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Chapter 1
Antioxidant Activity and Oxidative Stress: An Overview
Kyung-Jin Yeum, Robert M. Russell, and Giancarlo Aldini
INTRODUCTION
Oxidative stress is involved in the process of aging (Kregel and Zhang 2007) and various chronic diseases such as atherosclerosis (Fearon and Faux 2009), diabetes (Ceriello and Motz 2004), and eye disease (Li et al. 2009a), whereas fruit and vegetable diets rich in antioxidants such as polyphenols, vitamin C, and carotenoids are correlated with a reduced risk of such chronic diseases (Christen et al. 2008, Dauchet et al. 2006, Dherani et al. 2008). An excessive amount of reactive oxygen/nitrogen species (ROS/RNS) leading to an imbalance between antioxidants and oxidants can cause oxidative damage in vulnerable targets such as unsaturated fatty acyl chains in membranes, thiol groups in proteins, and nucleic acid bases in DNA (Ceconi et al. 2003). Such a state of "oxidative stress" is thought to contribute to the pathogenesis of a number of human diseases (Thannickal and Fanburg 2000).
Sensitive and specific biomarkers for antioxidant status/oxidative stress are essential to better understand the role of antioxidants and oxidative stress in human health and diseases, thereby maintaining health and establishing effective defense strategies against oxidative stress. Several assays to measure "t otal" antioxidant capacity of biological systems have been developed to investigate the involvement of oxidative stress in pathological conditions or to evaluate the functional bioavailability of dietary antioxidants. Conventional assays to determine antioxidant capacity primarily measure the antioxidant capacity in the aqueous compartment of plasma. Consequently, water-soluble antioxidants such as ascorbic acid, uric acid, and protein thiols mainly influence these assays, whereas fat-soluble antioxidants such as tocopherols and carotenoids show little influence over the many results. However, there are new approaches to define the total antioxidant capacity of plasma, which reflect the antioxidant network between waterand fat-soluble antioxidants. Revelation of the mechanism of action of antioxidants and their true antioxidant potential can lead to identifying proper strategies to optimize the antioxidant defense systems in the body.
Methodological aspects of various antioxidant capacity assays have been extensively discussed recently (Magalhaes et al. 2008). This chapter focuses on important antioxidants in biological systems, factors affecting bioavailability of antioxidants and, therefore, antioxidant capacity, and basic principles of various biomarkers for antioxidant capacity and their applications.
OXIDATIVE STRESS AND ANTIOXIDANTS IN A BIOLOGICAL SYSTEM
ROS are continuously generated by normal metabolism in the body (Gate et al. 1999) and these ROS are necessary to maintain biological homeostasis through various functions such as vasoregulation and various cellular signal transduction (Hensley and Floyd 2002). However, overproduction of these ROS can also cause damage to the macromolecules necessary for cell structure and function.
Cellular production of ROS such as superoxide anion (O2•–), hydroxyl radical (HO•), peroxyl radical (ROO•), and alkoxyl radical (RO•) occurs from both enzymatic and non-enzymatic reactions. Mitochondria appear to be the most important subcellular site of ROS production, in particular of O2•and H2O2 in mammalian organs. The electron transfer system of the mitochondrial inner membrane is a major source of superoxide production when molecular oxygen is reduced by a single electron. Superoxide can then dismutate to form hydrogen peroxide (H2O2), and then can further react to form the hydroxyl radical (HO•) and ultimately water.
In addition to intracellular membrane-associated oxidases, soluble enzymes such as xanthine oxidase, aldehyde oxidase, dihydroorotate dehydrogenase, flavoprotein dehydrogenase, and tryptophan dioxygenase can generate ROS during catalytic cycling. Auto-oxidation of small molecules such as dopamine, adrenaline (epinephrine), flavins, and quinols can be an important source of intracellular ROS production as well. In most cases, the direct product of such autooxidation reactions is the superoxide anion (Thannickal and Fanburg 2000).
Any compound that can inhibit oxidation of external oxidants is considered to be an antioxidant. This is a relatively simple definition but, at times, it becomes very difficult to evaluate whether a compound actually has an antioxidant action, particularly in vivo.
