THE OF CHEMISTRY Vol. 269, No. 42, Issue of 21, pp ... · THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc. Vol. 269,

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269, No. 42, Issue of October 21, pp. 2606626075, 1994 Printed in U.S.A.

Nitric Oxide Regulation of Superoxide and Peroxynitrite-dependent Lipid Peroxidation FORMATION OF NOVEL NITROGEN-CONTAINING OXIDIZED LIPID DERNATIVES*

(Received for publication, June 21, 1994)

Homero RubboSI, Rafael &dig, Madia TryiilloQ, Rossana Teneri$, Balaraman Kalyanaramann, Stephen Barnesll, Marion Kirk**, and Bruce A. Freeman$$ $$ $9 From the Departments of $Anesthesiology, $$Biochemistry and Molecular Genetics, §$Pediatrics, and IPharmacology and the **Mass Spectrometry Shared Facility, Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, Alabama 35233, §Departamento de Bioquimica, Facultad de Medicina, Universidad de la Republica, 11800 Montevideo, Uruguay, and the llBiophysics Research Institute, Medical College of Wisconsin, Milwaukee, Wisconsin 53226

Superoxide (O:), nitric oxide ('NO), and their reaction product peroxynitrite (ONOO-) have all been shown to independently exert toxic target molecule reactions. Be- cause these reactive species are often generated in excess during diverse inflammatory and other pathologic circumstances, we assessed the influence of 'NO on membrane lipid peroxidation induced by O;, H,O,, and 'OH derived from xanthine oxidase (XO) and by ONOO-. Experimental conditions in lipid oxidation systems were adjusted to yield different rates of delivery of 'NO, relative to rates of 0; and HaOz generation, by infusion of either 'NO or via 'NO released from S-nitroso-N- acetylpenicillamine or S-nitrosoglutathione. Peroxida- tion of phosphatidylcholine liposomes was assessed by formation of thiobarbituric acid-reactive products and by liquid chromatography-mass spectrometry. Lipo- somes exposed to XO-derived reactive species in the presence of 'NO exhibited both stimulation and inhibition of lipid peroxidation, depending on the ratio of the rates of reactive oxygen species production and 'NO in- troduction into reaction systems. Nitric oxide alone did not induce lipid peroxidation. Linolenic acid emulsions peroxidized by XO-derived reactive species showed similar dose-dependent regulation of lipid peroxidation by 'NO. Mass spectral analysis of oxidation products showed formation of nitrito-, nitro-, nitrosoperoxo-, andor nitrated lipid oxidation adducts, demonstrating that 'NO serves as a potent terminator of radical chain propagation reactions. Electron spin resonance (ESR) analysis of incubation mixtures provided no evidence for formation of paramagnetic iron-lipid-nitric oxide complexes in reaction systems. Peroxynitrite-dependent lipid peroxidation, which predominantly occurs by metal-independent mechanisms, was also inhibited by 'NO. Peroxynitrite-mediated benzoate hydroxylation was partially inhibited by 'NO, inferring reaction between 'NO and ONOOH. It is concluded that 'NO can both stimulate O;/H,O,/'OH-induced lipid oxidation and

Institutes of Health Grants Pol-HL48676 and R01-HL51245, and a * This work was supported by the Fulbright Foundation, National

grant from the Council for Tobacco Research (all to B. A. F.); by grants from the Universidad de la Republica and Consejo de Investigaciones Cientificas y Tecnicas (to R. R.); by National Institutes of Health Grant HL 47250 (to B. K.); and National Institutes of Health Grants SlORR06487 and P30-AI13148 (to S. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Anesthe-

THT, Birmingham, AL 35233-6810. "el.: 205-934-4234; Fax: 205-934- siology, University ofAlabama at Birmingham, 619 19th St. South, 946

7437.

mediate oxidant-protective reactions in membranes at higher rates of 'NO production, with the prooxidant uersus antioxidant outcome critically dependent on relative concentrations of individual reactive species, Prooxi- dant reactions of 'NO will occur after 0; reaction with 'NO to yield potent secondary oxidants such as ONOO- and the antioxidant effects of 'NO a consequence of direct reaction with alkoxy1 and peroxyl radical intermediates during lipid peroxidation, thus terminating lipid radical chain propagation reactions.

Nitric oxide ('NO)' is an endogenously produced free radical first characterized as part of the endothelial-derived relaxation factor (1, 2). Nitric oxide, produced by a variety of mammalian cells including endothelium, neuronal cells, smooth muscle cells, macrophages, neutrophils, platelets, fibroblasts, and type I1 pneumocytes, mediates biological actions ranging from va- sodilation, neurotransmission, inhibition of platelet adherence and aggregation, and macrophage- and neutrophil-mediated killing of pathogens (3).

