Reactive Species Detection in Biology

From Fluorescence to Electron Paramagnetic Resonance Spectroscopy

Frederick A. Villamena

• Publisher: Elsevier
• Year: 2016
• Print ISBN: 9780124200173
• eBook ISBN: 9780124200807
• Topic: Biological Sciences
• Subtopic: Biochemistry
• Index: Biological Sciences

Reactive Species Detection in Biology: From Fluorescence to Electron Paramagnetic Resonance Spectroscopy discusses the reactive oxygen species that have been implicated in the pathogenesis of various diseases, presenting theories, chemistries, methodologies, and various applications for the detection of reactive species in biological systems, both in-vitro and in-vivo.Techniques covered include fluorescence, high performance chromatography, mass spectrometry, immunochemistry, and electron paramagnetic resonance spectroscopy. Probe design and development are also reviewed in order to advance new approaches in radical detection through synthesis, computations, or experimental applications.- Reviews all current advances in radical detection- Emphasizes chemical structures and reaction schemes fundamental to radical detection and identification- Describes the uses, advantages, and disadvantages of various probe designs- Examines new approaches to radical probe development

Chapter 1

Introduction

Respiration has been commonly known for centuries as an essential process for survival because it provides the “fuel” for the normal functioning of animal organs. This fuel found in air was first known as dephlogisticated air on its discovery in the 1770s by scientists Joseph Priestly and Carl Wilhelm Scheele.^1,2 Antoine Lavoisier later coined the term oxygen and extended the theory of combustion to introduce the idea of respiration as a biological process in which inhaled oxygen is used in the oxidation of carbon and hydrogen from food to give carbonic acid, thus relating respiration as a combustion process.^3,4 Studies from 1940s and 1950s on the enzymatic metabolism of oxygen provided molecular bases for oxygen’s diverse biological functions through enzyme-catalyzed transfer of oxygen atom to a substrate, or electron-transfer reactions to oxygen leading to the formation of reactive oxygen species (ROS) or water.⁵ This enzyme-catalyzed reduction of oxygen was later found to have beneficial as well as detrimental effects on cellular function. Without a doubt, the progress made in understanding oxygen metabolism owes a debt to the development of electrochemical techniques for analyzing oxygen in biological fluids and tissues as well as whole animals. Oxygen sensor development was described⁶ dating as far back as 1938 when the first biological application of platinum electrodes was demonstrated for the purpose of monitoring oxygen to study photosynthesis, and this was followed by the use of a Clark-type electrode that allowed for the measurement of oxygen tension (pO₂) in an in vivo system, air, blood, and cell cultures, which then led to further innovation that exhibited high accuracy.

The absorption of oxygen and its transformation to carbonic anhydride (carbon dioxide) during respiration has thus been the paradigm of oxygen metabolism.⁷ In the early 1900s, the relationship between oxygen and disease was suggested by Todd,⁸ whereby the human body is in a state of chemical equilibrium between the processes of oxidation and reduction; when this equilibrium shifts toward the formation of more reduced species than oxidized ones, the body could lose resistance to diseases, and hence resupplying the body with oxygen in the form of ozonized air or oxidized oils is used as a therapeutic means of counteracting diseases such as tuberculosis or Bright’s disease. Oxygen therapy was employed for a variety of diseases; e.g., patients with respiratory disease such as pneumonia exhibited excellent therapeutic effect when such therapy was introduced soon after diagnosis.⁹ It also became apparent that oxygen exhibits toxicity against bacterial pneumococcus type I¹⁰ as well as in protozoans,¹¹ brain respiration,^12,13 or in whole animals causing pulmonary damage.¹⁴ In humans, oxygen results in the reduction of blood-flow rate to the brain when inhaled at high atmospheric pressure,¹⁵ causing cerebral complications as well as diminished overall cardiac output and changes in alveoli that result in edema, transudation, and fibrinous deposits.¹⁶

The formation of hydroxyl radicals from water under ionizing radiation had long been implicated for the radicals’ biological actions and toxicity.¹⁷ Soon thereafter, chemical agents that have radiation-like properties were implicated in the initiation¹⁸ of cancer or tumor inhibition via chromosome alteration through formation of free radicals.¹⁹ Evidence supported the idea that free radicals formed radiolytically were toxic because they were found to diffuse inside the cells when generated extracellularly.²⁰ Moreover, it was also demonstrated that x-ray radiation inhibited glutathione metabolism inside the cells, and this inhibition was decreased at low oxygen concentration and on addition of catalase, which suggested the involvement of oxygen-derived reactive species such as H₂O₂.²¹ The link between radiation and oxygen levels on their cellular toxicity had become more apparent by their inactivation of T₂ bacteriophage and by the observation that thiol compounds such as thiourea could compete with oxygen-derived radicals, thereby protecting the phages from radiation.²² Using electron paramagnetic resonance (EPR), radiation damage to DNA or RNA was reported to produce paramagnetic nucleic acids at 77K.²³ This finding was consistent with the increased effect of radiation on DNA inactivation in the presence of oxygen and protection in the presence of the thiol cysteamine^24,25 with mutations successfully induced in the cell nucleus of onion rootlets, e.g., by hydroxyl radical and x-irradiation.²⁶

