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1 Mary Nell and Ralph B. Rogers Magnetic Resonance Center, Department of Radiology, University of Texas Southwestern Medical Center, Dallas 75390-9085; 2 Department of Internal Medicine, University of Texas Southwestern Medical Center and Department of Veterans Affairs Medical Center, Dallas 75216; and 3 Department of Chemistry, University of Texas at Dallas, Richardson, Texas 75083-0688
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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This study was designed to test the hypothesis that indirect 1H[13C] detection of tricarboxylic acid (TCA) cycle intermediates using heteronuclear multiple quantum correlation-total correlation spectroscopy (HMQC-TOCSY) nuclear magnetic resonance (NMR) spectroscopy provides additional 13C isotopomer information that better describes the kinetic exchanges that occur between intracellular compartments than direct 13C NMR detection. NMR data were collected on extracts of rat hearts perfused at various times with combinations of [2-13C]acetate, propionate, the transaminase inhibitor aminooxyacetate, and 13C multiplet areas derived from spectra of tissue glutamate were fit to a standard kinetic model of the TCA cycle. Although the two NMR methods detect different populations of 13C isotopomers, similar values were found for TCA cycle and exchange fluxes by analyzing the two data sets. Perfusion of hearts with unlabeled propionate in addition to [2-13C]acetate resulted in an increase in the pool size of all four-carbon TCA cycle intermediates. This allowed the addition of isotopomer data from aspartate and malate in addition to the more abundant glutamate. This study illustrates that metabolic inhibitors can provide new insights into metabolic transport processes in intact tissues.
13C isotopomers; aminooxyacetate; metabolism; NMR; glutamate
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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STUDIES OF
INTERMEDIARY metabolism (7, 22) in intact tissues by
13C nuclear magnetic resonance (NMR) isotopomer analysis of
glutamate are most often made with the assumption that exchange between 
-ketoglutarate and glutamate is rapid compared with tricarboxylic acid (TCA) cycle flux. Recent NMR kinetic studies (25, 29, 30,
33) of perfused hearts have shown that the rate of the 13C appearance in glutamate in heart tissue is influenced
by an exchange process (F1 and
VX have both been used as a symbol of this flux)
likely related to transport across the inner mitochondrial membrane. We recently reported the effects of aminooxyacetate (AOA), a
widely used transaminase inhibitor (11, 28, 29), on the
rate of 13C incorporation into glutamate C4 and C3 from
[2-13C]acetate in isolated perfused rat hearts and showed
that inhibition of transaminases results in a decreased rate of
13C enrichment at both glutamate C4 and C3, with similar
rates of 13C incorporation at these two carbons
(25). These observations were consistent with a two
compartment model where a small glutamate pool exchanges more rapidly
with a metabolically active pool of -ketoglutarate (presumably
mitochondrial), followed by slower exchange of this 13C
enriched glutamate with a larger pool of unenriched glutamate (presumably cytosolic). Results from a subsequent study
(9) suggested that there may be an advantage to using
13C multiplets appearing in the glutamate spectrum over
13C enrichment data for determining TCA cycle kinetics.
In this study, we applied two-dimensional (2D) heteronuclear multiple quantum correlation-total correlation spectroscopy (HMQC-TOCSY) (15), to further examine the influence of AOA on the kinetics of 13C appearance in glutamate in rat hearts perfused with [2-13C]acetate. This method indirectly detects 13C enrichment via attached 1H but differs from direct 13C detection in that it reports different groups of 13C isotopomers (3). The 2D technique thereby offers the possibility of providing new insights into the distribution of 13C in various metabolites during isotopic enrichment of cycle intermediates. The method was validated by comparing TCA cycle kinetic constants derived from HMQC-TOCSY data with constants derived from 13C NMR isotopomer data collected on the same tissue extracts. Kinetic data from hearts perfused with 2 mM [2-13C]acetate plus 1.5 mM propionate were also evaluated. As propionate increases the level of all four-carbon TCA cycle intermediates (18, 24), 13C isotopomer data from glutamate, aspartate, and malate could then be included in the kinetic analysis.
