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The FASEB Journal Research Communication

Real-time assessment of Krebs cycle metabolism using


hyperpolarized 13C magnetic resonance spectroscopy
Marie A. Schroeder,* Helen J. Atherton,* Daniel R. Ball,* Mark A. Cole,*
Lisa C. Heather,* Julian L. Griffin, Kieran Clarke,* George K. Radda,* and
Damian J. Tyler*,1
*Cardiac Metabolism Research Group, Department of Physiology, Anatomy and Genetics, University
of Oxford, Oxford, UK; and Department of Biochemistry, University of Cambridge, Cambridge, UK
The Krebs cycle plays a fundamental
role in cardiac energy production and is often implicated in the energetic imbalance characteristic of heart
disease. In this study, we measured Krebs cycle flux in
real time in perfused rat hearts using hyperpolarized
magnetic resonance spectroscopy (MRS). [2-13C]Pyruvate was hyperpolarized and infused into isolated perfused hearts in both healthy and postischemic metabolic states. We followed the enzymatic conversion of
pyruvate to lactate, acetylcarnitine, citrate, and glutamate with 1 s temporal resolution. The appearance of
13
C-labeled glutamate was delayed compared with that
of other metabolites, indicating that Krebs cycle flux
can be measured directly. The production of 13Clabeled citrate and glutamate was decreased postischemia, as opposed to lactate, which was significantly
elevated. These results showed that the control and
fluxes of the Krebs cycle in heart disease can be studied
using hyperpolarized [2-13C]pyruvate.Schroeder, M. A.,
Atherton, H. J., Ball, D. R., Cole, M. A., Heather, L. C.,
Griffin, J. L., Clarke, K., Radda, G. K. Tyler, D. J.
Real-time assessment of Krebs cycle metabolism using
hyperpolarized 13C magnetic resonance spectroscopy.
FASEB J. 23, 2529 2538 (2009)
ABSTRACT

Key Words: dynamic nuclear polarization ischemia pyruvate dehydrogenase TCA cycle

The breakdown of adenosine triphosphate (ATP) is


the only immediate source of energy for the heart for
contraction, maintenance of active ion gradients, and
other vital functions. Cardiac ATP production is controlled largely by the rate at which the Krebs cycle
operates (1). The input to the Krebs cycle is the
2-carbon component of acetyl CoA, which is produced
from the oxidation of fatty acids, ketone bodies, or
glucose via glycolysis and the pyruvate dehydrogenase
(PDH) enzyme complex (2, 3). The rates of Krebs cycle
metabolism and oxidative phosphorylation are closely
coupled to the rate of contractile work and overall ATP
demand.
Many pathological states in the heart detrimentally
affect cardiac energetics by creating a mismatch between the anaerobic glycolytic pathway and oxidative
0892-6638/09/0023-2529 FASEB

metabolism via the Krebs cycle. For example, in myocardial ischemia, the glycolytic rate is maintained despite suppressed oxidative metabolism (4, 5). In the
hypertrophic heart, fatty acid oxidation is decreased,
with an increase in glycolysis (6, 7). However, increased
glucose oxidation via PDH does not compensate for
reduced fatty acid oxidation, which raises the question
as to how Krebs cycle flux and cardiac energetics can be
maintained in the state of hypertrophy (8).
The mechanisms that lead to energetic imbalances in
heart disease could be better studied by simultaneously
monitoring the source and fate of glucose-derived
acetyl CoA in the heart (glycolytic and Krebs cycle
metabolism). To date, Krebs cycle flux and substrate
selection have been measured using carbon-13 MR
spectroscopy (13C MRS) combined with isotopomer
analysis (8 10). This technique models the steady-state
incorporation of 13C label into the glutamate pool and
therefore has limited application in non-steady-state
situations (11) and in vivo studies of cardiac metabolism (12).
Metabolic imaging with hyperpolarized 13C MRS (13,
14) has enabled the measurement of normal and
abnormal metabolism in real time in several systems
(1519). In the heart, hyperpolarized [1-13C]pyruvate is
rapidly converted to [1-13C]lactate, [1-13C]alanine, and
H13CO3 (in pH-dependent equilibrium with 13CO2);
thus, glycolysis via lactate dehydrogenase (LDH) and
flux through the PDH enzyme complex can be assessed
(20, 21). However, since PDH-mediated oxidation of
hyperpolarized [1-13C]pyruvate releases the hyperpolarized 13C nucleus as 13CO2, the formation of acetyl
CoA and its incorporation into the Krebs cycle cannot
be followed.
In this study, we developed the use of hyperpolarized
[2-13C]pyruvate as a tracer to monitor Krebs cycle metabolism in the isolated perfused heart directly. Hyperpolarized [2-13C]pyruvate was infused into healthy hearts, and
the metabolic products that had sufficient MR signal to be
1
Correspondence: Cardiac Metabolism Research Group,
Department of Physiology, Anatomy and Genetics, Sherrington Bldg., University of Oxford, Parks Rd., Oxford, UK.,
OX1 3PT. E-mail: damian.tyler@dpag.ox.ac.uk
doi: 10.1096/fj.09-129171

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detected with high temporal resolution were identified.


