©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Steady-state in Vivo Glutamate Dehydrogenase Activity in Rat Brain Measured by N NMR (*)

(Received for publication, May 10, 1995; and in revised form, July 25, 1995)

Keiko Kanamori(§)(¶) Brian D. Ross (¶)

From the Magnetic Resonance Spectroscopy Laboratory, Huntington Medical Research Institutes, Pasadena, California 91105

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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(4). The in vivo rate was determined from the steady-state fractional N enrichment of brain ammonia, measured by selective observation of NH(4) protons in brain extract by ^1H-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.


INTRODUCTION

Glutamate dehydrogenase (GDH) (^1)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 ^14C-labeled glucose (15, 16) and the slow labeling of brain glutamate after short term NH(4) or NH(4) 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(4)-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(4) 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.


EXPERIMENTAL PROCEDURES

Animal Preparation and Ammonia Infusion

Male Wistar rats (250-300 g) were anesthetized by the intraperitoneal injection of sodium pentobarbital (Nembutal; 40 mg/kg body weight), and prepared for ammonia infusion through the femoral vein(21) . Two infusion protocols were used to achieve different steady-state brain ammonia concentrations for measurement of in vivo GDH activities. One group of rats (Group I) was given infusion of NH(4)Cl (Cambridge Isotopes: >97% enriched in N in 1 M aqueous solution at pH 7.4) at a rate of 2.3 ± 0.04 mmol/h/kg body weight. The infusion was continued (a) for the time indicated up to 6 h, or (b) for a fixed period of 3.1 ± 0.18 h, followed by ^14NH(4)Cl infusion at the same rate for leq3.2 h (chase period). In Group II, NH(4)Cl (2 M) was infused at a rate of 3.3 ± 0.07 mmol/h/kg for leq6 h. In this group, ammonia concentration was increased to 2 M to keep the infusate volume below 1 ml/h.

NMR

In vivo and in vitroN NMR spectra were obtained on a General Electric CSI-II spectrometer operating at 20.25 MHz for N, as described previously(19, 20) . N chemical shifts are reported in ppm from nitromethane, with the negative sign indicating an upfield shift. For in vitro study, the rat was sacrificed at the time indicated after NH(4) infusion or ^14NH(4) chase. With the anesthetized rat still breathing, the cranium was opened by scissor dissection and the brain was removed in toto and rapidly frozen in liquid nitrogen for preparation of a perchloric acid extract, as described previously(21) . This procedure takes <30 s and is as fast as similar dissection-freezing methods (22, 23) that have been shown to yield brain ammonia, glutamate, and glutamine concentrations that are in good agreement with those obtained by freeze-blowing(24) . [N]glutamate, [2-N]glutamine, and [5-N]glutamine in the brain extract were identified by N NMR and quantified from the observed peak intensity (measured as integrated peak area) by comparison with those of standards, as described previously(19, 20) . The NH(4) concentration in brain extract was measured by selective observation of the NH(4) protons by ^1H-N heteronuclear multiple quantum coherence NMR at 200 MHz for ^1H as described previously(20, 25) , with the following modification. A sample volume of 3.7 ml was used to accommodate the entire brain extract (4 ml/brain). To quantify brain NH(4), a new standard curve was constructed by measuring the ^1H peak intensities for 300, 500, and 700 nmol of NH(4)Cl dissolved in 3.7 ml of unlabeled brain extract at pH 3.3. For quantification of brain NH(4) and [2-N]glutamate + glutamine, the brain extract prepared from a single rat was used in each experiment, for the number of rats indicated in Table 1. For resolution of [N]glutamate and [2-N]glutamine at pH 9.1, the brain extract for each NMR experiment was prepared from a single rat (Group II), or pooled from 2 or 3 rats that were infused at the same rate for the same duration, to increase sensitivity (Group I). In the latter case, the number of separate experiments is shown in parentheses in Table 1. The concentrations of total [^14N+N]ammonia, -glutamate, and -glutamine in the brain extracts were measured enzymatically according to published procedures(26, 27, 28) .




RESULTS

Rate of Increase of Brain [2-N]Glu+Gln

Fig. 1A shows an in vivoN NMR spectrum obtained from the head of an anesthetized rat after 3 h of NH(4) infusion at the rate of 2.3 mmol/h/kg weight. We have previously shown that the peaks for [5-N]glutamine (-271 ppm) and [2-N]glutamate/glutamine (-342.1 ppm) arise exclusively from the brain(29) . Fig. 1B shows an N NMR spectrum of the perchloric acid extract of the brain of a rat after 4.2 h of NH(4) infusion at the rate of 3.3 mmol/h/kg weight, and Fig. 1C the spectrum of an extract after 2.9 h of infusion at the rate of 2.3 mmol/h/kg. In vivo and in extracts, at pH 7, the [N]glutamate peak overlaps the [2-N]glutamine peak at -342.1 ppm. The two peaks can be well resolved at pH 9.1, as described previously(20) , and will be shown later in this work. However, for measurement of in vivo GDH activity, we need to monitor the increase, not only of brain [N]glutamate but also of [2-N]glutamine, because a substantial portion of the [N]glutamate pool is subsequently converted to [2-N]glutamine by GS. Hence, the concentration of brain [2-N]glutamate + [2-N]glutamine (abbreviated hereafter to [2-N]Glx), was determined from the observed peak intensity at -342.1 ppm at various time points during the NH(4) infusion. [N]Aspartate (-343.5 ppm), previously shown to be easily resolved from [2-N]Glx at pH 7 (20) , was not detected.


