The Spectroscopic Investigation of Free Radicals and Related Species

There can be very few areas of medical research – particularly in the fields of cardiovascular disease, oncology, cell biology, toxicology, pharmacology and clinical nutrition – that have remained untouched by the explosion in the interest in free radicals seen over the past four decades. Indeed a quick search on PubMed using the term ‘free radical’ returns over 250,000 published articles to date. Similarly, almost 400,000 articles are returned using the search term ‘antioxidant’.

Despite the recognition of their importance in biology and medicine, the investigation of free radicals remains a specialized branch of chemistry, largely confined to physical organic chemists skilled in the investigation of highly reactive, short-lived species using specialized techniques, including EPR spectroscopy, pulse radiolysis and flash photolysis. The application of this chemistry to biological problems can be a very daunting task, indeed.

Dr Burkitt has some twenty years’ experience working in this area – specifically in the application of biophysical techniques and free radical chemistry to problems of biomedical importance. His experience in interdisciplinary research enables him to interact and work productively with chemists, physicists, biochemists and clinicians – as evident from the wide range of projects to which he has contributed (details below).

Whatever your research project or technological problem, if is involves free radical processes in a biological context Dr Burkitt will be able to assist. In particular, Dr Burkitt will help steer your project though the minefield of pitfalls and system artefacts that await those working in this field, ensuring your findings are robust and reliable.

An introductory overview of the area is given below, followed by details of Dr Burkitt’s experience in the field and details of specific projects to which he has contributed.

1. Background: Free Radicals in Biology and Medicine
The discovery of the Cu,Zn-superoxide dismutases (Cu,Zn-SOD) by Joe McCord and Irwin Fridovich in the late Sixties provided the first direct evidence that oxygen-derived radicals – in this case the superoxide radical anion (O2) – are generated by mammalian cells (J. M. McCord and I. Fridovich, J. Biol Chem. 244, 6049 – 6055, 1969). Over the intervening few decades, which have also seen the identification of nitric oxide (NO) as endothelium-derived growth factor, interest in the roles of both reactive oxygen species (ROS) and reactive nitrogen species (RNS) in biological processes has grown to the extent that today one would be hard-pressed to identify a disease in which such species have not been implicated in one way or another. This recognition of the key roles played by ROS and RNS in many of the most important human diseases (including cancer and diseases of the cardiovascular and nervous systems), as well as the ageing process itself, has been accompanied by the gradual realization that reactive species also perform important physiological functions, particularly as cell-signalling agents within the wider phenomenon commonly referred to as ‘redox regulation’.

Although both superoxide and nitric oxide exert many of their biological actions directly, they can also be converted into range of highly-reactive species, particularly under pathological situations. Thus the ROS/RNS ‘family’ includes hydrogen peroxide (H2O2), the hydroxyl radical (OH), peroxynitrate (ONOO), nitrogen dioxide (NO2), nitrosoperoxycarbonate (ONOOCO2) and the carbonate anion radical (CO3). Add to these the enormous range of thiyl (RS), peroxyl (ROO), alkoxyl (RO) and carbon-centred (R) radicals and alkyl hydroperoxides (ROOH) generated from endogenous biological molecules following their attack by ROS/RNS and one can begin to appreciate the difficulty in disentangling the biological actions of these intermediates, most of which have lifetimes measured in microseconds (10–6 s) and occur in vivo at submicromolar (10–6 mol dm–3) concentrations.

2. Detection, identification and investigation of reactive species
By definition, a free radical contains at least one unpaired electron (represented by the ‘radical dot’ in its formula). Several transition metals and their ions also contain unpaired electrons, but in the current context we are referring specifically to species with their unpaired electrons located on non-metallic atoms (typically carbon, oxygen, nitrogen and sulphur). Unpaired electrons confer upon free radicals magnetic properties, which can be exploited in their detection and identification using electron paramagnetic (EPR) resonance spectroscopy, which is often called electron spin resonance (ESR) spectroscopy. For this reason the detection, identification and investigation of radicals will be described initially, followed by a look at the methods used to investigate reactive species that are not free radicals (H2O2 and alkyl hydroperoxides, including ‘lipid peroxides’).

2.1 Free radicals
Owing to their high reactivity, most free radicals are very short-lived, making their detection extremely difficult. The hydroxyl radical (OH), for example, will react with anything at a rate determined (essentially) only by reactant concentration and diffusion rate. (In the language used by physical chemists, we would say that OH reacts at the diffusion-controlled limit, with a pseudo first-order rate constant of some 1010 s–1). Therefore if OH is formed in a cell it will, to all intents and purposes, ‘disappear’ immediately – the moment, in fact, it encounters another molecule, whether a protein, lipid, nucleic acid or a low-molecular-weight substance (such as glutathione).

2.1 (a) Fast-reaction techniques used in physical chemistry
In addition to EPR spectroscopy, free radicals can be ‘seen’ directly by UV-VIS spectroscopy (which, unlike EPR, does not require the presence of unpaired electrons). Both methodologies, however, require the use of specialized techniques to circumvent the transient nature of most radicals. In chemical systems – often designed to model or mimic biological processes – continuous-flow techniques, pulse radiolysis and flash photolysis can be coupled with EPR or UV-VIS detection systems. Pulse radiolysis and flash photolysis are particularly suited to the investigation of reaction kinetics; although continuous-flow EPR can also be used to extract rate data, the technique is particularly valued for the structural information it can provide on short-lived radicals (involving the analysis of hyperfine-coupling interactions, along lines very similar to those used in NMR).

