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Reaction of Uric Acid with Peroxynitrite and Implications for the Mechanism of Neuroprotection by Uric Acid
Archives of Biochemistry and Biophysics Vol. 376, No. 2, April 15, pp. 333–337, 2000 doi:10.1006/abbi.2000.1721, available online at http://www.idealibrary.com on
Reaction of Uric Acid with Peroxynitrite and Implications for the Mechanism of Neuroprotection by Uric Acid Giuseppe L. Squadrito,* ,1 Rafael Cueto,* Andres E. Splenser,* ,† Athanasios Valavanidis,‡ Houwen Zhang,* Rao M. Uppu,* and William A. Pryor* ,1 *Biodynamics Institute, 711 Choppin Hall, Louisiana State University, Baton Rouge, Louisiana 70803-1800; †Premedical Honors College, University of Texas Pan American, Edinburg, Texas 78539-2999; and ‡Department of Chemistry, University of Athens, University Campus Zografou, 15771 Athens, Greece
Received September 23, 1999
effects associated with peroxynitrite in many diseases. Peroxynitrite, a biological oxidant formed from the reaction of nitric oxide with the superoxide radical, is associated with many pathologies, including neurodegenerative diseases, such as multiple sclerosis (MS). Gout (hyperuricemic) and MS are almost mutually exclusive, and uric acid has therapeutic effects in mice with experimental allergic encephalomyelitis, an animal disease that models MS. This evidence suggests that uric acid may scavenge peroxynitrite and/or peroxynitrite-derived reactive species. Therefore, we studied the kinetics of the reactions of peroxynitrite with uric acid from pH 6.9 to 8.0. The data indicate that peroxynitrous acid (HOONO) reacts with the uric acid monoanion with k ⴝ 155 M ⴚ1 s ⴚ1 (T ⴝ 37°C, pH 7.4) giving a pseudo-first-order rate constant in blood plasma k Urate /plasma ⴝ 0.05 s ⴚ1 (T ⴝ 37°C, pH 7.4; assuming [uric acid] plasma ⴝ 0.3 mM). Among the biological molecules in human plasma whose rates of reaction with peroxynitrite have been reported, CO 2 is one of the fastest with a pseudo-first-order rate constant k CO2 /plasma ⴝ 46 s ⴚ1 (T ⴝ 37°C, pH 7.4; assuming [CO 2] plasma ⴝ 1 mM). Thus peroxynitrite reacts with CO 2 in human blood plasma nearly 920 times faster than with uric acid. Therefore, uric acid does not directly scavenge peroxynitrite because uric acid can not compete for peroxynitrite with CO 2. The therapeutic effects of uric acid may be related to the scavenging of the radicals CO 3•ⴚ and NO 2• that are formed from the reaction of peroxynitrite with CO 2. We suggest that trapping secondary radicals that result from the fast reaction of peroxynitrite with CO 2 may represent a new and viable approach for ameliorating the adverse
© 2000 Academic Press
1 To whom correspondence should be addressed. Fax: (225) 3884936. E-mail: [email protected] or [email protected].
2 Abbreviations used: MS, multiple sclerosis; DTPA, diethylenetriaminepentaacetic acid.
0003-9861/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
Key Words: uric acid; urate; peroxynitrite; carbon dioxide; nitric oxide; superoxide; oxidation; kinetics; free radical; antioxidant.
Uric acid is a natural antioxidant (1, 2) that is present in plasma in relatively high concentrations (in men, 302 ⫾ 60 M; in women, 234 ⫾ 52 M (3)). In patients with certain diseases, such as gout (hyperuricemic), plasma levels of uric acid are markedly higher than in normal subjects. Interestingly, gout and multiple sclerosis (MS) 2 are two diseases that are almost mutually exclusive (4). In MS, plaque areas in the brain have been associated with peroxynitrite-mediated damage (5). Peroxynitrite is an oxidant formed in vivo from the reaction of nitric oxide and the superoxide radical. In experimental allergic encephalomyelitis, an animal disease that models MS, administration of uric acid was found to have strong therapeutic effects (4). Thus, uric acid plays a protective role in these two diseases, where damage is mediated by peroxynitrite. Other diseases in which damage is currently thought to be mediated by peroxynitrite include arthritis (6), asthma (7), sepsis (8), inflammatory bowel disease (9), stroke (10), Alzheimer’s disease (11), Parkinson’s disease (12), amyotrophic lateral sclerosis (13), atherosclerosis (14), preeclampsia (15), and AIDS dementia (16). To probe the potential protective role of uric acid against peroxynitrite, we studied the kinetics of the
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reaction of uric acid with peroxynitrite using stoppedflow spectroscopy. MATERIALS AND METHODS Chemicals. Uric acid and sodium azide were purchased from Sigma (St. Louis, MO). All other chemicals were of analytical grade. Deionized water (⬎18 M⍀/cm) was used in the preparation of all buffers and reagents. Instrumentation. The stopped-flow instrument used for the measurement of reaction rates was an RSM 1000 from On-Line Instrument Systems (Bogart, GA) equipped with a UV/Vis rapid scan spectrophotometer and a 2-cm cell. The Sander 200 ozonizer used in the synthesis of peroxynitrite was from Erwin Sander (Uetze-Eltze, Germany). An HP8451A diode array UV/Vis spectrophotometer from Hewlett Packard (Willmington, DE) was used to monitor the concentration