A general model for analysis of the tricarboxylic acid cycle with use of [13C]glutamate isotopomer measurements

1994 ◽  
Vol 266 (6) ◽  
pp. E1012-E1022 ◽  
Author(s):  
J. A. Vogt ◽  
A. J. Fischman ◽  
M. Kempf ◽  
Y. M. Yu ◽  
R. G. Tompkins ◽  
...  
Keyword(s):  
13C Nmr ◽  
Tricarboxylic Acid Cycle ◽  
Nonlinear Least Squares ◽  
Tricarboxylic Acid ◽  
Acetyl Coa ◽  
Metabolic System ◽  
Acid Cycle ◽  
Nmr Data ◽  
13C Nuclear Magnetic Resonance ◽  
Model Equations

A generalized steady-state model was developed for determining tricarboxylic acid cycle fractional fluxes from 13C nuclear magnetic resonance (NMR) data. The model relates the measured mole fractions of [13C]glutamate isotopomers to the fractional fluxes and predicted mole fractions of isotopomers of oxaloacetate (OAA) and acetyl-CoA. This model includes cycling between OAA and fumarate. Fractional fluxes are determined by fitting the model equations to NMR parameters by use of nonlinear least squares. Although only fractional fluxes can be determined from 13C-NMR data, when they are combined with mass spectroscopic measurements, absolute values can be derived. A specific metabolic system represented by published 13C-NMR data from extracts of hearts perfused with [13C]acetate, [13C]pyruvate (PYR), and [13C]acetate plus [13C]PYR was used to test the model. The intensities of predicted 13C-NMR splitting patterns were compared with observed values, and there was excellent agreement between observed and predicted signal intensities. With this model, important physiological parameters, including the OAA-derived fraction of inflow to PYR, PYR-derived fraction of inflow to acetyl-CoA, citrate-derived fraction of inflow to OAA, and PYR-derived fraction of inflow to OAA, can be determined.

13C isotopomer model for estimation of anaplerotic substrate oxidation via acetyl-CoA

1996 ◽  
Vol 271 (4) ◽  
pp. E788-E799 ◽  
Author(s):  
F. M. Jeffrey ◽  
C. J. Storey ◽  
A. D. Sherry ◽  
C. R. Malloy
Keyword(s):  
Biochemical Analysis ◽  
Tricarboxylic Acid Cycle ◽  
Tricarboxylic Acid ◽  
Acetyl Coa ◽  
Acid Cycle ◽  
Propionate Metabolism ◽  
13C Nuclear Magnetic Resonance ◽  
Isotopomer Analysis ◽  
Tricarboxylic Acid Cycle Intermediates ◽  
Anaplerotic Pathway

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).

Analysis of tricarboxylic acid cycle of the heart using 13C isotope isomers

1990 ◽  
Vol 259 (3) ◽  
pp. H987-H995 ◽  
Author(s):  
C. R. Malloy ◽  
A. D. Sherry ◽  
F. M. Jeffrey
Keyword(s):  
13C Nmr ◽  
Nmr Spectra ◽  
Tricarboxylic Acid Cycle ◽  
Tricarboxylic Acid ◽  
13C Nmr Spectra ◽  
Acid Cycle ◽  
Rat Hearts ◽  
13C Nuclear Magnetic Resonance ◽  
Peak Areas

13C-nuclear magnetic resonance (NMR) spectroscopy provides a new approach to the analysis of metabolic pathways, because it detects an interaction between adjacent 13C nuclei. Previous models of isotope distribution in the tricarboxylic acid cycle were designed for analysis of radioisotope data and did not consider the information provided by 13C-13C coupling. A mathematical model of the tricarboxylic acid cycle was developed that preserves all isotope isomer (isotopomer) information and yields simple relationships between 13C-NMR spectra of glutamate and metabolic parameters under steady-state conditions. With the use of relative peak areas measured from the spectra of tissues supplied with 13C-enriched substrate(s), the relative fluxes through both oxidative (acetyl-CoA utilization) and nonoxidative (anaplerotic) pathways of the tricarboxylic acid cycle can be determined. Furthermore, with the judicious selection of 13C-labeling patterns in a mixture of substrates, direct substrate competition experiments can be performed. The perchloric acid extracts of Langendorff and working rat hearts oxidizing 13C-enriched fatty acids or carbohydrates are analyzed to illustrate this approach, and the importance of measuring the fractional enrichment of the available substrate is demonstrated. The technique can of course be used with all tissues, not just heart, and is applicable to the analysis of in vivo 13C-NMR spectra.