In many cases, 'NO is viewed to have a salutary role, by preventing vascular thrombosis (4), inflammatory cell-mediated injury (51, and reperfusion injury (6-8). Stimulation of tissue production of 'NO is also associated with adverse events such as hypotension (9), inhibition of intermediary metabolism (lo), and the production of the potent oxidant peroxynitrite (ONOO-) following radical-radical reaction with superoxide (O;, k = 6.7.109 mo1"d) (11-16). Despite extensive evidence that excess production of 'NO in the presence of 0; will lead to cytotoxic events, 'NO has also been reported to limit injury to target molecules or tissues during events associated with excess production of reactive oxygen species, by often undefined mechanisms. This includes inhibition of oxidative killing of Chinese hamster V79 cells (17), attenuation of low density lipoprotein oxidation (18-21), and prevention of central nervous system, splanchnic, and myocardial ischemia-reperfusion injury (6-8, 22, 23). The protective effects observed for 'NO with in vivo models of reperfusion injury, when administered as a bolus of nitrosothiol or other 'NO donors, are often ascribed to 'NO inhibition of inflammatory cell margination and function (7, 8, 24, 25).

anion; ONOO-, peroxynitrite anion; 'OH, hydroxyl radical; H,O,, hy- The abbreviations used here are: 'NO, nitric oxide; O,, superoxide

drogen peroxide; XO, xanthine oxidase; PC, phosphatidylcholine; DTPA, diethylenetriaminepentaacetic acid; TBA, 2-thiobarbituric acid; SNAP, S-nitroso-N-acetylpenicillamine; GSNO, S-nitrosoglutathione; MS, mass spectroscopy; Me,SO, dimethyl sulfoxide; GSH, glutathione; BHT, butylated hydroxytoluene.

26066

'NO Regulation of 0, / 0NOO"dependent Lipid Peroxidation 26067

We report herein that 'NO both stimulates and inhibits 0;-, H,O,-, and 0NOO"dependent lipid peroxidation and yields novel nitrogen-containing derivatives of oxidized lipids. From this, we conclude that in biological systems where co-generation of multiple reactive species can occur, 'NO can ( a ) exacer- bate oxidant injury via production of the potent oxidant ONOO- and ( b ) exert a protective role toward membranes or lipoproteins via redirection of 0,-mediated cytotoxic reactions to other oxidative pathways, by inhibition of 0NOO"dependent oxidation reactions, and by termination of free radical-dependent chain propagation reactions catalyzed by diverse initiating species.

EXPERIMENTAL PROCEDURES Materials-Egg phosphatidylcholine and 1-palmitoyl-2-arachidonyl-

sn-glycero-3-phosphocholine were from Avanti Polar Lipids (Pelham, AL). Linolenic acid was from Nu-Chek-Prep (Elysian, MI). EDTA, DTPA, potassium phosphate, 2-thiobarbituric acid (TBA), butylated hydroxytoluene, hypoxanthine, urate, mannitol, dimethyl sulfoxide, cata- lase, superoxide dismutase, cytochrome c, benzoate, salicylic acid, and glutathione were from Sigma. Bovine milk xanthine oxidase was from Calbiochem. S-Nitroso-N-acetylpenicillamine was from Biomol (Plym- outh Meeting, PA), and 1,1,3,3-tetramethoxypropane was from Aldrich. Acetaldehyde was prepared according to Ref. 26.

Preparation of Peroxynitrite, Nitric Oxide, and S-Nitrosoglu- tathione-Peroxynitrite was synthesized from sodium nitrite and hy- drogen peroxide using a quenched-flow reactor as previously (12, 14). Peroxynitrite concentration was determined spectrophotometrically at 302 nm (eM = 1.67 mM-km-l), with residual H,O, eliminated by elution of ONOO- on a MnO, column. Solutions of 'NO were prepared by bub- bling of 'NO gas (Matheson, Madison, WI) for 30 min into argon-satu- rated deionized water. Any 'NO, present was eliminated by first bub- bling 'NO through 5 M NaOH. Nitric oxide production rates and solution concentrations were measured by electrochemical detection using a 'NO sensor (Iso-NO, WPI, Inc., Sarasota, FL). S-Nitrosoglutathione (GSNO) was synthesized at 25 "C by combining equimolar (200 mM) concentrations of reduced glutathione with sodium nitrite in 0.5 M HCl(27). Stock solutions of SNAP were prepared in 100 mM H,SO, (28). Either SNAP or GSNO was added to 100 mM potassium phosphate, pH 7.4, at 20 "C, to measure 'NO release. For example, addition of 100 pd SNAP to air- equilibrated buffer systems resulted in 'NO production for approxi- mately 30 min, achieving a maximum concentration of 8.3 VM in the absence of 0;. In the case of 800 p GSNO, a constant rate of 'NO production was observed for more than 60 min, achieving a steady state concentration of 8 pd in the absence of 0;.

