The goal of this article is to indicate how the unique capabilities of EPR can be used very effectively to follow redox status/events in vivo. This will be done in the context of a general overview and two more detailed complementary mini-reviews of the current status of the use of in vivo EPR to measure free radicals and thiols. The article also seeks to delineate areas where developments are needed and the potential pathways to achieve them.
The ability to measure redox status and redox processes in vivo
is very attractive for several reasons. It has been clearly established that redox-active species are intrinsically involved in many physiological and pathophysiological processes. The roles of these species include being essential intermediates in key physiological reactions and, in many cases, being directly involved in the causal chain leading to pathology. The redox state is an important parameter for many key events in cell signaling (13
). Also, therapeutic approaches for many types of pathophysiology involve redox reactions (84
Whereas many useful insights can be achieved by measurements in model systems and cell cultures, the complexity of redox processes and physiology makes it difficult to understand fully the redox processes that occur in vivo without making direct measurements with the intact system. The dynamics of the vascular system, metabolism in different organs, and the complex web of oxidants and antioxidants, cannot be modeled readily. There are few techniques that are capable of making such measurements in vivo, but fortunately EPR can follow many of the most important aspects of these complex processes.
Some of the redox-active species are free radicals, and for these, EPR is the most direct and unambiguous method to measure them. Under some circumstances the concentration of these species or their magnetic properties may make it difficult to measure them directly with EPR. Therefore, a technique termed “spin trapping” has been developed in which a detecting molecule (usually termed a “spin trap”) is added to the system, which, upon reaction with a free radical, can form a product that is another free radical that is considerably more stable than the initial radical (). This provides at least a qualitative indication of the presence and often the type of free radical, and sometimes also may provide quantitative information. Under appropriate conditions, the resulting EPR-active product has spectral features that enable the original radical to be identified. As described below, this technique has been applied for efficient detection and identification of many different types of free radicals in vivo.
Although the “spin trap” is different, the same principle has been used to identify and, often, to quantitate nitric oxide, taking advantage of its free radical nature. In the case of nitric oxide, the trapping usually occurs by the localization of the unpaired electron in a nitrogen–metal complex.
Thiols have a key role in redox reactions in vivo. Redox equilibria between thiols (the reduced state) and disulfides (the oxidized state) are integrally involved in many processes such as cell signaling and enzymic mechanisms. Many redox processes affect the status of thiols and disulfides. Some thiols, such as glutathione, are by themselves key effectors. The structure of proteins can be profoundly affected by changes in thiols and disulfides. Therefore, methods to measure some of the key parameters in vivo, including the amount of glutathione and other small thiols, their oxidized derivatives, and thiols and disulfides in macromolecules could be extremely valuable. As noted below in detail, currently such measurements are very difficult to make, but EPR techniques appear to have the capability to obtain much of the needed data.
It also is potentially possible to detect the presence of free radicals and other redox-active species by following the status of paramagnetic molecules that are redox-active. This usually is done with nitroxides, measuring the changes in their intensity as a function of time and location. A reversible reduction of the paramagnetic nitroxide group to the nonparamagnetic hydroxylamine group is the principal redox reaction in vivo. The distribution of the nitroxides, and hence the compartment that they sample, can be selected by using appropriate structures; for example, charged nitroxides injected into the vascular system will stay in the vascular system; nitroxides whose structures enable them to cross the blood brain barrier can enter the brain; lipophilic nitroxides will localize in lipid-rich domains, including membranes. This can be a powerful tool but also requires considerable planning and controls to attain an unambiguous result. The rate of disappearance of the paramagnetism from a region can be due to a number of factors, including reduction of the nitroxide, reoxidation of the hydroxylamine to the paramagnetic nitroxide, excretion of either the nitroxide or hydroxylamine, and irreversible reactions that destroy the nitroxide group.
There are a number of other capabilities of in vivo EPR related to measurements of redox status that are not covered in this paper, but should be mentioned here because of their importance. The most important one for the subject of this paper is the ability to measure oxygen sensitively and repeatedly. The amount of available oxygen is clearly a critical factor for most redox processes, and in vivo EPR provides some unique capabilities to measure it, as illustrated in another article in this special issue (High Spatial Resolution Multi-Site EPR Oximetry of Transient Focal Cerebral Ischemia in the Rat; B Williams et al.). Other relevant capabilities of in vivo EPR that have implications for redox reactions include measuring a number of parameters that are difficult to measure in vivo by other means, such as: the structure of membranes (especially fluidity), pH, charge, viscosity, and motion of the macromolecules.
Finally, it is important to note that while in vivo EPR is indeed a very powerful technique that has been used extensively and successfully for the types of measurements discussed in this paper, there also are some important potential limitations that one needs to bear in mind. The in vivo EPR technique itself has some technical limitations in regards to sensitivity and the depth at which measurements can be made (the two, of course, are closely related). In the most typical in vivo EPR spectrometers, operating at 1,200 MHz, the depth that can be probed for most uses is ~10 mm. Other spectrometers have been used successfully with frequencies as low as 300 MHz, but as the frequency decreases the sensitivity also decreases. The specificity of EPR for species with unpaired electrons, while usually considered an important asset, of course does limit the measurements to only molecules with unpaired electrons. Because of the paucity of such molecules in vivo, most uses of in vivo EPR require the addition of paramagnetic materials or their precursors (e.g., spin traps). There are some potential perturbations from these substances, but in animal systems the perturbations are usually minor and, if considered carefully, impose few limits on their effective use. In studies with human subjects, however, the paramagnetic materials need to undergo the same time consuming and costly process, as is required for any drug to demonstrate that they are safe and efficacious for use in humans. The time and cost for this process may limit their use in clinical medicine despite the inherent low toxicity of most of these materials. While the measurements of free radicals either by direct observation or with spin traps usually provide data that can be interpreted readily, there are some important caveats. Free radical intermediates can be generated by a variety of processes, and therefore it is essential to understand the pathways that can occur in the context of the specific experiment that is carried out. It is important to recognize that spin traps can be converted to species that are identical to radical adducts through nonradical paths. It also is possible for the spin traps to perturb the process that they are being used to follow, especially if the amount of radicals that is trapped is high; the latter can be especially important in measuring nitric oxide where the spin traps can significantly alter the pathophysiology by effectively scavenging a significant portion of the nitric oxide. The interpretation of data on redox status by the use of nitroxides can be quite complex because of the many paths for reduction and oxidation of nitroxides and their potentially complex metabolism, distribution, and excretion. The measurement of thiols in vivo also can be quite complex because of potential perturbations from the consumption of free thiol groups. While this list of potential limitations seems long and formidable and also probably is incomplete, there are very effective and practical ways to overcome them. This can be done by the same process that is needed for virtually all experimental studies, for example:
- One needs to understand the potential perturbations that can occur;
- Appropriate controls need to be included to determine the occurrence and potential effects of the perturbations;
- Confirmatory experiments, often using other methods need to be carried out.
With such precautions, which really are simply good experimental techniques, in vivo EPR can be a very effective and powerful technique for following redox-related processes in vivo, bringing many unique capabilities to these important types of measurements.