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Competition of pyruvate with physiological substrates for oxidation by theheart: implications for studies with hyperpolarized [1-13C]pyruvate

Karlos X. Moreno,1 Scott M. Sabelhaus,1 Matthew E. Merritt,1,2 A. Dean Sherry,1,2,5

and Craig R. Malloy1,2,3,4

1Advanced Imaging Research Center, Departments of 2Radiology and 3Internal Medicine (Division of Cardiology), Universityof Texas Southwestern Medical Center, Dallas; 4Veterans Affairs North Texas Health Care System, Dallas; and 5Departmentof Chemistry, University of Texas at Dallas, Richardson, Texas

Submitted 14 July 2009; accepted in final form 3 March 2010

Moreno KX, Sabelhaus SM, Merritt ME, Sherry AD, Malloy CR.Competition of pyruvate with physiological substrates for oxidation bythe heart: implications for studies with hyperpolarized [1-13C]pyruvate.Am J Physiol Heart Circ Physiol 298: H1556–H1564, 2010. Firstpublished March 5, 2010; doi:10.1152/ajpheart.00656.2009.—Carbon13 nuclear magnetic resonance (NMR) isotopomer analysis was usedto measure the rates of oxidation of long-chain fatty acids, ketones,and pyruvate to determine the minimum pyruvate concentration([pyruvate]) needed to suppress oxidation of these alternative sub-strates. Substrate mixtures were chosen to represent either the fed orfasted state. At physiological [pyruvate], fatty acids and ketonessupplied the overwhelming majority of acetyl-CoA. Under conditionsmimicking the fed state, 3 mM pyruvate provided �80% of acetyl-CoA, but under fasting conditions 6 mM pyruvate contributed only33% of acetyl-CoA. Higher [pyruvate], 10–25 mM, was associatedwith transient reduced cardiac output, but overall hemodynamic per-formance was unchanged after equilibration. These observations sug-gested that 3–6 mM pyruvate in the coronary arteries would be anappropriate target for studies with hyperpolarized [1-13C]pyruvate.However, the metabolic products of 3 mM hyperpolarized[1-13C]pyruvate could not be detected in the isolated heart duringperfusion with a physiological mixture of substrates including 3%albumin. In the presence of albumin even at high concentrations ofpyruvate, 20 mM, hyperpolarized H13CO3

� could be detected only inthe absence of competing substrates. Highly purified albumin (but notalbumin from plasma) substantially reduced the longitudinal relax-ation time of [1-13C]pyruvate. In conclusion, studies of cardiacmetabolism using hyperpolarized [1-13C]pyruvate are sensitive to theeffects of competing substrates on pyruvate oxidation.

myocardium; pyruvate; dynamic nuclear polarization; substrate oxi-dation; isolated rat heart; carbon-13; nuclear magnetic resonance

FLUXES IN BIOCHEMICAL PATHWAYS are abnormal in many formsof heart disease. Metabolism in specific pathways, however, isdifficult to measure with confidence in vivo by positron emis-sion computed tomography (PET) or single photon emissioncomputed tomography (SPECT) because the chemical fate ofthe tracer is not detected. Consequently, there is intense interestin the demonstration by Golman and colleagues (14, 15) thatchemical information is retained in 13C images of the heartafter injection of hyperpolarized (HP) [1-13C]pyruvate. Pyru-vate is particularly attractive because products of its metabo-lism such as lactate and alanine provide information aboutenzyme activity in the cytosol, and the appearance of 13CO2 orH13CO3

� is due to flux through a single mitochondrial enzyme,

pyruvate dehydrogenase (PDH) (31). The ability to monitor13CO2 and H13CO3

� production is an important goal becausethe ratio 13CO2/H13CO3

� is a direct measure of pH (12), and theratio H13CO3

�/[1-13C]lactate may be an index of myocardialrecovery after ischemia (32). However, studies of 13CO2 ap-pearance in the heart have been limited to isolated tissueswhere substrate concentrations do not mimic the physiologicalsituation. In vivo, the heart is exposed to a complex mixture ofcompounds, including long-chain fatty acids, lactate, pyruvate,ketones, and glucose, and the myocardium readily switchesamong these energy sources (53). Furthermore, the concentra-tion of each compound varies considerably as a consequence ofdisease and nutritional state. Depending on their concentrationand other factors, any one of these molecules could suppressmetabolism of HP [1-13C]pyruvate.

Translation of 13C hyperpolarization technology for physi-ological studies and clinical applications will require under-standing the influence of circulating substrates on metabolismof injected HP pyruvate and specifically the minimum concen-tration of pyruvate necessary to overcome oxidation of com-peting substrates. At steady state, the rate of 13CO2 productionis calculated as the rate of acetyl-CoA production � thefractional contribution of [1-13C]pyruvate to acetyl-CoA. Be-cause the rate of acetyl-CoA production and consumption bythe myocardium under baseline conditions is relatively con-stant, this relationship simply means that, other factors beingequal, the appearance of 13CO2 or H13CO3

� in the heart isproportional to the fractional contribution of pyruvate toacetyl-CoA. Although pyruvate carboxylation is known tooccur within the heart, this contribution to overall TCA flux issmall and is not affected by changes in pyruvate concentration([pyruvate]) (8, 38, 39).