The time courses of the formation of each of these
metabolites gave kinetic information describing the relationships among cytosolic metabolism of [2-13C]pyruvate,
PDH-mediated oxidation of [2-13C]pyruvate, and its subsequent incorporation into the Krebs cycle. In addition,
hyperpolarized [2-13C]pyruvate was infused at the moment of reperfusion into globally ischemic hearts, to
identify differences in [2-13C]pyruvate metabolism in the
reperfused myocardium and to assess the potential of
hyperpolarized [2-13C]pyruvate as a metabolic tracer to
study ischemic heart disease.
We have demonstrated that Krebs cycle metabolism
can be directly and instantaneously monitored in the
perfused heart and that new information can be obtained about the coordination of glycolysis, pyruvate
oxidation, and Krebs cycle flux in the normal and
postischemic myocardium.

sure and 37C temperature, as described previously (22, 23).


Intraventricular pressure development resulting from thebesian artery drainage was minimized by the insertion of polyethylene tubing through the apex of the heart. A small
water-filled balloon was inserted into the left ventricular cavity
via the mitral valve, attached via tubing to a pressure transducer, which allowed left ventricular contractile function and
heart rate to be measured. End diastolic pressure was set to
4 mmHg. Heart rate and left ventricular systolic and diastolic pressure were continuously recorded using a PowerLab/4SP data acquisition system (ADInstruments Ltd., Chalgrove, UK). Myocardial contractile function was assessed by
calculating the product of heart rate and left ventricular
developed pressure to give the rate pressure product
(mmHg/min).
The Krebs-Henseleit perfusion buffer contained 118 mM
NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.75 mM CaCl2, 0.5 mM
Na2EDTA, 25 mM NaHCO3, 1.2 mM KH2PO4, 11 mM glucose, and 2.5 mM pyruvate and was aerated with a mixture of
95% oxygen (O2) to 5% carbon dioxide (CO2), to give a final
pH of 7.4 at 37C.
Hyperpolarized

13

C MR spectroscopy

MATERIALS AND METHODS


The [2-13C]pyruvic acid and all unlabeled compounds were
obtained from Sigma (Gillingham, UK). The trityl radical,
OX063, was obtained from GE-Healthcare (Amersham, Little
Chalfont, UK), and the gadolinium compound 1,3,5-tris-(N(DO3A-acetamido)-N-methyl-4-amino-2-methylphenyl)-(1,3,5)triazinane-2,4,6-trione, referred to here as 3-Gd, was obtained
from Imagnia AB (Malmo, Sweden). All investigations conformed to Home Office Guidance on the Operation of the
Animals (Scientific Procedures) Act, 1986 (HMSO) and to
institutional guidelines.
Overview of the experimental protocol
[2-13C]Pyruvate was hyperpolarized in a HyperSense system
(Oxford Instruments, Abingdon, UK). Six isolated perfused
rat hearts were infused with hyperpolarized [2-13C]pyruvate
in the normal and postischemic metabolic states. Each heart
was perfused in the Langendorff mode and placed in the bore
of an 11.7 T vertical bore MR scanner. Hyperpolarized
13
C-labeled metabolic tracer was infused into the heart while
it was functioning normally and data were acquired with 1 s
temporal resolution. A 10-min period of no-flow global ischemia was then initiated. A second dose of the same 13Clabeled hyperpolarized tracer was infused immediately on
reperfusion.
Peaks arising from hyperpolarized [2-13C]pyruvate were
identified using phantom experiments, high-resolution NMR
and/or reference to the literature. Differences between metabolism in normal and ischemic hearts, as reported by
hyperpolarized [2-13C]pyruvate, were quantified, and 13C
measurements of cardiac metabolism were compared with
cardiac function. Details of the heart perfusion, hyperpolarized 13C protocol, data processing, analysis, and [2-13C]pyruvate peak assignment are described below.
Isolated perfused rat heart
Six male Wistar rats (300 g) were anesthetized using a 0.5
ml intraperitoneal injection of pentobarbital sodium (200
mg/ml euthatal). The beating hearts were quickly removed
and arrested in ice-cold Krebs-Henseleit perfusion buffer, and
the aorta was cannulated for perfusion in a recirculating
retrograde Langendorff mode at a constant 85 mmHg pres2530

