Development and validation of a highly sensitive LC-MS/MS method for simultaneous quantitation of acetyl-CoA and malonyl-CoA in animal tissues

Gilibili, Ravindra Reddy; Kandaswamy, Murugesh; Sharma, Kuldeep; Giri, Sanjeev; Rajagopal, Sriram; Mullangi, Ramesh

doi:10.1002/bmc.1608

Cited by 27 publications

(22 citation statements)

References 7 publications

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“…LCMS analysis was performed in a Waters Acquity UPLC system attached to a Xevo Triple Quadrupole mass spectrometer equipped with an electrospray ionization source (ESI) (Waters Corp., Milford, MA) for acylcarnitines in plasma [32] and hepatic acetyl CoA [33] with slight modifications for sample preparation as described in Supplemental notes.…”

Section: Methodsmentioning

confidence: 99%

Role of the tumor suppressor IQGAP2 in metabolic homeostasis: possible link between diabetes and cancer

et al. 2014

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Deficiency of IQGAP2, a scaffolding protein expressed primarily in liver leads to rearrangements of hepatic protein compartmentalization and altered regulation of enzyme functions predisposing development of hepatocellular carcinoma and diabetes. Employing a systems approach with proteomics, metabolomics and fluxes characterizations, we examined the effects of IQGAP2 deficient proteomic changes on cellular metabolism and the overall metabolic phenotype. Iqgap2−/− mice demonstrated metabolic inflexibility, fasting hyperglycemia and obesity. Such phenotypic characteristics were associated with aberrant hepatic regulations of glycolysis/gluconeogenesis, glycogenolysis, lipid homeostasis and futile cycling corroborated with corresponding proteomic changes in cytosolic and mitochondrial compartments. IQGAP2 deficiency also led to truncated TCA-cycle, increased anaplerosis, increased supply of acetyl-CoA for de novo lipogenesis, and increased mitochondrial methyl-donor metabolism necessary for nucleotides synthesis. Our results suggest that changes in metabolic networks in IQGAP2 deficiency create a hepatic environment of a ‘pre-diabetic’ phenotype and a predisposition to non-alcoholic fatty liver disease (NAFLD) which has been linked to the development of hepatocellular carcinoma.

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Section: Methodsmentioning

confidence: 99%

Role of the tumor suppressor IQGAP2 in metabolic homeostasis: possible link between diabetes and cancer

et al. 2014

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“…To put our results into physiological context, we applied the data from our “at rest” M-CoA inhibition curves derived from isolated mitochondria and PMF (Figure 3A and B) to a one phase exponential decay equation to predict the % inhibition of LCFA oxidation in the presence of resting concentrations of M-CoA (Table 1). We assumed resting M-CoA concentrations to be 0.7 μM as previously documented in two independent studies using HPLC/MS [47] and HPLC/MS/MS [48]. As seen in Table 1, inhibition of LCFA oxidation is predicted to be >95% in isolated mitochondria at rest.…”

Section: Perspectives and Significancementioning

confidence: 99%

Identification of a novel malonyl-CoA IC50 for CPT-I: implications for predicting in vivo fatty acid oxidation rates

Smith

Perry

Koves

et al. 2012

Biochemical Journal

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Synopsis Published values regarding the sensitivity (IC50) of carnitine palmitoyl transferase I (CPT-I) to malonyl-CoA (M-CoA) inhibition in isolated mitochondria are inconsistent with predicted in vivo rates of fatty acid oxidation. Therefore, we have re-examined M-CoA inhibition kinetics under varying palmitoyl-CoA (P-CoA) concentrations in both isolated mitochondria and permeabilized muscle fibres (PMF). PMF have an 18-fold higher IC50 (0.61 vs 0.034 μM) in the presence of 25 μM P-CoA and a 13-fold higher IC50 (6.3 vs 0.49 μM) in the presence of 150 μM P-CoA compared to isolated mitochondria. M-CoA inhibition kinetics determined in PMF predicts that CPT-I activity is inhibited by 33% in resting muscle compared to >95% in isolated mitochondria. Additionally, the ability of M-CoA to inhibit CPT-I appears to be dependent on P-CoA concentration, as the relative inhibitory capacity of M-CoA is decreased with increasing P-CoA concentrations. Altogether, the use of PMF appears to provide a M-CoA IC50 that better reflects the predicted in vivo rates of fatty acid oxidation. These findings also demonstrate the ratio of [P-CoA]/[M-CoA] is critical for regulating CPT-I activity and may partially rectify the in vivo disconnect between M-CoA content and CPT-I flux within the context of exercise and type II diabetes.

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“…However, most methods only assay either short-, medium-, or long-chain acyl-CoAs (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19). The best approach for acyl-CoA quantitation, so far, is to measure the target acyl-CoAs from short to long carbon chain length in a single method ( 20,21 ).…”

Section: Lc-ms/ms Is the Most Sensitive Methods To Analyze Acylcoas Anmentioning

confidence: 99%

Novel approach in LC-MS/MS using MRM to generate a full profile of acyl-CoAs: discovery of acyl-dephospho-CoAs

Zhang

Berthiaume

et al. 2014

Journal of Lipid Research

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This article is available online at http://www.jlr.org Acyl-CoAs are a class of important molecules that play essential roles in many physiological processes ( 1 ), such as fatty acid oxidation, lipid synthesis/remodeling, ketone body synthesis, xenobiotic metabolism, and signaling pathways. Classically, acyl-CoAs such as free CoA, acetyl-CoA, and malonyl-CoA are recognized as regulators of metabolic fl ux. The ratio of acetyl-CoA versus free CoA tightly regulates glycolysis and fatty acid oxidation. Malonyl-CoA attenuates fatty acid oxidation by inhibiting acyl-CoA transport into the mitochondrion for oxidation and is utilized in fatty acid synthesis when its concentration is elevated ( 2 ). Many proteins and genes are dynamically regulated by deacylation and acylation via various acyl-CoAs, such as acetyl-CoA, succinyl-CoA, palmitoyl-CoA, etc. ( 3 ). However, acyl-CoAs present in the cell are diverse and may involve molecules beyond fatty acids and their oxidized derivatives. The metabolism of xenobiotics can also lead to the formation of acyl-CoAs, as demonstrated in our previous work on the metabolism of 4-hydroxy acids from C 4 to C 11 in perfused rat liver. The identifi cation of these novel acyl-CoAs extends our understanding of the new catabolic pathways involved in the disposal of 4-hydroxy acids including drugs of abuse and lipid peroxidation products ( 4-8 ).Acyl-CoAs are exclusive to the intracellular metabolites, and the profi le of these biomolecules is indicative of the local metabolic status. Each organ has its specifi c physiological roles with different energetic demands, and therefore, would be expected to exhibit a unique acyl-CoA profi le. The heart preferentially utilizes fatty acids to meet the ATP demand of mechanical contraction. Glucose and/or ketone bodies serve as the primary substrate of the brain for energy

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Development and validation of a highly sensitive LC-MS/MS method for simultaneous quantitation of acetyl-CoA and malonyl-CoA in animal tissues

Cited by 27 publications

References 7 publications

Role of the tumor suppressor IQGAP2 in metabolic homeostasis: possible link between diabetes and cancer

Role of the tumor suppressor IQGAP2 in metabolic homeostasis: possible link between diabetes and cancer

Identification of a novel malonyl-CoA IC50 for CPT-I: implications for predicting in vivo fatty acid oxidation rates

Novel approach in LC-MS/MS using MRM to generate a full profile of acyl-CoAs: discovery of acyl-dephospho-CoAs

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