detection of superoxide radicals and peroxynitrite by...

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 248, 211–215 (1998)ARTICLE NO. RC988936

Detection of Superoxide Radicals and Peroxynitriteby 1-Hydroxy-4-phosphonooxy-2,2,6,6-tetramethylpiperidine: Quantification of ExtracellularSuperoxide Radicals Formation

S. Dikalov,*,1 I. A. Grigor’ev,† M. Voinov,† and E. Bassenge‡*Institute of Chemical Kinetics & Combustion, Novosibirsk, Russia; †Institute of Organic Chemistry, Novosibirsk, Russia;and ‡Institute of Applied Physiology, University of Freiburg, Germany

Received May 18, 1998

Recently, we have reported that the use of stericallyThe reactions of the new sterically hindered hydrox- hindered hydroxylamines 1-hydroxy-2,2,6,6-tetramethyl-

ylamine 1-hydroxy-4-phosphonooxy-2,2,6,6-tetrameth- 4-oxo-piperidine (TEMPONE-H) and 1-hydroxy-3-car-ylpiperidine (PP-H) with superoxide radical and per- boxy-pyrrolidine (CP-H) (6, 7) gives the possibility tooxynitrite have been studied. These reactions produce quantify the formation of superoxide radical and peroxy-the nitroxide 4-phosphonooxy-2,2,6,6-tetramethyl-pip- nitrite by means of nitroxides 2,2,6,6-tetramethyl-4-oxo-eridinyloxy. The rate constant for reaction of superox-

piperidinyloxy and 3-carboxy-proxyl (CP) detection (8).ide with PP-H is determined as (8.4{0.6)r102 M01s01. ItThe use of hydroxylamines is shown to increases the sen-was found that PP-H provides almost the same spinsitivity of the superoxide radical and peroxynitrite detec-trapping efficacy as 1-hydroxy-3-carboxy-pyrrolidinetion about 10 times as compared with nitrone-based spin(CP-H). The background oxidation of PP-H in blood istraps (6).much less than for CP-H. The extremely slow PP-H

However, both TEMPONE-H and CP-H readily pene-penetration into the cells makes possible the study oftrate through the lipid membrane (9). The new stericallyextracellular formation of superoxide radical. Thehindered hydroxylamine 1-hydroxy-4-phosphonooxy-acute treatment of blood with nitroglycerin is shown2,2,6,6-tetramethylpiperidine (PP-H), being anion atto induce an extracellular superoxide radical forma-

tion. PP-H is more sensitive for detection of reactive physiological pH values, extremely slow penetrates intooxygen species as compared with CP-H. PP-H is an ef- the cells (10), that makes possible the study of extracel-fective scavenger of superoxide radical and of peroxy- lular formation of superoxide radical.nitrite, and can be used to quantify the extracellular In this work we have studied the reactions of 1-hy-formation of these reactive oxygen species. q 1998 droxy-4-phosphonooxy-2,2,6,6-tetramethylpiperidineAcademic Press with superoxide radical and peroxynitrite. The PP-H

and CP-H stability and efficacy of superoxide radicalscavenging in the whole blood were compared. It wasshown that acute treatment of blood with nitroglycerinSuperoxide radical and peroxynitrite are well known

to play an important role in the development of oxida- induces extracellular formation of superoxide radicals(Scheme 1).tive damage (1, 2). Studying the development of patho-

logical conditions mediated by oxidative damage it isvery important to quantify the formation of reactiveoxygen species. The electron spin resonance is the mostdirect and sensitive methods for detection of oxygenradicals (3). However, the quantitative determinationof superoxide radical and peroxynitrite in biologicalsystems is still problematical (4, 5).

1 To whom correspondence should be addressed. NIEHS, P.O. Box12233, MD F002, Durham, NC 27709. Fax: (919) 541-1043, E-mail:[email protected]. SCHEME 1. Chemical structures of PP-H and CP-H.

0006-291X/98 $25.00Copyright q 1998 by Academic PressAll rights of reproduction in any form reserved.

