NMR indirect detection of glutamate to measure citric acid cycle flux in the isolated perfused mouse heart.

(13)C-edited proton nuclear magnetic resonance (NMR) spectroscopy was used to follow enrichment of glutamate C3 and C4 with a temporal resolution of approximately 20 s in mouse hearts perfused with (13)C-enriched substrates. A fit of the NMR data to a kinetic model of the tricarboxylic acid (TCA) cycle and related exchange reactions yielded TCA cycle (V(tca)) and exchange (V(x)) fluxes between alpha-ketoglutarate and glutamate. These fluxes were substrate-dependent and decreased in the order acetate (V(tca)=14.1 micromol g(-1) min(-1); V(x)=26.5 micromol g(-1) min(-1))>octanoate (V(tca)=6.0 micromol g(-1) min(-1); V(x)=16.1 micromol g(-1) min(-1))>lactate (V(tca)=4.2 micromol g(-1) min(-1); V(x)=6.3 micromol g(-1) min(-1)).

An integrated (2)H and (13)C NMR study of gluconeogenesis and TCA cycle flux in humans.

Hepatic glucose synthesis from glycogen, glycerol, and the tricarboxylic acid (TCA) cycle was measured in five overnight-fasted subjects by (1)H, (2)H, and (13)C NMR analysis of blood glucose, urinary acetaminophen glucuronide, and urinary phenylacetylglutamine after administration of [1,6-(13)C(2)]glucose, (2)H(2)O, and [U-(13)C(3)]propionate. This combination of tracers allows three separate elements of hepatic glucose production (GP) to be probed simultaneously in a single study: 1) endogenous GP, 2) the contribution of glycogen, phosphoenolpyruvate (PEP), and glycerol to GP, and 3) flux through PEP carboxykinase, pyruvate recycling, and the TCA cycle. Isotope-dilution measurements of [1,6-(13)C(2)] glucose by (1)H and (13)C NMR indicated that GP in 16-h-fasted humans was 10.7 +/- 0.9 micromol.kg(-1).min(-1). (2)H NMR spectra of monoacetone glucose (derived from plasma glucose) provided the relative (2)H enrichment at glucose H-2, H-5, and H-6S, which, in turn, reflects the contribution of glycogen, PEP, and glycerol to total GP (5.5 +/- 0.7, 4.8 +/- 1.0, and 0.4 +/- 0.3 micromol.kg(-1).min(-1), respectively). Interestingly, (13)C NMR isotopomer analysis of phenylacetylglutamine and acetaminophen glucuronide reported different values for PEP carboxykinase flux (68.8 +/- 9.8 vs. 37.5 +/- 7.9 micromol.kg(-1).min(-1)), PEP recycling flux (59.1 +/- 9.8 vs. 27.8 +/- 6.8 micromol.kg(-1).min(-1)), and TCA cycle flux (10.9 +/- 1.4 vs. 5.4 +/- 1.4 micromol.kg(-1).min(-1)). These differences may reflect zonation of propionate metabolism in the liver.

TCA cycle kinetics in the rat heart by analysis of (13)C isotopomers using indirect (1)H.

This study was designed to test the hypothesis that indirect (1)H[(13)C] detection of tricarboxylic acid (TCA) cycle intermediates using heteronuclear multiple quantum correlation-total correlation spectroscopy (HMQC-TOCSY) nuclear magnetic resonance (NMR) spectroscopy provides additional (13)C isotopomer information that better describes the kinetic exchanges that occur between intracellular compartments than direct (13)C NMR detection. NMR data were collected on extracts of rat hearts perfused at various times with combinations of [2-(13)C]acetate, propionate, the transaminase inhibitor aminooxyacetate, and (13)C multiplet areas derived from spectra of tissue glutamate were fit to a standard kinetic model of the TCA cycle. Although the two NMR methods detect different populations of (13)C isotopomers, similar values were found for TCA cycle and exchange fluxes by analyzing the two data sets. Perfusion of hearts with unlabeled propionate in addition to [2-(13)C]acetate resulted in an increase in the pool size of all four-carbon TCA cycle intermediates. This allowed the addition of isotopomer data from aspartate and malate in addition to the more abundant glutamate. This study illustrates that metabolic inhibitors can provide new insights into metabolic transport processes in intact tissues.