Liposome Oxidation-Phosphatidylcholine liposomes were prepared from 6 ml of a 25.4 mM (20 mg.ml") lipid stock solution in chloroform as described previously (29, 30). Briefly, solvent was removed in uacuo at 45-55 "C and 6 ml of 10 m~ potassium phosphate, pH 7.4, was added. The suspension was placed in a 4 "C water bath and sonicated three times for 30 s each at 65 watts using a Branson sonifier. Liposomes were stored in the dark under argon and used within 24 h of preparation. Superoxide was generated by X 0 using hypoxanthine or acetaldehyde as substrate, measured by superoxide dismutase-inhibitable cytochrome c reduction at 550 nm (eM = 21 mM''.cm-'). Xanthine oxidase activity was determined at 20 "C by the rate of uric acid production at 295 nm (eM = 11,000 M'l.cm-l), or at 308.5 nm (eM = 2750 "l.cm") when light scattering from liposome suspensions prevented assay of urate formation at 295 nm. For some experiments, X 0 was dialyzed against 0.1 mM desferrioxamine in 50 mM Tris-HC1, pH 8.0 (31). This effectively removed transition metals from X0 and prevented formation of 'OH generated by the Fenton reaction between adventitiously bound iron and H,O, (32). The specific activity of X0 was unaffected by dialysis procedures, with the percentage of univalent flux (0; uersus H,O, formation by XO) from 10 milliunitsm" X 0 plus 150 p~ hypoxanthine being - 18%. Liposomes, suspended in 10 mM potassium phosphate, pH 7.4, were incubated with X 0 (0-10 milliunits.ml") alone and in the presence of 'NO generated by addition of 1-100 p~ SNAP or 0.5-1.0 mM GSNO, or by infusion of 'NO using a motor-driven microliter syringe. All incubations were continually stirred at 20 "C and aliquots removed at indicated time points for measurement of 2-thiobarbituric acid-reactive substances as previously (29, 30) using eM = 150 m"l.cm-l at 532 nm, calculated from reaction with known amounts of malonaldehyde generated by acid hydrolysis of 1,1,3,3-tetramethoxypropane. To pre- vent further peroxidation of lipid during assay procedures, butylated hydroxytoluene (0.025%, w/v) was added to the thiobarbituric acid reagent.

12 I 1

10 -

3

: s Y E 8 -

+ 6 -

2 t -----"- I 0 5 10 15 20 25 30

Time (mid

S-nitrosoglutathione on xanthine oxidase-dependent liposome FIG. 1. The intluence of S-nitroso-N-acetylpenicillamine and

membrane lipid peroxidation. PC liposomes (6.6 mg.ml"), in 10 mM potassium phosphate, pH 7.4, were oxidized for 30 min in stirred incubations at 20 "C. The reaction mixtures contained 10 milliunits.ml" XO, 150 p hypoxanthine and 100 pd EDTA-Fe3+ (0.11 mM EDTA plus 0.1 mM FeC1,) (0, A, 0) or 100 pd DTPA(O, a), and 100 PM SNAP (0,O) or 800 p GSNO (A). All assays were performed in triplicate using the same preparation of PC, with experiments repeated at least three times.

Biochemical Analyses-Lipids were extracted according to Ref. 33. Thin layer chromatography on Silica Gel G with a chloroform-methanol-H,O (65:25:4) solvent system was used for the separation of phos- pholipids and chloroform-methanol-H,O-acetic acid (90:10:0.5:0.5) for linolenic acid. Plates were developed by fluorescent detection with an aqueous solution of rhodamine 6G. Nitrogen-containing derivatives of lipid oxidation intermediates were assessed by mass spectrometry. Analyses were performed on an API I11 triple quadrupole mass spectrometer (PE-Sciex, Thornhill, Ontario, Canada) equipped with two MacIntosh Quadra 950 computers for data analysis. Linolenic acid and its NOx adducts were separated by reversed phase high pressure liquid chromatography on a 10 cm x 2.1 mm (inner diameter) Aquapore C, column at a flow rate of 0.2 mVmin using a linear 50-100% methanol gradient in 1% aqueous acetic acid. The column eluent was split 1:1, with 100 pl.min-' going to the Ionspray" interface. Negative ion mass spectra were recorded in this mode, with an orifice potential of -60 V. MS-MS experiments were carried out by selecting the parent ion with Q,. Collision of the parent ion in Q, with a mixture of 10% N,, 90% argon resulted in fragment ions that were separated in Q,.

