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The Reaction between Nitric Oxide and α-Tocopherol: A Reappraisal
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
224, 696–702 (1996)
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The Reaction between Nitric Oxide and a-Tocopherol: A Reappraisal Neil Hogg, Ravinder Jit Singh, Steven P. A. Goss, and B. Kalyanaraman Biophysics Research Institute, Medical College of Wisconsin, Milwaukee Wisconsin 53226 Received June 11, 1996 Recently Gorbunov et al. reported that nitric oxide (rNO) can directly oxidize a-tocopherol to atocopheroxyl radical (Gorbunov et al., Biochem. Biophys. Res. Commun., 219, 835–841, 1996). We have reinvestigated this reaction and report that a direct reaction between rNO and a-tocopherol does not occur. However, the reaction between rNO and oxygen generates an oxidant which oxidizes a-tocopherol to atocopheryl quinone. Exposure of a-tocopherol to a low flux of rNO generated from spermine NONOate (100 mM) results in no consumption of a-tocopherol under either aerobic or anaerobic conditions. A higher flux of rNO, generated from 1 mM spermine NONOate, oxidizes a-tocopherol only under aerobic conditions. Artifactual oxidation of a-tocopherol can be observed when using commercial rNO that is contaminated with higher oxides of nitrogen, such as dinitrogen trioxide and dinitrogen tetraoxide. q 1996 Academic Press, Inc.
It has recently been reported that rNO reacts directly with a-tocopherol to generate the atocopheroxyl radical (1). This observation is in agreement with reports by Janzen et al. (2) and de Groot et al. (3). However, a number of other publications (4-6) have shown that atocopherol is stable in the presence of rNO. In order to address this controversy we have reinvestigated the reaction between rNO and a-tocopherol in dilauryl phosphatidylcholine (DLPC) liposomes. If rNO were to oxidize a-tocopherol directly, as has been suggested (1-3), a-tocopherol would be intrinsically unstable in the vicinity of rNO synthesis. For example, a-tocopherol in endothelial cell membranes is continuously exposed to a low steady state concentration of rNO, derived form the basal activity of constitutive endothelial rNO synthase. On the other hand, if a-tocopherol oxidation is due to oxidized metabolites of rNO, then the relevance of this reaction is questionable. The reaction of rNO with oxygen, to form dinitrogen trioxide, is extremely slow, and perhaps irrelevant, under biological conditions (7). Here we present evidence that rNO does not react with a-tocopherol by either a one- or a two-electron mechanism. However, oxidation products of rNO (i.e., N2O3 or a related oxidant) will oxidize a-tocopherol to both a-tocopheroxyl radical and a-tocopheryl quinone. The discrepancy between our results and those of Gorbunov et al. (1) and others (2,3) emphasizes the pitfalls of using rNO gas to mimic the effects of rNO synthase. Improper handling of rNO gas solutions can lead to erroneous conclusions about the reactivity of rNO in biological systems. MATERIALS AND METHODS Materials. a-Tocopherol was purchased from Sigma Chemical Co. (St. Louis, MO). rNO was obtained from Mathesen Pure Gasses (Madison, WI). Nitrous oxide and argon were obtained from AIRCO (Murrey Hill, NJ).
