**2. Glutamine is the major source of ammonium produced in cultured cells**

Because the bulk (≈90%),[1,3-5] of the ammonium produced by cells in culture derives from glutamine's 2 nitrogen moieties (preformed DMEM media glutamate is <50uM) chemical measures of ammonium produced (after subtracting any ammonium produced in the absence of glutamine) and glutamine consumed offers an index of the GDH pathway activity. A ratio of 1, for example, would be consistent with glutamine metabolized by glutaminase with NH4+ released to the media and glutamate either released to the media or transaminated to amino acids e.g. alanine (**Figure 1, Rxn2**). In either case there is no net GDH flux and therefore no net generation of keto acids. In contrast a ratio of 2 NH4+/glutamine (**Figure 1, Rxn1**) is consistent with glutamine metabolized by glutaminase and the glutamate produced metabolized by GDH yielding NH4+ and net keto acid (anaplerosis) for the TCA cycle(running in either the normal forward or reverse direction,[ 6]). On the other hand, an NH4+ produced per glutamine consumed less than 1 (**Figure 1, Rxn3**) is consistent with glutaminase generating NH4+ from the amide nitrogen with reductive amination [3,4] catalyzed by GDH reducing the NH4+/Gln ratio to less than 1 producing glutamate and consuming net keto acid (alpha ketoglutarate); subsequent transamination yields amino acids(e.g. alanine, aspartate) consuming keto acids (cataplerosis) containing the amide nitrogen of glutamine[3,4]. Normal breast cell line exhibits an NH4+/glutamine ratio less than 1[7] whereas cancerous breast cell lines exhibit a ratio greater than 1[7] consistent with a quantitative difference in bidirectional GDH fluxes between normal (reductive amination) and tumorigenic cells (oxidative deamination). In addition, in vivo administered [2-(14)C] labeled glutamate was taken up by tumors with 14C distributed more into protein and lactate than in normal tissues [ 8 ]. In line with this early finding, a recent study[9] showed[U-(13)C] glucose contributed less than 50% of the acetyl COA pool in human brain tumors consistent with glutaminolysis and GDH's role in maintaining TCA pool homeostasis (anaplerosis).

30 Dehydrogenases

**cultured cells** 

alanine produced(**Figure1,Rxn2**).

an assessment of net GDH flux (**Figure 1,Rxn1)** as 15N NH4+ and, or, ALT flux as 15N

**Figure 1. GDH determines the fate of 15N glutamine**. Pathways of glutaminolysis, net keto acid production and NH4+ produced per glutamine consumed ratio. [1] Deamidation coupled to GDH deamination yielding 2NH4+/Gln and net keto acid (αKG). [2] Deamination coupled to ALT-mediated transamination yielding 1 NH4+/Gln and no net acid production. [3] Deamidation coupled to GDH

Because the bulk (≈90%),[1,3-5] of the ammonium produced by cells in culture derives from glutamine's 2 nitrogen moieties (preformed DMEM media glutamate is <50uM) chemical measures of ammonium produced (after subtracting any ammonium produced in the absence of glutamine) and glutamine consumed offers an index of the GDH pathway activity. A ratio of 1, for example, would be consistent with glutamine metabolized by glutaminase with NH4+ released to the media and glutamate either released to the media or transaminated to amino acids e.g. alanine (**Figure 1, Rxn2**). In either case there is no net GDH flux and therefore no net generation of keto acids. In contrast a ratio of 2 NH4+/glutamine (**Figure 1, Rxn1**) is consistent with glutamine metabolized by glutaminase and the glutamate produced metabolized by GDH yielding NH4+ and net keto acid (anaplerosis) for the TCA cycle(running in either the normal forward or reverse direction,[ 6]). On the other hand, an NH4+ produced per glutamine consumed less than 1 (**Figure 1, Rxn3**) is consistent with glutaminase generating NH4+ from the amide nitrogen with reductive amination [3,4] catalyzed by GDH reducing the NH4+/Gln ratio to less than 1 producing glutamate and consuming net keto acid (alpha ketoglutarate); subsequent transamination yields amino acids(e.g. alanine, aspartate) consuming keto acids (cataplerosis) containing the amide nitrogen of glutamine[3,4]. Normal breast cell line exhibits an NH4+/glutamine ratio less than 1[7] whereas cancerous breast cell lines exhibit a ratio greater than 1[7] consistent with a quantitative difference in bidirectional GDH fluxes between normal (reductive amination) and tumorigenic cells (oxidative deamination). In

reductive amination and transamination yielding < 1 NH4+/Gln and net keto acid

**2. Glutamine is the major source of ammonium produced in** 