of peroxynitrite during its preparation and also to measure its concentration in stock and working solutions. Peroxynitrite synthesis. Peroxynitrite was synthesized by ozonation of an aqueous solution of sodium azide (17, 18). Briefly, a solution of sodium azide (0.1 N, 100 mL; pH 12 adjusted with NaOH) was chilled to ⬍5°C and ozonized by feeding oxygen to the ozonator at a flow rate of 300 mL/min. The concentration of peroxynitrite was monitored by measuring A 302 nm of samples diluted 100 times with water adjusted to pH 12 with NaOH. It required about 2 h for the absorbance to reach the maximum (ca. 1.1), and the ozonation was stopped after the absorbance decreased to ca. 0.9 (18, 19). The resulting solution was purged for 10 min with nitrogen and then stored at ⫺12°C until used. Stopped-flow kinetics of peroxynitrite decomposition in aqueous phosphate buffers. The reactions were conducted on the stopped flow spectrophotometer thermostated at 25.0 ⫾ 0.1 or 37.0 ⫾ 0.1°C. Briefly, peroxynitrite solutions (0.2 mM, pH 11.5–12.0) were mixed with equal volumes of 100 mM sodium phosphate buffer, pH 6.7– 8.0, containing uric acid (from 0 to 5 mM). Sodium pyrophosphate buffers (100 mM) were used for pH ⬎ 8.0. The pH values were measured both before and after mixing of reactants. A small pH jump (ca. 0.2) was usually observed on mixing, and the final pH was reported unless otherwise stated. Data for the disappearance of peroxynitrite collected at wavelengths longer than 330 nm were analyzed using the OLIS software. [Uric acid absorbs strongly at wavelengths shorter than 330 nm and interferes with our measurements.] The low solubility of uric acid at low pH prevented measuring apparent second-order rate constants at pH values lower than 6.7. In addition, the solubility of uric acid is lower in pyrophosphate buffers and this further limited the working concentration range at pH ⬎ 8.0. Studies of the reaction kinetics at temperatures lower than 25°C were hampered by the low solubility of uric acid. At temperatures higher than 37°C, the uric acid-independent component (the first-order component) of the decomposition of peroxynitrite takes over due to the larger activation energy of this component relative to its uric aciddependent counterpart (the second-order component) and this precludes accurate measurement of the reaction rates of uric acid with peroxynitrite. The ionic strength was not controlled but this is of no consequence because increasing the ionic strength with NaCl above I ⫽ 0.09 M up to levels corresponding to the higher pH while keeping all other parameters unchanged, had insignificant effects on k obs. Also, k obs is relatively insensitive to changes in the concentration of the buffer from 10 to 200 mM. Table Curve 3D (Jandel Scientific Software) was used to analyze k obs as a function of pH and the uric acid concentration ([uric acid] ⬎ 0) with k 1 , k 2 , and K a as adjustable parameters (see Results and Discussion); 3D fitting has the advantage of being able to consider the exact value of the pH of every data point and this is important due to the relatively narrow working pH range of uric acid. Data points represent the average of at least three independent experiments. Data points with [uric acid] ⫽ 0 were
excluded because of a behavior similar to that previously observed for other substrates [such as methionine and 2-keto-4-thiomethylbutanoic acid (20), ascorbate (21), and tryptophan (22)] in which curvature is observed at low substrate concentrations. With all data points with [uric acid] ⬎ 0 the reaction is first order in both uric acid and peroxynitrite and these data points were used to determine the second-order rate constant. Uric acid oxidation yields. The appropriate amount of uric acid was dissolved in a minimal volume of NaOH (0.5 M) and diluted with phosphate buffer (pH 7.2, 0.10 M) containing 0.1 mM DTPA. Uric acid (0.2 mM) was allowed to react with increasing concentrations of peroxynitrite (0 to 0.8 mM) in a final volume of 2 mL. When appropriate, the reaction mixture also contained 10 mM sodium bicarbonate. Rapid mixing was accomplished with a vortex mixer. The uric acid oxidation yields were calculated by relating the areas under the unreacted uric acid peaks of the reaction mixtures HPLC traces. The samples were analyzed on a Perkin-Elmer Series 410 liquid chromatograph (Perkin-Elmer, Norwalk, CT) equipped with a PerkinElmer LC-95 UV/Vis spectrophotometer using a Phenomenex LUNA 5-m C18(2) 150 ⫻ 4.6-mm column and an isocratic solvent system consisting of 95% 25 mM KH 2PO 4/H 3PO 4 and 5% methanol at a flow of 1.0 mL/min. The unreacted uric acid acid eluted with a retention time of 3.2 min. The reaction products are in both cases complex mixtures that contain allantoin and parabanic acid, which are likely formed from the hydrolysis of the unstable dismutation product dehydrouric acid.
RESULTS AND DISCUSSION
Stopped-flow kinetics of the reaction of uric acid with peroxynitrite. Figures 1A and 1B show 3D plots of the
FIG. 1. Rate constants for the reaction of peroxynitrite with uric acid at (A) 25.0°C and (B) 37.0°C as a function of the pH and uric acid concentration.
