Cornelius von Morze1, Peder E Larson1, Michael A Ohliger1, Ralph E Hurd2, John Kurhanewicz1, and Daniel B Vigneron1
1Department of Radiology and Biomedical Imaging, University of California, San Francisco, San Francisco, CA, United States, 2GE Healthcare, Menlo Park, CA, United States
Synopsis
The purpose of this abstract was to investigate pool
size effects in experiments with hyperpolarized [13C]α-ketobutyrate (αKB), a molecular analog of pyruvate which also has
substantial activity with LDH. In contrast to pyruvate, formation of the
reduction product [13C]α-hydroxybutyrate (αHB) necessarily reflects net metabolic flux as opposed
to label exchange. We observed little change when co-injecting αHB but a large increase in the αHB-to-αKB ratio
when co-injecting lactate. This suggests that the observed conversion of αKB to αHB only reflects
net metabolic flux even in the presence of a large pool of reduction product.Introduction
The observed production of hyperpolarized (HP) [
13C]lactate
from [
13C]pyruvate substrate has been shown to reflect largely
exchange of the
13C label with the endogenous lactate pool, as
opposed to net metabolic flux, both in cells
1 and in vivo
2.
α-ketobutyrate (αKB) is a good alternative substrate to pyruvate for
LDH (especially for LDHB expressed subunits, but also active with LDHA), and
13C
hyperpolarization of αKB has
recently been demonstrated
3. HP αKB yields a similar spectrum as HP pyruvate after
injection, but the observed production of corresponding reduction product (α-hydroxybutyrate or αHB) by definition represents net metabolic flux as
opposed to exchange, since the endogenous pool of αHB is very small (low μm range)
4. The purpose of this study was to
investigate the effect of manipulating the pool sizes of LDH-catalyzed
reduction products (αHB and
lactate) on experiments with HP αKB, via co-injections of unlabeled material, to clarify
the nature of the metabolic conversion observed in experiments using HP αKB.
Methods
We performed 3D MRSI of HP [1-
13C]αKB with and without simultaneous co-injection of unlabeled
αHB or lactate, in a group of five rats (for a total of 15 HP experiments). For hyperpolarization, the neat acid form of [1-
13C]αKB (Sigma Isotec) was liquified by addition of a small
amount of H
2O (12% by weight) and mixed with 15mM trityl radical OX063
for polarization via DNP. Polarization of approximately 19% (time of
dissolution) was attained, with measured aqueous T
1= 73s at 3T. For data
acquisition, MRSI data (1cm isotropic 3D EPSI) was acquired in a clinical 3T
scanner at 30s after the start of a 12s injection of 2.5mL 80mM HP [1-
13C]αKB, with or without simultaneous co-injection of unlabeled
80mM αHB or lactate. The chronological order of injections in
each imaging session was alternated in an attempt to exclude effects from prior
injections, and each injection was separated by approximately one hour. The
mean αHB/αKB ratio averaged over the rat left kidney was
measured using SIVIC
5 and Osirix. Notably, substantial HP bicarbonate signal (e.g.
Fig. 1, folding into the EPSI window) was also observed in both the kidneys and the liver (more than
typically observed with HP pyruvate), presumably due to decarboxylation via PDH
or similar enzyme. αKB is
converted to propionyl-CoA following decarboxylation, feeding the TCA cycle via
succinyl-CoA. Transamination product was not detected in the localized spectra.
Results and Discussion
The appearance of HP αHB product after
injection of HP αKB only (Fig. 1B) is definitively due to net metabolic
flux via LDH, since the preexisting endogenous pool of αHB is very small.
Moreover, the HP αHB-to-αKB ratio was not much affected (change= -4.9%, p= 0.80) by co-injection of unlabeled αHB (e.g. Fig.
1C, results summarized in Fig. 2), indicating that the conversion of αKB to αHB does not
reflect label exchange even in the presence of a large reduction product pool
(i.e. not pool size limited), and further suggesting that, unlike lactate,
back-conversion of αHB to αKB is negligible. The αHB-to-αKB ratio on
the other hand increased by 74% (p=
0.057) with co-infusion of unlabeled lactate (e.g. Fig. 1D, Fig. 2). The added
lactate thus appears to exert an indirect pool size effect on the production of
αKB which is coupled via the
LDH system, as more of the supraphysiologic HP αKB (which is much more highly concentrated than either
of the unlabeled pools of oxidized substrate, pyruvate or αKB) is converted to HP αHB to balance the rapid conversion of the infused unlabeled
lactate to pyruvate as their pools equilibrate. However, according to the absence
of an effect from co-injecting unlabeled αHB, no αHB appears to be
converted back to αKB, and the
conversion of αKB to αHB still
solely reflects net metabolic flux. In this
analysis, we ignore any possible differences in transport between molecular
analogs since prior studies have indicated similar transport characteristics
6.
Another complication is the preferential activity of αKB with LDHB, although αKB also has significant activity with LDHA.
Acknowledgements
We gratefully acknowledge support from NIH K01DK099451 and P41EB013598.References
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