Formation of paramagnetic iron-lipid-nitric oxide complexes were analyzed by ESR spectroscopy (34, 35). Reaction systems were trans- ferred to a 4-mm quartz tube and immersed in a quartz Dewar flask containing liquid N,. ESR spectra were recorded at liquid N,temperature using a Varian E-109 spectrometer operating at X band frequencies and employing 100-kHz field modulation. Spectrometer conditions were: microwave power, 2 milliwatts; modulation amplitude, 10 gauss; scan time, 8 min; time constant, 0.5 s. ESR spectra were scanned between gauss = 2.10 and 2.00 region.

Oxygen consumption was measured polarographically at 37 "C using a 1.8-ml water-jacketed cell fitted with a Clark model YSI 4004 elec- trode (Clark, Yellow Springs, OH). Fluorescent measurement of benzoate hydroxylation was performed using A,, = 300 nm and A,, = 410 nm (36) with an SLM DMX-1000 fluorometer. Calibration of the fluorescence yield of benzoate hydroxylation was performed using standard solutions of salicylate.

Statistical analyses were performed after ANOVA and application of Duncan's multiple range test, with the level of significance defined as p < 0.05.

RESULTS

Xanthine Oxidase-dependent Liposomal Oxidation Zs Znhib- ited by 'NO-Incubation of PC liposomes with X 0 plus hypoxanthine in the presence of EDTA-Fe3+ yielded a significant increase in the production of TBA-reactive lipid oxidation products with time, which was inhibited by addition of 0.1 mM

26068 'NO Regulation of 0;) 0NOO"dependent Lipid Peroxidation

TABLE I

xanthine oxidase-dependent liposome and linolenic acid oxidation The influence of 5'-nitroso-N-acetylpenicillamine and substrate on

PC liposomes (6.6 mgml") or linolenic acid (1 mgml") were oxidized as in Fig. 1, using 150 p~ hypoxanthine or 10 mM acetaldehyde and 10 milliunitsml" X 0 2 100 p~ SNAP. Data represent mean -c S.D., n = 3.

Condition TBA + material

PM Control PC liposomes, EDTA-Fe3+ 1.9 f 0.3

+ Hypoxanthine 2.1 f 0.3 + Hypoxanthine, X 0 + Hypoxanthine, XO, SNAP

10.2 * 0.7 3.8 * 0.4

+ Acetaldehyde + Acetaldehyde, X 0

5.4 2 0.5

+ Acetaldehyde, XO, SNAP 19.6 = 0.8 7.6 f 0.6

Control linolenic acid, EDTA-Fe3+ 1.1 .c 0.3 + Hypoxanthine, X 0 9.6 -+ 0.5 + Hypoxanthine, XO, SNAP 3.3 -+ 0.4

DTPA(Fig. 1). Lipid peroxidation was also completely inhibited by 'NO derived from either 100 1.1~ SNAP or 800 1.1~ GSNO (Fig. 1). Although there is a recent suggestion indicating that high concentrations of 'NO partially inhibit X 0 activity (37), no significant inhibition of X 0 by 'NO was observed in control studies at steady state concentrations of 'NO of 100 or less, as indicated by rates of XO-dependent oxygen consumption and uric acid formation (not shown). Additionally, similar concentrations of acetylpenicillamine and GSH did not affect XO- dependent liposome oxidation.

Xanthine oxidase-dependent oxidation of liposomes and linolenic acid micelles was also inhibited by *NO derived from 100 w SNAP (Table I). Uric acid, a product of XO-mediated hypoxanthine oxidation, is a potent 'OH scavenger and an effective inhibitor of the 'OH-like reactivity of ONOOH (38). Since ONOO- is a product in reaction systems where 'NO and 0, are being co-generated, the influence of X 0 substrate on lipid peroxidation yields was investigated. Use of acetaldehyde as an X0 substrate gave 92% greater yields of XO-dependent lipid peroxidation products and demonstrated similar extents of inhibition of lipid peroxidation by 'NO (Table I).

Inhibition of Peroxynitrite-dependent Liposome Oxidation by 'NO-Peroxynitrite-dependent peroxidation of PC liposomes occurs by a predominantly iron-independent mechanism (30); thus, TBA-reactive product formation was only partially inhibited by addition of DTPA to reactions (Table 11). Peroxynitrite- dependent liposome lipid peroxidation was inhibited by either infusion of 'NO into reaction systems or addition of GSNO.

