INTRODUCTION

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CARBON-13 nuclear magnetic resonance (NMR) spectroscopy is proving to be a powerful tool for probing intermediary metabolism in intact tissues (1, 10, 13, 20, 22). Although many early studies were designed to probe linear pathways such as glycolysis or gluconeogenesis, recent emphasis has been placed on using this tool to probe metabolic pathways which reflect O₂ consumption and energy production. We have shown (14) that relative flux through various metabolic pathways associated with the Krebs citric acid (tricarboxylic acid, TCA) cycle can be derived from a single ¹³C spectrum collected at metabolic and isotopic steady state. Numerous recent reports have shown that it is also possible to obtain dynamic ¹³C NMR data. In particular, rates of ¹³C incorporation into glutamate have been used to estimate TCA cycle flux in brain (5, 17) and heart in vivo (19) and in isolated, perfused heart preparations (3, 4, 24, 25, 27, 28). One assumption common to early dynamic NMR studies was that exchange between -ketoglutarate (-KG) and glutamate was rapid compared with TCA cycle flux. This assumption was supported by results reported by Chance et al. (3), who modeled ¹³C fractional enrichment data from extracts of hearts freeze-clamped at various times after exposure to a ¹³C-enriched substrate. A fit of those data to an elaborate kinetic model indicated that transaminase flux was about threefold greater than TCA cycle flux and that exchange of intermediates across the mitochondrial membrane was fast compared with other fluxes in their model. However, more recent modeling studies challenged that result and concluded that the rate or ¹³C appearance in glutamate in heart tissue is influenced either by exchange through the transaminases or by mitochondrial-to-cytosolic transport (4, 25, 28). These combined observations suggest that dynamic ¹³C NMR methods which rely on glutamate ¹³C fractional enrichment as a direct index of TCA cycle should be interpreted with caution, at least in the myocardium.

There is a striking disparity among published theoretical predictions of the effects of altered aminotransferase activity on the rates of ¹³C incorporation into glutamate C4 and C3. First, Mason et al. (17) predicted that the enrichment curves for glutamate C4 and C3 should approach one another whenever V_x/V_TCA 1, where V_x is transaminase flux and V_TCA is TCA cycle flux. They presented theoretical plots for glutamate C3, showing that the rate of ¹³C enrichment at this carbon should increase with decreasing V_x/V_TCA [results shown in Fig. 5 of Mason et al. (17) must have been calculated holding V_x constant while increasing V_TCA, although this was not clearly stated]. Their prediction of more rapid appearance of ¹³C in glutamate C3 with decreasing V_x/V_TCA was later challenged by Weiss et al. (25), who concluded that ¹³C flux into glutamate C3 and C4 should decrease equally with decreasing aminotransaminase flux (at constant TCA cycle flux). This theoretical prediction, along with experimental data for one group of hearts exposed to 0.1 mM aminooxyacetate (AOA), led Weiss et al. (25) to conclude that the difference in time to reach half-maximal enrichment of C4 vs. C3 (t₅₀) is insensitive to altered transaminase activity.

In this report, we have examined in detail the effects of AOA on the rates of ¹³C incorporation into glutamate C4 and C3 from [2-¹³C]acetate in isolated, perfused rat hearts and conclude that this widely used transaminase inhibitor does indeed slow ¹³C incorporation into both glutamate carbons [as demonstrated by Weiss et al. (25)] but also attenuates the difference between the rates of ¹³C enrichment at glutamate C3 and C4 [as predicted by Mason et al. (17)]. We also demonstrate that AOA alters the ¹³C NMR visibility of glutamate in vivo and the temporal evolution of the glutamate C4 multiplets (C4S and C4D34). These observations indicate that AOA has multiple effects on metabolism in hearts, including a net redistribution of glutamate from the cytosol to the mitochondria and a decrease in the effective size of the intermediate pools undergoing ¹³C isotopic turnover in the TCA cycle.

