Elmer J. Rauckman

Spin trapping of superoxide and hydroxyl radical: practical aspects.

Reports are regularly appearing in the literature describing spin trapping of superoxide and hydroxyl radicals from various sources. Careful scrutiny of these reports will often reveal that insufficient controls have been run to properly validate the results. Nitrones are highly reactive compounds which can form nitroxides by mechanisms other than radical trapping. Thus, investigators using spin trapping should be cognizant of the many artifacts which accompany this technique, and take care to validate their results with satisfactory controls. We have described straightforward procedures to determine whether superoxide or hydroxyl radical trapping have occurred, and which can help verify the assignment of the radical adduct. Nitrones are the only spin traps currently suitable for detection of hydroxyl and superoxide radicals. The various nitrone spin traps in current use each have advantages and disadvantages. In general, the cyclic nitrone traps such as DMPO have greater reactivity with superoxide and hydroxyl radicals, are less readily hydrolyzed, but are more susceptible to oxygen and light, and thus have lesser shelf lives. Aryl nitrones such as 4-POBN or PBN have lesser reactivity with superoxide and hydroxyl radicals, are more readily hydrolyzed, but have greater shelf lives. The stability of DMPO-OH is also greater than that of 4-POBN-OH. Thus in general, DMPO appears to be the most versatile spin trap currently available. Spin trapping is an inefficient means of detecting superoxide, due to the low rate constants for spin trapping. Spin traps possessing a β-hydrogen will also form unstable superoxide adducts. Spin trapping will, however, undoubtedly prove useful in detecting superoxide under conditions where more conventional methods, such as cytochrome c reduction, cannot be used.

[23] Spin trapping of superoxide and hydroxyl radicals

Publisher Summary Many free radicals of biological interest are highly reactive and never reach a concentration high enough to be detected by electron paramagnetic resonance (EPR). An example of this is the hydroxyl radical, which reacts with itself or with most organic molecules at diffusion-controlled rates. Its rate of reaction is limited mainly by the frequency with which it collides with other species. Thus, the direct detection of hydroxyl radicals by EPR in a biologic system is impossible. For short-lived radicals of lesser reactivity compared to the hydroxyl radical, there are various means of detection using EPR. A simple method is to slow the rate of disappearance of the radical by rapidly freezing the sample. This has the disadvantage that the radical is no longer in a fluid environment, and the resultant anisotropic effects can obscure the identification of the radical. In theory, spin trapping can overcome many of these difficulties. This technique consists of using a spin trap––that is, a compound that forms a stable free radical by reacting covalently with an unstable free radical. This chapter discusses the spin trapping of the biologically important free radicals: superoxide and hydroxyl.

A method for the detection of superoxide in biological systems

Abstract The ability to detect superoxide in biological milieu is filled with a number of difficult problems. For example, the ferricytochrome c assay method cannot be used in the presence of NADPH-cytochrome P -450 reductase since cytochrome c is preferentially reduced by this enzyme. We have found that the superoxide-dependent oxidation of one particular hydroxylamine, 2-ethyl-1-hydroxy-2,5,5-trimethyl-3-oxazolidine, to its corresponding nitroxide, 2-ethyl-2,5,5-trimethyl-3-oxazolidinoxyl, can be used to quantitate superoxide production by hepatic microsomes and purified enzymes. We determined that this assay method is free from most of the problems inherent in other methods for the identification of superoxide.

Acetaminophen hepatotoxicity. An alternative mechanism.

Alcohol-fed hamsters were used to study the mechanism by which acetaminophen initiates hepatotoxicity. Animals maintained on an ethanol-containing diet (Group B) exhibited an increased mortality rate after administration of acetaminophen (400 mg/kg) as compared to control hamsters (Group A). However, in those animals in which the ethanol-containing diet had been replaced by the control diet 24 hr before receiving acetaminophen (Group C), significant protection against acetaminophen toxicity was observed as compared to control animals (Group A). This observation correlates well with the finding that Group C hamsters had higher levels of glutathione and catalase than was found in either Group A or Group B animals. It was also demonstrated that acetaminophen was oxidized by cytochrome P-450, producing acetaminophen free radical and hydrogen peroxide. The free radical in the presence of oxygen was found to generate superoxide and presumably N-acetyl-p-benzoquinone imine. Microsomal lipid peroxidation was found to be stimulated markedly in the presence of acetaminophen. The role of glutathione in protecting hamsters from acetaminophen-mediated hepatotoxicity is discussed.

Superoxide-dependent reduction of nitroxides by thiols

Abstract It was found that superoxide can reduce certain nitroxide free radicals to their corresponding hydroxylamines in the presence of most sulfhydryl-containing compounds. The stoichiometry of the reaction was found to be three nitroxides reduced per superoxide. Evidence is presented indicating that superoxide directly reacts with a nitroxide to yield a N-hydroxy-N-hydroperoxyl compound. This product rapidly decomposes, giving a hydroxylamine and an oxidized sulfhydryl compound, which is postulated to be a sulfenyl hydroperoxide. It is proposed that this sulfenyl hydroperoxide reduces two additional nitroxyl free radicals to account for the unusual stoichiometry.