13C isotopomer analysis of glutamate by J-resolved heteronuclear single quantum coherence spectroscopy.

13C NMR isotopomer analysis is a powerful method for measuring metabolic fluxes through pathways intersecting in the tricarboxylic acid cycle. However, the inherent insensitivity of 13C NMR spectroscopy makes application of isotopomer analysis to small tissue samples (mouse tissue, human biopsies, or cells grown in tissue culture) problematic. (1)H NMR is intrinsically more sensitive than 13C NMR and can potentially supply the same information via indirect detection of 13C providing that isotopomer information can be preserved. We report here the use of J-resolved HSQC (J-HSQC) for 13C isotopomer analysis of tissue samples. We show that J-HSQC reports isotopomer multiplet patterns identical to those reported by direct 13C detection but with improved sensitivity.

Effects of storage and reperfusion oxygen content on substrate metabolism in the isolated rat lung.

BACKGROUND: Lung transplantation requires a period of storage and ischemia; we examined the largely unknown effects of that period on intermediary metabolism. METHODS: Two groups of isolated rat lung blocks (n = 16 each) were flushed with Euro-Collins solution and harvested. The lung blocks were immediately ventilated and either perfused for 30 minutes with an erythrocyte-based solution containing carbon 13 labeled substrates (group 1) or stored for 6 hours at 1 degree C and then reperfused (group 2). Half of each group was reperfused at a physiologic Po2 the other half at high Po2. Analysis of carbon 13 isotopomers was performed to determine substrate utilization through aerobic pathways in lung tissue. RESULTS: Lungs from both groups oxidized all major substrates. The contribution of fatty acids to acetylcoenzyme acid oxidized in the citric acid cycle was significantly higher in group 2 than in group 1 (31.3% +/- 2.2% versus 22.0% +/- 2.1%, p < 0.05). Perfusate Po2 did not affect substrate preference. Gas exchange was worse in stored lungs. CONCLUSIONS: After a period of hypothermic ischemia and storage, substrate preference in lung tissue exhibits a switch towards fatty acids. As fatty acid oxidation occurring after ischemia is deleterious in other organs, strategies to inhibit this process in stored lungs may warrant further investigation.

Effect of exercise on (23)Na MRI and relaxation characteristics of the human calf muscle.

The acute affect of voluntary muscle contractions performed by healthy volunteers was evaluated using (23)Na nuclear magnetic resonance (NMR). Three-dimensional gradient-echo (23)Na images, pulse-acquired spectra, and transverse relaxation times were obtained before and after ankle flexion-extension exercise. The muscle sodium concentration was calculated from (23)Na images using a 40 mM NaCl standard and the measured T(2) values. Before exercise the muscle sodium concentration was 26+/-4 mmole/kg wet weight. This agrees closely with literature values, suggesting that muscle Na(+) is fully NMR visible. The (23)Na image intensity increased by 34%+/-7% in the exercised muscle and diminished with a half-life of 30+/-6 minutes. The pulse-acquired spectra, however, did not show any significant change in muscle signal intensity following exercise, but the relative contribution of the slow T(2) component increased. The calculated sodium concentration also did not change significantly after the exercise. We therefore infer that the changes in (23)Na magnetic resonance imaging (MRI) were due to a change in sodium-macromolecular interaction rather than a change in tissue sodium content. We believe that this report represents the first study of (23)Na MRI of skeletal muscle.

Quantitation of gluconeogenesis by (2)H nuclear magnetic resonance analysis of plasma glucose following ingestion of (2)H(2)O.

We present a simple (2)H NMR assay of the fractional contribution of gluconeogenesis to hepatic glucose output following ingestion of (2)H(2)O. The assay is based on the measurement of relative deuterium enrichment in hydrogens 2 and 3 of plasma glucose. Plasma glucose was enzymatically converted to gluconate, which displays fully resolved deuterium 2 and 3 resonances in its (2)H NMR spectrum at 14.1 T. The signal intensity of deuterium 3 relative to deuterium 2 in the gluconate derivative as quantitated by (2)H NMR was shown to provide a precise and accurate measurement of glucose enrichment in hydrogen 3 relative to hydrogen 2. This measurement was used to estimate the fractional contribution of gluconeogenesis to hepatic glucose output for two groups of rats; one group was fasted for 7 h and the other was fasted for 29 h. Rats were administered (2)H(2)O to enrich total body water to 5% over the last 4-5 h of each fasting period. For the 7-h fasted group, the hydrogen 3/hydrogen 2 enrichment ratio of plasma glucose was 0.32 +/- 0.09 (n = 7). This indicates that gluconeogenesis contributed 32 +/- 9% of total hepatic glucose output with glycogenolysis contributing the remainder. For the 29-h fasted group, the hydrogen 3/hydrogen 2 enrichment ratio of plasma glucose was 0.81 +/- 0.10 (n = 6), indicating that gluconeogenesis supplied the bulk of hepatic glucose output (81 +/- 10%).

