INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

DURING MYOCARDIAL HYPOXIA or mild ischemia, mitochondrial metabolism becomes impaired, and glycolytic flux increases to partially meet cellular ATP demands. Glycolytic ATP production, which often does not fully compensate for loss of mitochondrial energy production due to flux limitations primarily at the level of phosphofructokinase, can result in damage to the myocardium. One strategy, used experimentally to augment glycolytic ATP production during conditions of metabolic stress, is to provide exogenous fructose-1,6-bisphosphate (FDP). This compound is a naturally occurring glycolytic intermediate that enters glycolysis after the rate-limiting step of phosphofructokinase. A number of reports indicate FDP effectively protects and enhances recovery of tissue functions from oxygen-deficient conditions. For example, FDP provided to hypoxic or ischemic myocardium improved recovery of mechanical function after 20-min low-flow ischemia (9). Indeed, FDP administration to a wide variety of tissues during metabolic energy production deficiencies usually modestly improved tissue function and/or energetic state (1, 5, 9-11, 17, 18, 23, 25). That only modest effects were observed has been assumed to be due to low FDP utilization because of a limitation of its transport into the tissues (5).

Despite FDP's promise as a therapeutic agent, little has been done to assess its mechanism(s) of action. The first studies of how FDP might exert its salutary influence focused on its metabolic actions. It was shown that FDP reduced myocardial glycolytic rate, oxygen consumption, and glucose oxidation (22, 25). It also was found that FDP caused the myocardium to become reduced as indicated by increased adenine dinucleotide fluorescence (6). This is consistent with enhanced flux through glycolysis. However, these changes may have been a consequence of FDP depression of mechanical function via altered Ca²⁺ metabolism. Definitive results showing that FDP directly influences myocardial metabolism were obtained by Tavazzi et al. (25) who found that perfused rat hearts converted [¹⁴C]FDP to [¹⁴C]lactate and ¹⁴CO₂. Evidence that FDP entered cells rather than being converted to fructose was provided by data for metabolism of exogenous fructose by hearts, which was only about 10% that found with FDP (25), and fructose metabolism was undetectable in vascular smooth muscle (4).

FDP is a highly charged molecule at physiological pH, is not expected to readily cross cell membranes, and the mechanism of its transport is not known. One recent report showed FDP is capable of crossing artificial lipid bilayers in a dose-dependent manner (2). However, a membrane transporter has not been identified for FDP. Therefore, the purposes of this study were to verify that FDP is transported and metabolized in Langendorff-perfused rat hearts, test the hypothesis that FDP enters cardiomyocytes by a dicarboxylate transport system, and attempt to assess FDP maximum utilization rates. We found that perfused rat hearts were able to metabolize exogenous FDP and exogenous fumarate, a dicarboxylate, indicating that both were capable of entering cardiomyocytes. Furthermore, we discovered that fumarate inhibited FDP utilization by isolated perfused hearts consistent with translocation via a dicarboxylate transporter. We conclude that the keto (linear) form of FDP utilizes a dicarboxylate or analogous transporter to enter cells based on the structural similarity of this form of FDP and dicarboxylates. This is the first evidence in support of a specific transport mechanism for FDP into cells. We were able to increase FDP utilization rates to about three times that for glucose observed under physiological substrate conditions, but this rate was considered to be less than maximal. These observations may lead to insights on strategies to further increase FDP uptake and metabolism by metabolically stressed tissues and eventually FDP may become a useful intervention during ischemic or hypoxic conditions to maintain tissue-energetic state and enhance survivability.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

General approach. We employed two methods to address the nature of FDP transport and metabolism by isolated perfused rat hearts. We first used ¹³C nuclear magnetic resonance (NMR) to assess the ability of these hearts to metabolize [¹³C]FDP and [¹³C]fumarate as a representative dicarboxylate and then used [¹⁴C]FDP to quantify FDP metabolism and assess competition between it and fumarate.