URIC ACID OXIDATION BY PEROXYNITRITE-DERIVED RADICALS TABLE I
Values for k 1 , k 2 , and K a That Best Describe the Data Points Shown in Figs. 1a and 1b T (°C) 25.0 37.0
k 1 (s ⫺1)
k 2 (M ⫺1 s ⫺1)
Ka
pK a
1.5 5.2
380 540
7.9 ⫻ 10 ⫺8 1.0 ⫻ 10 ⫺7
7.1 7.0
observed rate constant (k obs) as a function of pH and uric acid concentration for data collected at 25.0 and 37.0°C, respectively. The data points were best fit by Eq. (1) using k 1 , k 2 , and K a as adjustable parameters. This indicates that (a) the reaction is first order in peroxynitrite and first order in uric acid, and (b) that peroxynitrous acid (HOONO) is the reactive species: k obs ⫽
共k a ⫹ k 2 ⫻ 关uric acid兴兲 ⫻ 关H ⫹ 兴 . K a ⫹ 关H ⫹ 兴
[1]
Uric acid has pK a 1 ⫽ 5.4 and pK a 2 ⫽ 9.8 (23) and exists largely as a single species (the monoanion) within the pH range that we study here (from 6.7 to 8.0). Thus, HOONO must react with the uric acid monoanion. The values for k 1 , k 2 , and K a that best describe each set of data points are shown in Table I. The values of pK a that we obtain here are in good agreement with values reported earlier for the pK a of HOONO (24). The pK a of HOONO is generally taken as 6.8 and the values we report here are 7.1 (25°C) and 7.0 (37°C). We estimate the 95% confidence interval for these values is 0.2 pK units. We feel that these values are in agreement with the value for the pK a of HOONO, conforming with our mechanism. The values of k 1 shown in Table I should be interpreted as the maximum rate constants for the firstorder decomposition of peroxynitrite, because uric acid traps intermediates and blocks reversible steps that could occur during the decomposition of peroxynitrite (25, 26). The values of k 2 in Table I represent the pH-independent second-order rate constants for the reaction of HOONO with the uric acid monoanion, as shown in Eq. (2); k 2 is relatively insensitive to changes in temperature. An estimation of the of the Arrhenius energy of activation using just two temperatures, 25.0 and 37.0°C, yields approximately 5– 6 Kcal/mol: k2
HOONO ⫹ urate ion O ¡ products.
[2]
Physiological impact of the reaction of uric acid with peroxynitrite: A kinetic analysis. The pseudo-first-order rate constant for the reaction of uric acid with peroxynitrite in human blood plasma (k Urate /plasma) can be
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calculated by using Eq. (3) and substituting for k 2 ⫽ 540 M ⫺1 s ⫺1and K a ⫽ 1.0 ⫻ 10 ⫺7 (the values obtained at 37°C shown in Table I), pH 7.4 ([H ⫹] ⫽ 4.0 ⫻ 10 ⫺8 M), and [uric acid] plasma ⫽ 3 ⫻ 10 ⫺4 M: k Urate/plasma ⫽ k 2 关uric acid兴 ⫻
关H ⫹ 兴 ⫽ 0.05 s ⫺1. [3] K a ⫹ 关H ⫹ 兴
This affords k Urate /plasma ⫽ 0.05 s ⫺1, a relatively low value owing to the relatively small second-order rate constant and low concentration of uric acid. The value for k Urate /plasma can be compared to the pseudo-first-order rate constant for the reaction of CO 2 with peroxynitrite in blood plasma (k CO2 /plasma ⫽ 46 s ⫺1) (27). Since the reaction of peroxynitrite with CO 2 is about 920 times faster than the corresponding reaction with uric acid, uric acid cannot directly scavenge peroxynitrite. Instead, uric acid’s role in preventing peroxynitrite-mediated damage must involve scavenging reactive intermediates generated from the reaction of peroxynitrite with carbon dioxide. Physiological role of uric acid as a scavenger of peroxynitrite. The reaction of peroxynitrite with CO 2 is an important reaction of peroxynitrite in blood. This is because of the fast reaction of peroxynitrite with CO 2 and the high concentration of CO 2 in blood (27). As shown in Fig. 2, the reaction of peroxynitrite with CO 2 involves the peroxynitrite anion and forms nitrosoperoxycarbonate (ONOOCO 2⫺), which then rapidly homolyzes to form a pair of caged radicals [CO 3•⫺ •NO 2]. The caged radicals can then diffuse apart and become free radicals or combine to form the nitrocarbonate (O 2NOCO 2⫺), which then decomposes to NO 3⫺ and CO 2. This last reaction is responsible for the catalytic action of CO 2 during the decomposition of peroxynitrite (27– 29). Uric acid can scavenge only the free radicals CO 3•⫺ and •NO 2, since ONOOCO 2⫺, O 2NOCO 2⫺, and the caged radicals [CO 3•⫺ •NO 2] are all too short-lived to be trapped by potential scavengers. Under conditions in which the reaction of peroxynitrite with CO 2 is the dominant reaction of peroxynitrite, the maximum yield of uric acid oxidation will depend on the yield of free radicals that escape from the solvent cage. This yield has been estimated to be ca. 20% (28, 30). Nitrogen dioxide reacts rapidly with uric acid in aqueous solution with a rate constant of 1.8 ⫾ 0.2 ⫻ 10 7 M ⫺1 s ⫺1 (23). The rate constant for the reaction of uric acid with CO 3•⫺ is unknown. However, CO 3•⫺ is likely to be even more reactive than •NO 2 toward uric acid, just as it is toward ascorbate (for CO 3•⫺ k ascorbate ⫽ 1 ⫻ 10 9 M ⫺1 s ⫺1; for •NO 2 k ascorbate ⫽ 3.5 ⫻ 10 7 M ⫺1 s ⫺1) (31). Thus, uric acid may provide biological systems with an efficient first line of defense toward the free radicals CO 3•⫺ and •NO 2 that are formed from the reaction of peroxynitrite with CO 2.
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FIG. 2.
Uric acid as a scavenger of free radicals derived from the reaction of peroxynitrite with CO 2.