	METHODS

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Materials. [2-¹³C]acetate sodium salt (99%) was purchased from Cambridge Isotope Laboratories (Andover, MA). AOA (hemihydrochloride), Dowex 50W resin (100-200 mesh, hydrogen form), and Dowex 1-X8 resin (100-200 mesh, chloride form) were purchased from Sigma Chemical (St. Louis, MO). All other reagents from commercially available sources were of the highest quality available. Male Sprague-Dawley rats, 250-300 g, were purchased from Sasco (Houston, TX).

Heart perfusions. Rats were anesthetized in an ether atmosphere, and 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. Typical coronary flow rates were 15-18 ml/min. A modified Krebs-Henseleit (KH) buffer containing 119.2 mM NaCl, 4.7 mM KCl, 1.25 mM CaCl₂, 1.2 mM MgSO₄, and 25 mM NaHCO₃ was bubbled with 95% O₂-5% CO₂. The entire recirculation system was jacketed and maintained at 37°C. After an initial washout period of 10-15 min, hearts were perfused with 300 ml of recirculating KH buffer for 30 min before addition of 2 mM [2-¹³C]acetate. In the inhibitor experiments, AOA was present during this entire 30-min period to ensure equilibration of the inhibitor into all cellular compartments. After addition of [2-¹³C]acetate, 14 3-min proton-decoupled ¹³C spectra were collected during the approach to isotopic steady state (42 min). Hearts were then freeze-clamped, extracted with 3.6% cold perchloric acid, neutralized with KOH, freeze-dried, and dissolved in 0.6 ml of deuterated water (²H₂O) for NMR analysis and glutamate assays.

NMR. Proton-decoupled ¹³C NMR spectra were obtained at 125.7 MHz on a GN-500 spectrometer. Intact heart spectra were collected in an 18-mm thin-walled NMR tube (Wilmad) with the heart bathed in perfusate, as previously described (15). The temperature of the heart was maintained at 37°C by control of the circulation perfusate temperature and by temperature control of the air surrounding the 18-mm NMR tube in the magnet using the GE VT accessory. A spherical bulb (~100 µl) containing [3-¹³C]propionate positioned near the heart provided an external concentration and chemical shift standard. Typically, spectra were signal averaged over periods of 3 min using a 45° observe pulse, a sweep width of ±14,000 Hz, 16K data points, and a 1-s delay between pulses and Waltz bilevel ¹H decoupling.

Myocardial O₂ consumption and tissue assays. Myocardial O₂ consumption was calculated from the difference in O₂ content of perfusion medium in the supply line and coronary effluent collected from the pulmonary artery as described previously (18). Total tissue glutamate was measured fluorometrically in tissue extracts (2). Homogenized tissue samples were prepared from isolated, perfused hearts by Polytron homogenization. Glutamic-oxalacetic transaminase [GOT; aspartate aminotransferase (AST); L-aspartate:2-oxoglutarate aminotransferase; EC 2.6.1.1] activity in homogenized heart tissue samples was assayed spectrophotometrically using techniques outlined by Bergmeyer and Bernt (see Ref. 2).

Isolation of glutamate. Glutamate was isolated from rat heart extracts using two short columns, one cationic (Dowex 50W hydrogen column) and one anionic (Dowex 1-X8 formate column) to separate the strongly acidic and neutral amino acids. 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 (final pH of eluant was between 5 and 6). A second column containing 3 ml of Dowex 1-X8 was assembled using 0.6-cm-diameter glass Pasteur pipette. This column was washed with 20 ml of 2 M formic acid followed by 20 ml of distilled water (final pH of eluant was near 7). Neutralized heart extract samples were dissolved in 2 ml of distilled water at pH 2.5-3.0 (adjusted by HCl) and applied to the Dowex 50W column. The column was washed with 25 ml of distilled water to remove carboxylic acids followed by 30 ml of 2 M NH₄OH to recover all amino acids. The amino acid fraction was freeze dried and redissolved into 2 ml of distilled water, and the pH was adjusted to 8 using KOH. This fraction was applied to the Dowex 1-X8 column and washed with 200 ml of distilled water to remove neutral amino acids and glycerol. Glutamate was eluted with 10 ml of 0.5 M formic acid, freeze dried to remove excess formic acid, and redissolved into 0.5 ml ²H₂O for ¹H NMR analysis.