211

AID BBRC 8936 / 6958$$$$21 06-23-98 16:58:16 bbrcgs AP: BBRC

Vol. 248, No. 2, 1998 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MATERIALS AND METHODS

1-Hydroxy-4-phosphonooxy-2,2,6,6-tetramethylpiperidine (PP-H)and 1-hydroxy-3-carboxy-pyrrolidine (CP-H) were supplied by AlexisCorporation (Switzerland). DMSO, deferoxamine mesylate, superox-ide dismutase from bovine erythrocyte (SOD), and xanthine wereobtained from Sigma (Germany). Peroxynitrite was obtained fromAlexis Corporation (USA). The water solutions of GTN, containingof 0.9% NaCl, was supplied by Pohl Boskamp (Hohenlockstedt, Ger-many). Xanthine oxidase was purchased from Fluka. Blood wasdrawn from the carotid artery of dogs into a citric acid solution (6:1vol/vol) (11).

Preparation of PP-H and CP-H stock solutions. PP-H and CP-Hwere dissolved in oxygen-free (nitrogen bubbled) 0.1M sodium phos-phate buffer (pH 7.4) in the presence of 0.9% NaCl and 50 mM defer-oxamine. Deferoxamine was used to decrease the self-oxidation ofhydroxylamines catalyzed by traces of transition metal ions. The FIG. 1. Formation of 4-Phosphonooxy-TEMPO in xanthine oxi-concentration of PP-H and CP-H in the stock solutions was 10mM. dase plus xanthine (XO/X) superoxide generating system in the fol-Prior to the experiments, stock solutions were kept frozen or in a lowing samples: 0.5mM PP-H (a); 0.5mM PP-H / XO/X (b); 0.5mMcool airtight place. Nitroxide content in the stock solution of hydrox- PP-H / SOD / XO/X (c). ESR settings were described in Materialsylamines was about 0.02%. and Methods. Hyperfine ESR splitting constant is aNÅ17.1 G, line

width is 1.7 G.Spin trapping experiments. All ESR samples were placed in 100ml glass tubes and were prepared using 0.1M sodium phosphatebuffer (pH 7.4) in the presence of 0.9% NaCl. In order to inhibit iron-catalysed reactions deferoxamine (50 mM) was added to all samples.

solution of PP-H contains the trace quantity of 4-phospho-The ESR measurements were performed at room temperature usingnooxy-TEMPO (Fig. 1a). A significant amount of ni-an EMX-A ESR spectrometer (Bruker). The ESR-settings were the

following: field sweep 60 G, microwave frequency 9.72 GHz, micro- troxide 4-phosphonooxy-TEMPO (PP) is formed in thewave power 20 mW, modulation amplitude 1 G, conversion time 655 reaction of PP-H with superoxide radical (Fig. 1b). Thems, detector time constant 1310 ms, receiver gain 105. ESR amplitude of 4-phosphonooxy-TEMPO was con-

Superoxide radical generation. Xanthine oxidase superoxide gen- stantly increasing as a result of continuous formation oferating system (12) contained xanthine oxidase (20mg/ml), xanthine superoxide radicals and their reaction with PP-H. The(50mM), deferoxamine (50 mM) in 0.1M sodium phosphate buffer (pH

SOD addition (100U/ml) completely inhibited the forma-7.4) in the presence of 0.9% NaCl. The rate of superoxide radicalgeneration was controlled by cytochrome C assay (13). tion of 4-phosphonooxy-TEMPO (Fig. 1c).