Abbreviations: DLPC, dilauryl phosphatidylcholine; N2O3 , dinitrogen trioxide; N2O4 , dinitrogen tetraoxide; HPLC, high performance liquid chromatography; HNO/NO0, nitroxyl; rNO, nitric oxide; N2O, nitrous oxide, SIN-1, 3morpholinosydnonimine-N-ethylcarbamide. 696 0006-291X/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Spermine NONOate was obtained from Cayman Chemical Co. (Ann Arbor, MI). DLPC was obtained from Avanti Lipids (Birmingham, AL). a-Tocopheryl quinone was synthesized as previously described (8). Preparation of rNO solution. rNO solution was prepared in phosphate buffer (200 mM, pH 7.4) in 8 ml glass vials that were sealed with a ‘suba-seal’ (Aldrich Chemical Co., Milwaukee, WI). The vial was degassed under vacuum and refilled with argon three times to remove oxygen and degassed a final time. It was then filled with rNO gas that had been passed through a sodium hydroxide scrubber to remove contaminating oxides of nitrogen. rNO concentration in stock solutions was determined to be 1.6-1.9 mM as previously described (9). Liposome preparation. Aliquots from stock solutions of DLPC (200 mM in chloroform) and a-tocopherol (100 mM in methanol) were mixed and dried under nitrogen. Phosphate buffer (100 mM) was added to give a final concentration of a-tocopherol (2.5 mM) and DLPC (250 mM). Liposomes were prepared by vortexing this mixture for 5 minutes. a-Tocopherol and a-tocopheryl quinone assays. The concentrations of a-tocopherol and a-tocopheryl quinone were determined by reverse phase high performance liquid chromatography (HPLC) as previously described (8). Anaerobic samples containing rNO were thoroughly vacuum degassed and flushed with argon to remove any residual rNO before exposure to air. Samples were extracted into heptane, dried under nitrogen and redissolved in methanol before injection onto a Partisil 10 ODS-3 reverse phase HPLC column (Whatman, NJ). The mobile phase consisted of methanol:water (95:5) for 10 minutes, a linear gradient to 100 % methanol over the next 5 minutes and an additional 5 minutes of 100 % methanol. a-Tocopherol was monitored by fluorescence (lex Å 275 nm and lem Å 320 nm) and a-tocopheryl quinone was monitored by UV (l Å 266 nm). Compounds were quantified using a standard curve generated with known concentrations of a-tocopherol and a-tocopheryl quinone. Measurement of a-tocopheroxyl radical. Formation of a-tocopheroxyl radical was monitored using electron spin resonance (ESR) spectroscopy. ESR was performed at room temperature using a Varian E-109 spectrometer operating at 9.5 GHz (X-band). Samples were prepared in a quartz flat cell, for ESR analysis, either under strict nitrogen atmosphere inside a glove box or under atmospheric oxygen. N2O measurements. N2O was measured using gas chromatography employing a thermal conductivity detector and quantified as previously described (10).
RESULTS
The Effects of rNO and Oxygen on the Oxidation of a-Tocopherol to a-Tocopheryl Quinone HPLC analysis of DLPC liposomes (5 mM) containing a-tocopherol (50 mM) after incubation under anaerobic conditions for one hour resulted in minimal destruction of a-tocopherol (Figure 1A, left panel). A small amount of a-tocopheryl quinone (0.6 mM) was observed (Figure 1A, right panel); however this was present in the original a-tocopherol solution and corresponds to only 1.2 % of the total a-tocopherol. Addition of a rNO solution (1.4 mM final concentration) to an identical preparation of liposomes, under anaerobic conditions, caused neither the depletion of a-tocopherol nor the formation of a-tocopheryl quinone after incubation for 1 hour (Figure 1B). A third sample of liposomes was vacuum degassed and the atmosphere replaced by rNO gas directly from the cylinder (i.e. without passing through a sodium hydroxide scrubber). This treatment caused no significant depletion of a-tocopherol, however the amount of a-tocopheryl quinone was slightly increased to 0.85 mM (1.7% of the original atocopherol concentration, Figure 1C). This slight increase in quinone is likely to be due to contaminating higher oxides of nitrogen in the rNO gas. A fourth sample of liposomes was exposed to rNO gas in the presence of oxygen. Under these conditions, total oxidation of atocopherol occurred and 69% of the original a-tocopherol was recovered as a-tocopheryl quinone (Figure 1D). A second unidentified peak was also observed with a retention time of 14.5 minutes. We have shown previously that slow-releasing rNO donors are unable to deplete the atocopherol content of low-density lipoprotein in the presence of atmospheric oxygen (4,5). Figure 2 shows that this is also the case in DLPC liposomes. Liposomes consisting of DLPC (5 mM) and a-tocopherol (50 mM) were incubated with spermine NONOate (100 mM), a compound that releases rNO slowly and spontaneously (11). Aliquots were removed at the specified time intervals and assayed for a-tocopherol and a-tocopheryl quinone. As shown in figure 2, no a-tocopherol depletion was observed during 3 hours of incubation. A higher 697
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FIG. 1. Oxidation of a-tocopherol to a-tocopheryl quinone by bolus addition of rNO. DLPC liposomes (50 mM) containing a-tocopherol (50 mM) in phosphate buffer (100 mM) were incubated at 377C for 1 hour (A) alone under anaerobic conditions, (B) in the presence of rNO (1.4 mM) under anaerobic conditions, (C) in the presence of unscrubbed rNO gas under anaerobic conditions, and (D) in the presence of rNO gas and air. After one hour all samples were thoroughly degassed before exposure to oxygen and analyzed by HPLC. The left panel shows the fluorescence HPLC chromatogram showing the presence of a-tocopherol (retention time 14 min.). The right panel shows the UV chromatogram showing the presence of a-tocopheryl quinone (retention time 11 min.).