Modeling. A kinetic model similar to that of Chance et al. (3) was constructed. Single pools of citrate, -KG, succinate, malate, oxalacetate, and glutamate were included in reactions that involved the Krebs TCA cycle, exchange between -KG and glutamate, and anaplerosis. Differential equations describing the time-dependent changes in concentration of the individual ¹³C isotopomers in each metabolite pool were solved numerically. This kinetic model was used to fit the ¹³C fractional enrichment curves of glutamate C4 and C3 to optimal values of V_x and V_TCA (where V_x is transaminase flux, mitochondrial export flux, or some combination thereof and V_TCA is TCA cycle flux) and to simulate time-dependent changes in the glutamate spectrum (both C3 and C4 fractional enrichments and C4 multiplet areas) as a function of V_x/V_TCA and total TCA cycle intermediate pool size.

Statistical analysis. All results are reported as means ± SD. The Student's t-test was used to compare means; P < 0.05 was considered significant.

	RESULTS

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Rates of ¹³C enrichment in glutamate with or without AOA. Temporal changes in proton-decoupled ¹³C NMR spectra of isolated, perfused rat hearts in the absence and presence of 0.5 mM AOA are compared in Fig. 1. The average glutamate C3 and C4 resonance areas for five or six hearts in each group are shown plotted vs. time in Fig. 2. There are three obvious differences between these data: first, ¹³C enrichment of glutamate C4 and C3 occurred more slowly in the heart perfused with the transaminase inhibitor; second, there was less glutamate detected by ¹³C NMR at steady state in the hearts perfused with AOA (intensities of glutamate resonances in these plots were standardized relative to an external [3-¹³C]propionate reference at 11 ppm); and third, the difference in rates of ¹³C incorporation into glutamate C4 and C3 was much less in the presence of inhibitor. Homogenized tissue from hearts perfused with 0.5 mM AOA had 60% less GOT activity than control hearts (in vitro assays), whereas coronary flow, heart rate, developed pressure, and O₂ consumption (in vivo assays) were unaffected by the presence of the inhibitor (see Table 1).

View larger version (37K): [in this window] [in a new window]   Fig. 1.   Dynamic 13C nuclear magnetic resonance (NMR) spectra of rat hearts perfused with 2 mM [2-13C]acetate with (A) and without (B) 0.5 mM aminooxyacetate (AOA). These expanded spectra show only glutamate C4 (~34.5 ppm) and C3 (~28 ppm) resonances.

View larger version (28K): [in this window] [in a new window]   Fig. 2.   A summary of average glutamate C4 (top curve, A and B) and C3 (bottom curve, A and B) resonance areas from hearts perfused as described in Fig. 1 either with (A) or without (B) 0.5 mM AOA (n = 5 in each group). Resonance areas were normalized using signal from [3-13C]propionate contained in an external reference bulb. Solid curves represent a fitting of data to a kinetic model described in text.

¹³C NMR spectra of intact hearts at steady state. Figure 3B compares glutamate signal intensities in three different intact hearts after perfusion to steady state with [2-¹³C]acetate and 0.5, 1, or 2 mM AOA. Although the amount of ¹³C-enriched glutamate in these three hearts appeared to be lower at the high levels of AOA, slight variations in heart size could have contributed to these differences. Thus we also compared glutamate intensities from a single heart, before and after exposure to AOA. The stacked plot shown in Fig. 3A compares the glutamate resonance intensities in a heart first perfused to steady state with [2-¹³C]acetate and then every 3 min after addition of 0.5 mM AOA (with [2-¹³C]acetate still present). The temporal intensity changes detected in all three glutamate resonances after addition of AOA showed quite clearly that less glutamate was detected by ¹³C NMR when the inhibitor was present.

