Summary
d-2-Hydroxyglutaric aciduria (d-2-HGA) is a neurometabolic disorder characterized by elevated levels of d-2-hydroxyglutarate (d-2-HG) in physiological fluids. Recent findings revealed that mutations in the D2HGDH gene, encoding d-2-hydroxyglutarate dehydrogenase, cause d-2-HGA. So far, a functionalenzyme assay to determine d-2-hydroxyglutarate dehydrogenase activity, converting d-2-HG into 2-ketoglutarate (2-KG), has been unavailable. We have now developed a unique enzyme assay for the determination of d-2-hydroxyglutarate dehydrogenase activity in cells derived from d-2-HGA patients and controls. The enzyme assay was performed using enantiomerically pure stable-isotope-labelled d-2-hydroxy[3,3,4,4-2H4]glutarate. This substrate is convertedby d-2-hydroxyglutarate dehydrogenase into 2-[3,3,4,4-2H4]ketoglutarate, which is subsequently converted into l-[3,3,4,4-2H4]glutamate by l-glutamate dehydrogenase, present in saturating amounts in cell homogenates. Enzyme activities were quantified using LC-MS/MS. The mean activities in control fibroblast and lymphoblast homogenates were 298 ± 207 and 1670 ± 940 pmol/h per mg protein, respectively. In fibroblast and lymphoblast cell lines derived from patients with pathogenic mutations in the D2HGDH gene, considerably decreased enzyme activities (e.g. <41 pmol/h per mg protein) were found compared with controls. This enzyme assay will have additional utility in further differentiating patients with d-2-HGA and l-2-HGA and in assessing the residual activities linked to pathogenic mutations in the D2HGDH gene.
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Abbreviations
- 2-KG:
-
2-ketoglutarate
- CV:
-
coefficient of variation
- d-2-HG:
-
d-2-hydroxyglutarate
- d-2-HGA:
-
d-2-hydroxyglutaric aciduria
- D2HGDH :
-
d-2-hydroxyglutarate dehydrogenase gene
- HPLC:
-
high-performance liquid chromatography
- l-2-HG:
-
l-2-hydroxyglutarate
- LC-MS/MS:
-
liquid chromatography–tandem mass spectrometry
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Acknowledgements
We thank Dr M. Alessandri and Dr G. Cioni (Italy), Dr W. Craigen, Dr C. Gaba and Dr C. Sansaricq (USA), Dr S. Korman (Israel), and the Birmingham Children’s Hospital for submission of the d-2-HGA patients’ cell lines.
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Communicating editor: Moacir Wajner
Competing interests: None declared
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Wickenhagen, W.V., Salomons, G.S., Gibson, K.M. et al. Measurement of d-2-hydroxyglutarate dehydrogenase activity in cell homogenates derived from d-2-hydroxyglutaric aciduria patients. J Inherit Metab Dis 32, 264–268 (2009). https://doi.org/10.1007/s10545-009-1104-1
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DOI: https://doi.org/10.1007/s10545-009-1104-1