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The perfused heart was placed in a 20 mm NMR sample tube


and positioned inside the bore of a 11.7-T Bruker spectrometer (500 MHz for 1H resonances; Bruker Biospin, Ettlingen,
Germany). The function of each heart was allowed to stabilize
in the bore of the magnet, while the heart was imaged to
localize it in the center of the RF coils, and a slice-selective
shim was implemented to reduce 1H linewidth to 50 Hz.
Modifications to the Langendorff mode
During image acquisition and shimming, flow rate to the
heart was measured from the recirculation line. Buffer delivery to the heart was then switched from the constant pressure
system to constant flow through a peristaltic pump (Gilson
Minipuls 3; Gilson, Middleton, WI, USA) set to the measured
flow rate. Buffer was rerouted from the oxygenation system
and 85-mmHg pressure head, via a second water-jacketed
umbilical, and delivered to the heart immediately above the
cannula. This modification to the Langendorff perfusion
setup was introduced to enable the rapid delivery of hyperpolarized buffer to the heart by limiting dead volume in the
perfusion line, with a view toward maximizing hyperpolarized signal during 13C infusions. Heart function was stabilized at constant flow prior to infusion of hyperpolarized
13
C-pyruvate.
Hyperpolarized [2-13C]pyruvate preparation
[2-13C]Pyruvate was prepared and polarized in the HyperSense 13C polarizer system with 15 mM OX063 concentration and a trace amount of 3-Gd. On dissolution, 6 ml of
effluent hyperpolarized tracer (80 mM sodium pyruvate, 0.27
mM Na2EDTA, 20 mM TRIS base buffer, 60 mM NaOH, pH
7.4, temperature 40C) was infused directly into 190 ml of
oxygenated perfusion buffer in a water-jacketed reservoir at
37C. This diluted the hyperpolarized [2-13C]pyruvate concentration to 2.5 mM pyruvate, the concentration used in the
initial KH perfusion buffer. The posthyperpolarized buffer
contained 118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.75
mM CaCl2, 0.49 mM Na2EDTA, 25 mM NaHCO3, 1.2 mM
KH2PO4, 11 mM glucose, and from the hyperpolarized infusion, 2.5 mM hyperpolarized [2-13C]pyruvate, and 0.63 mM
TRIS base. The hyperpolarized perfusion buffer was identical
to the Krebs-Henseleit perfusion buffer initially used to

The FASEB Journal

SCHROEDER ET AL.

perfuse each heart, except that the 2.5 mM pyruvate was


excluded. The primary differences between perfusion and
hyperpolarized buffer composition were the slightly decreased Na2EDTA concentration and the TRIS buffer added
to neutralize the pyruvic acid on dissolution in the HyperSense.
After a 2-s delay to allow for mixing, the constant-flow
peristaltic pump was switched to draw from the hyperpolarized [2-13C]pyruvate reservoir and begin perfusion of the
isolated heart with hyperpolarized [2-13C]pyruvate. Depending on the exact flow rate measured for each heart, which
ranged from 14 to 21 ml/min, hyperpolarized tracer reached
the heart in 1520 s.
13