Perfused hearts. Hearts were rapidly removed from pentobarbital anesthetized (30 mg/kg body wt ip), heparin injected (1,000 units intra-vena cava), 300-500 g male Wistar (Morini, S. Polo D'Enza; Rome, Italy) or Sprague-Dawley (Harlan, Indianapolis, IN) rats maintained on standard laboratory rat chow and ad libitum drinking water. Each heart was placed in ice-cold perfusion buffer solution and the aorta was cleaned of excess tissue and cannulated with a stainless-steel cannula connected to a perfusion buffer solution reservoir set to provide 80 cmH₂O perfusion pressure. Perfusion was begun as soon as the aorta cannula was patent. The entire procedure typically required <1 min after the thoracotomy was begun. The perfusate from the initial 10-min perfusion period was not recirculated. The perfusion buffer solution was then switched to one containing FDP, fumarate, or FDP and fumarate (as described below) and the perfusates were recirculated. For the [¹³C]FDP NMR studies, the total perfusion buffer volume was 12 to 14 ml and for the ¹⁴C studies the total recirculated volume was 50 (perfusions for 30 min or less) or 100 ml (perfusions for >30 min). Each heart was perfused with only one solution in addition to the basic one used in the initial perfusion. The basic perfusion buffer solution was modified Krebs-Henseleit-Ringer bicarbonate, pH 7.4 containing 12 IU insulin/l and (in mM) 119 NaCl, 2.5 CaCl₂, 4.8 KCl, 1.2 MgSO₄, 0.6 KH₂PO₄, 25 NaHCO₃, and 10 glucose. The perfusion solutions were equilibrated with 95% O₂-5% CO₂ at 37°C by a constant flow of gas through a bubbling stone ([¹⁴C]FDP studies) or falling film oxygenator (13) ([¹³C]FDP studies).

[1-¹³C]FDP synthesis and purification. The [1-¹³C]FDP was synthesized from [1-¹³C]fructose as previously described (5) and further purified by anion exchange column chromatography. Briefly, [1-¹³C]FDP was synthesized enzymatically from [1-¹³C]fructose using hexokinase and phosphofructokinase with the ATP supplied by an ATP-regenerating system consisting of phosphocreatine and creatine kinase. Neutralized products were applied to a 2.5- × 50-cm column of Dowex 1 × 8-200 at a flow rate of ~2 ml/min and eluted with a gradient of NH₄HCO₃ (0.75-1.5 M). Fractions, collected every 2 min, were evaporated in a Speed Vac (Savant Instruments; Holbrook, NY) until residual water and NH₄HCO₃ in the form of NH₃ and CO₂ were removed. Each fraction was assayed for ATP and FDP and fractions with a FDP/ATP of >50 were used for heart perfusions. The [1-¹³C]FDP was used at a concentration of ~2 mM in the heart perfusion buffer.

¹³C NMR Spectroscopy. Frozen perfusate samples (2.5 ml each) were lyophilized in a Speed Vac (Savant Instruments). The powder was suspended in 1 ml 99% D₂O with 25 mM 3-(trimethylsilyl)-1-propane-sulfonic acid (TMSPS) as a chemical shift reference. A 0.65-ml sample was transferred to a 5-mm NMR tube. ¹³C NMR spectroscopy was performed on a Bruker DRX 500 spectrometer with the following acquisition parameters: 300 scans with 16 dummy scans, 30 pulse angle at 125.77 MHz, 33,333-Hz sweep width, and a 1-s predelay. 32 K points were acquired and processed with 1-Hz line broadening before Fourier transform. All spectra were broadband proton decoupled and referenced with TMSPS at 0 ppm. NMR data were processed using Bruker software for peak amplitude determination.

