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?

Oxidative stress is known to play an important role in the pathogenesis of a number of diseases. In particular, it is linked to the etiology of Alzheimer's disease (AD), an age-related neurodegenerative disease and the most common cause of dementia in the elderly. Histopathological hallmarks of AD are intracellular neurofibrillary tangles and extracellular formation of senile plaques composed of the amyloid-beta peptide (Aβ) in aggregated form along with metal-ions such as copper, iron or zinc. Redox active metal ions, as for example copper, can catalyze the production of Reactive Oxygen Species (ROS) when bound to the amyloid-β (Aβ). The ROS thus produced, in particular the hydroxyl radical which is the most reactive one, may contribute to oxidative damage on both the Aβ peptide itself and on surrounding molecule (proteins, lipids, …). This review highlights the existing link between oxidative stress and AD, and the consequences towards the Aβ peptide and surrounding molecules in terms of oxidative damage. In addition, the implication of metal ions in AD, their interaction with the Aβ peptide and redox properties leading to ROS production are discussed, along with both in vitro and in vivo oxidation of the Aβ peptide, at the molecular level.

https://hal.archives-ouvertes.fr/hal-01635507/document

Oxidative stress and the amyloid beta peptide in Alzheimer's disease.

Lipid peroxidation: Physiological levels and dual biological effects

Meat and meat products are a fundamental part of the human diet. The protein and vitamin content, as well as essential fatty acids, gives them an appropriate composition to complete the nutritional requirements. However, meat constituents are susceptible to degradation processes. Among them, the most important, after microbial deterioration, are oxidative processes, which affect lipids, pigments, proteins and vitamins. During these reactions a sensory degradation of the product occurs, causing consumer rejection. In addition, there is a nutritional loss that leads to the formation of toxic substances, so the control of oxidative processes is of vital importance for the meat industry. Nonetheless, despite lipid oxidation being widely investigated for decades, the complex reactions involved in the process, as well as the different pathways and factors that influenced them, make that lipid oxidation mechanisms have not yet been completely understood. Thus, this article reviews the fundamental mechanisms of lipid oxidation, the most important oxidative reactions, the main factors that influence lipid oxidation, and the routine methods to measure compounds derived from lipid oxidation in meat.

https://www.mdpi.com/2076-3921/8/10/429/pdf

A Comprehensive Review on Lipid Oxidation in Meat and Meat Products.

Assessment of antioxidant capacity in vitro and in vivo.

Cholesterol oxidation products, termed oxysterols, are increasingly considered of potential interest in the pathogenesis of atherosclerotic lesions. Of dietary or endogenous origin, oxysterols may occur in significant amounts in low density lipoprotein (LDL) particles, especially in hypercholesterolemic subjects. They likely contribute to the uptake of modified LDL by scavenger receptors and some of them finally accumulate in the subintimal space of major arteries; here cholesterol oxides may favor the perpetuation of a chronic inflammatory state, through their ability to trigger irreversible damage of vascular cells with consequent activation of phagocytes. Furthermore, practically all oxysterols of major pathophysiologic interest have been shown to markedly up-regulate expression and synthesis of adhesion molecules, inflammatory cytokines and chemokines. Cholesterol oxidation thus appears to be an important biochemical pathway through which it exerts toxic, inflammatory and finally atherogenic effects.

Oxidized products of cholesterol: dietary and metabolic origin, and proatherosclerotic effects (review).

To date more than 4000 compounds are recognized to belong to the class of flavonoids. These natural phenolic drugs are poorly soluble in water and are rapidly degraded and metabolized in the human body, but nevertheless are very promising for their potential contribution to the prevention and therapy of major chronic diseases, including cardiovascular and neurodegenerative diseases and cancer. In recent years a number of flavanols (e.g. catechins), flavonols (e.g. quercetin, myricetin) and isoflavones (e.g. genistein, daidzein) have been confirmed to possess strong antioxidant, anti-inflammatory, anti-proliferative and anti-aging activities. Incorporation into lipidic or polymer-based nanoparticles appears to markedly help the oral delivery of flavonoids, as these particles can protect the drug from degradation in the gastrointestinal tract and, by virtue of their unique absorption mechanism through the lymphatic system, also from first-pass metabolism in the liver. In addition, both oral and parenteral administration of flavonoids exploits a pharmacologic delivery route that guarantees sustained release of the active principle at the desired site of action. A comprehensive review of studies currently available on the in vitro and in vivo experimental administration of flavonoids by means of nanovectors may be of use as a foundation for the development of advanced delivery systems for these powerful compounds, in view of their adoption in primary and secondary disease prevention.

Design and Development of Nanovehicle-Based Delivery Systems for Preventive or Therapeutic Supplementation with Flavonoids

Cholesterol oxidation products in the vascular remodeling due to atherosclerosis

Oxysterols are a family of 27-carbon cholesterol oxidation derivatives that may be absorbed with the diet or originated endogenously. These cholesterol metabolites are now considered to be potentially involved in the initiation and progression of major chronic diseases including atherosclerosis, neurodegenerative processes, diabetes, kidney failure, and ethanol intoxication. Thus we deemed it of interest to comprehensively analyze the actual relevance of oxysterols, acting through up-regulation of inflammation, apoptosis and fibrosis, to human pathology from cell signaling to disease expression; we also review the available literature on related therapeutic prospects. Oxysterols of pathophysiologic relevance generally possess a strong pro-oxidant effect, chiefly since they activate NAD(P)H oxidases. Further, stimulation of the MEK/ERK signaling pathway appears to be a common feature of the biochemical effects of this class of compounds. Selective metabolic inhibitors of NAD(P)H oxidase and the MAPK pathway might quench or even prevent the cytotoxic effects of pathological accumulation of cholesterol oxides in cells and tissues. The marked reduction of plasma oxysterols reported for statin-based therapy is interesting: it has been associated with a lower incidence and prevalence of Alzheimer's disease (AD) and vascular dementia. Quenching reactive oxygen species' generation seems the likely mechanism exploited by statins against AD incidence and development; intervention with antioxidants might thus also be re-considered as regards molecular "integrated" prevention and possible therapy of human "multifactorial" disease processes.

Cholesterol oxidation products and disease: an emerging topic of interest in medicinal chemistry.

Alzheimer's disease (AD), the most common form of dementia, is characterized by the progressive loss of neurons and synapses, and by extracellular deposits of amyloid-β (Aβ) as senile plaques, Aβ deposits in the cerebral blood vessels, and intracellular inclusions of hyperphosphorylated tau in the form of neurofibrillary tangles. Several mechanisms contribute to AD development and progression, and increasing epidemiological and molecular evidence suggests a key role of cholesterol in its initiation and progression. Altered cholesterol metabolism and hypercholesterolemia appear to play fundamental roles in amyloid plaque formation and tau hyperphosphorylation. Over the last decade, growing evidence supports the idea that cholesterol oxidation products, known as oxysterols, may be the missing link between altered brain cholesterol metabolism and AD pathogenesis, as their involvement in neurotoxicity, mainly by interacting with Aβ peptides, is reported.

/pdf/the-link-between-altered-cholesterol-metabolism-and-4ky1fo942y.pdf

Barbara Sottero

Papers

Oxidized products of cholesterol: dietary and metabolic origin, and proatherosclerotic effects (review).

Design and Development of Nanovehicle-Based Delivery Systems for Preventive or Therapeutic Supplementation with Flavonoids

Cholesterol oxidation products in the vascular remodeling due to atherosclerosis

Cholesterol oxidation products and disease: an emerging topic of interest in medicinal chemistry.

The link between altered cholesterol metabolism and Alzheimer's disease