The Journal of Nutrition Vol. 128 No. 2 February 1998, pp. 152-157

-Tocopherol in Rat Brain Subcellular Fractions Is Oxidized Rapidly during Incubations with Low Concentrations of Peroxynitrite^1,2

Govind T. Vatassery^{*, , **, 3}, W. Ed Smith^*, and Hung T. Quach^**

^*  Research Service and  GRECC, VA Medical Center, Minneapolis, MN 55417 and ^**  Department of Psychiatry, University of Minnesota, Minneapolis, MN 55455

	ABSTRACT

Abstract Introduction Methods Results Discussion References

The reaction of superoxide (a reactive oxygen species) and nitric oxide (one of the nitrogen oxides with numerous biological functions) results in the production of peroxynitrite. The characteristics of oxidation of -tocopherol (vitamin E) in synaptosomes (nerve ending particles) and mitochondria by peroxynitrite were studied. The subcellular fractions were isolated from brain hemispheres of 4-month-old male Fischer 344 rats by standard centrifugation procedures involving Ficoll gradients. Peroxynitrite treatment oxidized -tocopherol in <5 s. This reaction was selective because another membrane component, cholesterol, was not oxidized at the same time, as observed in our previous studies. Mitochondrial -tocopherol was more susceptible to peroxynitrite-induced oxidation than synaptosomal tocopherol. Measurable and significant (P < 0.05) oxidation of tocopherol occurred when mitochondria or synaptosomes were incubated with peroxynitrite in concentrations as low as 5 or 10 µmol/L, respectively. The oxidation could be readily monitored by estimating the production of tocopherolquinone. Oxidation of tocopherol induced by ferrous iron and ascorbate was much slower and the yield of tocopherolquinone lower than by peroxynitrite. The fast and selective oxidation of -tocopherol by peroxynitrite suggests that vitamin E may play an important role in preventing membrane oxidation induced by peroxynitrite. Literature reports indicate the existence of threshold concentrations of tocopherol below which functional alterations occur. Tocopherol oxidation by peroxynitrite could reduce tocopherol concentrations in tissues and subcellular structures to these threshold levels by different concentrations of peroxynitrite. Hence the sensitivity of tissues to peroxynitrite could vary over a wide range.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Nitric oxide is an important endogenous free radical that has many physiologic functions in the cardiovascular, immune and neuronal systems (Lowenstein et al. 1994). Superoxide is one of the oxygen free radicals that is produced by some biochemical enzymatic reactions as well as by the endogenous respiratory activities of subcellular organelles such as mitochondria. Superoxide reacts with nitric oxide to form peroxynitrite at a rate that is faster than the dismutation of superoxide catalyzed by superoxide dismutase (Beckman et al. 1993). The oxidation of several biochemical components such as thiols and vitamin C by peroxynitrite has been reported in the literature (Radi et al. 1991, Squadrito et al. 1995). Our laboratory has studied the oxidation of these biomolecules by peroxynitrite with the use of subcellular fractions to obtain information on the reactivity of peroxynitrite with scavengers in a biological environment. For example, we have observed that incubation of rat brain synaptosomes with peroxynitrite results in the oxidation of vitamin E, vitamin C and thiols (Vatassery 1996). Among these compounds, vitamin E (-tocopherol) is the major, if not the only lipid-soluble, chain-breaking antioxidant present in biological membranes. One characteristic of the peroxynitrite-induced oxidations was that -tocopherol was converted to tocopherolquinone in nearly quantitative yields. Similar observations have been made by other investigators (de Groot et al. 1993, Graham et al. 1993). In this investigation, we have examined the oxidation of -tocopherol in both synaptosomes and mitochondria from rat brain hemispheres by low concentrations of peroxynitrite. We have found that monitoring the production of tocopherolquinone can be a very useful index of the oxidative activity of peroxynitrite under in vitro conditions. In addition, the oxidation of tocopherol in synaptosomes by peroxynitrite takes place within a few seconds.

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

Chemicals.  The chemicals used were of reagent grade purity from standard sources. Solvents for chromatography were HPLC grade from Burdick and Jackson Laboratories, Muskegon, MI. Other special chemicals were purchased from the following sources: -tocopherol and -tocopherolquinone from Kodak Laboratory Chemicals, Rochester, NY; Ficoll (Type 400) from Sigma Chemical, St. Louis, MO. (Ficoll is a nonionic synthetic polymer of sucrose.) Absolute ethanol was obtained from Midwest Solvents Company, Pekin, IL and was redistilled before use. Most of the reagent grade chemicals were from Sigma Chemical.

