Stable isotope dilution microquantification of creatine metabolites in plasma, whole blood and dried blood spots for pharmacological studies in mouse models of creatine deficiency

C Tran; M Yazdanpanah; L Kyriakopoulou; V Levandovskiy; H Zahid; A Naufer; D Isbrandt; A Schulze

doi:10.1016/j.cca.2014.05.007

Stable isotope dilution microquantification of creatine metabolites in plasma, whole blood and dried blood spots for pharmacological studies in mouse models of creatine deficiency

Clin Chim Acta. 2014 Sep 25:436:160-8. doi: 10.1016/j.cca.2014.05.007. Epub 2014 May 28.

Authors

C Tran¹, M Yazdanpanah², L Kyriakopoulou², V Levandovskiy³, H Zahid³, A Naufer³, D Isbrandt⁴, A Schulze⁵

Affiliations

¹ Division of Clinical and Metabolic Genetics, The Hospital for Sick Children, Toronto, ON, Canada; University of Toronto, Toronto, ON, Canada.
² Department of Pediatric Laboratory Medicine, The Hospital for Sick Children, Toronto, ON, Canada.
³ Genetics and Genome Biology, The Research Institute, The Hospital for Sick Children, Toronto, ON, Canada.
⁴ University Medical Center Hamburg/Eppendorf, Germany.
⁵ 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.

PMID: 24877651
DOI: 10.1016/j.cca.2014.05.007

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.

Conclusion: The assay is fast and reliable to study creatine metabolism and pharmacokinetics in mouse models of creatine deficiency.

Keywords: Creatine deficiency syndromes; Creatine metabolites; Mouse models; Stable isotope dilution; Tandem mass spectrometry.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Amidinotransferases / blood
Amidinotransferases / deficiency*
Amidinotransferases / metabolism
Amino Acid Metabolism, Inborn Errors / blood*
Amino Acid Metabolism, Inborn Errors / metabolism
Animals
Chromatography, Liquid
Creatine / blood*
Creatine / deficiency*
Creatine / metabolism
Developmental Disabilities / blood
Developmental Disabilities / metabolism
Disease Models, Animal
Dried Blood Spot Testing / methods*
Guanidinoacetate N-Methyltransferase / blood
Guanidinoacetate N-Methyltransferase / deficiency*
Guanidinoacetate N-Methyltransferase / metabolism
Humans
Intellectual Disability / blood*
Intellectual Disability / metabolism
Isotopes / chemistry
Language Development Disorders / blood*
Language Development Disorders / metabolism
Limit of Detection
Linear Models
Mice
Movement Disorders / blood
Movement Disorders / congenital*
Movement Disorders / metabolism
Plasma / metabolism*
Reproducibility of Results
Speech Disorders / blood*
Speech Disorders / metabolism
Tandem Mass Spectrometry

Substances

Isotopes
Guanidinoacetate N-Methyltransferase
Amidinotransferases
Creatine

Supplementary concepts

Arginine-Glycine Amidinotransferase Deficiency
Guanidinoacetate methyltransferase deficiency