RESUMO
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.
Assuntos
Ácido Glutâmico/análise , Espectroscopia de Ressonância Magnética/métodos , Miocárdio/metabolismo , Consumo de Oxigênio/fisiologia , Acetatos/farmacologia , Animais , Isótopos de Carbono , Ciclo do Ácido Cítrico/fisiologia , Glucose/farmacologia , Técnicas de Cultura de Órgãos , Consumo de Oxigênio/efeitos dos fármacos , Prótons , Ácido Pirúvico/farmacologia , Ratos , Análise de RegressãoRESUMO
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.
Assuntos
Compostos de Anilina/farmacologia , Isquemia Miocárdica/metabolismo , Oxiemoglobinas/efeitos dos fármacos , Fosfatos/metabolismo , Propionatos/farmacologia , Animais , Técnicas In Vitro , Espectroscopia de Ressonância Magnética , Masculino , Isquemia Miocárdica/fisiopatologia , Oxiemoglobinas/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
A previous model using 13C nuclear magnetic resonance isotopomer analysis provided for direct measurement of the oxidation of 13C-enriched substrates in the tricarboxylic acid cycle and/or their entry via anaplerotic pathways. This model did not allow for recycling of labeled metabolites from tricarboxylic acid cycle intermediates into the acetyl-CoA pool. An extension of this model is now presented that incorporates carbon flow from oxaloacetate or malate to acetyl-CoA. This model was examined using propionate metabolism in the heart, in which previous observations indicated that all of the propionate consumed was oxidized to CO2 and water. Application of the new isotopomer model shows that 2 mM [3-13C]propionate entered the tricarboxylic acid cycle as succinyl-CoA (an anaplerotic pathway) at a rate equal to 52% of tricarboxylic acid cycle turnover and that all of this carbon entered the acetyl-CoA pool and was oxidized. This was verified using standard biochemical analysis; from the rate (mumol.min-1.g dry wt-1) of propionate uptake (4.0 +/- 0.7), the estimated oxygen consumption (24.8 +/- 5) matched that experimentally determined (24.4 +/- 3).
Assuntos
Acetilcoenzima A/metabolismo , Animais , Ácido Aspártico/metabolismo , Isótopos de Carbono , Ciclo do Ácido Cítrico , Ácido Glutâmico/metabolismo , Espectroscopia de Ressonância Magnética , Masculino , Modelos Biológicos , Oxaloacetatos/metabolismo , Oxirredução , Propionatos/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Several substrates are available in vivo for oxidation by the myocardium. Although substrate selection has been studied extensively in normoxic myocardium, relatively little is known about substrate preference very early during reperfusion after ischemia. Carbon-13 isotopomer analysis was used to study substrate usage by nonischemic and reperfused-ischemic myocardium in a working heart that was subjected to 15 min or regional ischemia and reperfused for 5 min. Compared with nonischemic myocardium, the contribution of acetoacetate to acetyl coenzyme A was increased in the reperfused-ischemic region, and the contribution of exogenous lactate was decreased. Free fatty acid oxidation, however, was not different in the two regions. These results indicate that (1) early during reperfusion, ketone body oxidation may be more significant than has been emphasized, (2) the relative contribution of fatty acids to acetyl coenzyme A is not sensitive to ischemia followed by reperfusion, and (3) Carbon-13 magnetic resonance spectroscopy methods may be used for analysis of spatial heterogeneity of metabolism in the heart.
Assuntos
Espectroscopia de Ressonância Magnética , Reperfusão Miocárdica , Miocárdio/metabolismo , Acetoacetatos/metabolismo , Acetilcoenzima A/metabolismo , Animais , Butiratos/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Glucose/metabolismo , Glutamatos/metabolismo , Técnicas In Vitro , Lactatos/metabolismo , Ácido Láctico , Masculino , CoelhosRESUMO
13C and 31P NMR were used to evaluate exogenous substrate utilization and endogenous phosphate metabolites in perfused rat hearts exposed to tert-butylhydroperoxide (tert-BOOH) and hydrogen peroxide (H2O2). Both reagents caused a reduction in developed pressure compared to controls and, in agreement with previous 31P NMR data, had different effects on intracellular high-energy phosphates and glycolysis. 13C Isotopomer analysis of tissue extracts showed that H2O2 and tert-BOOH also had significantly different effects on substrate utilization by the citric acid cycle. The contribution of exogenous lactate and glucose to acetyl-CoA was 43% in controls and increased to over 80% in the presence of either oxidant. With tert-BOOH, exogenous glucose and lactate were both significant contributors to acetyl-CoA (44 +/- 2 and 41 +/- 3%). However, with H2O2, exogenous lactate supplied a much higher fraction of acetyl-CoA (72 +/- 2%) than glucose (9 +/- 1%). Also, when [2-(13)C] glucose was supplied, accumulation of [2-(13)C] and [5-(13)C] fructose 1,6-bisphosphate was observed in the presence of H2O2, indicating inhibition of glyceraldehyde-3-phosphate dehydrogenase. These results indicate that despite this glycolytic inhibition, H2O2 increased the utilization of pyruvate precursors when lactate was present as an alternative carbohydrate substrate.
