(Circulation. 1999;99:975-978.)
© 1999 American Heart Association, Inc.
Correspondence |
Factors Influencing Isotope Equilibrium Rates Affect 11C PET Analysis
Departments of Radiology and Medicine
Department of Radiology Massachusetts General Hospital, Harvard Medical School, Boston, Mass
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, 
-ketoglutarate (-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 -KG and glutamate is rate limiting, owing
to -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 -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 -KG from mitochondria via
the -KG-malate transporter is 10- to 35-fold slower than cytosolic
GOT flux and is influenced by competition for -KG between the
transporter and -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 -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 -KG and glutamate.
References
1.
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.
2. Yu X, White LT, Doumen C, Damico LA, LaNoue KF, Alpert NM, Lewandowski ED. Kinetic analysis of dynamic 13C NMR spectra: metabolic flux, regulation, and compartmentation in hearts. Biophys J. 1995;69:2090–2102.[Medline] [Order article via Infotrieve]
3. 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.[Medline] [Order article via Infotrieve]
4. 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.
5.
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.
Response
Abteilung für Pathophysiologie, Zentrum für Innere Medizin, Universitätsklinikum Essen, Essen, Federal Republic of Germany
We appreciate the letter of Drs Lewandowski and Alpert addressing our article on 11C-acetate kinetics in short-term hibernating myocardium1 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 -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 -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 -ketoglutarate and glutamate might be similar to
that of the TCA cycle.2 4 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 aspartate4 5 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.1
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.
References
1. 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.
2. 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.
3. 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.
4. 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.
5. Wiesner RJ, Kreutzer U, Rösen P, Grieshaber MK. Subcellular distribution of malate-aspartate cycle intermediates during normoxia and anoxia in the heart. Biochim Biophys Acta. 1988;936:114–123.[Medline] [Order article via Infotrieve]
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