Journal article
A simple dried blood spot-method for in vivo measurement of ureagenesis by gas chromatography-mass spectrometry using stable isotopes.
- Allegri G Division of Metabolism and Children's Research Centre (CRC), University Children's Hospital Zurich, Switzerland.
- Deplazes S Division of Metabolism and Children's Research Centre (CRC), University Children's Hospital Zurich, Switzerland.
- Grisch-Chan HM Division of Metabolism and Children's Research Centre (CRC), University Children's Hospital Zurich, Switzerland.
- Mathis D Division of Clinical Chemistry and Biochemistry, University Children's Hospital Zurich, Switzerland.
- Fingerhut R 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.
- Häberle J 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.
- Thöny B 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.
- 2016-12-08
Published in:
- Clinica chimica acta; international journal of clinical chemistry. - 2017
Dried blood spots
GC–MS
Hyperammonemia
OTC
Orotic aciduria
Ureagenesis
Animals
Dried Blood Spot Testing
Fasting
Gas Chromatography-Mass Spectrometry
Isotopes
Male
Mice
Ornithine Carbamoyltransferase
Urea
English
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 spfash mice 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 spfash mice 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 spfash mice under fasting conditions (19% of wild-type). Five consecutive injections over a period of five weeks had no effect on body weight or ureagenesis.
CONCLUSION
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.
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 spfash mice 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 spfash mice 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 spfash mice under fasting conditions (19% of wild-type). Five consecutive injections over a period of five weeks had no effect on body weight or ureagenesis.
CONCLUSION
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.
- Language
-
- English
- Open access status
- closed
- Identifiers
-
- DOI 10.1016/j.cca.2016.11.038
- PMID 27923571
- Persistent URL
- https://folia.unifr.ch/global/documents/21463
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