Influence of 'NO on Peroxynitrite-mediated Benzoate Hydrox- ylation-The reaction of ONOO- with benzoic acid to yield salicylate reconfirms the 'OH-like reactivity of protonated ONOO-, peroxynitrous acid (ONOOH, Refs. 16 and 38). Addi- tion of SNAP or coinfusion of 'NO at the time of ONOO- infusion into a benzoate-containing reaction system partially inhibited benzoate hydroxylation to salicylic acid (Table 111). Benzoate hydroxylation was also partially inhibited by the 'OH scavengers mannitol and Me,SO, while DTPA had no effect. Similar concentrations of the thiol-containing SNAP precursor acetylpenicillamine minimally affected 0NOO"dependent benzoate hydroxylation. Control studies also showed that all employed 'OH scavengers, SNAP, and the infusion of 'NO did not quench salicylate fluorescence (not shown).

Influence of the Ratio of 'NO and 0; Production Rates on XO-dependent Lipid Peroxidation-The continuous infusion of 'NO (0-3 w.min-l) into liposome suspensions exposed to X 0 plus acetaldehyde (1 pwmin" 0; production) and EDTA-Fe3+ first stimulated and then inhibited formation of TBA-reactive products at greater rates of infusion (Fig. 2). The same re- sponses were observed for shorter XO-dependent liposome lipid

TABLE I1 Nitric oxide inhibition of peroxynitrite-dependent liposome oxidation Reaction mixtures contained PC liposomes (6.6 mgml") in 100 n-m

were made as indicated: DTPA, 100 w; EDTA-Fe3+, 100 PM; ONOO-, 33 potassium phosphate, pH 7.4, at 25 "C (control). The following additions

pwmin" infusion for 15 min; GSNO, 1 m; .NO, 2.7 pM.min" infusion for 15 min. Data represent mean f S.D., n = 3.

Condition TBA + material

PM

ONOO- 0.1 & 0.03

EDTA-Fe3+ 10.8 f 1.3

EDTA-Fe+3, ONOO- 0.3 f 0.05

12.5 2 1.8 EDTA-Fe3+, ONOO-, *NO EDTA-Fe3+, ONOO-, GSNO

5.4 f 0.9 5.8 f 0.9

DTPA, ONOO- 8.1 f 0.6

DTPA, ONOO-, GSNO 3.9 f 0.6

Control PC liposomes

DTPA, ONOO-, .NO 3.4 f 0.5

T ~ L E 111 The effect of nitric oxide and hydroxyl radical scavengers on

peroxynitrite-dependent benzoate hydroxylation A continuous infusion of ONOO- (6.7 *mid) to a solution of 10 m

benzoate in 50 m potassium phosphate, pH 6.8, at 18 "C was performed. The reaction mixtures contained as indicated 100 PM SNAP, 4.3 pwmin" .NO, 200 PM DTPA, 50 mM mannitol, or 50 lll~ Me,SO. Infu- sions were for 15 min and then assayed for benzoate hydroxylation to salicylic acid (Aex = 300 nm and A,, = 410 nm). Data represent mean f S.D., n = 5.

Condition Control relative fluorescence

%

ONOO- 145 f 18 SNAP 26 f 5 ONOO-, SNAP 89 z 11 ONOO-, acetylpenicillamine 118 z 19 *NO ONOO-, *NO 90 * 9

4 * 1

ONOO-, mannitol ONOO-, mannitol, SNAP

53 z 7

ONOO-, Me,SO 25 * 5

ONOO-, Me,SO, SNAP 71 9

ONOO-, DTPA 152 2 22 14 f 4

ONOO-, DTPA, SNAP 93 2 16

peroxidation reactions (1 min) to which 'NO was added as a bolus (not shown). Similar 'NO-dependent stimulation of XO- mediated lipid peroxidation, followed by inhibition of peroxidation a t greater rates of 'NO release, was observed when using GSNO as an 'NO source (Fig. 3). Nitric oxide stimulated XO- dependent liposome lipid peroxidation, when production rates of 'NO approached or were equivalent to rates of 0, production by X 0 (Figs. 2 and 3). When the pwmin" rate of 'NO production exceeded that of O;, lipid peroxidation was inhibited as before (Fig. 1, Tables I and 11).

Product Analysis of 'NO Reaction with Oxidizing Lipids- Production of novel lipid oxidation products, secondary to the 'NO-mediated inhibition of XO-dependent and 0NOO"dependent oxidation of linolenic acid emulsions, was first indicated by appearance of reaction products having lower R, values than untreated controls when separated by thin-layer chromatography following chloroform-methanol-H,O extraction (not shown).

