title: A simple dried blood spot-method for in vivo measurement of ureagenesis by gas chromatography-mass spectrometry using stable isotopes.
abstract: BACKGROUND
  Clinical management of inherited or acquired hyperammonemia depends mainly on the plasma ammonia level which is not a reliable indicator of urea cycle function as its concentrations largely fluctuate. The gold standard to assess ureagenesis in vivo is the use of stable isotopes.
  
  
  METHODS
  Here we developed and validated a simplified in vivo method with [15N]ammonium chloride ([15N]H4Cl) as a tracer. Non-labeled and [15N]urea were quantified by GC-MS after extraction and silylation.
  
  
  RESULTS
  Different matrices were evaluated for suitability of analysis. Ureagenesis was assessed in ornithine transcarbamylase (OTC)-deficient spfashmice with compromised urea cycle function during fasted and non-fasted feeding states, and after rAAV2/8-vector delivery expressing the murine OTC-cDNA in liver. Blood (5μL) was collected through tail vein puncture before and after [15N]H4Cl intraperitoneal injections over a two hour period. The tested matrices, blood, plasma and dried blood spots, can be used to quantify ureagenesis. Upon [15N]H4Cl challenge, urea production in spfashmice was reduced compared to wild-type and normalized following rAAV2/8-mediated gene therapeutic correction. The most significant difference in ureagenesis was at 30min after injection in untreated spfashmice under fasting conditions (19% of wild-type). Five consecutive injections over a period of five weeks had no effect on body weight or ureagenesis.
  
  
  CONCLUSIONS
  This method is simple, robust and with no apparent risk, offering a sensitive, minimal-invasive, and fast measurement of ureagenesis capacity using dried blood spots. The stable isotope-based quantification of ureagenesis can be applied for the efficacy-testing of novel molecular therapies.
authors.affiliation: Division of Metabolism and Children's Research Centre (CRC), University Children's Hospital Zurich, Switzerland.
authors.forename: Gabriella
authors.initials: G
authors.lastname: Allegri
authors.affiliation: Division of Metabolism and Children's Research Centre (CRC), University Children's Hospital Zurich, Switzerland.
authors.forename: Sereina
authors.initials: S
authors.lastname: Deplazes
authors.affiliation: Division of Metabolism and Children's Research Centre (CRC), University Children's Hospital Zurich, Switzerland.
authors.forename: Hiu Man
authors.initials: HM
authors.lastname: Grisch-Chan
authors.affiliation: Division of Clinical Chemistry and Biochemistry, University Children's Hospital Zurich, Switzerland.
authors.forename: Déborah
authors.initials: D
authors.lastname: Mathis
authors.affiliation: Division of Metabolism and Children's Research Centre (CRC), University Children's Hospital Zurich, Switzerland; Swiss Newborn Screening Laboratory, University Children's Hospital Zurich, Switzerland.
authors.forename: Ralph
authors.initials: R
authors.lastname: Fingerhut
authors.affiliation: Division of Metabolism and Children's Research Centre (CRC), University Children's Hospital Zurich, Switzerland; Zurich Centre for Integrative Human Physiology (ZIHP) and the Neuroscience Centre Zurich (ZNZ), Zurich, Switzerland.
authors.forename: Johannes
authors.initials: J
authors.lastname: Häberle
authors.affiliation: Division of Metabolism and Children's Research Centre (CRC), University Children's Hospital Zurich, Switzerland; Zurich Centre for Integrative Human Physiology (ZIHP) and the Neuroscience Centre Zurich (ZNZ), Zurich, Switzerland. Electronic address: beat.thony@kispi.uzh.ch.
authors.forename: Beat
authors.initials: B
authors.lastname: Thöny
mesh_terms: D000818:Animals; D059788:Dried Blood Spot Testing / Q000379:methods*; D005215:Fasting / Q000097:blood; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D007554:Isotopes; D008297:Male; D051379:Mice; D009954:Ornithine Carbamoyltransferase / Q000378:metabolism; D014508:Urea / Q000097:blood*
chemical_list: D007554:Isotopes; D014508:Urea; D009954:Ornithine Carbamoyltransferase
keywords: Dried blood spots; GC–MS; Hyperammonemia; OTC; Orotic aciduria; Ureagenesis