Oxidative Stress and Biomaterials

States

Preface

After decades of free radical biology research and development, the conclusions are clear; there are no simple solutions to oxidative stress related-disorders. Dietary antioxidants are not enough to stave off the countless diseases that involve excess production of reactive oxygen species. The failed clinical trials have shown that there is no panacea to use for all conditions. Much like the combustive processes involved in oxidation, such disappointments result in scorched earth that turns researchers off from continued development in the area. However, out of the wreckage of these shattered therapeutic dreams emerges a deeper understanding of the way the body deals with oxidation events, inflammation and attempts at reverting to homeostasis. As the entire field of biomaterials explores more deeply the ability to control the inflammatory response, improve material biocompatibility and even delve into the field of regenerative medicine, this understanding provides fertile ground for exploring new ways to improve the materials and devices we create for biomedical applications.

With the above paragraph in mind, this book, we believe, serves as a starting point to introduce investigators to the importance of oxidative stress to biomaterials and broaden the work being done in the area. To facilitate this objective, we separated the chapters into three main sections: (1) a background summary of oxidative biology and its links to inflammation and biomaterial biocompatibility; (2) an overview of analytical approaches currently available to characterize oxidative stress responses; and (3) a series of examples of in which researchers have taken advantage of oxidative stress biology to improve biomaterial function. We hope as you read this book you will agree with us that the future of biomaterials must include on some level, a mechanism by which such substances interact with cellular redox systems. We look forward to seeing what new, phoenix-like devices are created out these antioxidant ashes.

Sincerely,

Thomas Dziubla and D. Allan Butterfield

Chapter One

A Free Radical Primer

Prachi Gupta¹, Andrew Lakes¹ and Thomas Dziubla²,    ¹Chemical and Materials Engineering Department, University of Kentucky, Lexington, KY, United States,    ²Department of Chemical and Materials Engineering, University of Kentucky, Lexington, KY, United States

Abstract

Free radicals, reactive oxygen species, and reactive nitrogen species are the result of the oxidation-reduction reactions that are constantly taking place around us, such as combustion, photosynthesis, rusting, and cellular metabolism. In this chapter, the mechanisms of free radical production and their importance in natural chemical processes is discussed. Parallels are made between different systems as a means of underlining the similarities and fundamental relationships these reactions have in nature. Following the redox potential principles, the concept is extended to the cellular metabolic pathways, where different reactions in the cellular matrix result in the generation and consumption of free radicals or reactive intermediates, which define the redox sate of a cell. With this background, a deeper understanding on the relationship between free radicals, oxidative stress and biomaterials can be obtained.

Keywords

Free radical; thiol chemistry; redox potential; cellular enzymes; Fenton Chemistry; Haber-Weiss Reaction

1.1 Free Radical Biology—Importance

, are defined as any atom or molecule containing an unpaired electron, which has a strong tendency to gain another electron to achieve a nonradical state [1]. These molecules are considered to be highly reactive and are capable of reacting with a nonradical molecule in their quest for self-stabilization. Radical species can be formed through a variety of mechanisms, one of which is abstraction of an electron from an atom or a molecule. Radicals can also be generated by the splitting of a molecule at a very high-energy state. A classic example would be radiation-induced homolysis of a water molecule into a hydroxyl radical and a hydrogen atom [2] (Eq. (1.i))

(1.i)

As these radical ions exist in a high and unstable potential energy state, they can react in variety of ways. For example, two radical species can react with each other to form a nonradical molecule or one radical can donate an electron to another yielding two stable compounds [3].

As oxygen serves as the primary player in most biological free radical reactions, free radicals are also commonly known as reactive oxygen species (ROS). In a similar way, nitrogen base free radicals are called reactive nitrogen species (RNS). Table 1.1 lists some of the most common free radicals/oxidants/ROS important in biology. Most of the essential cellular matrix components (protein, cellular membrane, DNA, lipids, PUFAs (polyunsaturated fatty acids), etc.) in physiological systems are stable nonradical entities and perform their regular function of energy production in the form of ATP and maintain the cellular redox balance. But, the presence of more than the basal level of free radical molecules/ROS can lead to reactions of ROS with alternative cellular components in a quest to stabilizing themselves, damaging the chemical integrity of cellular biomolecules [4,5]. One well-known example is lipid membrane damage via lipid peroxidation, where the hydroxyls radical react with PUFAs of the cellular membrane, extracting an electron and yielding a lipid free radical. If this reaction phenomena is not controlled in a timely manner, it can initiate a chain reaction of free radical molecules with intact lipids resulting in overall cellular membrane damage [6] (Fig. 1.1).

