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A rapid and sensitive fluorometric method for determination of aldehyde oxidase activity
Nancy Apenova1, Hui Peng2, Markus Hecker3
1Toxicology Centre, University of Saskatchewan, 44 Campus Drive, Saskatoon, Saskatchewan S7N 5B3, Canada.
Toxicology and Applied Pharmacology
|January 14, 2018
Summary
Researchers developed a sensitive fluorometric assay for aldehyde oxidases (AOX) activity. This new method enhances xenobiotic metabolism studies in diverse vertebrate species, improving drug and environmental contaminant research.
Area of Science:
- Biochemistry
- Enzymology
- Xenobiotic Metabolism
Background:
- Aldehyde oxidases (AOX) are crucial for metabolizing drugs and environmental contaminants in mammals.
- Research on AOX activity in non-mammalian vertebrates is limited due to insensitive and low-throughput assay methods.
- Existing photometric assays for AOX suffer from cross-reactivity issues, hindering accurate measurements.
Purpose of the Study:
- To develop a novel, sensitive, and high-throughput fluorometric assay for aldehyde oxidase (AOX) activity.
- To overcome the limitations of current photometric methods for AOX research.
- To enable broader investigation of AOX function across different vertebrate species.
Main Methods:
- Developed a fluorometric assay utilizing the conversion of 4-(dimethyl)amino cinnamaldehyde to a fluorescent product by AOX.
- Evaluated the assay using partially purified hepatic cytosol from rat, human, and rainbow trout.
- Confirmed product identity using liquid chromatography and mass spectrometry.
Main Results:
- The novel fluorometric assay demonstrated 4- to 10-fold greater sensitivity compared to conventional absorbance-based methods.
- Purification of cytosol via heat treatment and ammonium sulfate precipitation increased specific AOX activity.
- Kinetic parameters (Km, Vmax) and inhibitor effects (hydralazine) were comparable to established methods.
Conclusions:
- Successfully developed a rapid, sensitive, and reliable fluorometric assay for determining AOX activity across vertebrate species.
- The new assay facilitates environmental, toxicological, and pharmacological studies on xenobiotic metabolism and AOX induction.
- This advancement provides a valuable tool for identifying AOX substrates and understanding their role in diverse organisms.
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