(Received for publication, May 10, 1995; and in revised form, July 25, 1995)
From the
The in vivo activity of glutamate dehydrogenase (GDH)
in the direction of reductive amination was measured in rat brain at
steady-state concentrations of brain ammonia and glutamate after
intravenous infusion of the substrate NH
. The in vivo rate
was determined from the steady-state fractional
N
enrichment of brain ammonia, measured by selective observation of
NH
protons in brain extract
by
H-
N heteronuclear multiple-quantum
coherence transfer NMR, and the rate of increase of brain
[
N]glutamate and
[2-
N]glutamine measured by
N NMR.
The in vivo GDH activity was 0.76-1.17 µmol/h/g at a
brain ammonia concentration of 0.87 ± 0.18 µmol/g, and
1.1-1.2 µmol/h/g at 1.0 ± 0.17 µmol/g. Comparison
of the observed in vivo GDH activity with the in vivo rates of glutamine synthesis and of phosphate-activated
glutaminase suggests that, under mild hyperammonemia, GDH-catalyzed de novo synthesis can provide a minimum of 19% of the
glutamate pool that is recycled from neurons to astrocytes through the
glutamate-glutamine cycle.
Glutamate dehydrogenase (GDH) ()catalyzes the
reversible reaction below.
This mitochondrial enzyme is present at a high level in rat brain, with an in vitro activity of 900 µmol/h/g(1) . GDH is believed to contribute to the synthesis of the metabolic and neurotransmitter pools of glutamate. While glutamine is also an important precursor of the neurotransmitter glutamate(2, 3, 4) , there is evidence to suggest that the glutamate-glutamine cycle is not operating in a stoichiometric manner(5) , and some de novo synthesis of glutamate from glucose is required to maintain the neurotransmitter pool(5, 6) . GDH is a likely candidate for this role, since the equilibrium of the reaction favors the formation of glutamate.
While GDH activities measured in cultured astrocytes and
synaptosomal preparations have provided useful
information(7, 8, 9) , it is also important
to measure the activity in intact brain, because the distribution of
this enzyme according to cell type is controversial. Biochemical and
histochemical studies show higher GDH levels in neurons (10, 11, 12) , while immunocytochemical
studies show highest GDH immunoreactivity in astrocytes (13) with only 15% reactivity in neurons(14) .
Moreover, a recent review of glutamate metabolism in mammalian brain (6) suggests that the rapid labeling of brain glutamate after
intravenous injection of C-labeled glucose (15, 16) and the slow labeling of brain glutamate
after short term
NH
or
NH
infusion (17, 18) give conflicting pictures of the role of GDH
in glutamate synthesis. The classical study by Berl et al. (17) on the
N labeling of brain glutamate and
glutamine in
NH
-infused cat
led to the important concept of compartmentation of brain glutamate
metabolism. A subsequent
N study in normal rat brain (18) confirmed the labeling pattern. However, these short term
(10-25 min) labeling experiments did not yield a rate for
GDH-catalyzed glutamate synthesis. To our knowledge, measurement of
steady-state in vivo GDH activity in mammalian brain has not
been reported. We report here measurement of in vivo GDH
activity in the direction of reductive amination at steady-state
concentrations of brain ammonia and glutamate in rats given
NH
infusion for 1-6 h,
using
N NMR in combination with biochemical techniques.
N NMR was previously used for noninvasive monitoring of
N-metabolites to determine in vivo glutamine
synthetase (GS) (19) and phosphate-activated glutaminase (20) activities in rat brain. In the present study, after
N enrichment in vivo, brain
[
N]glutamate and
[2-
N]glutamine were quantified in vitro for better spectral resolution. The results are discussed in
relation to the role of GDH in glutamate replenishment. An explanation
is offered for the apparent discrepancy between results
obtained with labeled glucose and ammonia on the role of GDH in
glutamate synthesis.
Figure 1:
In vivo and in vitroN NMR spectra of brain
N-metabolites in
NH
-infused rat, obtained with
proton-decoupling at 20.25 MHz for
N. The peaks are
inverted because proton-decoupling results in a negative nuclear
Overhauser effect for these
N nuclei. A, an in vivo spectrum obtained from the head of an anesthetized rat
after 3 h of
NH
infusion at
the rate of 2.3 mmol/h/kg weight. The peaks for
[5-
N]glutamine (-271 ppm) and
[2-
N]glutamate/glutamine (-342.1 ppm)
arise from the brain(29) . The peak at -306 ppm is
[
N]urea, which was synthesized in the liver and
circulating in the blood to enter various tissues in the head including
the brain. B-D, a spectrum of the perchloric acid
extract of the brain of a rat after
NH
infusion at the rate of
3.3 mmol/h/kg weight for 4.2 h (B), 2.3 mmol/h/kg weight for
3.0 ± 0.1 h (C), and
NH
infusion as in C,
followed by
NH
infusion for
2.2 h (D). The brain [
N]urea was not
detected in C, because the spectrum was acquired for a shorter
time (19,361 scans) than D (29,664 scans). In A-D, the pH was at 7.1 ± 0.1, and the
[
N]glutamate peak overlaps the
[2-
N]glutamine peak at -342.1 ppm. E, spectrum (taken at pH 9.1) of the pooled brain extract of 3
rats given
NH
infusion at the
rate of 2.3 mmol/h/g for 5.9 h. At this pH, the
[
N]glutamate peak (-343.6 ppm) is resolved
from [2-
N]glutamine peak (-345.6
ppm).