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METHODS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Materials
[2-13C]acetate sodium salt (99%) was purchased from Cambridge Isotope Laboratories (Andover, MA). AOA (hemihydrochloride), sodium propionate, Dowex 50W resin (100-200 mesh, hydrogen form), and Dowex 1-X8 resin (100-200 mesh, chloride form) were purchased from Sigma (St. Louis, MO). All other reagents from commercially available sources were of the highest quality. Male Sprague-Dawley rats, 250-300 g, were purchased from Sasco (Houston, TX).Heart Perfusions
After general anesthesia was performed, rat hearts were rapidly excised and placed in an ice-cold perfusion medium. The aorta was immediately cannulated, and hearts were perfused using standard Langendorff methods at a column height of 70 cmH2O. A modified Krebs-Hensleit (KH) buffer containing (in mM) 119.2 NaCl, 4.7 KCl, 1.25 CaCl2, 1.2 MgSO4, and 25 NaHCO3 was bubbled with 95% O2-5% CO2. The entire all-glass perfusion system was jacketed and maintained at 37°C. Before sodium [2-13C]acetate (2 mM) or a mixture of sodium [2-13C]acetate (2 mM) plus sodium propionate (1.5 mM) was added, hearts were perfused for 30 min with 300 ml of recirculating unenriched (either 2 mM acetate or a mixture of 2 mM acetate plus 1.5 mM propionate) KH buffer (control group) or unenriched KH buffer containing 0.5 mM AOA. This period ensured equilibration of the inhibitor into all cellular compartments (13). Hearts were then perfused with sodium [2-13C]acetate ± sodium propionate for variable periods of time (3, 6, 9, 12, 15, and 45 min) before being freeze-clamped in liquid N2. Frozen tissues were extracted in 3.6% perchloric acid (PCA), neutralized with KOH, freeze-dried, and dissolved in 0.6 ml of deuterated water (2H2O) for NMR analysis.13C NMR
Proton-decoupled 13C NMR spectra of tissue extracts at pH 7.2 were acquired at 125.7 MHz on a Varian INOVA spectrometer by using a 5-mm broad-band probe. The acquisition parameters consisted of a 45° pulse and a repetition time of 3 s. Broad-band proton decoupling was achieved by using Waltz decoupling at two power levels, and the temperature was maintained at 25°C. The relative areas of the multiplet components in each glutamate resonance were determined by deconvolution using the NUTS NMR program (Acorn; Fremont, CA).HMQC-TOCSY
2D HMQC-TOCSY spectra were acquired on the same tissue extracts after adjustment of the pH to 2.75 (3) using either the 500- or 600-MHz Varian INOVA spectrometer and 5-mm internal diameter probes. The positional 13C isotopomer information was obtained from the 1H-13C off-diagonal cross peaks, as reported previously (3). Integration of the cross-peak volumes was achieved using standard peak-picking Varian NMR software (ll2d subroutine).Isolation of Glutamate
After NMR spectra were collected, glutamate was purified from carboxylic acids and neutral amino acids using sequential ion-exchange columns. A column containing 6 ml of Dowex 50W in a 10-ml disposable syringe was washed with 50 ml of 2 N HCl, followed by washing with distilled water. A tissue extract (pH 2.75) was applied to the cation column, washed with 25 ml of distilled water to remove carboxylic acids, and then washed with 30 ml of 2 M NH4OH to recover all amino acids. A second column, prepared from 2.5 ml of Dowex 1-X8 in a 0.6-cm diameter glass Pasteur pipette, was washed with 50 ml of 2 M sodium acetate, followed by distilled water. The amino acid fraction from the first column was freeze-dried and redissolved in 2 ml of distilled water, and the pH was adjusted to 8, using KOH as necessary. This sample was applied to the second column and washed with 50 ml of distilled water to remove neutral amino acids and glycerol. Glutamate and aspartate were eluted from the column using 10 ml of 0.5 M acetic acid. This acetic acid fraction was subsequently freeze-dried and dissolved in 600 µl 2H2O for 1H NMR analysis. The pH was adjusted to 2.5-3.0 to simplify the 1H spectrum of the prochiral glutamate H3 protons (3).13C Fractional Enrichments
1H NMR spectra of purified glutamate were run on a Varian INOVA 600 MHz spectrometer using a 5-mm internal diameter probe. Relative fractional enrichments were measured by deconvolution of the glutamate 1H resonances using the NUTS software.HPLC
Krebs cycle intermediates (citrate, malate, succinate, fumarate, and-ketoglutarate) were determined by HPLC using a Dionex DX500
chromatography system (Dionex; Sunnyvale, CA) containing an IonPac
AS11 analytical column, an anion self-regenerating
suppressor in autosuppression external water mode, and a CD20
conductivity detector (29). With the use of a computer,
the column eluent was generated by the mixture of appropriate volumes
of aqueous NaOH (200 mM) and 16% methanol. The initial eluant
was composed of 2% NaOH-98% methanol. After an initial 5-min
equilibration period, NaOH was increased linearly from 2 to 35% over
25 min (flow rate of 2.0 ml/min.). This provided the best separation of
TCA cycle dicarboxylic and TCA. This method did not, however, resolve
aspartate and glutamate, so these were assayed independently with the
use of a fluorometric assay (17). Chromatographic peak areas were measured using the PeakNet software provided by Dionex and
converted to quantitative values by calibration with known standards.
Modeling
Metabolite pool sizes, 13C fractional enrichments, and HMQC-TOCSY and 13C NMR multiplet data were used in a kinetic model (tcaFLUX) to evaluate TCA cycle flux (VTCA), the exchange flux (VX), anaplerosis (y), and the fractional contribution of [2-13C]acetate to acetyl-CoA (FC2). The program tcaFLUX (9) is an extension of a kinetic model based on the study by Chance et al. (4).| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Hearts Perfused with [2-13C]acetate ± AOA
Tissue levels of TCA cycle intermediates.