The objective of this study was to take advantage of thisconvenient relationship to determine the minimum concentra-tion of 13C-enriched pyruvate required to compete effectivelywith physiological substrates for production of 13CO2 or,equivalently, acetyl-CoA. Intravenously administered pyruvatehas been extensively examined as a therapeutic intervention forcardiac (17, 18, 35), neurological (10, 33), and acid-basedisorders (28, 42). In these studies, the typical plasma concen-tration was 2–6 mM. In human subjects with congestive heartfailure, [pyruvate] up to 6 mM in the coronary arteries was safe(17, 18), but adverse effects of pyruvate such as arrhythmiasand fluctuations in blood pressure were reported at 9 mMpyruvate in dogs (24). The maximum efficacy of pyruvate inpostischemic guinea pig hearts was 5–10 mM (5) perhapssuggesting that pyruvate metabolism cannot be further stimu-lated at higher concentrations. In the current study, the con-

Address for reprint requests and other correspondence: C. R. Malloy,Advanced Imaging Research Center, 5323 Harry Hines Blvd., Dallas, TX75390 (e-mail: [email protected]).

Am J Physiol Heart Circ Physiol 298: H1556–H1564, 2010.First published March 5, 2010; doi:10.1152/ajpheart.00656.2009.

http://www.ajpheart.orgH1556

centration range selected for detailed studies of pyruvate oxi-dation, 0.1–6 mM, was chosen based on a safe range describedin these literature reports.

In this study, hearts were exposed to mixtures of ketones,long-chain fatty acids, lactate, pyruvate, and glucose. Althoughthe isolated heart does not duplicate all features of the envi-ronment in vivo, it allows precise control of the concentrationof each substrate as well as full hemodynamic monitoring,including cardiac output during changes in substrate concen-trations. The concentration of each substrate was chosen tomimic plasma concentrations of these substrates in either thefed or fasted state (20) because fasting is common amongpatients, and increases in the concentration of fatty acids andketones frequently occur among patients with diabetes or withheart disease. Although our fasting conditions do not com-pletely model the complicated events of fasting, we are study-ing one component: how plasma substrate concentration willaffect pyruvate utilization. Under conditions mimicking the fedstate, 3 mM pyruvate was sufficient to produce �80% of theacetyl-CoA. At substrate concentrations present in fasting,pyruvate at 6 mM was oxidized at a significantly higher ratethan at baseline, but oxidation of fats and ketones still providedthe majority of acetyl-CoA. The hemodynamic effects ofhigher concentrations of pyruvate were also examined. Al-though transiently reduced cardiac output was observed at[pyruvate] �10 mM, hearts quickly recovered normal O2

consumption and function even at 25 mM pyruvate. Based onthese studies, a target [pyruvate] for cardiac studies is �3–6mM in the coronary arteries.

METHODS

Materials. [3-13C]sodium pyruvate (99% enriched) was purchasedfrom Isotec Laboratories. U-13C long-chain fatty acids, [1-13C]sodiumpyruvate, and [1,3-13C2]ethylacetoacetate (99% enriched) were pur-chased from Cambridge Isotope Laboratories (Andover, MA). Thetrityl radical tris[8-carboxyl-2,2,6,6-tetra-[2-(1-hydroxyethyl)]-benzo-(1,2-d:4,5-d)-bis-(1,3)-dithiole-4-yl]methyl sodium salt was purchasedfrom Oxford Molecular Biotools (Abingdon, Oxfordshire, UK). All otherchemicals were obtained from Sigma-Aldrich (St. Louis, MO) at thehighest quality available. BSA was fatty acid free and was purchased inthe anhydrous form. Male Sprague-Dawley rats (260–330 g) wereobtained from Charles River Laboratories (Wilmington, MA) and givenfree access to standard rat chow and water until the day of death. Thestudies were performed under a protocol approved by the University ofTexas Southwestern Medical Center Animal Care and Use Committee.

Working heart preparation. Hearts were rapidly excised from ratsunder general anesthesia. The aorta was immediately cannulated forretrograde Langendorff perfusion at 100 cm of hydrostatic pressureand later converted to a working preparation as described below. Theinitial perfusate was a modified Krebs-Henseleit medium maintainedat 37°C that contained (in mM): 25 NaHCO3, 118 NaCl, 4.7 KCl, 1.2MgSO4, 1.2 KH2PO4, 10 glucose, and 1.25 CaCl2. The buffer wassaturated with a 95:5 mixture of O2-CO2. Polyethylene tubing at-tached to a pressure transducer was inserted in the left ventriclethrough the mitral valve. This enabled the continuous measurement ofleft ventricular developed pressure and heart rate throughout theexperiment. Myocardial O2 consumption (MV̇O2) was calculated fromthe difference in O2 tension (measured with a blood gas analyzer)between the perfusion medium in the arterial supply line and thecoronary effluent, which was collected via a cannula placed in thepulmonary artery. Coronary flow and aortic output were measuredusing a stopwatch and a graduated cylinder. After �5–10 min ofperfusion in the Langendorff mode, the hearts were switched to a

working mode at a left atrial pressure of 15 cmH2O and supplied witha buffered medium containing the same mixture of electrolytes butwith one of the following mixtures of oxidizable substrates. The “fed”mixture contained glucose (10 mM, unlabeled), lactate (1.2 mM,unlabeled), pyruvate (0.12 mM, unlabeled), [1,3-13C2]acetoacetate(0.17 mM), long-chain fatty acids [total 0.4 mM, a mixture ofpalmitate, palmitoleic, stearic, oleate, and linoleic in physiologicalconcentrations, all U-13C (see Refs. 19 and 20 for details)], and 3%BSA. The “fasted” mixture contained glucose (4.9 mM, unlabeled),lactate (0.89 mM, unlabeled), pyruvate (0.08 mM, unlabeled), [1,3-13C2]acetoacetate (1.2 mM), long-chain fatty acids (total 0.85 mM, amixture of palmitate, palmitoleic, stearic, oleate, and linoleic inphysiological concentrations, all U-13C), and 3% BSA. These sub-strate concentrations duplicate the plasma concentrations in a fed orfasted rat. In all experiments, the medium was oxygenated with athin-film multibulb oxygenator, filtered, and recirculated (34).

