Free radicals and other reactive species (RS) are thought to play an important role in many human diseases. Establishing their precise role requires the ability to measure them and the oxidative damage that they cause.


This article first reviews what is meant by the terms free radical, RS, antioxidant, oxidative damage and oxidative stress.


It then critically examines methods used to trap RS, including spin trapping and aromatic hydroxylation, with a particular emphasis on those methods applicable to human studies.


Methods used to measure oxidative damage to DNA, lipids and proteins and methods used to detect RS in cell culture, especially the various fluorescent ‘probes' of RS, are also critically reviewed.


The emphasis throughout is on the caution that is needed in applying these methods in view of possible errors and artifacts in interpreting the results.





Keywords: Cell culture, free radical, reactive species, antioxidant, oxidative stress, oxidative damage, fluorescent probe, lipid peroxidation, superoxide


Introduction
Free radicals and other ‘reactive oxygen (ROS)/nitrogen/chlorine species' (for an explanation of these terms see Table 1) are widely believed to contribute to the development of several age-related diseases, and perhaps, even to the aging process itself (Halliwell & Gutteridge, 1999; Sohal et al., 2002) by causing ‘oxidative stress' and ‘oxidative damage' (terms explained in Table 2). For example, many studies have shown increased oxidative damage to all the major classes of biomolecules in the brains of Alzheimer's patients (Halliwell, 2001; Butterfield, 2002; Liu et al., 2003). Other diseases in which oxidative damage has been implicated include cancer, atherosclerosis, other neurodegenerative diseases and diabetes (Hagen et al., 1994; Chowienczyk et al., 2000; Halliwell, 2000a, 2001, 2002a, 2002b; Parthasarathy et al., 2000). If oxidative damage contributes significantly to disease pathology (Table 3 lists the criteria needed to establish this), then actions that decrease it should be therapeutically beneficial (Halliwell, 2001; Lee et al., 2002a; Liu et al., 2003). If the oxidative damage is involved in the origin of a disease, then successful antioxidant treatment should delay or prevent the onset of that disease (Halliwell, 1991, 2002a, 2002b; Galli et al., 2002; Steinberg & Witztum, 2002). To establish the role of oxidative damage (Table 3), it is therefore essential to be able to measure it accurately. For example, the failure of interventions with antioxidants such as vitamin E, β-carotene or ascorbate to decrease disease incidence in several human intervention trials may have simply been due to the failure of these compounds to decrease oxidative damage in the subjects tested (Halliwell, 1999a, 2000c; Levine et al., 2001; Meagher et al., 2001). In this review, we will examine the methods available to measure reactive species (RS) and oxidative damage, with a particular emphasis on those applicable to human studies.



Table 1

Nomenclature of reactive species





Table 2

Some key definitions





Table 3

Criteria for implicating RS as a significant mechanism of tissue injury in human disease




Measuring RS in vivo: basic principles

Some fascinating techniques such as L-band electron spin resonance (ESR) with nitroxyl probes and magnetic resonance imaging spin trapping are under development to measure RS directly in whole animals (e.g. Berliner et al., 2001; Han et al., 2001; Utsumi & Yamada, 2003), but no probes are currently suitable for human use. Most RS persist for only a short time in vivo and cannot be measured directly. There are a few exceptions: examples include H2O2 (discussed below), and perhaps, NO•, in the sense that serum levels of NO2− have been claimed to measure vascular endothelial NO• synthesis (Kelm et al., 1999), despite the fact that NO2− is quickly oxidized to NO3− in vivo (Kelm et al., 1999; Oldreive & Rice-Evans, 2001). Essentially, there are two approaches to detecting transient RS: 


attempting to trap these species and measure the levels of the trapped molecules and


measuring the levels of the damage done by RS, that is, the amount of oxidative damage.




Sometimes other approaches are used. They include measurements of erythrocyte antioxidant defences and of total antioxidant activity of body fluids; falls in these parameters are often taken as evidence of oxidative stress. Erythrocytes cannot synthesize proteins, however, and their antioxidant enzyme levels may drop as they ‘age' in the circulation (Denton et al., 1975). Thus changes in their levels are more likely to reflect changes in the rates of red blood cell turnover: if this slows down, the circulating erythrocytes will be older on average and so levels of antioxidant enzymes in them will appear to fall. Vice versa, if an intervention accelerates red cell removal or increases erythropoiesis, levels of antioxidants in red cells will seem to rise. Hence, such data should be interpreted with caution.

