Acta Neuropathologica

, Volume 124, Issue 6, pp 883–891

Enzymatic assay for quantitative analysis of (d)-2-hydroxyglutarate

  • Jörg Balss
  • Stefan Pusch
  • Ann-Christin Beck
  • Christel Herold-Mende
  • Alwin Krämer
  • Christian Thiede
  • Wolfgang Buckel
  • Claus-Dieter Langhans
  • Jürgen G. Okun
  • Andreas von Deimling
Methods Paper

DOI: 10.1007/s00401-012-1060-y

Cite this article as:
Balss, J., Pusch, S., Beck, AC. et al. Acta Neuropathol (2012) 124: 883. doi:10.1007/s00401-012-1060-y

Abstract

Levels of (d)-2-hydroxyglutarate [D2HG, (R)-2-hydroxyglutarate] are increased in some metabolic diseases and in neoplasms with mutations in the isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2) genes. Determination of D2HG is of relevance to diagnosis and monitoring of disease. Standard detection methods of D2HG levels are liquid-chromatography–mass spectrometry or gas-chromatography–mass spectrometry. Here we present a rapid, inexpensive and sensitive enzymatic assay for the detection of D2HG levels. The assay is based on the conversion of D2HG to α-ketoglutarate (αKG) in the presence of the enzyme (d)-2-hydroxyglutarate dehydrogenase (HGDH) and nicotinamide adenine dinucleotide (NAD+). Determination of D2HG concentration is based on the detection of stoichiometrically generated NADH. The quantification limit of the enzymatic assay for D2HG in tumor tissue is 0.44 μM and in serum 2.77 μM. These limits enable detection of basal D2HG levels in human tumor tissues and serum without IDH mutations. Levels of D2HG in frozen and paraffin-embedded tumor tissues containing IDH mutations or in serum from acute myeloid leukemia patients with IDH mutations are significantly higher and can be easily identified with this assay. In conclusion, the assay presented is useful for differentiating basal from elevated D2HG levels in tumor tissue, serum, urine, cultured cells and culture supernatants.

Supplementary material

401_2012_1060_MOESM1_ESM.pptx (121 kb)
Supplementary material 1 (PPTX 121 kb)
401_2012_1060_MOESM2_ESM.pptx (126 kb)
Supplementary material 2 (PPTX 125 kb)
401_2012_1060_MOESM3_ESM.pptx (100 kb)
Supplementary material 3 (PPTX 99 kb)
401_2012_1060_MOESM4_ESM.docx (23 kb)
Supplementary material 4 (DOCX 22 kb)

Copyright information

© Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  • Jörg Balss
    • 1
    • 2
  • Stefan Pusch
    • 1
    • 2
  • Ann-Christin Beck
    • 2
  • Christel Herold-Mende
    • 3
  • Alwin Krämer
    • 4
  • Christian Thiede
    • 5
  • Wolfgang Buckel
    • 6
    • 7
  • Claus-Dieter Langhans
    • 8
  • Jürgen G. Okun
    • 8
  • Andreas von Deimling
    • 1
    • 2
  1. 1.Department of Neuropathology, Institute of PathologyRuprecht-Karls-Universität HeidelbergHeidelbergGermany
  2. 2.Clinical Cooperation Unit NeuropathologyGerman Cancer Research Center (DKFZ)HeidelbergGermany
  3. 3.NeurosurgeryRuprecht-Karls-Universität HeidelbergHeidelbergGermany
  4. 4.Clinical Cooperation Unit Molecular Hematology/Oncology, German Cancer Research Center and Department of Internal Medicine VUniversity of HeidelbergHeidelbergGermany
  5. 5.Medizinische Klinik und Poliklinik 1Universitätsklinikum Carl Gustav Carus an der Technischen Universität DresdenDresdenGermany
  6. 6.Laboratory of Microbial BiochemistryPhilipps-Universität MarburgMarburgGermany
  7. 7.Max-Planck-Institut für Terrestrische MikrobiologieMarburgGermany
  8. 8.Department of General PediatricsUniversity Children’s HospitalHeidelbergGermany