DRUG FORMULATIONS AND CLINICAL METHODS A Highly Sensitive RP-HPLC-Fluorescence Method to Study Aldehyde Oxidase Activity MOHAMMAD-REZA RASHIDI Tabriz University of Medical Sciences, School of Pharmacy, Department of Medicinal Chemistry, 51664-14766 Tabriz, Iran KAVEH AMINI 1 and MOHAMMAD-YASER KHANI University of Tabriz, Faculty of Chemistry, Department of Analytical Chemistry, 51666-16471 Tabriz, Iran AKRAM FARIDI and JALAL HANAEE Tabriz University of Medical Sciences, School of Pharmacy, Department of Medicinal Chemistry, 51664-14766 Tabriz, Iran MOHAMMAD-HOSSEIN SOROURADDIN University of Tabriz, Faculty of Chemistry, Department of Analytical Chemistry, 51666-16471 Tabriz, Iran Aldehyde oxidase is a widely distributed enzyme that is involved in the metabolism of an extensive range of aldehydes and N-heterocyclic compounds with physiological, pharmacological, and toxicological relevance. In the present study, a highly sensitive RP-HPLC-fluorescence method based on the oxidation of phenanthridine to phenanthridinone has been developed and validated to assay aldehyde oxidase activity in biological samples. Determination of phenanthridinone was achieved on a C18 column using 10 mmol/L phosphate buffer (pH 5.0) containing 0.1 mmol/L EDTA–acetonitrile (40 + 60, v/v) as the mobile phase. The fluorescence intensity of phenanthridinone was measured at 364 nm with excitation at 236 nm. The proposed method was precise, accurate, specific and rapid (analysis time, approximately 8 min) with a mean RSD of 2.54%. Peak responses were linear from 0.5 to 100 nmol/L, with an LOD of 0.125 nmol/L. The applicability of the method was demonstrated by measurement of aldehyde oxidase activity in rat liver, kidney, ovary, and heart fractions. A ldehyde oxidase (EC 1.2.3.1: aldehyde: oxygen oxidoreductase) is a large homodimeric enzyme that is widely distributed in nature, being found in various species, from different plants to mammals such as sheep, camels, and humans (1–4). The enzyme is capable of oxidizing an extensive range of aldehydes and N-heterocyclic compounds with physiological, pharmacological, and toxicological relevance, such as N-methylnicotinamide (5), retinal (6), allopurinol (7), famciclovir (8), methotrexate (9), 6-mercaptopurine (10), citalopram (11), and tamoxifen (12). The reaction catalyzed by aldehyde oxidase involves production of two electrons that are transferred to molecular oxygen as the electron acceptor. Therefore, the reactive oxygen species generated during the oxidative activity of aldehyde oxidase may be relevant in various disease conditions. Aldehyde oxidase can be detected in various mammalian tissues, but the distribution of the enzyme is species dependent (4, 13). For example, in guinea pigs, the maximum specific activity of aldehyde oxidase is found in the liver, whereas the stomach is the richest source of the enzyme in camels (2). Some tissues have been reported to be devoid of aldehyde oxidase activity (2). The enzyme activity has also been detected in different tissues of some plants (14, 15). Due to very low activity of aldehyde oxidase in some plant or animal tissues, sometimes the enzyme activity has to be measured by immunological techniques (16). It is possible that the failure of detection of aldehyde oxidase activity in some tissues arises from the low sensitivity of the method applied to measure the enzyme activity. Although aldehyde oxidase is able to metabolize a broad range of aldehydes and N-heterocyclic compounds, only few of these compounds have been used as a substrate to determine the enzyme activity. Phenanthridine is one of these compounds for which spectroscopic measurement of its oxidation to phenanthridinone has been used in many studies for monitoring aldehyde oxidase activity (8, 17–22). However, the method is not sensitive enough to measure activity when the enzyme is present at very low levels. In the present study, a highly sensitive method based on RP-HPLC coupled with fluorescence detection has been developed and validated for the evaluation of aldehyde oxidase activity based on the oxidation of phenanthridine to phenanthridinone (Figure 1). The applicability of the proposed method has been examined and verified by measurement of the enzyme activity in rat liver, kidney, heart, and ovary tissues. Experimental Chemicals All experiments were performed with analytical reagent grade chemicals. Phenanthridine and phenanthridinone were 550 RASHIDI ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 94, NO. 2, 2011 Received October 28, 2009. Accepted by SW May 31, 2010. Corresponding author’s e-mail: rashidi@tbzmed.ac.ir 1 Present address: York University, Department of Chemistry, Toronto, ON, M3J 1L3, Canada. Downloaded from https://academic.oup.com/jaoac/article/94/2/550/5655475 by guest on 05 July 2022