scholarly journals Development of a LC-MS/MS Method for the Simultaneous Detection of Tricarboxylic Acid Cycle Intermediates in a Range of Biological Matrices

2017 ◽  
Vol 2017 ◽  
pp. 1-12 ◽  
Author(s):  
Omar Al Kadhi ◽  
Antonietta Melchini ◽  
Richard Mithen ◽  
Shikha Saha
Keyword(s):  
Ex Vivo ◽  
Tricarboxylic Acid Cycle ◽  
Simultaneous Detection ◽  
Tca Cycle ◽  
Tricarboxylic Acid ◽  
In Vivo Studies ◽  
Biological Matrices ◽  
Wide Range ◽  
Tca Cycle Intermediates

It is now well-established that perturbations in the tricarboxylic acid (TCA) cycle play an important role in the metabolic transformation occurring in cancer including that of the prostate. A method for simultaneous qualitative and quantitative analysis of TCA cycle intermediates in body fluids, tissues, and cultured cell lines of human origin was developed using a common C18 reversed-phase column by LC-MS/MS technique. This LC-MS/MS method for profiling TCA cycle intermediates offers significant advantages including simple and fast preparation of a wide range of human biological samples. The analytical method was validated according to the guideline of the Royal Society of Chemistry Analytical Methods Committee. The limits of detection were below 60 nM for most of the TCA intermediates with the exception of lactic and fumaric acids. The calibration curves of all TCA analytes showed linearity with correlation coefficientsr2>0.9998. Recoveries were >95% for all TCA analytes. This method was established taking into consideration problems and limitations of existing techniques. We envisage that its application to different biological matrices will facilitate deeper understanding of the metabolic changes in the TCA cycle from in vitro, ex vivo, and in vivo studies.

Tricarboxylic acid cycle intermediates in human muscle at rest and during prolonged cycling

1997 ◽  
Vol 272 (2) ◽  
pp. E239-E244 ◽  
Author(s):  
M. J. Gibala ◽  
M. A. Tarnopolsky ◽  
T. E. Graham
Keyword(s):  
Fiber Type ◽  
Tricarboxylic Acid Cycle ◽  
Vastus Lateralis ◽  
Tca Cycle ◽  
Tricarboxylic Acid ◽  
Human Muscle ◽  
Pool Size ◽  
Recruitment Pattern ◽  
Total Pool ◽  
Tca Cycle Intermediates

Previous studies have used the muscle concentration of citrate + malate + fumarate to estimate tricarboxylic acid (TCA) cycle pool size in humans [e.g., Am. J. Physiol. 259 (Cell Physiol. 28): C834-C841, 1990]. Our purpose was to quantify changes in individual TCA cycle intermediates (TCAI) and total pool size by measuring the concentrations of the eight TCAI in human muscle. Eight males cycled to exhaustion (Exh) at approximately 70% of their maximal oxygen uptake, and biopsies were obtained from the vastus lateralis at rest and during exercise. Succinyl-CoA was not consistently detectable, but the sum of the other seven TCAI was 1.23 +/- 0.04 mmol/kg dry wt at rest, 4.80 +/- 0.25 and 4.87 +/- 0.30 mmol/kg after 5 and 15 min of exercise, respectively, and 3.08 +/- 0.15 mmol/kg at Exh. Pool size during exercise was approximately 50% higher than that seen in rodent muscle after intense electrical stimulation (Eur. J. Biochem. 110: 371-377, 1980). Relative changes in individual TCAI were not uniform, and no one intermediate was "representative" of the changes in total pool size. We conclude that changes in specific intermediates or total pool size cannot be used as indicators of cycle flux and that the apparent species differences in total pool size may reflect differences in fiber type composition, recruitment pattern, or relative intensity of contraction.

scholarly journals Tricarboxylic Acid (TCA) Cycle Intermediates: Regulators of Immune Responses

