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

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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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Authors and Affiliations

  1. Department of Neuropathology, Institute of Pathology, Ruprecht-Karls-Universität Heidelberg, INF 224, 69120, Heidelberg, Germany

    Jörg Balss, Stefan Pusch & Andreas von Deimling

  2. Clinical Cooperation Unit Neuropathology, German Cancer Research Center (DKFZ), 69120, Heidelberg, Germany

    Jörg Balss, Stefan Pusch, Ann-Christin Beck & Andreas von Deimling

  3. Neurosurgery, Ruprecht-Karls-Universität Heidelberg, 69120, Heidelberg, Germany

    Christel Herold-Mende

  4. Clinical Cooperation Unit Molecular Hematology/Oncology, German Cancer Research Center and Department of Internal Medicine V, University of Heidelberg, 69120, Heidelberg, Germany

    Alwin Krämer

  5. Medizinische Klinik und Poliklinik 1, Universitätsklinikum Carl Gustav Carus an der Technischen Universität Dresden, 01307, Dresden, Germany

    Christian Thiede

  6. Laboratory of Microbial Biochemistry, Philipps-Universität Marburg, 35035, Marburg, Germany

    Wolfgang Buckel

  7. Max-Planck-Institut für Terrestrische Mikrobiologie, 35043, Marburg, Germany

    Wolfgang Buckel

  8. Department of General Pediatrics, University Children’s Hospital, 69120, Heidelberg, Germany

    Claus-Dieter Langhans & Jürgen G. Okun

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  1. Jörg Balss
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Correspondence to Andreas von Deimling.

Electronic supplementary material

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Supplementary figure 1: Optimization of the D2HG assay. (1a) Assay performed with four different buffers. Best performance is realized using HEPES buffer. Hence, following experiments were performed only with HEPES buffer. (1b)To determine the optimal pH conditions several pH values in the range from 7.1 to 8.0 were tested. As expected from previous data [4] pH 8.0 gives the best output but assay could also be performed at pH 7.4 and 7.7 if necessary with only a slight loss in performance. (1c + d) Best concentrations of HGDH and diaphorase were determined with 0.1 μg HGDH and 0.1 U diaphorase per well considering sensitivity and economic reasons.

Supplementary figure 2: Resazurin concentration in the D2HG assay was optimized for different amounts of D2HG (0.5 μM, 5 μM and 50 μM). (2a + b) For low concentrations of D2HG (0.5 μM and 5 μM) best signal-to-noise ratio was seen for concentrations of 1 μM resazurin. (2c + d) Higher D2HG concentrations required higher resazurin levels. For 50 μM D2HG best signal-to-noise ratio was seen with 5 μM resazurin.

Supplementary figure 3: (3a) Comparison of D2HG standard solved in water, human serum and urine detected with diaphorase/resazurin. (3b) D2HG solved in water detected with PMS/XTT. Absorption was measured at 450 nm. (3c) Testing stereoselectivity of HGDH with 5 μM and 50 μM D2HG and L2HG.

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Balss, J., Pusch, S., Beck, AC. et al. Enzymatic assay for quantitative analysis of (d)-2-hydroxyglutarate. Acta Neuropathol 124, 883–891 (2012). https://doi.org/10.1007/s00401-012-1060-y

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