Investigating the free radical trapping ability of NXY-059, S-PBN and PBN

The spin trapping ability of the nitrones 2,4-disulphophenyl-N-tert-butyl nitrone (NXY-059), 2-sulphophenyl-N-tert-butyl nitrone (S-PBN) and alpha-phenyl-N-tert-butyl nitrone (PBN) for both hydroxyl and methanol radicals was investigated using electron paramagnetic resonance (EPR) spectroscopy. The radicals of interest were generated in situ in the spectrometer under constant flow conditions in the presence of each nitrone. The spin adducts formed were detected by EPR spectroscopy. This approach allowed for quantitative comparison of the EPR spectra of the spin adducts of each nitrone. The results obtained showed that NXY-059 trapped a greater number of hydroxyl and methanol radicals than the other two nitrones, under the conditions studied.     

Comparison of the radical trapping ability of PBN,S-PBN and NXY-059

The nitrones α-phenyl-N-tert-butyl nitrone (PBN), sodium 2-sulfophenyl-N-tert-butyl nitrone (S-PBN) and disodium 2,4-disulfophenyl-N-tert-butyl nitrone (NXY-059) are neuroprotective in a variety of rodent models. The objective of the current studies was to compare the ability of PBN, S-PBN, and NXY-059 to form radical adducts and to prevent salicylate oxidation in an aqueous system. For the electron spin resonance (ESR) studies, hydroxyl radicals were generated with ultraviolet (UV) light and hydrogen peroxide. Secondary radicals were then produced by the addition of methanol, ethanol, isopropanol, dimethylsulfoxide, tetrahydrofuran or 1,4-dioxane. In addition, competition spin trapping studies were performed using PBN-α-¹³C and either S-PBN or NXY-059. In the salicylate studies, PBN, S-PBN and NXY-059 were compared to a variety of other antioxidants and reference compounds (cysteine, glutathione, ascorbate, uric acid, Tempo, Trolox, and Tirilizad) for their ability to prevent 2,3- and 2,5-dihydroxybenzoic acid formation induced by hydroxyl radical generating systems. All 3 nitrones trapped carbon- and oxygen-centered radicals to produce ESR-detectable radical adducts. Each nitrone also prevented salicylate oxidation, with PBN being the most effective. The ability of these 3 nitrones to prevent salicylate oxidation resembled that of most of the other compounds tested.     

Comparison of the radical trapping ability of PBN, S-PPBN and NXY-059

The nitrones alpha-phenyl-N-tert-butyl nitrone (PBN), sodium 2-sulfophenyl-N-tert-butyl nitrone (S-PBN) and disodium 2,4-disulfophenyl-N-tert-butyl nitrone (NXY-059) are neuroprotective in a variety of rodent models. The objective of the current studies was to compare the ability of PBN, S-PBN, and NXY-059 to form radical adducts and to prevent salicylate oxidation in an aqueous system. For the electron spin resonance (ESR) studies, hydroxyl radicals were generated with ultraviolet (UV) light and hydrogen peroxide. Secondary radicals were then produced by the addition of methanol, ethanol, isopropanol, dimethylsulfoxide, tetrahydrofuran or 1,4-dioxane. In addition, competition spin trapping studies were performed using PBN-alpha-(13) C and either S-PBN or NXY-059. In the salicylate studies, PBN, S-PBN and NXY-059 were compared to a variety of other antioxidants and reference compounds (cysteine, glutathione, ascorbate, uric acid, Tempo, Trolox, and Tirilizad) for their ability to prevent 2,3- and 2,5-dihydroxybenzoic acid formation induced by hydroxyl radical generating systems. All 3 nitrones trapped carbon- and oxygen-centered radicals to produce ESR-detectable radical adducts. Each nitrone also prevented salicylate oxidation, with PBN being the most effective. The ability of these 3 nitrones to prevent salicylate oxidation resembled that of most of the other compounds tested.     