2020 ◽  
Author(s):  
Inseok Choi ◽  
Hyewon Son ◽  
Jea-Hyun Baek
Keyword(s):  
Immune Responses ◽  
Molecular Mechanisms ◽  
Tricarboxylic Acid Cycle ◽  
Tca Cycle ◽  
Tricarboxylic Acid ◽  
Cell Stress ◽  
Danger Signals ◽  
Cellular Processes ◽  
The Tca Cycle ◽  
Tca Cycle Intermediates

Tricarboxylic acid cycle (TCA) is a series of chemical reactions in aerobic organisms used to generate energy via the oxidation of acetyl-CoA derived from carbohydrates, fatty acids, and proteins. In the eukaryotic system, the TCA cycle completely occurs in mitochondria, while the intermediates of the TCA cycle are retained in mitochondria due to their polarity and hydrophilicity. Under conditions of cell stress, mitochondria become disrupted and release their contents, which act as danger signals in the cytosol. Of note, the TCA cycle intermediates may also leak from dysfunctioning mitochondria and regulate cellular processes. Increasing evidence shows that the metabolites of the TCA cycle are substantially involved in the regulation of immune responses. In this review, we aimed to provide a comprehensive systematic overview of the molecular mechanisms of each TCA cycle intermediate that may play key roles in regulating cellular immunity in cell stress and discuss their implications for immune activation and suppression.

HPLC–UV method for temozolomide determination in complex biological matrices: Application for in vitro , ex vivo and in vivo studies

2019 ◽  
Vol 33 (10) ◽  
Author(s):  
Luana R. Michels ◽  
Flávia N.S. Fachel ◽  
Juliana H. Azambuja ◽  
Nicolly E. Gelsleichter ◽  
Elizandra Braganhol ◽  
...  
Keyword(s):  
Ex Vivo ◽  
In Vivo Studies ◽  
Biological Matrices

scholarly journals Tricarboxylic Acid (TCA) Cycle Intermediates: Regulators of Immune Responses

Life ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 69
Author(s):  
Inseok Choi ◽  
Hyewon Son ◽  
Jea-Hyun Baek
Keyword(s):  
Immune Responses ◽  
Molecular Mechanisms ◽  
Tricarboxylic Acid Cycle ◽  
Tca Cycle ◽  
Tricarboxylic Acid ◽  
Cell Stress ◽  
Danger Signals ◽  
Cellular Processes ◽  
The Tca Cycle ◽  
Tca Cycle Intermediates

The tricarboxylic acid cycle (TCA) is a series of chemical reactions used in aerobic organisms to generate energy via the oxidation of acetylcoenzyme A (CoA) derived from carbohydrates, fatty acids and proteins. In the eukaryotic system, the TCA cycle occurs completely in mitochondria, while the intermediates of the TCA cycle are retained inside mitochondria due to their polarity and hydrophilicity. Under cell stress conditions, mitochondria can become disrupted and release their contents, which act as danger signals in the cytosol. Of note, the TCA cycle intermediates may also leak from dysfunctioning mitochondria and regulate cellular processes. Increasing evidence shows that the metabolites of the TCA cycle are substantially involved in the regulation of immune responses. In this review, we aimed to provide a comprehensive systematic overview of the molecular mechanisms of each TCA cycle intermediate that may play key roles in regulating cellular immunity in cell stress and discuss its implication for immune activation and suppression.

Analysis of tricarboxylic acid cycle using [14C]citrate specific activity ratios

1985 ◽  
Vol 248 (2) ◽  
pp. E252-E260 ◽  
Author(s):  
J. K. Kelleher
Keyword(s):  
Steady State ◽  
Specific Activity ◽  
Tricarboxylic Acid Cycle ◽  
Specific Radioactivity ◽  
Tca Cycle ◽  
Tricarboxylic Acid ◽  
State Equations ◽  
Futile Cycle ◽  
Net Flux ◽  
Tca Cycle Intermediates