Assuntos
Miocárdio/metabolismo , Animais , Ciclo do Ácido Cítrico/fisiologia , Ácido Glutâmico/metabolismo , Glutationa/metabolismo , Técnicas In Vitro , Espectroscopia de Ressonância Magnética , Masculino , Contração Miocárdica/fisiologia , NADP/metabolismo , Oxirredução , Perfusão , Fósforo , Ratos , Ratos Sprague-DawleyRESUMO
A spin-echo method is presented for obtaining high resolution, 13C coupled, proton spectra of lactate and alanine in intact, beating rat hearts. All hearts were depleted of glycogen prior to prolonged perfusion with either 10 mM unenriched glucose or [1-13C]glucose to restore glycogen. These two groups of hearts were then examined by 1H NMR during prolonged global (zero flow) or low pressure (low flow) ischemia. During global ischemia, lactate was derived from both glucose and glycogen, with endogenous glycogen contributing twice as much lactate as exogenous glucose. During low perfusion pressure ischemia, however, lactate was derived exclusively from exogenous glucose. The entire pool of lactate (both 12C and 13C) was visible by NMR in intact, glucose perfused hearts while alanine was not detected. However, upon adding 10 mM pyruvate to the perfusate, the entire alanine pool became NMR visible while some of the lactate became NMR invisible. These observations indicate that the NMR visibility of small, usually highly mobile metabolites such as alanine and lactate is not always 100% in intact hearts and that the NMR visibility of these molecules may depend upon which exogenous substrate is presented to the heart.
Assuntos
Alanina/análise , Lactatos/análise , Espectroscopia de Ressonância Magnética , Isquemia Miocárdica/metabolismo , Miocárdio/metabolismo , Animais , Glicogênio/análise , Coração/efeitos dos fármacos , Coração/fisiologia , Isoproterenol/farmacologia , Ácido Láctico , Espectroscopia de Ressonância Magnética/métodos , Masculino , Perfusão , Ratos , Ratos Sprague-DawleyRESUMO
A new 13C NMR technique for measuring substrate utilization by the citric acid cycle based on an analysis of succinate 13C isotopomers is presented. The relative contribution of up to three different labeling patterns in acetyl-CoA entering the citric acid cycle may be determined under non-steady-state conditions. We present experimental data from perfused rat hearts subjected to a brief period of ischemia, where both succinate and glutamate resonances were observed in the 13C spectrum. The contributions of labeled exogenous acetate and lactate and unlabeled sources to the acetyl-CoA pool were compared using this succinate analysis and a previously published glutamate analysis [Malloy et al. (1990) Biochemistry 29, 6756-6761], and the two methods give identical results. This indicates that the succinate and glutamate isotopomers originated from a common alpha-ketoglutarate pool, verifying that glutamate is in isotopomeric equilibrium with alpha-ketoglutarate under these conditions.
Assuntos
Acetilcoenzima A/metabolismo , Ciclo do Ácido Cítrico , Succinatos , Animais , Isótopos de Carbono , Espectroscopia de Ressonância Magnética , Masculino , Prótons , Ratos , Ratos Sprague-Dawley , Ácido SuccínicoRESUMO
The effect of aspartate and glutamate on myocardial function during reperfusion is controversial. A beneficial effect has been attributed to altered delivery of carbon into the citric acid cycle via substrate oxidation or by stimulation of anaplerosis, but these hypotheses have not been directly tested. 13C isotopomer analysis is well suited to the study of myocardial metabolism, particularly where isotopic and metabolic steady state cannot be established. This technique was used to evaluate the effects of aspartate and glutamate (amino acids, AA) on anaplerosis and substrate selection in the isolated rat heart after 25 min of ischemia followed by 30 or 45 min of reperfusion. Five groups of hearts (n = 8) provided with a mixture of [1,2-13C]acetate, [3-13C]lactate, and unlabeled glucose were studied: control, control plus AA, ischemia followed by 30 min of reperfusion, ischemia plus AA followed by 30 min of reperfusion, and ischemia followed by 45 min of reperfusion. The contribution of lactate to acetyl-CoA was decreased in postischemic myocardium (with a significant increase in acetate), and anaplerosis was stimulated. Metabolism of 13C-labeled aspartate or glutamate could not be detected, however, and there was no effect of AA on functional recovery, substrate selection, or anaplerosis. Thus, in contrast to earlier reports, aspartate and glutamate have no effect on either functional recovery from ischemia or on metabolic pathways feeding the citric acid cycle.