It is still not clear what kinds of ROS play a role in the pathogenesis of human disease and where the major sites of ROS action occur. There is, however, convincing evidence that lipid peroxidation is related to human pathology, such as in atherosclerosis (Valkonen and Kuusi 1997). The actions of antioxidants in biological systems depend on the nature of oxidants or ROS imposed on the systems, and the activities and amounts of antioxidants present and their cooperative/synergistic interactions in these systems.
Numerous epidemiological studies have indicated that diets rich in fruits and vegetables are correlated with a reduced risk of chronic diseases (Czernichow et al. 2009, Hung et al. 2004, Liu et al. 2001, Liu et al. 2000). It is probable that antioxidants, present in the fruits and vegetables such as polyphenols, carotenoids, and vitamin C, prevent damage from harmful reactive oxygen species, which either are continuously produced in the body during normal cellular functioning or are derived from exogenous sources (Gate et al. 1999). The possible protective effect of antioxidants in fruits and vegetables against ROS has led people to consume antioxidant supplements such as β-carotene, α-tocopherol, and/or multivitamins. It is not surprising to note that more than 11% of US adults age 20 years or older consume at least 400 IU of vitamin E per day from supplements (Ford et al. 2005). However, intervention studies have failed to show a consistent beneficial effect of antioxidant supplements such as vitamin E (Lee et al. 2005) or β-carotene (Baron et al. 2003, Omenn et al. 1996) against chronic diseases. How can we explain these apparent contradictory results between observational studies and intervention trials?
It is interesting to note that although seven and a half years of supplementation with a combination of antioxidants (vitamin C, β-carotene, zinc, and selenium) did not affect the risk of metabolic syndrome, baseline concentrations of serum vitamin C and β-carotene were negatively associated with metabolic syndrome in a generally well-nourished population (Czernichow et al. 2009). It is probable that the generally well -nourished population maintains optimal ranges of antioxidants through a balanced dietary fruit and vegetable intake. However, high doses of a single or limited mixture of antioxidant supplements may not affect the already saturated in vivo antioxidant network, but rather could result in an imbalance in the antioxidant network and could possibly even act as pro-oxidants. A recent prospective study showing an inverse association of baseline plasma antioxidant concentrations with the risk of heart disease and cancer also supports the beneficial effect of a balanced antioxidant status, which can be attained by eating diets high in fruits and vegetables (Buijsse et al. 2005).
MARKERS OF ANTIOXIDANT CAPACITY IN A BIOLOGICAL SYSTEM
Several human studies have failed to show a direct correlation between the physiologic consumption of dietary fat-soluble antioxidants and subsequent changes in antioxidant capacity (Castenmiller et al. 1999, Pellegrini et al. 2000). For example, it has even been suggested that carotenoids may not act as antioxidants) in vivo (Rice-Evans et al. 1997). These suggestions derive from the lack of proper analytical methods for measuring antioxidant capacity. Inasmuch as conventional methods, such as total radical trapping antioxidant parameter (TRAP), oxygen radical absorbance capacity (ORAC), etc., use primarily hydrophilic radical generators and measure primarily antioxidant capacity in the aqueous compartment of plasma, they are unable to determine the antioxidant capacity of the lipid compartment (Cao et al. 1993) Lussignoli et al. 1999). Therefore, it is not surprising that most of the methods used to measure purported "total antioxidant capacity" of plasma are not affected by lipophilic antioxidants, such as carotenoids (Cao et al. 1998b, Castenmiller et al. 1999, Pellegrini et al. 2000).
This can be explained by the fact that plasma carotenoids, which are deeply embedded in the core of lipoproteins, are not available for reaction with aqueous radical species or ferric complexes used in these assays. In addition, an assay to measure total antioxidant capacity in a biological sample such as plasma must consider the heterogeneity of the sample, which consists of both hydrophilic and lipophilic compartments that contain water-soluble and fatsoluble antioxidants, respectively. Possible cooperative/synergistic interactions among antioxidants in biological samples should not be overlooked.
Azo initiators are a class of radical inducers (which contain the —N=N— group) widely used in experiments in vitro to generate radical species. The azo initiators decompose at a temperature-controlled rate to give carbon-centered radicals, which react rapidly with O2 to yield the peroxyl radical (ROO•).