Metabolic rate measurements. FDP metabolism was assessed by measuring [¹⁴C]lactate and ¹⁴CO₂ production from [U-¹⁴C]FDP. The [¹⁴C]FDP (2.5 to 10 µCi to give radiospecific activities of 4 × 10³ to 4 × 10⁴ disintegrations per minute/µmol) was added to the perfusion buffer just before beginning perfusion of hearts with the FDP-containing perfusion solution. Separation of [¹⁴C]lactate from [¹⁴C]FDP was carried out using ion-pairing HPLC. For this purpose, perfusate samples were collected at the desired times from the reservoir and immediately deproteinized by adding ice-cold 70% HClO₄ (1:10; vol/vol) and centrifugation. After neutralization with 5 M K₂CO₃ and subsequent centrifugation for 10 min at 4°C to remove precipitated potassium perchlorate, perfusate samples were filtered through a 0.45-µm Millipore filter and then loaded (200 µl) onto a Kromasil C-18, 5-µm particle size, 4.6 × 250-mm HPLC column. The column was previously equilibrated with buffer having the following composition: 10 mM tetrabutylammonium hydroxide (as the pairing reagent), 10 mM KH₂PO₄, 0.25% methanol, pH 7.00. Lactate (both cold and [¹⁴C]) was isocratically eluted with a retention time of 6.3 min [capacity factor, k' = 4.22, which is defined as (V_iV_o)/V_o, where V_i is the retention volume of peak i, and V_o is the void volume of the column] at a flow rate of 1.2 ml/min and a constant column temperature of 23°C. Because FDP was retained by the stationary phase during lactate elution, before any new sample injection, the column was cleaned with methanol/water (40:60, vol/vol) for 15-20 min at a flow rate of 1.2 ml/min. HPLC apparatus consisted of a Constametric 3500 dual pump system (ThermoQuest, Rodano; Milan, Italy) connected to a SpectraSystem UV6000LP spectrophotometric diode array detector (ThermoQuest) set up between 200 and 300 nm wavelengths for chromatogram acquisition. The detector sensitivity was increased with a 5-cm light path flow cell. Acquisition and analysis of chromatographic data were performed on a personal computer using ChromQuest software supplied by the HPLC manufacturers. Lactate peak recognition in perfusates was effected at 210 nm by comparing the retention times of known quantities of standard lactate. After verifying enzymatically that FDP was not present, and hence no [¹⁴C]FDP was present, 2 ml of the collected lactate peak of perfusate samples were then added to 8 ml of liquid scintillation cocktail (Aquasol-2, DuPont NEN; Boston, MA) and radioactivities were determined by liquid scintillation spectroscopy.

Statistics. Statistical significances were determined using a two-tailed paired Student's t-test assuming unequal variances. A P value <0.05 was accepted as significant.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

FDP and dicarboxylates are highly charged at physiological pH, and it was of interest to assess that these molecules cross cardiac sarcolemma. We examined the fates of [1-¹³C]FDP and [U-¹³C]fumarate included in the perfusates of isolated Langendorff-perfused rat hearts. One advantage of ¹³C NMR spectroscopy is the ability to identify all metabolic products from the metabolism of a ¹³C-labeled substrate. Shown in Fig. 1 are results from perfusing hearts with [¹³C]FDP. The perfusate contained [3-¹³C]lactate as the primary metabolite as would be expected if FDP crossed the cell membrane and acted as a substrate for glycolysis. It is important to note that no new resonances (between 66 and 66.5 ppm) corresponding to [1-¹³C]fructose appear. Therefore, no detectable conversion of FDP to fructose appears to occur outside the cell, and thus the phosphorylated form (FDP) appears to be the form transported and metabolized.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   13C nuclear magnetic resonance (NMR) spectrum of perfusate from a heart perfused with [1-13C]fructose-1,6-bisphosphate (FDP) and unlabeled pyruvate. Note the appearance of [3-13C]lactate indicating uptake and metabolism of [1-13C]FDP during the perfusion period. Concentrations of [1-13C]FDP and pyruvate were 2 and 5 mM, respectively; perfusion time = 30 min. Several peaks remain unidentified between 70 and 80 ppm and are not likely to be from unlabeled buffer components or substrates used in the FDP synthesis (e.g., Tris) but may be heretofore unidentified metabolic products of [1-13C]FDP metabolism in the heart. This region includes 2 hydroxyl carbons adjacent to a carboxylate group. Candidates include [2-13C]malate, although identification is speculative. TMSPS, 3-(trimethylsilyl)-1-propane-sulfonic acid.