View larger version (32K): [in this window] [in a new window]   Fig. 3.   A: set of 13C NMR spectra of glutamate from a single heart perfused to steady state with [2-13C]acetate before addition of AOA (front spectrum in stack) and every 5 min after addition of 0.5 mM AOA. B: 13C NMR spectra of glutamate in separate hearts perfused to steady state with [2-13C]acetate and 0.5, 1.0, or 2.0 mM AOA.

¹³C NMR spectra of extracts of tissue at isotopic steady state. The observations described above from intact, perfused hearts suggested that either 1) less [2-¹³C]acetate entered the TCA cycle as [2-¹³C]acetyl-CoA when the transaminase inhibitor was present, 2) addition of inhibitor resulted in a decrease in total tissue glutamate, or 3) glutamate was sequestered in some cellular compartment in intact hearts perfused with AOA that rendered it at least partially invisible by ¹³C NMR. High-resolution ¹³C spectra of extracts of hearts perfused to steady state with or without AOA were not significantly different (not shown). A steady-state isotopomer analysis (14) of those spectra indicated that the fraction of acetyl-CoA derived from [2-¹³C]acetate (F_C2) and relative anaplerosis (y) was identical in the presence and absence of inhibitor (Table 2). Total tissue glutamate (as measured enzymatically and from ¹³C NMR spectra of extracts) was also independent of inhibitor. ¹H NMR spectra of isolated, purified glutamate from these tissues provided an independent measure of the ¹³C fractional enrichment glutamate C4. Those values, reported in the last column of Table 2, were significantly less than F_C2 as measured by ¹³C NMR. This indicated that there was a small pool of glutamate in these hearts (~20% of total glutamate) that was not in exchange with TCA cycle intermediates and that the size of this nonexchanging pool was not affected by the presence of AOA (Table 2).

Time-dependent evolution of glutamate multiplets. ¹³C NMR spectra such as those shown in Fig. 1 had sufficiently good signal-to-noise to allow deconvolution of the singlet (S) and doublet (D34) components of glutamate C4. These data, shown in Fig. 4, indicate there was a dramatic difference in the temporal evolution of C4S and C4D34 (and other glutamate resonance multiplets as well; not shown) in hearts perfused with or without AOA. Although C4S was high and C4D34 low in the first 3-min spectrum of hearts in the absence of AOA, C4D34 was already higher than C4S in the first 3-min spectrum of hearts exposed to AOA. This was confirmed in extracts of hearts freeze-clamped at 5 min after addition of [2-¹³C]acetate with and without AOA. The glutamate C4 resonance from two representative spectra are shown in Fig. 5. The average C4D34/C4S from four such experiments was 0.64 ± 0.11 in hearts without AOA and 1.30 ± 0.12 in hearts with AOA. Because O₂ consumption and hence TCA cycle flux were identical in the two groups of hearts, the larger C4D34/C4S and the faster enrichment of glutamate C3 (relative to C4, see Fig. 2) in the AOA group suggested that the size of the TCA cycle intermediate pools in exchange with glutamate was smaller in hearts exposed to AOA than in control hearts. This was confirmed by ¹H spectroscopy of these same samples (Fig. 5). The larger ¹³C satellite wings around the glutamate H4 resonance centered at 2.34 ppm verified that a larger fraction of total tissue glutamate had exchanged with -KG in hearts without AOA. The average C4F at 5 min was 0.65 ± 0.05 in hearts without AOA vs. 0.22 ± 0.01 in hearts with AOA (n = 4 for each group). This confirmed that the kinetics of exchange among TCA cycle intermediate pools were different in the two experiments even though glutamate reached the same level of ¹³C fractional enrichment and isotopomeric composition after a period of 40-50 min in both groups of hearts (compare F_C2 values for 2 groups at steady state; Table 2).

