Improved method to detect nitric oxide in biological systems

Abstract

In this work an improved method is described for using organic solvent extracting to detect nitric oxide. The partition coefficients of the diethylthiocarbamate (DETC)-Fe 2+ complex between different organic solvents and water, the signal intensity of the same NO trapping complex concentration in different organic solvents, and the extracting abilities of the organic solvents were determined. It was found that ethyl acetate was the optimal organic solvent. With ethyl acetate as extracting solvent, the (DETC) 2 -Fe"-NO complex was extracted from water phase to organic phase, and low concentration of nitric oxide in large volume could be detected by electron spin resonance (ESR) at room tem -p e r a t u r e . T h e E S R s i g n a l i n t e n s i t y h a d a g o o d l i n e a r r e l a t i o n s h i p w i t h t h e c o n c e n t r a t i o n o f n i t r i c o x i d e , at a signal-to-noise ratio of 2. The detection threshold of this method was improved to lower than 50 nM, which was more sensitive than the usually used method. The (DETC)z FeZ*-NO complex was stable in the dark at 0-4°C, and there was little change after days. Nitric oxide produced by cardiomyocytes cultured in media and other biological systems was firstly detected with this method.

Electron paramagnetic resonance (EPR) spin trapping of biological nitric oxide

“…Because the intensity of EPR signal is always proportional to the amount of NO-Fe(DETC) 2 present in the sample (there is no extinction coefficient), the construction of a calibration curve is not necessary. Recent studies have demonstrated that NO-Fe(DETC) 2 generated in vascular tissue can be quantified by EPR not only at liquid nitrogen temperature, but also at ambient temperature if extraction of NO-Fe(DETC) 2 by ethyl acetate is employed [86].…”

Evidence of nuclear localization of neuronal nitric oxide synthase in cultured astrocytes of rats

“…In brief, L-arginine, CaCl 2 , NADPH, FeSO 4 , Na 2 S 2 O 4 and diethyldithiocarbamate (DETC) were added to the cytoplasmic and nuclear protein extract (1 Ag ) to reach the final concentration of 50 AM of L-arginine, 500 AM of Ca 2 + , 800 AM of NADPH, 2 mM of Fe 2 + , 10 mM of Na 2 S 2 O 4 and 10 mM of DETC, and incubated in dark for 0.5 h at 37 jC. The (DETC) 2 -Fe(II)-NO complex was extracted with 1:5 (v/v) ethyl acetate for 4 hours (Zhang et al, 2001(Zhang et al, , 2002. Finally, ESR samples were measured in a quartz tube at room temperature with an ESP-300 spectrometer (Bruker, Germany) using normal measuremental parameters.…”

Protective Effect of Natural Antioxidants on Heart Against Ischemia-Reperfusion Damage

“…It can form a stable paramagnetic mononitrosyl iron complex-(NOFe 2+ DETC) with diethyldithiocarbamate (DETC) and iron. This complex shows characteristic peaks at g=2.035 and g=2.020 with triplet hyperfine structure in EPR spectrum , therefore Fe 2+ DETC has been used as NO trapping reagents to detect NO specifically in tissues [26,27]. Using DETC/ Fe 2+ , we studied the NO free radical generation in rat heart during ischemia-reperfusion.…”