nitrogen to be converted to urea in the liver enters this pathway predominantly as NH+4 rather than a large mixture of amino acids, a feature of our experimental setting. One other point merits emphasis. Although GNG and urea synthesis represent a single metabolic process (Fig. 1), this process has "flexibility" depending on the metabolic conditions. For example, the component of GNG (fumarate to glucose) need not occur when there is a relatively large load of NH+4 to be detoxified and little need for glucose synthesis (the absence of hypoglycemia). Nevertheless, there is a price to pay for this metabolic benefit, the need to supply the second nitrogen for urea from an endogenous source (largely via alanine, Tables 2 and 3).

Brosnan et al. (2) recently reported the mass isotopomer distribution of urea made in rat livers perfused with 15NH4Cl. These authors found that a substantial fraction of the urea molecules were doubly labeled, and they demonstrated labeling of citrulline and aspartate. In our in vivo experiments, aspartate was very poorly 15N enriched, and urea was probably singly labeled. In humans infused with 15NH4Cl, Patterson et al. (25) also reported that urea was singly labeled and that the 15N enrichment of aspartate was much lower than enrichments of NH+4 and the amide nitrogen of glutamine. Under in vivo conditions, the nitrogen incorporated into urea via aspartate reaches the liver in the form of nonglutamine amino acids (mostly alanine), with low 15N enrichment. In contrast, in isolated livers perfused with 15NH4Cl and no amino acids, aspartate becomes more labeled from 15NH+4.

Concluding remarks. The combination of traditional chemical balances in experiments conducted in vivo, a sensitive assay for the MPE of 15NH+4, and an appreciation of the stoichiometry of nitrogenous precursors for the combined hepatic GNG/ureagenesis pathway led to the following novel interpretations. First, deamidation of glutamine in the intestinal tract was evident in fasted dogs. Although this generates potentially toxic NH+4, it could provide the precursor for an activator of NH+4 removal in the liver (N-acetylglutamate) and the precursor for the extrahepatic synthesis of arginine (ornithine + citrulline). Second, all of the NH+4 produced in the portal drainage system is cleared by the liver in a single pass (Table 2). Third, of interest to the hypothesis concerning hepatic zonation (17) that has the requirement for initial metabolism of glutamine in the liver (13), our data do not support this as a universal view because little glutamine was extracted by the liver, and the aspartate nitrogen was derived from peripheral tissues and metabolized in a compartment that did not equilibrate with 15NH+4. Overall, intestinal production of NH+4 seemed to require some net catabolism of body proteins in the fasted state.

Perspectives. Given that urea undergoes an enterohepatic cycle in which close to 20-25% of its usual production rate is converted to NH+4 and HCO-3 via GI bacterial urease (7, 8, 12, 14, 15, 21, 28), there should be a continuous supply of NH+4 for the liver via the portal vein. Two major factors may increase this delivery of NH+4. First, more urea could be made available to bacterial urease. If urea gains access to the lumen of the GI tract by facilitated diffusion via a urea transporter from the interstitial fluid compartment (14), it is possible that more urea would diffuse into the lumen when its concentration in plasma is higher if this urea transporter were not regulated. A high plasma urea concentration occurs when there is a high-protein diet and/or a low glomerular filtration rate. Second, there could be more urease in the lumen of the GI tract. This, by keeping the luminal urea concentration even lower, could increase net diffusion of urea and thereby NH+4 formation. A higher urease activity could be the result of an altered bacterial flora in the GI tract (9) or the introduction of bacteria with urease in a new location, such as H. Pylori in the stomach (20).

Two additional points are worthy of emphasis. First, if the delivery of NH+4 to the liver via urease is high and NH+4 would be toxic if it escaped into the general circulation, there must be total extraction of NH+4 in a single pass through the liver. This requirement would be difficult to achieve if the only control in the liver were a kinetic one via the [NH+4] in portal venous blood. Therefore, having an activator of CPS (N-acetylglutamate) (23) accompany the delivery of NH+4 would achieve this objective. This could be accomplished by the hydrolysis of the amido and not the amino nitrogen of glutamine in cells of the intestinal tract. It follows that a patient with both chronic renal (high blood urea nitrogen) and hepatic (e.g., viral hepatitis) disease might not be able to achieve complete, single-pass extraction of NH+4 by the liver, and this could lead to and/or aggravate the degree of encephalopathy. Second, the fact that the concentration of urea in plasma is much higher with a high-protein diet could serve a very useful nonrenal function. Given the high amount of amino acid ingested in the form of protein, one also needs a larger amount of NH+4 delivered to the liver (Fig. 1), because there is an energetic constraint in the liver that should limit hepatic formation of NH+4 via glutamate dehydrogenase (16). Moreover, this source of NH+4 should be provided either by the liver itself or via the portal drainage system, because now NH+4 would not be delivered to the brain. Therefore, it follows that a high production of NH+4 via the hydrolysis of urea in the GI tract would be a physiologically advantageous event. Moreover, spreading this supply of NH+4 out over most of the 24-h period is an additional means of having smaller fluctuations in the daily fluxes in the GNG/ureagenesis pathway, given its limitation by the rate of ATP turnover in hepatocytes (16). Cheema-Dhadli et al. (4) have provided indirect evidence to support the view that protein synthesis in the liver occurs at a rapid rate, whereas its degradation (and urea formation) occurs at a much slower, but steady rate, thereby avoiding a sudden input of urea and, as a result, an unwanted urea-induced osmotic diuresis.


    ACKNOWLEDGEMENTS

The assistance of John Koshy and Feiwen Yu is acknowledged with thanks.


    FOOTNOTES

This work was supported by grants from the National Institute of Diabetes and Digestive and Kidney Diseases (DK-35543) and the Cleveland Mt. Sinai Medical Center.

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.

1 Although the conversion of lactate to glucose is part of the Cori cycle (i.e., glucose right-arrow L-lactate right-arrow glucose) and uses some of the reactions of GNG, it does not synthesize new glucose molecules for the body. The only important nonprotein net contributors of gluconeogenic carbon are glycerol, derived from lipolysis, and muscle glycogen breakdown with release of L-lactate. The former accounts for ~10-15% of the usual daily net GNG (19).

Address for reprint requests and other correspondence: H. Brunengraber, Department of Nutrition, Mt. Sinai Medical Center, Cleveland, OH 44106-4198.

Received 18 November 1998; accepted in final form 14 October 1999.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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