Pharmacokinetics of Nitroxide Nmr Contrast Agents

Pharmacokinetics of the nitroxide stable free radical functionality of compounds containing this moiety were evaluated in the rat. The agents were injected i.v. at either high (1.75 mmoles/kg) or low (10 mumoles/kg) dose, and timed blood samples were drawn and assayed for nitroxide concentration by EPR spectrometry. Similarly, various organs and tissues were removed at specified times after injection and homogenized for determination of nitroxide concentration. Urine was collected by catheter for estimation of urinary excretion of the intact nitroxide free radical. At high doses, the various nitroxides exhibited an initial rapid disposition phase, followed by a terminal disposition phase with disappearance from the blood showing apparent log-linear half-lives of about 5 to 30 minutes. Generally, 20 to 60% of the dose was recovered in the urine. At low doses, dissimilar results were obtained. Blood levels again showed biphasic decay; however, blood concentrations at all times were much lower than those predicted by the high dose kinetics, indicating probably nonlinear pharmacokinetic behavior. Tissue homogenate studies showed low or nondetectable levels of nitroxide signal, demonstrating that the low blood concentrations could not be accounted for by a rapid uptake into specific tissues. Moreover, only 2 to 6% of the nitroxide could be recovered in the urine. Additional studies demonstrated that at the low dose a rapid in vivo bioreduction occurred which appeared to be saturable at the higher dose.

Studies of the mobility of maleimide spin labels within the erythrocyte membrane

We have confirmed a method yielding reproducible and reliable spectrometric parameters derived from spin-labeled erythrocyte ghosts using nitroxide derivatives of maleimide compounds. The disorder parameter, W/S, was shown to vary with changes in the structure of the label, the conditions utilized for labeling such as ionic strength and erythrocyte age and the presence of drugs such as alcohol and acetaminophen. The nitroxide spectrum was also found to change with increasing and decreasing temperature in an irreversible manner. These findings should permit increased reliance to be placed on the spin-labeling technique when used to monitor changes in membrane lipid or protein assembly.

Acute cocaine-induced hepatotoxicity in dba/2ha male mice.

Abstract Cocaine produced hepatotoxicity in nonpretreated mice of at least one strain, DBA 2 Ha . This hepatic damage involved a wide variety of hepatic enzymes, including protective and drug-metabolizing enzyme systems, and was demonstrated histologically by centrilobular necrosis. Depression of hepatic cytochrome P-450 was seen as an early, reliable, and sensitive indicator of cocaine-induced hepatic injury. This liver damage was both dose and time dependent with significant damage to enzyme systems occurring as early as 2 hr after cocaine administration. Phenobarbital enhanced this cocaine-induced hepatic injury, while metyrapone decreased the extent of damage. This nonpretreated DBA 2 Ha mouse model can serve as a useful model for the in vivo and in vitro study of cocaine-mediated hepatotoxicity, both in descriptive and mechanistic studies.

Improved Methods for the Oxidation of Secondary Amines to Nitroxides

Abstract Biologically active spin labeled compounds have recently gained significant prominance as molecular probes in several areas of research1–4. The most generally useful method described in the literature for oxidizing a secondary amine to a stable nitroxyl free radical requires the use of a water solution of hydrogen peroxide with a catalytic amount of sodium tungstate4. Unfortunately, this procedure fails in the case of secondary amines with minimal water solubility. A number of alternative oxidative procedures have been described in the literature. For example, the oxides of lead, mercury and silver as well as an alkaline solution of potassium ferricyanide have been employed5. In the case of aromatic amines, which are not generally oxidized by tungstate salts, t-butyl hydroperoxide (in the presence of catalytic amounts of cobalt stearate) as well as p-nitroperbenzoic acid in dioxane6 have given the desired product.

Evidence of enhanced in vivo lipid peroxidation after acute cocaine administration

An acute intraperitoneal dose (60 mg/kg) of cocaine to DBA/2Ha male mice results in enhanced lipid peroxidation in vivo, as measured by an increase in conjugated diene absorption in hepatic microsomal lipids. The initiation of this lipid peroxidation is an early consequence of cocaine administration; as early as 1 h after cocaine, peroxidized lipids are significantly greater in treated animals than in controls. This cocaine-induced lipid peroxidation remains at a maximal level from 2 to 4 h and returns approximately to control levels by 8 h. The metabolites of cocaine also produce lipid peroxidation in vitro. Liver microsomes from phenobarbital-treated DBA/2Ha male mice, incubated aerobically in the presence of NADPH, cocaine or the cocaine oxidative metabolites, norcocaine and norcocaine nitroxide, induced lipid peroxidation as measured by an increase in the production of thiobarbituric acid (TBA)-reactive products. The extent of lipid peroxidation is greater for the oxidative metabolites of cocaine than for cocaine itself.