Comparison of three analytical methods for superoxide produced by activated immune cells

Abstract

Superoxide plays a key role in normal immune function and inflammatory diseases. In order to evaluate normal immune function or screen inhibitors of superoxide production for treating inflammatory diseases, it is very important to detect superoxide with good accuracy, sensitivity, and flexibility. In present study, we investigated three analysis methods of superoxide, colorimetric assay by WST-8, fluorescence assay by dihydroethidium and chemiluminescence assay by lucigenin, compared their precisions, specificities, sensitivities and time curve characteristics in superoxide analysis, and then validate their values in the screening of anti-inflammatory compounds. The results reveal that three analysis methods of superoxide all have good precisions and high specificities but have different sensitivities and time curve characteristics, which suggest their different applications. In addition, they can all be used in the screening of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors and anti-inflammatory compounds.

Introduction

Superoxide (O₂^{• –}) is a short-lived radical generated by the addition of an electron to oxygen. Superoxide plays a key role in the immune system, protecting the animal from infectious organisms. The highly reactive superoxide is released by stimulated leukocytes including neutrophils, macrophages, and monocytes. In immune cells, the superoxide is mainly synthesized by the enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Superoxide not only kills bacteria and fungi directly by inactivating critical metabolic enzymes and initiating lipid peroxidation, but also defends microbial pathogens by other downstream reactive oxygen species (Bagaitkar, Matute, Austin, Arias, & Dinauer, 2012; Guzik, Korbut, & Adamek-Guzik, 2003; Wang et al., 2012). However, in persistent and recurrent episodes of inflammation mediated by aberrant activation of innate and acquired immunity, overproduced superoxide also attack normal cellular components, causing damage to lipids, proteins, and DNA. This can initiate numerous diseases, including rheumatoid arthritis, diabetes, atherosclerosis, central nervous system disorders, and cancer (Kim & Byzova, 2014; Miller, Gutterman, Rios, Heistad, & Davidson, 1998; Seredenina et al., 2016; Victor & De La Fuente, 2003).

In order to evaluate normal immune function or screen inhibitors of superoxide production for treating inflammatory diseases, it is very important to detect superoxide with good accuracy, sensitivity, and flexibility. At the earliest time, superoxide production by activated immune cells was usually quantified by colorimetric determination with ferricytochrome c. However, this method has not been widely applied because of poor signal/noise ratio which is resulting from the relatively high background absorbance of ferricytochrome c (Babior, Kipnes, & Curnutte, 1973). Subsequently, a tetrazolium salt, WST-1 was found that it could be reduced specifically to produce a soluble formazan by superoxide and was used to measure respiratory burst of neutrophils and screen anti-inflammatory agents by simple colorimetric assay (Tan & Berridge, 2000). In addition, dihydroethidium (DHE) has been used increasingly as a probe for superoxide in biological systems. DHE is a hydrophobic uncharged compound that is able to cross cell membrane and, upon oxidation by superoxide, becomes positively charged fluorescent ethidium and accumulates in cells by intercalating into DNA. Thus, DHE is often used as superoxide detector in flow cytometry (Back, Matthijssens, Vanfleteren, & Braeckman, 2012; Benov, Sztejnberg, & Fridovich, 1998; Bindokas, Jordan, Lee, & Miller, 1996; Laurindo, Fernandes, & Santos, 2008; Peshavariva, Dusting, & Selemidis, 2007; Zhao et al., 2003). Furthermore, there is also a chemiluminescence probe used in superoxide detection. Lucigenin, a di-acridinium compound, can emit light on reaction with superoxide (Gyllenhammar, 1987; Kervinen, Patsi, Finel, & Hassinen, 2004; Kirchner et al., 2012; Myhre, Andersen, Aarnes, & Fonnum, 2003; Stevens & Hong, 1984).

In the present study, we will perform a comparative study of colorimetric, fluorescence, and chemiluminescence determinations for superoxide. On the basis of conforming precisions and specificities of these methods in superoxide analysis, we will compare their sensitivities and time curve characteristics and validate their values in the screening of anti-inflammatory compounds.