Experimental procedures.  The protocols for animal usage were approved by the Subcommittee on Animal Studies of the Minneapolis Veterans Administration Medical Center. The animals were fed nonpurified diet (Teklad rodent diet: stock #8604) obtained from Harlan Teklad Laboratories, Madison, WI.

All experiments were repeated and confirmed with brain samples from separate animals on different days. Data from typical experiments are reported.

View larger version (16K): [in this window] [in a new window]   View larger version (65K): [in this window] [in a new window]   Fig 1. Oxidation of synaptosomal -tocopherol by peroxynitrite. A) Influence of low concentrations of peroxynitrite on the oxidation of -tocopherol when rat brain synaptosomes were incubated with peroxynitrite at 37°C. The synaptosomes (250 µg protein/mL medium) were preincubated in modified Krebs medium at 37°C for 10 min; after the addition of appropriate amounts of peroxynitrite, the tubes were incubated for 10 min. Then the samples were placed on ice and centrifuged to isolate the synaptosomes. The tocopherol compounds in the sediment were assayed by HPLC. Each point is the mean ± SD of three determinations. B) The production of tocopherolquinone during the experiment described in Figure 1A. *The mean value is significantly different from control mean value, P < 0.05.

View larger version (16K): [in this window] [in a new window]   View larger version (57K): [in this window] [in a new window]   Fig 2. Oxidation of mitochondrial -tocopherol by peroxynitrite. A) Effect of low concentrations of peroxynitrite on the oxidation of -tocopherol when rat brain mitochondria were incubated with peroxynitrite at 37°C. The mitochondria (250 µg protein/mL medium) were preincubated for 10 min in a medium of 25 mmol/L HEPES and 115 mmol/L KCl at pH 7.4. After the addition of appropriate amounts of peroxynitrite, the tubes were incubated for 10 min. The samples were then placed on ice and centrifuged to isolate the mitochondria. The tocopherol compounds in the sediment were assayed by HPLC. Each point is the mean ± SD of three determinations. B) The production of tocopherolquinone during the experiment described in Figure 2A. *The mean value is significantly different from control mean value with P < 0.05.

Preparation of peroxynitrite.  Peroxynitrite was prepared by the method of Keith and Powell (1969). Briefly, a mixture of sodium nitrite (35 mmol) and hydrogen peroxide (35 mmol) in 150 mL of ice-cold distilled water was acidified with hydrochloric acid (25 mL, 1 mol/L) and sodium hydroxide (40 mL, 1 mol/L) was added within 1-2 s to neutralize the acid and make the solution alkaline. The excess hydrogen peroxide was destroyed by passing the solution through manganese dioxide. The solution was then frozen. A darker yellow solution enriched in peroxynitrite separated out, was removed and used in all experiments. The peroxynitrite concentration in the stock solutions used on each day was estimated by using an extinction coefficient of 1670 cm¹ (mol/L)¹ at 302 nm (Hughes and Nicklin 1968). The peroxynitrite solution prepared was usually very basic (pH ~ 12). When larger volumes of peroxynitrite were added, part of the excess base was neutralized on the day of the experiment. This peroxynitrite solution of lower basicity was checked for its absorption at the beginning and end of the study to ascertain that the peroxynitrite had not decomposed during the time required for incubations. The incubation tubes were also routinely checked to make sure that the final pH did not change after the addition of peroxynitrite.