Table 1 summarizes analytic data for a
few TCA cycle intermediates plus glutamate and aspartate. Only
aspartate and citrate were significantly higher in hearts perfused with
AOA versus controls.
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13C NMR spectra.
[2-13C]acetate is efficiently oxidized by heart tissue
and this is reflected in isotopic scrambling of the 13C
tracer into all possible positions of TCA cycle intermediates and all
other metabolites in exchange with those intermediates (i.e., aspartate
and glutamate) (4). This 13C isotopic
scrambling is evident in the time-dependent evolution of glutamate
multiplets (4, 9, 18). For example, the glutamate C3
resonance evolves as a singlet (C3S, reflecting isotopomers enriched at
C3 but not at C2 or C4), a doublet (C3D, reflecting isotopomers
enriched at C3 and either C2 or C4), and a triplet (C3T, reflecting
isotopomers enriched at C2, C3, and C4). The C3 resonances shown in
Fig. 1 collected at two early perfusion times illustrate the sensitivity of these multiplets to partial inhibition of transaminases by AOA. The graphs on the right
illustrate the complete temporal evolution of the glutamate C3D and C3T
over 45 min after exposure to [2-13C]acetate ± AOA.
For hearts with AOA, C3D was always lower than C3T, whereas in control
hearts, C3D was greater than C3T at both 3 and 6 min. The glutamate C4
and C2 multiplets also evolve with time of perfusion and reach an
apparent isotopic steady state at 45 min (data not shown).
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HMQC-TOCSY spectra.
Figure 2 illustrates HMQC-TOCSY spectra
of extracts of hearts perfused for 6 min with 2 mM
[2-13C]acetate ± AOA. These 2D spectra show several
1H-13C cross peaks that can be attributed to
groups of glutamate 13C isotopomers that differ from those
reported by the one-dimensional 13C spectra
(3). For example, the triad of cross peaks labeled C3H4
reflect two groups of glutamate 13C isotopomers, those
enriched in both C3 and C4 (given by the outer C3H4D cross peaks) and
those enriched in C3 but not C4 (given by the central C3H4S cross
peak). The graphs above each HMQC-TOCSY spectrum report the C3H4D and
C3H2D cross-peak volumes as a function of perfusion time. As
illustrated for only two HMQC-TOCSY multiplets, these spectra are also
sensitive to the presence or absence of AOA. Similar graphs were
obtained for all other HMQC-TOCSY cross-peak multiplet volumes.
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1H NMR spectra of isolated glutamate.
Figure 3 shows expanded regions of the
1H NMR spectra of glutamate isolated from hearts perfused
with sodium [2-13C]acetate ± AOA for 6 min.
Clearly, hearts perfused without AOA achieve a much higher level of
13C enrichment than do hearts perfused with the inhibitor
at this early time point. The graphs above the two spectra in Fig. 3
show the 13C-fractional enrichments of glutamate C3 and C4
as a function of perfusion time. Both C3 and C4 reach steady-state
13C fractional enrichment at ~30 min in control hearts
but these values appear to be only approaching steady state at 45 min
in hearts perfused with AOA. Nevertheless, glutamate reaches the same
level of 13C enrichment at isotopic steady state in both
experiments (±AOA) as evidenced by the identical FC2
values (see Tables 2 and 3).
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Kinetic modeling using tcaFLUX. Table 2 reports values for VTCA, VX, y, and the contribution [2-13C]acetate to acetyl-CoA (FC2) for control and AOA perfused hearts obtained by fitting the 13C NMR multiplet data to tcaFLUX. Values in parentheses are 5-95% confidence limits determined for each parameter. VTCA was significantly higher (~1.5-2-fold), whereas VX was dramatically lower (~10-fold), in hearts perfused with AOA compared with controls. FC2 and y were essentially unaffected by the inhibitor. These values are consistent with previous reports (25, 29).
Table 3 summarizes flux values and other parameters determined by fitting the HMQC-TOCSY cross-peak multiplet data to the same kinetic model. There was excellent agreement between all parameters determined by the two NMR methods (none of the parameters of Table 3 differ significantly from the corresponding parameter of Table 2). Again, VTCA tended to be higher (although the confidence intervals do overlap), whereas VX was lower, in hearts perfused with AOA. This comparison shows that HMQC-TOCSY data, although reflecting different groups of isotopomers than 13C NMR, is sensitive to TCA cycle and related pathway kinetics. The solid lines drawn through the data of Figs. 1-3 represent the best fit of all data, 13C NMR multiplets, HMQC-TOCSY cross-peak volumes, and 13C fractional enrichments to the kinetic model. In general, the agreement is quite good for all of the data shown; the largest deviation was found for the limiting fractional 13C enrichments in control hearts as determined by 1H NMR (Fig. 3).Hearts Perfused With [2-13C]acetate and Propionate
Tissue levels of TCA cycle intermediates.
