Measurement of Acetyl‐CoA turnover ( ≈citric acid cycle flux) in perfused rat hearts by isotope dilution

Prior evidence has suggested that propionyl-L-carnitine improves function in ischemic hearts by providing carnitine for dissipation of acyl-CoA derivatives and propionate for enrichment of the citric acid cycle. Because contractile failure in hearts oxidizing ketone bodies is due to sequestration of free coenzyme A, which can be reversed by the addition of anaplerotic substrates that enrich the citric acid cycle, experiments were performed to determine whether the addition of propionyl-L-carnitine (2 mM) can improve performance in working rat hearts utilizing acetoacetate (7.5 mM). Whereas the addition of propionyl-L-carnitine to acetoacetate resulted in a sustained improvement in the work output of the heart, the addition of propionate (2 mM) or L-carnitine (2 mM) alone to acetoacetate had negligible effects on contractile function. Propionyl-L-carnitine increased the uptake of acetoacetate by 130%, whereas beta-hydroxybutyrate release was minimal and unchanged compared with other groups. These observations show that rates of acetoacetate oxidation are increased commensurate with increased contractile function. Tissue metabolite data indicate that the utilization of propionyl-L-carnitine did not lead to accumulation of citric acid cycle intermediates in the span from citrate to 2-oxoglutarate but to an increase in the tissue content of malate. The results show that addition of propionyl-L-carnitine in hearts oxidizing acetoacetate results in improved mechanical performance that is comparable to the mechanical performance of hearts perfused with glucose as the only substrate. This improvement is most likely conferred by anaplerosis, as suggested by enhanced rates of acetoacetate utilization and citric acid flux.

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Metabolism of [3-13C]pyruvate and [3-13C]propionate in normal and ischaemic rat heart in vivo: 1H- and 13C-NMR studies

Biochemical Journal ◽

10.1042/bj3120075 ◽

1995 ◽

Vol 312 (1) ◽

pp. 75-81 ◽

Cited By ~ 17

Author(s):

B Sumegi ◽

B Podanyi ◽

P Forgo ◽

K E Kover

Keyword(s):

Citric Acid ◽

13C Nmr ◽

Rat Heart ◽

Citric Acid Cycle ◽

Nmr Studies ◽

Acetyl Coa ◽

1H And 13C Nmr ◽

Acid Cycle ◽

Propionyl Carnitine

The oxidation of [3-13C]pyruvate and [3-13C]propionate was studied in vivo in infused rats. The infused [3-13C]pyruvate was quickly converted to [3-13C]lactate in the blood, and the [3-13C]lactate formed was well metabolized in both normoxic and ischaemic hearts. Large differences (200-600%) in the 13C enrichment of alanine (C-3) and acetyl-CoA (C-2) compared with lactate (C-3) were found in both normoxic and ischaemic hearts, suggesting that the extracellular [3-13C]lactate preferentially entered a region of the cytoplasm which specifically transfers the labelled pyruvate (formed from [3-13C]lactate) to the mitochondria. The highly enriched mitochondrial pyruvate gave high enrichment in alanine and acetyl-CoA, which was detected by 1H- and 13C-NMR spectroscopy. Ischaemia increased 13C incorporation into the main cytoplasmic lactate pool and decreased 13C incorporation into citric acid cycle intermediates, mainly decreasing the pyruvate anaplerosis. Isoprenaline-induced ischaemia of the heart caused only a slight decrease in pyruvate oxidation. In contrast to the decreased anaplerosis of pyruvate, the anaplerosis of propionate (and propionyl-carnitine) increased significantly in ischaemic hearts, which may contribute to the protective effect of propionyl-carnitine seen in ischaemia. In addition, we found that [3-13C]propionate preferentially labelled aspartate C-3 in rat heart, suggesting incomplete randomization of label in the succinyl-CoA-malate span of the citric acid cycle. These data show that proton observed 13C edited spectroscopic methods, i.e. heteronuclear spin-echo and the one-dimensional heteronuclear multiple quantum coherence sequence, can be successfully used to study heart metabolism in vivo.