Liquid chromatography-mass spectroscopic analysis of reaction systems, performed immediately after addition of methanol to give a final concentration of 40% (v/v), revealed molecular compositional characteristics of novel derivatives produced by 'NO reactions with oxidizing linolenic acid (Figs. 4, A and B, and 5, A and B). Quantitative yields are summarized in Table n! Linolenic acid oxidation induced by hypoxanthine, XO, and EDTA-Fe3+ yielded two principal oxidation products, in addi-

'NO Regulation of 0, J 0NOO"dependent Lipid Peroxidation 26069

20 I T

0

0 ' 1 1 1 I I I I I 0 0.5 1 1.5 2 2.5 3

'NO (VM-min-') FIG. 2. The influence of 'NO infusion on xanthine oxidase-de-

pendent liposome oxidation. PC liposomes (6.6 mg.rnl-') were oxidized with 1.5 milliunits. ml-l XO, 10 mM acetaldehyde, and 100 p~ EDTA-Fe3+. Infusion of 'NO (0.1,0.25,0.5,0.7,1,2, and 3 pM.min") was performed simultaneously (0). 0 represents incubation of reaction mixture in the absence of XO. Data represent the mean of duplicate deter- minations and were representative of three separate experiments.

I I 1 I 1 I I

0 0.5 1 1.5 2 2.5 3 .NO (pM mmin") 3 2.5 2 1.5 1 0.5 0 02" (pM.rnin")

FIG. 3. The effect of continuous variation of rates of both 0: and 'NO generation on liposome oxidation. PC liposomes (6.6 mgml") were oxidized for 30 min in stirred incubations containing 50 mM potassium phosphate, pH 7.4, 100 PM EDTA-Fe3+ (0) or 100 p~ DTPA (B), 10 mM acetaldehyde, with X 0 and GSNO concentrations (up to 5 milliunitsm" X 0 and 660 p~ GSNO) calibrated by separate assay to give the noted rates of 0, and 'NO production. 0 represents liposome oxidation in the absence of GSNO.

tion to the ["HI- ion (mlz = 277) of the native fatty acid. These species had a molecular mass of 310 (9- and/or 16-hydroperoxolinolenate) and 342 (9, 16-dihydroperoxolinolenate). Lower quantities of 9- andor 16-hydroxylinolenate were also formed (molecular mass = 294). MS-MS analysis of the species having a molecular mass of 310 yielded a loss of 33 mass units, indicating the presence of -0OH. In lipid oxidation reactions simultaneously exposed to 'NO derived from SNAP, two additional ions were observed for novel products having molecular masses of 355 and 371 (Fig. 4B).

Peroxynitrite oxidation of linolenic acid yielded significant formation of hydroperoxolinolenate, with this product exhibit- ing a heterogeneous elution profile due to formation of multiple alkyl hydroperoxide isomers. Trace amounts of 'NO-dependent oxidized lipid adduct formation (ions with a molecular mass of 323, 339, 355, and 371) were also detected (Fig. 5 A ) . Lower quantities of hydroxyl-, hydroxyhydroperoxyl-, and nitrogen-

containing adducts of ONOO--oxidized linolenate were also detected (molecular masses = 294,326,342,323, and 339; Fig. 5A, Table IV). Co-exposure of fatty acid emulsions to SNAP-derived 'NO during infusion of ONOO- led to even greater quantities of ions having a molecular mass of 323,339,355, and 371 (Fig. 5B, Table IV). In both the XO- and 0NOO"dependent oxidation systems, a similar product distribution was observed when GSNO was substituted for SNAP as the source of 'NO. The presence of 'NO in peroxidation reactions inhibited formation of hydroperoxolinolenate, as well as reducing net yields of TBA- reactive oxidation products. Further incubation of 'NO-containing lipid oxidation systems for 3 h led to the loss of appar- ently reactive or unstable nitrogen-containing oxidized lipid species and a continued generation of peroxidation products (Table IV).

DISCUSSION Nitric oxide has proven to be a ubiquitous signal transduc-

tion molecule and a potent mediator of tissue injury because of its low molecular mass, volatility, lipophilicity, free radical nature, and diverse reactivities. Because of the transient nature of free radical species and their often broad range of reactivities, it becomes challenging to define the mechanisms of tissue injury in processes of oxidant stress when a diverse spectrum of reactive species is produced. It has recently been established that 'NO will react with 0, to yield ONOO- at almost diffusion- limited rates ( 6 . 7 ~ 1 0 ~ M-'.s', Ref. 11). Once formed in biological systems, ONOO- can exert direct and hydroxyl radical-like oxidation reactions, as well as metal-catalyzed nitration reactions (15, 16, 38, 39). A principal goal of this study was to examine the influences of the independent and combined production of these reactive species on a model membrane system and to define mechanisms accounting for the protective effects of 'NO sometimes observed in pathological events associated with excess production of reactive oxygen species.

