Alpha ketoglutaric acid

Manufactured by Merck Group

Sourced in United States

Alpha-ketoglutaric acid is a chemical compound used in various laboratory applications. It is a precursor for the synthesis of other organic compounds and has biochemical functions in the citric acid cycle. The product is available in different purities and packaging sizes to meet the needs of various research and analytical applications.

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Ketoglutaric acid (3 citations)

4 protocols using alpha ketoglutaric acid

Metabolic Flux Analysis of Fibroblasts

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Mitochondrial and glycolytic stress test analysis in fibroblasts were performed on an XF24 Seahorse bioanalyser as previously described (Allen et al., 2014 (link), 2015 (link), 2019a (link); Raman et al., 2015 (link)). Specifically for metabolic substitution assays, fibroblast media was supplemented with 0.3 mM glutamine and 5 mM glucose or either 5 mM pyruvic acid, butyric acid, alpha ketoglutaric acid, succinamic acid or adenosine (Sigma) for 40 h prior to metabolic flux analysis. Flux analysis was performed using XF basal media (Agilent) supplemented with the combination of metabolic substrates listed above. Metabolic flux analysis under physiological and stress conditions were assessed following sequential addition of the mitochondrial inhibitors oligomycin, FCCP and antimycin/rotenone (Sigma) as previously described (Allen et al., 2014 (link), 2015 (link)). Flux data were normalized to CyQUANT fluorescence in each well as a proxy for cell number following the manufacturer's instructions as previously described (Allen et al., 2019a (link)).

Allen S.P., Al Sultan A., Kabucho Kibirige E., Tonkiss E., Hamer K.J., Castelli L.M., Lin Y.H., Roscoe S., Stefanidis N., Mead R.J., Highley J.R., Cooper-Knock J., Hautbergue G.M., Heath P.R., Kirby J, & Shaw P.J. (2023). A Y374X TDP43 truncation leads to an altered metabolic profile in amyotrophic lateral sclerosis fibroblasts driven by pyruvate and TCA cycle intermediate alterations. Frontiers in Aging Neuroscience, 15, 1151848.

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LC-MS Metabolite Quantification Protocol

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LC-MS grade methanol (MeOH) formic acid (FA) and acetonitrile (ACN) were from Fisher Scientific (Pittsburgh, PA, USA). Water was of ultrapure grade (EMD Millipore Co., Billerica, MA, USA). Deuterated internal standards (IS) D5-Glutamic acid, D5-Phenyl alanine and D4-Succinic acid were from Cambridge Isotope Laboratories (Tewksbury, MA, USA). [U-¹³C₅¹⁵N₂]-Glutamine, [5-¹³C]-Glutamine and [1-¹³C]-Glutamine were from Cortecnet (Paris, France). Glutamine, glutamic acid, [U-¹³C₆]-glucose, ornithine, fumaric acid, aspartic acid, alpha-ketoglutaric acid, alanine and lactic acid were from Sigma (Sigma-Aldrich, St. Louis, MO, USA). Commercial negative/positive calibration and reference (lock masses) solutions for the MS device were from Agilent Technologies (Santa Clara, CA, USA).

Ballester M., Sentandreu E., Luongo G., Santamaria R., Bolonio M., Alcoriza-Balaguer M.I., Palomino-Schätzlein M., Pineda-Lucena A., Castell J., Lahoz A, & Bort R. (2019). Glutamine/glutamate metabolism rewiring in reprogrammed human hepatocyte-like cells. Scientific Reports, 9, 17978.

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Calibration Curve and QC Solutions Preparation

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Working solution (WS) for calibration curves and quality control solutions were prepared from two separate mother solutions (100 µg/ml in water) of each quantified compound: L-glutamic acid, L-aspartic acid, L-glutamine, succinic acid, alpha-ketoglutaric acid, trans-aconitic acid, L-(-)-malic acid, D,L-iso citric acid, D-glyceric acid, fumaric acid, citric acid, pyruvic acid, D-alpha-hydroxyglutaric acid disodium salt, D-(-)-lactic acid, D-(-)−3-phosphoglyceric acid, phosphoenolpyruvic acid and itaconic acid (all from Sigma). Several diluted solutions of calibration standard solutions (CSS) and quality control solutions (QCS) were prepared by successive two-fold dilutions of WS in ultrapure water. Then, a three-fold dilution in a BSA solution (7200 µg/mL), of each previous diluted solution (CSS1-8 and QCS1-3) was applied to prepare standards for the calibration curve (from 33.33 to 0.26 µg/mL), and quality control (from 53.33 to 1.51 µg/mL). A volume of 350 µL of cold methanol was added to each calibration curve and quality control solution and followed the metabolite extraction process.

Orliaguet L., Ejlalmanesh T., Humbert A., Ballaire R., Diedisheim M., Julla J.B., Chokr D., Cuenco J., Michieletto J., Charbit J., Lindén D., Boucher J., Potier C., Hamimi A., Lemoine S., Blugeon C., Legoix P., Lameiras S., Baudrin L.G., Baulande S., Soprani A., Castelli F.A., Fenaille F., Riveline J.P., Dalmas E., Rieusset J., Gautier J.F., Venteclef N, & Alzaid F. (2022). Early macrophage response to obesity encompasses Interferon Regulatory Factor 5 regulated mitochondrial architecture remodelling. Nature Communications, 13, 5089.

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Analytical Methods for Fermentation Metabolites

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Cell concentration was measured by the 721 visible spectrophotometer (APL Instrument, Shanghai) at 600 nm with 2 mL fermentation broth added in a cuvette. To measure metabolites, fermentation broth was centrifuged at 12,000 rpm for 10 min and filtered through 0.22-μm membrane filter to remove bacteria. Residual glucose concentration was measured every 3 h by an SBA biosensor analyzer (Institute of Biology, Shandong Academy of Sciences). Aconitic acid, citric acid, alpha-ketoglutaric acid, lactic acid, and acetic acid in supernatant were analyzed by high performance liquid chromatography (HPLC) system (Shimazu, Japan) equipped with a C18 column and an SPD-20A UV detector at 210 nm. Column temperature was 25 °C. Mobile phase was 0.03% phosphoric acid at a flow rate of 0.8 mL/min. Analytical pure aconitic acid, citric acid, alpha-ketoglutaric acid, lactic acid, and acetic acid (Sigma-Aldrich, U.S.) were used as standards for quantification. F-test of two samples for variance was performed, and significance of the differences (P-values) was calculated using unpaired two-tailed t-tests for equal or unequal variance. All tests were performed by software GraphPad Prism 5.0.

Li Q., Zhao P., Yin H., Liu Z., Zhao H, & Tian P. (2020). CRISPR interference-guided modulation of glucose pathways to boost aconitic acid production in Escherichia coli. Microbial Cell Factories, 19, 174.

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