Recently we have studied the reaction of CP-H withSpin trapping of peroxynitrite. As a source of peroxynitrite thestock solution of ONOONa (1mM stabilised in 0.3M NaOH) was used. superoxide radical (7). The rate constant of this reac-Concentration of peroxynitrite in 0.3M NaOH was determined spec- tion was determined by using SOD as a competitivetrophotometrically using the extinction coefficient e (302nm)Å1670 inhibitor of the reaction of CP-H with superoxide radi-M01 cm01 (14).

cal (7). The rate constants for the reactions of PP-HQuantification of superoxide radical and peroxynitrite. The abso- with superoxide radical were calculated by using thelute rates of superoxide radical formation in xanthine/xanthine oxi-

same method from the dependence of the rate of ni-dase system were determined in the presence of PP-H and CP-H (0.5mM) as a SOD (100 U/ml) inhibited formation of nitroxide radicals. troxide formation versus SOD concentrations (see Fig.The reaction of peroxynitrite with PP-H and CP-H was performed 2): (Vo/V)01Åk*PP-Hr[PP-H]/kSODr[SOD], where Vo andby quick mixing of hydroxylamine solution (0.5 mM) with small ali- V represent the rates of formation of nitroxide in thequot of peroxynitrite (in 0.3M NaOH). Amount of peroxynitrite

absence and in the presence of SOD, respectively. Thetrapped was quantified from nitroxide radical formation. Final pH ofk*PP-H and kSOD are the second order rate constants ofmixtures was controlled (pHÅ7.4). In all experiments hydroxylamine

was in excess to nitroxide radical. Relative concentration of ni- the superoxide radical reactions with PP-H and SOD,troxides during experiments was in range from 0.02% till 1% to the respectively. According to the above equation the rateconcentration of hydroxylamine. constant for reaction of superoxide with PP-H was de-

Determination of rate constants by competitive reactions. The rate termined as (8.4{0.6)r102 M01s01. This value is less asconstant for reaction of PP-H with superoxide radical was deter- compared with previously reported for CP-H (3.21103mined by competitive kinetics as described in (5) using SOD as com-

M01s01) (7). Nevertheless, the amounts of superoxidepetitive reagent. The rate constant for the reaction of PP-H withradical trapped were almost the same both for 0.5mMperoxynitrite was determined using DMSO as competitive reagent

for peroxynitrite (6). All measurements were done at pHÅ7.4. CP-H and 0.5mM PP-H.Both PP-H and CP-H are found to provide much more

RESULTS AND DISCUSSION superoxide radical scavenging efficacy as comparedwith spin trap DMPO. The rate constant for the reac-

Reaction with Superoxide Radical tion of superoxide radical with DMPO is only 35 M01s01

(15), which is hundreds times smaller the value for PP-Reaction of PP-H with superoxide radical was studiedin xanthine oxidase superoxide generating system. Stock H and CP-H. Moreover, PP-H provides much higher

212



FIG. 4. Quantification of superoxide radical formation in thexanthine oxidase plus xanthine superoxide generating system inFIG. 2. Determination of the rate constant for the reaction ofthe absence and in the presence of the whole blood (80%) measuredPP-H with superoxide radical using SOD as competitive inhibitor.as the rate of formation of the 3-carboxy-proxyl or 4-phosphonooxy-The rate constant was calculated from the following equation: (Vo/TEMPO using CP-H and PP-H, respectively. Standard deviationsV)01Åk*PP-Hr[PP-H]/kSODr[SOD], where Vo and V represent thefor columns are shown (nÅ6; *põ0.05 vs XO/X / CP-H, **põ0.01rate of nitroxide formation in the absence and presence of SOD,vs. XO/X / PP-H).respectively. The k*PP-H and kSOD are the second order rate con-

stants of the superoxide radical reactions with PP-H and SOD,respectively.

reactivity in the reaction with peroxynitrite. The ratioof the PP-H and DMSO oxidation rate constants was

sensitivity of the superoxide radical detection method calculated from the dependence of nitroxide 4-phos-due to the larger life-time of nitroxide 4-phosphonooxy- phonooxy-TEMPO resulting concentration versus theTEMPO as compared with DMPO-OH spin-adduct. DMSO concentration (Fig. 3) by using the following

equation:Reaction of PP-H with Peroxynitrite

The reaction of PP-H with peroxynitrite afforded the (Ao/A)01ÅkPP-Hr[PP-H]/kDMSOr[DMSO],stable nitroxide 4-phosphonooxy-TEMPO (data notshown). In our previous study of the reaction of CP-H where Ao and A represent the amplitudes of the low-with peroxynitrite (6, 7) DMSO was used as a competi- field component of the ESR spectrum of PP in the ab-tive inhibitor of CP-H oxidation by peroxynitrite. We sence and in the presence of DMSO, respectively. Theapplyed the same method to compare PP-H and DMSO kPP-H and kDMSO are the second order rate constants for

the reactions of peroxynitrite with PP-H and DMSO,respectively.