In order to test if uric acid scavenges CO 3•⫺ and/or NO 2 generated from the reaction of peroxynitrite with carbon dioxide, we measured the yields of oxidation of uric acid (200 M) by peroxynitrite (0 – 800 M) in the presence of bicarbonate (10 mM) at pH 7.2 and 25°C. (Fig. 3). Under these conditions, approximately 98% of the peroxynitrite reacts with CO 2, about 2% decomposes in a reaction that is zero order in uric acid, and 0.3% reacts directly with uric acid. However, uric acid is oxidized in 17 mol% yield relative to peroxynitrite (r 2 ⫽ 0.99), proving that uric acid reacts with reactive intermediates from the reaction of peroxynitrite with CO 2, which we suggest are the free radicals CO 3•⫺ and • NO 2. The yield of oxidized uric acid (17%) is in good agreement with the maximum yield of radicals (ca. 20%) produced by the reaction of peroxynitrite with carbon dioxide (28, 30). These results strongly suggest that any protection from peroxynitrite-mediated damage that may be attributed to uric acid is due to the ability of uric acid to scavenge the free radicals CO 3•⫺ and •NO 2 and not to uric acid scavenging peroxynitrite directly. The yields of uric acid oxidation obtained under these conditions without adding bicarbonate are 15% and are also shown in Fig. 3. Uric acid oxidation effected under these conditions arises from the reactions of uric acid with activated species that are produced during the decomposition of HOONO, such as the free radicals HO • and •NO 2 formed from the homolysis of the peroxo bond (25) and also from the direct reaction of uric acid with peroxynitrite. However, all these reactions are unlikely to occur in the physiological environment because both the self-decomposition of peroxynitrite and the direct reaction of peroxynitrite with •
uric acid are too slow to compete with the reaction of peroxynitrite with CO 2. SUMMARY AND CONCLUSIONS
Potential direct scavengers of peroxynitrite cannot effectively compete with CO 2 in the physiological environment (27). Uric acid reacts relatively slowly with peroxynitrite itself but, strikingly, it efficiently scavenges the free radicals CO 3•⫺ and •NO 2, which are produced following the fast reaction of peroxynitrite with CO 2. These observations suggest that targeting secondary radicals from the fast reaction of peroxynitrite
FIG. 3. Yields of uric acid oxidation by peroxynitrite in the presence (F) or absence (Œ) of added bicarbonate.
URIC ACID OXIDATION BY PEROXYNITRITE-DERIVED RADICALS
with CO 2 may represent a new and viable approach toward fighting the adverse effects associated with peroxynitrite in many diseases. ACKNOWLEDGMENTS This publication was made possible in part by Grant ES-06754 from the National Institute of Environmental Health Sciences, NIH (to W.A.P.), and by a Howard Hughes Medical Institute grant through the Undergraduate Biological Sciences Education Program to Louisiana State University (to A.E.S.). We thank Dr. Koppenol for the gift of the stopped-flow instrument.
REFERENCES 1. Halliwell, B. (1996) Uric acid: An example of antioxidant evaluation, in Handbook of antioxidants (Cadenas, E., and Packer, L., Eds.), pp. 243–255, Dekker, New York. 2. Becker, B. F. (1993) Free Rad. Biol. Med. 14, 615– 631. 3. Lentner, C. (1984) Physical Chemistry, Composition of Blood, Hematology, and Somatometric Data, Geigy Scientific Tables, Vol. 3, p. 103, CIBA-GEIGY, West Caldwell, NJ. 4. Hooper, D. C., Spitsin, S., Kean, R. B., Champion, J. M., Dickson, G. M., Chaudhry, I., and Koprowski, H. (1998) Proc. Natl. Acad. Sci. USA 95, 675– 680. 5. Hooper, D. C., Bagasra, O., Marini, J. C., Zborek, A., Ohnishi, S. T., Kean, R., Champion, J. M., Sarker, A. B., Bobroski, L., Farber, J. L., Akaike, T., Maeda, H., and Koprowski, H. (1997) Proc. Natl. Acad. Sci. USA 94, 2528 –2533. 6. Kaur, H., and Halliwell, B. (1994) FEBS Lett. 350, 9 –12. 7. Saleh, D., Ernst, P., Lim, S., Barnes, P. J., and Giaid, A. (1998) FASEB J. 12, 929 –937. 8. Szabo´, C., Salzman, A. L., and Ischiropoulos, H. (1995) FEBS Lett. 363, 235–238. 9. Rachmilewitz, D., Stamler, J. S., Karmeli, F., Mullins, M. E., Singel, D. J., Loscalzo, J., Xavier, R. J., and Podolsky, D. K. (1993) Gastroenterology 105, 1681–1688. 10. Oury, T. D., Piantadosi, C. A., and Crapo, J. D. (1993) J. Biol. Chem. 268, 15394 –15398. 11. Smith, M. A., Harris, P. L. R., Sayre, L. M., Beckman, J. S., and Perry, G. (1997) J. Neurosci. 17, 2653–2657. 12. Good, P. F., Hsu, A., Werner, P., Perl, D. P., and Olanow, C. W. (1998) J. Neuropathol. Exp. Neurol. 57, 338 –342.