C MR data acquisition

Immediately after infusion of hyperpolarized [2-13C]pyruvate, MR acquisition of 13C-pyruvate and its metabolic derivatives was initiated. Metabolites were detected with a 1-s
temporal resolution over the course of 4 min (TR1 s,
excitation flip angle30, pulse length30 s, 240 acquisitions). Over a bandwidth of 180 ppm, 4096 points were
acquired. Spectra were centered at 125 ppm.
Total global ischemia
Global ischemia was initiated in perfused hearts by shutting
off the peristaltic pump to stop buffer flow to the heart.
Hearts were maintained in this state for 10 min, at which
point hyperpolarized [2-13C]pyruvate was infused into the
buffer reservoir and the pump was switched on (at the same flow
rate previously measured) to restart buffer flow. The perfusion
line was briefly disconnected immediately above the magnet to
wash out nonhyperpolarized buffer from the dead volume, such
that each heart was reperfused with nonhyperpolarized buffer
for no more than 5 s. By this means, the hyperpolarized
pyruvate experiments enabled rapid measurement of changes
to the myocardium almost immediately on reperfusion, following no-flow ischemia.
Data analysis
Cardiac 13C MR spectra were analyzed using the AMARES
algorithm as implemented in the jMRUI software package
(24). Spectra were DC-offset corrected based on the last half
of acquired points and peaks corresponding with [2-13C]pyruvate and its metabolic derivatives (quantifiable at 1-s temporal
resolution) were fitted assuming a Gaussian line shape and
initial peak frequencies, relative phases and linewidths. Quantified peak areas were plotted against time in Microsoft Excel
(Microsoft Corporation, Redmond, WA, USA). The averaged
maximum peak area of each metabolite over the 60 s of
acquisition was determined for each series of spectra, as
described previously (21) along with the area under the
metabolic progression curve (AUC). The initial rate of signal
production for each metabolite, in arbitrary units per second
(s1), was measured as the slope of its average metabolite
progression plot over the first 5 s following metabolite
appearance when the detected signal increased linearly. The
time to appearance of signal from each metabolite was
defined as the delay between pyruvate appearance in that
particular experiment and the first time point that was 10%
of the maximum peak area. This corrected for the variation in
hyperpolarized pyruvate arrival at the heart due to the
variable flow rate at which tracer was delivered. In addition,
an exponential signal decay term was fit to each metabolite
progression plot from the point of maximum signal over the
course of signal decay.
MONITORING INSTANTANEOUS KREBS CYCLE METABOLISM

Statistical analysis
Data are reported as means se. Statistical significance
between healthy and ischemic groups was assessed using a
paired Students t test. Statistical significance was considered
at P 0.05.
Peak assignment
[2-13C]pyruvate phantom
Infusion of hyperpolarized [2-13C]pyruvate into an empty
perfusion rig was performed according to the same protocol
as infusion into the isolated perfused heart. 13C data were
acquired and processed identically for comparison to perfused heart spectra.
Metabolite extraction
One heart was perfused further for 30 min with the
Krebs-Henseleit perfusion buffer described above containing
both 80 mM [1-13C]pyruvate and [2-13C]pyruvate, after which
time the heart was immediately excised and frozen in liquid
nitrogen before being stored at 80C until analysis. Metabolites were extracted from heart tissue using methanol/
chloroform/water as described previously (25). Briefly, frozen tissue (100 mg) was placed in methanol/chloroform
(2:1, 600 l) and homogenized. Samples were then sonicated
for 5 min before chloroform/water (1:1) was added (200 l
of each). Samples were centrifuged (13,500 rpm, 20 min),
and the aqueous layer was removed and dried overnight in an
evacuated centrifuge (Eppendorf, Hamburg, Germany).
NMR spectroscopy
Dried extracts were rehydrated in 600 l of D2O and buffered
in 0.24 M sodium phosphate (pH 7.0) containing 1 mM
sodium-3-(trimethylsilyl)-2,2,3,3-tetradeuteriopropionate (TSP;
Cambridge Isotope Laboratories, Andover, MA, USA) as an
internal standard. The samples were analyzed using an Avance
II spectrometer operating at 500 MHz for the 1H frequency
(Bruker) equipped with a 5-mm broadband TXI automatic
tuning and matching (ATMA) probe. One-dimensional spectra
were collected using a solvent suppression pulse sequence based
on a 1-D nuclear Overhauser effect spectroscopy pulse sequence
to saturate the residual [1H] water proton signal (relaxation
delay2 s, t1 3 s, mixing time150 ms, solvent presaturation
applied during the relaxation time and the mixing time,
NS128, SW12 ppm, T37C). Two-dimensional heteronuclear multiple bond correlations (HMBCs; ref. 26) were also
performed to correlate 1H and 13C resonances [hmbcgplpndqf
Bruker pulse program, TD4096 (F1) and 256 (F2), NS256,
D12.5 s, SW(1H)12 ppm, SW(13C)250 ppm, AQ0.31 s).
Peak assignments were based on their 13C chemical shifts
relative to the known 1-D 1H peaks using the 2-D NMR spectrum.

RESULTS
Polarization of [2-13C]pyruvate
[2-13C]Pyruvic acid polarized similarly to the [1-13C]pyruvic
acid used previously (20). [2-13C]Pyruvate polarization
levels were slightly below the 30% polarization
reached by [1-13C]pyruvate and were estimated by
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comparison of solid-state polarization data to be 27%.