Tricarboxylic acid cycle inhibition by Li+ in the human neuroblastoma SH-SY5Y cell line: A 13C NMR isotopomer analysis

2005 ◽  
Vol 47 (6) ◽  
pp. 385-393 ◽  
Author(s):  
Carla P. Fonseca ◽  
John G. Jones ◽  
Rui A. Carvalho ◽  
F. Mark H. Jeffrey ◽  
Liliana P. Montezinho ◽  
...  
Keyword(s):  
Cell Line ◽  
13C Nmr ◽  
Tricarboxylic Acid Cycle ◽  
Tricarboxylic Acid ◽  
Human Neuroblastoma ◽  
Acid Cycle ◽  
Isotopomer Analysis

Effect ofEchinococcus multilocularison the origin of acetyl-CoA entering the tricarboxylic acid cycle in host liver

2002 ◽  
Vol 76 (1) ◽  
pp. 31-36 ◽  
Author(s):  
C. Kepron ◽  
M. Novak ◽  
B.J. Blackburn
Keyword(s):  
Carbohydrate Metabolism ◽  
Tricarboxylic Acid Cycle ◽  
Tricarboxylic Acid ◽  
Meriones Unguiculatus ◽  
Host Liver ◽  
Acetyl Coa ◽  
Acid Cycle ◽  
A Value ◽  
Alveolar Hydatid Disease ◽  
And Control

AbstractCarbon-13 nuclear magnetic resonance (NMR) spectroscopy was employed to investigate alterations in hepatic carbohydrate metabolism inMeriones unguiculatusinfected withEchinococcus multilocularis. Following portal vein injections of an equimolar mixture of ]#x005B;1,2-13C2]acetate and [3-13C]lactate, perchloric acid extracts of the livers were prepared and NMR spectra obtained. Isotopomer analysis using glutamate resonances in these spectra showed that the relative contributions of endogenous and exogenous substrates to the acetyl-CoA entering the tricarboxylic acid cycle differed significantly between infected and control groups. The mole fraction of acetyl-CoA that was derived from endogenous, unlabelled sources (FU) was 0.50±0.10 in controls compared to 0.34±0.04 in infected animals. However, the fraction of acetyl-CoA derived from [3-13C]lactate (FLL) was larger in livers of infected animals than those from controls with values of 0.27±0.04 and 0.18±0.04, respectively. Similarly, the fraction of acetyl-CoA derived from [1,2-13C2]acetate (FLA) was larger in livers of infected animals compared to those in controls; the fractions were 0.38±0.01 and 0.32±0.07, respectively. The ratio of FLA:FLLwas significantly smaller in the infected group with a value of 1.42±0.18 compared to 1.74±0.09 for the controls. These results indicate that alveolar hydatid disease has a pronounced effect on the partitioning of substrates within the pathways of carbohydrate metabolism in the host liver.

Sources of acetyl-CoA entering the tricarboxylic acid cycle as determined by analysis of succinate carbon-13 isotopomers

Biochemistry ◽  
1993 ◽  
Vol 32 (45) ◽  
pp. 12240-12244 ◽  
Author(s):  
John G. Jones ◽  
A. Dean Sherry ◽  
F. Mark H. Jeffrey ◽  
Charles J. Storey ◽  
Craig R. Malloy
Keyword(s):  
Tricarboxylic Acid Cycle ◽  
Tricarboxylic Acid ◽  
Acetyl Coa ◽  
Acid Cycle

scholarly journals Computational estimation of tricarboxylic acid cycle fluxes using noisy NMR data from cardiac biopsies

2013 ◽  
Vol 7 (1) ◽  
pp. 82 ◽  
Author(s):  
Hannes Hettling ◽  
David J C Alders ◽  
Jaap Heringa ◽  
Thomas W Binsl ◽  
A B Groeneveld ◽  
...  
Keyword(s):  
Tricarboxylic Acid Cycle ◽  
Tricarboxylic Acid ◽  
Acid Cycle ◽  
Computational Estimation ◽  
Nmr Data

scholarly journals The activities of 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase in hearts and mammary glands from ruminants and non-ruminants

