Kinetics of superoxide-induced exchange among nitroxide antioxidants and their oxidized and reduced forms

Free Radical Biology & Medicine, Vol. 26, Nos. 9/10, pp. 1245–1252, 1999 Copyright © 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/99/$–see front matter

PII S0891-5849(98)00328-1

Original Contribution KINETICS OF SUPEROXIDE-INDUCED EXCHANGE AMONG NITROXIDE ANTIOXIDANTS AND THEIR OXIDIZED AND REDUCED FORMS RENLIANG ZHANG,* SARA GOLDSTEIN,†

and

AMRAM SAMUNI*

†

*Department of Molecular Biology, School of Medicine and Department of Physical Chemistry, The Hebrew University of Jerusalem, Jerusalem 91120, Israel (Received 14 September 1998; Revised 10 November 1998; Accepted 10 November 1998)

Abstract—Nitroxide stable radicals generally serve for probing molecular motion in membranes and whole cells, transmembrane potential, intracellular oxygen and pH, and are tested as contrast agents for magnetic resonance imaging. Recently nitroxides were found to protect against oxidative stress. Unlike most low molecular weight antioxidants (LMWA) which are depleted while attenuating oxidative damage, nitroxides can be recycled. In many cases the antioxidative activity of nitroxides is associated with switching between their oxidized and reduced forms. In the present work, superoxide radicals were generated either radiolytically or enzymatically using hypoxanthine/xanthine oxidase. Electron paramagnetic resonance (EPR) spectrometry was used to follow the exchange between the nitroxide radical and its reduced form; whereas, pulse radiolysis was employed to study the kinetics of hydroxylamine oxidation. The results indicate that: a) The rate constant of superoxide reaction with cyclic hydroxylamines is pH-independent and is lower by several orders of magnitude than the rate constant of superoxide reaction with nitroxides; b) The oxidation of hydroxylamine by superoxide is primarily responsible for the non-enzymatic recycling of nitroxides; c) The rate of nitroxides restoration decreases as the pH decreases because nitroxides remove superoxide more efficiently than is hydroxylamine oxidation; d) The hydroxylamine reaction with oxidized nitroxide (comproportionation) might participate in the exchange among the three oxidation states of nitroxide. However, simulation of the time-dependence and pH-dependence of the exchange suggests that such a comproportionation is too slow to affect the rate of non-enzymatic nitroxide restoration. We conclude that the protective activity of nitroxides in vitro can be distinguished from that of common LMWA due to hydroxylamine oxidation by superoxide, which allows nitroxide recycling and enables its catalytic activity. © 1999 Elsevier Science Inc. Keywords—Oxidative stress, Free radicals, Electron paramagnetic resonance, Pulse radiolysis

INTRODUCTION

Initially nitroxide stable free radicals have mainly been used to probe cellular metabolism, molecular motion in membranes and whole cells [1,2], transmembrane potential, and intracellular oxygen and pH [3]. Nitroxides were also tested as contrast agents for nuclear magnetic resonance imaging [4,5], and their reactions in chemical and biological systems were extensively investigated [6]. Nitroxides undergo oneelectron redox reactions to yield hydroxylamines and oxoammonium cations, as shown below (Scheme 1) for 2,2,6,6,-tetramethyl-piperidinoxyl (TPO).

Aside from a few unstable nitroxide derivatives, which reportedly disproportionate to yield hydroxylamine and oxo-ammonium cation [7], the radical form is generally more stable. In vivo, however, the redox reactions of nitroxides are primarily enzymatic processes [8] and the predominant species is not the nitroxide radical but rather its reduced form. With the exception of ascorbate, most endogenous reductants have been found to be incapable of nonenzymatic reduction of nitroxides [7]. More recently, nitroxides have been shown to atten-

Address correspondence to: Amram Samuni, Molecular Biology, Medical School, Hebrew University, Jerusalem, 91120, Israel; Tel: 1972(2)675-8244; Fax: 1972(2)678-4010; E-Mail: [email protected]. 1245