Peroxyl radicals derived from azo initiators can induce the lipid peroxidation cascade and can also damage proteins. Depending on the lipophilicity of the azo initiators [2,2'-azobis-(2amidinopropane) dihydrochloride (AAPH) is water soluble whereas 2,2I-azobis(2,4-dimethylvaleronitrile (AMVN) and,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile) (MeO-AMVN) are lipophilic], the peroxyl radicals are generated in the aqueous or lipid phase of the sample, respectively. The choice of the site of radical generation is of great importance, because the activities of antioxidants present in both the lipid and aqueous compartments depend on the localization of the attacking radical species (Yeum et al. 2003).
Table 1.1 shows the currently available assays to determine antioxidant capacity in hydrophilic and lipophilic environments in biological samples such as plasma. When used alone, those assays (Cao et al. 1993, Valkonen and Kuusi 1997) that use hydrophilic radical initiators and probes are insufficient for determining the antioxidant activity of carotenoids, which are deeply embedded in the lipoprotein core of biological samples. There have been attempts to determine the activity of fat-soluble antioxidants by measuring the antioxidant activity of lipid extracts dissolved in an organic solvent (Prior et al. 2003). This approach, however, cannot appreciate the possible interactions between the fat-soluble and water-soluble antioxidants. The alternative approach of producing radicals in the lipid compartment of whole plasma and monitoring lipid peroxidation by a lipophilic probe (Aldini et al. 2001) allows measurement of the actual "total" antioxidant activity including possible interactions among antioxidants located in the hydrophilic and lipophilic compartments, because the interference of large amounts of protein (e.g. albumin) in the hydrophilic compartment can be overcome by this approach.
HYDROPHILIC ANTIOXIDANT CAPACITY ASSAYS
There are mainly two hydrophilic approaches to determine the antioxidant capacity in plasma. The first approach measures the antioxidant capacity in plasma using hydrophilic assays in the presence of oxidants that act as prooxidants. These assays determine the susceptibility of plasma against oxidation induced by added pro-oxidants (radical inducers) and monitored by an exogenous oxidizable substrate (probe). The oxidation of the probe is theoretically inhibited by the antioxidants present in plasma during the induction period. The TRAP and ORAC assays are presently the most widely used methods for measuring antioxidant capacity in biological systems such as serum and tissues. Dichlorofluorescein-diacetate, phycoerythrin (R-Pe), and crocin-based assays also are included in this category of assays. Specifically, plasma or serum, when challenged with a hydrophilic radical inducer such as 2,2'-azobis(2,4-amidinopropane) dihydrochloride (AAPH), can be monitored by a hydrophilic oxidizable substrate such as 2',7'-dichlorodihydrofluorescein (DCFH) (Valkonen and Kuusi 1997), crocin (Kampa et al. 2002, Tubaro et al. 1998), or R-Pe (Cao and Prior 1999). Antioxidant capacity can be expressed in various ways such as lag phase, area under the curve, or competition kinetics.
AAPH is a hydrophilic azo-compound that spontaneously decomposes at 37°C with a known rate constant (Ri = 1.36 X 10-6 [AAPH] mol/liter/sec), giving rise to carbon-centered radicals that then react with oxygen, yielding the corresponding peroxyl radicals. DCFH, which can be oxidized to highly fluorescent (Exc 480 nm, Em 526 nm) dichlorofluorescein by peroxyl radicals, is used as an oxidizable substrate in the TRAP assay (Valkonen and Kuusi 1997). R-Pe is a protein isolated from Corallina officinalis, and is used as the oxidizable substrate in the TRAP (Ghiselli et al. 1995) and ORAC (Cao and Prior 1999) assays. R-Pe is a fluorescent protein that emits in the visible region (Exc 495 nm, Em 595 nm) and is characterized by fluorescence quenching upon reaction with peroxyl radicals. Crocin, isolated from saffron and characterized by a polyene chain with a high extinction coefficient, has been used as an oxidizable substrate in the assay developed by Tubaro (Tubaro et al. 1998) and then automated by Kampa (Kampa et al. 2002) in the crocin bleaching assay. The reaction of crocin with peroxyl radical leads to a loss of the double bond conjugation and hence to bleaching that can be readily monitored at 445 nm.
