Transaminase reaction rates, transport activities and TCA cycle analysis by post‐steady state ¹³C NMR

Abstract

In this work, we present the post-steady state analysis of the TCA cycle and a closed form solution to the rate of label washout from the C4 carbon of glutamic acid through the transaminases and the malate-aspartate shuttle and then through alpha-ketoglutarate dehydrogenase. We demonstrate using a model of this problem that the rate of label washout depends not only on the flux through alpha-ketoglutarate dehydrogenase, but most importantly on the activity of the malate-aspartate shuttle as determined by the forward and reverse fluxes through the transaminases and by the rate of transport of glutamate and alpha-ketoglutarate across the mitochondrial membrane.

In‐vivo Myocardial Substrate Alteration during Perfused Ventricular Fibrillation

“…6 A reduction in glutamate pool labeling was noted as workload increased. The authors postulated that the change at high workload from acetate selection to some other endogenous substrate was most likely due to an increased switch to fatty acids from triacylglycerol breakdown.…”

“…6,13 [2][3][4][5][6][7][8][9][10][11][12][13] C] labeled acetate is freely taken up across the membrane and incorporated into acetyl-CoA (2-13 C acetyl-CoA), subsequently into 4-13 C citrate, 4-13 C ␣-ketoglutarate, and then 4-13 C glutamate across the mitochondrial membrane. This transfer across the mitochondrial membrane is effected through the malate-aspartate shuttle.…”

“…Labeling of the glutamate pool is thought to be dependent not only on TCA cycle flux but also on the interconversion rate of ␣-ketoglutarate to glutamate, which involves transport across the mitochondrial membrane to the cytosol. 6 Recent evidence in the ex-vivo perfused rabbit heart suggests the rate of interconversion and transport across the mitochondrial membrane through the malateaspartate shuttle is driven by the cytosolic redox state. 15 An alternative explanation for the decrease in fractional enrichment of glutamate is a decrease in the glutamate pool size with dobutamine or perfused VF.…”

“…All NMR measurements were performed using a 4.7-tesla, 40-cm bore spectrometer (GE Omega) equipped with 26-cm actively shielded gradients as previously described. 5,6,10 During normal sinus rhythm, cardiac and respiratory motions were synchronized to minimize motion artifacts. 11 The LV pressure waveform was used for cardiac gating.…”

“…The oxidation of 13 C labeled substrates within the TCA cycle results in the labeling of the cytosolic glutamate pool, which is in equilibrium with ␣-ketoglutarate. 5,6 The glutamate pool, in turn, is readily observable with 13 C NMR methods. Monitoring of the cytosolic glutamate C4 resonance has been considered a convenient marker of labeled substrate oxidation within the TCA cycle.…”