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uate oxidative damage in experimental models such as cells [9 –12], hyperoxia-induced brain damage [13], experimental pancreatitis [14], lipid peroxidation in liver microsomes [15], thymocyte apoptosis [16], xenobiotics toxicity [17], intestinal and gastric lesions [18], and closed head injury [19]. Evidently, a better understanding of their chemistry is important for the elucidation of the mechanism(s) underlying the observed antioxidative activity. Several mechanisms by which nitroxides attenuate oxidative damage have been proposed including oxidation of reduced transition metal ions, detoxification of intracellular radicals such as alkyl, alkoxy, and alkylperoxyl radicals, and dismutation of O2•2. Previous studies have shown that both five- and six-membered ring nitroxides react with O2•2 in a catalytic fashion mimicking that of superoxide dismutase (SOD) [20,21]. Oxazolidine derivatives oxidize O2•2 to O2, and the respective EPR-silent hydroxylamine reduces O2•2 to H2O2. During this process, the concentration of the nitroxide is continuously restored while effectively dismutating O2•2 radicals [22]. On the other hand, piperidinyl and pyrrolidinyl derivatives catalyze superoxide dismutation by switching between the nitroxide radical and oxo-ammonium cation [23]. In several experimental models of oxidative stress both the nitroxide and the hydroxylamine [15,24] function as antioxidants. Since nitroxide reduction and reoxidation, whether enzymatic or nonenzymatic, can occur simultaneously [25], those two antioxidants are practically recycled. The exchange between the oxidation states of nitroxides challenges the elucidation of the kinetics of individual redox reactions. In the present work, EPR spectrometry and pulse radiolysis technique were used to study kinetics of the nonenzymatic processes that mediate exchange between nitroxide and its respective hydroxylamine and oxoammonium cation. The results indicate that comproportionation does not facilitate the nonenzymatic restoration of nitroxides. Instead, a direct oxidation of hydroxylamine by superoxide restores the nitroxide, and allows it to act catalytically, unlike common LMWA, which act in a stoichiometric fashion. MATERIALS AND METHODS

N-[2-hydroxyethyl]piperazine-N9-[2-ethanesulfonic acid] (HEPES), hypoxanthine (HX), reduced nicotinamide adenine dinucleotide (NADH), and superoxide dismutase (SOD; EC 1.15.1.1) were obtained from Sigma (St. Louis, MO, USA). The reduced form of TPL, 4-OH-2,2,6,6,tetramethyl-N-hydroxypiperidine (TPL-H), and of other nitroxides was prepared by catalytic reduction using H2 bubbled over Pt powder or by bubbling HCl gas through ethanolic solution of the respective nitroxide followed by drying [26]. Because hydroxylamine is readily oxidized, particularly in the presence of transition metals, it was kept below 0°C and fresh solutions were prepared immediately before each experiment. Solutions were prepared with distilled water that was passed through a Milli-Q water purification system. All experiments were performed at room temperature.

Kinetics study of fast reactions Pulse radiolysis experiments were carried out with the Varian 7715 linear accelerator with 5 MeV electron pulses of 0.4 –1.5 ms and 200 mA. The dose per pulse ranged from 5 to 25 Gy as determined by the hexacyanoferrate(II) dosimeter (5 mM K4Fe(CN)6 in N2O-saturated water) [27]. A 200 W Xe-Hg lamp produced the analyzing light. Irradiation was carried out at room temperature using a 4-cm spectrosil cuvette and three passes of the analyzing light.

Formation of superoxide When air-saturated solutions containing formate ions at pH . 3 are irradiated, all the primary radicals formed by the radiation of water are converted into superoxide (Eq. 1): • • H 2O 3 e 2 aq (2.6), OH (2.7), H (0.6), H 2 (0.45),

H2O2 (0.7), H3O1 (2.6)

(The numbers in parenthesis are G-values which represent the number of molecules formed per 100 eV energy absorbed by pure water [Equations 2– 6] [28]:) •2 e2 aq 1 O 2 3 O 2

k 2 5 2 3 10 10 M21 s21 [28]

(2)

H• 1 O2 3 HO•2

k 3 5 2 3 10 10 M21 s21 [28]

(3)

Chemicals Catalase (EC 1.11.1.6) and xanthine oxidase (XO) (EC 1.1.3.22) were purchased from Boehringer Biochemicals. 3-Carbamoyl-2,2,5,5,-tetramethylpyrrolidinoxyl, 2,2,6,6,-tetramethyl-piperidinoxyl (TPO), and 4-OH2,2,6,6,-tetramethylpiperidinoxyl (TPL) were purchased from Aldrich. Diethylenetriaminopentaacetic acid (DTPA),

(1)

•

•2 OH 1 HCO2 2 3 H 2O 1 CO 2

k 4 5 3.5 3 10 9 M21 s21 [28]

(4)

