title: Guanidinoacetate Methyltransferase Activity in Lymphocytes, for a Fast Diagnosis.
abstract: BACKGROUND
  Guanidinoacetate methyltransferase (GAMT) deficiency is an inborn error of metabolism (IEM), clinically characterized by intellectual disability, developmental delay, seizures, and movement disorders. Biochemical diagnosis of GAMT deficiency is based on the measurement of creatine and guanidinoacetate in urine, plasma, or CSF and is confirmed genetically by DNA analysis or by enzyme assay in lymphoblasts or fibroblasts. To obtain enough cells, these cells need to be cultured for at least 1 month. A less time-consuming diagnostic functional test is needed, since GAMT deficiency is a candidate for newborn screening (NBS) programs, to be able to confirm or rule out this IEM after an initial positive result in the NBS.
  
  
  METHODS
  Stable-isotope-labeled13C2-guanidinoacetate and2H3-S-adenosylmethionine (SAM) were used, which are converted by GAMT present in lymphocyte extracts into2H3-13C2-creatine. The formed2H3-13C2-creatine was butylated and subsequently measured by liquid chromatography tandem mass-spectrometry (LC-MS/MS).
  
  
  RESULTS
  We measured GAMT enzyme activity in lymphocyte extracts of 24 controls, 3 GAMT deficient patients and of 2 parents proven to be carrier. Because GAMT activity decreases when isolation time after venipuncture increases, reference values were obtained for 2 control groups: isolation on the day of venipuncture (27-130 pmol/h/mg) and 1 day afterwards (15-146 pmol/h/mg). Deficient patients had no detectable GAMT activity. The two carriers had GAMT activity within the normal range.
  
  
  CONCLUSIONS
  We designed a fast, less invasive, and valid method to measure GAMT activity in lymphocytes using LC-MS/MS analysis without the need of time-consuming and laborious cell culture.
authors.affiliation: Metabolic Laboratory, Department of Clinical Chemistry, Amsterdam Neuroscience, VU Medical Center, Amsterdam, The Netherlands.
authors.forename: Lisette M
authors.initials: LM
authors.lastname: Berends
authors.affiliation: Metabolic Laboratory, Department of Clinical Chemistry, Amsterdam Neuroscience, VU Medical Center, Amsterdam, The Netherlands.
authors.forename: Eduard A
authors.initials: EA
authors.lastname: Struys
authors.affiliation: Metabolic Laboratory, Department of Clinical Chemistry, Amsterdam Neuroscience, VU Medical Center, Amsterdam, The Netherlands.
authors.forename: Birthe
authors.initials: B
authors.lastname: Roos
authors.affiliation: Metabolic Laboratory, Department of Clinical Chemistry, Amsterdam Neuroscience, VU Medical Center, Amsterdam, The Netherlands.
authors.forename: Ulbe
authors.initials: U
authors.lastname: Holwerda
authors.affiliation: Metabolic Laboratory, Department of Clinical Chemistry, Amsterdam Neuroscience, VU Medical Center, Amsterdam, The Netherlands.
authors.forename: Erwin E W
authors.initials: EEW
authors.lastname: Jansen
authors.affiliation: Metabolic Laboratory, Department of Clinical Chemistry, Amsterdam Neuroscience, VU Medical Center, Amsterdam, The Netherlands.
authors.forename: Gajja S
authors.initials: GS
authors.lastname: Salomons
authors.affiliation: Metabolic Laboratory, Department of Clinical Chemistry, Amsterdam Neuroscience, VU Medical Center, Amsterdam, The Netherlands. m.wamelink@vumc.nl.
authors.forename: Mirjam M C
authors.initials: MMC
authors.lastname: Wamelink
keywords: Enzyme assay; GAMT; LC-MS/MS; Lymphocytes