Six groups of hearts (n � 3–5 in each group) were studied. Eachgroup was exposed to a different concentration of [3-13C]pyruvate, inaddition to the other substrates described above: group 1, fed mixtureplus 0.12 mM [3-13C]pyruvate; group 2, fed mixture plus 1 mM[3-13C]pyruvate; group 3, fed mixture plus 3 mM [3-13C]pyruvate;group 4, fed mixture plus 6 mM [3-13C]pyruvate; group 5, fastedmixture plus 3 mM [3-13C]pyruvate; and group 6, fasted mixture plus6 mM [3-13C]pyruvate. The lower concentrations of pyruvate werenot studied with the fasted mixture because ketones and long-chainfatty acids are already known to be the dominant energy source at lowconcentrations of pyruvate (20).

The hearts were perfused for 15 min with the fed or fasted mediumwithout [3-13C]pyruvate. After collection of baseline hemodynamicdata and O2 consumption, an appropriate amount of 500 mM[3-13C]pyruvate was added at a rate of 0.04 ml/s to the left atrialreservoir. The hearts were perfused for an additional 40 min. O2

consumption and hemodynamics were measured every 15 min. At theend of the perfusion period, the heart was freeze-clamped usingaluminum tongs precooled in liquid N2 (LN2). A small portion of thefrozen tissue was used for determination of the wet-to-dry ratio byweighing the tissue before and after slow oven drying. The remainderof the frozen tissue was pulverized into a fine powder under LN2,extracted with 6% perchloric acid, centrifuged at 15,000 g for 15 minat 4°C, neutralized with KOH, and centrifuged a second time. Thesupernatant was freeze-dried, and the lyophilized heart extract wasdissolved in 0.60 ml of 2H2O and pH corrected to 7.1 for nuclearmagnetic resonance (NMR) analysis.

In a separate set of experiments, working hearts were studied asdescribed above using the fed mixture of substrates except that thesubstrates were not 13C enriched. In these experiments, hearts wereexposed to graded concentrations of pyruvate (10, 15, and 25 mM).One group of hearts was exposed to 25 mM NaCl in addition to thenormal concentration of 143 mM sodium to observe the effect ofexcess sodium from the pyruvate salt on the heart. The hearts wereperfused for 15 min before the slow addition, 0.04 ml/s, of 2.5 Munlabeled pyruvate. The hearts were perfused for an additional 40min. O2 consumption and hemodynamics were measured every 15min. At the end of the perfusion period, the heart was freeze-clampedusing aluminum tongs precooled in LN2. A small portion of the frozentissue was used for determination of the wet-to-dry ratio by weighingthe tissue before and after slow oven drying.

HP 13C NMR spectroscopy of the isolated heart. Hearts wererapidly excised from rats under general anesthesia and perfusedretrograde through the aorta at 37°C and 100 cmH2O using standardLangendorff methods. Heart rate and developed pressure were mon-itored through a catheter in the left ventricle. The heart was suppliedwith a nonrecirculating medium and placed in a 20-mm NMR tube.The perfusing medium was a modified Krebs-Henseleit buffer con-taining the electrolytes described above plus 10 mM glucose andbubbled continuously with a 95:5 mixture of O2-CO2. The water-jacketed glass perfusion apparatus was placed in the bore of a Varian

H1557PYRUVATE SUPPRESSES FATTY ACID AND KETONE OXIDATION

AJP-Heart Circ Physiol • VOL 298 • MAY 2010 • www.ajpheart.org

Inova 9.4 T magnet. During a preparation and stabilization time of�20 min, the NMR probe was tuned, and the field homogeneity wasoptimized using the 23Na free induction decay (FID). A line width of15 Hz was typically obtained. Shimming on 23Na approximates the13C frequency and shimmed volume, whereas the short spin-spinrelaxation time (T2) values of 23Na allow for quicker pulsing. Afterstable mechanical performance was established, the perfusate wasswitched to the fed mixture described above (long-chain fatty acidsbound to albumin, lactate, pyruvate, ketones, and glucose, all not 13Cenriched), filtered through a 5-�m pore cellulose acetate filter, andrecirculated. 13C NMR spectra were acquired at 100 MHz in a 20-mmVarian broadband probe using 66° pulses. The first FIDs were ac-quired a few seconds before injection of the HP solution, describedbelow. Serial, single FIDs were acquired with 16 K complex datapoints over a � 21,000 Hz bandwidth with proton decoupling usingGARP (47). Acquisition time was �1 s, giving a time to repeat of 1s for each scan. Typically 20–30 separate FIDs were collected. Thesedata were zero-filled before Fourier transformation. The relative peakareas were measured by integration using the VNMR software.