Preparation of subcellular fractions from rat brain.  Male Fischer 344 rats (4-month old) from Harlan Sprague Dawley, Indianapolis, IN, were used. The rats were starved overnight and killed by decapitation on the day of the experiment. Samples of brain hemispheres were dissected out and subcellular fractions isolated by standard centrifugation methods (Lai and Clark 1989). The Ficoll solution used was purified by dialyzing a 400 g/L aqueous solution against water for 3 h. The final Ficoll concentration was estimated by using a graph relating density and concentration. Brain tissue was homogenized in 10 volumes of ice-cold isolation medium containing 0.32 mol/L sucrose, 10 mmol/L HEPES, and 1 mmol/L EDTA at pH 7.4, using a glass-glass homogenizer. The homogenate was centrifuged at 1300 × g for 3 min and the supernatant saved. The pellet was resuspended in 10 mL of the isolation medium, rehomogenized and centrifuged at 1300 × g for 3 min. The pooled supernatants were centrifuged at 17,000 × g for 10 min to obtain the crude mitochondrial fraction. The resulting pellet was resuspended in 15 mL of isolation medium. Half of this suspension was layered over 11 mL of 75 g/L Ficoll medium, which had been layered over 11 mL of 100 g/L Ficoll medium. The tubes were centrifuged in a Beckman SW 28 rotor at 99,000 × g for 45 min. The fraction at the interface between the two Ficoll solutions was removed, diluted 1:5 with isolation medium and centrifuged for 10 min at 17,000 × g to isolate the synaptosomes. The purity of the synaptosomal fraction was tested by estimating the activities of the marker enzyme Na-K-ATPase (Maynard et al. 1982). The activity of the ATPase was usually enriched two- to threefold in synaptosomes compared with the crude homogenate. The synaptosomes isolated were metabolically active and have been used in studies of transmitter uptake and release. The mitochondria that sedimented at the bottom of the Ficoll gradient tube were also removed, suspended in a small volume (~2 mL) of isolation medium and centrifuged for 10 min at 17,000 × g. The purity of mitochondria was checked by assaying for the marker enzyme succinate dehydrogenase (Pennington 1961), which was enriched eight- to tenfold in the mitochondria compared with crude homogenate.

Protocol for incubations.  Synaptosomes were incubated at 37°C in a medium (pH 7.4) simulating plasma and had the following composition: 135 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L MgCl₂, 1 mmol/L CaCl₂, 1 mmol/L sodium phosphate and 10 mmol/L glucose. HEPES (10 mmol/L) was also added for additional buffering capacity. Each incubation tube had synaptosomes containing 250 µg of total protein suspended in 1 mL of the above buffer.

The peroxynitrite concentrations reported here refer to the concentrations of the compound at the time of addition of the stock peroxynitrite solution. This concentration was calculated by dividing the stock concentration by the dilution factor. The stock solutions were prepared so that the dilution factors were at least 40-fold or more so as to avoid the addition of excess amounts of base. The final pH of the medium was verified to be 7.4.

Test tubes containing buffer and the synaptosomal fractions were equilibrated at 37°C for 10 min and the peroxynitrite added while mixing. After an additional 10 min of incubation at 37°C, the tubes were placed on ice. The mixture was then centrifuged for 15 min at 35,000 × g in a Sorvall SS 34 rotor to sediment the synaptosomes. The sedimented fractions were then analyzed for various components.

Experiments with mitochondria were very similar except that the mitochondria were incubated in a pH 7.4 buffer containing 25 mmol/L HEPES and 115 mmol/L potassium chloride. As with synaptosomes, each incubation tube contained mitochondria (250 µg total protein) suspended in 1 mL of the above buffer solution. The procedures for incubation, centrifugation and analyses were identical to those with synaptosomes.

When the effect of incubation time on -tocopherol oxidation was studied, the subcellular fraction was separated by filtration. After treatment with peroxynitrite, the mixture was rapidly filtered using a manifold kept under vacuum. Whatman glass microfiber filters (GF/F) were used for filtration, and subcellular fractions collected on the filters were assayed for tocopherol compounds.

Because peroxynitrite decomposes spontaneously in buffer within 1 or 2 s, it was possible to run peroxynitrite-depleted controls. These control tubes were incubated with peroxynitrite for 10 min to decompose all of the peroxynitrite, and then aliquots of the subcellular fraction were added. These samples are referred to as peroxynitrite-depleted controls.