concentration of spermine NONOate (1 mM), under aerobic conditions, caused a significant and time-dependent depletion of a-tocopherol over 3 hours. This was accompanied by an increase in a-tocopheryl quinone (Figure 2). Under anaerobic conditions, however, spermine NONOate (1 mM) caused no oxidation of a-tocopherol. Formation of a-Tocopheroxyl Radical during the Reaction between a-Tocopherol and rNO It has been previously demonstrated that the reaction between rNO and a-tocopherol generates an ESR signal, characteristic of the a-tocopheroxyl radical (1,2). Using ESR, we monitored the formation of a-tocopheroxyl radical under various conditions as shown in Figure 3. Incubation of DLPC liposomes (125 mM) containing a-tocopherol (1.25 mM) with rNO (0.9 mM) resulted in no ESR signal (Figure 3A). UV irradiation of liposomes containing a-tocopherol gave an ESR spectrum consistent with the formation of a-tocopheroxyl radical (Figure 3B). The magnitude of this radical was approximately the same in the presence and absence of rNO (Figure 3C) indicating that rNO does not significantly affect the steady-state concentration of the a-tocopheroxyl radical. Under aerobic conditions, low levels of a-tocopheroxyl radical 698
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FIG. 2. Oxidation of a-tocopherol to a-tocopheryl quinone by slow release of rNO. DLPC liposomes (50 mM) containing a-tocopherol (50 mM) in phosphate buffer (100 mM) were incubated at 377C for 3 hours (A) alone (l, s), (B) in the presence of spermine NONOate (100 mM) under aerobic conditions (j, h), (C) in the presence of spermine NONOate (1 mM) under aerobic conditions (l, L), and (D) in the presence of spermine NONOate (1 mM) under anaerobic conditions (m, n). Samples were removed every hour and assayed for a-tocopherol (filled symbols) and a-tocopheryl quinone (open symbols). Data points represent means{S.D. (nÅ3).
were observed when a-tocopherol was treated either with rNO solution (Figure 3D) or bubbled with rNO gas (Figure 3E). N2O Formation during the Reaction between a-Tocopherol and rNO The direct reduction of rNO by a-tocopherol would result in the formation of nitroxyl (HNO) and a-tocopheroxyl radical (Equation 1). HNO, under anaerobic a-TOH / rNO r a-TOr / HNO
(1)
2NHO r N2O / H2O
(2)
conditions, undergoes rapid dismutation to form N2O (Equation 2). Incubation of DLPC liposomes (50 mM) containing a-tocopherol (500 mM) with rNO (1.7 mM) resulted in no significant formation of N 2O over 6 hours (Figure 4). DISCUSSION
Literature Evidence for and against a Direct Reaction between rNO and a-Tocopherol Three independent studies (1-3) have reported that rNO can directly react with a-tocopherol to generate a-tocopheroxyl radical and a-tocopheryl quinone. The common link between all of these studies is that rNO gas was used as the source of rNO. In contrast to these reports are a number of studies (4-6) that show rNO does not react with a-tocopherol. In these cases rNO was slowly released from rNO donor compounds into either LDL or a liposomal preparation of a-tocopherol. Only in the presence of superoxide was rNO able to oxidize atocopherol to a-tocopheryl quinone (4). In these conditions, peroxynitrite is formed and causes 699
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FIG. 3. Oxidation of a-tocopherol to a-tocopheroxyl radical by rNO. DLPC (125 mM) containing a-tocopherol (1.5 mM) were incubated (A) with rNO solution (0.9 mM) under anaerobic conditions, (B) as A but upon irradiation with UV light, (C) under anaerobic conditions and irradiated with UV light, (D) as A under aerobic conditions, and (E) after bubbling with rNO gas (1 ml). Spectrometer conditions; microwave power, 50 mW; modulation amplitude, 2 G; time constant, 0.128 s; scan time, 1 min. Each spectrum is the average of 6 scans.