Nitric oxide has a relatively low reactivity for a free radical species, resulting in a long biological half-life (t,,, = 5-30 s, Ref. 40). In spite of this, 'NO still undergoes numerous reactions. For example, 'NO is an iron ligand, forming paramagnetic species after reaction of 'NO with ferrous ions containing one or more additional coordinating anionic ligands (41, 42). The tox- icity of 'NO is also often attributed to 'NO reaction with iron- sulfur containing mitochondrial enzymes (10) and the inhibition of DNA or protein synthesis (43). Heme proteins such as guanylate cyclase are activated by low 'NO Concentrations (44), while cytochrome P-450 (45) and nonheme iron proteins such as aconitase (10) and lipoxygenase (46) are inhibited by 'NO, al- beit often a t much greater concentrations of 'NO than employed herein or present in tissues.

Nitric oxide reacts with molecular oxygen in aqueous solution, yielding nitrite (NO;) and nitrate (NO;) as products of N,O, and N,O, decomposition (47, 48). This reaction is second order for 'NO, with tissue 'NO levels in the range of 1-1000 nM, and first order for 0, ( k = 6-8 x lo6 M-'.s-'), thus this reaction does not account for the long tu, of 'NO or serve as a principal pathway for 'NO reaction in vivo. A number of investigators have reported that less reactive nitrosothiol intermediates are formed in vivo from reaction of some oxidation state of NO with protein or nonprotein thiols, which can increase the effective tissue t,,, of 'NO (44). Since tissue 'NO concentrations are low (<I p~), it should be noted that significant reactivity with heme, iron-sulfur, and thiol-containing proteins often require high concentrations of 'NO or 'NO-generating agents (49). The rapid bimolecular reaction of 'NO with 0, to yield ONOO- is more than 3 times faster than the enzymatic dismutation of 0, catalyzed by superoxide dismutase (kSoD = 2 x lo9 Ref. 50). Thus, ONOO- formation represents a major potential

26070 ’NO Regulation of 02;1 ONOO--dependent Lipid Peroxidation

341

J 309

I

6.50 7.50 8.50 9.50 10.50 11.50

c

Time (min)/Scan

6.50 7.50 8.50 9.50 10.50 11 S O

Time (min)/Scan FIG. 4. Liquid chromatography-mass spectroscopic analysis of xanthine oxidase-dependent linolenic acid oxidation products in

the absence (A) and presence ( B ) of ‘NO derived from S-nitroso-N-acetylpenicillamine. The ions detected represent [“HI- ions.

pathway of ‘NO reactivity, which depends on rates of tissue 0; reactivity of ‘OH via a metal-independent mechanism (12, 14). production, with steady state concentrations ranging from 10 Peroxynitrite in pure form will cause oxidative damage to pro- PM under basal conditions (51) to 0.01-0.1 p~ during tissue tein (12), lipid (30), carbohydrate (14) DNA (551, subcellular pathologic states (52) . The reaction of ‘NO with Oi, initially organelles (56), and cell systems (57). Peroxynitrite will also viewed as a route for ’NO “inactivation” (53,54), instead yields react with metal centers, yielding a species with the reactivity the potent oxidant ONOO-, which will protonate to peroxyni- of nitronium cation (NO;), an oxidizing and nitrating interme- trous acid (ONOOH, pK, = 6.8) to yield an oxidant with the diate (15, 16). There i s growing evidence that ‘NO-mediated

'NO Regulation of 0; 1 0NOO"dependent Lipid Peroxidation

30:

26071

7.00 8.00 9.00 10.00 1 1 .oo 12.00 370 423 476 529 582 634

Time (min)/Scan

3759

281 9 1 .

1879.

