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

• Verfasst von: Balß, Jörg [VerfasserIn]
• Verfasst von: Pusch, Stefan [VerfasserIn]
• Verfasst von: Herold-Mende, Christel [VerfasserIn]
• Verfasst von: Krämer, Alwin [VerfasserIn]
• Verfasst von: Langhans, Claus-Dieter [VerfasserIn]
• Verfasst von: Okun, Jürgen G. [VerfasserIn]
• Verfasst von: Deimling, Andreas von [VerfasserIn]
• Titel: Enzymatic assay for quantitative analysis of (D)-2-hydroxyglutarate
• Verf.angabe: 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
• E-Jahr: 2012
• Jahr: 2 November 2012
• Umfang: 9 S.
• Teil: volume:124
• Teil: year:2012
• Teil: number:6
• Teil: pages:883-891
• Teil: extent:9
• Fussnoten: Gesehen am 25.09.2018
• Titel Quelle: Enthalten in: Acta neuropathologica
• Ort Quelle: Berlin : Springer, 1961
• Jahr Quelle: 2012
• Band/Heft Quelle: 124(2012), 6, Seite 883-891
• ISSN Quelle: 1432-0533
• DOI: doi:10.1007/s00401-012-1060-y
• Datenträger: Online-Ressource
• Sprache: eng
• Sach-SW: Enzymatic Test
• Sach-SW: Fluorescence Intensity Unit
• Sach-SW: Fresh Freeze Tissue
• Sach-SW: IDH1 R132H
• Sach-SW: Resazurin
• K10plus-PPN: 1581269994

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.