Factors Influencing Isotope Equilibrium Rates Affect 11C PET Analysis
To the Editor:
Schulz et al1 contribute to a very important application of positron emission tomography (PET), the assessment of oxidative flux via 11C uptake. Unfortunately, the authors err in their assumption that equilibrium between the citric acid cycle, namely, α-ketoglutarate (α-KG), and glutamate is sufficiently rapid as to have no effect on glutamate-labeling rates. This assumption is invalid, as recently shown,2 3 4 5 because isotope exchange between α-KG and glutamate is rate limiting, owing to α-KG transport across the mitochondrial membrane before exchange with a large pool of glutamate that is 90% cytosolic.2 3 4 This exchange reflects reversible α-KG efflux from mitochondria,4 and in approximating citric acid cycle flux, it is sufficiently slow to influence glutamate labeling.2 Whereas the reaction catalyzed by glutamate-oxaloacetate transaminase (GOT) is much faster than citric acid cycle flux,2 efflux of α-KG from mitochondria via the α-KG-malate transporter is 10- to 35-fold slower than cytosolic GOT flux and is influenced by competition for α-KG between the transporter and α-KG dehydrogenase.2 4
In citing our work,2 Schulz et al overlook the very premise of slow transport versus rapid transamination (9 versus 223 μm · min−1 · g−1 dry weight) and the point that transport is rate determining in glutamate labeling. Contrary to their suggestion on page 1014, our report did not address effects of glucose utilization on isotope transfer. A subsequent study3 showed how high cytosolic redox state (NADH/NAD+) drives the malate-aspartate shuttle, and consequently the α-KG-malate transporter, to increase the isotope exchange rate. Data from reperfused hearts showing reduced exchange rates5 suggest that the authors account for this transport before applying inaccurate assumptions to hibernating myocardium.
Schulz et al base their assumption of rapid isotope exchange on early work that never directly tested the exchange rate but instead also made the same assumption. A particular problem with assessing 11CO2 loss is that 11C at the 5-position of glutamate must reenter the mitochondria by the same rate-limiting transport before being liberated as 11CO2.
Correlation of myocardial oxygen consumption and the rate constant (Kmono) is a useful empirical standard, but it holds no explicit relationship to the citric acid cycle. A deeper understanding from PET requires an appropriate kinetic model, incorporating known biochemistry and accounting for the kinetic relationship between α-KG and glutamate.
- Copyright © 1999 by American Heart Association
Schulz R, Kappeler C, Coenen H, Bockisch A, Heusch G. Positron emission tomography analysis of [1-11C]acetate kinetics in short-term hibernating myocardium. Circulation. 1998;97:1009–1016.
O’Donnell JM, Doumen C, LaNoue KF, White LT, Yu X, Alpert NM, Lewandowski ED. Dehydrogenase regulation of metabolite oxidation and efflux from mitochondria of intact hearts. Am J Physiol. 1998;274:H467–H476.
Lewandowski ED, Yu X, LaNoue KF, White LT, Doumen C, O’Donnell JM. Altered metabolite exchange between subcellular compartments in intact postischemic rabbit hearts. Circ Res. 1997;81:165–175.
We appreciate the letter of Drs Lewandowski and Alpert addressing our article on 11C-acetate kinetics in short-term hibernating myocardiumR1 and the opportunity to respond to it.
In our manuscript (page 1009, left column, lines 14 to 15), we stated that “the exchange rate between α-ketoglutarate and glutamate is equal to or higher than the TCA cycle rate itself.” For the data analysis, we then assumed the flux rate of α-ketoglutarate to glutamate to be faster than that along the tricarboxylic acid (TCA) cycle, not considering some more recent work that indicates that the flux rate between α-ketoglutarate and glutamate might be similar to that of the TCA cycle.R2 R4 We are convinced, however, that even the assumption of equal flux rates does not alter the conclusion of our study, for the following reasons:
1. The activity measured within the myocardium—after termination of the bolus input—relates to activity retained within TCA cycle intermediates or within compartments in exchange with the TCA cycle, such as the mitochondrial and cytosolic glutamate and aspartate concentrations. Since the absolute concentrations of glutamate and aspartateR4 R5 are substantially higher than those of TCA cycle intermediates, the measured decay of activity originated most likely from the glutamate and aspartate compartments.
2. A decreased transport/exchange rate of label into the activity-retaining cytosolic compartments during late ischemia could also result in a more rapid washout rate. The label input into the activity-retaining compartments, however, appeared to be constant during normoperfusion and hypoperfusion, because the peak count rate measured within the myocardium closely correlated to the sum of the glutamate and aspartate concentrations.
3. The efflux of label from the activity-retaining compartments will determine the observed decay of activity from the myocardium. Because the major activity-retaining compartments appear to be glutamate and aspartate, the flux rate through the TCA cycle after termination of tracer input can be simplified as the sum of the glutamate and aspartate concentrations times the washout rate kmono. Alterations in the glutamate and aspartate concentrations, at constant regional myocardial oxygen consumption and thus flux rate through the TCA cycle, will then inversely affect kmono, as we have indeed demonstrated.R1
We realize that we provided an empirical approach and certainly agree with Drs Lewandowski and Alpert’s statement that a deeper understanding of metabolic pathways from PET has to rely on known biochemistry, specific biochemical measurements, and appropriate kinetic models.
Schulz R, Kappeler C, Coenen HH, Bockisch A, Heusch G. Positron emission tomography analysis of (1-11C) acetate kinetics in short-term hibernating myocardium. Circulation. 1998;97:1009–1016.
Yu X, White LT, Alpert NM, Lewandowski ED. Subcellular metabolite transport and carbon isotope kinetics in the intramyocardial glutamate pool. Biochemistry. 1996;35:6963–6968.
O’Donnell JM, Doumen C, LaNoue KF, White LT, Yu X, Alpert NM, Lewandowski ED. Dehydrogenase regulation of metabolite oxidation and efflux from mitochondria in intact hearts. Am J Physiol. 1998;43:H467–H476.
Lewandowski ED, Yu X, LaNoue KF, White LT, Doumen C, O’Donnell JM. Altered metabolic exchange between subcellular compartments in intact postischemic rabbit hearts. Circ Res. 1997;81:165–175.