Figure 1: In vivo and in vitroN NMR spectra of brain N-metabolites in NH(4)-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(4) 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(4) 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(4) infusion as in C, followed by ^14NH(4) 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(4) 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(4) at the rates of 2.3 and 3.3 mmol/h/kg weight (Groups I and II) for leq6 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(4) infusion at the rate of 2.3 (Group I, bullet) 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.



N Enrichment of Brain Ammonia

Table 1shows the concentration and the N enrichment of brain ammonia and the concentration of brain glutamate [N+^14N] after 0.9, 2.4, and 4.3 h of NH(4) infusion for Group I. Clearly, during the observation period, the concentration and the N enrichment of the substrate ammonia and the concentration of product glutamate were at steady state. The cerebral concentration of 2-oxoglut-arate, the second substrate of the GDH reaction, is reported to be unaffected by acute or chronic hyperammonemia(30, 31) , and hence can reasonably be assumed to be at steady state under our experimental condition.

Calculation of in Vivo GDH Activity

The in vivo GDH activity in the direction of reductive amination in the brain was estimated from the observed rate of increase of [2-N]Glx (Fig. 2) and the steady-state N enrichment of brain ammonia (Table 1) to be 0.295/(0.39 ± 0.046) = 0.756 ± 0.089 µmol/h/g for Group I and 0.50/(0.43 ± 0.02) = 1.16 ± 0.054 µmol/h/g for Group II. This method of rate determination is based on the assumption that the rate of efflux of [2-N]glutamate into glutamate-utilizing pathways (other than GS) was negligible compared to the rate of glutamate synthesis during the observation period. This assumption is reasonable because the observed N enrichments of brain glutamate and glutamine are low (6-18%; Table 1). This in turn strongly suggests that the effluxing metabolites consisted mainly of endogenous [2-^14N]glutamate/glutamine. At brain glutamine levels observed in these rats, 8-10 µmol/g(32) , efflux of glutamine from the brain has been shown not to occur (23) as discussed in detail previously(19, 32) . [N]Aspartate, although below the limit of detection by N NMR, is likely to have been formed by transamination from [N]glutamate. However, the maximum quantity of brain [N]aspartate observed in NH(4)-infused rat was 15% of that of [N]glutamate(20) , and hence about 10% of [2-N]Glx, as expected from the observation that, in rat brain, the concentration of aspartate is only about 20% of that of glutamate(31) . Hence, underestimate of the GDH activity resulting from the efflux of N into aspartate, is at most 10%.

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 ^14N 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(4) infusion at the rate of 2.3 mmol/h/kg for 3.1 h, followed by ^14NH(4) 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(4) 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(3) to -NH(2) causes an 8 ppm upfield shift and pK(a) 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 [^14N+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(4) infusion, the N enrichment of brain glutamate is higher than that of glutamine 2-N.


DISCUSSION

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(m) values of the enzyme, 10-18 mM for NH(4) and 0.2-1.5 mM for 2-oxoglutarate(33, 34) .

Carbon Versus Nitrogen Labeling

It has been suggested that the rapid labeling of brain glutamate after intravenous injection of [^14C]glucose (15, 16, 35) and the slow labeling of brain glutamate after NH(4) or NH(4) administration (17, 18) lead to conflicting conclusions on the role of GDH in glutamate synthesis(6) . In reality, both carbon and nitrogen labeling experiments are correct, but use of labeled ammonia leads to measurement of GDH activity while use of labeled glucose yields the rate of 2-oxoglutarate-glutamate exchange, as shown by Mason et al. (36, 37) using in vivoC NMR. This exchange is catalyzed by transaminases, including aspartate aminotransferase, as well as by GDH. Aspartate aminotransferase is present in the brain at a much higher level than GDH and is near equilibrium(38) . Balázs and Haslam (39) showed that the rapid ^14C labeling of brain glutamate from labeled glucose mainly reflects aspartate aminotransferase-catalyzed isotopic exchange between 2-oxoglutarate and glutamate. After intravenous injection of [^14C]glucose, formation of [^14C]glutamate from [^14C]-2-oxoglutarate and [^14N]aspartate, concomitant with the conversion of unlabeled glutamate to 2-oxoglutarate, results in rapid labeling of the glutamate pool until isotopic equilibrium is reached, without net glutamate synthesis(39) . Labeling with ammonia, on the other hand, permits measurement of the rate of GDH-catalyzed glutamate synthesis from 2-oxoglutarate and ammonia.