direct one- and two-electron oxidation of a-tocopherol (8). SIN-1, a compound that generates rNO and superoxide simultaneously, also oxidizes a-tocopherol to a-tocopheryl quinone. The addition of superoxide dismutase, which scavenges superoxide and consequently increases the level of available rNO, inhibited a-tocopherol oxidation (4). The Pitfalls of Improper Use of rNO Gas We show here that there is no fundamental difference between rNO donor compounds and rNO gas with respect to its reactivity with a-tocopherol, if appropriate precautions are taken in the preparation of rNO solutions. The artifactual oxidation of a-tocopherol in previous studies (1-3) most likely occurred as a result of oxygen contamination or a failure to remove contaminating nitrogen oxides from commercial rNO gas. The rate of the reaction between rNO and oxygen is proportional to the second power of the rNO concentration (7,12). Consequently this reaction will be greatly favored at the high, non-biological, concentrations of rNO generated by bubbling rNO gas through solutions (7, 13). In organic solvents, the higher oxides of rNO are more persistent, due to the absence of hydrolysis reactions, and will oxidize target molecules to a greater extent than in aqueous systems (14). We show here that rNO donor compounds, when used judiciously do not exhibit the chemistry of N2O3 even under aerobic conditions. This is likely to be due to the fact that 700
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FIG. 4. Formation of N2O from the reaction between a-tocopherol and rNO. DLPC liposomes (50 mM) in phosphate buffer (100 mM, pH 7.4) were incubated in sealed vials at 377C for 6 hours in the presence of rNO (1.7 mM), in either the absence (l) or presence (l) of a-tocopherol (50 mM). Headspace (100 ml) was removed every two hours and N2O measured by gas chromatography. Data represent means{S.D. (nÅ3).
a low steady state concentration of N2O3 will favor hydrolysis to nitrite (effectively a first order reaction in aqueous solution) over any second order oxidation reactions. However, at a higher concentration of rNO donor compound oxidative chemistry was observed indicating that one should be cautious when extrapolating such experimental results to the biological effects of rNO. Biological Considerations rNO has been proposed to be an important endogenous anti-atherogenic agent (14). The mechanism for this has not been elucidated. However it has been proposed that, at least in part, the ability of rNO to inhibit the propagation of lipid peroxidation may be responsible for the anti-atherogenic effect (15,16). In both chemical and cellular systems, rNO has been shown to inhibit the oxidative modification of LDL to an atherogenic form (15-19). If rNO were to directly oxidize a-tocopherol it would be expected to have a pro-oxidant effect. To date, all the evidence indicates that when rNO is slowly delivered to a system containing lipid and a-tocopherol (either within LDL or liposomes), a-tocopherol is not oxidized. rNO has a potent inhibitory effect on the rate of a-tocopherol oxidation during lipid peroxidation (5,6). In conclusion, in contrast to previous reports (1-3), we show that rNO does not directly react with a-tocopherol whether it is added as an aqueous solution or it is slowly generated in situ using an rNO donor compound. ACKNOWLEDGMENTS This research was supported by HL47250 from the National Heart, Lung and Blood Institute and RR01008.
REFERENCES 1. Gorbunov, N. V., Osipov, A. N., Sweetland, M. A., Day, B. W., Elsayed, N. M., and Kagan, V. E. (1996) Biochem. Biophys. Res. Commun. 219, 835–841. 2. Janzen, E. G., Wilcox, A. L., and Manoharan, V. (1993) J. Org. Chem. 58, 3597–3599. 701