The information content of citrate carbon specific radioactivity ratios in steady-state 14C isotopic studies has been analyzed. Sixteen steady-state equations resulted containing five unknowns, 14 equations in terms of citrate carbon specific radioactivity ratios, and two modified forms of the “CO2 ratio” equation. Although each of the 16 equations is not independent, there is more than one independent equation for each variable. These additional equations may be used to test the assumptions on which the model is based. Each of the five unknowns is defined as the probability of flux around a complete cycle, either the tricarboxylic acid (TCA) cycle or a futile cycle such as pyruvate--oxaloacetate--pyruvate. To solve these equations for the five unknowns, an investigator need only measure the specific radioactivity of various citrate carbons and the 14CO2 production rate. The study did not yield a direct expression for net flux between pyruvate and 4-carbon TCA cycle intermediates. However, these equations do place certain constraints on the net flux through this important pathway.

scholarly journals Thioredoxin, a master regulator of the tricarboxylic acid cycle in plant mitochondria

2015 ◽  
Vol 112 (11) ◽  
pp. E1392-E1400 ◽  
Author(s):  
Danilo M. Daloso ◽  
Karolin Müller ◽  
Toshihiro Obata ◽  
Alexandra Florian ◽  
Takayuki Tohge ◽  
...  
Keyword(s):  
Tricarboxylic Acid Cycle ◽  
Plant Mitochondria ◽  
Tca Cycle ◽  
Tricarboxylic Acid ◽  
Mitochondrial Enzymes ◽  
Atp Citrate Lyase ◽  
Cycle Enzyme ◽  
The Tca Cycle

Plant mitochondria have a fully operational tricarboxylic acid (TCA) cycle that plays a central role in generating ATP and providing carbon skeletons for a range of biosynthetic processes in both heterotrophic and photosynthetic tissues. The cycle enzyme-encoding genes have been well characterized in terms of transcriptional and effector-mediated regulation and have also been subjected to reverse genetic analysis. However, despite this wealth of attention, a central question remains unanswered: “What regulates flux through this pathway in vivo?” Previous proteomic experiments withArabidopsisdiscussed below have revealed that a number of mitochondrial enzymes, including members of the TCA cycle and affiliated pathways, harbor thioredoxin (TRX)-binding sites and are potentially redox-regulated. We have followed up on this possibility and found TRX to be a redox-sensitive mediator of TCA cycle flux. In this investigation, we first characterized, at the enzyme and metabolite levels, mutants of the mitochondrial TRX pathway inArabidopsis: theNADP-TRX reductasea and b double mutant (ntra ntrb) and the mitochondrially locatedthioredoxin o1(trxo1) mutant. These studies were followed by a comparative evaluation of the redistribution of isotopes when13C-glucose,13C-malate, or13C-pyruvate was provided as a substrate to leaves of mutant or WT plants. In a complementary approach, we evaluated the in vitro activities of a range of TCA cycle and associated enzymes under varying redox states. The combined dataset suggests that TRX may deactivate both mitochondrial succinate dehydrogenase and fumarase and activate the cytosolic ATP-citrate lyase in vivo, acting as a direct regulator of carbon flow through the TCA cycle and providing a mechanism for the coordination of cellular function.

scholarly journals Involvement of enzymes of amino acid metabolism and tricarboxylic acid cycle in bovine oocyte maturation in vitro

Reproduction ◽  
2003 ◽  
pp. 753-763 ◽  
Author(s):  
P Cetica ◽  
L Pintos ◽  
G Dalvit ◽  
M Beconi
Keyword(s):  
Amino Acid ◽  
Isocitrate Dehydrogenase ◽  
Amino Acid Metabolism ◽  
Acid Metabolism ◽  
Tricarboxylic Acid Cycle ◽  
Tca Cycle ◽  
Cumulus Cells ◽  
Tricarboxylic Acid ◽  
Maturation In Vitro

