title: Enzymatic assay for quantitative analysis of (D)-2-hydroxyglutarate.
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.
authors.affiliation: Department of Neuropathology, Institute of Pathology, Ruprecht-Karls-Universität Heidelberg, Heidelberg, Germany.
authors.forename: Jörg
authors.initials: J
authors.lastname: Balss
authors.forename: Stefan
authors.initials: S
authors.lastname: Pusch
authors.forename: Ann-Christin
authors.initials: AC
authors.lastname: Beck
authors.forename: Christel
authors.initials: C
authors.lastname: Herold-Mende
authors.forename: Alwin
authors.initials: A
authors.lastname: Krämer
authors.forename: Christian
authors.initials: C
authors.lastname: Thiede
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Buckel
authors.forename: Claus-Dieter
authors.initials: CD
authors.lastname: Langhans
authors.forename: Jürgen G
authors.initials: JG
authors.lastname: Okun
authors.forename: Andreas
authors.initials: A
authors.lastname: von Deimling
mesh_terms: D000429:Alcohol Oxidoreductases / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D014408:Biomarkers, Tumor / Q000032:analysis / Q000235:genetics; D057075:Enzyme Assays / Q000379:methods*; D005977:Glutarates / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D007521:Isocitrate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D015470:Leukemia, Myeloid, Acute / Q000175:diagnosis* / Q000235:genetics; D009154:Mutation / Q000235:genetics
chemical_list: D014408:Biomarkers, Tumor; D005977:Glutarates; C019417:alpha-hydroxyglutarate; D000429:Alcohol Oxidoreductases; D007521:Isocitrate Dehydrogenase; C023858:2-hydroxyglutarate dehydrogenase