Determination of tocopherol and tocopherolquinone.  The method for determination of tocopherols and tocopherolquinone by liquid chromatography has been published (Vatassery et al. 1993). Briefly, 2 mL ethanol containing 0.25 g/L butylated hydroxytoluene (BHT) and 0.1 mL of 300 g/L ascorbic acid were pipetted into tubes containing samples for tocopherol analyses. The mixture was saponified at 60°C for 30 min after the addition of 1 mL of 100 g/L potassium hydroxide solution. (In our experience saponification under these conditions results in the oxidation of very small amounts of tocopherol as shown by the minute amounts of tocopherolquinone found in the control samples.) Tubes were cooled and 2 mL of water was added followed by 2 mL of hexane containing 0.25 g/L BHT. Tocopherols and tocopherolquinone were extracted into the hexane phase by vortexing for 1 min. The hexane phase was separated and evaporated under a stream of nitrogen. The residue was redissolved in mobile phase and analyzed by reverse-phase liquid chromatography using the following conditions: column, ultrasphere ODS, 5 µm, 4.6 × 150 mm (Beckman Instruments); mobile phase, methanol/water, (94.5:5.5) with 7.5 mmol/L sodium dihydrogen phosphate (final concentration); flow rate, 2.7 mL/min. The tocopherols and tocopherolquinone were detected electrochemically by using the following equipment and conditions: Coulochem 5100 A detector, 5011 analytical cell with detector 1 at 0.25 V and detector 2 at +0.55 V and 5021 conditioning cell at 0.75 V.

Biochemical assays.  Concentration of total protein was determined by the Lowry technique as modified by Markwell et al. (1978).

Statistical analyses.  Statistical significance of the production of tocopherolquinone (shown in Figs. 1B and 2B) was estimated as follows. Independent group t tests with Bonferroni corrections for multiple comparisons were used to compare production of quinone at control vs. each concentration of peroxynitrite. If P < 0.05 after applying the Bonferroni correction, means were considered significantly different.

	RESULTS

Abstract Introduction Methods Results Discussion References

Synaptosomes isolated from 4-month-old Fischer rats were incubated with varying concentrations of peroxynitrite, and then the concentrations of -tocopherol and tocopherolquinone in these samples were determined (Fig. 1A). Treatments with low concentrations of peroxynitrite caused only small nonsignificant differences in -tocopherol concentrations. The production of tocopherolquinone when synaptosomes were incubated with low concentrations of peroxynitrite are shown in Figure 1B. The production of tocopherolquinone was significantly higher than for controls when the peroxynitrite concentrations were at least 10 µmol/L (P < 0.05). Thus, the oxidation of tocopherol by low concentrations of peroxynitrite can be demonstrated easily by following the production of tocopherolquinone. It is important to note that incubations in the presence of peroxynitrite-depleted solutions did not cause any oxidation of tocopherol or formation of tocopherolquinone.

A similar experiment was conducted with isolated mitochondria and the results are shown in Figure 2. The production of tocopherolquinone was significant even at 5 µmol/L peroxynitrite (Fig. 2B, P < 0.05). This was expected because -tocopherol in mitochondria is generally more oxidizable than that in synaptosomes (see below).

The oxidation of -tocopherol by peroxynitrite in both synaptosomes and mitochondria was compared in the next experiment. The results are shown in Figure 3. The data were analyzed statistically as follows. The 95% confidence intervals for mean tocopherol concentrations in mitochondria after treatment with the different peroxynitrite concentrations were calculated. Then the 95% confidence limits for the regression line of tocopherol concentration vs. peroxynitrite were calculated for the synaptosomal samples. These confidence limits did not overlap. Thus mitochondrial tocopherol undergoes significantly more oxidation (P < 0.05) than synaptosomal tocopherol at all tested concentrations of peroxynitrite. This could be due to the differences in accessibility of tocopherol to peroxynitrite in the medium. Synaptosomes are more complex structurally and contain mitochondria within the plasma membrane envelope. It is possible that some of the peroxynitrite is decomposed before it reaches the interior membranes within the synaptosomes.

View larger version (15K): [in this window] [in a new window]   Fig 3. Comparison of peroxynitrite-induced oxidation of -tocopherol in rat brain mitochondria and synaptosomes. The synaptosomal samples (250 mg protein/L medium) were incubated at 37°C in modified Krebs buffer and mitochondria (250 µg protein/mL medium) in 25 mmol/L HEPES and 115 mmol/L KCl at pH 7.4. The preincubation time was 10 min; after the addition of appropriate amounts of peroxynitrite, the tubes were incubated for 10 min. The samples were then placed on ice and centrifuged to isolate the synaptosomes or mitochondria. -Tocopherol in the pellets was assayed by HPLC. Each point is the mean ± SD of three determinations.

