Enzymatic assay for quantitative analysis of (D)-2-hydroxyglutarate
Enzymatic assay for quantitative analysis of (D)-2-hydroxyglutarate
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DOI：
10.1007/s00401-012-1060-y
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发表时间：
2012-12-01
影响因子：
12.7
通讯作者：
von Deimling, Andreas
中科院分区：
文献类型：
--
作者：
Balss, Joerg;Pusch, Stefan;von Deimling, Andreas
在某些代谢性疾病和异柠檬酸脱氢酶1(IDH1)和异柠檬酸脱氢酶2(IDH2)基因突变的肿瘤中，(D)-2-羟基戊二酸[D2HG，(R)-2-羟基戊二酸]水平升高。D2HG的测定对疾病的诊断和监测具有重要意义。D2HG水平的标准检测方法是液-质联用或气-质联用。在这里，我们提出了一种快速、廉价和灵敏的酶法来检测D2HG水平。该分析是基于D2HG在酶(D)-2-羟基戊二酸脱氢酶(HGDH)和烟酰胺腺嘌呤二核苷酸(NAD(+))存在下转化为α-酮戊二酸(α-KG)。D2HG浓度的测定是基于对化学计量生成的NADH的检测。酶法检测肿瘤组织中D2HG的定量下限为0.44 mU M，血清中D2HG的定量下限为2.77 mU M。这些限值使检测人肿瘤组织和血清中D2HG的基础水平成为可能。冰冻和石蜡包埋的含IDH突变的肿瘤组织或IDH突变的急性髓系白血病患者血清中的D2HG水平显著升高，用该方法可以很容易地识别D2HG。总之，该方法有助于区分肿瘤组织、血清、尿液、培养细胞和培养上清液中D2HG的基础水平和升高水平。
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 alpha-ketoglutarate (alpha KG) 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 mu M and in serum 2.77 mu M. 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.