title: Reliable analysis of phenylalanine and tyrosine in a minimal volume of blood.
abstract: OBJECTIVE
  Phenylketonuria (PKU) is an inborn error of phenylalanine metabolism due to a defect in phenylalanine hydroxylase (PAH). Treatment principle is to reduce phenylalanine concentration sufficiently to prevent neuropathological effects. Dietary management is performed and the effect of treatment is monitored by regular analysis of phenylalanine and tyrosine. The aim of the study was to develop a rapid method to routinely measure both metabolites in minimal bloodspot volume (1.5 mm Ø, corresponding with a volume of 1.3 μL blood).
  
  
  METHODS
  Whole blood was spiked with phenylalanine and tyrosine at 24 different concentrations. Dried blood spots (DBS) were prepared, after which punches of 1.5 mm Ø and 6 mm Ø (corresponding with a volume of 12.4 μL) were taken. Additionally, punches of both sizes were prepared from DBS of PKU-patients (n=77). All samples were analyzed by tandem mass-spectrometry and results between both punches were compared.
  
  
  RESULTS
  A good correlation between concentrations of phenylalanine and tyrosine in 1.5 and 6 mm punches was found (r(2)=0.9917 and r(2)=0.9892, respectively). Analysis of phenylalanine and tyrosine in punches of PKU-patients (n=77) showed similar results and fitted within the procentual range of the between run variation.
  
  
  CONCLUSIONS
  We developed an accurate and rapid method to analyze phenylalanine and tyrosine concentrations in a 1.5 mm Ø bloodspot punch with an estimated whole blood volume of 1.3 μL. This technical improvement does not only result in a 10 fold reduction in required patients' material, but also in a 30-60 min time saving in sample preparation.
authors.affiliation: Section Metabolic Diagnostics, Department of Medical Genetics, University Medical Center Utrecht, KC.02.069.1, Lundlaan 6, 3584 EA Utrecht, The Netherlands. b.prinsen@umcutrecht.nl
authors.forename: Hubertus C M T
authors.initials: HC
authors.lastname: Prinsen
authors.forename: Nellie E
authors.initials: NE
authors.lastname: Holwerda-Loof
authors.forename: Monique G M
authors.initials: MG
authors.lastname: de Sain-van der Velden
authors.forename: Gepke
authors.initials: G
authors.lastname: Visser
authors.forename: Nanda M
authors.initials: NM
authors.lastname: Verhoeven-Duif
mesh_terms: D059788:Dried Blood Spot Testing; D006801:Humans; D010649:Phenylalanine / Q000097:blood*; D010651:Phenylalanine Hydroxylase / Q000235:genetics; D010661:Phenylketonurias / Q000097:blood / Q000175:diagnosis* / Q000473:pathology; D053719:Tandem Mass Spectrometry; D014443:Tyrosine / Q000097:blood*
chemical_list: D014443:Tyrosine; D010649:Phenylalanine; D010651:Phenylalanine Hydroxylase
keywords: DBS; Dried blood spots; Dried blood spots (DBS); PAH; PKU; Phenylalanine; Phenylalanine hydroxylase; Phenylketonuria; Tandem mass spectrometry; Tyrosine