View larger version (29K): [in this window] [in a new window]   Fig. 4.   Glutamate C4 resonance multiplet areas obtained with resolution enhancement and deconvolution of spectra such as those shown in Fig. 1. C4S represents singlet component and C4D34 represents doublet component, each expressed as a fraction of total C4 resonance area. Data in B are from hearts perfused without inhibitor, whereas those in A are from hearts perfused with 0.5 mM AOA (n = 5 in each group).

View larger version (17K): [in this window] [in a new window]   Fig. 5.   13C and 1H NMR spectra (left and right, respectively) of extracts of hearts freeze-clamped after 5 min of exposure to 2 mM [2-13C]acetate (0.5 mM AOA). These hearts were first perfused with unlabeled acetate with (top) or without AOA (bottom) for 30 min and then perfused with [2-13C]acetate with or without AOA for 5 min before freeze clamping.

	DISCUSSION

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Dynamic NMR measurements of ¹³C enrichment in glutamate C3 and C4 offer considerable potential for estimating TCA cycle flux in vivo; yet the assumptions involved in such measurements and their validity have generated considerable scientific debate. The first concern is whether the rate of ¹³C appearance in glutamate is limited to any significant extent by an exchange [-KGglutamate (Glu)] or transport (MitoCyto) process. In an early kinetic study using ¹³C fractional enrichment data derived from spectra of tissue extracts, Chance et al. (3) estimated that exchange between -KG and glutamate was about threefold faster than TCA cycle flux in hearts perfused with acetate. They concluded that the rate of exchange between cytosolic and mitochondrial metabolite pools was fast relative to other fluxes and that the size of each metabolite pool in exchange in the cycle was equal to that measured enzymatically in tissue extracts. A few years later, the first in vivo NMR demonstration of ¹³C kinetics to measure TCA cycle flux was elegantly demonstrated by Fitzpatrick et al. (5). They fitted the ¹³C enrichment curves of glutamate C4 and C3 in rat brain obtained by ¹H observe-¹³C edited spectroscopy to a kinetic model that also assumed that the entire cerebral glutamate pool was in rapid exchange with -KG. This gave a reasonable 1.4 µmol · g¹ · min¹ value for TCA cycle flux, in agreement with values determined using other techniques. This kinetic model was later extensively validated (16, 17) and applied to ¹³C NMR data collected from human brain during infusion of [1-¹³C]glucose (6). It was concluded that exchange between -KG and glutamate (V_x) was 72 times faster than V_TCA in human brain (16).