Hyperpolarization and delivery of 13C-enriched pyruvate to hearts.The polarization process was started �90 min before each experi-ment. The details have been described previously (31, 32). Briefly,�20 �l of 1.5 M [1-13C]pyruvate and 16.6 mM trityl radical preparedin 50:50 glycerol-deionized H2O was polarized using a HyperSenseinstrument (Oxford Instruments Molecular Biotools, Abingdon, UK).�90 min of microwave irradiation at 1.4 K typically yielded �15%polarization. The polarization of 15% was measured by separatedissolution experiments in the liquid state. The SD of these measure-ments is �1% on a day-to-day basis. The frozen, polarized samplewas rapidly thawed by dissolution in 4 ml of hot water (�180°C)containing 0.85 mM Na2EDTA. The resulting solution (3 ml) wasfurther diluted in 20 ml of perfusing medium containing BSA,long-chain fatty acids, etc., as described above (all substrates not 13Cenriched) to achieve a final concentration of 3 mM HP [1-13C]pyru-vate. The total time required for dissolution, ejection, and furtherdilution was �10 s. The solution was injected gently and continuouslyby catheter in the perfusion column directly above the heart at a rateof �0.3 ml/s. The temperature of all solutions entering the heart was37°C, and the pH was 7.3–7.4. The NMR console was triggered tostart acquisition at the end of the dissolution process. To inject a 20mM HP [1-13C]pyruvate solution to the heart, 100 �l of the[1-13C]pyruvate/trityl radical solution was polarized. Following dis-solution with hot (�180°C) 0.85 mM Na2EDTA (aqueous) solution,3 ml of the resulting solution were diluted with 3 ml of the perfusionmedium described above and gently injected to the heart at a rate of�0.3 ml/s.

13C NMR spectroscopy of solutions. Proton-decoupled 13C spectraof heart extracts at 25°C were acquired at 150 MHz on a VarianVNMRS spectrometer (Varian Instruments, Palo Alto, CA) using a5-mm broadband NMR probe, a 45° observe pulse, and a 3-s delaybetween pulses. Broadband proton decoupling was achieved usingWALTZ (48). Relative peak areas for each multiplet component weredetermined by deconvolution using ACDLabs SpecManager (Ad-vanced Chemistry Development, Toronto, Canada). Substrate 13Cfractional enrichments and 13C multiplet data were used in a meta-bolic model (tcaCALC) to measure the relative rates of oxidation ofeach substrate, and anaplerosis (29). To measure the effects ofalbumin on 13C relaxation times, [1-13C]pyruvate (2.5 mM in PBS �D2O for a lock) was added to eight different concentrations of BSA(0–3%) with and without long-chain physiological fatty acids at afixed ratio of 0.4 mM fatty acids/3% BSA. 13C spin-lattice relaxationtimes (T1) were measured at 150 MHz using standard inversion-recovery methods at 37°C.

Statistical analysis. Data are presented as means � 1 SD. Two-tailed t-tests were performed assuming unequal variance using Mi-crosoft Excel for Figs. 4 and 5, A and C. For Fig. 3, the pyruvate, fattyacid, ketone body oxidation, and PDH flux, response to increased

[pyruvate] was assessed with one-way ANOVA followed by Dun-nett’s procedure for comparing 1, 3, and 6 mM [pyruvate] with the 0.1concentration while adjusting for multiple testing. For Fig. 5B, thedeveloped pressure was compared using a mixed-model repeated-measures analysis that included concentration and pre vs. post com-parisons in the model. Within each concentration, pre vs. post changeswere made with least-squared means contrasts that were derived fromthe mixed model and adjusted for multiple testing using the Bonfer-roni-Holm method. Statistical analyses for Figs. 3 and 5B wereperformed with SAS version 9.2 (SAS Institute, Cary, NC).

RESULTS

Pyruvate oxidation in the fed vs. fasted substrate mixtures.The glutamate multiplets observed in 13C NMR spectra wereused to determine the labeling pattern of acetyl-CoA enteringthe TCA cycle (26–30, 50). A typical spectrum of a heartextract is shown in Fig. 1, with the insets displaying the13C-13C spin-spin coupling in glutamate. In the glutamatecarbon-4 resonance (Fig. 2), the quartet (Q, due to glutamatelabeled in positions 3, 4, and 5) and the doublet D45 (due toglutamate labeled in positions 4 and 5 but not 3) are due tooxidation of U-13C long-chain fatty acids, whereas the doublet,D34, and the singlet, S, are due to oxidation of [3-13C]pyru-vate. The effects of graded concentrations of [3-13C]pyruvateon the glutamate spectrum are shown in Fig. 2. Because theratio of long-chain fatty acid oxidation relative to pyruvateoxidation is simply the ratio of multiple areas (Q � D45)/(S �D34) (50), it is evident that, at physiological [pyruvate], fattyacids overwhelm pyruvate as an energy source. At higher[pyruvate], the situation is reversed: pyruvate is by far thepreferred substrate for oxidation.

