In who detection of free radical metabolites

During the past two decades, the ESR spin-trapping technique has been used extensively to detect free radicals generated by metabolism of xenobiotic compounds in vitro. Although this ESR technique has proven useful in vitro, it has only recently been applied to drug metabolism in vivo. Spin trapping has successfully been employed to study the metabolism of halocarbons, hydrazines and hydroperoxides in perfused organs and in whole animals. The results of these current studies are reviewed herein. INTRODUCTION The production of free radical metabolites has been thoroughly studied in vitro using ESR techniques; detection of these reactive species in vivo has only recently been undertaken. There are several reasons for the late development of this area. Although many drugs and industrial chemicals are metabolized through free radical intermediates, most biochemicals are metabolized via two electron processes. When free radical metabolites are produced, their detection is difficult due to the transitory nature of these species. The problem of detection is confounded by the slow rate of production of free radicals in animals relative to that observed in chemical systems; therefore, it is very important to obtain the highest possible sensitivity. Unfortunately, the molar sensitivity of biological samples is decreased because they contain a large fraction of water so only small samples can be studied. Water is the worst solvent for ESR due to its high dielectric constant. Although the detection of free radical metabolites in vivo is a challenging task, their existence must be demonstrated in a whole animal or there will always be some question as to their actual existence in biology. Several techniques have been used to overcome the problem of working with aqueous samples. The dielectric constant of water can be lowered by freezing so that larger samples can be analyzed. The freeze quench method is useful for enzymatic systems where solutions can be frozen in milliseconds (ref. 1). This method is not as applicable to tissues because frozen tissues must be ground to fit into ESR sample tubes, and this leads to mechanically induced radicals or artifacts (ref. 2). In addition, the resulting powder spectra are poorly resolved, and their interpretation in complex biological systems is very difficult. The same problems of artifacts and poor resolution are observed when tissue is lyophilized (refs. 3,4). Larger samples can be analyzed using low-frequency ESR; this method could theoretically be used to study radical production directly in small animals (i.e., in vivo spectroscopy). Unfortunately, sensitivity is directly dependent on frequency; thus, lowfrequency instruments are unlikely to achieve the molar sensitivity needed to directly detect the low concentrations of free radical metabolites generated in vivo. Spin trapping appears to be the most convenient approach to the detection of free radicals in & because it facilitates a higher steady-state concentration of free radicals (as free radical adducts) and therefore overcomes the sensitivity problems inherent to detection of radicals in biological systems. Since the concentration of endogenous radicals in biological tissues is generally near the sensitivity limit of ESR spectroscopy, the spintrapping technique is not limited by background signals. SPIN TRAPPING IN VlVO The technique of spin trapping involves the addition of a primary free radical across the double bond of a diamagnetic compound (the spin trap) to form a radical adduct more stable than the primary free radical. This technique involves the indirect detection of primary free radicals that cannot be directly observed by conventional ESR due to low steady-state concentrations or to very short relaxation times, which lead to very broad lines (ref. 5). All of the reported in vivo spin trapping investigations have used the nitrone spin traps, phenyl---butylnitrone (PBN), a-2,4,6-trimethoxy-PBN (!CH30)3PBN) and 5,5-dimethyl-lpyrroline &oxide (DMPO). Radical adducts of nitrone spin traps typically exhibit six-