Use of a single (13)C NMR resonance of glutamate for measuring oxygen consumption in tissue.

A kinetic model of the citric acid cycle for calculating oxygen consumption from (13)C nuclear magnetic resonance (NMR) multiplet data has been developed. Measured oxygen consumption (MVO(2)) was compared with MVO(2) predicted by the model with (13)C NMR data obtained from rat hearts perfused with glucose and either [2-(13)C]acetate or [3-(13)C]pyruvate. The accuracy of MVO(2) measured from three subsets of NMR data was compared: glutamate C-4 and C-3 resonance areas; the doublet C4D34 (expressed as a fraction of C-4 area); and C-4 and C-3 areas plus several multiplets of C-2, C-3, and C-4. MVO(2) determined by set 2 (C4D34 only) gave the same degree of accuracy as set 3 (complete data); both were superior to set 1 (C-4 and C-3 areas). Analysis of the latter suffers from the correlation between citric acid cycle flux and exchange between alpha-ketoglutarate and glutamate, resulting in greater error in estimating MVO(2). Analysis of C4D34 is less influenced by correlation between parameters, and this single measurement provides the best opportunity for a noninvasive measurement of oxygen consumption.

Multiple bond 13C-13C spin-spin coupling provides complementary information in a 13C NMR isotopomer analysis of glutamate.

Most 13C nuclear magnetic resonance (NMR) isotopomer analyses relate a metabolic index of interest to populations of 13C isotopomers as reported by one-bond 13C-13C spin-spin couplings. Metabolic conditions that produce highly enriched citric acid cycle intermediates often lead to 13C NMR spectra of metabolites such as glutamate that show extra multiplets due to long-range couplings. It can be demonstrated from 13C NMR spectra of hearts perfused with mixtures of acetate plus propionate that multiplets in glutamate C2 arising from 3J25 coupling provide a direct readout of acetyl-CoA fractional enrichment (FC1 and FC3), while multiplets in glutamate C5 arising from 2J35 and 3J25 couplings quantitatively reflect enrichment of the anaplerotic substrate.

Multiple quantum filtered 23Na NMR spectroscopy of the isolated, perfused rat liver.

Isolated, perfused rat livers were examined by single-quantum (SQ) and double-quantum-filtered (DQ-filtered) 23Na spectroscopy during prolonged global ischemia and during perfusion with ouabain, low-buffer potassium, or lithium-enriched buffer. Baseline separation of the intracellular (Na(i)+) and extracellular (Na(e)+) sodium resonances using TmDOTP5- allowed a direct comparison of temporal changes in SQ versus DQ-filtered Na(i)+. The SQ Na(i)+ signal increased approximately 150% during the first 15 min of global ischemia and then remained relatively constant over the next 45 min, while the DQ-filtered signal steadily increased approximately 400% over the same 60 min period. In similar experiments in which all perfusate sodium was replaced by lithium, the DQ-filtered Na(i)+ signal increased approximately 180% over a similar period of ischemia. Exposure of livers to ouabain also resulted in larger increases in DQ-filtered versus SQ signal of Na(i)+. The approximately 290% increase in DQ-filtered sodium observed during perfusion of livers with a hypokalemic buffer (1.2 mM K+) could be completely reversed by continued perfusion with a buffer containing normal levels of K+ (4.7 mM). These data suggest that the DQ-filtered Na(i)+ signal of liver does not simply report an increase in [Na(i)+], but may be exquisitely sensitive to other intracellular events initiated by altered physiology.

Distribution of TmDOTP5- in rat tissues: TmDOTP5- vs. CoEDTA- as markers of extracellular tissue space.