When perfused with the fed buffer, the fraction of acetyl-CoA from pyruvate, ketones, and fatty acids, determined froma full isotopomer analysis (29, 30), are shown in Fig. 3A, andthe effect of increasing [pyruvate] on flux through PDH deter-mined using 13C isotopomer analysis and MV̇O2 is shown inFig. 3B. At a physiological concentration of pyruvate, 0.12mM, �50% of the heart’s energy is supplied by fatty acids,�20% from ketones, and the remainder from lactate, pyruvate,glucose, and stored sources. At 1 mM [pyruvate] (�10�physiological), both fatty acid and ketone oxidation werereduced and pyruvate oxidation increased to supply �45% ofacetyl-CoA. At 3 mM pyruvate and 6 mM pyruvate, thecontribution of fatty acids and ketones to acetyl-CoA wassmall, and pyruvate provided �80% of acetyl-CoA. Anaple-rosis relative to TCA cycle flux was �8–10% for all concen-trations of pyruvate.

The sources of energy in the heart were quite different whenexposed to the fasted substrate mixtures (Fig. 4). At 3 mMpyruvate under fasted conditions, ketones supplied most of theacetyl-CoA, �65%, and fatty acids and pyruvate supplied�23% and �7%, respectively. Doubling the concentration ofpyruvate to 6 mM inhibited fatty acid and ketone oxidationsomewhat, but ketones remained the preferred substrate forproduction of acetyl-CoA even at 6 mM pyruvate (Fig. 4). Fluxthrough PDH when pyruvate was present at 6 mM in the fastedmixture was roughly equal to PDH flux at 1 mM pyruvate inthe fed mixture.

Effect of graded [pyruvate] on cardiac function. The effectsof adding high concentrations of pyruvate to the perfusingmedium (6, 10, 15, or 25 mM) in the presence of a fed mixture

H1558 PYRUVATE SUPPRESSES FATTY ACID AND KETONE OXIDATION


of fatty acids, ketones, lactate, and glucose is shown in Fig. 5.There was no effect of pyruvate in this concentration range onheart rate (Fig. 5, top), developed pressure (Fig. 5, middle), orMV̇O2 (Fig. 5, bottom) when measured 10–15 min after ad-ministration of pyruvate. However, at [pyruvate] �10 mM,there was a variable decrease in aortic flow during the first 2–3min after exposure to pyruvate without a change in developedpressure or heart rate (data not shown). All hearts recoveredbaseline function within 5 min. Hearts exposed to 25 mM NaCldid not show any variation in heart rate, developed pressure, orMV̇O2 (data not shown).

13C NMR spectra of the isolated heart. 13C NMR spectraof the isolated rat heart collected after injection of HP[1-13C]pyruvate are shown in Fig. 6. In the presence of fattyacid-free BSA and no other substrates, only a very small signalfrom HP [1-13C]pyruvate could be detected after administra-tion of 3 mM HP [1-13C]pyruvate (Fig. 6A). In the presence ofketones and long-chain fatty acids bound to albumin, HP[1-13C]lactate and HP [1-13C]pyruvate hydrate, in addition toHP [1-13C]pyruvate, could be detected after administration of3 mM HP [1-13C]pyruvate (Fig. 6B). Because the concentra-tions of pyruvate and BSA are the same in Fig. 6, A and B, thedifferences between the spectra must be because of an effect offatty acids or ketones on pyruvate metabolism, an interactionamong substrates and BSA to reduce polarization of HP[1-13C]pyruvate, or some combination of the two. The effect ofa higher concentration of HP [1-13C]pyruvate in the presenceof BSA but no other substrates was tested, and the results areshown in Fig. 6C. A product of oxidation of HP [1-13C]pyru-vate, HP H13CO3

�, can be detected at �161 ppm. However, inthe presence of competing substrates, fatty acids and ketones,this HP H13CO3

� signal disappears as would be expected iffatty acids or ketones are being oxidized (Fig. 6D) in prefer-ence to HP [1-13C]pyruvate. Interestingly, in the presence of

fatty acids and BSA (Fig. 6, B and D), both HP [1-13C]lactateand HP [1-13C]pyruvate hydrate were detected in approxi-mately the same ratio, regardless of the concentration ofadministered pyruvate, but, in the absence of fatty acids, HP[1-13C]lactate could not be detected (Fig. 6, A and C). In allstudies, the HP signals were lost in the presence of albuminwithin �30 s, and the overall signal to noise was attenuatedcompared with previous studies (31, 32).

Together, these experiments demonstrate that, in the pres-ence of albumin, there is a complex interaction among alter-native physiological substrates (ketones and fatty acids) andthe 13C NMR spectrum of HP pyruvate and its metabolicproducts. Based on the spectrum in Fig. 6D, it appears that HP[1-13C]pyruvate is not being oxidized since the HP H13CO3

�

signal could not be detected. Common illnesses and brieffasting cause variation of plasma ketones and fatty acids, aswells as plasma albumin, and for this reason the effectiveconcentration of pyruvate for 13C cardiac imaging will besensitive to the condition of the subject. In principle, suffi-ciently high concentrations of pyruvate in the coronary arteriesshould overcome competing substrates, but high concentra-tions of pyruvate may have adverse effects (24, 25).