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13. Crow, J. P., Sampson, J. B., Zhuang, Y., Thompson, J. A., and Beckman, J. S. (1997) J. Neurochem. 69, 1936 –1944. 14. Beckman, J. S., Ye, Y. Z., Anderson, P., Chen, J., Accavitti, M. A., Tarpey, M. M., and White, C. R. (1994) Biol. Chem. Hoppe-Seyler 375, 81– 88. 15. Roggensack, A. M., Zhang, Y., and Davidge, S. T. (1999) Hypertension 33, 83– 89. 16. Hensley, K., Tabatabaie, T., Stewart, C. A., Pye, Q., and Floyd, R. A. (1997) Chem. Res. Toxicol. 10, 527–532. 17. Pryor, W. A., Cueto, R., Jin, X., Koppenol, W. H., Ngu-Schwemlein, M., Squadrito, G. L., Uppu, P. L., and Uppu, R. M. (1995) Free Rad. Biol. Med. 18, 75– 83. 18. Uppu, R. M., Squadrito, G. L., Cueto, R., and Pryor, W. A. (1996) Methods Enzymol. 269, 311–321. 19. Zhang, H., Squadrito, G. L., Uppu, R. M., Lemercier, J.-N., Cueto, R., and Pryor, W. A. (1997) Arch. Biochem. Biophys. 339, 183–189. 20. Pryor, W. A., Jin, X., and Squadrito, G. L. (1994) Proc. Natl. Acad. Sci. USA 91, 11173–11177. 21. Squadrito, G. L., Jin, X., and Pryor, W. A. (1995) Arch. Biochem. Biophys. 322, 53–59. 22. Alvarez, B., Rubbo, H., Kirk, M., Barnes, S., Freeman, B. A., and Radi, R. (1996) Chem. Res. Toxicol. 9, 390 –396. 23. Simic, M. G., and Jovanovic, S. V. (1989) J. Am. Chem. Soc. 111, 5778 –5782. 24. Pryor, W. A., and Squadrito, G. L. (1995) Am. J. Physiol. (Lung Cell. Mol. Physiol. 12) 268, L699 –L722. 25. Coddington, J. W., Hurst, J. K., and Lymar, S. V. (1999) J. Am. Chem. Soc. 121, 2438 –2443. 26. Goldstein, S., Meyerstein, D., van Eldik, R., and Czapski, G. (1999) J. Phys. Chem. A 103, 6587– 6590. 27. Squadrito, G. L., and Pryor, W. A. (1998) Chem. Res. Toxicol. 11, 718 –719. 28. Pryor, W. A., Lemercier, J.-N., Zhang, H., Uppu, R. M., and Squadrito, G. L. (1997) Free Rad. Biol. Med. 23, 331–338. 29. Squadrito, G. L., and Pryor, W. A. (1998) Free Rad. Biol. Med. 25, 392– 403. 30. Zhang, H., Squadrito, G. L., and Pryor, W. A. (1997) Nitric Oxide 1, 301–307. 31. Ross, A. B., Mallard, W. G., Helman, W. P., Buxton, G. V., Huie, R. E., and Neta, P. (1998) NDRL-NIST Solution Kinetics Database Ver. 3.0 NIST, Gaithersburg, MD.
Reaction of Uric Acid with Peroxynitrite and Implications for the Mechanism of Neuroprotection by Uric Acid Giuseppe L. Squadrito,* ,1 Rafael Cueto,* Andres E. Splenser,* ,† Athanasios Valavanidis,‡ Houwen Zhang,* Rao M. Uppu,* and William A. Pryor* ,1 *Biodynamics Institute, 711 Choppin Hall, Louisiana State University, Baton Rouge, Louisiana 70803-1800; †Premedical Honors College, University of Texas Pan American, Edinburg, Texas 78539-2999; and ‡Department of Chemistry, University of Athens, University Campus Zografou, 15771 Athens, Greece
Received September 23, 1999
effects associated with peroxynitrite in many diseases. Peroxynitrite, a biological oxidant formed from the reaction of nitric oxide with the superoxide radical, is associated with many pathologies, including neurodegenerative diseases, such as multiple sclerosis (MS). Gout (hyperuricemic) and MS are almost mutually exclusive, and uric acid has therapeutic effects in mice with experimental allergic encephalomyelitis, an animal disease that models MS. This evidence suggests that uric acid may scavenge peroxynitrite and/or peroxynitrite-derived reactive species. Therefore, we studied the kinetics of the reactions of peroxynitrite with uric acid from pH 6.9 to 8.0. The data indicate that peroxynitrous acid (HOONO) reacts with the uric acid monoanion with k ⴝ 155 M ⴚ1 s ⴚ1 (T ⴝ 37°C, pH 7.4) giving a pseudo-first-order rate constant in blood plasma k Urate /plasma ⴝ 0.05 s ⴚ1 (T ⴝ 37°C, pH 7.4; assuming [uric acid] plasma ⴝ 0.3 mM). Among the biological molecules in human plasma whose rates of reaction with peroxynitrite have been reported, CO 2 is one of the fastest with a pseudo-first-order rate constant k CO2 /plasma ⴝ 46 s ⴚ1 (T ⴝ 37°C, pH 7.4; assuming [CO 2] plasma ⴝ 1 mM). Thus peroxynitrite reacts with CO 2 in human blood plasma nearly 920 times faster than with uric acid. Therefore, uric acid does not directly scavenge peroxynitrite because uric acid can not compete for peroxynitrite with CO 2. The therapeutic effects of uric acid may be related to the scavenging of the radicals CO 3•ⴚ and NO 2• that are formed from the reaction of peroxynitrite with CO 2. We suggest that trapping secondary radicals that result from the fast reaction of peroxynitrite with CO 2 may represent a new and viable approach for ameliorating the adverse
© 2000 Academic Press
1 To whom correspondence should be addressed. Fax: (225) 3884936. E-mail: [email protected] or [email protected].
2 Abbreviations used: MS, multiple sclerosis; DTPA, diethylenetriaminepentaacetic acid.
0003-9861/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
Key Words: uric acid; urate; peroxynitrite; carbon dioxide; nitric oxide; superoxide; oxidation; kinetics; free radical; antioxidant.