Garfinkel (40) combined data from ^14C-glucose labeling (15, 35, 41) and NH(4) 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.

Net Glutamate Synthesis by GDH

The GDH reaction is reversible and the whole-brain glutamate level is observed to be at steady state. It is therefore important to consider whether (a) the observed GDH-catalyzed [N]glutamate synthesis is offset by oxidative deamination of [^14N]glutamate at an equal rate, resulting in no net GDH-catalyzed glutamate synthesis, or (b) there is net synthesis by GDH but glutamate is utilized by other pathways, such as GS (conversion to glutamine) and glutamate decarboxylase (conversion to GABA). The following considerations suggest that GDH-catalyzed glutamate catabolism is negligible in the brain. The equilibrium of GDH reaction favors glutamate formation, particularly in brain compartments with a low glutamate level such as astrocytes and GABAergic neurons (42) . Glutamatergic neurons have a high glutamate level, but the highest concentration occurs in the presynaptic terminal where the transmitter glutamate pool is sequestered in vesicles(43) . In neuronal perikaryon, mitochondria do not show a gradient of glutamate-like reactivity over the cytoplasm(43) . Neuronal GDH level, on the other hand, is highest in somatic zones and dendritic processes and lower in axon terminals(12) . Hence, glutamate concentration at the site of GDH in mitochondria is probably not high enough to overcome the unfavorable equilibrium. Rat brain mitochondria incubated with 10 mM glutamate produced no detectable ammonia(44) , while synaptosomal preparation incubated with [N]glutamate produced NH(4) at the rate of only 0.2 nmol/mg protein in 30 min(9) . In contrast, in intact brain, NH(4) and NH(4) label GABA and glutathione nitrogens(17, 18) . These considerations strongly suggest that the GDH-catalyzed [N]glutamate synthesis reported here reflects net synthesis, which can replenish the metabolic and neurotransmitter pools of glutamate.

Role of GDH in Glutamate Replenishment

The glutamate-glutamine cycle is not operating in a stoichiometric manner (5) , and some de novo synthesis of glutamate from glucose is needed to maintain the neurotransmitter pool(5, 6) . The relative contributions of glutamate synthesized de novo by GDH and of glutamine-derived glutamate to the pool can be estimated by comparing the observed in vivo GDH activity, 0.76-1.17 µmol/h/g, with the in vivo rate of glutamine synthesis and utilization, 3.3 ± 0.3 µmol/h/g, also measured under identical mildly hyperammonemic condition(32) , and of phosphate-activated glutaminase, 1.1 ± 0.2 µmol/h/g(20) . These in vivo rates show that glutamate is converted to glutamine in astrocytes at the rate of 3.3 ± 0.3 µmol/h/g. After migration to neurons, glutamine is reconverted to glutamate by phosphate-activated glutaminase at the rate of 1.1 ± 0.2 µmol/h/g. Amidotransferases involved in purine and pyrimidine synthesis also release glutamate from glutamine. The maximum possible rate of glutamine conversion to glutamate is the experimentally measured rate of total glutamine utilization, which, at steady state, is equal to the rate of glutamine synthesis(32) . Hence the in vivo rate of glutamine conversion to glutamate is between 1.1 ± 0.2 µmol/h/g (phosphate-activated glutaminase) and 3.3 ± 0.3 µmol/h/g (total glutamine utilization rate). Comparison with the observed in vivo GDH activity shows that GDH-catalyzed de novo synthesis can provide at least 0.76/(0.76 + 3.3) times 100% = 19% of the glutamate pool that is recycled from neurons to astrocytes through the glutamate-glutamine cycle. Thus, the role of GDH in glutamate replenishment can be significant. The source of carbon for the GDH reaction is likely to be glucose, but the rate of net conversion of 2-oxoglutarate to glutamate cannot be significantly greater than that identified here for GDH.


FOOTNOTES

*
This work was supported by Grant 1RO1 NS29048 from the National Institutes of Health and an instrumentation grant from Schulte Research Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and requests for reprints should be addressed: Magnetic Resonance Spectroscopy Laboratory, Huntington Medical Research Institutes, 660 S. Fair Oaks Ave., Pasadena, CA 91105. Tel.: 818-397-8532; Fax: 818-397-3332.

Visiting Associate in the Division of Chemistry and Chemical Engineering at California Institute of Technology.

(^1)
The abbreviations used are: GDH, glutamate dehydrogenase; GS, glutamine synthetase; GABA, -aminobutyric acid; Glx, glutamate + glutamine.


ACKNOWLEDGEMENTS

We are grateful to Emily L. Kuo for assistance with perchloric acid extraction and enzymatic assay for brain ammonia.


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