The second approach to measure antioxidant capacity in plasma using a hydrophilic assay is to quench a stable and pre-formed radical that does not act as a pro-oxidant. The trolox equivalent antioxidant capacity (TEAC) assay, which was reported by Miller et al.) 1993) ' determines the antioxidant capacity of plasma by measuring the ability of plasma to quench the radical cation of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS). The quenching reaction is monitored by measuring the decay of the radical cation at 734 nm. The ferric reducing ability of plasma (FRAP) assay has received a great deal of attention because of its quick and simple methodology (Benzie and Strain 1996). The FRAP assay measures the reduction of the ferric ion to ferrous ion at low pH, which causes a colored ferrous-fripyridyltriazine complex to form. FRAP values can be obtained by comparing the absorbance change at 593 nm in test reaction mixtures with those containing the ferrous ion in a known concentration.
LIPOPHILIC ANTIOXIDANT CAPACITY ASSAYS
Two decades ago, Niki) 1990) introduced AAPH and AMVN as the sources of water- and lipid-soluble peroxyl radicals respectively. As shown in the work of Massaeli et al. (1999)) where preincubation of LDL with fat-soluble antioxidants increased the protective effect against free radicals while preincubation with watersoluble antioxidants did not show any effect, the importance of lipophilicity vs. hydrophilicity in antioxidants and free radical generating systems for determining antioxidant capacity has been recognized. It has also been demonstrated (Yeum et al. 2003) that the activities of antioxidants present in both the lipid and aqueous compartments depend on the localization of the attacking radical species.
In an effort to understand the biological significance of lipophilic antioxidants, several recent studies have paid attention to the antioxidant capacity in the lipid compartment of plasma. Mayer et al. (2001) proposed a continuous spectroscopic method using selective fluorescence markers to monitor the aqueous and lipid phases in human serum. In particular, diphenylhexatrienel abeled proprionic acid was used as an appropriate probe for the aqueous phase because it preferentially binds to albumin, while diphenylhexatriene-labeled phosphatidylcholine, which incorporates into lipoproteins, monitors the lipid compartment oxidizability. AAPH was selected as the radical inducer for both compartments.
By using this method, the authors reported that supplementation of human serum with quercetin, rutin, vitamins E and C, or total apple phenolics in vitro led to a decrease in oxidizability depending on the oxidation marker and the hydrophobicity of the antioxidant. That is, fat) soluble antioxidants such as quercetin and vitamin E showed higher protective effects against lipoprotein oxidation, whereas water-soluble lutin and vitamin C more efficiently protected the aqueous phase.
An improved TEAC assay has been reported by Re et al. (1999). By using a pre-formed radical mono-cation of ABTS and an appropriate solvent system, the assay is applicable to both hydrophilic and lipophilic systems. The ORAC assay has also been expanded to reflect lipophilic antioxidants by using randomly methylated β-cyclodextrin (RMCD) as a solubility enhancer, AAPH as a radical initiator, and fluorescein as an oxidizable substrate (Huang et al. 2002). Recently, this updated ORAC assay was applied to human plasma (Prior et al. 2003) and the authors reported that lipophilic antioxidants represent less than 30% of the total antioxidant capacity of the protein-free plasma. For the lipophilic ORAC assay, lipophilic antioxidants were extracted by hexane, dried, and resuspended in 7% RMCD solution (50% acetone/50% water, v/v). However, this assay, which partitioned hydrophilic and lipophilic antioxidants, may not be relevant to a true biological system in which active communication occurs among hydrophilic and lipophilic antioxidants.
Aldini et al. (2001) reported a method that measures antioxidant capacity in both the hydrophilic and lipophilic compartments of plasma and allows for interaction between the antioxidants in the two compartments. A lipophilic radical generator coupled with a selective fluorescent probe capable of detecting lipid peroxidation was used to measure the lipid compartment. 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile) (MeO-AMVN), which decomposes at 37°C, was selected as a lipid-soluble radical ...
