Marli Dercksen1, Marinus Duran2, Lodewijk IJlst2, Wim Kulik2, Jos P N Ruiter2, Arno van Cruchten2, Mendel Tuchman3, Ronald J A Wanders2. 1. Laboratory Genetic Metabolic Diseases, Departments of Pediatrics and Clinical Chemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; Human Metabonomics, North-West University, Potchefstroom Campus, South Africa. Electronic address: marli.dercksen@nwu.ac.za. 2. Laboratory Genetic Metabolic Diseases, Departments of Pediatrics and Clinical Chemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. 3. Children's National Medical Center, The George Washington University, Washington, DC, USA.
Abstract
BACKGROUND: N-acetylglutamate synthase (NAGS) plays a key role in the removal of ammonia via the urea cycle by catalyzing the synthesis of N-acetylglutamate (NAG), the obligatory cofactor in the carbamyl phosphate synthetase 1 reaction. Enzymatic analysis of NAGS in liver homogenates has remained insensitive and inaccurate, which prompted the development of a novel method. METHODS: UPLC-MS/MS was used in conjunction with stable isotope (N-acetylglutamic-2,3,3,4,4-d5 acid) dilution for the quantitative detection of NAG produced by the NAGS enzyme. The assay conditions were optimized using purified human NAGS and the optimized enzyme conditions were used to measure the activity in mouse liver homogenates. RESULTS: A low signal-to-noise ratio in liver tissue samples was observed due to non-enzymatic formation of N-acetylglutamate and low specific activity, which interfered with quantitative analysis. Quenching of acetyl-CoA immediately after the incubation circumvented this analytical difficulty and allowed accurate and sensitive determination of mammalian NAGS activity. The specificity of the assay was validated by demonstrating a complete deficiency of NAGS in liver homogenates from Nags -/- mice. CONCLUSION: The novel NAGS enzyme assay reported herein can be used for the diagnosis of inherited NAGS deficiency and may also be of value in the study of secondary hyperammonemia present in various inborn errors of metabolism as well as drug treatment.
BACKGROUND:N-acetylglutamate synthase (NAGS) plays a key role in the removal of ammonia via the urea cycle by catalyzing the synthesis of N-acetylglutamate (NAG), the obligatory cofactor in the carbamyl phosphate synthetase 1 reaction. Enzymatic analysis of NAGS in liver homogenates has remained insensitive and inaccurate, which prompted the development of a novel method. METHODS: UPLC-MS/MS was used in conjunction with stable isotope (N-acetylglutamic-2,3,3,4,4-d5 acid) dilution for the quantitative detection of NAG produced by the NAGS enzyme. The assay conditions were optimized using purified humanNAGS and the optimized enzyme conditions were used to measure the activity in mouse liver homogenates. RESULTS: A low signal-to-noise ratio in liver tissue samples was observed due to non-enzymatic formation of N-acetylglutamate and low specific activity, which interfered with quantitative analysis. Quenching of acetyl-CoA immediately after the incubation circumvented this analytical difficulty and allowed accurate and sensitive determination of mammalianNAGS activity. The specificity of the assay was validated by demonstrating a complete deficiency of NAGS in liver homogenates from Nags -/- mice. CONCLUSION: The novel NAGS enzyme assay reported herein can be used for the diagnosis of inherited NAGS deficiency and may also be of value in the study of secondary hyperammonemia present in various inborn errors of metabolism as well as drug treatment.