Effect of albumin on the longitudinal relaxation time of[1-13C]pyruvate. The T1 of carbon-1 of pyruvate was �44 s at2.5 mM, 37°C, and 150 MHz in aqueous solution in theabsence of albumin. In the presence of albumin mixed withlong-chain fatty acids, there was a graded and approximatelylinear effect of albumin concentration ([albumin]) on T1 ofcarbon-1 of pyruvate � 44.7 � 6.6[albumin], where T1 is inseconds, [albumin] is in percent, r2 � 0.91 (Fig. 7). At 3%albumin, the T1 of pyruvate carbon-1 was 25 � 2 s. In thepresence of fatty acid-free albumin, the T1 of pyruvate car-bon-1 was further reduced to �10 s at 3% [albumin]. Numer-ous small molecules competitively bind albumin, and these

Fig. 1. 1H-decoupled 13C nuclear magnetic resonance (NMR) spectrum of a heart extract. The working heart was supplied with a mixture of 13C-enrichedlong-chain fatty acids, ketones, lactate, pyruvate, and glucose in concentrations typical of a fed, rested animal. The multiplets of glutamate carbons 1–5, shownas insets, are the result of 13C-13C spin-spin coupling and reflect the relative rates of oxidation of these substrates. Ala, alanine; HB, -hydroxybutyrate; Lac,lactate; Ac, acetate; Suc, succinate; Asp, aspartate; Mal, malate; Cit, citrate; D, doublet; Q, quartet; S, singlet; T, triplet.



studies with fatty acid-free albumin or with albumin bound tofatty acids do not duplicate conditions in vivo. Therefore,plasma was prepared from fed rats pretreated with heparin. TheT1 of carbon-1 of [1-13C]pyruvate, 25 mM, in rat plasma was�38 s at 37°C and 150 MHz.

Fig. 2. Influence of pyruvate concentration ([pyruvate]; [Pyr]) on the carbon-4resonance of glutamate. These 13C spectra were acquired from hearts exposedto the “fed” mixture of substrates at graded [pyruvate], 0.12 mM (bottom) or1.0, 3.0, and 6 mM (top). The fraction of acetyl-CoA derived from pyruvaterelative to the fraction from long-chain fatty acids is indicated by the ratio (S �D34)/(Q � D45). At 3 mM pyruvate, the oxidation of long-chain fatty acids isinhibited. F, resonances arising from oxidation of 13C-enriched long-chainfatty acids; P, resonances arising from oxidation of 13C-enriched pyruvate, Q,quartet due to J45 and J34 coupling; D34, doublet due to J34 coupling; D45,doublet due to J45; S, singlet due to glutamate with 13C in position 4 but notposition 3 or 5. J is standard notation for the spin-spin coupling constant.

Fig. 3. Influence of [pyruvate] on sources of acetyl-CoA and flux throughpyruvate dehydrogenase (PDH) using a fed buffer. A: relative rates of oxida-tion of ketones (dotted line), long-chain fatty acids (broken line), or pyruvate(solid line) are shown. At 3 mM pyruvate, oxidation of competing substratesis largely suppressed. B: flux through PDH. Flux through PDH is maximal at3–6 mM pyruvate. Data are means � SD. Analysis of variance revealedstatistically significant responses to increased [pyruvate] for pyruvate (P 0.0001), fatty acid (P � 0.004), ketone body oxidation (P � 0.006), and PDHflux (P � 0.0004). *P 0.05 [pyruvate] vs. [pyruvate] � 0.12 mM. $P 0.05for fatty acids and ketone bodies vs. respective 0.12 mM data point.

Fig. 4. Effect of 6 mM pyruvate on the contribution of ketones (KB),long-chain fatty acids (FA), and pyruvate (Pyr) to acetyl-CoA. Hearts exposedto the fed mixture derived �80% of acetyl-CoA from 6 mM pyruvate (openbar); the contribution of ketones (gray bar) or fatty acids (solid bar) wasnegligible. However, hearts exposed to the “fasted” mixture preferentiallyoxidized ketones. Pyruvate contributed only 33% of the acetyl-CoA. Data aremeans � SD. *P 0.05 vs. respective Pyr.



DISCUSSION

In the presence of long-chain fatty acids bound to albuminplus other substrates in physiological concentrations and understeady-state conditions, the contribution of pyruvate to acetyl-CoA and therefore to CO2 and bicarbonate is very sensitive tothe concentration of pyruvate. At 3 mM pyruvate, �80% ofacetyl-CoA was derived from pyruvate under conditions mim-icking the fed state. In the presence of competing substratesmimicking the fasted state, only �33% of acetyl-CoA wasderived from pyruvate even at 6 mM. These results are con-sistent with the known regulation of flux through PDH by bothend-product inhibition and phosphorylation-dephosphoryla-

tion. PDH kinase phosphorylates PDH, thereby inactivating theenzyme, and, since pyruvate inhibits PDH kinase (9, 21, 40,44), the presence of pyruvate would be expected to stimulatepyruvate oxidation. Earlier reports have shown that oxidationof pyruvate is suppressed by fatty acids and ketones and thatthe effects of fatty acids on pyruvate oxidation are overcomeby increasing [pyruvate] (2, 4, 7, 8, 11, 20, 21, 23, 24, 36, 39,

Fig. 5. Influence of [pyruvate] on hemodynamic performance of the isolatedworking heart. “Pre” and “Post” refer to measurements immediately before and5 min after switching to 6, 10, 15, or 25 mM pyruvate, respectively. Within the1st min of switchover, there was a variable decrease in developed pressure thatrecovered quickly. Heart rate, developed pressure, and O2 consumption werenot altered substantially by exposure to graded [pyruvate]. Data are means �SD. All pre vs. post data sets were not significantly different from each otherfor heart rate and myocardial O2 consumption (MV̇O2). Developed pressuredecreased significantly with all concentrations included in the repeated-mea-sures model (pre vs. post difference 13.3 mmHg, 95% confidence interval,8.8–17.9 mmHg, P 0.0001). However, no significant pre vs. post differenceswere observed within any concentration, analyzed with Bonferroni-Holmadjusted multiple comparisons.