Spin Trapping Study in the Lungs and Liver of F344 Rats after Exposure to Ozone

Fischer 344 rats were injected with the spin traps C-phenyl N-tert-butyl nitrone (PBN, 150 mg/kg bw, ip) or 4-pyridine-N-oxide N-tert -butyl nitrone (POBN, 775 mg/kg bw, ip), and exposed to clean air or 2 ppm ozone for two hours. The presence of spin adducts was determined by electron paramagnetic resonance (EPR) spectroscopy of chloroform extracts of lung and liver homogenates. No significant levels of adducts were detected in the lungs of air control animals. Benzoyl N-tert-butyl aminoxyl, attributed to direct reaction of ozone with PBN, and tert-butyl hydroaminoxyl, the scission product of the hydroxyl adduct of PBN, were detected in the lungs of ozone exposed rats. EPR signals for carbon-centred alkoxyl and alkyl adducts were also detected with PBN in the lungs and liver of animals exposed to ozone. With POBN, only carbon-centred alkyl radicals were detected. Senescent, 24 months old rats were found to retain about twice more ¹⁴C-PBN in blood, heart and lungs by comparison to juvenile, 2 months old animals. Accordingly, the EPR signals were generally stronger in the lungs of the senescent rats by comparison to juvenile rats. Together, the observations were consistent with the previously proposed notion that a significant flux of hydrogen peroxide produced from the reaction of ozone with lipids of the extracellular lining, or from activated macrophages in the lungs could be a source of biologically relevant amounts of hydroxyl radical.     

Spin trap evidence for production of superoxide radical anions by purified NADPH-cytochrome P-450 reductase

Previous evidence for superoxide radicals as initial reduction products of oxygen by NADPH cytochrome P-450 reductase has been indirect. In this paper a technique is described to spin trap radicals produced in incubations of oxygen and reductase. Reference spin trap adducts were synthesized by adding phenyl-$\text{t}$-butyl nitrone (PBN) to superoxide radicals (PBN-OOH) or to hydroxyl radicals (PBN-OH). Both PBN adducts are stable in water or ethyl acetate for hours. Electron Paramagnetic Resonance (EPR) spectra measured in N₂-saturated ethyl acetate allow clear resolution of the hyperfine extrema of PBN-OH and PBN-OOH (2.1 and 4.5 G splitting, respectively). Comparison of EPR spectra from reductase and oxygen incubations with those of synthetic PBN-OOH suggest that superoxide radicals are the major primary reduction product of oxygen.     

Induction of free radicals in hepatocytes,mitochondria and microsomes of rats by ochratoxin A and its analogs

Oxidative damage may be one of the manifestations of cellular damage in the toxicity of ochratoxin A (OA). OA; its three natural analogs, OB, OC and Oα; and three synthetic analogs, the ethyl amide of OA (OE-OA), O-methylated OA (OM-OA), and the lactone-opened OA (OP-OA) were used to study free radical generation in hepatocytes, mitochondria and microsomes from rats. Electron paramagnetic resonance spectroscopy (EPR) using α-(4-pyridyl-1-oxide)-N-tert-butyl nitrone (4-POBN) as a spin trapping agent showed an enhanced free radical generation due to the addition of NADPH to the microsomes. An EPR signal was not observed in the mitochondria and hepatocyte samples when they were treated with a variety of agents. Addition of OM-OA together with NADPH and Fe³⁺ to the microsomes resulted in a strong EPR signal compared with the other analogs, whereas the signal could be quenched by the addition of catalase. OM-OA does not have a dissociable phenolate group and does not chelate Fe³⁺. The spin adduct hyperfine splitting constants indicated the presence of α-hydroxyethyl radicals resulting from generated hydroxyl radicals, which were trapped by 4-POBN. The results also suggested that the production of hydroxyl radicals by OA does not require a dissociable phenolate group or the prior formation of an OA-Fe complex.     

Serendipitous findings while researching oxygen free radicals

This review is based on the honor of receiving the Discovery Award from the Society of Free Radical Biology and Medicine. The review is reflective and presents our thinking that led to experiments that yielded novel observations. Critical questioning of our understanding of oxygen free radicals in biomedical problems led us to use and develop more direct and extremely sensitive methods. This included nitrone free radical spin trapping and HPLC–electrochemical detection. This technology led to the pioneering use of salicylate to trap hydroxyl free radicals and show increased flux in ischemia/reperfused brain regions and also to first sensitively detect 8-hydroxyl-2-deoxyguanosine in oxidatively damaged DNA and help assess its role in cancer development. We demonstrated that methylene blue (MB) photoinduces formation of 8-hydroxyguanine in DNA and RNA and discovered that MB sensitively photoinactivates RNA viruses, including HIV and the West Nile virus. Studies in experimental stroke led us serendipitously to discover that α-phenyl-tert-butylnitrone (PBN) was neuroprotective if given after the stroke. This led to extensive commercial development of NXY-059, a PBN derivative, for the treatment of stroke. More recently we discovered that PBN nitrones have potent anti-cancer activity and are active in preventing hearing loss caused by acute acoustical trauma.     

