Measurement of D: -2-hydroxyglutarate dehydrogenase activity in cell homogenates derived from D: -2-hydroxyglutaric aciduria patients.
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-(2)H(4)]glutarate. This substrate is convertedby D: -2-hydroxyglutarate dehydrogenase into 2-[3,3,4,4-(2)H(4)]ketoglutarate, which is subsequently converted into L: -[3,3,4,4-(2)H(4)]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.
Wickenhagen
W V
WV
Metabolic Unit, Department of Clinical Chemistry, VU University Medical Center, De Boelelaan 1117, 1081 HV, Amsterdam, The Netherlands.
Salomons
G S
GS
Gibson
K M
KM
Jakobs
C
C
Struys
E A
EA
D000429:Alcohol Oxidoreductases / Q000235:genetics* / Q000378:metabolism*; D000465:Algorithms; D000592:Amino Acid Metabolism, Inborn Errors / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D005347:Fibroblasts / Q000201:enzymology; D005977:Glutarates / Q000302:isolation & purification / Q000652:urine*; D006207:Half-Life; D006801:Humans; D008214:Lymphocytes / Q000201:enzymology; D013058:Mass Spectrometry; D013237:Stereoisomerism
D005977:Glutarates; C019417:alpha-hydroxyglutarate; D000429:Alcohol Oxidoreductases; C023858:2-hydroxyglutarate dehydrogenase