CO2•2 1 O2 3 CO2 1 O2•2 k 5 5 3.5 3 10 9 M21 s21 [28]

(5)

Kinetics of nitroxide reactions

HO•2 ^ H1 1 O2•2

pKa 5 4.8 @29#

1247

(6)

Under such conditions, the initial concentration of O2•2, formed by the pulse, ranged from 3 to 15 mM. In the absence of other reactants and with 10 –20 mM DTPA, the superoxide decayed via a pH-dependent second-order reaction (Eq. 7) [29]: HO /O2 1 HO /O2 • 2

•2

• 2

•2

H1 O ¡ H 2O 2 1 O 2

(7)

Electron paramagnetic resonance (EPR) measurements To determine the nitroxide concentration, samples were drawn into a gas-permeable, teflon capillary with inner diameter of 0.81 mm. The capillary was inserted into a quartz tube open at both sides which was placed within the EPR cavity. Alternatively, the sample was injected through a teflon tube inserted in a quartz tube, held in the cavity, without disturbing the alignment of the tube within the spectrometer. EPR spectra were recorded on a JES-RE3X ESR spectrometer of JEOL working at X band with center field set at 3362 G, 100 kHz modulation frequency, 0.25 G modulation amplitude, and nonsaturating incident microwave power.

RESULTS

Fig. 1. TPL-H oxidation induced by superoxide. The formation of TPL in 50 mM HEPES (pH 7.4) containing 0.1 mM DTPA was followed by incubating 0.1 mM TPL-H with 0.5 mM HX and 16 mU/ml XO at 22°C in the presence of various additives, taking samples at various time periods and monitoring the EPR signal of TPL before and after adding 1 mM K3Fe(CN)6. Sham, without HX/XO (E); control, HX/XO alone (F); 200 U/ml catalase (h); 200 U/ml SOD (■). Error bars are shown when greater than the symbol.

TPL-H, thus indicating that O2•2/HO•2 radicals are predominantly responsible for TPL-H oxidation (Eq. 8):

TPL-H 1 O2

H1 /HO O ¡ TPL 1 H2O2 • 2

(8)

However, additional reactions which might affect the nitroxide/hydroxylamine exchange should be considered. While the reaction of TPL-H with H2O2 hardly contributes towards hydroxylamine oxidation, the three oxidation states of nitroxide, TPL-H, TPL, and TPL1, effectively compete for O2•2/HO•2 (Eqs. 9 and 10):

Hydroxylamine oxidation In the present study, HX/XO was used to generate O2 and H2O2. To avoid metal-catalyzed oxidation of the hydroxylamine, 100 mM DTPA was always included [30]. After adding 20 –100 mM of hydroxylamine, the EPR signal of the nitroxide was monitored at various points of time before and after adding ferricyanide to oxidize TPL-H. Thus, the levels of both TPL and TPL-H could be assayed. The progressive accumulation of TPL in a typical experiment at pH 7.4 is displayed in Fig. 1. When the experiment was repeated with 200 U/ml catalase, only a marginal effect on the rate of TPL-H oxidation was observed (Fig. 1), which indicates that H2O2 hardly affects the rate of TPL-H oxidation. Further support for the lack of H2O2 contribution was provided by the observation that 1 mM H2O2 induces a marginal oxidation of 100 mM TPL-H under similar experimental conditions (data not shown). On the other hand, the addition of 200 U/ml SOD inhibited the oxidation of

•2

TPL 1 O2

•2

H1 /HO | -0 TPL1 1 H2O2 • 2

•2

k 9 is pH-dependent [31]

(9)

TPL1 1 O2•2 3 TPL 1 O2 k 10 5 1.5 3 10 10 M21 s21 [6]

(10)

The comproportionation reaction 11 (Eq. 11), which was previously suggested [32], might also affect the conversion between TPL-H and TPL: 2H1 TPL 1 TPL-H O ¡ TPL 1 TPL 1

(11)

Effect of pH The relative concentration of HO•2, that is a stronger oxidant than O2•2, increases as the pH decreases (pKa of

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Fig. 2. Effect of pH on superoxide-induced oxidation of TPL-H. The time dependence of the EPR signal of TPL formed upon incubation of 40 mM TPL-H in 50 mM phosphate of different pH containing 0.1 mM DTPA, 1 mM HX and 16 mU/ml XO at 22°C. The total concentration of TPL and TPL-H was determined by monitoring the EPR signal after adding 1 mM K3Fe(CN)6. Inset: The pH-dependence of the absolute initial rates of TPL-H oxidation measured during the first minute of the EPR experiment. The initial rates were corrected with respect to pH 7.8 for the different flux of superoxide generated by XO at pH , 7.8.