Table 1.1

Potential Free Radical Species

Figure 1.1 .

Although most of the research concerning the role of ROS/RNS has been done towards the potential cellular damage and subsequent pathological events, they do serve beneficial role to the body under certain conditions. When present at optimum concentrations, they help maintain the redox homeostasis in the cellular environment that regulates cell functioning and cell signaling and responds to endogenous and exogenous stimuli [7]. Under normal physiological conditions, a balance between generation and elimination of ROS/RNS via endogenous antioxidant enzymes/molecules helps the redox-sensitive signaling proteins to function properly [8]. Endogenous ROS generating enzymes like myeloperoxidase and NADPH oxidase (NOX), also known as phagocyte oxidase, actually help the neutrophils perform their phagocytic function against microbial intrusion. In a process known as the respiratory burst, NOX-catalyzed superoxide production further forms hydrogen peroxide with the help of superoxide dismutase (SOD), which in turn converts to hypochlorus acid (HOCl), which has strong bactericidal properties [9,10].

(1.ii)

Indeed, the dual nature of biological oxidation and reduction processes is the very reason for its importance in biomaterial/tissue interaction. Through a proper understanding of the underlying chemistry, it is possible to design next generation materials which can harness this intrinsic biological signaling mechanism. This chapter will cover the key points in free radical chemistry, the various pathways and vocabulary and finally, the way these processes occur in a biological setting.

1.2 RED/OX Chemistry

1.2.1 Oxidation/Reduction Reactions and Voltage Potentials

Originally, the term oxidation was described as a process of any element, primarily metals, to combine with oxygen to form metal oxides and reduction defined as a process that will convert the metal oxide back to pure metal. For example, conversion of magnesium (Mg) to magnesium oxide is oxidation, while smelting of magnesium oxide to magnesium at high temperature in presence of carbon is reduction. Later, the discovery of electrons changed the definition of oxidation–reduction to the transfer of electrons from one species to another. As per the law of conservation of mass applied to electrons, oxidation and reduction are always linked to one another. Meaning, if one species is oxidized, the counter reactant species will be reduced [11,12].

(1.iii)

(1.iv)

By definition, oxidation of any given element or molecule will involve a loss of electron and the element/molecule will be known as a reducing agent while the reduction of any molecule will be a gain of electron and that molecule will be known as an oxidizing agent [13]. In reference to free radicals or ROS they are commonly known to be strong oxidizing agents and have a tendency to get reduced [14,15]. This is because most radicals have one unpaired electron, and the addition of another electron (a.k.a. getting reduced) with the opposite electron spin would help in stabilizing the electron pair, taking them to a more inert state. However, this is not always true. Oxidizing tendency of any free radical will depend on its affinity to gain an electron or its own reduction potential against the affinity or potential of the coreactant. It is possible that free radical A gets reduced in presence of B but might get oxidized itself by losing another electron in presence of C because the reduction potential or electron gaining affinity of C is greater than A.

1.2.1.1 Types of Redox Reactions

1.2.1.1.1 Corrosion and Rusting

Iron has served as a common example of corrosion, which is the electrochemical oxidation of metals in presence of oxygen to form respective oxides. In reference to iron, it is specifically termed as formation of rust (Eq. (1.v)) [16].

(1.v)

In presence of an acid, iron (II) is oxidized to iron (III) by reaction with hydrogen peroxide, which acts as an oxidizing agent [17], although iron (III) can be reduced back to iron (II) in the presence of stronger reducing agents or free radicals, such as superoxide anion [18]. This oxidation–reduction chemistry of iron with hydrogen peroxide is popularly known as the Fenton and Haber–Weiss reaction mechanisms (see Section 2.4) and are critical in maintaining the redox state of the cell.

(1.vi)

1.2.1.1.2 Nitrification and Denitrification

Nitrification often occurs naturally and is a biologically oxidative process where ammonia is oxidized to nitrite followed by formation of nitrate by nitrifying bacteria. On the other hand, reduction of nitrate to nitrogen in the presence of an acid is termed as denitrification and is often used as water purification process [19]. These nitrates have the ability to diffuse through the cellular membrane and play a significant role in the production of RNS in a cellular environment.

(1.vii)

(1.viii)

(1.ix)

1.2.1.1.3 Dismutation Reaction

Dismutation or disproportionation is a specific kind of redox reaction, where both oxidized and reduced forms of a chemical species are produced. For example, superoxide free radicals produced in mitochondria dismutate to hydrogen peroxide and oxygen (Eq. (1.x)) [20] or ascorbyl radical to ascorbate (vitamin C) and dehydroascorbate (DHA) (Eq. (1.xi)) in order to maintain intracellular nutrient requirements for the cells [21,22].