Table 4 summarizes analytic data for
selected TCA cycle intermediates, glutamate, and aspartate for hearts
perfused with a combination of acetate and propionate. Total tissue
glutamate was lower by ~3-fold, aspartate was ~10-fold higher,
while malate was ~120-fold higher, for hearts perfused with a
combination of acetate plus propionate compared with acetate alone
(24). The sum of all tissue metabolites shown was ~33%
higher in hearts perfused with acetate plus propionate compared with
acetate alone. These changes reflect an altered metabolic steady state
resulting from influx of propionate into the TCA cycle pools via
succinyl-CoA and matched by an equivalent disposal flux of a
four-carbon intermediate, likely malate to pyruvate [flux of this
pyruvate into acetyl-CoA and the TCA cycle, previously given the symbol
yox (in Ref. 10), is therefore
assumed equal to y].
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HMQC-TOCSY spectra.
Figure 4 shows selected regions of
HMQC-TOCSY spectra of extracts of hearts perfused for 6 min with 2.0 mM
[2-13C]acetate plus 1.5 mM propionate ± 0.5 mM AOA.
These regions illustrate that isotopomer data can be derived for at
least two four-carbon intermediates associated with the TCA cycle
(malate and aspartate) in addition to glutamate. The triad cross peaks
of aspartate C3H2 and malate C3H2 show that both four-carbon
intermediates at the 6-min time-point are made up of isotopomers with
13C enrichment at both C3 and C2 (the doublets) and
isotopomers with 13C enrichment at C3 but not C2 (the
singlets). Clearly, the doublet component in each cross peak is higher
in hearts perfused with AOA than without AOA. Also, the doublet
components of aspartate C3H2 and malate C3H2 were equal in spectra
collected at 6 min in control hearts, but the malate C3H2 doublet was
higher than the aspartate C3H2 doublet at this same time-point in
hearts with AOA. This illustrates that AOA has differential effects on
13C enrichment of the four-carbon TCA cycle intermediate,
malate, compared with the four-carbon intermediate that is in exchange with cycle intermediates, aspartate. The graphs illustrate how these
and other HMQC-TOCSY cross-peak volumes change as a function of
perfusion time.
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Kinetic modeling.
The HMQC-TOCSY data derived from hearts perfused with acetate plus
propionate were fit to the same kinetic model as described above for
hearts perfused with acetate alone. Because propionate is essentially
completely oxidized via pyruvate and acetyl-CoA in heart tissue
(10, 24), it was assumed that y = yox, i.e., flux of propionate through the TCA
cycle and into acetyl-CoA. Table 5
summarizes the best-fit parameters and their 5-95% confidence limits. As anticipated, VTCA was ~2-fold lower
and y was ~10-fold higher in hearts perfused with acetate
plus propionate compared with acetate alone (compare Tables 3 and 5).
Furthermore, the contribution of acetate to acetyl-CoA was considerably
reduced in the presence of propionate (from 92 to 57%).
VX was comparable to VTCA
in this group, although the uncertainty in VX
was noticeably higher. AOA had similar metabolic effects in hearts
perfused with the acetate-propionate substrate combination compared
with acetate alone; VTCA increased in response
to AOA (almost twofold), VX was again lower (but
less so that without propionate), anaplerosis was stimulated by AOA,
whereas the contribution made by [2-13C]acetate to
acetyl-CoA (the FC2 parameter) was unaffected by AOA.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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This study demonstrates that 13C isotopomer data
derived from multiplet areas in 13C NMR spectra or from
multiplet volumes in HMQC-TOSCY spectra of heart extracts exposed to
13C-enriched substrates for variable time periods can be
used as input to kinetic models of the TCA cycle. In hearts perfused
with 2 mM [2-13C]acetate alone,
VTCA was 11.7 µmol · min
1 · g dry wt1
(average of 2 values), whereas VX was 15.6 µmol · min1 · g dry wt1
(average of 2 values) as estimated using either NMR data set. The value
of VTCA determined here was similar to values
reported previously for hearts perfused with acetate [7.5
(25); 10.1 (33); 10.67 (31)]
but VX determined here appears to be somewhat higher than the corresponding flux reported previously [7.5
(25); 9.3 (33); 10.18 (31)].