The ratio of the rate constants kPP-H/kDMSO is foundto be 3.2{0.1 that is very close to the value kCP-H/kDMSOÅ2.6{0.2. These data testify that PP-H is an ef-fective scavenger of peroxynitrite.

Quantification of Extracellular Formationof Superoxide Radical

The hydroxylamine PP-H, possessing the negativecharge on phosphonooxy moiety at physiological pHvalues, extremely slow penetrates into the cell (10) thatmakes possible the study of extracellular formation of

FIG. 3. Determination of the rate constant for the reaction of superoxide radical. In order to demonstrate the possi-PP-H with peroxynitrite using DMSO as competitive inhibitor. bilities of this method for the determination of superox-The rate constant was calculated from the following equation:(Ao/ ide radical extracellular formation the following experi-A)01ÅkPP-Hr[PP-H]/kDMSOr[DMSO], where Ao and A represent

ments were performed:the amplitude of the low-field component of the ESR spectrum of4-Phosphonooxy-TEMPO in the absence and presence of DMSO, 1. The efficacy of the reaction of PP-H with superox-respectively. The kPP-H and kDMSO are the second order rate con-

ide radical extracellularly generated by xanthine oxi-stants for the reactions of peroxynitrite with PP-H and DMSO,respectively. dase in the whole blood was determined;

213



superoxide radical formation (Fig. 5, difference be-tween control and nitroglycerin columns). Thus, acutetreatment of the blood with the nitroglycerin inducesthe extracellular superoxide formation in the great ex-tent than intracellular one.

The nitroxides 3-carboxy-proxyl and 4-phospho-nooxy-TEMPO, being placed to the biological system,can be reduced back to the ESR silent hydroxylaminesCP-H and PP-H. This background process could causethe underestimation of superoxide formation and thedecrease of detection limits. Luckily, both 3-carboxy-proxyl and 4-phosphonooxy-TEMPO are much morestable in blood as compared with uncharged nitroxides

FIG. 5. Quantification of GTN induced superoxide radical forma- (7, 16).tion in the whole blood measured as the rate of formation of the 3- Thus, PP-H provides a high sensitivity for the quan-carboxy-proxyl or 4-phosphonooxy-TEMPO using CP-H and PP-H, tification both of superoxide radical and peroxynitriterespectively. Standard deviations for columns are shown (nÅ9; formation and makes possible the detection of extracel-*põ0.05 vs. control, **põ0.01 vs. GTN).

lular superoxide radical formation.

CONCLUSION2. The superoxide radical extracellular formationduring the acute treatment of blood with nitroglycerin

PP-H is considered to be an effective scavenger ofwas quantified.superoxide radical and of peroxynitrite. PP-H pro-

CP-H is reported to be an effective scavenger of su- vides a high sensitivity for the quantification of for-peroxide radical even in the whole blood (8). The mation both of superoxide radical and peroxynitrite.amount of superoxides trapped by PP-H in the pure The use of PP-H is shown to make possible the studyxanthine oxidase system is found to be about the same of extracellular formation of superoxide radical. Theas for CP-H (Fig. 4). The presence of the whole blood acute treatment of blood with nitroglycerin is foundresults in the 3-fold decrease of the superoxide radical to induce an extracellular formation of superoxidescavenging efficacy because of competition between hy- radical.droxylamines and blood antioxidant system. The The results, received in this work, allow for the state-amount of superoxide radicals trapped by PP-H in the ment, that PP-H can be used for spin trapping experi-whole blood is similar to the value for CP-H (Fig. 4). ments both in vitro and ex vivo.Therefore, PP-H is an effective scavenger of superoxideradical even in the presence of extracellular antioxi-dant system of the blood. ACKNOWLEDGMENTS