(1.x)

(1.xi)

1.2.1.1.4 Cellular Respiration

Oxidation of glucose to carbon dioxide with simultaneous reduction of oxygen to water is another example of natural oxidation–reduction reaction, and is required for energy production in living organisms [23].

(1.xii)

All the reactions shown (Eqs. (1.v)– (1.xii)) and their tendency to either undergo oxidation or reduction are controlled by their reduction potential. Reduction potential (Eo) is defined as a tendency of a chemical species to be reduced by gaining an electron and is defined with electrochemical reference of hydrogen, which is globally given the reduction potential of zero [24]. As this is an electric potential, it is measured in volts and each chemical species has its own intrinsic reduction potential. Numerically, the more positive the potential, the stronger the affinity of the species is to acquire an electron and get reduced.

1.2.1.2 Redox Potential

A normal redox reaction example could be as given below (Eq. (1.xiii)):

(1.xiii)

and this can be broken down into two parts:

(1.xiv)

(1.xv)

For a combined redox reaction, overall redox potential is estimated by

(1.xvi)

of the reaction:

(1.xvii)

n, number of electrons associated with the reaction

F, Faraday’s constant.

should be positive. In Eq. (1.xv), Cu is the electron donor with redox potential of Cu²+ to Cu+ is +0.16 V and that of Fe+ to Fe²+ is 0.77 V. Therefore, overall redox potential becomes +0.61 V.

To state this simply, for a system to undergo a redox reaction, the redox potential of a species to be reduced should be higher than the species to be oxidized. Table 1.2 lists some of the common redox potential of certain half-cell redox couples and of some important biomolecules important to physiological redox environment.

Table 1.2

Half-Cell Reduction Potentials of Biologically Significant Molecules

1.2.2 Thermodynamic Treatment (Ellingham Diagram)

is also a function of temperature (Eq. (1.xviii)). An alternative method to determine the direction of a reaction can be obtained by a simplified thermodynamic analysis of the reaction equilibrium.

(1.xviii)

where

T, reaction temperature

ΔS, entropy change

ΔH, enthalpy change.

The temperature dependence of a reaction using Eq. (1.xviii) is also utilized in redox chemistry to drive the direction of reactions of metal oxides and sulfides to pure metal. This phenomenon is usually illustrated in the form of Ellingham’s diagram, which represents the stability of a metal oxide as a function of temperature in reference to ΔG (Figure 1.2). Free energy versus temperature plot for a particular metal shows the free energy values for its metal oxide formation where slope depicts the ΔS values while the y-intercept gives ΔH. Therefore, magnesium can reduce aluminum oxide to metallic aluminum. Looking at the diagram, all the metal oxide formations have a positive slope while carbon oxidation to carbon monoxide has a negative slope and it cuts across many of the metals at particular temperatures. Therefore, carbon becomes a very useful reducing agent for metal oxides at higher temperatures. For example, carbon can reduce manganese oxide (MnO) to its metallic form Mn once the reaction temperature goes above 1400°C while it will be able to reduce TiO2 to Ti above 1600°C [25–27].

Figure 1.2 Ellingham’s diagram. The figure is reproduced with permission from the University of Cambridge Dissemination of IT for the Promotion of Materials Science (DoITPoMS) website. (www.doitpoms.ac.uk).

As highlighted here, while the reduction potential of two reacting molecules decides their affinity to get reduced or oxidized, it is important to remember that the change in other system properties (eg, temperature) can alter the fate of one species getting reduced in presence of another.

1.2.3 Combustion Sequences and/or Metal Oxides

Combustion is a classic example of free radical reactions and generation. It is a high temperature exothermic process involving multiple redox reactions of a fuel (hydrocarbons) with an oxidant mostly oxygen resulting in oxidized products primarily carbon dioxide and water, to generate heat and light. The overall combustion process of oils is described by the reaction:

(1.xix)

While this equation is a useful simplification of the combustion process, the exact chemistry of combustion is highly complex, multifaceted, and not easy to describe.

Molecular oxygen in its ground state is a very stable molecule and unreactive to hydrocarbons until a catalyst is introduced. However, at elevated temperatures as high as 2200°C, oxygen converts into highly reactive singlet oxygen (O2¹) radicals, and carbon monoxide [29,30]. As for hydrocarbon pyrolysis, the process involves generation of various aliphatic and aromatic radicals.

Below are some of the common series of radical reactions that can occur during combustion process:

(1.xx)

(1.xxi)

(1.xxii)

(1.xxiii)

(1.xxiv)

(1.xxv)

(1.xxvi)

(1.xxvii)

Decay tounsaturated hydrocarbons

, CH2, H2CO finally oxidize to CO2 and H2O.