The transaminase inhibitor AOA had multiple complex effects on the
kinetic data. First, the multiplets arising from multiply labeled
isotopomers (see, for example, C3T in Fig. 1 and C3H2D in Fig. 2)
appeared more rapidly with time in both NMR data sets. This is
indicative of more rapid turnover of a smaller pool of tissue
metabolites in the presence of AOA, consistent with rapid 
KG
Glu
exchange within the mitochondrial matrix, followed by slower exchange
of both metabolites with their larger cytosolic components
(25). As anticipated, VX was
substantially lower in the presence of AOA, decreasing ~10-fold from
15.6 to 1.74 µmol · min1 · g dry
wt1. As discussed below,
VX determined in the presence of AOA likely reflects a rate- limiting transaminase flux and not a metabolite exchange flux. Again, both NMR data sets reported similar values for
VX. The decrease in VX
brought about by 0.5 mM AOA in these experiments (10-fold) was much
larger than that found by Weiss et al. (29) using only 0.1 mM AOA (twofold).
One unanticipated result was an increase in VTCA
found for all hearts perfused with AOA. A fit of the 13C
NMR data set indicated that VTCA increased from
10.9 to 19.4 µmol · min1 · g dry
wt1, whereas a fit of the 2D data set predicted an
increase from 12.5 to 16.3 µmol · min1 · g dry wt1.
This 30-70% increase in VTCA brought about
by partial inhibition of transaminases likely reflects a redistribution
of metabolites between the cytosol and mitochondrial. We have shown
previously that tissue glutamate is not fully visible by NMR in
isolated hearts exposed to AOA and ascribed this to a net shift in
glutamate from the cytosol to the mitochondrial matrix
(25). This could also occur with other components of the
malate-aspartate shuttle and thereby give the cell less ability to
transport reducing equivalents from the cytosol to the mitochondria.
Thus the increase in VTCA brought about by
partial inhibition of transaminases indicates that the contribution of
cytosolic reduced nicotinamide adenine dinucleotide to overall energy
production is lower in hearts perfused with acetate plus AOA. We
demonstrated previously that AOA does not alter O2
consumption by heart tissue (25), so any decrease in
contribution of cytosolic reducing equivalents would require a higher
flux through the oxidative TCA cycle.
Hearts perfused with [2-13C]acetate plus propionate
showed substantial kinetic differences compared with those perfused
with [2-13C]acetate alone. We have shown in previous
reports that propionate is efficiently oxidized in heart tissue via the
TCA cycle, pyruvate, and subsequently acetyl-CoA (10, 24).
Thus, given that additional FADH2 is produced by propionate
at the level of succinate dehydrogenase plus two additional reducing
equivalents are formed at the level of the malic enzyme and pyruvate
dehydrogenase (PDH), one would anticipate that
VTCA would be significantly reduced in hearts perfused with propionate. Clearly, VTCA
was significantly reduced when propionate was present, compared with
acetate alone (5.3 vs. 11.7 µmol · min1 · g dry wt1)
because of the three extra reducing equivalents produced along this
oxidative pathway (succinyl-CoA 
malate pyruvate acetyl-CoA). It has been shown (S. C. Burgess, E. Babcock, A. D. Sherry, and C. R. Malloy, unpublished observations, 32, 33)
that perfusion of hearts with any substrate that contributes non-TCA
cycle-reducing equivalents (i.e., butyrate, lactate, or octanoate)
results in a decrease in VTCA relative to hearts
perfused with acetate alone. Given that y was ~35% of
VTCA in hearts perfused with acetate plus
propionate, one would predict that propionate (the only anaplerotic substrate present) would contribute one (0.35 × 3 = 1.05)
extra reducing equivalent for each turn of the TCA cycle (4 reducing equivalents). Given this stoichiometry and the assumption that O2 consumption is not altered by propionate,
VTCA should be ~25% lower in the presence of
propionate. The fact that we observed an ~50% decrease in
VTCA indicates that propionate has more complex effects on metabolism. Latipää et al. (14)
have shown that propionate fully activates the pyruvate dehydrogenase
complex via phosphorylation yet flux through PDH is reduced in the
presence of propionate due to the combined effects of low-tissue CoA
and partial inhibition of PDH by the high tissue levels of
propionyl-CoA and methylmalonyl-CoA. This suggests that hearts may turn
toward alternative, nonglycolytic sources of acetyl-CoA in the presence of propionate, consistent with a lowering of the contribution of
[2-13C]acetate to acetyl-CoA (the
FC2 parameter) observed here. A lowering of free
CoA by propionate could also directly alter VTCA
by lowering flux through -ketoglutarate dehydrogenase, an enzyme
that has a strict requirement for free CoA.
Interestingly, VX was also substantially lower
in the presence of propionate (6.3 vs. 15.7 µmol · min1 · g dry wt1
for acetate alone). Although one might anticipate that the "extra" reducing equivalents produced by oxidation of propionate would stimulate the malate-aspartate shuttle and hence increase
VX as suggested before for lactate
(32), the exact opposite was observed. This indicates
again that propionate has multiple contrasting effects on metabolism.
