Reductive amination of α-Ketoglutarate in metabolite extracts results in glutamate overestimation

J Chromatogr A. 2020 Jul 19;1623:461169. doi: 10.1016/j.chroma.2020.461169. Epub 2020 Apr 27.

Abstract

Artifacts due to metabolite extraction, derivatization, and detection techniques can result in aberrant observations that are not accurate representations of actual cell metabolism. Here, we show that α-ketoglutarate (α-KG) is reductively aminated to glutamate in methanol:water metabolite extracts, which introduces an artifact into metabolomics studies. We also identify pyridoxamine and urea as amine donors for α-KG to produce glutamate in methanol:water buffer in vitro, and we demonstrate that the addition of ninhydrin to the methanol:water buffer suppresses the reductive amination of α-KG to glutamate in vitro and in metabolite extracts. Finally, we calculate that glutamate levels have been overestimated by 10-50%, depending on cell line, due to α-KG reductive amination. These findings suggest that precautions to account for α-KG reductive amination should be taken for the accurate quantification of glutamate in metabolomics studies.

MeSH terms

  • Amination
  • Animals
  • Artifacts*
  • Cell Line
  • Glutamic Acid / analysis*
  • Glutamic Acid / chemistry
  • Ketoglutaric Acids / chemistry*
  • Metabolomics*
  • Methanol / chemistry
  • Ninhydrin / chemistry
  • Pyridoxamine
  • Urea / chemistry

Substances

  • Ketoglutaric Acids
  • Glutamic Acid
  • Pyridoxamine
  • Urea
  • Ninhydrin
  • Methanol