Uric acid is a natural antioxidant (1, 2) that is present in plasma in relatively high concentrations (in men, 302 ⫾ 60 M; in women, 234 ⫾ 52 M (3)). In patients with certain diseases, such as gout (hyperuricemic), plasma levels of uric acid are markedly higher than in normal subjects. Interestingly, gout and multiple sclerosis (MS) 2 are two diseases that are almost mutually exclusive (4). In MS, plaque areas in the brain have been associated with peroxynitrite-mediated damage (5). Peroxynitrite is an oxidant formed in vivo from the reaction of nitric oxide and the superoxide radical. In experimental allergic encephalomyelitis, an animal disease that models MS, administration of uric acid was found to have strong therapeutic effects (4). Thus, uric acid plays a protective role in these two diseases, where damage is mediated by peroxynitrite. Other diseases in which damage is currently thought to be mediated by peroxynitrite include arthritis (6), asthma (7), sepsis (8), inflammatory bowel disease (9), stroke (10), Alzheimer’s disease (11), Parkinson’s disease (12), amyotrophic lateral sclerosis (13), atherosclerosis (14), preeclampsia (15), and AIDS dementia (16). To probe the potential protective role of uric acid against peroxynitrite, we studied the kinetics of the
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reaction of uric acid with peroxynitrite using stoppedflow spectroscopy. MATERIALS AND METHODS Chemicals. Uric acid and sodium azide were purchased from Sigma (St. Louis, MO). All other chemicals were of analytical grade. Deionized water (⬎18 M⍀/cm) was used in the preparation of all buffers and reagents. Instrumentation. The stopped-flow instrument used for the measurement of reaction rates was an RSM 1000 from On-Line Instrument Systems (Bogart, GA) equipped with a UV/Vis rapid scan spectrophotometer and a 2-cm cell. The Sander 200 ozonizer used in the synthesis of peroxynitrite was from Erwin Sander (Uetze-Eltze, Germany). An HP8451A diode array UV/Vis spectrophotometer from Hewlett Packard (Willmington, DE) was used to monitor the concentration of peroxynitrite during its preparation and also to measure its concentration in stock and working solutions. Peroxynitrite synthesis. Peroxynitrite was synthesized by ozonation of an aqueous solution of sodium azide (17, 18). Briefly, a solution of sodium azide (0.1 N, 100 mL; pH 12 adjusted with NaOH) was chilled to ⬍5°C and ozonized by feeding oxygen to the ozonator at a flow rate of 300 mL/min. The concentration of peroxynitrite was monitored by measuring A 302 nm of samples diluted 100 times with water adjusted to pH 12 with NaOH. It required about 2 h for the absorbance to reach the maximum (ca. 1.1), and the ozonation was stopped after the absorbance decreased to ca. 0.9 (18, 19). The resulting solution was purged for 10 min with nitrogen and then stored at ⫺12°C until used. Stopped-flow kinetics of peroxynitrite decomposition in aqueous phosphate buffers. The reactions were conducted on the stopped flow spectrophotometer thermostated at 25.0 ⫾ 0.1 or 37.0 ⫾ 0.1°C. Briefly, peroxynitrite solutions (0.2 mM, pH 11.5–12.0) were mixed with equal volumes of 100 mM sodium phosphate buffer, pH 6.7– 8.0, containing uric acid (from 0 to 5 mM). Sodium pyrophosphate buffers (100 mM) were used for pH ⬎ 8.0. The pH values were measured both before and after mixing of reactants. A small pH jump (ca. 0.2) was usually observed on mixing, and the final pH was reported unless otherwise stated. Data for the disappearance of peroxynitrite collected at wavelengths longer than 330 nm were analyzed using the OLIS software. [Uric acid absorbs strongly at wavelengths shorter than 330 nm and interferes with our measurements.] The low solubility of uric acid at low pH prevented measuring apparent second-order rate constants at pH values lower than 6.7. In addition, the solubility of uric acid is lower in pyrophosphate buffers and this further limited the working concentration range at pH ⬎ 8.0. Studies of the reaction kinetics at temperatures lower than 25°C were hampered by the low solubility of uric acid. At temperatures higher than 37°C, the uric acid-independent component (the first-order component) of the decomposition of peroxynitrite takes over due to the larger activation energy of this component relative to its uric aciddependent counterpart (the second-order component) and this precludes accurate measurement of the reaction rates of uric acid with peroxynitrite. The ionic strength was not controlled but this is of no consequence because increasing the ionic strength with NaCl above I ⫽ 0.09 M up to levels corresponding to the higher pH while keeping all other parameters unchanged, had insignificant effects on k obs. Also, k obs is relatively insensitive to changes in the concentration of the buffer from 10 to 200 mM. Table Curve 3D (Jandel Scientific Software) was used to analyze k obs as a function of pH and the uric acid concentration ([uric acid] ⬎ 0) with k 1 , k 2 , and K a as adjustable parameters (see Results and Discussion); 3D fitting has the advantage of being able to consider the exact value of the pH of every data point and this is important due to the relatively narrow working pH range of uric acid. Data points represent the average of at least three independent experiments. Data points with [uric acid] ⫽ 0 were