On the basis of the similarities in planar structures, we hypothesized that the linear form of FDP (the keto form) utilizes a dicarboxylate transporter to cross the plasma membrane. To study whether dicarboxylates are transported into myocardial cells, we included 2 mM of [U-¹³C]fumarate in the perfusion solution for rat hearts. An example of ¹³C NMR spectrum of the perfusate after 20 min of perfusion is shown in Fig. 2. The only metabolic product of [U-¹³C]fumarate we are able to detect is [U-¹³C]malate. Because fumarate is converted to malate and malate appears in the perfusate, we conclude that the fumarate that enters cardiac myocytes is catabolized by fumarase in the mitochondria, and the product, malate, is transported out of the myocytes. Therefore, fumarate and malate can both cross the plasma membrane of cardiac myocytes, indicating the presence of a dicarboxylate transport system in heart sarcolemma.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   13C NMR spectrum of perfusate from a heart perfused with [1,2,3,4-13C]fumarate (Fum). Note the appearance of malate in the perfusate indicating uptake and metabolism of Fum and transport of malate out of the myocytes. Fum concentration = 5 mM; perfusion time = 45 min.

We then ascertained whether FDP is transported by the same mechanism as dicarboxylates by determining whether metabolism is inhibited by the dicarboxylic acid fumarate. Because dicarboxylate transporters typically are characterized by the ability of other dicarboxylates to compete for transport and by having an apparent Michaelis-Menton constant (K_m) for transport in the millimolar range, we examined the uptake and oxidation of 5 mM [U-¹⁴C]FDP in the presence and absence of 10 mM unlabeled fumarate. Shown in Fig. 3 is the total ¹⁴CO₂ production (from gas trapping and dissolved in perfusate) from [U-¹⁴C]FDP at the indicated FDP concentrations and with fumarate. Included in this figure are data from experiments in which we included 10 µM 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS) a band 3 inhibitor. Because the mechanism for FDP transport into cells was unknown, we also hypothesized that the band 3 anion tranporter may be a transport mechanism for FDP to enter myocardial cells. First, there is a concentration-dependent oxidation of FDP the rate at 20 mM being about six times the rate at 5 mM. Second, uptake and oxidation of FDP are significantly inhibited about 70% by 10 mM of fumarate, indicating possible competition of transport between FDP and fumarate and suggesting a common transport system. Third, DNDS does not significantly affect ¹⁴CO₂ production from [U-¹⁴C]FDP, indicating FDP uptake is not via the band 3 anion exchanger.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Total myocardial 14CO2 production from [U-14C]FDP. FDP = 20 mM (squares) or 5 mM (circles, diamonds, and triangles) in the absence (diamonds) or presence (circles) of 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS) (an inhibitor of the anion exchange transporter, see text for details) or in the presence of 10 mM Fum (triangles).

Because myocardial FDP metabolism is expected to primarily yield both ¹⁴CO₂ and [¹⁴C]lactate, we separated [¹⁴C]lactate from [¹⁴C]FDP (as described in MATERIALS AND METHODS). FDP conversion to lactate accounted for 32% of total lactate produced by the heart at 5 and 10 mM FDP and for 60% at 20 mM FDP. The sums of [¹⁴C]lactate and ¹⁴CO₂ production from [¹⁴C]FDP are shown in Fig. 4. As with ¹⁴CO₂ production, DNDS does not and fumarate does inhibit lactate production from FDP. The relationships between FDP metabolism and its inhibition by fumarate at 10 and 20 mM FDP and 5 to 20 mM fumarate are shown in Fig. 5. At a ratio of fumarate to FDP of 4:1, FDP utilization is nearly completely inhibited (85%). Fumarate causes proportional decreases in both lactate and CO₂ production from FDP compared with when fumarate is absent with an apparent IC₅₀ of 8 mM at 5 mM FDP. The fraction of total FDP metabolized to lactate remains constant in hearts perfused with 5 mM FDP in the presence or absence of either fumarate or DNDS (Fig. 6). Therefore, fumarate inhibits FDP transport rather than metabolism because inhibition of metabolism would change the ratio of lactate/CO₂ production from FDP (see DISCUSSION). However, at 20 mM FDP, the fraction of FDP converted to lactate increases significantly. The high total glycolytic rate observed with 20 mM FDP (Fig. 4) appears to result in a pyruvate production rate in excess of the capacity of pyruvate dehydrogenase to convert it to acetyl CoA under the conditions of the experiment. The rate of FDP transport/metabolism does not approach a maximum at 20 mM FDP.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   [14C]lactate and [14C]CO2 production from [U-14C]FDP. FDP concentration = 5 mM, perfusion = 60 min. FDP concentration 20 mM, perfusion time = 30 min. The latter values were multiplied by 2 for comparison to the results with 5 mM FDP.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Fum competition for FDP transport and metabolism. FDP transport and utilization is inhibited by increasing concentrations of Fum consistent with competition for the same transport mechanism.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Fraction of the total [14C]FDP metabolized to [14C]lactate. Note that the fraction of total FDP (5 mM) converted to lactate remains constant despite inhibition of transport by Fum. See text for full explanation.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