Some of the enzymes required for complete oxidation of propionate are
mitochondrial in origin (succinyl-CoA malate) so propionate
directly contributes mitochondrial-reducing equivalents without
invoking the malate-aspartate shuttle. It is perhaps then not
surprising that less shuttle activity is required for propionate
oxidation and, hence, VX decreases. The
metabolic consequences of propionate are complex; it reduces the TCA
cycle energy requirement by directly producing mitochondrial-reducing equivalents yet, at the same time, it reduces the level of free CoA
available to mitochondrial enzymes. The net metabolic result is a
reduction in both VTCA and
VX. We have observed (unpublished observations)
that hearts do not perform well when perfused with propionate as their
sole substrate for an extended period of time (~2 h), consistent with
limited energy production by mitochondrial reactions.
The addition of AOA to hearts perfused with acetate plus propionate had
predictable effects on both VTCA and
VX. VX was further decreased in the presence of AOA and, interestingly, to nearly the same
level as in hearts perfused with acetate alone (compare 1.5 vs. 2.3 µmol · min1 · g dry wt1
for acetate vs. acetate plus propionate, respectively). This is
consistent with VX reflecting a different
rate-limiting step in the presence of AOA than in its absence. The fact
that VX dropped to the same level on addition of
AOA to acetate versus acetate plus propionate hearts is consistent with
a rate limited by transaminase flux. Interestingly, AOA partially
reverses the deleterious effect propionate has on
VTCA, exactly as expected for a metabolic system that may be limited by levels of mitochondrial -KG. This again is
consistent with a net shift in intermediates from the cytosol to the
mitochondria in the presence of AOA, thereby stimulating VTCA and energy production.
What additional kinetic information, if any, is provided by the
malate and aspartate isotopomers detected in HMQC-TOCSY spectra (Fig.
4) of hearts perfused with acetate plus propionate compared with
glutamate isotopomers alone? Table 6
compares the spread in 5-95% confidence limits for
VTCA and VX that one
finds by fitting glutamate isotopomer data alone, glutamate plus
aspartate isotopomer data, glutamate plus malate isotopomer data, and
isotopomer data from all three metabolites combined. The time-dependent
appearance of aspartate isotopomers basically parallels that seen in
glutamate because both molecules are enriched via transamination of TCA cycle intermediates. The data in Table 6 show that inclusion of both
glutamate and aspartate data does little to improve the confidence
limits for VTCA but does improve slightly the
confidence limits for VX. A similar improvement
in the confidence limits for VTCA and
VX was observed by comparing the fits of
glutamate data alone versus the glutamate plus malate data in control
hearts. In AOA-perfused hearts, however, inclusion of the malate data makes a marked improvement on the confidence limits for
VX.
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One would predict a priori that malate would become enriched somewhat faster in the presence of AOA because the size of the exchanging aspartate and glutamate pools would be effectively smaller during isotopic turnover. Plots of the temporal appearance of malate and aspartate C3H2D are compared in Fig. 4. The solid lines through the data reflect the isotopomer populations predicted by the kinetic model described in Table 5. Here, one sees that the malate and aspartate C3H2D volumes essentially track one another in control hearts although enrichment of aspartate requires an additional transaminase step. In the presence of AOA, both malate and aspartate become enriched more quickly, as predicted for smaller exchanging pool sizes, but the malate also clearly approaches steady-state enrichment more rapidly than aspartate. Here, the kinetic model reproduces the aspartate and glutamate data reasonably well but rather poorly predicts the isotopomer distribution in malate. This indicates that VX reported by the kinetic analysis of these hearts reflects transaminase flux, whereas a fit of the malate data alone would give a different VX value reflecting instead a transport step. All of our observations are consistent with the hypothesis offered by Yu et al. (33) that VX in control hearts reflects a transport process involving exchange of a metabolite labeled in the TCA cycle with a larger counterpart in the cytosol. In hearts perfused with AOA, VX is apparently limited by flux through the transaminases rather than a transport flux so a separate fit of malate isotopomer data may allow the determination of both the transaminase and transport fluxes.
This study has shown that equivalent kinetic data is derived from 13C isotopomer data obtained by either direct 13C observe or indirect HMQC-TOCSY NMR spectroscopy. Thus the possible advantages offered by the greater sensitivity of the 2D method were not realized in this study because the amount of tissue available for analysis was not limiting. However, the HMQC-TOCSY method could prove important when analyzing kinetic data in situations where tissue samples may be limiting (i.e., tissue biopsies or perfused mouse hearts) or if a high-field NMR spectrometer is not available. The reported ~5-fold increased sensitivity of the method (3) does offer the possibility of detecting more metabolites than direct 13C observe spectroscopy and such data should provide added value in fitting 13C isotopomer data to ever increasingly more complex kinetic models.
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ACKNOWLEDGEMENTS |
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This work was supported in part by National Institutes of Health Grants HL-34557 and RR-02584. R. A. Carvalho was supported by Portuguese Foundation for Science and Technology (PRAXIS XXI) Grant BD-3604/94.