excluded because of a behavior similar to that previously observed for other substrates [such as methionine and 2-keto-4-thiomethylbutanoic acid (20), ascorbate (21), and tryptophan (22)] in which curvature is observed at low substrate concentrations. With all data points with [uric acid] ⬎ 0 the reaction is first order in both uric acid and peroxynitrite and these data points were used to determine the second-order rate constant. Uric acid oxidation yields. The appropriate amount of uric acid was dissolved in a minimal volume of NaOH (0.5 M) and diluted with phosphate buffer (pH 7.2, 0.10 M) containing 0.1 mM DTPA. Uric acid (0.2 mM) was allowed to react with increasing concentrations of peroxynitrite (0 to 0.8 mM) in a final volume of 2 mL. When appropriate, the reaction mixture also contained 10 mM sodium bicarbonate. Rapid mixing was accomplished with a vortex mixer. The uric acid oxidation yields were calculated by relating the areas under the unreacted uric acid peaks of the reaction mixtures HPLC traces. The samples were analyzed on a Perkin-Elmer Series 410 liquid chromatograph (Perkin-Elmer, Norwalk, CT) equipped with a PerkinElmer LC-95 UV/Vis spectrophotometer using a Phenomenex LUNA 5-m C18(2) 150 ⫻ 4.6-mm column and an isocratic solvent system consisting of 95% 25 mM KH 2PO 4/H 3PO 4 and 5% methanol at a flow of 1.0 mL/min. The unreacted uric acid acid eluted with a retention time of 3.2 min. The reaction products are in both cases complex mixtures that contain allantoin and parabanic acid, which are likely formed from the hydrolysis of the unstable dismutation product dehydrouric acid.
RESULTS AND DISCUSSION
Stopped-flow kinetics of the reaction of uric acid with peroxynitrite. Figures 1A and 1B show 3D plots of the
FIG. 1. Rate constants for the reaction of peroxynitrite with uric acid at (A) 25.0°C and (B) 37.0°C as a function of the pH and uric acid concentration.
URIC ACID OXIDATION BY PEROXYNITRITE-DERIVED RADICALS TABLE I
Values for k 1 , k 2 , and K a That Best Describe the Data Points Shown in Figs. 1a and 1b T (°C) 25.0 37.0
k 1 (s ⫺1)
k 2 (M ⫺1 s ⫺1)
Ka
pK a
1.5 5.2
380 540
7.9 ⫻ 10 ⫺8 1.0 ⫻ 10 ⫺7
7.1 7.0
observed rate constant (k obs) as a function of pH and uric acid concentration for data collected at 25.0 and 37.0°C, respectively. The data points were best fit by Eq. (1) using k 1 , k 2 , and K a as adjustable parameters. This indicates that (a) the reaction is first order in peroxynitrite and first order in uric acid, and (b) that peroxynitrous acid (HOONO) is the reactive species: k obs ⫽
共k a ⫹ k 2 ⫻ 关uric acid兴兲 ⫻ 关H ⫹ 兴 . K a ⫹ 关H ⫹ 兴
[1]
Uric acid has pK a 1 ⫽ 5.4 and pK a 2 ⫽ 9.8 (23) and exists largely as a single species (the monoanion) within the pH range that we study here (from 6.7 to 8.0). Thus, HOONO must react with the uric acid monoanion. The values for k 1 , k 2 , and K a that best describe each set of data points are shown in Table I. The values of pK a that we obtain here are in good agreement with values reported earlier for the pK a of HOONO (24). The pK a of HOONO is generally taken as 6.8 and the values we report here are 7.1 (25°C) and 7.0 (37°C). We estimate the 95% confidence interval for these values is 0.2 pK units. We feel that these values are in agreement with the value for the pK a of HOONO, conforming with our mechanism. The values of k 1 shown in Table I should be interpreted as the maximum rate constants for the firstorder decomposition of peroxynitrite, because uric acid traps intermediates and blocks reversible steps that could occur during the decomposition of peroxynitrite (25, 26). The values of k 2 in Table I represent the pH-independent second-order rate constants for the reaction of HOONO with the uric acid monoanion, as shown in Eq. (2); k 2 is relatively insensitive to changes in temperature. An estimation of the of the Arrhenius energy of activation using just two temperatures, 25.0 and 37.0°C, yields approximately 5– 6 Kcal/mol: k2
HOONO ⫹ urate ion O ¡ products.