Formation of radicals during heating lysine and glucose in solution with an intermediate water activity

Abstract

Heating glucose with lysine under alkaline conditions (pH 7.0–10.0) was found to take place with consumption of oxygen together with formation of brown-colored compounds. Highly reactive intermediary radicals were detected when lysine and glucose were heated at intermediate water activity at pH 7.0 and 8.0. The detection was based on initial trapping of highly reactive radicals by ethanol followed by spin trapping of 1-hydroxyethylradicals with α-(4-pyridyl N-oxide)-N-tert-butylnitrone (POBN) and Electron Spin Resonance (ESR) spectroscopy. The generation of reactive intermediary radicals from the Maillard reactions was favored by enhancing alkaline conditions (pH 8.0) and stimulated by presence of the transition metal ion Fe²⁺. The stability of the nitrone spin traps, N-tert-butyl-α-phenylnitrone and POBN was examined in buffered aqueous solutions within the pH range 1–12, and found to be less temperature dependent at acidic pH compared to alkaline conditions. A low rate (k_obs) of hydrolysis of POBN was found at the used experimental conditions of 70°C and pH 7.0 and 8.0, which made this spin trap method suitable for the detection of radicals in the Maillard reaction system.     

Synthesis and ESR studies of a novel cyclic nitrone spin trap attached to a phosphonium group-a suitable trap for mitochondria-generated ROS?

In this study we report the synthesis and biological application of a novel cyclic nitrone spin trap containing a phosphonium cation. This new spin trap ([4-(2-methyl-1-oxy-3, 4-dihydro-2H-pyrrole-2-carbonyloxy)-butyl]-triphenyl-phosphonium bromide, MitoBMPOBr) is a derivative of the cyclic nitrone, 5-tert-butoxycarbonyl 5-methyl-1-pyrroline N-oxide (BMPO). MitoBMPOBr forms radical adducts upon trapping of superoxide and hydroxyl radicals that exhibit highly distinct and characteristic EPR spectra. The stability of these adducts is comparable to those of BMPO. Because of the presence of a positively-charged phosphonium moiety, MitoBMPOBr may be suitable for trapping reactive oxygen species (ROS) in the mitochondria.     

Spin Trapping of Ibuprofen Radicals: Evidence That Ibuprofen is a Hydroxyl Radical Scavenger

The purpose of this study was to use electron paramagnetic resonance (EPR) spectroscopy to determine if ibuprofen, [2–(4-isobutylphenyl) propanoic acid], a potent nonsterodial anti-inflammatory agent, could modify hydroxyl radicals generation in vim. Ibuprofen (IBU; 0.1–50 mM) in water or water alone was added to EPR tubes containing ferrous sulfate (0.5–2.0mM). and either 5.5-dimethyl-l-pyrroline-N-oxide (DMPO; 40mM) or a-phenyl N-tert-butyl nitrone (PBN; 48 mM). Hydrogen peroxide (l mM) was added to inititate the Fenton reaction, and the systems were then analyzed by EPR spectroscopy to determine the type and relative quantity of free radical(s) produced. IBU caused a dose-dependent decrease of signal intensity of the hydroxyl radical adduct of DMPO (DMPO-OH) which is an indication that IBU either scavenges the hydroxyl radical and/or chelates iron. In addition, other radicals (presumably IBU radicals) produced in these systems were trapped by both DMPO (a_N = 16.1G, a^H_β = 24.0G) and PBN (a_N = 15.7G. a^H_β = 4.4G and a_N = 17.0G, a^H_β = 2.1 G). The signal height of these IBU radicals increased in systems containing ferrous sulfate (l mM), hydrogen peroxide (lmM), PBN (48mM), and increasing IBU concentrations. Therefore. we conclude that IBU scavenges the hydroxyl radical. If IBU chelated iron, then less hydroxyl radicals would be generated, less IBU radicals formed and the signal height of IBU radicals trapped by PBN would have decreased. However, these data do not fully exclude the possiblity that IBU may, to some extent. also chelate iron. Scavenging of hydroxyl radicals may be one of the mechanisms responsible for the beneficial action of IBU during the management of several rheumatic diseases. However, the IBU radicals produced when IBU scavenges hydroxyl radicals are reactive. and may be associated with the reported toxicity of this therapeutic agent.     