While the effect of irradiation-mediated free radical formation on nucleotides was not as pronounced as in proteins and peptides, it became clear that free radicals formed enzymatically could have profound consequences on protein function. Metabolic hydroxylation of aromatic amino acids has long been suspected as a biosynthetic process for the conversation of phenylalanine to tyrosine, tyrosine to 3,4-dihydroxyphenylalanine, kynurenine to 3-hydroxykynurenine, and tryptophan to 5-hydroxytryptophan.²⁷ In the 1950s, metabolic hydroxylation of aromatic compounds such as N-2-fluorenylacetamide in guinea pigs and rats was demonstrated and believed to be a detoxification mechanism.²⁸ This conversion was duplicated in cell-free in vitro studies involving ferrous ion, ascorbic acid, oxygen, and a chelating agent (ethylenediaminetetraacetic acid) under physiological conditions, showing that hydroxylation of aromatic compounds could indeed be mediated by free radial reaction, specifically that of hydroxyl radicals.²⁹ Not long after, it was proposed that biological hydroxylation occurs via activation of oxygen by peroxidase^30,31 and by other hydroxylating systems found in liver microsomes that require triphosphopyridine nucleotides and oxygen for their activity.³² Altogether, Harman³³ proposed the role of oxygen, metals ions, oxidative enzymes, and radiation on the development of age-related and degenerative diseases through generation of reactive oxygen species, and these propositions became the foundation of today’s widely accepted free radical theory of aging. Subsequently, the link between free radicals and the development of atherosclerosis,^34,35 cancer,³⁶ and neurodegeneration³⁷ was proposed, and the role of proper nutrition and lower metabolic demand were seen as essential for the slower progression of free radical–mediated reactions in the body.^38,39 While free radicals such as semiquinones were identified using EPR as integral components of the mitochondrial respiratory chain,^40,41 evidence for the production of ROS such as superoxide and hydrogen peroxide by mitochondria through electron transfer had become more compelling.^42–44

Although the existence of superoxide as an inorganic species is known dating as far back as the late 1890s, its paramagnetic character was not established until the 1930s.⁴⁵ For the next four decades, studies on superoxide were mostly focused on their chemistry with metals and nonmetals and, for the first time, its characterization through EPR spectroscopy.⁴⁶ Only in the late 1960s did the idea become acceptable that superoxide could also be generated in biological system, an idea helped by the discovery of superoxide generation from enzymatic systems. The idea was first introduced by McCord and Fridovich,⁴⁷ whose seminal study demonstrated the production of superoxide from xanthine oxidase and xanthine; they found this formed species capable of reducing cytochrome c and initiating the sulfite oxidation reaction. In further support of this evidence, superoxide formation from xanthine oxidase was confirmed using EPR spectroscopy at pH 10; signal intensity was shown to be dependent on oxygen concentration and not the enzyme itself.⁴⁸ The oxygen origin of superoxide from xanthine oxidase was unequivocally confirmed using ¹⁷O-labeled O₂ and EPR spectroscopy giving the 11-line or 6-line EPR spectra for the formed ¹⁷O₂^•– or ¹⁷O¹⁶O^•–, respectively.⁴⁹ Generation of superoxide from oxygen was achieved through electrolytic reduction of oxygen in aqueous solution.⁵⁰ In addition, electrochemically generated chemiluminescence of lucigenin showed evidence of superoxide-mediated light emission, paving the way for the development of chemiluminescence probes for superoxide.⁵¹ Excited-state oxygen resulting from the oxidation of xanthine by xanthine oxidase can also induce chemiluminescence via recombination of ROS, probably that of superoxide,⁵² and this finding was further supported by evidence showing that singlet oxygen–sensitized fluorescence from organic compounds is mediated by superoxide, which suggests the possible enzyme-mediated formation of singlet oxygen.⁵³

The chemistry of superoxide enzymatic formation and decomposition then became of interest to investigators who wanted to know whether the mechanism is ligand mediated or metal mediated. It was demonstrated that superoxide is decomposed by erythrocuprein and ferricytochrome c and is formed during the oxidation of reduced flavin⁵⁴ rather the iron heme of flavoproteins.⁵⁵ This was later supported by studies showing that the one-electron reduction of oxygen by reduced flavins and quinones results in the formation superoxide.⁵⁶ However, the formation of superoxide from the reaction of oxygen with reduced iron-sulfur proteins from plant ferredoxins that are flavin free was also reported,⁵⁷ indicating that oxygen reduction can occur via electron transfer not only from organic radicals but also from low-valent metal ions.⁵⁶ The enzyme superoxide dismutase (SOD) was then proposed to catalyze the dismutation of superoxide to oxygen and hydrogen peroxide and had been a gold standard as a competitive inhibitor for the investigation of superoxide-mediated reactions such as the oxidation of epinephrine to adrenochrome by xanthine oxidase and the reduction of ferricytochrome c or tetranitromethane.⁵⁸ The reduction of ferricy...

Table of contents

• Cover image
• Title page
• Table of Contents
• Copyright
• Dedication
• Preface
• Chapter 1. Introduction
• Chapter 2. Chemistry of Reactive Species
• Chapter 3. Reactive Species in Biological Systems
• Chapter 4. Fluorescence Technique
• Chapter 5. EPR Spin Trapping
• Chapter 6. UV–Vis Absorption and Chemiluminescence Techniques
• Chapter 7. Electrochemical, Mass Spectroscopic, Immunochemical, and Nuclear Magnetic Resonance Techniques
• Index