Next, the rapidity of oxidation of tocopherol within the membranes by peroxynitrite was tested. The synaptosomes were incubated with 50 or 100 µmol/L peroxynitrite for lengths of times ranging from 5 to 40 s and the reaction was stopped by rapid filtration as discussed under methods. The subcellular fraction retained on the filter was analyzed for -tocopherol and tocopherolquinone. It should be pointed out that the addition of peroxynitrite to the synaptosomes and the mixing and separation of the fractions by membrane filtration required a minimum of 5 s. The results given in Figure 4 show that the oxidation of tocopherol was complete within 5 s or possibly even sooner. Therefore, it is quite likely that -tocopherol within biological membranes rapidly neutralizes peroxynitrite.

View larger version (16K): [in this window] [in a new window]   Fig 4. Oxidation of -tocopherol with production of tocopherolquinone when rat brain synaptosomes (250 mg protein/L medium) were incubated with 50 or 100 µmol/L peroxynitrite for different times. The samples were preincubated at 37°C in modified Krebs medium; after the addition of peroxynitrite, the mixture was incubated for the stated times. The subcellular fraction was then rapidly recovered by filtration using a vacuum manifold. The filter with the synaptosomes was assayed for the tocopherol compounds by HPLC. Each point is the mean ± SD of three determinations.

It is instructive to compare the oxidation of -tocopherol induced by peroxynitrite with that induced by a mixture of ferrous iron and ascorbate. In this experiment, synaptosomes were incubated for varying amounts of time with a mixture of 25 µmol/L ferrous iron and 75 µmol/L ascorbate. The loss of tocopherol and the production of tocopherolquinone were monitored. The results shown in Figure 5 show that the loss of tocopherol is nearly linear for the 12 min; this is in sharp contrast to the nearly instantaneous oxidation of tocopherol by peroxynitrite shown in Figure 4.

View larger version (13K): [in this window] [in a new window]   Fig 5. Oxidation of -tocopherol and production of tocopherolquinone when rat brain synaptosomes were incubated with a mixture of 25 µmol/L ferrous iron and 75 µmol/L ascorbate for various times. The synaptosomes (250 mg protein/L medium) were incubated in modified Krebs buffer at 37°C; after the addition of oxidant, the samples were incubated for the stated times. The synaptosomes were then separated by filtration using a vacuum manifold. The tocopherol compounds on the filter were assayed by HPLC. Each point is the mean ± SD of three determinations.

The relative amounts of tocopherolquinone that were produced upon treatment of synaptosomes with peroxynitrite or ferrous iron and ascorbate were compared. The percentage yield of tocopherolquinone formed in each case was calculated by dividing the tocopherolquinone formed by the amount of -tocopherol lost and then multiplying the result by 100. The average maximum yield of tocopherolquinone after 6, 9 or 12 min of incubation of synaptosomes with ferrous iron and ascorbate was 30 ± 3.8% (SD, n = 3) (calculated from data in Figure 5). The mean yield of tocopherolquinone in synaptosomes incubated with 300, 700 and 900 µmol/L peroxynitrite shown in Figure 3 was 95.5 ± 7.4% (SD, n = 3). Thus, the percentage-yields of tocopherolquinone are strikingly different when synaptosomes were incubated with either peroxynitrite or ferrous iron plus ascorbate, suggesting that the mechanisms of oxidation are also quite different.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

Numerous reports have appeared in the literature dealing with the biological effects of peroxynitrite. Concentrations of peroxynitrite ranging from nanomolar to a few millimolar have been used in these experiments. In one of the earliest studies, Beckman and coworkers examined the toxicity of peroxynitrite to Escherichia coli (Beckman and Crow 1993). They found that the LD₅₀ for killing of the bacteria was 250 µmol/L peroxynitrite, whereas the same concentration of hydrogen peroxide did not have any effect. Mayer et al. (1995) observed that peroxynitrite stimulated purified soluble guanyl cyclase in the presence of 2 mmol/L glutathione. The concentration of peroxynitrite required to increase the cyclase activity to half the maximal velocity was 20 µmol/L. Villa et al. (1994) studied coronary perfusion pressure and vasodilation induced by acetyl choline and isoproteranol. They found that peroxynitrite in concentrations as low as 3 µmol/L caused inhibition of the vasodilation. One of the most potent biological activities of peroxynitrite seems to be its effect upon prostacyclin biosynthesis. Zou and Ullrich (1996) studied the effect of peroxynitrite addition to microsomal preparations from bovine aorta and found that 50 nmol/L concentrations of the compound caused 50% inhibition of the prostacyclin synthase activity. These authors propose that the inhibition of the synthase proceeds by nitration of the enzyme molecule rather than by oxidation. In this study, we found that incubation of brain subcellular fractions with peroxynitrite in the range of 5-10 µmol/L resulted in the production of measurable amounts of tocopherolquinone from oxidation of tocopherol in the membranes.