Fig. 2shows the
progressive increase in the cerebral concentration of
[2-N]Glx in rats infused with
NH
at the rates of 2.3 and
3.3 mmol/h/kg weight (Groups I and II) for
6 h. Brain
[2-
N]Glx increased linearly with time in both
groups. From the slope of the least-squares line through the plots, the
rate of increase was determined to be 0.295 µmol/h per g of brain
for Group I and 0.50 µmol/h/g for Group II.
Figure 2:
Increase in the cerebral concentration
(µmol/g) of [N]glutamate +
[2-
N]glutamine, abbreviated to
[2-
N]Glx, in rats given
NH
infusion at the rate of
2.3 (Group I,
) or 3.3 mmol/h/kg weight (Group II,
). Each
data point is the mean ± S.E. for 3-5 rats. From the
equation of the least-squares line through the plots, y = 0.295x for Group I and y =
0.50x for Group II, the rate of increase of brain
[2-
N]Glx was determined for each
group.
Another method of estimating the in
vivo GDH activity is to determine the rate of N flux
through Glx 2-N relative to that through glutamine 5-N
(catalyzed by GS) when
N is chased by
N at
steady state. This flux ratio, combined with the known in vivo GS activity in rat brain(32) , permits estimation of the
GDH activity. Fig. 1D shows an
N spectrum
of the brain extract of a rat given
NH
infusion at the rate of
2.3 mmol/h/kg for 3.1 h, followed by
NH
chase for 2.2 h.
Comparison of the peak intensity of [2-
N]Glx
(-342 ppm) with that of [5-
N]Gln
(-271 ppm) shows that the
[2-
N]/[5-
N]peak intensity
ratio changed substantially between prechase (Fig. 1C)
and postchase (Fig. 1D) period. This suggests that the
flux of
N through 2-N is much slower than that through
5-N. Table 1shows the progressive increase in
[2-
N]Glx/[5-
N]Gln
concentration ratio (determined from their observed concentrations in
brain extracts), during the chase period for Group I. The concentration
ratio changed from 0.38 ± 0.06 (n = 4) before
the chase to 1.45 ± 0.33 (n = 3) after the 3.2 h
chase. This 3.8-fold increase shows that the GDH-catalyzed flux of
N through 2-N was 3.8-fold slower than the GS-catalyzed
flux through 5-
N during the steady-state chase period. The in vivo GS activity, determined from the rate of decrease of
[5-
N]glutamine during the same chase period, was
3.3-4.4 µmol/h/g(32) . Hence, in vivo GDH
activity estimated by this method is 0.86-1.17 µmol/h/g. This
rate is in reasonable agreement with the in vivo rate of GDH
obtained by the first method for Group I. For Group II, the second
method was not attempted because of insufficient numbers of postchase
data. The in vivo rates of GDH reaction for each group,
determined as described above, are listed in Table 1.
N Enrichments of Glutamate and Glutamine
2-N-Fig. 1E shows an
N
spectrum, obtained at pH 9.1, of the brain extract of rats given
NH
infusion at the rate of
2.3 mmol/h/kg weight for 5.9 h. At this pH, the
[
N]glutamate peak (-343.6 ppm) is well
resolved from the [2-
N]glutamine peak
(-345.6 ppm) because deprotonation of -NH
to -NH
causes an 8 ppm upfield shift and
pK
values differ for glutamate (9.6) and glutamine
(8.9). From the concentrations of [
N]glutamate
and [2-
N]glutamine determined by NMR and the
total [
N+
N]glutamate and
glutamine levels measured enzymatically, the
N enrichments
of brain glutamate and glutamine 2-N in Groups I and II were calculated
and are shown in Table 1. It was previously shown that total
brain glutamine reaches steady-state levels of 8.5 ± 0.96 (Group
I) and 9.8 ± 0.86 µmol/g (Group II) after 3-4 h of
ammonia infusion(32) . It is interesting that, after 3-4
h of
NH
infusion, the
N enrichment of brain glutamate is higher than that of
glutamine 2-N.
Our results show that in vivo GDH activity in the
direction of reductive amination in rat brain is 0.76-1.17
µmol/h/g at steady-state brain ammonia level of 0.87 ± 0.18
µmol/g, and 1.1-1.2 µmol/h/g at 1.0 ± 0.17
µmol/g. The low in vivo activity compared to the reported in vitro activity measured at enzyme-saturating concentrations
of the substrates, 900 µmol/h/g (1) , is most probably the
result of the low in situ concentrations of ammonia and
2-oxoglutarate (0.23 ± 0.05 mM) (30, 31) relative to the K values
of the enzyme, 10-18 mM for NH
and 0.2-1.5 mM for
2-oxoglutarate(33, 34) .
Garfinkel (40) combined data from C-glucose labeling (15, 35, 41) and
NH
labeling experiments (17) to calculate the rates of 104 reactions involved in the
tricarboxylic acid cycle and amino acid metabolism. The rates were
adjusted to provide the best overall fit to the reported specific
isotopic enrichments of the brain metabolites, using a two-compartment
brain model. The calculated aspartate aminotransferase activity was
very high, 27-240 µmol/h/g, while GDH activity (reductive
amination) was 1.08 µmol/h/g for the large compartment and 7.8
µmol/h/g for the small compartment. The large compartment was
thought to contain 4-10 times as much of the metabolites as the
small compartment. On that assumption, GDH activity for the whole brain
is expected to be about 1.7-2.8 µmol/h/g, which is only
slightly higher than the rate reported here. These considerations,
together with the experimental result reported here, strongly suggest
that in vivo GDH activity in the direction of reductive
amination in rat brain is of the order of 1 µmol/h/g of brain.