A novel UPLC-MS/MS based method to determine the activity of N-acetylglutamate synthase in liver tissue.
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.
['Dercksen|Marli|M|', 'Duran|Marinus|M|', 'IJlst|Lodewijk|L|', 'Kulik|Wim|W|', 'Ruiter|Jos P N|JP|', 'van Cruchten|Arno|A|', 'Tuchman|Mendel|M|', 'Wanders|Ronald J A|RJ|']
D000105:Acetyl Coenzyme A D051046:Amino-Acid N-Acetyltransferase D000818:Animals D002222:Carbamoyl-Phosphate Synthase (Ammonia) D006801:Humans D022124:Hyperammonemia D008099:Liver D051379:Mice D018345:Mice, Knockout D053719:Tandem Mass Spectrometry D056806:Urea Cycle Disorders, Inborn
N-acetylglutamate Hyperammonia Mass spectrometry NAGS-deficiency