These studies clearly show that FDP is transported and metabolized to CO₂ and lactate. The flux of FDP through the glycolytic pathway is 3 µmol · g¹ · min¹ at 20 mM FDP, which is about 3.5 times the flux of glucose with 11 mM glucose as the only exogenous substrate (20). Because this flux rate is approximately sixfold higher than the rate observed with 5 mM FDP, the maximum flux with FDP is not achieved at 5 mM FDP. We suspect the increase in flux is due to a proportional increase in keto-FDP and that at 20 mM the ~0.4 mM keto-FDP is less than the K_m for transport. It is likely that if the maximum transport rate (V_max) were to be achieved, FDP metabolic flux would simply mirror transport, because glycolytic enzymes are relatively unregulated from FDP on, except during ischemia, and under strongly reducing conditions (20).

For several decades, the glycolytic intermediate FDP has been administered in attempts to improve cellular energetic state during periods of ischemia or hypoxia. The rationale for the use of FDP has been that because oxidative metabolism is inhibited, glycolytic ATP production will become substantially more important in meeting cellular ATP demands. The ATP yield of the glycolytic oxidation of FDP is four molecules of ATP per molecule of FDP compared with two molecules of ATP per molecule of glucose. However, a key rate-limiting step in glycolysis is phosphofructokinase, and during ischemia, its activity becomes more restricted at low pH as does that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (20). Therefore, if FDP could enter the cell, glycolysis would no longer be regulated by phosphofructokinase activity, more substrate would be available for the remainder of the pathway, and the rate of glycolytic ATP production could be increased. Even under severely ischemic conditions, FDP likely would increase flux through GAPDH simply by mass action as the activity of this enzyme is influenced by substrate concentration, H⁺, lactate, and NADH. However, the ultimate limitation to flux through GAPDH would be NAD⁺ availability.

FDP provided during periods of ischemia or hypoxia has resulted in modest improvement of tissue function in a wide variety of tissues studied. Possible limitations to the usefulness of FDP during ischemia may include limited transport of FDP into cells and inadequate lactate removal may limit FDP metabolism when the cell is very reduced. In Langendorff-perfused rat hearts, FDP was shown to preserve high-energy metabolites during anoxia, restore myocardial metabolism and contractility during reperfusion (9), and improve myocardial efficiency under well-perfused and well-oxygenated conditions (22). Improvement in tissue metabolic state during metabolic stress has been reported in a number of other tissue types, including kidney (1), liver (19), brain (26), erythrocytes (8), smooth muscle (5), and intestine (24). Many of these effects were likely due to FDP entering cells, but also due to the known calcium chelating activity of FDP (see Ref. 6). It has been demonstrated that FDP is converted to lactate by heart and vascular smooth muscle, and thus some of the effect of FDP in improving tissue energetic state must have resulted from glycolysis of FDP (5, 25). However, the magnitude of the improvement has typically been modest. Because FDP is a highly charged molecule at physiological pH, it has generally been presumed that the transport of FDP has limited its utilization and hence its effectiveness. However, the mechanism of FDP transport has remained unknown.

In the current study, we have shown that FDP can be metabolized to lactate in normal Langendorff-perfused rat heart indicating transport into myocytes and subsequent metabolism of FDP by glycolysis. We also demonstrated that the dicarboxylic acid fumarate can be converted to malate, indicating transport of both malate and fumarate across the plasmalemma of the myocyte. We hypothesized that keto-FDP shares enough structural similarities to dicarboxylic acids that the keto-FDP could be transported using dicarboxylate transporters.