In both compounds, the rate of polarization buildup
had a time constant of 850 s.
Cardiac function throughout the protocol
Figure 1 shows cardiac function throughout the protocol, in terms of developed pressure, heart rate, and rate
pressure product. The perfused rat hearts (n6) had a
rate pressure product of 34,000 5000 mmHg/min.
During constant pressure perfusion, buffer flow to the
heart was 19 3 ml/min and was set at this rate for the
remainder of the perfusion protocol. Infusion of hyperpolarized pyruvate tended to decrease transiently developed pressure and, therefore, the rate pressure
product, without affecting heart rate. Global ischemia
decreased heart function to negligible levels within 5
min. At 45 s after reperfusion with hyperpolarized
pyruvate, the rate pressure product recovered to
10,000 4000 mmHg/min. At 5 min following reperfusion, the rate pressure product recovered to 56% of
its original value.
[2-13C]pyruvate and metabolite peak assignment
Figure 2 shows an example of stacked spectra acquired
from a healthy heart over the first 60 s of data acquisition. The signal from [2-13C]pyruvate had the highest
intensity at 207.8 ppm. Other identifiable peaks are
shown inset (averaged over 10 acquisitions) and annotated. All chemical shifts were referenced to the doublet of [2-13C]lactate (peak 7) at 71.2 ppm. Based on
previous work with [1-13C]pyruvate and literature values, peaks 4, 5, and 8 were assigned to natural abundance [1-13C]pyruvate at 172.8 ppm, [2-13C]pyruvate
hydrate at 96.5 ppm, and [2-13C]alanine at 53.3 ppm,
respectively. Two impurities in the [2-13C]pyruvate
preparation, located at 149 and 89 ppm (peak 6),
were assigned by comparison of the spectra acquired

from a healthy heart with that acquired from an


infusion into an empty perfusion rig. A very small acetyl
CoA peak was also detected at 202 ppm (only visible
when all data were summed). In addition, 3 singlet
peaks with sufficiently high SNR to be quantified in
single scans were detected at 183.7 ppm (peak 1), 181.0
ppm (peak 2), and 175.2 ppm (peak 3).
To assign these peaks, 2-D HMBCs were also performed on one additional heart, which had been
infused with [1,2-13C]pyruvate for 30 min to correlate
1
H and 13C resonances. The annotated results are
shown in Fig. 3, which confirmed that the 13C label was
taken up into the Krebs cycle, based on the coordinated
appearance of Krebs cycle intermediates in both 1H
and 13C spectra. Inspection of the expanded 1H/13C
spectral region of interest, shown inset, confirmed that
myocardial perfusion with [2-13C]pyruvate produced
[5-13C]glutamate at 183.5 ppm (peak 1), [1-13C]citrate at 181.2 ppm (peak 2) and [1-13C]acetyl carnitine at 175.0 ppm (peak 3). To further the assignment of peak 3 (Fig. 2) confirmation, previous work
regarding peak assignment following infusion of hyperpolarized [1-13C]acetate was referenced (27). When
infused in vivo, hyperpolarized [1-13C]acetate generates
a low-amplitude acetyl CoA peak and a higher-magnitude [1-13C]acetyl carnitine peak at 175 ppm.
These findings confirm the utility of hyperpolarized
[2-13C]pyruvate as a metabolic tracer to follow Krebs
cycle metabolism with high temporal resolution.
Characterization of spectra derived from
[2-13C]pyruvate
In the healthy perfused hearts examined, many peaks
derived from [2-13C]pyruvate were of sufficiently high
magnitude to be quantified with 1-s temporal resolution, which thereby allowed kinetic analyses. Figure 4
shows the metabolic progression plots of [2-13C]lactate,
[1-13C]acetyl carnitine, [1-13C]citrate, [2-13C]alanine,

Figure 1. A representative recording of cardiac


function throughout the perfusion protocol,
showing developed pressure, heart rate and
rate pressure product. Left panel: effects of
hyperpolarized pyruvate infusion during the
first 2 min of MR data acquisition. Right panel:
ischemia and 5 min of postischemic recovery.

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SCHROEDER ET AL.