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FOOTNOTES |
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Present address of R. A. Carvalho: Department of Biochemistry, University of Coimbra, 3000 Coimbra, Portugal.
Address for reprint requests and other correspondence: A. D. Sherry, Mary Nell and Ralph B. Rogers Magnetic Resonance Center, 5801 Forest Park Rd., Dallas, TX 75390-9085 (E-mail: dean.sherry{at}utsouthwestern.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 20 March 2001; accepted in final form 13 May 2001.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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1.
Bhattacharya, M,
Fuhrman L,
Ingram A,
Nickerson KW,
and
Conway T.
Single-run separations and detection of multiple metabolic intermediates by anion-exchange high-performance liquid chromatography and application to cell pool extracts prepared from Escherichia coli.
Anal Biochem
232:
98-106,
1995[Web of Science][Medline].
3.
Carvalho, RA,
Jeffrey FMH,
Sherry AD,
and
Malloy CR.
13C isotopomer analysis of glutamate by heteronuclear multiple quantum coherence-total correlation spectroscopy (HMQC-TOCSY).
FEBS Lett
440:
382-386,
1998[Web of Science][Medline].
4.
Chance, EM,
Seeholzer SH,
Kobayashi K,
and
Williamson JR.
Mathematical analysis of isotope labeling in the citric acid cycle with applications to 13C NMR studies in perfused rat hearts.
J Biol Chem
258:
13785-13794,
1983
5.
Chatham, JC,
Forder JR,
Glickson JD,
and
Chance EM.
Calculation of absolute metabolic flux and the elucidation of the pathways of glutamate labeling in perfused rat heart by 13C NMR spectroscopy and nonlinear least square analysis.
J Biol Chem
270:
7999-8008,
1995
6.
Chatham JC and Chance EM. Biological magnetic resonance. In:
In Vivo Carbon-13 NMR, edited by Berliner LJ and
Robitaille P-M. New York: Kluwer-Plenum, ch. 3, vol. 15, p.
99-116, 1998.
7.
Fitzpatrick, SM,
Heterington HP,
Behar KL,
and
Shulman RG.
The flux from glucose to glutamate in the rat brain in vivo as determined by 1H-observed, 13C-edited NMR spectroscopy.
J Cereb Blood Flow Metab
10:
170-179,
1990[Web of Science][Medline].
8.
Gruetter, R,
Novotny EJ,
Boulware SD,
Mason GF,
Rothman DL,
Shulman GI,
Prichard JW,
and
Shulman RG.
Localized 13C NMR spectroscopy in the human brain of amino acid labeling from D-[1-13C]glucose.
J Neurochem
63:
1377-1385,
1994[Web of Science][Medline].
9.
Jeffrey, FMH,
Reshetov A,
Storey CJ,
Carvalho RA,
Sherry AD,
and
Malloy CR.
Use of a single resonance of glutamate for measuring oxygen consumption in intact tissues.
Am J Physiol Endocrinol Metab
277:
E1103-E1110,
1999
10.
Jeffrey, FMH,
Storey CJ,
Sherry AD,
and
Malloy CR.
13C isotopomer model for estimation of anaplerotic substrate oxidation via acetyl-CoA.
Am J Physiol Endocrinol Metab
271:
E788-E799,
1996
11.
Kauppinen, RA,
Sihra TS,
and
Nicholls DG.
Aminooxyacetic acid inhibits the malate-aspartate shuttle in isolated nerve terminals and prevents the mitochondria from utilizaing glycolytic substrates.
Biochim Biophys Acta
930:
173-178,
1987[Medline].
12.
Kihara, M,
and
Kubo T.
Aspartate aminotransferase for synthesis of transmitter glutamate in the medulla oblongata: effect of aminooxyacetic acid and 2-oxoglutarate.
J Neurochem
52:
1127-1134,
1989[Web of Science][Medline].
13.
LaNoue, KF,
Nicklas WJ,
and
Williamson JR.
Control of citric acid cycle activity in rat heart mitochondria.
J Biol Chem
245:
102-111,
1970
14.
Latipää, PM,
Peuhkurinen KJ,
Hiltunen JK,
and
Hassinen IE.
Regulation of pyruvate dehydrogenase during infusion of fatty acids of varying chain lengths in the perfused rat heart.
J Mol Cell Cardiol
17:
1161-1171,
1985[Web of Science][Medline].
15.
Lerner, L,
and
Bax A.
Sensitivity-enhanced two-dimensional heteronuclear relayed coherence transfer NMR spectroscopy.
J Magn Reson
69:
375-380,
1986.
16.
Lewandowski ED. Biological magnetic resonance. In: In Vivo
Carbon-13 NMR, edited by Berliner LJ and Robitaille P-M. New York:
Kluwer-Plenum, ch. 4, vol. 15, p. 117-159, 1998.
17.
Lowry, OH,
and
Passonneau JV.
Typical fluorometric procedures for metabolic assays.