O2•2 equals 4.8) [29]. Therefore, the rate of TPL-H oxidation was anticipated to increase as the pH decreases. To examine it, the experiment has been repeated at several pH values and the results are displayed in Fig. 2. The pH-dependence of the rate of TPL-H oxidation was determined by measuring the initial rate during the first minute of the EPR experiment. The initial rates were corrected with respect to pH 7.8 for any different flux of superoxide generated by XO at pH Þ 7.8. The results, displayed in Fig. 2 inset, show that the rate of hydroxylamine oxidation decreases as the pH decreases.

NADH effect on nitroxide/hydroxylamine distribution When hydroxylamines were incubated with HX/XO, 100 mM DTPA and 0.4 mM NADH, the accumulation of nitroxides stopped shortly after the start of the reaction. Figure 3 shows that a steady state distribution between the nitroxide and the hydroxylamine was rapidly established and maintained thereafter, as reflected by the time-invariance of the EPR signal. Evidently, in the presence of NADH, additional reactions might affect the nitroxide/hydroxylamine distribution (Eqs. 12–14): NADH 1 HO•2 3 NAD• 1 H2O2 k 12 5 1.8 3 10 5 M21 s21

(12)

Fig. 3. Superoxide-induced hydroxylamine/nitroxide exchange. Conversion between nitroxide and its reduced form upon incubation of 68 mM TPO (F), 68 mM TPO-H (E), 43 mM TPL (■) or 20 mM TPL-H (h) in 50 mM HEPES pH 7.4 containing 100 mM DTPA, 400 mM NADH, 1 mM HX and 16 mU/ml XO at 22°C. The reaction mixture was sampled at various points of time and the nitroxide EPR signal was recorded before and after adding 1 mM K3Fe(CN)6.

NADH 1 O2

•2

H1 O ¡ NAD• 1 H2O2 k 12a , 27 M21 s21

(12a)

NAD• 1 O2 3 NAD1 1 O2•2 k 13 5 1.9 3 10 9 M21 s21 [33] 1

TPL 1 NADH 3 TPL-H 1 NAD

1

(13) (14)

The time-invariant distribution was the same when the nitroxide, rather than the hydroxylamine, served as the starting reagent (Fig. 3). Independently, the oxidation of NADH to NAD1 was followed spectrophotometrically at 340 nm. The loss of OD340 did not stop when a final steady-state distribution was achieved, but continued whether nitroxide or hydroxylamine served as the starting reagent. On the other hand, SOD at 100 U/ml inhibited the nitroxide/hydroxylamine conversion and the oxidation of NADH (data not shown). The final steady-state distribution was independent of [NADH], [hydroxylamine], and [nitroxide] (data not shown), but depended on pH. Figure 4 shows that both {[TPL]/[TPL-H]}steady state and {[TPO]/[TPO-H]}steady state decrease as the pH decreases. Fast reaction kinetics The reaction of superoxide with several hydroxylamines was studied using pulse-radiolysis. All solutions were air-saturated and contained 2 mM phosphate buffer,

Kinetics of nitroxide reactions

Fig. 4. The pH dependence of the distribution between nitroxide and hydroxylamine. The ratio of nitroxide to hydroxylamine concentrations was determined following 30 min incubation of 68 mM TPL-H (squares) or TPO-H (circles) with 1 mM HX, 16 mU/ml XO, 0.1 mM DTP, and 1 mM NADH, in 50 mM phosphate at various pH values, by monitoring the nitroxide EPR signal before and after adding 1 mM K3Fe(CN)6.

50 mM DTPA, 50 mM formate and various concentration of each hydroxylamine. Typical traces of the decay of the transient absorbance change at 290 nm, where O2•2 and the nitroxide absorb differently, are given in Fig. 5. The decay of superoxide combined with the conversion of hydroxylamine to nitroxide obeyed first order kinetics. The respective k obs for the various hydroxylamines were linearly dependent on [hydroxylamine] and independent of pH in the range 6 –9 (Fig. 6). The second order rate constant, k 8 , was evaluated for three nitroxides from the

Fig. 5. The oxidation of TPL-H by superoxide determined using pulse radiolysis. Kinetic traces of DOD measured at l 5 290 nm when air-saturated solutions containing 100 mM formate and 2 mM phosphate at pH 8 are irradiated at room temperature in the presence of 1.3, 2.6, and 5.2 mM TPL-H.