Dicarboxylate transport activity in the plasmalemma has been extensively examined in kidney cells (14, 15, 23), intestinal cells (27, 28), and liver cells (29). However, it has long been known that substrates such as succinate and fumarate can enter cardiovascular cells (3, 7, 16, 17). Therefore, dicarboxylate transport activity likely exists in a wide variety of cell types. Typically, the K_m for transport of dicarboxylic acids has been reported to be in the range of ~0.4 mM to 34 mM (14, 27, 29) with the sodium-dependent type having a lower K_m than the sodium-independent type. From our data, neither the K_m, nor the sodium dependence of dicarboxylate transport can be determined (see discussion below). However, if keto-FDP [2% of total (12)] is the transported form, then concentrations of keto-FDP that were used would be 0.1, 0.4, and 1 mM and thus likely would be below or near the K_m for the transporter. This is consistent with our data in Figs. 3 and 4, showing that transport and metabolism of FDP increased at least linearly as total FDP was increased from 5 to 20 mM.

FDP transport/metabolism is inhibited by fumarate, indicating competition between the two. Fumarate is transported into the cell and subsequently into the mitochondria where it is converted to malate from where it is transported into the sarcoplasm and into the extracellular space. It is likely this efflux is to achieve transsarcolemmal equilibrium, but the extracellular fluid contains no malate, and equilibrium is not obtained because of the large "extracellular volume" of the perfused heart. Alternatively, malate efflux is in exchange for extracellular fumarate. The observed inhibition of myocardial FDP utilization by fumarate might occur at either transport or metabolism. The fact that fumarate does not change the ratio of FDP-derived CO₂ and lactate indicates fumarate does not compete with FDP for metabolism. If it did, the ¹⁴CO₂/[¹⁴C]lactate ratio would decrease with fumarate as substrate. At 5 mM FDP, it does not, but it does at higher concentrations of FDP, regardless of whether or not fumarate is present. The latter findings indicate flux through pyruvate decarboxylase (pyruvate dehydrogenase complex) is restricted relative to pyruvate conversion to lactate at FDP concentrations above 5 mM. Flux of FDP-derived pyruvate to acetyl CoA likely would be higher without insulin as a stimulator of glucose transport and without glucose as additional exogenous substrate but we have not determined this.

The mechanism for inhibition of FDP transport by fumarate, like the kinetics of FDP transport, cannot be accurately discerned from these data, because at FDP concentrations are much lower than K_m. The plot of rate versus FDP for all data is linear. We attempted several approximations including fit of the data to v = V_max · S/(K_m + K_m · I/K_i), where v is the rate, S is the substrate concentration, I is the inhibitor concentration, and K_i is the inhibition constant. A plot of 1/slope of this line versus I gives a K_m/V_max = 0.2 and an estimate of K_i = 1.5. This analysis does not provide insight into the nature of the inhibition by fumarate. Fumarate certainly changes the slope of v versus S indicating fumarate is not uncompetitive, but competitive and noncompetitive inhibition both predict changes in V_max/K_m. Based solely on structural similarities between FDP and fumarate, we would expect inhibition to be competitive.

It is possible that rather than a metabolic action, FDP protects against the untoward effects of hypoxia and ischemia in several tissues by chelating extracellular Ca²⁺. This would be particularly germane to the heart, because reduced Ca²⁺ decreases cardiac mechanical performance and oxygen consumption, and the ischemic insult is lessened because of a more favorable oxygen supply/demand ratio. Even if the extracellular Ca²⁺ concentration is adjusted to compensate for added FDP, it is possible FDP protects via chelation of intracellular Ca²⁺. In order for this to happen, free FDP would need to accumulate inside cells. This is unlikely, because FDP transported into cells is metabolically active and all enzymatic steps beyond phosphofructokinase are near equilibrium, which ensures a relatively high flux from aldolase on with little change in pathway substrate concentrations. Also, aldolase concentration is about 800 µM (21), and the calculated total FDP concentrations in aerobic, and ischemic hearts are 90 and 250 µM, respectively (17) indicating that the free FDP concentration is very low. In view of these considerations and based on the results of the present studies, it is more plausible that when extracellular Ca²⁺ concentrations are controlled, FDP confers protection to oxygen-deficient tissues via a metabolic action.

The present studies determine the extent to which the heart metabolizes FDP and test the possibilities that FDP is transported either via the band 3 transport system or via a dicarboxylate transporter. The data are consistent with transport of the keto form of FDP, because its utilization continues to increase at the highest concentration we can effectively test, and its transport is inhibited by the dicarboxylate fumarate, which probably enters cells via the same transporter as FDP.