title: Development and implementation of a novel assay for L-2-hydroxyglutarate dehydrogenase (L-2-HGDH) in cell lysates: L-2-HGDH deficiency in 15 patients with L-2-hydroxyglutaric aciduria.
abstract: 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.
authors.affiliation: Metabolic Unit, Department of Clinical Chemistry, PK 1X 014, VU University Medical Center, De Boelelaan 1117, 1081 HV, Amsterdam, The Netherlands.
authors.forename: M
authors.initials: M
authors.lastname: Kranendijk
authors.affiliation: Metabolic Unit, Department of Clinical Chemistry, PK 1X 014, VU University Medical Center, De Boelelaan 1117, 1081 HV, Amsterdam, The Netherlands.
authors.forename: G S
authors.initials: GS
authors.lastname: Salomons
authors.affiliation: Department of Biological Sciences, Michigan Technological University, Houghton, MI, USA.
authors.forename: K M
authors.initials: KM
authors.lastname: Gibson
authors.affiliation: Division of Metabolic Diseases and Nutrition, Cerrahpasa Medical Faculty, University of Istanbul, Istanbul, Turkey.
authors.forename: C
authors.initials: C
authors.lastname: Aktuglu-Zeybek
authors.affiliation: Laboratoire de Biochimie Médicale, Rouen University Hospital, University of Rouen, Rouen, France.
authors.forename: S
authors.initials: S
authors.lastname: Bekri
authors.affiliation: Department of Clinical Genetics, Rigshospitalet, University Hospital of Copenhagen, Copenhagen, Denmark.
authors.forename: E
authors.initials: E
authors.lastname: Christensen
authors.affiliation: Division of Clinical Genetics, Hospital for Sick Children, Toronto, ON, Canada.
authors.forename: J
authors.initials: J
authors.lastname: Clarke
authors.affiliation: Department of Neuropediatrics, Justus-Liebig University, Giessen, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Hahn
authors.affiliation: Department of Human Genetics and Metabolic Diseases, Hadassah - Hebrew University Medical Center, Jerusalem, Israel.
authors.forename: S H
authors.initials: SH
authors.lastname: Korman
authors.affiliation: Child Neurology, Children's Hospital Zagreb, University of Zagreb Medical School, Zagreb, Croatia.
authors.forename: V
authors.initials: V
authors.lastname: Mejaski-Bosnjak
authors.affiliation: Department of Pediatrics, University of Freiburg, Freiburg, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Superti-Furga
authors.affiliation: Service Maladies Héréditaires du Métabolisme and INSERM U820, Centre de Biologie Est, CHU Lyon, France.
authors.forename: C
authors.initials: C
authors.lastname: Vianey-Saban
authors.affiliation: Paediatric Neurology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: M S
authors.initials: MS
authors.lastname: van der Knaap
authors.affiliation: Metabolic Unit, Department of Clinical Chemistry, PK 1X 014, VU University Medical Center, De Boelelaan 1117, 1081 HV, Amsterdam, The Netherlands. c.jakobs@vumc.nl.
authors.forename: C
authors.initials: C
authors.lastname: Jakobs
authors.affiliation: Metabolic Unit, Department of Clinical Chemistry, PK 1X 014, VU University Medical Center, De Boelelaan 1117, 1081 HV, Amsterdam, The Netherlands.
authors.forename: E A
authors.initials: EA
authors.lastname: Struys
mesh_terms: D000429:Alcohol Oxidoreductases / Q000032:analysis* / Q000134:cerebrospinal fluid / Q000172:deficiency*; D000818:Animals; D020739:Brain Diseases, Metabolic, Inborn / Q000134:cerebrospinal fluid / Q000175:diagnosis* / Q000473:pathology; D002138:Calibration; D002457:Cell Extracts / Q000032:analysis / Q000737:chemistry*; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid / Q000379:methods; D057075:Enzyme Assays / Q000379:methods* / Q000592:standards; D005347:Fibroblasts / Q000737:chemistry / Q000201:enzymology; D006801:Humans; D008214:Lymphocytes / Q000737:chemistry / Q000201:enzymology; D008954:Models, Biological; D008958:Models, Molecular; D051381:Rats; D012107:Research Design; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D002457:Cell Extracts; D000429:Alcohol Oxidoreductases; C506763:L2HGDH protein, human