The distribution of TmDOTP5- in rat tissue was compared with CoEDTA-, an anionic complex previously used as a marker of extracellular space. Heart, liver, muscle, blood, and urine were collected from rats after infusion of either complex and were quantitatively analyzed by atomic absorption spectroscopy. Although total TmDOTP5- in blood and tissue was consistently lower (0.88 +/- 0.04; n = 6) than CoEDTA- after an identical infusion protocol (presumably because of some association of the phosphonate complex with bone), a comparison of blood and tissue contents indicated that the two anionic complexes distributed into identical extracellular spaces. Relative extracellular space in the in vivo liver, as determined by TmDOTP5- and CoEDTA-, was 0.18 +/- 0.02 and 0.15 +/- 0.01, respectively. The corresponding relative extracellular space values for the in vivo heart reported by the two agents were identical (0. 11 +/- 0.02). Experiments were also performed to evaluate the washout kinetics of TmDOTP5- from anesthesized rats. In rats given a total dose of 0.16 mmol TmDOTP5-, 81% appeared in urine by 180 min, <2% was found in all remaining soft tissue, leaving approximately 18% undetected. The rate of Tm appearance in urine was fit to a standard pharmacokinetic model that included four tissue compartments: plasma, one fast equilbrating space, one slow equilibrating space, and one very slow equilibrating space (presumably bone). The best fit result suggests that the highly charged TmDOTP5- complex is cleared from plasma more rapidly than is the typical lower charged Gd-based contrast agents and that release from bone is slow compared with renal clearance.

Quantitation of intracellular [Na+] in vivo by using TmDOTP5- as an NMR shift reagent and extracellular marker.

A method is presented to measure the absolute concentration of intracellular Na+ ([Na+]i) in vivo by using interleaved 23Na- and 31P-nuclear magnetic resonance (NMR) spectroscopy and TmDOTP5- as shift reagent and chemical marker of tissue extracellular space (ECS). The technique was used to determine [Na+]i and relative ECS in livers of control rats (21 +/- 3 and 0.11 +/- 0.02 mM, respectively) and in rats exposed to carbon tetrachloride (103 +/- 29 and 0.23 +/- 0.03 mM, respectively). The NMR measurements were confirmed independently on excised tissue samples by using atomic absorption spectroscopy. The results confirm that TmDOTP5- can be used as a combined cation shift reagent and ECS marker, thereby allowing quantitation of [Na+]i in vivo by NMR.

13C NMR measurements of human gluconeogenic fluxes after ingestion of [U-13C]propionate, phenylacetate, and acetaminophen.

Anaplerotic, pyruvate recycling, and gluconeogenic fluxes were measured by 13C isotopomer analysis of plasma glucose, urinary phenylacetylglutamine, and urinary glucuronide in normal, 24-h-fasted individuals after ingestion of [U-13C]propionate, phenylacetate, and acetaminophen. Plasma glucose isotopomer analysis reported a total anaplerotic flux of 5.92 +/- 1.03 (SD) relative to citrate synthase. This was not significantly different from glucuronide and phenylacetylglutamine analyses (6.08 +/- 1.16 and 7. 14 +/- 1.94, respectively). Estimates of pyruvate recycling from glucose and glucuronide isotopomer distributions were almost identical (3.55 +/- 0.99 and 3.66 +/- 1.11, respectively), whereas phenylacetylglutamine reported a significantly higher estimate (5.74 +/- 2.13). As a consequence, net gluconeogenic flux reported by phenylacetylglutamine (1.41 +/- 0.28) was significantly less than that reported by glucose (2.37 +/- 0.64) and glucuronide (2.42 +/- 0. 76). This difference in fluxes detected by analysis of phenylacetylglutamine vs. hexose is likely due to compartmentation of hepatic metabolism of propionate. Net gluconeogenic flux estimates made by use of this stable isotope method are in good agreement with recent measurements in humans with [14C]propionate.

39K NMR measurement of intracellular potassium during ischemia in the perfused guinea pig heart.

