A rapid and sensitive fluorometric method for determination of aldehyde oxidase activity

Apenova, Nancy; Peng, Hui; Hecker, Markus; Brinkmann, Markus

A rapid and sensitive fluorometric method for determination of aldehyde oxidase activity

dc.contributor.author	Apenova, Nancy
dc.contributor.author	Peng, Hui
dc.contributor.author	Hecker, Markus
dc.contributor.author	Brinkmann, Markus
dc.date.accessioned	2023-10-17T19:37:32Z
dc.date.available	2023-10-17T19:37:32Z
dc.date.issued	2018
dc.description	CC BY-NC-ND Accepted Version	en_US
dc.description.abstract	Previous research has characterized the important role of aldehyde oxidases (AOX) in biotransformation of N-heterocyclic therapeutic drugs and environmental contaminants in mammals. Research pertaining to AOX activity in non-mammalian vertebrates, however, is scarce, despite its biological role as a potentially important metabolic pathway for xenobiotics. One of the limiting factors of research on AOX is that available photometric methods are relatively insensitive, limited in throughput, and prone to cross-reactivity from other enzymes. Therefore, this study aimed to develop a novel and improved fluorometric AOX assay. This assay is based on the conversion of the exogenous aldehyde substrate 4-(dimethyl)amino cinnamaldehyde to its corresponding fluorescent acid by AOX, and was evaluated using partially purified hepatic cytosol from rat, human, and rainbow trout. Purification of native cytosol by heat treatment and ammonium sulfate precipitation resulted in increased specific activity of AOX. Michaelis-Menten kinetic parameters (Km and Vmax) were comparable to values previously generated by photometric methods. Furthermore, effects of the inhibitor hydralazine on AOX activity revealed half maximal inhibitory concentrations comparable to those generated using conventional methods. Product identity was confirmed by liquid chromatography and mass spectrometry. In summary, this study successfully developed a rapid and sensitive assay for determination of AOX activity in across different vertebrate species that is 4- to 10-fold more sensitive compared to conventional absorbance-based methods. It can be applied in environmental, toxicological, and pharmacological studies relating to identification of AOX substrates, as well as the induction of AOX expression through drugs and environmental contaminants.	en_US
dc.description.sponsorship	The research was supported through the Canada Research Chair program and an NSERC Discovery Grant (Grant #371854-20) to Markus Hecker. Dr. Brinkmann received support through a Banting Postdoctoral Fellowship of the Natural Sciences and Engineering Research Council of Canada (NSERC)	en_US
dc.description.version	Peer Reviewed	en_US
dc.identifier.citation	Apenova, N., Peng, H., Hecker, M., & Brinkmann, M. (2018). A rapid and sensitive fluorometric method for determination of aldehyde oxidase activity. Toxicology and Applied Pharmacology, 341, 30–37. https://doi.org/10.1016/J.TAAP.2018.01.006	en
dc.identifier.doi	10.1016/j.taap.2018.01.006
dc.identifier.uri	https://hdl.handle.net/10388/15143
dc.language.iso	en	en_US
dc.publisher	Elsevier	en_US
dc.rights	Attribution-NonCommercial-NoDerivs 2.5 Canada	*
dc.rights.uri	http://creativecommons.org/licenses/by-nc-nd/2.5/ca/	*
dc.subject	Biotransformation	en_US
dc.subject	molybdenum oxidases	en_US
dc.subject	fluorometric assay	en_US
dc.subject	cytosol	en_US
dc.subject	aquatic species	en_US
dc.title	A rapid and sensitive fluorometric method for determination of aldehyde oxidase activity	en_US
dc.type	Article	en_US