[2]
Physiological impact of the reaction of uric acid with peroxynitrite: A kinetic analysis. The pseudo-first-order rate constant for the reaction of uric acid with peroxynitrite in human blood plasma (k Urate /plasma) can be
335
calculated by using Eq. (3) and substituting for k 2 ⫽ 540 M ⫺1 s ⫺1and K a ⫽ 1.0 ⫻ 10 ⫺7 (the values obtained at 37°C shown in Table I), pH 7.4 ([H ⫹] ⫽ 4.0 ⫻ 10 ⫺8 M), and [uric acid] plasma ⫽ 3 ⫻ 10 ⫺4 M: k Urate/plasma ⫽ k 2 关uric acid兴 ⫻
关H ⫹ 兴 ⫽ 0.05 s ⫺1. [3] K a ⫹ 关H ⫹ 兴
This affords k Urate /plasma ⫽ 0.05 s ⫺1, a relatively low value owing to the relatively small second-order rate constant and low concentration of uric acid. The value for k Urate /plasma can be compared to the pseudo-first-order rate constant for the reaction of CO 2 with peroxynitrite in blood plasma (k CO2 /plasma ⫽ 46 s ⫺1) (27). Since the reaction of peroxynitrite with CO 2 is about 920 times faster than the corresponding reaction with uric acid, uric acid cannot directly scavenge peroxynitrite. Instead, uric acid’s role in preventing peroxynitrite-mediated damage must involve scavenging reactive intermediates generated from the reaction of peroxynitrite with carbon dioxide. Physiological role of uric acid as a scavenger of peroxynitrite. The reaction of peroxynitrite with CO 2 is an important reaction of peroxynitrite in blood. This is because of the fast reaction of peroxynitrite with CO 2 and the high concentration of CO 2 in blood (27). As shown in Fig. 2, the reaction of peroxynitrite with CO 2 involves the peroxynitrite anion and forms nitrosoperoxycarbonate (ONOOCO 2⫺), which then rapidly homolyzes to form a pair of caged radicals [CO 3•⫺ •NO 2]. The caged radicals can then diffuse apart and become free radicals or combine to form the nitrocarbonate (O 2NOCO 2⫺), which then decomposes to NO 3⫺ and CO 2. This last reaction is responsible for the catalytic action of CO 2 during the decomposition of peroxynitrite (27– 29). Uric acid can scavenge only the free radicals CO 3•⫺ and •NO 2, since ONOOCO 2⫺, O 2NOCO 2⫺, and the caged radicals [CO 3•⫺ •NO 2] are all too short-lived to be trapped by potential scavengers. Under conditions in which the reaction of peroxynitrite with CO 2 is the dominant reaction of peroxynitrite, the maximum yield of uric acid oxidation will depend on the yield of free radicals that escape from the solvent cage. This yield has been estimated to be ca. 20% (28, 30). Nitrogen dioxide reacts rapidly with uric acid in aqueous solution with a rate constant of 1.8 ⫾ 0.2 ⫻ 10 7 M ⫺1 s ⫺1 (23). The rate constant for the reaction of uric acid with CO 3•⫺ is unknown. However, CO 3•⫺ is likely to be even more reactive than •NO 2 toward uric acid, just as it is toward ascorbate (for CO 3•⫺ k ascorbate ⫽ 1 ⫻ 10 9 M ⫺1 s ⫺1; for •NO 2 k ascorbate ⫽ 3.5 ⫻ 10 7 M ⫺1 s ⫺1) (31). Thus, uric acid may provide biological systems with an efficient first line of defense toward the free radicals CO 3•⫺ and •NO 2 that are formed from the reaction of peroxynitrite with CO 2.
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FIG. 2.
Uric acid as a scavenger of free radicals derived from the reaction of peroxynitrite with CO 2.
In order to test if uric acid scavenges CO 3•⫺ and/or NO 2 generated from the reaction of peroxynitrite with carbon dioxide, we measured the yields of oxidation of uric acid (200 M) by peroxynitrite (0 – 800 M) in the presence of bicarbonate (10 mM) at pH 7.2 and 25°C. (Fig. 3). Under these conditions, approximately 98% of the peroxynitrite reacts with CO 2, about 2% decomposes in a reaction that is zero order in uric acid, and 0.3% reacts directly with uric acid. However, uric acid is oxidized in 17 mol% yield relative to peroxynitrite (r 2 ⫽ 0.99), proving that uric acid reacts with reactive intermediates from the reaction of peroxynitrite with CO 2, which we suggest are the free radicals CO 3•⫺ and • NO 2. The yield of oxidized uric acid (17%) is in good agreement with the maximum yield of radicals (ca. 20%) produced by the reaction of peroxynitrite with carbon dioxide (28, 30). These results strongly suggest that any protection from peroxynitrite-mediated damage that may be attributed to uric acid is due to the ability of uric acid to scavenge the free radicals CO 3•⫺ and •NO 2 and not to uric acid scavenging peroxynitrite directly. The yields of uric acid oxidation obtained under these conditions without adding bicarbonate are 15% and are also shown in Fig. 3. Uric acid oxidation effected under these conditions arises from the reactions of uric acid with activated species that are produced during the decomposition of HOONO, such as the free radicals HO • and •NO 2 formed from the homolysis of the peroxo bond (25) and also from the direct reaction of uric acid with peroxynitrite. However, all these reactions are unlikely to occur in the physiological environment because both the self-decomposition of peroxynitrite and the direct reaction of peroxynitrite with •
uric acid are too slow to compete with the reaction of peroxynitrite with CO 2. SUMMARY AND CONCLUSIONS
Potential direct scavengers of peroxynitrite cannot effectively compete with CO 2 in the physiological environment (27). Uric acid reacts relatively slowly with peroxynitrite itself but, strikingly, it efficiently scavenges the free radicals CO 3•⫺ and •NO 2, which are produced following the fast reaction of peroxynitrite with CO 2. These observations suggest that targeting secondary radicals from the fast reaction of peroxynitrite
FIG. 3. Yields of uric acid oxidation by peroxynitrite in the presence (F) or absence (Œ) of added bicarbonate.
URIC ACID OXIDATION BY PEROXYNITRITE-DERIVED RADICALS
with CO 2 may represent a new and viable approach toward fighting the adverse effects associated with peroxynitrite in many diseases. ACKNOWLEDGMENTS This publication was made possible in part by Grant ES-06754 from the National Institute of Environmental Health Sciences, NIH (to W.A.P.), and by a Howard Hughes Medical Institute grant through the Undergraduate Biological Sciences Education Program to Louisiana State University (to A.E.S.). We thank Dr. Koppenol for the gift of the stopped-flow instrument.
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