The hyperfine shift reagent, TmDOTP5-, was used to resolve the 39K NMR resonances of intra- (Ki+) and extracellular (Ke+) potassium in isolated, perfused guinea pig hearts. [Ki+] as measured by 39K NMR was 25.9 +/- 10.3 mM, compared with 114.4 +/- 10.8 mM as measured by atomic absorption spectroscopy (AAS) using TmDOTP5- as a marker of extracellular space. Thus, only approximately 23% of intracellular potassium was detected by 39K NMR using our experimental conditions. The area of the Ki+ signal increased during early ischemia then returned to baseline levels during reperfusion. In an effort to learn more about the Ki+ not detected by 39K NMR, hearts were perfused with a Rb+-enriched, K+-depleted buffer for an extended period. This resulted in loss of the entire 39K NMR signal, and Ki+, as measured by AAS, decreased from approximately 60 to approximately 6 to 7 micromol/g wet weight. When K+-depleted hearts were subjected to global ischemia, a small 39K NMR signal reappeared, suggesting that at least a portion of the nonexchangeable Ki+ becomes detectable by NMR during ischemia. This newly visible K+ signal subsequently dissipated during reperfusion of ischemic hearts. We conclude that ischemia induces changes in the NMR visibility of 39K in perfused guinea pig hearts.

Measurement of hepatic glucose output, krebs cycle, and gluconeogenic fluxes by NMR analysis of a single plasma glucose sample.

13C and 1H NMR spectroscopy of plasma glucose was used to resolve the isotopomer contributions from tracer levels of [1,6-13C2]glucose, a novel tracer of glucose carbon skeleton turnover, and [U-13C]propionate, a tracer of hepatic citric acid cycle metabolism. This allowed simultaneous measurements of hepatic glucose production and citric acid cycle fluxes from the NMR analysis of a single plasma glucose sample in fasted animals. Glucose carbon skeleton turnover, as reported by the dilution of [1,6-13C2]glucose, was 56 +/- 2 micromol/kg/min in the presence of labeling from [U-13C]propionate and 53 +/- 4 micromol/kg/min in its absence. Therefore, as expected, the labeling contributions from [U-13C]propionate metabolism did not have a significant effect on the measurement of glucose turnover. For the group infused with both tracers, citric acid cycle flux estimates from the analysis of glucose C2 isotopomer ratios were consistent with those from our recent experiments where only [U-13C]propionate was infused, verifying that the presence of [1,6-13C2]glucose did not interfere with these measurements. This integrated analysis of hepatic glucose output and citric acid cycle fluxes from plasma glucose isotopomers yielded a noninvasive estimate of hepatic citrate synthase flux of 74 +/- 12 micromol/kg/min for 24-h fasted rats.

Dissociation of intracellular sodium from contractile state in guinea-pig hearts treated with ouabain.

The positive inotropic effect of cardiac glycosides has been attributed to inhibition of the Na-K-ATPase, accumulation of intracellular sodium and enhanced calcium availability due to Na-Ca exchange. However, few measurements of intracellular sodium in the functioning left ventricle following ouabain exposure at therapeutic doses are available. Our experimental objective was to quantitate the relationship between contractile state and intracellular sodium measured by 23Na nuclear magnetic resonance spectroscopy or atomic absorption in the intact heart. Isolated guinea-pig hearts, perfused in the Langendorff mode, were paced and then exposed to ouabain (3x10(-7)m) for 30 min. Left-ventricular pressure was monitored continuously. Intracellular sodium was measured either at 1-min intervals throughout the perfusion by shift reagent-aided 23Na nuclear magnetic resonance spectroscopy in the beating heart or following 30 minutes of perfusion by atomic absorption in myocardial tissue. While treatment with ouabain was associated with almost a two-fold rise in developed pressure, there was no significant increase in intracellular sodium measured by either technique. Thus, the positive inotropic effect of ouabain in this model is not associated with significant changes in bulk intracellular sodium. However, these results do not exclude the possibility of shifts between intracellular pools which would not be detected in bulk measurements, or changes in NMR-invisible intracellular pools which are not detectable by single quantum spectroscopy techniques.

Effects of aminooxyacetate on glutamate compartmentation and TCA cycle kinetics in rat hearts.