Peroxynitrite is known to decay spontaneously with a half-life of ~1 s. The very short half-life of peroxynitrite makes it very difficult to assign the active concentration of peroxynitrite that is taking part in the biochemical oxidation being studied. Hu et al. (1994) calculated that peroxynitrite decomposes at pH 7.4 and 37°C with a pseudo-first-order rate constant of 38 min¹. The lowest concentration of peroxynitrite that caused measurable oxidation of -tocopherol was 5 µmol/L. This reaction took place within 5 s and might even be faster because we could not measure oxidation experimentally in <5 s. Using the rate constant noted above, the concentration of peroxynitrite at the end of 5 s would be 5 e^38t where t is 5/60 min. This is equal to 5 × 0.042 or 0.21 µmol/L. Thus the concentration of peroxynitrite that caused the oxidation of tocopherol must have been between 0.21 and 5 µmol/L.

The rapidity of oxidation of tocopherol is quite remarkable. In addition, we have reported earlier that membrane cholesterol is not oxidized by peroxynitrite treatment (Vatassery 1996). This has been confirmed in all of the experiments reported in this paper (data not included). Thus, peroxynitrite selectively attacks -tocopherol and the reaction is quite rapid. Therefore, it can be postulated that -tocopherol plays a very important role in protecting biological membranes against peroxynitrite-induced oxidative damage.

Literature reports have shown that membrane peroxidation occurs only when -tocopherol concentrations in membranes fall below a critical level. For example, Fukuzawa et al. (1985) found that -tocopherol did not prevent peroxidation of phosphatidylcholine liposomes until its concentration had reached a critical level. Using a similar model, Liebler et al. (1986) also found that lipid oxy-radical propagation within liposomes takes place only when the concentration of -tocopherol is <0.2 mol/100 mol (based on phospholipid content). This critical threshold level of -tocopherol is expected to depend upon the membrane location, composition and the specific enzyme activity. Our data show that peroxynitrite treatment results in a concentration-dependent reduction of -tocopherol (Fig. 3). Furthermore, mitochondrial tocopherol is more easily oxidized than synaptosomal tocopherol (Fig. 3). Alterations in membrane or enzymatic function are expected to occur only when tocopherol levels fall below critical threshold levels. The peroxynitrite concentration that brings about such changes could be different in various tissues, cells and subcellular organelles.

In summary, our results show that peroxynitrite induces rapid and selective oxidation of -tocopherol in brain mitochondria and synaptosomes under in vitro conditions. This oxidative reaction can be monitored by estimating the quantity of tocopherolquinone formed. Vitamin E plays a critical role in protecting biological membranes from oxidative stress induced by peroxynitrite especially in the membranes of those cells in which the simultaneous production of nitric oxide and superoxide is known to occur. Different types of cells or subcellular organelles could exhibit differential susceptibilities of peroxynitrite depending upon the ease with which the tocopherol concentrations fall below threshold levels.

	ACKNOWLEDGMENT

We thank Michael A. Kuskowski for help with the statistical analyses of data.

	FOOTNOTES

Manuscript received 7 July 1997. Initial reviews completed 19 August 1997. Revision accepted 16 September 1997.