Depending on the method that is used to measure it, the plasma or serum ‘total antioxidant capacity' (TAC) usually involves major contributions from urate, ascorbate and sometimes albumin −SH groups (Benzie & Strain, 1996; Halliwell & Gutteridge, 1999; Prior & Cao, 1999; Rice-Evans, 2000; Bartosz, 2003), although different methods measure different things (Schlesier et al., 2002; Bartosz, 2003). Thus, for example, if plasma albumin levels fall, TAC will fall. If urate levels rise, TAC will rise. The multiple changes in blood chemistry that occur in sick people mean that TAC changes should be interpreted with caution. TAC is also influenced by diet, often because consumption of certain foods may produce changes in plasma ascorbate and/or urate levels (Halliwell, 2003b).

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Measuring reactive species and oxidative damage in vivo and in cell culture: how should you do it and what do the results mean?

Biomarkers of free radical damage: Applications in experimental animals and in humans

http://www.lqtc.fcien.edu.uy/cursos/Fq2/2010/Proyectos2010/Proyecto9/Paper1.pdf

Structure and function of xanthine oxidoreductase: where are we now?

I Abbreviations used: ACTH, adrenocorticotropic hormone; ADCC, antibody-dependent cellular cytotoxicity; Bma .. total number of binding sites; BREM, bremazocine, an agonist with modest selectivity for K sites; CCK, cholecystokinin; ConA, concanavalin A; DADLE, [D-Ala2,D-LeuS]enkephalin, an agonist somewhat selective for /j �ites; DEX, dextrorphan, inert enantiomer of LEV; DHM, dihydromorphine, a Jl-selective agonist; DIP, dipre­ norphine, a relatively nonselective antagonist; DYN, dynorphin, a peptide product of the dynorphin gene, selective for K sites; EKC, ethylketazocine, a benzomorphan agonist with modest selectivity for K sites; END, endorphin; a peptide product of the pro-opio­ melanocortin gene; <Xand y-endorphin are fragments of fJ-endorphin; fJ-endorphin is selective for e sites; ETO, etorphine, a relatively nonselective agonist; fMLP, formyl-methio­ nyl-leucinyl-phenylalanine; granulo, granulocyte(s); h, human; ICso, concentration of a com­ peting ligand that reduces specific binding of radioligand by 50%; icv, intracerebro­ ventricular; IFN, interferon; ir-, immunoreactive; Kd, K;, equilibrium dissociation con­ stants, determined by binding isotherm or by competition, respectively; LENK, [Leu]en­ kephalin, a minor product of the pro-enkephalin gene, an agonist somewhat selective for {) sites; leuko, leukocyte(s); LEV, levorphanol, a Jl-selective agonist; Iympho, Iymphocyte(s); m, mouse; M.p, macrophage(s); MENK, [Met]enkephalin, major product of the pro-enkephalin gene, a /j-selective agonist; MNC, mononuclear cell(s); mono, monocyte(s); MOR, morphine, a Jl-selective agonist; NA, not applicable; NAL, naloxone, a moderately jl-selective antag­ onist; NK, natural killer; NR, not reported; NTX, naltrexone, a long-lasting Jl-selective antagonist closely related to naloxone; PBL, peripheral blood Iymphocyte(s); PBMNC, peripheral blood mononuclear cell(s); PHA, phytohemagglutinin; PMNL, polymorpho­ nuclear leukocyte(s); r, rat; RIA, radioimmunoassay; spleno, splenocyte; SRBC, sheep red blood cells; VIP, vasoactive intestinal peptide.

Opioid Peptides and Opioid Receptors in Cells of the Immune System

Localization of xanthine oxidase in mammary-gland epithelium and capillary endothelium.

Morphine was demonstrated to exacerbate infections. Experiments were performed to evaluate variations of phagocytic physiology during morphine treatment. In mice, morphine drastically reduced reticuloendothelial system activity, phagocyte count, phagocytic index, killing properties, and superoxide anion production in polymorphonuclear leukocytes and macrophages. Similar effects on alveolar macrophage count, phagocytosis, and killing were found in rabbits, a result which suggested a lack of species specificity. Additional experiments demonstrated that morphine (1) induces a reduction of lymphoid organ weight, (2) impairs the ability to eradicate infections and (3) is counteracted in its depressing activity on phagocytic physiology by small amounts of Corynebacterium parvum. The results suggest that there is a close relationship between the fact that morphine exacerbates infections and the fact that morphine depresses phagocytic functions; therefore, the negative effect of morphine on phagocytosis is at least one of the reasons for its negative effect on the development of infections.

Ezio Tubaro

Papers

Effect of morphine on resistance to infection.

Morphine and methadone impact on human phagocytic physiology

Liver xanthine oxidase increase in mice in three patholgoical models. A possible defence mechanism.

Methadone vs morphine: comparison of their effect on phagocytic functions.

Xanthine oxidase increase in polymorphonuclear leucocytes and macrophages in mice in three pathological situations.