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Fig. 6. The pH-dependence of the bimolecular rate constant of O2•2/ HO•2 reaction with nitroxides. The reaction was initiated by pulse radiolyzing (1 MeV) at RT aerated solution of 2 mM phosphate of different pH’s containing 50 mM DTPA, 50 mM formate containing either TPO-H at pH 8.1 (‚), pH 7.54 (h), and pH 7.1 (E); or TPL-H at pH 8.9 (Œ), pH 8.1 (■), and pH 7.1 (F). The change in OD was followed at 290 nm. The data were interfaced to a computer which provides a best fit of first order kinetics and evaluates k obs (see Materials and Methods).

slopes of the lines obtained when k obs was plotted vs. [hydroxylamine] (Fig. 6), and found to be (1.1 6 0.1) 3 103 M21 s21 for TPO-H, (2.1 6 0.2) 3 103 M21 s21 for TPL-H, and (4.9 6 0.5) 3 103 M21 s21 for the 5-membered hydroxylamine of 3-carbamoyl-2,2,5,5-tetramethylpyrrolidinoxyl.

DISCUSSION

The oxidation of several cyclic hydroxylamines by O2•2 has been previously studied using competition techniques. The respective second order rate constants were determined as 4 3 102 M21 s21 for TPL-H at pH 7.8 [23], 1.7 3 103 M21 s21 for 2-ethyl,2,5,5-trimethyl-Nhydroxyoxazolidin at pH 7.8 [30], 3.2 3 103 M21 s21 for 3-carboxy-proxyl [34], and 1.2 3 104 M21 s21 for 4-oxo-2,2,6,6-tetramethyl-N-hydroxypiperidine at pH 7.4 [35]. However, in some of the studies, the simultaneous and generally much faster reaction of superoxide with the oxidation product, namely nitroxide, was not considered. Evidently, several reactions concomitantly affect the nitroxide/hydroxylamine exchange. The pHdependence of the accumulation of nitroxide upon exposure of hydroxylamine to O2•2/HO•2 (Fig. 2) reflects the fact that both TPL-H and TPL compete for O2•2/HO•2. Since k 8 was found to be pH-independent as evidenced by the pulse radiolysis experiments (Figs. 5, 6), whereas k 9 increases as the pH decreases [31], reaction 9 predominates and less O2•2/HO•2 is left to react through reaction 8.

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Apparently, only reaction 8 contributes to TPL-H oxidation, and under the conditions where k 9 3 [TPL] . k 8 3 [TPL-H], the oxidation of TPL-H is anticipated to cease as [TPL] increases; whereas reaction 8 becomes negligible compared with reaction 9. As seen in Fig. 1, the rate of TPL-H oxidation decreases progressively as [TPL] increases, which effectively catalyzes the dismutation of O2•2. Therefore, less superoxide is available for the reaction with the hydroxylamine. The competition between the concurrent reactions is reflected also in the steady state distribution of the nitroxide and its reduced form. Under our experimental conditions the reaction of NADH with superoxide can be ignored (pKa of HO•2 equals 4.8) [29] and in the presence of sufficiently high [NADH], the intermediate TPL1 disappears via reaction 14, rather than 10, to yield TPL-H. As seen in Fig. 3, TPL accumulation stopped since NADH not only generates TPL-H (reaction 14) but also pre-empts to a large extent the formation of TPL through reaction 8. Hence, the distribution [TPL]/[TPLH] reflects a balance achieved between two opposing processes, where both TPL-H and TPL compete for O2•2/HO•2. When steady state distribution is reached, the rates of reactions 8 and 9 balance each other, which implies that {[TPL]/[TPL-H]}steady state reflects k 8 /k 9 . The rate constant for reaction 9 has been previously determined for TPO and TPL, and k 9 was found to increase as the pH decreases [31]. Comparison of the pH-dependence of k 9 with that of k 9 /k 8 , implied from the dependence seen in Fig. 4, agrees with the finding that reaction 8 is independent of pH (Fig. 6), and substantiates the conclusion that hydroxylamine reactions of O2•2 and HO•2 have similar rate constants. The simulation of the time-dependence and pH-dependence of the nitroxide/hydroxylamine exchange Apparently the competition between the hydroxylamine and O2•2 for the oxo-ammonium cation should also affect the rate of hydroxylamine oxidation. Although k 10 approaches the diffusion-controlled limit, hydroxylamine oxidation in the presence of sufficiently large [TPL-H] could be attributed to reaction 11 rather than 8. Simulations of reactions 7–11 under the experimental conditions of Fig. 1 (pH 7.4, 0.1 mM TPL-H, superoxide flux of 16 mM/min, 30 min reaction duration) were performed. An excellent fit of the simulated timedependence of TPL formation to the experimentally observed values (given in Fig. 1) was obtained using k 7 5 2.5 3 10 5 M21 s21, k 8 5 2.1 3 10 3 M21 s21, k 9 5 1.1 3 10 5 M21 s21 and k 10 5 1.5 3 10 10 M21 s21. The simulated time-dependent accumulation of TPL was found to be independent of reaction 11 as long as k 11 ,

