13N as a tracer for studying glutamate metabolism

Abstract

This mini-review summarizes studies my associates and I carried out that are relevant to the topic of the present volume [i.e. glutamate dehydrogenase (GDH)] using radioactive (13)N (t(1/2) 9.96 min) as a biological tracer. These studies revealed the previously unrecognized rapidity with which nitrogen is exchanged among certain metabolites in vivo. For example, our work demonstrated that (a) the t(1/2) for conversion of portal vein ammonia to urea in the rat liver is ∼10-11s, despite the need for five enzyme-catalyzed steps and two mitochondrial transport steps, (b) the residence time for ammonia in the blood of anesthetized rats is ≤7-8s, (c) the t(1/2) for incorporation of blood-borne ammonia into glutamine in the normal rat brain is <3s, and (d) equilibration between glutamate and aspartate nitrogen in rat liver is extremely rapid (seconds), a reflection of the fact that the components of the hepatic aspartate aminotransferase reaction are in thermodynamic equilibrium. Our work emphasizes the importance of the GDH reaction in rat liver as a conduit for dissimilating or assimilating ammonia as needed. In contrast, our work shows that the GDH reaction in rat brain appears to operate mostly in the direction of ammonia production (dissimilation). The importance of the GDH reaction as an endogenous source of ammonia in the brain and the relation of GDH to the brain glutamine cycle is discussed. Finally, our work integrates with the increasing use of positron emission tomography (PET) and nuclear magnetic resonance (NMR) to study brain ammonia uptake and brain glutamine, respectively, in normal individuals and in patients with liver disease or other diseases associated with hyperammonemia.

Fig. 1

Decay-corrected HPLC elution profile of ¹³N-metabolites obtained from deproteinized liver 16 s after a bolus injection of tracer quantities of [¹³N]ammonia into the portal vein of an anesthetized adult male rat. Modified from Cooper et al. (1987).

Fig. 2

Concentration (RMBC) of ¹³N activity in arterial rat blood versus time after injection. A bolus (0.3–0.4 ml) of [¹³N]ammonia solution was injected into the femoral vein of each of three anesthetized 250 g rats and blood samples were collected from a cannula in the tail artery every second for 30 s and every 5 s thereafter for the next 90 s. Using a distinct symbol for each rat, RMBC measurements are plotted against time (the midpoint of each collection interval). (A) and (B) show the measurements for the first 15 s and the first 2 min, respectively. Smoothed curves have been fitted through the data points for each rat. RMBC is “the ratio to mean body concentration” and is defined as the decay-corrected fraction of injected tracer recovered in a specimen divided by the fraction of body weight contained in that specimen. From Freed and Cooper (2005) with permission.

Fig. 3

Cerebral ammonia metabolism. Under normal conditions, ammonia enters the brain mostly by diffusion of the free base (NH₃). This ammonia, and that derived from endogenous reactions, is metabolized primarily via incorporation into the amide position of L-glutamine in a reaction catalyzed by astrocytic glutamine synthetase (reaction 1). Although the GDH reaction is freely reversible, the evidence from our ¹³N-tracer studies suggests that this enzyme is a source of ammonia (reaction 2) rather than a sink for ammonia removal. The glutamate required for glutamine synthesis is derived in part from glutamate released from neurons during neurotransmission. Some of this glutamine may be released to the circulation to maintain nitrogen homeostasis. Another portion may be returned to the neurons, wherein it is converted back to glutamate by the action of glutaminase(s) (reaction 4). The sequence GLU (neurons) → GLU (astrocytes) → GLN (astrocytes) → GLN (neurons) → GLU (neurons) is known as the brain glutamine cycle. Anaplerotic reactions occur in the brain and may be used to replenish 5-C units. Such anaplerotic reactions include CO₂ fixation by pyruvate carboxylase (→, reaction 6) and metabolism of branched-chain amino acids (→, reaction 5). BCAAs, branched-chain amino acids; ECS, extracellular space; TCA_CA, TCA cycle in the astrocytic compartment; TCA_NA, TCA cycle in the neuronal compartment. From Brusilow et al. (2010) with permission.

Scheme 1

Flow of nitrogen through coupled aminotransferases and the GDH reaction in the liver. 1, Alanine aminotransferase; 2, aspartate aminotransferase; 3, GDH; 4, urea cycle enzymes.