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01-12-2012 | Methods Paper

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

Authors: 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

Published in: Acta Neuropathologica | Issue 6/2012

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.

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Title: Enzymatic assay for quantitative analysis of (d)-2-hydroxyglutarate
Authors: 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
Publication date: 01-12-2012
Publisher: Springer-Verlag
Published in: Acta Neuropathologica / Issue 6/2012
Print ISSN: 0001-6322
Electronic ISSN: 1432-0533
DOI: https://doi.org/10.1007/s00401-012-1060-y

At a glance: The ONWARDS insulin icodec trials

Springer Medicine

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

Abstract

At a glance: The ONWARDS insulin icodec trials

Springer Medicine

Abstract

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