Fig. 6. 13C Spectra of isolated hearts exposed to HP [1-13C]pyruvate. A: 3 mMHP [1-13C]pyruvate injection in heart in the presence of 3% BSA and no fattyacids. B: 3 mM HP [1-13C]pyruvate injection in heart in the presence of 3%BSA and 0.4 mM fatty acids. C: 20 mM HP [1-13C]pyruvate injection in heartin the presence of 3% BSA and no fatty acids. D: 20 mM HP [1-13C]pyruvateinjection in heart in the presence of 3% BSA and 0.4 mM fatty acids.

Fig. 7. Effect of albumin on the spin-lattice relaxation time (T1) of 2.5 mM[1-13C]pyruvate. �, Fatty acids present; �, no fatty acids present. When fattyacids were not present, T1 times were not able to be determined at [albumin]�2%. The two data sets were significantly different from each other at[albumin] �0.5%.



43, 49, 54, 55). However, competition between pyruvate andphysiological mixtures of competing substrates for oxidation inthe heart during graded changes in [pyruvate] has not beencarefully examined previously.

The current results demonstrate that, even at 1 mM, pyruvatebegins to suppress oxidation of both fatty acid and ketones. Ator above 3 mM, pyruvate is the dominant oxidative fuel for theheart when the competing substrates are typical of the fed state.These findings confirm those of Schroeder et al. (45), whichshowed H13CO3

� production leveling off with an infusion ofHP [1-13C]pyruvate in a rat heart between 40 and 80 mM. Theauthors calculated that an 80 mM pyruvate infusion equated a5.3 mM myocardial [pyruvate] based on a 10-s infusion timeand 15 ml of blood passing through the heart. Drake et al. (11)and Laughlin et al. (24) have shown similar findings thatincreased lactate concentration or [pyruvate] can be the pre-ferred substrate for the heart over fatty acids or glucose.However, the effect of 6 mM pyruvate on ketone and fatty acidoxidation is blunted under conditions mimicking the fastingstate, which is similar to in vivo results obtained by Schroederet al. (46). The authors report an �74% decrease in H13CO3

�

production in fasted animals following an 80 mM HP[1-13C]pyruvate injection (46). Similarly, the current studiesshow an �60% reduction in the utilization of pyruvate inisolated rat hearts perfused under fasted conditions. Theseresults relate well to studies by Olson (37) and Bassenge et al.(3) that showed elevated ketone body concentration duringfasting will suppress carbohydrate utilization in the heart. Thisconcentration of pyruvate, 6 mM, is at the upper limit ofsteady-state plasma [pyruvate] used in many previous studies(10, 16–18, 22, 24, 45, 56).

The isolated heart preparation is well suited to studies ofsubstrate competition because all concentrations can be pre-cisely controlled. Furthermore, the general features of substrateutilization measured in the isolated heart match results in vivo.For example, long-chain fatty acids are preferred over carbo-hydrates in vivo and in isolated hearts (2, 4, 43, 51). Ketonebodies inhibit fatty acid and glucose oxidation in both prepa-rations during fasting (3, 37), and elevated pyruvate or lactateeffectively competes with both fatty acids and glucose in bothpreparations as well (11, 24). Although there is a good generalagreement between in vivo studies and results from isolatedhearts, the relevance of results in isolated tissues must beconfirmed in vivo. From the current results, we anticipate thatintravenously administered HP pyruvate may not provide thesame quality images under conditions of hyperketonemia com-pared with control.

The nutritional state of the heart is not the only factor thataffects substrate utilization, but the disease state will as well.Several studies have shown decreased H13CO3

� production in adiseased state after administration of HP [1-13C]pyruvate (15,32, 46). Schroeder et al. (46) demonstrated that streptozotocin-treated rats have decreased PDH flux compared with controls.Golman et al. (15) and Merritt et al. (32) determined that PDHflux is near zero after ischemia. All three studies fall within theoptimal range of myocardial [pyruvate] determined by Schr-oeder et al. (45) and this work for imaging.

These experiments illustrate a fundamental difference be-tween studies of cardiac metabolism by HP [1-13C]pyruvatecompared with SPECT or PET. Radiotracers are administeredin such low concentration that the effect of the labeled sub-

strate on metabolism is negligible. However, for detection ofHP H13CO3

�, a significant mass of HP [1-13C]pyruvate must beadministered to overcome the presence of competing sub-strates, and the mass of administered pyruvate itself influencesmetabolism. Consequently, metabolic exams with [1-13C]pyru-vate are not tracer studies in the conventional sense.