1 3 104 M21 s21. The only factor according to the simulation model that substantially affected the yield of TPL was k9 which is pH-dependent. The value of k9 5 1.1 3 105 M21 s21, determined by the simulation, is similar to that found earlier using competition kinetics [31]. Simulation of the time-dependence of TPL reduction which takes into account the presence of 43 mM TPL and 0.4 mM NADH yielded an excellent fit to the experimentally determined results given in Fig. 3 using k 9 5 2.0 3 10 5 M21 s21, k 12 5 450 M21 s21 [29], k 13 5 1.9 3 10 10 M21 s21 [33] and k 14 5 1.0 3 10 6 M21

s21. The value of k 14 has not yet been determined and, therefore, could not be verified, whereas, k 9 is identical to that determined earlier [28]. In summary, unlike most LMWA that are depleted while attenuating oxidative damage, nitroxides can be recycled. The nonenzymatic processes affecting the exchange between the nitroxides and their respective cyclic hydroxylamines and oxoammonium cations can be summarized as schematically displayed for TPO (Scheme 2). Evidently, the recycling distinguishes nitroxides from common LMWA and enables them to act catalytically. However, the extrapolation of this mechanism to intracellular recycling of nitroxides is not straightforward but rather depends on the level of other reagents in specific cellular compartments. Where SOD, NO or [4Fe– 4S] clusters efficiently compete for superoxide radicals even the direct oxidation of hydroxylamines by O2•2/HO•2 is not anticipated to significantly contribute to nitroxide recycling. Instead, hydroxylamine oxidation is most likely enzymatic [8] or mediated by strong oxidants such

Kinetics of nitroxide reactions

as peroxynitrite, formed by the reaction of superoxide with NO. In conclusion, the present results indicate that under superoxide flux in vitro: a) the rate constant of the reaction of superoxide with cyclic hydroxylamines is pH-independent and is lower by several orders of magnitude than the rate constant of the reaction of superoxide with the nitroxides; b) the oxidation of hydroxylamine by superoxide is primarily responsible for the non-enzymatic recycling of nitroxides, having bimolecular rate constants of (1.1 6 0.1) 3 103 M21 s21, (2.1 6 0.2) 3 103 M21 s21, and (4.9 6 0.5) 3 103 M21 s21 for TPO-H, TPL-H and the hydroxylamine of 3-carbamoyl2,2,5,5-tetramethylpyrrolidinoxyl; c) the rate of nitroxides regeneration decreases as pH decreases because nitroxides remove superoxide more efficiently, when compared to the slow oxidation of hydroxylamines; d) the comproportionation reaction may also facilitate the exchange among nitroxide and its oxidized and reduced forms, but simulation of the time-dependence and pH-dependence of such an exchange suggests that comproportionation is too slow to affect the rate of nitroxide formation.

[11]

[12] [13]

[14]

[15]

[16]

[17]

[18] Acknowledgement — This research was supported (A.S.) by grant 95-00287 from the USA-Israel Binational Science Foundation.

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ABBREVIATIONS

EPR— electron paramagnetic resonance HX— hypoxanthine LMWA—low molecular weight antioxidant SOD—superoxide dismutase TPL— 4-OH-2,2,6,6-tetramethyl-piperidinoxyl TPL-H— 4-OH-2,2,6,6-tetramethyl-N-hydroxypiperidine TPO—2,2,6,6-tetramethyl-piperidinoxyl TPO-H—2,2,6,6-tetramethyl-N-hydroxypiperidine XO—xanthine oxidase