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A simple dried blood spot-method for in vivo measurement of ureagenesis by gas chromatography–mass spectrometry using stable isotopes


Allegri, Gabriella; Deplazes, Sereina; Grisch-Chan, Hiu Man; Mathis, Déborah; Fingerhut, Ralph; Häberle, Johannes; Thöny, Beat (2017). A simple dried blood spot-method for in vivo measurement of ureagenesis by gas chromatography–mass spectrometry using stable isotopes. Clinica Chimica Acta, 464:236-243.

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 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 (5 μL) 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 30 min 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.

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 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 (5 μL) 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 30 min 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.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > University Children's Hospital Zurich > Medical Clinic
Dewey Decimal Classification:610 Medicine & health
Language:English
Date:2017
Deposited On:09 Jan 2017 11:32
Last Modified:08 Dec 2017 21:49
Publisher:Elsevier
ISSN:0009-8981
Publisher DOI:https://doi.org/10.1016/j.cca.2016.11.038
PubMed ID:27923571

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