Furthermore, the potential toxicity of pyruvate must beconsidered. The metabolic consequences of high-dose pyruvateare likely to be complex in the heart, since the cytosol willbecome more oxidized (decreased NADH/NAD�) because offlux of pyruvate through lactate dehydrogenase. Pyruvate ox-idation in the mitochondria will have the opposite effect, anincrease in NADH/NAD�. In spite of these metabolic effects,in the current experiments, there was no evidence for adversehemodynamic events up to �10 mM, and even at higherconcentrations there was only a transient decrease in cardiacoutput and no change in developed pressure. It is not possibleto extrapolate these data to in vivo conditions, especially in thesetting of heart disease or depressed cardiac output, and theisolated heart model by definition excludes possible effects ofpyruvate on the peripheral circulation. Nevertheless, theseresults are consistent with earlier studies that emphasized a rolefor pyruvate as a therapeutic agent. Typically, the target con-centration was between 2 and 6 mM, but a significant cardio-protective effect was reported among patients exposed to a 10mM pyruvate-fortified cardioplegia solution during coronaryartery bypass surgery (35). A preliminary conclusion is that areasonable target concentration in the coronary arteries is 3–6mM to suppress oxidation of competing substrates and that theheart may tolerate exposure to significantly higher concentra-tions.

These observations from 13C isotopomer analysis of tissueextracts suggested that HP 13CO2 and H13CO3

� should bereadily observed in isolated hearts supplied with the samemixture of substrates. This anticipated result would be consis-tent with the finding by Golman and colleagues (13–15) thatimaging of HP H13CO3

� in the heart is feasible in vivo.Because the bolus dose of HP [1-13C]pyruvate was small inthose studies (0.3 mmol/kg) compared with earlier studies oftherapeutic pyruvate [0.9 mmol/kg (22) or 1.5 mmol/kg (56)],one might anticipate that the concentration of HP pyruvate wasmodest in the coronary arteries in those studies. Consequently,it was not anticipated that H13CO3

� could not be detected fromthe isolated heart supplied with albumin and competing sub-strates. Because the concentration of pyruvate was clearlysufficient to provide much of the acetyl-CoA oxidized in theTCA cycle, this finding cannot be the result of simply inhibi-tion of pyruvate oxidation by competing substrates. Two fac-tors likely play a role.

Attenuation of the pyruvate signal (Fig. 6A) in the presenceof fatty acid-free albumin demonstrated that the longitudinalrelaxation rate of pyruvate was significantly increased byalbumin, an observation confirmed in separate experimentswith graded concentrations of fatty acid-free albumin (Fig. 7).The mechanism of the adverse effect of albumin on pyruvateT1 has not been investigated extensively. Albumin has anaffinity for numerous small molecules (41). The albumin typ-ically used in isolated tissue experiments is extensively pro-cessed with the specific objective of removing most of theseligands. In cardiac studies, albumin is generally used to deliverphysiological long-chain fatty acids to the heart since these



substrates are otherwise insoluble. However, these experimentsillustrate that highly purified albumin may adversely affectexperiments with HP pyruvate. Chatham and Forder (6) re-ported disappearance of the 1H NMR signal of pyruvate withthe addition of BSA, and Stepuro et al. (52) described analbumin-catalyzed exchange of pyruvate methyl protons withthe solvent. These earlier experiments demonstrate that, undersome conditions, pyruvate binds to albumin. The current re-sults demonstrate that the magnitude of the effect of albuminon pyruvate T1 is sensitive to the presence of albumin ligands.It is likely that the effect is less significant in vivo wherenumerous other ligands such as bilirubin, porphyrins, andmetal ions are present, and it is conceivable that the concen-tration of fatty acids is often higher in vivo than used in thecurrent study.

A second factor is the duration of exposure of the heart tohigh concentrations of pyruvate. A stepped but transient in-crease in [pyruvate] as would be expected from a bolusinjection may produce a different set of metabolic conditionscompared with a sustained change in concentration of pyruvateover 20–30 min. Under the latter conditions, designed toproduce metabolic steady state, fluxes through all reactionsfeeding the TCA cycle are fixed. Although the rate of phos-phorylation-dephosphorylation of PDH is very rapid (21, 36),the input function of a bolus of pyruvate may have a complexeffect on the concentration of the injectate in the coronaryarteries (1) and on fluxes through PDH during the first fewseconds as the [pyruvate] changes.

Together, these experiments plus earlier studies with iso-lated hearts demonstrate that the appearance of HP 13CO2 andHP H13CO3

� after exposure to HP [1-13C]pyruvate is reducedby competing substrates and by exposure to albumin. Botheffects are concentration dependent. These results suggest aconceptual framework for designing studies in isolated heartswith HP [1-13C]pyruvate. At low concentration of competingsubstrates, most of the CO2 production will be the result ofoxidation of pyruvate, and the rate of 13CO2 appearance will benear-maximal even at relatively low [pyruvate], �3 mM. Asthe concentration of competing substrates increases or as theneurohumoral environment changes, the rate of CO2 produc-tion from pyruvate will be reduced but can be recovered athigher [pyruvate] in the coronary arteries. Eventually, how-ever, the delivery of high concentrations of pyruvate will belimited by practicalities such as osmolality or toxicity. Tech-nical factors such as the effects of highly purified albumin onthe T1 of pyruvate must also be considered. In vivo, the effectsof albumin are likely to be less significant, but the concentra-tion of competing substrates will significantly influence theappearance of HP 13CO2 and HP H13CO3

�.

ACKNOWLEDGMENTS

Angela Milde and Charles Storey provided outstanding technical support.We appreciate expert advice by William Mander from Oxford InstrumentsMolecular Biotools and Beverley Huet from the Division of Biostatistics at theUniversity of Texas Southwestern Medical Center.

GRANTS

This study was supported by National Institutes of Health Grants RR-02584and HL-34557.

DISCLOSURES

No conflicts of interest are declared by the authors.

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