A novel UPLC-MS/MS based method to determine the activity of N-acetylglutamate synthase in liver tissue

Marli Dercksen; Marinus Duran; Lodewijk IJlst; Wim Kulik; Jos P N Ruiter; Arno van Cruchten; Mendel Tuchman; Ronald J A Wanders

doi:10.1016/j.ymgme.2016.10.004

A novel UPLC-MS/MS based method to determine the activity of N-acetylglutamate synthase in liver tissue

Mol Genet Metab. 2016 Dec;119(4):307-310. doi: 10.1016/j.ymgme.2016.10.004. Epub 2016 Oct 13.

Authors

Marli Dercksen¹, Marinus Duran², Lodewijk IJlst², Wim Kulik², Jos P N Ruiter², Arno van Cruchten², Mendel Tuchman³, Ronald J A Wanders²

Affiliations

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.

PMID: 27771289
DOI: 10.1016/j.ymgme.2016.10.004

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.

Keywords: Hyperammonia; Mass spectrometry; N-acetylglutamate; NAGS-deficiency.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Acetyl Coenzyme A / metabolism
Amino-Acid N-Acetyltransferase / genetics*
Amino-Acid N-Acetyltransferase / metabolism
Animals
Carbamoyl-Phosphate Synthase (Ammonia) / deficiency
Carbamoyl-Phosphate Synthase (Ammonia) / genetics*
Humans
Hyperammonemia / diagnosis*
Hyperammonemia / genetics
Hyperammonemia / metabolism
Hyperammonemia / physiopathology
Liver / enzymology
Mice
Mice, Knockout
Tandem Mass Spectrometry
Urea Cycle Disorders, Inborn / diagnosis*
Urea Cycle Disorders, Inborn / genetics
Urea Cycle Disorders, Inborn / metabolism
Urea Cycle Disorders, Inborn / physiopathology

Substances

Acetyl Coenzyme A
Amino-Acid N-Acetyltransferase
NAGS protein, human
Carbamoyl-Phosphate Synthase (Ammonia)

Supplementary concepts

N-acetyl glutamate synthetase deficiency