Assuntos
Aminoácidos/metabolismo , Ácido Aspártico/farmacologia , Glutamatos/farmacologia , Coração/efeitos dos fármacos , Reperfusão Miocárdica , Miocárdio/metabolismo , Acetilcoenzima A/metabolismo , Alanina/metabolismo , Análise de Variância , Animais , Ácido Glutâmico , Técnicas In Vitro , Lactatos/metabolismo , Espectroscopia de Ressonância Magnética , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Depletion of high-energy phosphates, accumulation of inorganic phosphate and intracellular acidosis have each been proposed as important events in the transition from reversible to irreversible ischemic injury. To assess whether each variable is predictive of functional recovery on reperfusion, these were measured in the isolated isovolumic rat heart using 31P NMR. Perfused hearts were subjected to either 10, 12 or 40 min of normothermic ischemia followed by 40 min of reperfusion. Hearts were then freeze-clamped for further analysis of phosphate metabolites by NMR and ion chromatography. High-energy phosphates, Pi, phosphomonoesters and pH were measured by 31P NMR spectroscopy at 2 minute intervals. Heart rate and developed pressure were monitored simultaneously. All hearts undergoing 10 min of ischemia and 40% of hearts subjected to 12 min of ischemia demonstrated good functional recovery. The remainder of hearts ischemic for 12 min went into contracture on reperfusion with little return of function. Hearts subject to 40 min of ischemia went into ischemic contracture and showed no recovery on reperfusion. Intracellular pH, [ATP], and [Pi] measured prior to reperfusion did not predict the extent of recovery. However, phosphomonoesters were detected prior to reperfusion in all hearts that did not recover well, but were not observed in hearts that showed good mechanical recovery. Analysis of tissue extracts by 31P NMR and ion chromatography indicated that the most prominent components of the phosphomonoesters were glucose 6-phosphate, alpha-glycerol phosphate and AMP. In conclusion, of the various phosphorus metabolites that can be measured by 31P NMR, only one group, the phosphomonoesters, was predictive of functional recovery.
Assuntos
Isquemia Miocárdica/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Hemodinâmica , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Espectroscopia de Ressonância Magnética , Masculino , Isquemia Miocárdica/fisiopatologia , Isquemia Miocárdica/terapia , Reperfusão Miocárdica , Fosfatos/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Rabbit hearts were perfused with Krebs-Henseleit bicarbonate buffer supplemented with 15 mM glucose and 10 mU/ml of insulin +/- Pi. At the end of 60 min the hearts were freeze-clamped and the content of ATP, creatine phosphate, creatine, lactate, pyruvate, DHAP and 3-P glycerate were determined enzymatically in neutralized perchloric acid tissue extracts. The free cytosolic ADP and Pi and the cytosolic NAD+ redox and phosphorylation potentials were calculated from the measured metabolite concentrations. Pi free perfusion resulted in increased creatine, free cytosolic ADP and cytosolic phosphorylation potential, decreased calculated free Pi and no change in cardiac ATP and creatine phosphate content. The increase in the cytosolic phosphorylation potential was due to the lowering of cytosolic free Pi. The increase in ADP was due to the increase in creatine. The increase in creatine appeared to be due to an inhibition of creatine efflux from the heart during Pi free perfusion which was mediated by an enhanced Na+ electrochemical gradient.
Assuntos
Creatina/metabolismo , Miocárdio/metabolismo , Fosfatos/metabolismo , Difosfato de Adenosina/metabolismo , Animais , Soluções Tampão , Citosol/metabolismo , Técnicas In Vitro , Masculino , NAD/metabolismo , Oxirredução , Perfusão , Fosfocreatina/metabolismo , Fosforilação , CoelhosRESUMO
Phosphorus NMR spectroscopy is an important technique for the investigation of metabolism in tissues and intact organisms (including man). However, quantitation of the signals from an NMR experiment is difficult because it is not known from which regions of a cell metabolites are detected. It is generally believed that only metabolites free in the cytosol are observed. In this study a comparison of concentration measurements obtained by NMR and after freeze extraction was made in the normoxic and ischemic rat heart. The influence of ischemia was examined because of its potential effect on the level of phosphate metabolites in various compartments. The same fraction of ATP always appears visible to NMR, whereas inorganic phosphate is largely NMR invisible until after a period of ischemia and the phosphomonoesters are only partially observed early in ischemia.