(1.xxviii)

(1.xxix)

(1.xxx)

Looking at all the reactions that are involved in the process of producing heat energy via oxidation of long/short chain hydrocarbon fuels, it is clear that a milieu of free radicals are generated. Yet, a spatial relationship also exists in radical production, which is described by the zone theory. This concept divides the combustion process into four zones: zone 1-free flame zone (fuel zone), zone 2-high temperature flame zone (>1200°C), zone 3-postflame thermal zone (600–1200°C), and zone 4-gas quench cool and surface catalysis zone (<600°C) [31]. Predominantly, radical formation and consumption takes place during zone 3 and zone 4 combustion but as the process reaches the lower temperature in the cool down zone in the temperature range of 150–400°C, free radicals, mostly phenoxyl and semiquinone exists, which have resonating electrons and hence tend to stabilize via surface mediated reactions with any surrounding transition metal ions. They tend to form a metal oxide complex and are known as persistent free radicals. These radicals are sometimes very stable in ambient conditions and can become environmentally persistent free radicals (EPFRs) [32]. These aromatic compounds generated during combustion also known as organic aerosols mostly come from biomass ignition process. The fact that EPFRs are stabilized due to interaction with metal oxides, their half-lives consistently depend upon the kind of metal oxide they interact with. As per the studies, EPFRs associated with zinc oxide (ZnO) were found to have longest half-lives ranging from 3 to 73 days depending on the adsorbate [32].

Analogously, while the process is a more controlled slow burn, a host of free radicals are also produced in the process of carbohydrate, glucose, or fructose metabolism to generate cellular energy in the form ATP. These free radicals play a critical part in maintaining the cell function depending on their local and overall concentrations. Combination of radicals/oxidants generated during cellular metabolism and subsequent reaction combinations with the transition metal ions also play similar role in accelerating the production of more radicals just like EPFRs. .

Figure 1.3 Analogous comparison of various stages of combustion zone theory with cellular respiration during energy production along with free radical generation [27].

1.2.4 Fenton/Haber–Weiss Chemistry

radical by a reaction between Iron (II) (Fe²+) and hydrogen peroxide (H2O2) catalyzed by iron [34].

(1.xxxi)

(1.xxxii)

ions, converting ferrous back to ferric ion.

(1.xxxiii)

(1.xxxiv)

Henry J.H. Fenton reported for the first time the oxidation power of H2O2 and Fe²+ towards tartaric acid in 1876 but never mentioned the existence of the hydroxyl radical intermediate in the oxidation process, although the reaction was named after him in the cellular redox chemistry.

, superoxide (O2−), etc. during various chain initiation and propagation reactions shown in ) producing just hydrogen peroxide, water, and oxygen as shown in Eqs. (1.xlii) and (1.xliii) [37]. The existence and propagation of any of these reactions highly depends on the density of iron in its required oxidation state as well as the rate constant. An important condition for iron-induced reduction of hydrogen peroxide is the low pH requirement between 3 and 6.

(1.xxxv)

(1.xxxvi)

(1.xxxvii)

(1.xxxviii)

(1.xxxix)

(1.xl)

(1.xli)

(1.xlii)

(1.xliii)

Chain propagation and termination reactions associated with iron and hydrogen peroxide with rate constant measured at pH=5 [38–40].

Fenton chemistry is commercially utilized to treat water pollution, contaminated soils, sludge, etc. by oxidizing the pollutants such as benzene, formaldehyde, rubber chemicals, and pesticides. The rate constant for the initial reaction between Fe²+ and H2O2 is generally observed to be around 10² M−1 s−1 but in biological systems, this rate of reaction with free Fe²+ is not enough for the oxidation to occur. However, when bound to ADP, ATP, or citrate, the oxidation rate increases by at least 2 orders of magnitude, resulting in a reaction rate fast enough for the iron-catalyzed redox process to occur [41].

1.2.5 Thiol Chemistry (–SH;–SS–)

Thiol–disulfide reactions are one of the most important biological redox reactions. In biology, oxidation/reduction governs the metabolic redox state of a cell. Thiol–disulfide interchange/exchange reactions are significant in itself where a thiol (RSH) reacts with another disulfide containing molecule (R′SSR″) to give a new oxidized disulfide (R′SSR) and the corresponding reduced thiol (R″SH) [42]. The reaction is base catalyzed and is proposed to proceed through SN2, 2 step reactions as follows [43,44]:

(1.xliv)

(1.xlv)

(1.xlvi)

(1.xlvii)

(1.xlviii)

(1.xlix)

where

R, attacking group; R′, central group; R″, leaving group

, calculated rate constant dependent on thiolate concentration but independent of pH

kobs, observed rate constant dependent on total thiol concentration and pH.