More recent ¹³C NMR kinetic investigations on isolated, perfused hearts have reported that the rates at which ¹³C appears in glutamate C3 and C4 may be influenced by processes other than TCA cycle flux (4, 28). Chatham et al. (4) followed ¹³C incorporation into glutamate C3 and C4 in isolated rat hearts perfused with [2-¹³C]acetate and fitted those data using a similar kinetic model as described earlier by Chance et al. (3). However, they found that -KGGlu exchange was 18% that of TCA cycle flux, a result quite different from the earlier Chance et al. report, in which the data were derived from spectra of tissue extracts. Somewhat later, Yu et al. (28) applied a mathematically simplified kinetic model to ¹³C fractional enrichment data from intact rabbit hearts perfused with [2-¹³C]acetate and reported that two rate-limiting processes, TCA cycle flux (i.e., V_TCA) and -KGGlu exchange (F₁), contribute nearly equally to the glutamate enrichment kinetics. A comparison of their kinetically determined F₁ flux variable with estimates of total cytosolic transaminase flux (GOT_Cyto) from in vitro assays led them to conclude that flux through GOT_Cyto was much too high to contribute to the NMR observables, and thus F₁ probably reflects some transport process, perhaps transport of -KG from the mitochondria to the cytosol. No attempt was made to estimate mitochondrial transaminase flux (GOT_Mito) in that study probably due to the uncertainties in the distribution of -KG, glutamate, aspartate, and oxalacetate among the cytosolic and mitochondrial compartments. Finally, Weiss et al. (25), using a kinetic model similar to that used by Chance et al. (3) and Chatham et al. (4), reported that -KGGlu exchange via transamination was nearly equal to TCA cycle flux in isovolumic, paced rat hearts perfused with [2-¹³C]acetate. In each of these studies, total tissue glutamate, as measured enzymatically (3, 28) or as detected by ¹³C NMR (4, 25), was used to fit the kinetic data. Thus four independent ¹³C kinetic modeling studies of hearts perfused with [2-¹³C]acetate have yielded somewhat disparate results. Chance et al. (3) concluded that flux through the transaminases was a factor of 3 larger than TCA cycle flux, Chatham et al. (4) concluded that transaminase flux (as part of malate-aspartate shuttle) was smaller than TCA cycle flux, and Weiss et al. (25) concluded that transaminase flux and TCA cycle flux were comparable, whereas Yu et al. (28), on the basis of direct experimental evidence for high GOT_Cyto flux, suggested that flux of -KG from the mitochondria to the cytosol (but not transaminase flux) was comparable to TCA cycle flux.

A kinetic model (Jeffrey, unpublished results) similar to that of Chance et al. (3) was used to fit the ¹³C fractional enrichment data of Fig. 2 to obtain best values of V_x (flux through an undefined process involving interchange of -KG and glutamate carbons) and V_TCA for both groups of hearts (AOA). The solid lines drawn through the data of Fig. 2 represent the best fit of the data to a model that fixed F_C2 at 0.93, y at 0.05, total tissue glutamate at 22 µmol/g dry wt, and the remaining TCA cycle intermediates at values reported by Chance et al. (3) for acetate-perfused hearts. The fit of the data in the absence of inhibitor gave V_x = 7.5 µmol · min¹ · g dry wt¹ and V_TCA = 7.5 µmol · min¹ · g dry wt¹, quite similar to the values reported by Yu et al. (28) for acetate-perfused rabbit hearts. Although our fitted value of V_TCA was somewhat lower than that predicted by our O₂ consumption measurements (expected value was 19/2 = 9.5 µmol · min¹ · g dry wt¹), the agreement is reasonable considering the assumptions involving the sizes of the all TCA cycle intermediate pools. Importantly, our data in the absence of inhibitor indicate that V_x  V_TCA, in agreement with Weiss et al. (V_x/V_TCA = 0.86; Ref. 25) and Yu et al. (V_x/V_TCA = 0.92; Ref. 28) but clearly different from the conclusions reached by Chance et al. (V_x/V_TCA = 2.8; Ref. 3) and Chatham et al. (V_x/V_TCA = 0.18; Ref. 4).