The nonspecific transaminase inhibitor aminooxyacetate (AOA) has multiple influences on the dynamics of 13C appearance in glutamate in rat hearts as measured by 13C nuclear magnetic resonance (NMR) without altering O2 consumption or tricarboxylic acid (TCA) cycle flux. These include the following: 1) a reduced rate of 13C enrichment at glutamate C3 and C4; 2) a near coalescence of the C3 and C4 fractional enrichment curves; 3) a dramatic alteration in the time-dependent evolution of the glutamate C4 multiplets, C4S and C4D34; and 4) a decrease in the NMR visibility of glutamate. A fit of the 13C fractional enrichment curves of glutamate C4 and C3 in the absence of inhibitor to a kinetic model of the TCA cycle gave values for transaminase flux of 7.5 mumol.min-1.g dry wt-1 and TCA cycle flux of 7.5 mumol.min-1.g dry wt-1, thereby confirming reports by others that the kinetics of 13C enrichment of glutamate C3 and C4 in heart tissue is significantly affected by flux through reactions other than TCA cycle. The 13C fractional enrichment data collected in the presence of 0.5 mM AOA could not be fitted using this same kinetic model. However, kinetic simulations demonstrated that the time-dependent changes in C4S and C4D34 are only consistent with a 10-fold reduction in the size of intermediate pools undergoing rapid turnover in the TCA cycle. We conclude that inhibition of glutamic-oxalacetic transaminase by AOA effectively reduces the size of the alpha-ketoglutarate pool in rapid exchange with the TCA cycle. Our data indicate that changes in glutamate multiplet areas in the 13C NMR spectra of heart (as demonstrated by glutamate C4S and C4D34) are more sensitive to alterations in metabolic pool sizes in exchange with the TCA cycle than are measurements of 13C fractional enrichment at glutamate C3 and C4.

Effects of dichloroacetate on mechanical recovery and oxidation of physiologic substrates after ischemia and reperfusion in the isolated heart.

The effects of dichloroacetate (DCA) on fatty acid oxidation and flux through pyruvate dehydrogenase (PDH) were studied in ischemic, reperfused myocardium supplied with glucose, long-chain fatty acids, lactate, pyruvate, and acetoacetate. The oxidation rates of all substrates were determined by combined 13C nuclear magnetic resonance (NMR) spectroscopy and oxygen-consumption measurements, and PDH flux was assessed by lactate plus pyruvate oxidation. In nonischemic control hearts, DCA increased PDH flux more than eightfold (from 0.68 +/- 0.28 to 5.81 +/- 1.16 micromol/min/g dry weight; n = 8 each group; p < 0.05) and significantly inhibited the oxidation of acetoacetate and fatty acids. DCA also improved mechanical recovery after 30 min of ischemia plus 30 min of reperfusion but did not significantly increase PDH flux measured at the end of the reperfusion period (1.35 +/- 0.42 micromol/min/g dry weight) compared with untreated ischemic hearts (0.87 +/- 0.28 micromol/min/g dry weight; n = 8 each group; p = NS). Although DCA had a modest effect on functional recovery in the reperfused myocardium, this beneficial effect was not associated with either marked stimulation of PDH flux or inhibition of fatty acid oxidation.

Right-shifting the oxyhemoglobin dissociation curve with RSR13: effects on high-energy phosphates and myocardial recovery after low-flow ischemia.

RSR13[2-(4[[(3,5-Dimethylanilino)carbonyl] methyl] phenoxy)-2-methyl propionic acid], a synthetic allosteric modifier of hemoglobin, increases O2 release from hemoglobin at low oxygen tension. The isolated blood-perfused rat heart was examined during potassium-arrest to determine the effects of RSR13 on the concentration of phosphocreatine (PCr) and adenosine triphosphate (ATP) by using 31P nuclear magnetic resonance (NMR) spectroscopy throughout an episode of low-flow ischemia. All hearts were perfused at constant flow during control (2.0 ml/min) and low-flow (0.2 ml/min) conditions. In normoxic hearts, RSR13 had no effect on either the 31P NMR spectrum or the rate-pressure product. In hearts subjected to 30 min of reduced flow, treatment with RSR13 improved mechanical function on reperfusion (p = 0.026 after 20 min; p = 0.032 after 25 min; and p = 0.045 after 30 min) at 2.0 ml/min with normokalemic blood perfusate. In potassium-arrested hearts, the rate of decrease of [ATP] was reduced in hearts exposed to RSR13 (p < or = 0.05 between 10 and 35.8 min of ischemia except at 28.4 min) during low flow. These results indicate a protective effect of RSR13 on high-energy phosphates during low-flow ischemia and mechanical recovery after reperfusion.