Figure 2. Example stacked spectra acquired in the first 60 s following [2-13C]pyruvate infusion into a perfused rat heart.
[2-13C]Pyruvate was observed at 207.8 ppm. Peaks 1, 2, and 3 represent the metabolic products [5-13C]glutamate (183.7 ppm),
[1-13C]citrate (181.0 ppm), and [1-13C]acetylcarnitine (175.2 ppm), respectively. [1-13C]Pyruvate derived from natural
abundance 13C was seen as a quartet at 172.8 ppm (peak 4, left inset). [2-13C]Pyruvate hydrate, which is in chemical equilibrium
with pyruvate, was detected at 96.5 ppm (peak 5, right inset). Impurities in the [2-13C]pyruvic acid preparation were observed
at 149 and 89 ppm (peak 6, right inset). [2-13C]Lactate and [2-13C]alanine could also be observed (peaks 7 and 8,
respectively).

and [5-13C]glutamate; Fig. 5 details the fitted parameters for each metabolite.
The [2-13C]lactate peak was first detected 4 1 s
after pyruvate arrival and was the highest amplitude
peak (maximum peak area0.050.01, relative to maximum [2-13C]pyruvate peak area). The [1-13C]acetyl
carnitine and [1-13C]citrate peaks were first detected at
5 1 s and 4 1 s after pyruvate arrival and with
maximum normalized peak areas of 0.029 0.004 and
0.014 0.002 respectively. The [5-13C]glutamate peak
appeared significantly later than the other metabolites, at
7 1 s following pyruvate arrival (P0.001 vs. citrate
arrival) and had the lowest maximum normalized peak
area (0.0090.002).
The time constant of [2-13C]lactate MR signal decay
was calculated to be 24 1 s, whereas the decay time
constant of hyperpolarized [1-13C]acetyl carnitine was
the longest of all [2-13C]pyruvate-derived metabolites at
30 1 s. The decay time constants of [5-13C]glutamate
and [1-13C]citrate were calculated to be 17 1 and
26 5 s, respectively.
MONITORING INSTANTANEOUS KREBS CYCLE METABOLISM

Hyperpolarized [2-13C]pyruvate in the ischemic


myocardium
Figure 6 shows the averaged metabolic time courses for
[2-13C]lactate, [1-13C]acetyl carnitine, [1-13C]citrate,
and [5-13C]glutamate for all hearts in the preischemic
and postischemic states. On reperfusion with [2-13C]pyruvate, the ischemic myocardium produced significantly
more [2-13C]lactate than the healthy myocardium (area
under curve (AUC) 3.80.6 postischemia vs. 1.60.3
in the healthy heart, P0.001). Also, the decay time
constant of [2-13C]lactate MR signal decreased by 23%
in the postischemic myocardium.
No significant difference was found in the level of
[1-13C]acetyl carnitine production following ischemia
(AUC0.70.1 postischemia vs. 1.00.1 in the healthy
heart, P0.1). However, the initial rate of [1-13C]acetyl
carnitine signal production was significantly reduced
on reperfusion by 50% (P0.005). In contrast to lactate, the signal production of [1-13C]citrate and
[5-13C]glutamate were both significantly reduced fol2533

Figure 3. Annotated 2-D heteronuclear multiple bond correlations. The y axis shows 13C
chemical shifts, and the x axis shows 1H chemical shifts. Shaded regions within the plot indicate nuclei for which coupling between 1H and
13
1
C resonances had been detected. Peaks were assigned based on both H and 13C resonances. Inset: spectral region
corresponding with previously unassigned hyperpolarized 13C peaks detected in the isolated perfused heart.

lowing ischemia (AUC0.230.04 vs. 0.420.06,


P0.02 and AUC0.150.05 vs. 0.270.06, P0.01,
respectively).

DISCUSSION
This work has demonstrated the use of hyperpolarized
[2-13C]pyruvate as a metabolic tracer. Observation of
[2-13C]pyruvate metabolism in real time in the perfused heart has provided simultaneous information on
glycolysis, via [2-13C]lactate appearance; PDH flux and

energy demand, via [1-13C]acetyl carnitine; and a direct assessment of Krebs cycle metabolism. Detection of
[1-13C]citrate and [5-13C]glutamate has enabled the
first instantaneous measurements of oxidative metabolism in whole organs. The simultaneous appearance of
[1-13C]citrate, [2-13C]lactate, and [1-13C]acetyl carnitine revealed that pyruvate-derived acetyl CoA was
incorporated into the Krebs cycle within 1 s of both its
production by PDH and the cytosolic conversion of
pyruvate to lactate. All of these enzymatic conversions
occurred within 5 s of [2-13C]pyruvate arrival at the
coronary arteries.
Direct assessment of Krebs cycle flux

Figure 4. Progression of the metabolic products of [2-13C]pyruvate, for healthy perfused hearts (n6). Data were acquired with
a 1-s temporal resolution, using a 30 RF pulse.
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The detection of [5-13C]glutamate 3 s later than


[1-13C]citrate appearance has followed the oxidative metabolic fate of [2-13C]pyruvate carbon atoms across a
defined part of the Krebs cycle. Within these 3 s, [1-13C]citrate was converted reversibly to isocitrate by aconitase
and converted irreversibly to -ketoglutarate by isocitrate
dehydrogenase. This irreversible reaction would have also
generated a molecule of NADH, released a carbon nucleus as CO2 (Fig. 5), and served as a potential point of
Krebs cycle control (30). In addition, prior to [5-13C]glutamate detection, the labeled carbon must have accumulated to a detectable level in the glutamate pool, via
glutamate dehydrogenase-mediated exchange between
-ketoglutarate and glutamate. Integration of hyperpolarized MR measurements of [5-13C]glutamate production

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SCHROEDER ET AL.