In: Flexible System of Enzymatic Assays. New York: Academic, 1972.
18.
Malloy, CR,
Sherry AD,
and
Jeffrey FMH
Evaluation of carbon flux and substrate selection through alternate pathways involving the citric acid cycle of the heart by 13C NMR spectroscopy.
J Biol Chem
263:
6964-6971,
1988
19.
Mason, GF,
Gruetter Rothman DL,
Behar KL,
Shulman RG,
and
Novotny EJ.
Simultaneous determination of the rates of the TCA cycle, glucose utilization, -ketoglutarate/glutamate exchange, and glutamine synthesis in human brain by NMR.
J Cereb Blood Flow Metab
15:
12-25,
1995[Web of Science][Medline].
20.
Mason, GF,
Rothman DL,
Behar KL,
and
Shulman RG.
NMR determination of the TCA cycle rate and -ketoglutarate/glutamate exchange in rat brain.
J Cereb Blood Flow Metab
12:
434-447,
1992[Web of Science][Medline].
21.
O'Donnell, JM,
Doumen C,
LaNoue KF,
White LT,
Yu X,
Alpert NM,
and
Lewandowski ED.
Dehydrogenase regulation of metabolite oxidation and efflux from mitochondria in intact hearts.
Am J Physiol Heart Circ Physiol
274:
H467-H476,
1998
22.
Robitaille, PML,
Rath DP,
Skinner TE,
Abduljalil AM,
and
Hamlin RL.
Transaminase reaction rates, transport activities and TCA cycle analysis by post-steady state 13C NMR.
Magn Reson Med
30:
262-266,
1993[Web of Science][Medline].
23.
Shen, J,
Petersen KF,
Behar KL,
Brown P,
Nixon TW,
Mason GF,
Petroff OA,
Shulman GI,
Shulman RG,
and
Rothman DL.
Determination of the rate of the glutamate/glutamine cycle in the human brain by in vivo 13C NMR.
Proc Natl Acad Sci USA
96:
8235-8240,
1999
24.
Sherry, AD,
Malloy CR,
Roby RE,
Rajagopal A,
and
Jeffrey FMH
Propionate metabolism in the rat heart by 13C NMR spectroscopy.
Biochem J
254:
593-598,
1988[Web of Science][Medline].
25.
Sherry, AD,
Zhao P,
Wiethoff AJ,
Jeffrey FMH,
and
Malloy CR.
Effects of aminooxyacetate on glutamate compartmentation and TCA cycle kinetics in rat hearts.
Am J Physiol Heart Circ Physiol
274:
H591-H599,
1998
26.
Sibson, NR,
Dhankhar A,
Mason GF,
Rothman DL,
Behar KL,
and
Shulman RG.
Stoichiometric coupling of brain glucose metabolism and glutamatergic neuronal activity.
Proc Natl Acad Sci USA
95:
316-321,
1998
27.
Sibson, NR,
Dhankhar A,
Mason GF,
Behar KL,
Rothman DL,
and
Shulman RG.
In vivo 13C NMR measurements of cerebral glutamine synthesis as evidence for glutamate-glutamine cycling.
Proc Natl Acad Sci USA
94:
2699-2704,
1997
28.
Smith, SB,
Briggs S,
Triebwasser KC,
and
Freedland RA.
Re-evaluation of amino-oxyacetate as an inhibitor.
Biochem J
162:
453-455,
1977[Web of Science][Medline].
29.
Weiss, RG,
Stern MD,
De Albuquerque CP,
Vandegaer K,
Chacko VP,
and
Gerstenblith G.
Consequences of altered aspartate aminotransferase activity on 13C-glutamate labelling by the tricarboxylic acid cycle in intact rat hearts.
Biochim Biophys Acta
1243:
543-548,
1995[Medline].
30.
White, LT,
O'Donnell M,
Griffin J,
and
Lewandowski ED.
Cytosolic redox state mediates postischemic response to pyruvate dehydrogenase stimulation.
Am J Physiol Heart Circ Physiol
277:
H626-H634,
1999
31.
Yu, X,
Alpert NM,
and
Lewandowski ED.
Modeling enrichment kinetics from dynamic 13C NMR spectra: theoretical analysis and practical considerations.
Am J Physiol Cell Physiol
272:
C2037-C2048,
1997
32.
Yu, X,
White LT,
Alpert NM,
and
Lewandowski ED.
Subcellular metabolite transport and carbon isotope kinetics in the intramyocardial glutamate pool.
Biochemistry
35:
6963-6968,
1996[Medline].
33.
Yu, X,
White LT,
Doumen C,
Damico LA,
LaNoue KF,
Alpert NM,
and
Lewandowski ED.
Kinetic analysis of dynamic 13C NMR spectra: metabolic flux, regulation, and compartmentation in hearts.
Biophys J
69:
2090-2102,
1995[Web of Science][Medline].
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