In Vivo Detection of Free Radical Metabolites by Spin Trapping

BY R. P. MASON, K. R. MAPLES AND K. T. KNECHT

1 Introduction

1.1 The Problem.-In vivo detection of free radical metabolites is a very challenging task that has only recently been undertaken. One reason for the late development of this area is that most biochemicals, as opposed to drugs and industrial chemicals, are not easily metabolized through free radical intermediates. In addition, detection of something as ephemeral as a free radical inside a whole animal is inherently not easy. Production rates of free radicals in animals are slow in comparison to chemical systems; therefore, the highest possible sensitivity is of paramount importance. water, with its high dielectric constant, is the worst solvent for ESR spectroscopy in that only very small samples can be studied. This decreases the molar sensitivity of biological samples just when sensitivity is needed most. However, unless free radical metabolites can be demonstrated with a whole animal, there will always be some question as to their actual existence in biology.

1.2 Approaches.-Over the last thirty years, several approaches have been tried to circumvent the problem of working with aqueous samples. Freezing water lowers its dielectric constant so that larger samples can be studied. The freeze quench technique is useful for enzymes where solutions can be frozen in milliseconds. Frozen tissues, however, must be ground to fit into ESR sample tubes, and this leads to mechanically induced radicals or artifacts. In addition, the resulting powder spectra are poorly resolved, and their interpretation in complex biological systems is very difficult, if not impossible. Lyophilized, or freeze-dried tissue is plagued by the same problems of artifacts and poor resolution. Low-frequency ESR enables the study of larger samples, and perhaps even small animals could be studied directly, that is, with in vivo spectroscopy. Unfortunately, sensitivity is strongly dependent on frequency, and low-frequency instruments are unlikely to achieve the molar sensitivity of the standard X-band instruments. Spin trapping, in that it ideally integrates free radicals formed over time, appears to be the most attractive approach to the detection of free radicals in vivo. Since the concentration of naturally occurring radicals in body tissues is generally near the sensitivity limit of ESR spectroscopy, the spintrapping technique is not limited by background signals.

2 Spin Trapping In Vivo

2.1 Spin Trapping.-The technique of spin trapping involves the addition of a reactive, primary free radical across the double bond of a diamagnetic compound, the spin trap, to form a more persistent, secondary free radical, the radical adduct. This technique allows 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 radical relaxation times, which lead to very broad lines.

To date, all in vivo spin trapping investigations have used the nitrone spin traps, phenyl-tert-butylnitrone (PBN), α-2,4,6-trimethoxy-PBN ((MeO)3PBN) and 5,5-dimethyl-l-pyrroline N-oxide (DMPO). In most cases, radical adducts of nitrone spin traps produce six-line ESR spectra. The hyperfine splittings arise from the nitrogen and β-hydrogen of the spin trap rather than from atoms of the primary radical. Identification of the trapped radical species, therefore, depends on a careful comparison of hyperfine splitting values with those of reference nitroxides analyzed under exactly the same experimental conditions. If the radical is trapped at a nucleus with nonzero spin such as 14N, then additional hyperfine splittings occur which greatly facilitate the identification of the radical adduct. A very useful approach for the radical adducts of C-and a-centered free radicals is isotopic substitution at this center in the radical precursor with 13C or 17O, respectively.

2.2 Difficulties.-Production of a radical adduct stable enough to be detected in biological samples is a major difficulty, but other factors must also be considered. When using the spin-trapping technique in whole animals, spin traps may interfere with the experimental system by inhibiting or stimulating enzymes, or by producing toxicity. The latter possibility has not seemed...