Spin Trapping Study in the Lungs and Liver of F344 Rats after Exposure to Ozone

Fischer 344 rats were injected with the spin traps C-phenyl N-tert-butyl nitrone (PBN, 150 mg/kg bw, ip) or 4-pyridine-N-oxide N-tert -butyl nitrone (POBN, 775 mg/kg bw, ip), and exposed to clean air or 2 ppm ozone for two hours. The presence of spin adducts was determined by electron paramagnetic resonance (EPR) spectroscopy of chloroform extracts of lung and liver homogenates. No significant levels of adducts were detected in the lungs of air control animals. Benzoyl N-tert-butyl aminoxyl, attributed to direct reaction of ozone with PBN, and tert-butyl hydroaminoxyl, the scission product of the hydroxyl adduct of PBN, were detected in the lungs of ozone exposed rats. EPR signals for carbon-centred alkoxyl and alkyl adducts were also detected with PBN in the lungs and liver of animals exposed to ozone. With POBN, only carbon-centred alkyl radicals were detected. Senescent, 24 months old rats were found to retain about twice more ¹⁴C-PBN in blood, heart and lungs by comparison to juvenile, 2 months old animals. Accordingly, the EPR signals were generally stronger in the lungs of the senescent rats by comparison to juvenile rats. Together, the observations were consistent with the previously proposed notion that a significant flux of hydrogen peroxide produced from the reaction of ozone with lipids of the extracellular lining, or from activated macrophages in the lungs could be a source of biologically relevant amounts of hydroxyl radical.     

Detection of Free Radicals Generated from the in vitro Metabolism of Carbon Tetrachloride Using Improved ESR Spin Trapping Techniques

The spin trapping chemistry of carbon tetrachloride has been previously investigated in rat liver, both in vitro and in vivo. In addition to the trichloromethyl radical, both a 'carbon-centred' and an 'oxygen-centred' radical have been detected in vitro. These spin adducts have been assigned to 'lipid' and 'lipid oxyl' radicals. However, no specific structural characterization has been provided to date. The spin trapping chemistry of this system was reinvestigated with the use of deuterated α-phenyl N-tert-butyl nitrones to obtain better spectral resolution. Results indicate that the PBN trapped carbon-centred lipid radical is of a primary alkyl type.     

The production of oxygen-centered radicals by Bacillus-Calmette-Guerin-activated macrophages: An electron paramagnetic resonance study of the response to phorbol myristate acetate

The spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide of free radicals formed from Bacillus-Calmette-Guerin elicited peritoneal macrophages stimulated with phorbol myristate acetate resulted in the formation of a superoxide and hydroxyl spin adducts. The formation of both spin adducts was inhibited by copper/zinc superoxide dismutase. Only 70% of the hydroxyl spin adduct could be inhibited by catalase or the scavenger dimethyl sulfoxide. This suggests that the production of hydroxyl radicals involves prior formation of both superoxide radicals and hydrogen peroxide, implicating a Fenton catalysed Haber-Weiss reaction. The metal scavenger desferrioxamine also reduced the hydroxyl radical signal by 70%. The unaccounted 30% hydroxyl radical-like signals are probably due to carbon-centered free radicals formed by the lipoxygenase reaction. Spin trapping in the presence of the lipid-soluble spin trap, 5-octadecyl-5,3,3-trimethyl-1-pyrroline-N-oxide, resulted in a spectrum consistent with the presence of an oxaziridine nitroxide. This results from the free radical-induced cyclisation of a nitrone with an unsaturated fatty acid.     

