( Non-cy...)Luminol-enhanced Assay for Superoxide Anion (O2 face="Symbol" ,,,)

Non-cytotoxic chemiluminescent enhancer for increased sensitivity

Luminol-enhanced Assay for Superoxide Anion (O₂ )

Danny Q. Hoang
Stratagene

Lorraine C. Pfefferkorn
Dartmouth School of Medicine, Dept. of Microbiology, Hanover, NH

Stratagene introduces the LumiMax detection assay, a simple and unique chemiluminescent-based method to qualitatively detect the presence of superoxide anions in cell culture. In addition, the kit provides a specific enhancement reagent to amplify chemiluminescent signal output that is noncytotoxic and nondenaturing to live cells. Superoxide anion (O₂ ), a potent oxidant, is a short-lived oxygen radical that is released into the extracellular environment of stimulated leukocytes (monocytes, macrophages, and polymorphonuclear leukocytes).¹ Superoxide anion is synthesized when NADPH-oxidase, a plasma membrane enzyme complex containing cytochrome b 558, transfers an electron to molecular oxygen. Superoxide anions are implicated in several key cellular processes, including oxidative stress damage,² tumor promotion,³ and, most recently, cell growth and DNA synthesis via their involvement in the cell signaling pathway of proto-oncogene ras.⁴

At present, superoxide may be detected by measuring the reduction of exogenously supplied cytochrome c,⁵ the uptake of oxygen from the medium (measured by the Clark electrode⁶), or luminol-mediated chemiluminescence.⁷ The first method has limited sensitivity and is complicated by the reoxidation of superoxide-reduced cytochrome c by contaminants and cell lysis. The oxygen-uptake method is not only less sensitive than cytochrome c reduction but also requires a large number of cells (10⁶) per assay. The third method, luminol-mediated chemiluminescence, is the most sensitive method of all; accurate readings can extend three orders of magnitude over the signal range.

The Luminol Chemiluminescence Reaction

The chain of events for the luminol chemiluminescence (CL) assay is believed to involve cell surface receptors, such as those binding to complex immunoglobulin G (Fc receptors) or bacterial formylated peptides (f-met-leu-phe receptors), which are stimulated by their specific agonists. The activated receptor triggers the production or activation of NADPH oxidase, leading to the production of superoxide anion. Superoxide anion is subsequently released into the extracellular environment where it can oxidize luminol and available lipids and proteins. This oxidation results in chemiluminescence, a release of photons (light), which can be measured by a luminometer. Figure 1 illustrates Stratagenes enhanced chemiluminescent procedure for measuring superoxide anion.

When concentrations of superoxide anion are very low, assay sensitivity may need to be enhanced. Some enhancement reagents, such as iodophenol, are phenolic-based compounds. Unfortunately, such enhancers are toxic to live cells and denaturing to some components of subcellular systems; hence, they cannot be used for in situ assays. However, Stratagenes specific enhancement reagent increases photon output but is noncytotoxic and does not denature cellular components used in assaying live cell activity.

Assay for Superoxide Anions in U-937 cells

Cell cultures were provided with an analyte suspected of being able to generate superoxide anion; luminol and enhancement reagents were then added. Superoxide anion generated was measured as a result of luminol oxidation.

As a model, U-937 cells (monocytic-like, histocytic human lymphoma) were cultured in RPMI supplemented growth media with human gamma interferon (IFN-g) to induce cell differentiation. U-937 cells were assayed for superoxide anions after 2 to 5 days of differentiation with IFN-g. After 2 days of differentiation, superoxide anion activity was minimal (data not shown). However, after 3 days of differentiation, U-937 cells demonstrated superoxide anion activity upon stimulation with 100 ng/ml phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator and a potent stimulant of NADPH oxidase. Reaction samples were aliquoted into polystyrene round-bottomed tubes and placed into a luminometer to measure photon emission.

The luminometer was programmed to output raw data at 30-second counts. Detection of superoxide anion with luminol, enhancer, or PMA independently generated background numbers. Background values were determined against a control flask containing cells without differentiation.

Figure 2 shows the relative light units (RLUs) recorded by the luminometer for samples incubated at varying time points after 3 days of differentiation and control samples containing induced cells with luminol plus PMA, induced cells with luminol plus enhancer, induced cells with PMA plus enhancer, and induced cells alone. The results indicated that luminol oxidation by superoxide anion is time dependent. Superoxide anion activity was also assayed after 4 and 5 days of differentiation with IFN-g. The resulting RLUs yielded lower total RLUs and required a longer incubation period before substantial RLU values were established. The decrease in photon output coupled with the longer incubation period may be attributed to the health of the cultured cells.

Superoxide Generating System: Xanthine-Xanthine Oxidase

To demonstrate that superoxide anions interact with and oxidize luminol, we assayed a known superoxide anion generating system comprised of xanthine oxidase and xanthine.⁸ In addition to assaying for superoxide anion activity, we suppressed its activity by introducing superoxide dismutase (SOD). Since SOD catalyzes the dismutation of O₂ radicals into O₂ and H₂O₂, the addition of SOD to xanthine-xanthine oxidase solution results in a decreased O₂ concentration, which, in turn, leads to decreased oxidation of luminol or the chemiluminescent signal. Figure 3 shows the chemiluminescence assay of O₂ generated by the control xanthine-xanthine oxidase reaction.

Conclusions

LumiMax is a simple, sensitive, and unique kit for researchers performing luminol-enhanced chemiluminescent assays. It has many applications including clinical assessment of leukocytes in patients who have altered oxidative means to control infections and biochemical research involving the analysis of leukocyte signal transduction pathwayscomponents comprising NADPH oxidase and requirements of the respiratory burst. In addition, the assay may be used to study the generation of oxygen radicals in AIDS patients⁹ and control their opportunistic infections; to detect, measure, or monitor cell aging or damage in selected agricultural crops;¹⁰ and to monitor cell damage by pesticides or environmental pollution.

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