Nitroglycerin induced extracellular formation of su-peroxide radical in the whole blood was studied as well. This study was supported by the scholarship from German Aca-

demic Exchange Service (Deutsche Akademische Austauschdienst,For this purpose the rates of nitroxides formation wereBonn, Germany) for Dr. S. Dikalov and Young Scientist Grant fromdetermined in the control experiment without nitro-Siberian Branch of Russian Academy of Science. The authors areglycerin and in the presence of 0.2mM of nitroglycerin grateful to Dr. B. Fink for supplying blood samples and to Dr. M.

(Fig. 5). Skatchkov for fruitful discussion.The background oxidation of PP-H in blood is much

less than for CP-H (Fig. 5). It is likely to be due to theCP-H being oxidized inside of the erythrocytes while REFERENCESPP-H does not penetrate through the lipid membrane.

1. Halliwell, B., and Gutterige, J. M. C. (1990) in Methods in Enzy-Therefore, PP-H is more sensitive than CP-H for detec-mology (Packer, L., and Glazer, A. N., Eds.) 186, pp. 1–85, Aca-tion of reactive oxygen species.demic Press, London.Acute treatment of blood with nitroglycerin lead to

2. Pryor, W. A., Jin, X., and Squadrito, G. L. (1994) Proc. Natl.increase of nitroxides formation (Fig.5). As a result ofAcad. Sci. USA 91, 1173–1177.

extremely slow cell penetration PP-H traps only extra-3. Janzen, E. G., and Hare, D. L. (1990) Advances in Radical Chem-cellularly formed superoxide radical, testifying the ex- istry 1, 253–295.

tracellular mode of superoxide radical formation upon 4. Lemercier, J. N., Squadrio, G. L., and Pryor, W. A. (1995) Arch.the acute treatment of blood with nitroglycerin. It Biochem. Biophys. 321, 31–39.should be noted that both PP-H and CP-H give about 5. Rosen, G. M., Finkelstein, E., and Rauckman, E. J. (1982) Arch.

Biochem. Biophys. 215, 367–378.the same values for the rate of nitroglycerin induced

214



6. Dikalov, S., Skatchkov, M., and Bassenge, E. (1997) Biochem. 12. Fridovich, I. (1986) in CRC Handbook of Methods for OxygenRadical Research (Greenwald, R. A., Ed.) pp. 51–53, CRC Press,Biophys. Res. Commun. 230, 54–57.New York.7. Dikalov, S., Skatchkov, M., and Bassenge, E. (1997) Biochem.

13. Fridovich, I. (1986) in CRC Handbook of Methods for OxygenBiophys. Res. Commun. 231, 701–704.Radical Research (Greenwald, R. A., Ed.) pp. 121–122, CRC8. Dikalov, S., Skatchkov, M., Fink, B., and Bassenge, E. (1997)Press, New York.Nitric Oxide: Biology and Chemistry 1(5), 423–431.

14. Beckman, J. S., Beckman, T. W., Chen, J., Marchall, P. A., and9. Swartz, H. M., Sentjurc, M., and Morse, P. H. (1986) Biochem. Freeman, B. A. (1990) Proc. Natl. Acad. Sci. USA 87, 1620–1624.

Biophys. Acta 888, 82–90. 15. Rosen, G. M., and Rauckman, E. J. (1984) Methods in Enzymol-10. Ross, A. H., and McConnell, H. (1975) Biochemistry 14, 2793– ogy 105, 198–209.

2798. 16. Couet, W. R., Brasch, R. C., Sosnovsky, G., Lukszo, J., Prakash,I., Gnewuch, C. T., and Tozer, T. N. (1985) Tetrahedron 41,11. Bassenge, E., and Fink, B. (1996) Naunyn-Schmiedeberg’s Arch.

Pharmacol. 353, 363–367. 1165–1172.

215


detection of superoxide radicals and peroxynitrite by...

Documents