Effects of AOA on pre-steady-state ¹³C enrichment of glutamate C3 and C4. We have shown in this study that an inhibition of total cellular transaminase activity by 60% (as measured in vitro) has a dramatic effect on the rates of ¹³C appearance in glutamate C3 and C4. Inasmuch as flux through GOT_Cyto has been estimated at 20 times greater than TCA cycle flux in isolated rabbit hearts perfused with acetate (28), how can AOA at these levels have such a dramatic effect on the rates of ¹³C enrichment at glutamate C3 and C4? If GOT_Cyto flux is indeed much greater than TCA cycle flux in the rat heart, then one must consider a possible role of GOT_Mito in this process. Using the kinetic parameters for GOT_Mito and metabolite concentrations reported by Yu et al. (28) and making the assumption that 20% of total tissue aspartate and -KG is mitochondrial and 10% of total tissue water is mitochondrial, one can arrive at an estimate of 90 µmol · min¹ · g dry wt¹ for GOT_Mito flux. Clearly, this estimate may suffer from the likely oversimplifying assumption that the kinetic parameters of GOT_Mito in situ are the same as those measured in vitro (28). Nevertheless, the calculation suggests that neither GOT_Cyto nor GOT_Mito may limit the rate of ¹³C appearance in glutamate C3 and C4 in the absence of inhibitor. If one further assumes that AOA does nothing more than inhibit both GOT_Cyto and GOT_Mito by 60%, then we should not have detected the dramatic changes in the rate of ¹³C appearance in glutamate C3 and C4 shown in Fig. 2. This suggests that AOA must be affecting other metabolic processes in addition to partial inhibition of the transaminases. This conclusion was supported in our attempts to fit the ¹³C fractional enrichment data for hearts perfused with 0.5 mM AOA (Fig. 2A) using the same kinetic model as described above. The solid lines through the inhibitor data of Fig. 2 show the best fit using the same assumptions as stated above plus V_TCA fixed at 7.5 µmol · min¹ · g dry wt¹. Although V_x tended to decrease in all calculations (curves shown are for a value of 5.8 µmol · min¹ · g dry wt¹), the poor agreement between experimental and calculated data indicates that this kinetic model is too simplistic. Again, the near coincidence of the C4 and C3 enrichment curves indicates that much smaller pool(s) of intermediates were in rapid exchange with the TCA cycle reactions in this group of hearts (see modeling results below).

Evolution of glutamate multiplets with time. We also demonstrated for the first time that the evolution of glutamate ¹³C isotopomers, as reported by time-dependent evolution of C4S and C4D34, provides additional kinetic evidence about exchanging pools that is not easily detected in ¹³C fractional enrichment measurements. To illustrate this point, we have used our kinetic model to simulate the changes expected in the shape of the ¹³C enrichment curves of glutamate C4 and C3 as -KGGlu exchange (V_x) is reduced at a fixed V_TCA. Figure 6 shows the resulting simulations at two intermediate pool sizes (differing by a factor of 10) and for values of V_x/V_TCA = 2 and 0.1, with V_TCA held constant. As originally predicted Mason et al. (17) and demonstrated here experimentally, the simulation shows that the intensities of glutamate C3 and C4 should approach one another as V_x  V_TCA. This is quite intuitive. By slowing communication (exchange) between a relatively small pool of TCA cycle intermediates and the relatively large glutamate pool, we have effectively increased the rate at which ¹³C reaches the C3 position relative to C4 without increasing V_TCA. A comparison of Fig. 6, left and right, indicates that the shapes of the C3 and C4 kinetic curve are relatively insensitive to the size of the TCA cycle intermediate pool undergoing rapid ¹³C turnover, both for V_x/V_TCA = 2 (simulating our control hearts) and for V_x/V_TCA = 0.1 (simulating hearts inhibited by AOA). This same conclusion was also reached by Chatham et al. (4) and Yu et al. (28) for control hearts. Because the curves shown in Fig. 6A for both sets of conditions did not change as the total TCA cycle intermediate pool size was decreased by a factor of 10, we must conclude that it is not possible to judge the size of the exchanging pools based upon C4 and C3 fractional enrichment data alone. However, this situation is quite different for the glutamate multiplets. Figure 6, bottom, illustrates that the glutamate C4 multiplet, C4D34, is quite sensitive to both V_x/V_TCA and intermediate pool sizes. In particular, C4D34 reaches its maximum much more rapidly when V_x/V_TCA and the combined size of the TCA cycle intermediate pools are both small.

View larger version (31K): [in this window] [in a new window]   Fig. 6.   Predicted effects of changing total tricarboxylic acid (TCA) cycle intermediate pool size (A vs. B) and -ketoglutarate (-KG)glutamate (Glu) flux (Vx) on fractional 13C enrichment of glutamate C4 and C3 (top) and evolution of glutamate C4 doublet (C4D34; bottom) vs. time using a kinetic model similar to that described by Chance et al. (3). VTCA was held constant in all calculations. Vx/VTCA was fixed at 2 (solid lines) or 0.1 (dashed lines). Total tissue glutamate was fixed at 20 µmol/g dry wt in all calculations, and all remaining exchanging TCA cycle intermediate pool sizes were either set equal to those reported by Chance et al. (3) (data shown on A) or uniformly decreased by a factor of 10 (data shown on B).