Few studies demonstrate at a biochemical level the metabolic profile of both cumulus cells and the oocyte during maturation. The aim of the present study was to investigate the differential participation of enzymatic activity in cumulus cells and in the oocyte during in vitro maturation (IVM) by studying the activity of enzymes involved in the control of amino acid metabolism, alanine aminotransferase (ALT) and aspartate aminotransferase (AST); and the tricarboxylic acid (TCA) cycle, isocitrate dehydrogenase (IDH) and malate dehydrogenase (MDH). No NAD-dependent isocitrate dehydrogenase (NAD-IDH) activity was recorded in cumulus-oocyte complexes (COCs). ALT, AST, NADP-dependent isocitrate dehydrogenase (NADP-IDH) and MDH enzymatic units remained constant in cumulus cells and oocytes during IVM. Specific activities increased in oocytes and decreased in cumulus cells as a result of IVM (P<0.05). Similar activity of both transaminases was detected in cumulus cells, unlike in the oocyte, in which activity of AST was 4.4 times greater than that of ALT (P<0.05). High NADP-IDH and MDH activity was detected in the oocyte. Addition of alanine, aspartate, isocitrate + NADP, oxaloacetate or malate + NAD to maturation media increased the percentage of denuded oocytes reaching maturation (P<0.05), in contrast to COCs in which differences were not observed by addition of these substrates and co-enzymes. The activity of studied enzymes and the use of oxidative substrates denotes a major participation of transaminations and the TCA cycle in the process of gamete maturation. The oocyte thus seems versatile in the use of several oxidative substrates depending on the redox state.

scholarly journals Multiple Factors Affecting Cellular Redox Status and Energy Metabolism Modulate Hypoxia-Inducible Factor Prolyl Hydroxylase Activity In Vivo and In Vitro

2006 ◽  
Vol 27 (3) ◽  
pp. 912-925 ◽  
Author(s):  
Yi Pan ◽  
Kyle D. Mansfield ◽  
Cara C. Bertozzi ◽  
Viktoriya Rudenko ◽  
Denise A. Chan ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Hypoxia Inducible Factor ◽  
Simultaneous Detection ◽  
Tca Cycle ◽  
Cellular Responses ◽  
Prolyl Hydroxylase ◽  
In Vitro Assays ◽  
Tca Cycle Intermediates

ABSTRACT Prolyl hydroxylation of hypoxible-inducible factor alpha (HIF-α) proteins is essential for their recognition by pVHL containing ubiquitin ligase complexes and subsequent degradation in oxygen (O2)-replete cells. Therefore, HIF prolyl hydroxylase (PHD) enzymatic activity is critical for the regulation of cellular responses to O2 deprivation (hypoxia). Using a fusion protein containing the human HIF-1α O2-dependent degradation domain (ODD), we monitored PHD activity both in vivo and in cell-free systems. This novel assay allows the simultaneous detection of both hydroxylated and nonhydroxylated PHD substrates in cells and during in vitro reactions. Importantly, the ODD fusion protein is regulated with kinetics identical to endogenous HIF-1α during cellular hypoxia and reoxygenation. Using in vitro assays, we demonstrated that the levels of iron (Fe), ascorbate, and various tricarboxylic acid (TCA) cycle intermediates affect PHD activity. The intracellular levels of these factors also modulate PHD function and HIF-1α accumulation in vivo. Furthermore, cells treated with mitochondrial inhibitors, such as rotenone and myxothiazol, provided direct evidence that PHDs remain active in hypoxic cells lacking functional mitochondria. Our results suggest that multiple mitochondrial products, including TCA cycle intermediates and reactive oxygen species, can coordinate PHD activity, HIF stabilization, and cellular responses to O2 depletion.

scholarly journals Analysis of tricarboxylic acid-cycle metabolism of hepatoma cells by comparison of 14CO2 ratios

1987 ◽  
Vol 246 (3) ◽  
pp. 633-639 ◽  
Author(s):  
J K Kelleher ◽  
B M Bryan ◽  
R T Mallet ◽  
A L Holleran ◽  
A N Murphy ◽  
...  
Keyword(s):  
Steady State ◽  
Tricarboxylic Acid Cycle ◽  
Tca Cycle ◽  
Tricarboxylic Acid ◽  
Hepatoma Cells ◽  
Acid Cycle ◽  
The Tca Cycle ◽  
14Co2 Production ◽  
Metabolic Properties ◽  
Tca Cycle Intermediates

