Using the size of the VM-M3 cells and cell count used for the extracts, and quantifying the amount of ITA in the extracts using MS methods, we estimate the intracellular concentration of this compound to be 1.33☐.16 mM. The 1H resonances arising from itaconate were also confirmed by doping cell extracts with an itaconate standard. The 13C-labeled ITA was present at sufficiently high levels that it could easily be identified using 2D-gHSQC ( Fig. glutamine resulted in detection of proton resonances at 3.15 ppm characteristic of the ITA signal for the – 13CH 2– moiety glucose incubation gave rise to resonances consistent with labeled – 13CH 2– and = 13CH 2 (at 5.37 and 5.85 ppm) groups 13. 11, 12 We detected itaconic acid (ITA) in 1D-gHSQC NMR experiments (obtained on a Varian 600 MHz VNMRS equipped with a triple resonance probe) with methanol/water (80:20) extracts of VM-M3 cells that had been incubated with glutamine or glucose. 10 This cell line, reported to be metastatic, is similar in morphology to the well-characterized RAW 264.7 murine macrophage cell line, and both share properties of macrophages including gene expression and phagocytic capability. The VM-M3 murine tumor cell line is derived from a spontaneously arising brain tumor in a VM mouse. Despite the overall lack of study of ITA as a mammalian metabolite, there is evidence that ITA can be catabolized by both guinea pig and rat liver mitochondria. In those few cases, whether it was synthesized by mammalian cells or endogenous flora, or arose merely as an adventitious contaminant, was unknown. 3, 4 More recently, itaconic acid (ITA) has been identified in a small number of cases of metabolomic analysis of mammalian tissue specimens 5– 7. 1, 2 The biosynthesis of this dicarboxylic acid has been of interest because it can be used as a starting material for chemical synthesis of polymers. A case in point is itaconic acid (methylenesuccinic acid) – a metabolite made and secreted by the fungal organism Aspergillus terreus. Small molecule effectors of cells are often overlooked.
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