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
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ABSTRACT |
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
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INTRODUCTION |
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.
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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.
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METHODS |
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.
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RESULTS |
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.
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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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.
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.
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.
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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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DISCUSSION |
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. KG, -ketoglutarate; PEP,
phosphoenolpyruvate; GDH, glutamate
dehydrogenase.
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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
-ketoglutarate (
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
L-lactate
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.
 |
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