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Carbon Isotope Fractionation by Autotrophic Bacteria with Three Different C02 Fixation Pathways

Zeitschrift für Naturforschung C ◽

10.1515/znc-1989-5-610 ◽

1989 ◽

Vol 44 (5-6) ◽

pp. 397-402 ◽

Cited By ~ 82

Author(s):

Andrea Preuß ◽

Rolf Schauder ◽

Georg Fuchs ◽

Willibald Stichler

Keyword(s):

Citric Acid ◽

Carbon Isotope ◽

Isotope Fractionation ◽

Citric Acid Cycle ◽

Pentose Phosphate ◽

Acetobacterium Woodii ◽

Acetyl Coa ◽

Acid Cycle ◽

Acetyl Coa Pathway ◽

Cell Carbon

Abstract Carbon isotope fractionation during autotrophic growth o f different bacteria which possess different autotrophic CO2 fixation pathways has been studied. 13C /12C -Ratios in the cell carbon of the following bacteria were determined (CO2 fixation pathway suggested or proven in parentheses): Alkaligenes eutrophus (reductive pentose phosphate cycle), Desulfobacterium autotrophicum and Acetobacterium woodii (reductive acetyl-CoA pathway), Desulfobacter hydrogenophilus and Thermoproteus neutrophilus (reductive citric acid cycle). The Δδ13C values, which indicate the per mille deviation of the 13C content of cell carbon from that of the CO : used as the sole carbon source, range from - 10%° (reductive citric acid cycle) over - 26%° (reductive pentose phosphate cycle) to -36%° (reductive acetyl-CoA pathway). Acetate formed via the acetyl-CoA pathway by the acetogenic Acetobacterium woodii showed a Δδ13C = -40%°. These data are discussed in view of the different CO2 fixation reactions used by the bacteria and especially with regard to the isotopic composition of sedimentary carbon through time.

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

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1999.277.6.e1111 ◽

1999 ◽

Vol 277 (6) ◽

pp. E1111-E1121 ◽

Cited By ~ 5

Author(s):

F. Mark H. Jeffrey ◽

Alexander Reshetov ◽

Charles J. Storey ◽

Rui A. Carvalho ◽

A. Dean Sherry ◽

...

Keyword(s):

Nuclear Magnetic Resonance ◽

Oxygen Consumption ◽

Citric Acid ◽

Magnetic Resonance ◽

Citric Acid Cycle ◽

Single Measurement ◽

Acid Cycle ◽

Nmr Data ◽

Rat Hearts ◽

C Nmr

A kinetic model of the citric acid cycle for calculating oxygen consumption from13C nuclear magnetic resonance (NMR) multiplet data has been developed. Measured oxygen consumption (MV˙o 2) was compared with MV˙o 2 predicted by the model with 13C NMR data obtained from rat hearts perfused with glucose and either [2-13C]acetate or [3-13C]pyruvate. The accuracy of MV˙o 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. MV˙o 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 α-ketoglutarate and glutamate, resulting in greater error in estimating MV˙o 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.

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Differences between mouse and rat pancreatic islets: succinate responsiveness, malic enzyme, and anaplerosis

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00041.2002 ◽

2002 ◽

Vol 283 (2) ◽

pp. E302-E310 ◽

Cited By ~ 44

Author(s):

Michael J. MacDonald

Keyword(s):