NMR visibility of glutamate. This study has shown that the NMR visibility of glutamate can be altered by a common metabolic inhibitor, without changing total tissue glutamate, heart rate, developed pressure, or O₂ consumption. Safer et al. (21) have also reported that 0.4 mM AOA does not markedly affect O₂ consumption by hearts but does alter the cytosolic-mitochondrial distribution of several metabolites associated with the malate-aspartate shuttle. It has been shown by Safer et al. in heart (21) and Kauppinen et al. in brain (7) that AOA at these levels increases the ratio of cytosolic NADH/NAD⁺, which in turn increases cytosolic malate and decreases cytosolic aspartate. This would in turn induce aspartate efflux from mitochondria and glutamate influx into mitochondria (21), perhaps leading to a net redistribution of glutamate toward the mitochondrial compartment at equilibrium. Our observation that the ¹³C-enriched glutamate signal decreases by ~30% in hearts perfused with AOA is entirely consistent with these prior metabolic observations. This is important because kinetic models generally assume that all glutamate in exchange with the TCA cycle is NMR visible and that it equals total tissue glutamate as measured in extracts. In most cases, this appears to be a reasonable assumption (4, 16, 17, 24, 28), but our study demonstrates that there may be metabolic situations in which glutamate can become partially NMR invisible, perhaps by redistributing among subcellular compartments.

Summary. Dynamic ¹³C NMR studies of hearts perfused with [2-¹³C]acetate with or without the transaminase inhibitor, AOA, have shown that this inhibitor has multiple effects on metabolism in the heart. We have shown that as little as 0.5 mM AOA inhibits total transaminase activity by 60% and effectively slows communication between the mitochondrial and cytosolic spaces of the myocardium without altering TCA cycle flux. One unanticipated finding was that AOA also altered the amount of glutamate detected by NMR. The NMR data (both extract spectra and in vivo spectra) indicate that a small exchanging pool of metabolites turns over quite rapidly in the presence of inhibitor. This small, presumably mitochondrial, pool then exchanges with mitochondrial and cytosolic glutamate (Glu_Mito and Glu_Cyto, respectively) at rates determined by residual transaminase activity and transport processes. This was reflected in an unusually high C3/C4 and unusally large C4D34 early during cycle turnover. The sizes of Glu_Mito and Glu_Cyto cannot be firmly established by our experimental data nor can we determine whether multiple subcompartmental pools exist within the cytosolic and mitochondrial spaces. Glu_Mito has been reported to be as high as 30% (26) and as low as 10% (8) of total tissue glutamate in normoxic hearts; so if our NMR observations in the presence of AOA indeed reflect a redistribution of ~20% of Glu_Cyto to Glu_Mito, most of the total tissue glutamate would still remain in the cytosol. Given that the spin-lattice relaxation times and nuclear Overhauser enhancements of ¹³C-enriched glutamate in hearts perfused with [2-¹³C]acetate are nearly identical to those measured in aqueous saline at the same temperature (22), we assume that all glutamate detected by ¹³C NMR in vivo is Glu_Cyto and that Glu_Mito could be either invisible or less visible due to slower diffusion of molecules in the highly viscous mitochondrial matrix (23). Furthermore, our observation that ~20% of total cellular glutamate is totally sequestered and never becomes enriched with ¹³C in acetate-perfused hearts (in agreement with previous reports; Refs. 11, 12) illustrates that multiple glutamate pools can indeed exist in hearts, so that dividing total glutamate into further "kinetic" subcellular compartments may not be totally unreasonable.