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de Groote, H., Hegi, U., and Sies, H. (1993) FEBS Lett. 315, 139–142. Hogg, N., Darley-Usmar, V. M., Wilson, M. T., and Moncada, S. (1993) FEBS Lett. 326, 199–203. Goss, S. P. A., Hogg, N., and Kalyanaraman, B. (1995) Chem. Res. Toxicol. 8, 800–806. Hayashi, K., Noguchi, N., and Niki, E. (1995) FEBS Lett. 370, 37–40. Wink, D. A., Darbyshire, J. F., Nims, R. W., Saavedra, J. E., and Ford, P. C. (1993) Chem. Res. Toxicol. 6, 23– 17. Hogg, N., Joseph, J., and Kalyanaraman, B. (1994) Arch. Biochem. Biophys. 314, 153–158. Hogg, N., Singh, R. J., Joseph, J., Neese, F., and Kalyanaraman, B. (1995) Free Rad. Res. 22, 47–56. Singh, R. J., Hogg, N., and Kalyanaraman, B. (1995) Arch. Biochem. Biophys. 324, 367–373. Maragos, C. M., Morley, D., Wink, D. A., Dunams, T. M., Saavedra, J. E., Hoffman, A., Bove, A. A., Isaac, L., Hrabie, J. A., and Keefer, L. K. (1991) J. Med. Chem. 34, 3242–3247. Kharitinov, V. G., Sundquist, A. R., and Sharma, V. J. (1994) J. Biol. Chem. 269, 5881–5883. Chang, G. J., Woo, P., Hinda, H., Ignarro, L. J., Young, L., Berliner, J. A., and Demer, L. L. (1994) Arterioscler. Thromb. 14, 1808–1814. Cooke, J. P., and Tsao, P. S. (1994) Arterioscler. Thromb. 14, 653–655. Hogg, N., Kalyanaraman, B., Joseph, J., Struck, A., and Parthasarathy, S. (1993) FEBS Lett. 324, 170–174. Rubbo, H., Parthasarathy, S., Barnes, S., Kirk, M., Kalyanaraman, B., and Freeman, B. (1995) Arch. Biochem. Biophys. 324, 15–25. Jessup, W., Mohr, D., Gieseg, S. P., Dean, R. T., and Stocker, R. (1992) Biochim. Biophys. Acta 1180, 73–82. Mali-Ranta, U., Yla-Herttuala, S., Metsa-Ketala, T., Jaakkola, O., Moilanen, E., Vuorinen, P., and Nikkari, T. (1994) FEBS Lett. 337, 179–183. Hogg, N., Struck, A., Goss, S. P. A., Santanam, N., Joseph, J., Parthasarathy, S., and Kalyanaraman, B. (1995) J. Lipid Res. 36, 1756–1762.
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The Reaction between Nitric Oxide and a-Tocopherol: A Reappraisal Neil Hogg, Ravinder Jit Singh, Steven P. A. Goss, and B. Kalyanaraman Biophysics Research Institute, Medical College of Wisconsin, Milwaukee Wisconsin 53226 Received June 11, 1996 Recently Gorbunov et al. reported that nitric oxide (rNO) can directly oxidize a-tocopherol to atocopheroxyl radical (Gorbunov et al., Biochem. Biophys. Res. Commun., 219, 835–841, 1996). We have reinvestigated this reaction and report that a direct reaction between rNO and a-tocopherol does not occur. However, the reaction between rNO and oxygen generates an oxidant which oxidizes a-tocopherol to atocopheryl quinone. Exposure of a-tocopherol to a low flux of rNO generated from spermine NONOate (100 mM) results in no consumption of a-tocopherol under either aerobic or anaerobic conditions. A higher flux of rNO, generated from 1 mM spermine NONOate, oxidizes a-tocopherol only under aerobic conditions. Artifactual oxidation of a-tocopherol can be observed when using commercial rNO that is contaminated with higher oxides of nitrogen, such as dinitrogen trioxide and dinitrogen tetraoxide. q 1996 Academic Press, Inc.
It has recently been reported that rNO reacts directly with a-tocopherol to generate the atocopheroxyl radical (1). This observation is in agreement with reports by Janzen et al. (2) and de Groot et al. (3). However, a number of other publications (4-6) have shown that atocopherol is stable in the presence of rNO. In order to address this controversy we have reinvestigated the reaction between rNO and a-tocopherol in dilauryl phosphatidylcholine (DLPC) liposomes. If rNO were to oxidize a-tocopherol directly, as has been suggested (1-3), a-tocopherol would be intrinsically unstable in the vicinity of rNO synthesis. For example, a-tocopherol in endothelial cell membranes is continuously exposed to a low steady state concentration of rNO, derived form the basal activity of constitutive endothelial rNO synthase. On the other hand, if a-tocopherol oxidation is due to oxidized metabolites of rNO, then the relevance of this reaction is questionable. The reaction of rNO with oxygen, to form dinitrogen trioxide, is extremely slow, and perhaps irrelevant, under biological conditions (7). Here we present evidence that rNO does not react with a-tocopherol by either a one- or a two-electron mechanism. However, oxidation products of rNO (i.e., N2O3 or a related oxidant) will oxidize a-tocopherol to both a-tocopheroxyl radical and a-tocopheryl quinone. The discrepancy between our results and those of Gorbunov et al. (1) and others (2,3) emphasizes the pitfalls of using rNO gas to mimic the effects of rNO synthase. Improper handling of rNO gas solutions can lead to erroneous conclusions about the reactivity of rNO in biological systems. MATERIALS AND METHODS Materials. a-Tocopherol was purchased from Sigma Chemical Co. (St. Louis, MO). rNO was obtained from Mathesen Pure Gasses (Madison, WI). Nitrous oxide and argon were obtained from AIRCO (Murrey Hill, NJ).