Citric Acid ◽

Pancreatic Islets ◽

Insulin Release ◽

Malic Enzyme ◽

Citric Acid Cycle ◽

Acetyl Coa ◽

Acid Cycle ◽

Rat Islets ◽

Rat Pancreatic Islets ◽

Mouse Islets

Succinic acid methyl esters are potent insulin secretagogues in rat pancreatic islets, but they do not stimulate insulin release in mouse islets. Unlike rat and human islets, mouse islets lack malic enzyme and, therefore, are unable to form pyruvate from succinate-derived malate for net synthesis of acetyl-CoA. Dimethyl-[2,3-14C]succinate is metabolized in the citric acid cycle in mouse islets to the same extent as in rat islets, indicating that endogenous acetyl-CoA condenses with oxaloacetate derived from succinate. However, without malic enzyme, the net synthesis from succinate of the citric acid cycle intermediates citrate, isocitrate, and α-ketoglutarate cannot occur. Glucose and other nutrients that augment α-ketoglutarate formation are secretagogues in mouse islets with potencies similar to those in rat islets. All cycle intermediates can be net-synthesized from α-ketoglutarate. Rotenone, an inhibitor of site I of the electron transport chain, inhibits methyl succinate-induced insulin release in rat islets even though succinate oxidation forms ATP at sites II and III of the respiratory chain. Thus generating ATP, NADH, and anaplerosis of succinyl-CoA plus the four-carbon dicarboxylic acids of the cycle and its metabolism in the citric acid cycle is insufficient for a fuel to be insulinotropic; it must additionally promote anaplerosis of α-ketoglutarate or two intermediates interconvertible with α-ketoglutarate, citrate, and isocitrate.

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Changes in the contents of adenine nucleotides and intermediates of glycolysis and citric acid cycle in flight muscle of the locust upon flight and their relationship to the control of the cycle

Biochemical Journal ◽

10.1042/bj1780209 ◽

1979 ◽

Vol 178 (1) ◽

pp. 209-216 ◽

Cited By ~ 48

Author(s):

Andrew N. Rowan ◽

Eric A. Newsholme

Keyword(s):

Citric Acid ◽

Citric Acid Cycle ◽

Flight Muscle ◽

Citrate Synthase ◽

Adenine Nucleotides ◽

Total Content ◽

Acetyl Coa ◽

Free Concentration ◽

Acid Cycle ◽

Early Stages

1. The contents of some intermediates of glycolysis, the citric acid cycle and adenine nucleotides have been measured in the freeze-clamped locust flight muscle at rest and after 10s and 3min flight. The contents of glucose 6-phosphate, pyruvate, alanine and especially fructose bisphosphate and triose phosphates increased markedly upon flight. The content of acetyl-CoA is decreased after 3min flight whereas that of acetylcarnitine is decreased markedly after 10s flight, but returns towards the resting value after 3min flight. The content of citrate is markedly decreased after both 10s and 3min flight, whereas that of isocitrate is changed very little after 10s and is increased by 50% after 3min. The content of oxaloacetate is very low in insect flight muscle and hence it was measured by a sensitive radiochemical assay. The content of oxaloacetate increased about 2-fold after 3min flight. A similar change was observed in the content of malate. The content of ATP decreased about 15%, whereas those of ADP and AMP increased about 2-fold after 3min flight. 2. Calculations based on O2 uptake of the intact insect indicate that the rate of the citric acid cycle must be increased >100-fold during flight. Consequently, if citrate synthase catalyses a non-equilibrium reaction, the activity of the enzyme must increase >100-fold during flight. However, changes in the concentrations of possible regulators of citrate synthase, oxaloacetate, acetyl-CoA and citrate (which is an allosteric inhibitor), are not sufficient to account for this change in activity. It is concluded that there may be much larger changes in the free concentration of oxaloacetate than are indicated by the changes in the total content of this metabolite or that other unknown factors must play an additional role in the regulation of citrate synthase activity. 3. The increased content of oxaloacetate could be produced via pyruvate carboxylase, which may be stimulated during the early stages of flight by the increased concentration of pyruvate. 4. The decreases in the concentrations of citrate and α-oxoglutarate indicate that isocitrate dehydrogenase and oxoglutarate dehydrogenase may be stimulated by factors other than their pathway substrates during the early stages of flight. 5. Calculated mitochondrial and cytosolic NAD+/NADH ratios are both increased upon flight. The change in the mitochondrial ratio indicates the importance of the intramitochondrial ATP/ADP concentration ratio in the regulation of the rate of electron transfer in this muscle.

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