	LITERATURE CITED

Abstract Introduction Methods Results Discussion References

• Beckman, J. S., Carson, M., Smith, C. D. & Koppenol, W. H. (1993) ALS, SOD and peroxynitrite. Nature (Lond.) 364: 584.
• Beckman J. S., Crow J. P. Pathological implications of nitric oxide, superoxide and peroxynitrite formation. Biochem. Soc. Trans. 1993; 21:330-334 [Medline]
• de Groot H., Hegi U., Sies H. Loss of alpha tocopherol upon exposure to nitric oxide or the sydnonimine SIN-1. FEBS Lett. 1993; 315:139-142 [Medline]
• Fukuzawa K., Takase S., Tsukatani H. The effect of concentration on the antioxidant effectiveness of alpha tocopherol in lipid peroxidation induced by superoxide free radical. Arch. Biochem. Biophys. 1985; 240:117-120 [Medline]
• Graham A., Hogg N., Kalyanaraman B., O Leary, V., Darley-Usmar V., Moncada S. Peroxynitrite modification of low-density lipoprotein leads to recognition by the macrophage scavenger receptor. FEBS Lett. 1993; 330:181-185 [Medline]
• Hu P., Ischiropoulos H., Beckman J. S., Matalon S. Peroxynitrite inhibition of oxygen consumption and sodium transport in alveolar type II cells. Am. J. Physiol. 1994; 266:L628-L634 [Abstract/Free Full Text]
• Hughes M. N., Nicklin H. G. The chemistry of pernitrites. Part I. Kinetics of decomposition of pernitrous acid. J. Chem. Soc. A 1968; 2:450-452
• Keith, W. G. & Powell, R. E. (1969) Kinetics of decomposition of peroxynitrous acid. J. Chem. Soc. A 1: 90.
• Lai, J.C.K. & Clark, J. B. (1989) Isolation and characterization of synaptic and nonsynaptic mitochondria from mammalian brain. In: Neuromethods, Volume 11: Carbohydrates and Energy Metabolism (Boulton, A. A. & Baker, G. B., eds.), pp. 43-97. Humana Press, Clifton, NJ.
• Liebler D. C., Kling D. S., Reed D. J. Antioxidant protection of phospholipid bilayers by alpha tocopherol. Control of alpha tocopherol status and lipid peroxidation by ascorbic acid and glutathione. J. Biol. Chem. 1986; 261:12114-12119 [Abstract/Free Full Text]
• Lowenstein C. J., Dinerman J. L., Snyder S. H. Nitric oxide: a physiological messenger. Ann. Intern. Med. 1994; 120:227-237 [Abstract/Free Full Text]
• Markwell M.A.K., Haas S. M., Bieber L. L., Tolbert N. E. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal. Biochem. 1978; 87:206-210 ^[Medline]
• Mayer B., Schrammel A., Klatt P., Koesling D., Schmidt K. Peroxynitrite induced accumulation of cyclic GMP in endothelial cells and stimulation of purified soluble guanyl cyclase. J. Biol. Chem. 1995; 270:17355-17360 [Abstract/Free Full Text]
• Maynard R. R., Fullerton D. S., Ahmed K. A. A simple method for the purification of rat brain Na, K adenosine triphosphatase. J. Pharmacol. Methods 1982; 7:279-288 ^[Medline]
• Pennington R. J. Biochemistry of dystrophic muscle. Mitochondrial succinate-tetrazolium reductase and adenosine triphosphatase. Biochem. J. 1961; 80:649-654 ^[Medline]
• Radi R., Beckman J. S., Bush K. M., Freeman B. A. Peroxynitrite oxidation of sulfhydryls. J. Biol. Chem. 1991; 266:4244-4250 [Abstract/Free Full Text]
• Squadrito G. L., Jin X., Pryor W. A. Stopped-flow kinetic study of the reaction of ascorbic acid with peroxynitrite. Arch. Biochem. Biophys. 1995; 322:53-59 [Medline]
• Vatassery G. T. Oxidation of vitamin E, vitamin C, and thiols in rat brain synaptosomes by peroxynitrite. Biochem. Pharmacol. 1996; 52:579-586 [Medline]
• Vatassery G. T., Smith W. E., Quach H. T. A liquid chromatographic method for the simultaneous determination of alpha tocopherol and tocopherolquinone in human red blood cells and other biological samples where tocopherol is easily oxidized during sample treatment. Anal. Biochem. 1993; 214:426-430 [Medline]
• Villa L. M., Salas E., Darley-Usmar V. M., Radomski M. W., Moncada S. Peroxynitrite induces both vasodilatation and impaired vascular relaxation in the isolated perfused rat heart. Proc. Natl. Acad. Sci. U.S.A. 1994; 91:12383-12387 [Abstract/Free Full Text]
• Zou M. H., Ullrich V. Peroxynitrite formed by the simultaneous generation of nitric oxide and superoxide selectively inhibits bovine aortic prostacyclin synthase. FEBS Lett. 1996; 382:101-104 [Medline]

0022-3166/98 $3.00 ©1998 American Society for Nutritional Sciences