Metabolic Unit, Department of Clinical Chemistry, PK 1X 014, VU University Medical Center, De Boelelaan 1117, 1081 HV, Amsterdam, The Netherlands.
Department of Biological Sciences, Michigan Technological University, Houghton, MI, USA.
J Inherit Metab Dis. 2009 Dec;32(6):713. doi: 10.1007/s10545-009-1282-x. Epub 2009 Oct 10.
L-2-hydroxyglutaric aciduria (L-2-HGA) is a rare inherited autosomal recessive neurometabolic disorder caused by mutations in the gene encoding L-2-hydroxyglutarate dehydrogenase. An assay to evaluate L-2-hydroxyglutarate dehydrogenase (L-2-HGDH) activity in fibroblast, lymphoblast and/or lymphocyte lysates has hitherto been unavailable. We developed an L-2-HGDH enzyme assay in cell lysates based on the conversion of stable-isotope-labelled L-2-hydroxyglutarate to 2-ketoglutarate, which is converted into L-glutamate in situ. The formation of stable isotope labelled L-glutamate is therefore a direct measure of L-2-HGDH activity, and this product is detected by liquid chromatography-tandem mass spectrometry. A deficiency of L-2-HGDH activity was detected in cell lysates from 15 out of 15 L-2-HGA patients. Therefore, this specific assay confirmed the diagnosis unambiguously affirming the relationship between molecular and biochemical observations. Residual activity was detected in cells derived from one L-2-HGA patient. The L-2-HGDH assay will be valuable for examining in vitro riboflavin/FAD therapy to rescue L-2-HGDH activity.
L-2-羟戊二酸尿症（L-2-HGA）是一种罕见的遗传性常染色体隐性神经代谢疾病，由编码 L-2-羟戊二酸脱氢酶的基因突变引起。目前尚无法在成纤维细胞、淋巴母细胞和/或淋巴细胞裂解物中评估 L-2-羟戊二酸脱氢酶（L-2-HGDH）活性的测定方法。我们在细胞裂解物中开发了一种 L-2-HGDH 酶测定方法，该方法基于稳定同位素标记的 L-2-羟戊二酸转化为 2-酮戊二酸，后者在原位转化为 L-谷氨酸。因此，稳定同位素标记的 L-谷氨酸的形成是 L-2-HGDH 活性的直接测量指标，该产物通过液相色谱-串联质谱法检测。在 15 名 L-2-HGA 患者的 15 个细胞裂解物中检测到 L-2-HGDH 活性缺乏。因此，该特定测定方法明确地确认了分子和生化观察之间的关系，从而明确了诊断。在一名 L-2-HGA 患者来源的细胞中检测到残留活性。L-2-HGDH 测定方法将有助于检查体外核黄素/FAD 治疗以恢复 L-2-HGDH 活性。
