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Proc. Nati. Acad. Sci. USA Vol. 87, pp. 1620-1624, February 1990 Medical Sciences Apparent hydroxyl radical production by peroxynitrite: Implications for endothelial injury from nitric oxide and superoxide (endothelium-derived relaxing factor/desferrioxamine/ischemia/superoxide dismutase) JOSEPH S. BECKMAN*t, TANYA W. BECKMAN*, JUN CHEN*, PATRICIA A. MARSHALL*, AND BRUCE A. FREEMAN*t Departments of *Anesthesiology and *Biochemistry, University of Alabama at Birmingham, Birmingham, AL 35233 Communicated by Irwin Fridovich, December 4, 1989 (received for review October 4, 1989) ABSTRACT Superoxide dismutase reduces injury in many because of efficient scavenging systems. The rate constant disease processes, implicating superoxide anion radical (02 ) as for the reduction of Fe3" by O2 is only 1 x 106 M-1 s- (4), a toxic species in vivo. A critical target of superoxide may be while other cellular compounds such as ascorbate can reduce nitric oxide (NO-) produced by endothelium, macrophages, iron and are present in much higher concentrations than °2- neutrophils, and brain synaptosomes. Superoxide and NO- are (5). Thus, the contribution of °2 to HOP formation by the known to rapidly react to form the stable peroxynitrite anion iron-catalyzed Haber-Weiss reaction may be limited in vivo, (ONOO-). We have shown that peroxynitrite has a pKa of7.49 suggesting that other reactions may be important in under- ± 0.06 at 3TC and rapidly decomposes once protonated with standing O-j toxicity to endothelium. We propose that nitric a half-life of 1.9 sec at pH 7.4. Peroxynitrite decomposition oxide (NO-), a stable free radical, reacts with O-j in many generates a strong oxidant with reactivity similar to hydroxyl pathological states to yield secondary cytotoxic species. radical, as assessed by the oxidation ofdeoxyribose or dimethyl Recently, endothelium, macrophages, and brain synapto- sulfoxide. Product yields indicative of hydroxyl radical were some preparations have been shown to produce NOR by 5.1 ± 0.1% and 24.3 + 1.0%, respectively, of added perox- oxidizing arginine by a calcium-activated NADPH-depen- ynitrite. Product formation was not affected by the metal dent enzyme (6-9). NOR appears to be a major form of the chelator diethyltriaminepentaacetic acid, suggesting that iron endothelium-derived relaxing factor (EDRF) (10). Vasodila- was not required to catalyze oxidation. In contrast, desferri- tory agents such as acetylcholine, ATP, and bradykinin oxamine was a potent, competitive inhibitor of peroxynitrite- initiate a receptor-mediated influx of Ca2 , triggering the initiated oxidation because of a direct reaction between des- production and extracellular release of NO-, which then ferrioxamine and peroxynitrite rather than by iron chelation. activates soluble heme-containing guanylate cyclases to pro- We propose that superoxide dismutase may protect vascular duce cGMP in vascular smooth muscle and platelets. In- tissue stimulated to produce superoxide and NON under patho- creased cGMP promotes relaxation in vascular smooth mus- logical conditions by preventing the formation ofperoxynitrite. cle and inhibits platelet aggregation as well as adhesion of platelets to endothelium (11). Macrophages produce NOR as Vascular injury secondary to ischemia/reperfusion, inflam- part of their cytotoxic armamentarium (6). mation, xenobiotic metabolism, hyperoxic exposure, and The half-life of EDRF and NON ranges from 4 to 50 sec (12), other diseases results in loss of endothelial barrier function, which is approximately doubled by SOD (13, 14). NOR does adhesion of platelets, and abnormal vasoregulation. The not bind directly to the copper of SOD (15), suggesting that ability of superoxide dismutase (SOD) to often reduce endo- stabilization involves the scavenging of O- . Because NOR thelial injury indirectly implicates the participation of super- contains an unpaired electron and is paramagnetic, it rapidly oxide anion radical (Oj-) with many pathological processes reacts with °-- to form peroxynitrite anion (ONOO-) in high (1). While O-' can be directly toxic (2), it has a limited yield (16). In alkaline solutions, ONOO- is stable but has a reactivity with most biological molecules, raising questions PKa of 6.6 at 0C (17) and decays rapidly once protonated. about its toxicity per se (3). To account for the apparent toxicity of O-j in vivo, the secondary production of the + NO-* ONOO- + H+= ONOOH far-more-reactive hydroxyl radical (HO-) is frequently pro- posed to occur by the iron-catalyzed Haber-Weiss reaction: ->HO- + N02-* NN37 + HW. [4] 20- + 2H+ H202 + 02 [1] In the gaseous phase, decomposition of peroxynitrous acid to form HO- and nitrogen dioxide (NO29) is important in the O°2+ Fe3+ 02 + Fe2+ [21 formation of smog and acid rain. Although HOP and NO2 can recombine to form nitric acid, the rate constant for this Fe2+ + H202-> HOP + OH- + Fe3+. [3] reaction in solution (>3 x 104 M-1-s-; refs. 18 and 19) is much slower than most