An enzymatic fluorimetric assay for determination of N-acetylaspartate

N-acetylaspartate (NAA) is an abundant metabolite in the mammalian brain and a precursor of the neuropeptide N-acetylaspartylglutamate (NAAG). The physiological role of NAA is not fully understood and requires further studies. We here describe the development of a coupled enzymatic fluorimetric assay for the determination of NAA in biological samples. Deproteinized tissue extracts are first passed through a strong cation exchange col-umn to remove aspartate. NAA in the sample is hydrolysed by aspartoacylase and released aspartate oxidized using L-aspartate oxidase. Generated H2O2 is measured with peroxidase in a fluorimetric assay using Ampliflu Red. The limit of detection and the lower limit of quantification are 1.0 mu M (10 pmol/well) and 3.3 mu M (33 pmol/ well), respectively, with a linear range to 100 mu M. Specificity of the assay was confirmed using samples from mice deficient in NAA synthase Nat8l that were spiked with NAA. Analysis of samples from aspartoacylase-deficient mice showed a 2 to 3-fold increase in brain NAA concentration, in line with previous reports. Mice lacking NAAG synthetases had a slightly reduced (-10%) brain NAA level. Thus, the new fluorimetric enzymatic assay is useful to perform sensitive and large scale quantification of NAA in biological samples without the need for expensive equipment.

Automated summary

In this study, a coupled enzymatic fluorimetric assay was developed for the quantification of NAA in biological samples. The assay involves the removal of aspartate using strong cation exchange column, hydrolysis of NAA by aspartoacylase and oxidation of released aspartate by L-aspartate oxidase. The generated H2O2 is then measured using peroxidase in a fluorimetric assay with Ampliflu Red. The assay is sensitive, cost-effective and suitable for large-scale quantification of NAA in biological samples.