title: Stable isotope dilution microquantification of creatine metabolites in plasma, whole blood and dried blood spots for pharmacological studies in mouse models of creatine deficiency.
abstract: BACKGROUND
  To develop an accurate stable isotope dilution assay for simultaneous quantification of creatine metabolites ornithine, arginine, creatine, creatinine, and guanidinoacetate in very small blood sample volumes to study creatine metabolism in mice.
  
  
  METHODS
  Liquid-chromatography (C18) tandem mass spectrometry with butylation was performed in positive ionization mode. Stable isotope dilution assay with external calibration was applied to three different specimen types, plasma, whole blood and dried blood spot (DBS).
  
  
  RESULTS
  Analytical separation, sensitivity, accuracy, and linearity of the assay were adequate. The stable isotope dilution assay in plasma revealed no significant bias to gold standard methods for the respective analytes. Compared to plasma, we observed an overestimate of creatine and creatinine (2- to 5-fold and 1.2- to 2-fold, respectively) in whole-blood and DBS, and an underestimate of arginine (2.5-fold) in DBS. Validation of the assay in mouse models of creatine deficiency revealed plasma creatine metabolite pattern in good accordance with those observed in human GAMT and AGAT deficiency. Single dose intraperitoneal application of ornithine in wild-type mice lead to fast ornithine uptake (Tmax ≤ 10 min) and elimination (T1/2=24 min), and a decline of guanidinoacetate.
  
  
  CONCLUSIONS
  The assay is fast and reliable to study creatine metabolism and pharmacokinetics in mouse models of creatine deficiency.
authors.affiliation: Division of Clinical and Metabolic Genetics, The Hospital for Sick Children, Toronto, ON, Canada; University of Toronto, Toronto, ON, Canada.
authors.forename: C
authors.initials: C
authors.lastname: Tran
authors.affiliation: Department of Pediatric Laboratory Medicine, The Hospital for Sick Children, Toronto, ON, Canada.
authors.forename: M
authors.initials: M
authors.lastname: Yazdanpanah
authors.affiliation: Department of Pediatric Laboratory Medicine, The Hospital for Sick Children, Toronto, ON, Canada.
authors.forename: L
authors.initials: L
authors.lastname: Kyriakopoulou
authors.affiliation: Genetics and Genome Biology, The Research Institute, The Hospital for Sick Children, Toronto, ON, Canada.
authors.forename: V
authors.initials: V
authors.lastname: Levandovskiy
authors.affiliation: Genetics and Genome Biology, The Research Institute, The Hospital for Sick Children, Toronto, ON, Canada.
authors.forename: H
authors.initials: H
authors.lastname: Zahid
authors.affiliation: Genetics and Genome Biology, The Research Institute, The Hospital for Sick Children, Toronto, ON, Canada.
authors.forename: A
authors.initials: A
authors.lastname: Naufer
authors.affiliation: University Medical Center Hamburg/Eppendorf, Germany.
authors.forename: D
authors.initials: D
authors.lastname: Isbrandt
authors.affiliation: Division of Clinical and Metabolic Genetics, The Hospital for Sick Children, Toronto, ON, Canada; University of Toronto, Toronto, ON, Canada; Genetics and Genome Biology, The Research Institute, The Hospital for Sick Children, Toronto, ON, Canada.
authors.forename: A
authors.initials: A
authors.lastname: Schulze
mesh_terms: D000579:Amidinotransferases / Q000097:blood / Q000172:deficiency* / Q000378:metabolism; D000592:Amino Acid Metabolism, Inborn Errors / Q000097:blood* / Q000378:metabolism; D000818:Animals; D002853:Chromatography, Liquid; D003401:Creatine / Q000097:blood* / Q000172:deficiency* / Q000378:metabolism; D002658:Developmental Disabilities / Q000097:blood / Q000378:metabolism; D004195:Disease Models, Animal; D059788:Dried Blood Spot Testing / Q000379:methods*; D050937:Guanidinoacetate N-Methyltransferase / Q000097:blood / Q000172:deficiency* / Q000378:metabolism; D006801:Humans; D008607:Intellectual Disability / Q000097:blood* / Q000378:metabolism; D007554:Isotopes / Q000737:chemistry; D007805:Language Development Disorders / Q000097:blood* / Q000378:metabolism; D057230:Limit of Detection; D016014:Linear Models; D051379:Mice; D009069:Movement Disorders / Q000097:blood / Q000151:congenital* / Q000378:metabolism; D010949:Plasma / Q000378:metabolism*; D015203:Reproducibility of Results; D013064:Speech Disorders / Q000097:blood* / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D007554:Isotopes; D050937:Guanidinoacetate N-Methyltransferase; D000579:Amidinotransferases; D003401:Creatine
keywords: Creatine deficiency syndromes; Creatine metabolites; Mouse models; Stable isotope dilution; Tandem mass spectrometry