Figure 5. Summary of the metabolic fate of infused [2-13C]pyruvate along with the measured parameters of the observed
metabolites.

with steady-state Krebs cycle data, acquired with thermal


equilibrium 13C MRS, may enable validated modeling of
these steps of mitochondrial metabolism with unique
levels of temporal detail.
MONITORING INSTANTANEOUS KREBS CYCLE METABOLISM

Investigation of PDH flux


These experiments have provided new insight into the
nature of both [2-13C]pyruvate and [1-13C]pyruvate
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Figure 6. Comparison of the average metabolic time courses for lactate, acetylcarnitine, citrate, and glutamate in both the
healthy and postischemic hearts. Image shows a significant elevation in the lactate signal following a 10 min ischemic period,
along with a significant reduction in the citrate and glutamate signals.

metabolism, as assessed in numerous hyperpolarized


MR studies. Previous work from this group (20, 21) and
others (15, 37) have used the conversion of hyperpolarized [1-13C]pyruvate into H13CO3 to measure flux
through the PDH enzyme complex. While this application enables assessment of an important indicator of
cardiac substrate selection, cardiac H13CO3 production should not be used as an absolute marker of
glucose oxidation, or pyruvate incorporation into the
Krebs cycle. The maximal [1-13C]acetyl carnitine MR
signal produced following [2-13C]pyruvate infusion was
2-fold greater than the [1-13C]citrate signal produced. Though MR signal production is affected by
both substrate concentration and hyperpolarized signal
decay, the high magnitude of the [1-13C]acetyl carnitine peak has qualitatively indicated that even in
healthy hearts, the majority of pyruvate-derived acetyl
CoA may not be immediately taken up into the Krebs
cycle (29).
Acetyl carnitine production is mediated by the carnitine acetyl transferase (CAT) system, which is stimulated by abundant mitochondrial acetyl CoA and carnitine (4). When available acetyl CoA exceeds the Krebs
cycle capacity for ATP production, the acetyl carnitine
pathway enables acetyl group storage for later utilization when fuel supply is reduced (28). Further, Lysiak
et al. (29) have reported that in heart mitochondria,
acetyl CoA derived from pyruvate is particularly accessible to CAT, and that a greater proportion of acetyl
carnitine is produced from pyruvate-derived acetyl CoA
than from fatty acid-derived acetyl CoA.
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Metabolism in the ischemic myocardium


Hyperpolarized [2-13C]pyruvate can also be used to
study the metabolic consequences of myocardial ischemia. Global ischemia is characterized by accumulation
of the anaerobic metabolic products lactate, NADH,
and H, as glycolysis is maintained while oxidative phosphorylation is decreased (5, 30, 31). In the postischemic
myocardium, the 2.4-fold increase in [2-13C]lactate most
likely indicates lactate accumulation, as hyperpolarized
13
C-lactate production has been demonstrated to reflect
LDH-mediated exchange of the 13C-label (16, 32). In
addition, the 23% decrease in the time constant of
[2-13C]lactate MR signal decay provided information
about lactate washout on reperfusion (33).
The reduced rate of [1-13C]acetyl carnitine production observed in postischemic hearts indicated a reduced rate of acetyl CoA production from [2-13C]pyruvate during the early reperfusion period. This
reduction may have resulted from residual PDH inhibition, due to accumulation of intracellular NADH
during ischemia (5, 30, 34). The magnitude of the
maximal [1-13C]acetyl carnitine signal was not significantly changed, but it was delayed by 10 s, such that
the peak [1-13C]acetyl carnitine MR signal was not
observed until 2530 s after [2-13C]pyruvate arrival.
Therefore, it seemed that PDH flux was normalized
within 30 s of reperfusion.
The decrease in oxidative metabolism compared with
glycolysis, characteristic of ischemia, has also been
linked to substrate depletion of the Krebs cycle inter-

The FASEB Journal

SCHROEDER ET AL.