Abbreviations: DLPC, dilauryl phosphatidylcholine; N2O3 , dinitrogen trioxide; N2O4 , dinitrogen tetraoxide; HPLC, high performance liquid chromatography; HNO/NO0, nitroxyl; rNO, nitric oxide; N2O, nitrous oxide, SIN-1, 3morpholinosydnonimine-N-ethylcarbamide. 696 0006-291X/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Spermine NONOate was obtained from Cayman Chemical Co. (Ann Arbor, MI). DLPC was obtained from Avanti Lipids (Birmingham, AL). a-Tocopheryl quinone was synthesized as previously described (8). Preparation of rNO solution. rNO solution was prepared in phosphate buffer (200 mM, pH 7.4) in 8 ml glass vials that were sealed with a ‘suba-seal’ (Aldrich Chemical Co., Milwaukee, WI). The vial was degassed under vacuum and refilled with argon three times to remove oxygen and degassed a final time. It was then filled with rNO gas that had been passed through a sodium hydroxide scrubber to remove contaminating oxides of nitrogen. rNO concentration in stock solutions was determined to be 1.6-1.9 mM as previously described (9). Liposome preparation. Aliquots from stock solutions of DLPC (200 mM in chloroform) and a-tocopherol (100 mM in methanol) were mixed and dried under nitrogen. Phosphate buffer (100 mM) was added to give a final concentration of a-tocopherol (2.5 mM) and DLPC (250 mM). Liposomes were prepared by vortexing this mixture for 5 minutes. a-Tocopherol and a-tocopheryl quinone assays. The concentrations of a-tocopherol and a-tocopheryl quinone were determined by reverse phase high performance liquid chromatography (HPLC) as previously described (8). Anaerobic samples containing rNO were thoroughly vacuum degassed and flushed with argon to remove any residual rNO before exposure to air. Samples were extracted into heptane, dried under nitrogen and redissolved in methanol before injection onto a Partisil 10 ODS-3 reverse phase HPLC column (Whatman, NJ). The mobile phase consisted of methanol:water (95:5) for 10 minutes, a linear gradient to 100 % methanol over the next 5 minutes and an additional 5 minutes of 100 % methanol. a-Tocopherol was monitored by fluorescence (lex Å 275 nm and lem Å 320 nm) and a-tocopheryl quinone was monitored by UV (l Å 266 nm). Compounds were quantified using a standard curve generated with known concentrations of a-tocopherol and a-tocopheryl quinone. Measurement of a-tocopheroxyl radical. Formation of a-tocopheroxyl radical was monitored using electron spin resonance (ESR) spectroscopy. ESR was performed at room temperature using a Varian E-109 spectrometer operating at 9.5 GHz (X-band). Samples were prepared in a quartz flat cell, for ESR analysis, either under strict nitrogen atmosphere inside a glove box or under atmospheric oxygen. N2O measurements. N2O was measured using gas chromatography employing a thermal conductivity detector and quantified as previously described (10).
