fumaric-acid and isocitric-acid

The tricarboxylic acid (TCA) cycle is one of the most important pathways of energy metabolism, and the profiles of its components are influenced by factors such as diseases and diets. Therefore, the differences in metabolic profile of TCA cycle between healthy and cancer cells have been the focus of studies to understand pathological conditions. In this study, we developed a quantitative method to measure TCA cycle metabolites using LC-MS/MS to obtain useful metabolic profiles for development of diagnostic and therapeutic methods for cancer. We successfully analyzed 11 TCA cycle metabolites by LC MS/MS with high reproducibility by using a PFP column with 0.5% formic acid as a mobile phase. Next, we analyzed the concentration of TCA cycle metabolites in human cell lines (HaCaT: normal skin keratinocytes; A431: skin squamous carcinoma cells; SW480: colorectal cancer cells). We observed reduced concentration of succinate and increased concentration of citrate, 2-hydroxyglutarate, and glutamine in A431 cells as compared with HaCaT cells. On the other hand, decreased concentration of isocitrate, fumarate, and α-ketoglutarate and increased concentration of malate, glutamine, and glutamate in A431 cells were observed in comparison with SW480 cells. These findings suggested the possibility of identifying disease-specific metabolites and/or organ-specific metabolites by using this targeted metabolomic analysis.

Topics: Cell Line, Tumor; Cells, Cultured; Chromatography, Liquid; Citric Acid Cycle; Energy Metabolism; Fumarates; Humans; Isocitrates; Ketoglutaric Acids; Malates; Metabolome; Metabolomics; Neoplasms; Reproducibility of Results; Tandem Mass Spectrometry

We describe a method for the analysis of organic acids, including those of the tricarboxylic acid cycle (TCA cycle), by mixed-mode reversed-phase chromatography, on a CSH Phenyl-Hexyl column, to accomplish mixed-mode anion-exchange separations, which results in increased retention for acids without the need for ion-pairing reagents or other mobile phase additives. The developed method exhibited good retention time reproducibility for over 650 injections or more than 5 days of continuous operation. Additionally, it showed excellent resolution of the critical pairs, isocitric acid and citric acid as well as malic acid and fumaric acid, among others. The use of hybrid organic-inorganic surface technology incorporated into the hardware of the column not only improved the mass spectral quality and subsequent database match scoring but also increased the recovery of the analytes, showing particular benefit for low concentrations of phosphorylated species. The method was applied to the comparative metabolomic analysis of urine samples from healthy controls and breast cancer positive subjects. Unsupervised PCA analysis showed distinct grouping of samples from healthy and diseased subjects, with excellent reproducibility of respective injection clusters. Finally, abundance plots of selected analytes from the tricarboxylic acid cycle revealed differences between healthy control and disease groups.

Topics: Body Fluids; Chromatography, High Pressure Liquid; Citric Acid; Citric Acid Cycle; Fumarates; Humans; Isocitrates; Malates; Mass Spectrometry; Molecular Structure

The rates of p-aminohippurate (PAH) and of uric acid uptake by basolateral membrane vesicles isolated from proximal tubules of rat kidney have been investigated. Accumulation of both substrates against the concentration gradient within the vesicles was shown to occur in the presence of alpha-ketoglutarate (alpha-KG) and Na+ gradient in the incubation medium. The mechanism of the coupling between Na(+)-dicarboxylate symport and organic anion transport is discussed based on the differences between the rates of PAH and of uric acid uptake. It is proposed that the limiting step of coupling between two transporters is the step of alpha-KG-organic anion exchange.

Topics: Animals; Anion Transport Proteins; Anions; Biological Transport; Carrier Proteins; Cell Membrane; Cell Polarity; Citric Acid; Dicarboxylic Acids; Fumarates; Glutarates; Ion Transport; Isocitrates; Ketoglutaric Acids; Kidney Tubules, Proximal; Oxaloacetic Acid; p-Aminohippuric Acid; Rats; Sodium; Sodium Channels; Succinic Acid; Uric Acid

A procedure in which anionic analytes, trapped on ion exchange resin, are simultaneously methylated and released using methyl iodide in either supercritical carbon dioxide or acetonitrile has been extended to polyfunctional organic acids. The combined SFE methylation of fruit juice acids trapped onto ion exchange resin proceeds in good yield producing the methyl esters of fumaric, succinic, malic, tartaric, isocitric and citric acids which are readily separated by GC. Using this procedure low concentrations of one acid can be detected and quantitated in the presence of very high concentrations of another. This new method detects tartaric acid at levels of 10 ppm in juices containing 10,000 ppm citric acid. Quantitation was performed either by using GC-FID with triethyl citrate or diethyl tartrate as internal standards or with the element specific calibration capability of the GC-AED. A simple new technique for the determination of citric/isocitric acid ratio is now available. Also, in contrast to HPLC methods, the identity of an analyte is readily confirmed by GC-MS.

Topics: Beverages; Citric Acid; Fruit; Fumarates; Gas Chromatography-Mass Spectrometry; Hydrogen-Ion Concentration; Isocitrates; Malates; Methylation; Succinic Acid; Tartrates