mediates and the glutamate pool (35, 36). To promote


metabolic and functional recovery of the reversibly
ischemic heart, pyruvate may be consumed as an
anaplerotic substrate to maintain mitochondrial levels
of malate and oxaloacetate (36). Taegtmeyers findings
are consistent with our observations of Krebs cycle
metabolism in the postischemic heart. The delay in
[1-13C]acetyl carnitine production that we observed on
reperfusion may have reflected an initial diversion of
hyperpolarized [2-13C]pyruvate from PDH-mediated
oxidation to anaplerotic pathways. In addition, the
reduction of the [5-13C]glutamate and [1-13C]citrate
peak areas indicated a decline in hyperpolarized
[2-13C]pyruvate incorporation into Krebs cycle and
depletion of the glutamate pool.
General discussion
Our study has led to several conclusions on the ischemic
mismatch between glycolysis and Krebs cycle metabolism
and the utility of hyperpolarized [2-13C]pyruvate as a
metabolic tracer. However, our perfusion protocol
omitted fatty acids as a source of fuel to the heart, to
limit competition for [2-13C]pyruvate metabolism and
thus maximize MR signal from [2-13C]pyruvate-derived
metabolites. Acetyl CoA produced from -oxidation may
reduce PDH activity, thereby affecting [1-13C]acetyl carnitine production from hyperpolarized [2-13C]pyruvate
(29). Also, fatty acid metabolism contributes to Krebs
cycle recovery following ischemia (28, 37). Therefore,
these relationships should be explored in a more
physiological perfusion system and in in vivo models of
cardiac dysfunction.
Hyperpolarized [2-13C]pyruvate remains a challenging metabolic tracer to use. Only those metabolites that
accumulate to a sufficiently high concentration and
retain enough polarization can be visualized with 1-s
temporal resolution. For example, [5-13C]glutamate
and [1-13C]citrate, the most valuable metabolic products of [2-13C]pyruvate, were detected with low signal
amplitudes in this study. Future work will determine
the temporal resolution and length of data acquisition
possible to enhance the study of these metabolites.
Work will also focus on the development of a suitable
protocol for in vivo [2-13C]pyruvate experiments. The
higher cardiac workload observed in vivo, compared
with the Langendorff perfused heart, may compensate
for the increased sensitivity of the perfused heart
system, enabling [2-13C]pyruvate hyperpolarized MR
studies in living animals. Alternately, systems in which
cardiac workload is elevated, for example after infusion
of a -adrenergic agonist, may be useful for in vivo
studies with [2-13C]pyruvate.
Finally, several cardiac metabolic states exhibit elevated citrate levels, including fasting and experimental
diabetes (38), and modulation of citrate levels has also
been observed in tumor cells (39, 40). These metabolic
states may also warrant investigation with hyperpolarized [2-13C]pyruvate.
In summary, initial investigations in the isolated perMONITORING INSTANTANEOUS KREBS CYCLE METABOLISM

fused heart with hyperpolarized [2-13C]pyruvate have


supplied the first direct, whole-organ measurements of
instantaneous Krebs cycle metabolism. Simultaneous
measurement of [1-13C]citrate and [1-13C]acetyl carnitine
metabolism have provided an indication of the coupling
between glycolytic and oxidative metabolism in normal
hearts and after a period of global ischemia. Visualization
of 13C incorporation into the glutamate pool has enabled
real-time assessment of flux through a defined portion of
the Krebs cycle and may enable validation of hyperpolarized MR measurements based on thermal equilibrium 13C
MRS and isotopomer analysis. Further investigations with
[2-13C]pyruvate in a variety of physiological and pathological situations, both in the perfused heart and in vivo, may
yield a sensitive diagnostic test for impaired heart function
and enhance basic understanding of the mechanisms
regulating Krebs cycle metabolism.
The authors acknowledge previous work from scientists at
Imagnia AB (Malmo, Sweden), which aided peak assignment in
this study. We also thank Dr. Matthew Merritt and Professor
Craig Malloy for helpful discussions regarding perfused heart
techniques. M. S. gratefully acknowledges the Newton Abraham
Scholarship Foundation for her D.Phil. studentship, and NIH
grant 1-F31-EB006692-01A1. This work was funded by research
grants from the Medical Research Council (MRC grant
G0601490) and the British Heart Foundation (BHF grant PG/
07/070/23365) and through equipment support from GEHealthcare and Oxford Instruments Molecular Biotools.

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Received for publication January 8, 2009.


Accepted for publication February 19, 2009.

SCHROEDER ET AL.