2.1 (b) Detection of radicals by EPR in biological systems
Due to the low extinction coefficients of most radicals, their low concentrations and interference from other light-absorbing species, UV-VIS spectroscopy cannot realistically be used in the direct observation of short-lived radicals in biological systems (and certainly not when coupled with pulse radiolysis or flash photolysis). There are, however, approaches by which short-lived free radicals can be detected and investigated in biological systems based on EPR. Indeed some of the more stable radicals, such as the ascorbate radical from vitamin C, can be observed directly in biological samples at room temperature and many others can be seen upon freezing to 4 K. However, by far the most versatile EPR method for the investigation of radicals in biological systems at ambient temperatures is that of ‘spin-trapping’.

Using this approach, a spin trap (typically a nitrone compound) is included in the reaction system, which can be anything from an enzyme preparation to a whole animal. Nitrones are not free radicals (and are therefore EPR-silent), but upon reaction with radicals they form adducts, known as ‘radical adducts’, which are free radicals (they retain the unpaired electron of the added radical). The radical adducts generated from nitrones by spin trapping are nitroxides, which are a family of remarkably stable radicals. It is a relatively simple matter to record the EPR spectra of nitroxides, enabling one to identify the parent radical that has added to the spin trap.

Although simple in principle, EPR spin-trapping is fraught with difficulties, which can include:

2.2 Indirect methods for the detection of free radicals and other ROS/RNS in biological systems
In addition to the direct detection of radicals using EPR spectroscopy or UV-VIS spectrophotometry, which are not always feasible in biological systems, there are numerous indirect methods for the investigation of processes believed to involve radicals. In one way or another, these methods are all based on product analyses, involving either:

  1. the analysis of biological molecules for damage by free radicals (e.g. the products of lipid peroxidation or oxidized DNA bases); or

  2. monitoring the oxidation of a ‘probe’ by free radicals (typically using fluorescence spectroscopy).

Due to the indirect nature of these methodologies, it is not always possible to identify the free radical responsible for the chemical change being monitored. For example, a whole range of reactive species can initiate lipid peroxidation or protein oxidation. It is, for example, very difficult to distinguish between oxidation brought about peroxidise compound I type-intermediates and oxygen free radicals. For such reasons, biochemists and cell biologists often use these methods – especially those employing fluorescent probes – to measure general changes in ‘oxidative stress’.

Many researchers often use these indirect methods as ‘off the shelf assays’ – in much the same way as they would employ any one of the standard enzyme assays, many of which are available in ‘kit’ form and involve coupling the reaction of interest to either the oxidation or reduction of NAD(P)H or NAD(P)+, respectively, which can be readily followed at 340 nm. Unfortunately the investigation of processes involving free radicals – and also the wide range of other ROS/RNS that are not radicals – rarely lends itself to this ‘black box’ approach: there are simply too many artefacts and pitfalls waiting to catch out the unwary.

To use these methods reliably, the assay must be tailored specifically to the system under study: in each case, it is necessary to go back to the drawing board, ensuring the assay has been fully characterized and explored under the conditions of study. There are few better illustrations of this point than that provided by the probe dichlorofluorescin, the oxidation of which to the fluorescent dichlorofluoresein is widely used as a measure of ROS in cells. Indeed much of the evidence suggesting that ROS act as cell-signalling agents during apoptosis (and, indeed, that the anti-apopototic oncoprotein Bcl-2 is an antioxidant) is based on experiments involving the use of this probe.

Dr Burkitt and colleagues have shown that cytochrome c, which is released from mitochondria during apoptosis, is a potent catalyst of dichlorofluoresin oxidation by hydrogen peroxide. Although the release of cyt c from mitochondria is believed to enhance their production of superoxide radicals (which are converted to H2O2), neither superoxide nor H2O2 can oxidize the probe directly. Cyt c interacts with H2O2 to form a peroxidise compound-I intermediate, which is the species responsible for dichlorofluoresin oxidation in cells undergoing apoptosis. These studies showed very convincingly that dichlorofluoresein fluorescence in apoptotic cells is not a measure of ‘ROS’, but rather of cytosolic cyt c levels.

Further studies showed how cellular levels of glutathione can also affect the rate of dichlorofluoresin oxidation by competing with the probe for oxidation by the peroxidise compound-I intermediate. This finding has important implications for the interpretation of the changes in cellular ‘redox status’ – often expressed as a reduction potential based on the concentrations of the reduced and oxidized forms of glutathione – observed in cells undergoing apoptosis. Without this thorough understanding of the dichlorofluoresin assay for ‘ROS’, it would be easy to reach inappropriate conclusions on the role of ROS in apoptosis. Similar reasoning applies to the use of other probes.

3. Expertise and services offered by Westcott Research and Consulting
Dr Burkitt has over twenty years’ of experience in research at the forefront of each of the areas described above. This experience has been gained in world-leading centres of excellence in the respective areas, including:

3.1 Selected publications showing the scope of Dr Burkitt's experience in this area