In this reaction mechanism, the thiolate anion (RS−) acts as an active nucleophile or reactive species to propagate the reaction. In the cytosol, glutathione plays a key role in the thiol redox chemistry as it is the most abundant small molecule cellular antioxidant (see Section 1.3.2 for an in-depth discussion on glutathione redox reactions). One of the important factors that contribute towards the occurrence of this thiol exchange reaction is the pKa of the corresponding thiol and the pH of the reaction environment [45]. For example, a thiol with pKa value of 10, 0.1% of the total thiol will form thiolate at pH 7 while at pH 8, 10 times higher thiolate ions would be present in comparison [43]. In other words, we can say that thiol–disulfide interchange is most favorable at pH near to the pKa values of the thiol. Oxidation of glutathione (GSH) (pKa = 9.33) to its oxidized form (GSSG) is faster at higher pH of 9.43 with rate constant k = 45 L/mol s as compared to at pH of 8.46 with k = 9.1 L/mol s [46]. As the pKa is a function of the structure of thiol molecule, chemical properties of thiol molecules involved in the reaction will dictate the extent of reaction and their existence in reduced or oxidized form. For instance, the reaction of 2-mercaptoethanol (pKa = 10.14) with Ellman’s disulfide (5-(3-carboxy-4-nitrophenyl)disulfanyl-2-nitrobenzoic acid) (pKa = 4.5) is faster than mercaptoethanol with oxidized glutathione (GSSG) (pKa = 9.33) by the order of 10⁴ in water [47,48]. Also, the exchange reaction is faster when the pKa of a nucleophilic thiol (RSH) is as high as possible and that of the corresponding disulfide molecule (R′SH/R″SH) is as low as possible. Stearic interference is another factor that determines the rate constants of the thiol–disulfide exchange reactions. It is most pronounced when there is any carbon substitution at α-position to sulfur. For instance, reaction of bis (t-butyl) disulfide with 1-butylthiolate (k = 0.26 M−1 s−1) is 10⁶ times faster than that with t-butylthiolate (k = 10−7 M−1 s−1). Similarly, charge on the thiolate anion also affects the rate constants [49]. The effect is higher when the charged entity is near to the sulfur group [50].

The rate of reaction is often correlated with the Brønsted plot, which displays the relationship between pKa of the reacting thiols/disulfide and the rate constant. The slope of the plot is defined by the Brønsted coefficient (β), which normally lies between 0.4 and 0.5 for different thiol–disulfide exchange reactions. values for the thiolate content [51] and Fig. 1.4 shows the graphical correlation between pKat pH 7 for a thiol–disulfide exchange reaction. Fig. 1.4 also shows the degree of dissociation (θthat is equivalent to the pH of the solution, pH = 7 in the figure.

(1.l)

(1.li)

Figure 1.4 Correlation between pKa of reacting thiol and rate constant (kRS−)/degree of dissociation (θ) where kRS− was calculated using Eq. (1.l), considering pKa of R′SH and R″SH groups as 8.5. This figure is reproduced with permission from [43].

1.3 Biological Oxidation Events

1.3.1 Oxygen and Nitrogen Currency

Oxygen and nitrogen are the most abundant diatomic gaseous molecules in the atmosphere and play a significant role in regulating both, human and plant metabolism. Nitrogen is known to be highly inert and even oxygen (O2) in its ground state is a stable molecule. But O2 at a higher energy state form such as singlet oxygen (O2¹) or phenoxyl radicals [59]. This radical has a unique selectivity towards its oxidizing properties. For example, it does not readily react with free NADH/NAD+ but can easily oxidize enzyme-bound (lactate dehydrogenase) NADH to NAD+ [60]. Apart from being an oxidizing agent (oxidation of ascorbate to ascorbyl radical), superoxide can also serve as a reducing agent where it can reduce cytochrome c or Fe³+ to Fe²+ which is one of the steps in cellular redox cycling discussed later [61,62].

In reference to biological free radical production, oxygen nucleated free radicals are produced from all the oxygen that is inhaled or consumed by organisms and mitochondria is considered to be the main source of superoxide radical production due to oxygen leakage in electron transport chain during ATP production and is reported to be produced by complexes I and III [63–65]. Reported in vitro studies show that about 4% of the oxygen consumed is converted into superoxide, though in vivo ) (Fig. 1.5).

Figure 1.5 Free radical production/reactive oxygen species with oxygen as a precursor