α-Aryl-N-aryl nitrones: Synthesis and screening of a new scaffold for cellular protection against an oxidative toxic stimulus

Nitrone-containing compounds are commonly employed as spin traps of free radical species in chemical and biological studies. Some molecules as α-phenyl-N-t-butyl nitrone (PBN) and its derivatives have been tested as potential drugs to treat oxidative stress related diseases, as Alzheimer and stroke for example. In this work we report the design and the synthesis of α-aryl-N-aryl nitrones and their cytoprotection profile on human neuroblastoma cells (SH-SY5Y) under induced oxidative stress. All the nine synthesized nitrones showed a significant response at low micromolar concentration. The selected compound 8 (α-phenyl-N-phenyl nitrone) increased the reduced glutathione (GSH) levels by 65% and lowered the necrotic cell death from 25.8% to 3.8%. Based on our data, the designed highly conjugated nitrone double-bond skeleton can be considered as a good scaffold for further studies regarding oxidative stress-related diseases.     

Use of Spin Traps in Intact Animals Undergoing Myocardial Ischemia/Reperfusion: A New Approach to Assessing the Role of Oxygen Radicals in Myocardial “Stunning”

Numerous studies have indirectly, suggested that oxygen-derived free radicals play an important path-ogenetic role in the prolonged depression of contractile function observed in myocardium reperfused after reversible ischemia (myocardial “stunning”). In order to provide direct evidence for the oxy-radical hypothesis of stunning, we administered the spin trap, α-phenyl N-tert-butyl nitrone (PBN), to open-chest dogs undergoing a 15-min coronary artery occlusion followed by reperfusion. Plasma of local coronary venous blood was analyzed by electron paramagnetic resonance (EPR) spectroscopy. EPR signals characteristic of radical adducts of PBN appeared during ischemia and increased dramatically in the first few minutes after reperfusion. After this initial burst, the production of adducts abated but did not cease, persisting up to 3 h after reflow. The production of PBN adducts after reperfusion was inversely related to collateral flow during ischemia. PBN itself enhanced recovery of contractile function. indicating that the radicals trapped may play a pathogenetic role in myocardial stunning. Superoxide dismutase plus catalase attenuated PBN adduct production and, at the same time, improved recovery of contractile function. Antioxidant therapy given 1 min before reperfusion suppressed PBN adduct production and improved contractile recovery; however, the same therapy given 1 min after reperfusion did not suppress early radical production and did not attenuate contractile dysfunction. After i.v. administration, the elimination half-life of PBN was estimated to be approximately 4–5 h. The results demonstrate that 1) free radicals are produced in the stunned myocardium in intact animals; 2) inhibition of free radical production results in improved contractile recovery; and 3) the free radicals important in causing dysfunction are produced in the first few minutes of reperfusion. Taken together, these studies provide cogent evidence supporting the oxy-radical hypothesis of stunning in open-chest dogs. It is now critical to determine whether these results can be reproduced in conscious animal preparations.     

α-phenyl-tert-butyl-nitrone inhibits free radical release in brain concussion

Traumatic brain injury (TBI) is one of the important causes of mortality and morbidity. The pathogenesis of the underlying brain dysfunction is poorly understood. Recent data have suggested that oxygen free radicals play a key role in the primary and secondary processes of acute TBI. We report direct electron spin resonance (ESR) evidence of hydroxyl (·OH) radical generation in closed-head injury of rats. Moderate brain concussion was produced by controlled and reproducible mechanical, fixed, closed-head injury. A cortical cup was placed over one cerebral hemisphere within 20 min of the concussion, perfused with artificial cerebrospinal fluid (aCSF) containing the spin trap agent pyridyl-N-oxide-tert-butyl nitrone (POBN, 100 mM), and superfusate samples collected at 10 min intervals for a duration up to 130 min post brain trauma. In addition, POBN was administered systematically (50 mg/kg body wt.) 10 min pretrauma and 20 min posttrauma to improve our ability to detect free radicals. ESR analysis of the superfusate samples revealed six line spectra (α_N = 15.4 and α^β_H = 2.5 G) characteristic of POBN-OH radical adducts, the intensity of which peaked 40 min posttrauma. The signal was undetectable after 120 min. Administration of α-phenyl-tert-butyl-nitrone (PBN), a spin adduct forming agent systemically (100 mg/kg body wt. IP 10 min prior to concussion) alone or along with topical PBN (100 mM PBN in aCSF),6significantly (P< 0.001) attenuated the ESR signal, suggesting its possible role in the treatment of TBI.     

