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

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 [N-15]ammonium chloride ([(NH4Cl)-N-15) as a tracer. Non-labeled and [N-15]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 spf(ash) 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 mu L) was collected through tail vein puncture before and after [N-15]H4Cl intraperitoneal injections over a two hour period. The tested matrices, blood, plasma and dried blood spots, can be used to quantify ureagenesis. Upon [N-15]H4Cl challenge, urea production in spf(ash) 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 spf(ash) 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. (C) 2016 Elsevier B.V. All rights reserved.