1977 ◽  
Vol 164 (2) ◽  
pp. 349-355 ◽  
Author(s):  
G Read ◽  
B Crabtree ◽  
G H Smith
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Tricarboxylic Acid Cycle ◽  
Mammary Glands ◽  
Tricarboxylic Acid ◽  
Acetyl Coa ◽  
Simple Method ◽  
Acid Cycle ◽  
Maximum Flux ◽  
Oxoglutarate Dehydrogenase

1. The activities of 2-oxoglutarate dehydrogenase (EC 1.2.4.2) were measured in hearts and mammary glands of rats, mice, rabbits, guinea pigs, cows, sheep, goats and in the flight muscles of several Hymenoptera. 2. The activity of 2-oxoglutarate dehydrogenase was similar to the maximum flux through the tricarboxylic acid cycle in vivo. Therefore measuring the activity of this enzyme may provide a simple method for estimating the maximum flux through the cycle for comparative investigations. 3. The activities of pyruvate dehydrogenase (EC 1.2.4.1) in mammalian hearts were similar to those of 2-oxoglutarate dehydrogenase, suggesting that in these tissues the tricarboxylic acid cycle can be supplied (under some conditions) by acetyl-CoA derived from pyruvate alone. 4. In the lactating mammary glands of the rat and mouse, the activities of pyruvate dehydrogenase exceeded those of 2-oxoglutarate dehydrogenase, reflecting a flux of pyruvate to acetyl-CoA for fatty acid synthesis in addition to that of oxidation via the tricarboxylic acid cycle. In ruminant mammary glands the activities of pyruvate dehydrogenase were similar to those of 2-oxoglutarate dehydrogenase, reflecting the absence of a significant flux of pyruvate to fatty acids in these tissues.

scholarly journals Pyruvate Dehydrogenase and Tricarboxylic Acid Cycle Enzymes Are Sensitive Targets of Traumatic Brain Injury Induced Metabolic Derangement

2019 ◽  
Vol 20 (22) ◽  
pp. 5774 ◽  
Author(s):  
Giacomo Lazzarino ◽  
Angela Maria Amorini ◽  
Stefano Signoretti ◽  
Giuseppe Musumeci ◽  
Giuseppe Lazzarino ◽  
...  
Keyword(s):  
Traumatic Brain Injury ◽  
Brain Injury ◽  
Pyruvate Dehydrogenase ◽  
Coenzyme A ◽  
Tricarboxylic Acid Cycle ◽  
Tricarboxylic Acid ◽  
Gene Expressions ◽  
Acetyl Coa ◽  
Metabolic Derangement ◽  
Acid Cycle

Using a closed-head impact acceleration model of mild or severe traumatic brain injury (mTBI or sTBI, respectively) in rats, we evaluated the effects of graded head impacts on the gene and protein expressions of pyruvate dehydrogenase (PDH), as well as major enzymes of mitochondrial tricarboxylic acid cycle (TCA). TBI was induced in anaesthetized rats by dropping 450 g from 1 (mTBI) or 2 m height (sTBI). After 6 h, 12 h, 24 h, 48 h, and 120 h gene expressions of enzymes and subunits of PDH. PDH kinases and phosphatases (PDK1-4 and PDP1-2, respectively), citrate synthase (CS), isocitrate dehydrogenase (IDH), oxoglutarate dehydrogenase (OGDH), succinate dehydrogenase (SDH), succinyl-CoA synthase (SUCLG), and malate dehydrogenase (MDH) were determined in whole brain extracts (n = 6 rats at each time for both TBI levels). In the same samples, the high performance liquid chromatographic (HPLC) determination of acetyl-coenzyme A (acetyl-CoA) and free coenzyme A (CoA-SH) was performed. Sham-operated animals (n = 6) were used as controls. After mTBI, the results indicated a general transient decrease, followed by significant increases, in PDH and TCA gene expressions. Conversely, permanent PDH and TCA downregulation occurred following sTBI. The inhibitory conditions of PDH (caused by PDP1-2 downregulations and PDK1-4 overexpression) and SDH appeared to operate only after sTBI. This produced almost no change in acetyl-CoA and free CoA-SH following mTBI and a remarkable depletion of both compounds after sTBI. These results again demonstrated temporary or steady mitochondrial malfunctioning, causing minimal or profound modifications to energy-related metabolites, following mTBI or sTBI, respectively. Additionally, PDH and SDH appeared to be highly sensitive to traumatic insults and are deeply involved in mitochondrial-related energy metabolism imbalance.

Sign in / Sign up

Export Citation Format

Share Document