reactions involving HOP. Still, HOP Although low molecular weight scavengers ofHO-, such as formed by homolytic decomposition of HOONO in aqueous mannitol, dimethylthiourea, and dimethyl sulfoxide (DMSO), solutions may not escape beyond the solvent cage before and the iron chelator desferrioxamine reduce oxidant injury, reacting with NO2', making HOP undetectable. However, generation of strong oxidants by the iron-catalyzed Haber- and nitrite at Weiss reaction is not an entirely satisfactory explanation for peroxynitrite formed by the reaction ofH202 pH SOD-inhibitable injury in vivo. Formation of HOP by this °22.0 will hydroxylate benzene rings and polymerize methyl pathway requires the interaction of Oj-, H202, and suitably chelated iron-all maintained at low concentrations in vivo Abbreviations: DMSO, dimethyl sulfoxide; DTPA, diethyltriamine- pentaacetic acid; EDRF, endothelium-derived relaxing factor; MDA, malonyldialdehyde; SOD, superoxide dismutase. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed at: Department of payment. This article must therefore be hereby marked "advertisement" Anesthesiology, University of Alabama at Birmingham, Birming- in accordance with 18 U.S.C. §1734 solely to indicate this fact. ham, AL 35294. 1620 Downloaded by guest on October 2, 2021 Medical Sciences: Beckman et al. Proc. Natl. Acad. Sci. USA 87 (1990) 1621 methacrylate, reactions characteristic of a free-radical pro- used to find the amount of HO trapped in the presence of cess (20, 21). By measuring oxygen evolution from HOG infinite detector concentrations (5). If the rate constant of oxidation of H202, a 32% yield of HO- could be detected HO reacting with a given concentration ofdetector D to form during the reaction of H202 and nitrite at pH 5.0 (18). a product P is kd and the pooled rate constants of HO- Furthermore, oxygen evolution was inhibited by the HO0 undergoing all other reactions are summed to give kr, then the scavengers ethanol and benzoate. We have extended these molar concentration ofproduct [P] detected in an assay using to more observations physiological conditions and show that a detector molar concentration [D] may be written as peroxynitrite initiates many reactions currently used to infer the action of HO. We propose that the formation of peroxy- nitrite from O2* and NO- is important in recognizing potential kd[D] [5] mechanisms of SOD-inhibitable oxidant injury to endothe- kd[DI + kr lium. Eq. 5 can be linearized to give the equivalent form of a Scatchard plot in which METHODS [P]/[D] = + [6] Peroxynitrite Synthesis. Peroxynitrite was synthesized in a -(kd/kr)[P] (kd/kr)[HO°]. quenched-flow reactor (22). Solutions of(i) 0.6 M NaNO2 and In a plot of [P]/[D] versus [P] for a range of detector (ii) 0.6 M HCl/0.7 M H202 were pumped at 26 ml/min into concentrations, the x axis intercept equals the HO- concen- a tee-junction and mixed in a 3-mm diameter by 2.5-cm glass tration that can be trapped at infinite detector concentrations. tube. The acid-catalyzed reaction of nitrous acid with H202 The inhibition ofapparent HO- production, estimated from to form acid was 1.5 peroxynitrous quenched by pumping M the effect of various scavengers on yield, was calculated as NaOH at the same rate into a second tee-junction at the end described Winterbourn If is the fraction of inhi- of the Excess was by (28). Fi glass tubing. H202 removed by passage bition from a i at over a 1 5 cm column filled with 4 of scavenger concentration Si, then simple x g granular MnO2. The kinetics solution was frozen at -200C for as long as a week. Peroxy- competitive predict nitrite tends to form a yellow top due to freeze frac- layer Fj[D] tionation, which was scraped for further studies. This top ks5 [7] layer typically contained 170-220 mM peroxynitrite as de- kD (1 - Fi)[St] termined by absorbance at 302 nm in 1 M NaOH (E302 nm = 1670 M-1cm-1; ref. 23). Interference by other absorbing where ks, and kD are the respective rate constants for reaction compounds (e.g., nitrite) was corrected by subtracting the of HO- with scavenger and detector. The ratio ks,/kD is also final absorbance after adding peroxynitrite to 100 mM po- the concentration ofdetector divided by the concentration of tassium phosphate (pH 7.4). scavenger i required to give 50% inhibition of product yield. HO- Assays. The production of HOG from peroxynitrite If scavenging occurs by simple competition, values of FA[DJ/ decomposition was assayed by the oxidation of DMSO to (1 - F,)[S1] for each scavenger plotted against the known rate formaldehyde (24) and deoxyribose to malonyldialdehyde constants of ks, for reaction of HO- with scavenger Si will (MDA; ref. 25). All assays were conducted at 370C and yield a linear relationship of slope 1/kD (see Fig. 5). incubated for 3 min to allow peroxynitrite to fully decom- pose. Catalase (10 units/ml final concentration; Worthing- ton) was added at the end of each reaction to remove trace RESULTS H202 left after MnO2 treatment.

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