Oxidation of lipids. XI. Spin trapping and identification of peroxy and alkoxy radicals of methyl linoleate

Spin trapping of peroxy and alkoxy radicals generated from the hydroperoxide of methyl linoleate was studied using methyl-N-duryl nitrone (MDN) and phenyl-N-tert-butyl nitrone (PBN) as spin traps. The conjugated dienyl carbon radical was also generated from methyl linoleate and spin-trapped. The spin adducts of peroxy, alkoxy, and dienyl carbon radicals were observed by ESR and their hyperfine splitting constants were determined. The spin adducts of peroxy and alkoxy radicals could be distinguished clearly with MDN.     

Spin Trapping of Inorganic Radicals

The use of nitrose compounds and nitrones as spin traps for the detection of short-lived inorganic radicals is discussed. To a certain degree nitrones and nitroso compounds are complementary. While nitroso compounds are superior with respect to spin trapping metal-centred radicals, nitrones form more persistent spin adducts with most small inorganic radicals.

Erroneous results may be obtained when hydrolysis and redox reactions involving the spin adducts are ignored. Spin trapping of pseudohalide radicals (·N_j· ·CN, ·SCN) are discussed in more detail.     

The line asymmetry of electron spin resonance spectra as a tool to determine the cis:trans ratio for spin-trapping adducts of chiral pyrrolines N-oxides: the mechanism of formation of hydroxyl radical adducts of EMPO, DEPMPO, and DIPPMPO in the ischemic-reperfused rat liver

Nonstereospecific addition of free radicals to chiral nitrones yields cis/trans diastereoisomeric nitroxides often displaying different electron spin resonance (ESR) characteristics. Glutathione peroxidase-glutathione (GPx-GSH) reaction was applied to reduce the superoxide adducts (nitrone/*OOH) to the corresponding hydroxyl radical (HO*) adducts (nitrone/*OH) of two nitrones increasingly used in biological spin trapping, namely 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) and 5-ethoxycarbonyl-5-methyl-1-pyrroline N-oxide, and of 5-diisopropoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DIPPMPO), a sterically hindered DEPMPO analogue. The method offered improved conditions to record highly resolved ESR spectra and by accurate simulation of line asymmetry we obtained clear evidence for the existence of previously unrecognized isomer pairs of cis- and trans-[DEPMPO/*OH] and [DIPPMPO/*OH]. Additional nitrone/*OH generation methods were used, i.e. photolysis of hydrogen peroxide and the Fenton reaction. We developed a kinetic model involving first- and second-order decay and a secondary conversion of trans to cis isomer to fully account for the strongly configuration-dependent behavior of nitrone/*OH. In the reductive system and, to a lower extent, in the Fenton or photolytic systems cis-nitrone/*OH was the more stable diastereoisomer. In various biologically relevant milieu, we found that the cis:trans-nitrone/*OH ratio determined right after the spin adduct formation significantly differed upon the GPx-GSH vs (Fenton or photolytic) systems of formation. This new mechanistic ESR index consistently showed for all nitrones that nitrone/*OH signals detected in the postischemic effluents of ischemic isolated rat livers are the reduction products of primary nitrone/*OOH. Thus, ESR deconvolution of cis/trans diastereoisomers is of great interest in the study of HO* formation in biological systems.     

Metabolism of Ethanol to 1-Hydroxyethyl Radicals in Rat Liver Microsomes: Comparative Studies with Three Spin Trapping Agents

Metabolism of ethanol to 1-hydroxyethyl radicals by rat liver microsomes was studied with three nitrone spin trapping agents (POBN, PBN, and DMPO) under essentially comparable conditions. The data indicate that POBN was the superior spin trapping agent for 1-hydroxyethyl radicals, and that DMPO was least efficient. Addition of deferoxamine completely prevented detection of 1-hydroxyethyl radicals with PBN or DMPO, but caused only 50% decrease in EPR signals when POBN was the spin trap. However, superoxide dismutase only decreased 1-hydroxyethyl radical formation when POBN was the spin trap. Other experiments demonstrated that POBN was the most effective of these nitrones for reduction of Fe(III) in aqueous solutions. Furthermore, 1-hydroxyethyl radical adducts were formed when POBN was added to mixtures of ethanol, phosphate buffer, POBN and FeCl₃, but this effect did not occur with either PBN or DMPO. Thus, these data indicate that undesirable effects of POBN on iron chemistry may influence results of spin trapping experiments, and complicate interpretation of the resulting data.