The CO2-ratios method is applied to the analysis of abnormalities of TCA (tricarboxylic acid)-cycle metabolism in AS-30D rat ascites-hepatoma cells. This method utilizes steady-state 14CO2-production rates from pairs of tracers of the same compound to evaluate TCA-cycle flux patterns. Equations are presented that quantitatively convert CO2 ratios into estimates of probability of flux through TCA-cycle-related pathways. Results of this study indicated that the ratio of 14CO2 produced from [1,4-14C]succinate to 14CO2 produced from [2,3-14C]succinate was increased by the addition of glutamine (5 mM) to the medium. An increase in the succinate CO2 ratio is quantitatively related to an increased flux of unlabelled carbon into the TCA-cycle-intermediate pools. Analysis of 14C distribution in [14C]citrate derived from [2,3-14C]succinate indicated that flux from the TCA cycle to the acetyl-CoA-derived carbons of citrate was insignificant. Thus the increased succinate CO2 ratio observed in the presence of glutamine could only result from an increased flux of carbon into the span of the TCA cycle from citrate to oxaloacetate. This result is consistent with increased flux of glutamine to alpha-oxoglutarate in the incubation medium containing exogenous glutamine. Comparison of the pyruvate CO2 ratio, steady-state 14CO2 production from [2-14C]pyruvate versus [3-14C]pyruvate, with the succinate 14CO2 ratio detected flux of pyruvate to C4 TCA-cycle intermediates in the medium containing glutamine. This result was consistent with the observation that [14C]aspartate derived from [2-14C]pyruvate was labelled in C-2 and C-3. 14C analysis also produced evidence for flux of TCA-cycle carbon to alanine. This study demonstrates that the CO2-ratios method is applicable in the analysis of the metabolic properties of AS-30D cells. This methodology has verified that the atypical TCA-cycle metabolism previously described for AS-30D-cell mitochondria occurs in intact AS-30D rat hepatoma cells.

Sepsis does not impair tricarboxylic acid cycle in the heart

1991 ◽  
Vol 260 (1) ◽  
pp. C50-C57 ◽  
Author(s):  
R. S. Hotchkiss ◽  
S. K. Song ◽  
J. J. Neil ◽  
R. D. Chen ◽  
J. K. Manchester ◽  
...  
Keyword(s):  
Tricarboxylic Acid Cycle ◽  
Cecal Ligation ◽  
Tca Cycle ◽  
High Energy ◽  
Tricarboxylic Acid ◽  
Female Rats ◽  
Resonance Spectroscopy ◽  
High Energy Phosphates ◽  
The Tca Cycle ◽  
Tca Cycle Intermediates

Sepsis has been reported to cause mitochondrial dysfunction and inhibition of key enzymes that regulate the tricarboxylic acid (TCA) cycle. We investigated the effect of sepsis on high-energy phosphates, glycolytic and TCA cycle intermediates, and specific amino acids that are involved in regulating the size of the TCA cycle pool during changes in metabolic state of the heart. Sepsis was induced in 12 female rats by the cecal ligation and perforation technique under halothane anesthesia; seven control rats underwent cecal manipulation without ligation. At 36-42 h postsurgery, the rats were reanesthetized, the chest was opened, and the hearts were freeze-clamped. Perchloric acid extracts of the hearts were analyzed with fluorometric enzymatic methods and 31P nuclear magnetic resonance spectroscopy. There were no significant differences in the levels of the TCA cycle intermediates or high-energy phosphates between the septic and control rats. The major metabolic changes were the 28% decrease in alanine and the 31% decrease in glutamate in the septic hearts compared with control (P less than 0.05 and P less than 0.005, respectively). Phosphocholine, a component of membrane phospholipids, was increased by 91% in the septic hearts (P less than 0.01). We conclude that sepsis does not impair the TCA cycle or induce significant cellular ischemia in the heart. The increase in phosphocholine may represent significant cellular membrane disruption during sepsis.

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