Improved Method for EPR Detection of DEPMPO- Superoxide Radicals by Liquid Nitrogen Freezing M. Dambrova,* ,1 L. Baumane,* I. Kalvinsh,* and J. E. S. Wikberg† *Department of Medicinal Chemistry, Latvian Institute of Organic Synthesis, Aizkraukles St. 21, Riga, LV-1006, Latvia; and †Department of Pharmaceutical Biosciences, Division of Pharmacology, Uppsala University, Uppsala, Sweden Received August 2, 2000 5-Diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) is frequently used as a spin trap for the measurement of superoxide by EPR spectrometry. However, its half life is fairly short in room tempera- ture. We here show that superoxide radicals trapped by DEPMPO can be successfully recorded at 196°C. Moreover, we show that the signal intensity remains unaltered for up to 7 days, when the samples are stored in liquid nitrogen. Our new approach for mea- surement of superoxide should greatly simplify the studies of this important radical in biological systems. © 2000 Academic Press Key Words: DEPMPO; xanthine oxidase; superoxide; EPR; superoxide dismutase; allopurinol; liquid nitro- gene. Electron paramagnetic resonance (EPR) spin- trapping techniques have been used to study the oxy- gen free radicals produced by various cellular mecha- nisms that may represent key elements in the development of a wide range of clinically important diseases (1–3). A novel phosphorylated nitrone DEPMPO (5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide) was demonstrated to form stable oxyradical adducts (4, 5) and it has been used as an efficient spin trap for applications both in vitro and in vivo (6 – 8). DEPMPO is considered to be a potentially good candi- date for quantifying and trapping superoxide radicals in biological (9) and cellular systems (10, 11). However, spin-adducts are generally very short lived; e.g., the superoxide-DEPMPO adduct shows a half-life of only about 13 min at room temperature (5). Since EPR spectrometers are not always immediately accessible at the site of experimentation it is highly desired to find a method that can preserve a spin ad- duct over longer periods of time, as well as such a method should avoid the problem of the adduct decom- posing during the measurements. In this study we show that it is possible to measure DEPMPO- superoxide spin adducts when frozen in liquid nitrogen in aqueous solution. Moreover, we show that these adducts remain stable for long periods of time in the frozen state. MATERIALS AND METHODS Materials. Bovine milk xanthine oxidase, bovine kidney superox- ide dismutase, xanthine, DTPA (diethylenetriaminepentaacetic acid) and allopurinol were purchased from Sigma Chemical Company (St. Louis, MO). DEPMPO (5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide) was obtained from OXIS International, Inc. Sample preparation. Xanthine oxidase reactions were carried out at room temperature under atmospheric oxygen using incubation mixtures containing 20 mM DEPMPO, 0.25 mM xanthine, 1 mM DTPA, 0.08 U/ml xanthine oxidase in 1 ml of 50 mM PBS, pH 7.5. Allopurinol (0.2 mM) and superoxide dismutase (2000 U/ml) were included when indicated in the text. After 0.5– 60 min of incubation samples were withdrawn from the reaction mixture. While some of these samples were immediately recorded at room temperature, as described below, some were frozen by placing 0.3 ml of the reaction mixture into glass tubes (4-mm inner diameter) and immersing in liquid nitrogen. After freezing, the sample was extruded from the tube as a solid rod by using a glass bar. The sample was then stored in liquid nitrogen until placed in an EPR cell for analysis. EPR recordings. EPR spectra of liquid samples were recorded by using a flat quartz cell inserted into a TM 110-type cavity of an SE/X 2547 Radiopan (Poland) spectrometer operating at X-band with 100 kHz modulation frequency (12). For recordings at -196°C the frozen sample was placed into a Quartz Dewar (Bruker) cuvette cooled with liqid nitrogen. For the room temperature recordings the magnitude of the EPR signal was estimated from the height of the fourth component of the DEPMPO-superoxide signal I l (Fig. 1A). For the frozen samples it was measured from the height of the I f signal (Fig. 1B). Microwave frequencies and magnetic fields were measured by using a Radiopan MCM 101 microwave frequency counter and a JTM 247 NMR magnetometer, respectively. EPR spectrometer recording settings were as follows: microwave frequency, 9.24 MHz for liquid samples and 9.08 MHz for frozen samples; microwave power, 2.5 mW; modulation frequency, 100 kHz, modulation amplitude, 1 G; receiver gain, 1.0  10 4 for liquid samples and 0.5  10 4 for frozen samples; time constant, 1 s. Abbreviations used: DEPMPO, 5-diethoxyphosphoryl-5-methyl-1- pyrroline N-oxide; EPR, electron paramagnetic resonance; DTPA, diethylenetriaminepentaacetic acid; PBS, phosphate buffered saline. 1 To whom correspondence should be addressed. Fax: +371 7541644. E-mail: dambrova@biomed.lu.lv. Biochemical and Biophysical Research Communications 275, 895– 898 (2000) doi:10.1006/bbrc.2000.3387, available online at http://www.idealibrary.com on 895 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.