RESULTS
The Effects of rNO and Oxygen on the Oxidation of a-Tocopherol to a-Tocopheryl Quinone HPLC analysis of DLPC liposomes (5 mM) containing a-tocopherol (50 mM) after incubation under anaerobic conditions for one hour resulted in minimal destruction of a-tocopherol (Figure 1A, left panel). A small amount of a-tocopheryl quinone (0.6 mM) was observed (Figure 1A, right panel); however this was present in the original a-tocopherol solution and corresponds to only 1.2 % of the total a-tocopherol. Addition of a rNO solution (1.4 mM final concentration) to an identical preparation of liposomes, under anaerobic conditions, caused neither the depletion of a-tocopherol nor the formation of a-tocopheryl quinone after incubation for 1 hour (Figure 1B). A third sample of liposomes was vacuum degassed and the atmosphere replaced by rNO gas directly from the cylinder (i.e. without passing through a sodium hydroxide scrubber). This treatment caused no significant depletion of a-tocopherol, however the amount of a-tocopheryl quinone was slightly increased to 0.85 mM (1.7% of the original atocopherol concentration, Figure 1C). This slight increase in quinone is likely to be due to contaminating higher oxides of nitrogen in the rNO gas. A fourth sample of liposomes was exposed to rNO gas in the presence of oxygen. Under these conditions, total oxidation of atocopherol occurred and 69% of the original a-tocopherol was recovered as a-tocopheryl quinone (Figure 1D). A second unidentified peak was also observed with a retention time of 14.5 minutes. We have shown previously that slow-releasing rNO donors are unable to deplete the atocopherol content of low-density lipoprotein in the presence of atmospheric oxygen (4,5). Figure 2 shows that this is also the case in DLPC liposomes. Liposomes consisting of DLPC (5 mM) and a-tocopherol (50 mM) were incubated with spermine NONOate (100 mM), a compound that releases rNO slowly and spontaneously (11). Aliquots were removed at the specified time intervals and assayed for a-tocopherol and a-tocopheryl quinone. As shown in figure 2, no a-tocopherol depletion was observed during 3 hours of incubation. A higher 697
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FIG. 1. Oxidation of a-tocopherol to a-tocopheryl quinone by bolus addition of rNO. DLPC liposomes (50 mM) containing a-tocopherol (50 mM) in phosphate buffer (100 mM) were incubated at 377C for 1 hour (A) alone under anaerobic conditions, (B) in the presence of rNO (1.4 mM) under anaerobic conditions, (C) in the presence of unscrubbed rNO gas under anaerobic conditions, and (D) in the presence of rNO gas and air. After one hour all samples were thoroughly degassed before exposure to oxygen and analyzed by HPLC. The left panel shows the fluorescence HPLC chromatogram showing the presence of a-tocopherol (retention time 14 min.). The right panel shows the UV chromatogram showing the presence of a-tocopheryl quinone (retention time 11 min.).
concentration of spermine NONOate (1 mM), under aerobic conditions, caused a significant and time-dependent depletion of a-tocopherol over 3 hours. This was accompanied by an increase in a-tocopheryl quinone (Figure 2). Under anaerobic conditions, however, spermine NONOate (1 mM) caused no oxidation of a-tocopherol. Formation of a-Tocopheroxyl Radical during the Reaction between a-Tocopherol and rNO It has been previously demonstrated that the reaction between rNO and a-tocopherol generates an ESR signal, characteristic of the a-tocopheroxyl radical (1,2). Using ESR, we monitored the formation of a-tocopheroxyl radical under various conditions as shown in Figure 3. Incubation of DLPC liposomes (125 mM) containing a-tocopherol (1.25 mM) with rNO (0.9 mM) resulted in no ESR signal (Figure 3A). UV irradiation of liposomes containing a-tocopherol gave an ESR spectrum consistent with the formation of a-tocopheroxyl radical (Figure 3B). The magnitude of this radical was approximately the same in the presence and absence of rNO (Figure 3C) indicating that rNO does not significantly affect the steady-state concentration of the a-tocopheroxyl radical. Under aerobic conditions, low levels of a-tocopheroxyl radical 698
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FIG. 2. Oxidation of a-tocopherol to a-tocopheryl quinone by slow release of rNO. DLPC liposomes (50 mM) containing a-tocopherol (50 mM) in phosphate buffer (100 mM) were incubated at 377C for 3 hours (A) alone (l, s), (B) in the presence of spermine NONOate (100 mM) under aerobic conditions (j, h), (C) in the presence of spermine NONOate (1 mM) under aerobic conditions (l, L), and (D) in the presence of spermine NONOate (1 mM) under anaerobic conditions (m, n). Samples were removed every hour and assayed for a-tocopherol (filled symbols) and a-tocopheryl quinone (open symbols). Data points represent means{S.D. (nÅ3).
