Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

Summary

Electron paramagnetic resonance (EPR) spectroscopy was employed to detect nitric oxide from bovine aortic endothelial cells and superoxide radical anion from human neutrophils using iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)₂ and 5,5-dimethyl-1-pyroroline-N-oxide, DMPO, respectively.

Abstract

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in unregulated concentrations are detrimental to cell viability^{1, 2}. While living systems have evolved with endogenous and dietary antioxidant defense mechanisms to regulate ROS generation, ROS are produced continuously as natural by-products of normal metabolism of oxygen and can cause oxidative damage to biomolecules resulting in loss of protein function, DNA cleavage, or lipid peroxidation³, and ultimately to oxidative stress leading to cell injury or death⁴.

Superoxide radical anion (O₂•-) is the major precursor of some of the most highly oxidizing species known to exist in biological systems such as peroxynitrite and hydroxyl radical. The generation of O₂•- signals the first sign of oxidative burst, and therefore, its detection and/or sequestration in biological systems is important. In this demonstration, O₂•- was generated from polymorphonuclear neutrophils (PMNs). Through chemotactic stimulation with phorbol-12-myristate-13-acetate (PMA), PMN generates O₂•- via activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase⁵.

Nitric oxide (NO) synthase which comes in three isoforms, as inducible-, neuronal- and endothelial-NOS, or iNOS, nNOS or eNOS, respectively, catalyzes the conversion of L- arginine to L-citrulline, using NADPH to produce NO⁶. Here, we generated NO from endothelial cells. Under oxidative stress conditions, eNOS for example can switch from producing NO to O₂•- in a process called uncoupling, which is believed to be caused by oxidation of heme⁷ or the co-factor, tetrahydrobiopterin (BH₄)⁸.

There are only few reliable methods for the detection of free radicals in biological systems but are limited by specificity and sensitivity. Spin trapping is commonly used for the identification of free radicals and involves the addition reaction of a radical to a spin trap forming a persistent spin adduct which can be detected by electron paramagnetic resonance (EPR) spectroscopy. The various radical adducts exhibit distinctive spectrum which can be used to identify the radicals being generated and can provide a wealth of information about the nature and kinetics of radical production⁹.

The cyclic nitrones, 5,5-dimethyl-pyrroline-N-oxide, DMPO¹⁰, the phosphoryl-substituted DEPMPO¹¹, and the ester-substituted, EMPO¹² and BMPO¹³, have been widely employed as spin traps--the latter spin traps exhibiting longer half-lives for O₂•- adduct. Iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)₂ is commonly used to trap NO due to high rate of adduct formation and the high stability of the spin adduct¹⁴.

Protocol

1. Culture of Bovine Aortic Endothelial Cells (BAEC)

1. Proper aseptic techniques were followed.
2. In a water bath, warm medium without antibiotics at 37 °C.

Note: The medium consists of phenol free Dulbecco's modified Eagle's medium (DMEM) with 4.5 g/L D-glucose, 4 mM L-glutamine, 1% non-essential amino acids, supplemented with 10% fetal bovine serum (FBS) and 2.5 mg/L endothelial growth factor.

3. Remove the T75 flask containing cells from the incubator and clean the surface of the flask with 70% ethanol before placing it inside the hood.
4. Remove the old medium us....

Discussion

EPR spin trapping has been employed in a wide range of biomedical applications for quantifying and identifying free radicals. Spin trapping is highly sensitive, capable of detecting radicals at concentrations ranging from nM to μM thus making it suitable for application in biological systems. The formation of the paramagnetic adduct, NO-Fe²⁺-MGD, is the basis of NO detection via EPR. Fe²⁺-MGD reacts with NO rapidly¹⁸ at a rate of ~ 10⁶ M^-1 s^-1........

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Phenol free DMEM medium High glucose 1X	GIBCO	31053
0.25% Trypsin- EDTA	GIBCO	25200
L-Glutamine	Fisher Scientific	BP379-100
MEM Non Essential Amino acids	GIBCO	11140
Fetal Bovine serum	Atlanta Biologicals	S11550
Endothelial Growth factor	Millipore	02-102
CaI	Enzo Life Sciences	A-23187	Dissolve in DMSO
SIN-1	Enzo Life Sciences	BML-CN245-0020
DMPO	Dojindo Laboratories	D048-10
FeSO4.7H2O	Sigma Aldrich	215422-250G	Dissolve in PBS with Ca and Mg
MGD	Enzo Life Sciences	ALX-400-014-M050	Dissolve in PBS with Ca2+ and Mg2+
BAEC cells	Cell Systems	2B2-C75
DMSO	Fisher Scientific	BP231-100
DPBS	Sigma Aldrich	D8537
DPBS with CaCl2 and MgCl2	Sigma Aldrich	D8662
Phorbol-myristate acetate (PMA)	Sigma Aldrich	79346-1MG