Notes & Tips Determination of nitrite in biological fluids by use of electron spin resonance Kayoko Minakata, * Naoko Okamoto, Hideki Nozawa, Kanako Watanabe, and Osamu Suzuki Department of Legal Medicine, Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu 431-3192, Japan Received 19 August 2003 Several methods such as colorimetry [1], fluorometry [2], and chemiluminescence [3] have been used for the determination of nitrite. Colorimetry and fluorometry need several milliliters of sample and are often interfered with by substances in biological samples [2]. Chemilu- minescence is limited by the lack of specificity and/or by the requirement for a transfer of NO produced from sample solution to its detecting environment [3]. Appli- cation of electron spin resonance (ESR) for the detec- tion of nitrite is examined in the present work, since NO produced from nitrite is a radical and can be detected selectively by ESR in a large amount of diamagnetic matrix with high sensitivity [4]. We used a complex of Fe 2þ with diethyldithiocarbamate (DDC; 1 (C 2 H 5 ) 2 NCSS  ), Fe(DDC) 2 , as a trap of NO and detected a stable NO–Fe(DDC) 2 [5] in cyclohexanol. To reduce nitrite to NO, ascorbic acid (AsA) was chosen since it could stabilize NO in water [5]. The trap Fe(DDC) 2 was separated from the water to the upper cyclohexanol layer to avoid the reaction of Fe with NO in water [5]. Without sample pretreatments, nitrite in plasma, urine, or saliva was quantitated as NO–Fe(DDC) 2 at the detection limit of 50 pg within 5 min. Materials and methods 3-Carboxy-proxyl was obtained from Sigma Chemi- cal Co. USA, and other chemicals were of analytical grade from Wako Pure Chemical Ltd., Japan. ESR measurement was performed by using a JEOL JES- FE2XG ESR spectrometer [4]. As shown in Fig. 1A, ESR spectrum of NO–Fe(DDC) 2 was characterized by three lines with the hyperfine splitting of 1.27 mT [6] and the g value of 2.040. Modulation width of 0.8 mT and microwave power of 65 mW were adopted. To examine the efficiency of the production, the spin amounts of NO–Fe(DDC) 2 produced were compared with the spin amounts of 3-carboxy-proxyl. The accuracy of the ESR method was also compared with that of a colorimetry based on Griess reaction [1] by using a Shimadzu UV 2200. Two solutions, A, 2 mM Fe 2þ and 10 mM DDC in cyclohexanol, and B, 0.5 M AsA at maximum solubility, were prepared every day. When the pH of the reacting solution (10 vol of sample solution added with 2–20 vol of B) is higher than 3, suitable amount of HCl is added to B since AsA works at pH below 3. Nitrite is assayed within 5 min as follows: (a) take 10 lL (or 2–20 lL) of B in a tube (0.2 mL) and place 20 lL of A on it; (b) add 10 lL of sample (nitrite standard solution, plasma, ur- ine, or saliva) into the bottom of tube by a syringe and cap the tube; (c) wait for 1 min to trap NO completely and then take 10 lL of the upper cyclohexanol layer in a capillary [4] and seal the top and bottom of the capillary with putty; most substances in sample solutions remain in the aqueous layer; (d) measure the ESR spectrum of the cyclohexanol solution in the capillary at room temperature. Results and discussion NO is one of the least water-soluble gases with approximate partition coefficient of 20 [3]. The trap Fe(DDC) 2 should be placed in the upper layer rather than the water. Alcohols are less dense than water and * Corresponding author. Fax: +81-53-435-2233. E-mail address: kminakat@hama-med.ac.jp (K. Minakata). 1 Abbreviations used: DDC, diethyldithiocarbamate; ASA, ascorbic acid. 0003-2697/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2003.10.018 Analytical Biochemistry 325 (2004) 168–170 ANALYTICAL BIOCHEMISTRY www.elsevier.com/locate/yabio