were observed when a-tocopherol was treated either with rNO solution (Figure 3D) or bubbled with rNO gas (Figure 3E). N2O Formation during the Reaction between a-Tocopherol and rNO The direct reduction of rNO by a-tocopherol would result in the formation of nitroxyl (HNO) and a-tocopheroxyl radical (Equation 1). HNO, under anaerobic a-TOH / rNO r a-TOr / HNO
(1)
2NHO r N2O / H2O
(2)
conditions, undergoes rapid dismutation to form N2O (Equation 2). Incubation of DLPC liposomes (50 mM) containing a-tocopherol (500 mM) with rNO (1.7 mM) resulted in no significant formation of N 2O over 6 hours (Figure 4). DISCUSSION
Literature Evidence for and against a Direct Reaction between rNO and a-Tocopherol Three independent studies (1-3) have reported that rNO can directly react with a-tocopherol to generate a-tocopheroxyl radical and a-tocopheryl quinone. The common link between all of these studies is that rNO gas was used as the source of rNO. In contrast to these reports are a number of studies (4-6) that show rNO does not react with a-tocopherol. In these cases rNO was slowly released from rNO donor compounds into either LDL or a liposomal preparation of a-tocopherol. Only in the presence of superoxide was rNO able to oxidize atocopherol to a-tocopheryl quinone (4). In these conditions, peroxynitrite is formed and causes 699
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FIG. 3. Oxidation of a-tocopherol to a-tocopheroxyl radical by rNO. DLPC (125 mM) containing a-tocopherol (1.5 mM) were incubated (A) with rNO solution (0.9 mM) under anaerobic conditions, (B) as A but upon irradiation with UV light, (C) under anaerobic conditions and irradiated with UV light, (D) as A under aerobic conditions, and (E) after bubbling with rNO gas (1 ml). Spectrometer conditions; microwave power, 50 mW; modulation amplitude, 2 G; time constant, 0.128 s; scan time, 1 min. Each spectrum is the average of 6 scans.
direct one- and two-electron oxidation of a-tocopherol (8). SIN-1, a compound that generates rNO and superoxide simultaneously, also oxidizes a-tocopherol to a-tocopheryl quinone. The addition of superoxide dismutase, which scavenges superoxide and consequently increases the level of available rNO, inhibited a-tocopherol oxidation (4). The Pitfalls of Improper Use of rNO Gas We show here that there is no fundamental difference between rNO donor compounds and rNO gas with respect to its reactivity with a-tocopherol, if appropriate precautions are taken in the preparation of rNO solutions. The artifactual oxidation of a-tocopherol in previous studies (1-3) most likely occurred as a result of oxygen contamination or a failure to remove contaminating nitrogen oxides from commercial rNO gas. The rate of the reaction between rNO and oxygen is proportional to the second power of the rNO concentration (7,12). Consequently this reaction will be greatly favored at the high, non-biological, concentrations of rNO generated by bubbling rNO gas through solutions (7, 13). In organic solvents, the higher oxides of rNO are more persistent, due to the absence of hydrolysis reactions, and will oxidize target molecules to a greater extent than in aqueous systems (14). We show here that rNO donor compounds, when used judiciously do not exhibit the chemistry of N2O3 even under aerobic conditions. This is likely to be due to the fact that 700
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FIG. 4. Formation of N2O from the reaction between a-tocopherol and rNO. DLPC liposomes (50 mM) in phosphate buffer (100 mM, pH 7.4) were incubated in sealed vials at 377C for 6 hours in the presence of rNO (1.7 mM), in either the absence (l) or presence (l) of a-tocopherol (50 mM). Headspace (100 ml) was removed every two hours and N2O measured by gas chromatography. Data represent means{S.D. (nÅ3).
a low steady state concentration of N2O3 will favor hydrolysis to nitrite (effectively a first order reaction in aqueous solution) over any second order oxidation reactions. However, at a higher concentration of rNO donor compound oxidative chemistry was observed indicating that one should be cautious when extrapolating such experimental results to the biological effects of rNO. Biological Considerations rNO has been proposed to be an important endogenous anti-atherogenic agent (14). The mechanism for this has not been elucidated. However it has been proposed that, at least in part, the ability of rNO to inhibit the propagation of lipid peroxidation may be responsible for the anti-atherogenic effect (15,16). In both chemical and cellular systems, rNO has been shown to inhibit the oxidative modification of LDL to an atherogenic form (15-19). If rNO were to directly oxidize a-tocopherol it would be expected to have a pro-oxidant effect. To date, all the evidence indicates that when rNO is slowly delivered to a system containing lipid and a-tocopherol (either within LDL or liposomes), a-tocopherol is not oxidized. rNO has a potent inhibitory effect on the rate of a-tocopherol oxidation during lipid peroxidation (5,6). In conclusion, in contrast to previous reports (1-3), we show that rNO does not directly react with a-tocopherol whether it is added as an aqueous solution or it is slowly generated in situ using an rNO donor compound. ACKNOWLEDGMENTS This research was supported by HL47250 from the National Heart, Lung and Blood Institute and RR01008.
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