309

309 I \ 322

7.00 8.00 9.00 10.00 368 421 473 526 579

1 1 .oo

Time (min)/Scan

FIG. 5. Liquid chromatography-mass spectroscopic analysis of linolenic acid oxidation products generated by ONOO- infusion ( A ) and ONOO- infusion plus 'NO derived from S-nitroso-N-acetylpenicillamine (B) . The ions detected represent ["HI- ions.

production of ONOO- occurs in vivo (15, 22,39, 58-60), under- injury, as observed in models of cardiac ischemia-reperfusion scoring the importance of understanding the target molecule injury (64), N-methyl-D-aspartate-induced central nervous sys- reactions of the coordinated production of oxygen and nitrogen- tem injury (651, and immune complex-induced pulmonary containing reactive species. edema (66). In these models, 'NO was proposed to directly exert

Nitric oxide is a macrophage-derived effector molecule that is toxic reactions, as well as via secondary products such as toxic to bacteria, tumor cells, and parasites (61-63). Excess ONOO-. Contradictorily, 'NO has been observed to serve a pro- 'NO production by inflammatory cell and tissues often leads to tective role in pathologic events associated with excess produc-

26072 'NO Regulation of O~/ONOO--dependent Lipid Peroxidation

TABLE IV The influence of S-nitroso-N-acetylpenicillamine on linolenic oxidation products

Linolenic acid (0.1 mg), emulsifed in 50 mM potassium phosphate, pH 7.4, was oxidized as in Fig. 1. After oxidation, methanol was added to emulsions and mass spectroscopic analysis was performed as described under "Experimental Procedures."

%

Condition LOH LOOH L(OOH), LON0 LOONO "?$go LOONO Total 9c 294 310 342 323 339 355 (OOH) 371 oxidized 326

18:3, EDTA-Fe 0 9 0 1 0 0 0 10 4

0 21 + 10 milliunitdml X 0 0 3 0 0 0 0

+ XO, 100 I.~M SNAP 0 7 0 1 1 1 1.5 3 28

+ XO, 500 I.~M SNAP 0 2 0 0 1 2.5 1.5 3 10 14.5

+ XO, 100 I.~M SNAP" 0 37 0 13 0 0 0 0 50 + 18:3, 1 mM ONOO- 11 36 5 6 0.2 0.5 0 0

5 22 + 100 VM SNAP 2 1 4 8 3 3 48 58.7

+ 100 pM SNAP" 6 60 2 2 0 0 0 0 70

a Represents product distribution after incubation of samples for 3 h at 20 "C. Data represent percent distribution of individual products, with values below the structural identification denoting molecular mass. L denotes linolenic acid, and second substituents are in parentheses.

tion and reaction of partially reduced oxygen species. Numer- ous studies of cell or metal-induced lipoprotein oxidation reactions, as well as hepatic, cerebrovascular, pulmonary and myocardial inflammatory and ischemia-reperfusion injury models also show that stimulation of endogenous 'NO production or exogenous administration of 'NO will inhibit oxidant- related mechanisms and blunt the ultimate expression of molecular or tissue injury (4-8, 17-21,23-25, 67,681. In some of these models, inhibition of 'NO synthesis enhanced injury as well, emphasizing the salutary role sometimes observed for 'NO in oxidant injury-related processes.

Addition of 100 1.1~ SNAP will give a 100% yield of ONOO- from the 0, produced by hypoxanthine plus XO, secondary to 0, reaction with 'NO. We have observed herein that three different 'NO-generating systems (SNAP, GSNO, and infusion of 'NO) result in the 'NO-mediated inhibition of 0 - and 0NOO"dependent liposomal and fatty acid oxidation under conditions where both 0; and ONOO- are formed (Figs. 1-3, Tables I and 11). More detailed dose-response studies conducted by (a) increasing rates 'NO infusion into a liposome suspension subjected to a constant rate of XO-derived reactive species (Fig. 2) or ( b ) the simultaneous co-variance of both the rate of XO- derived reactive species and 'NO generation in liposome suspensions according to Job's analysis of continuous variation (Fig. 3, Ref. 69) demonstrated that 'NO will significantly en- hance lipid peroxidation when rates of 'NO production ap- proach or are equivalent to rates of 0, production by XO. This occurred even in the presence of iron-replete buffers (Fig. 31, implying that the 'NO (hence ONOO-)-mediated pathway for O~/H,O,-dependent lipid peroxidation predominates over the Haber-Weiss-mediated production of 'OH in this reaction system. One explanation would be that ONOO- can diffuse further into membrane bilayers than 'OH to initiate oxidative events. These results show that there can be a complex balance expected between the apparent tissue-protective and injurious effects of 'NO in vivo and during pathological processes. The final outcome of biological reactions of 'NO will ultimately depend on a multiplicity of factors, including the rate of 'NO production, the partitioning of 'NO into different tissue compartments, and the extent or rate of 'NO diffusion, which will be affected by the molecular environment (i.e. local 0, and 0; concentrations, the presence of 'NO-reactive molecules such as heme-containing proteins, and the distance to be traversed by 'NO). It has recently been shown that when 'NO is delivered to in vitro test systems in a fashion more reflective of biological production rates over extended periods of time (<m.min"), the cytotoxic and mutagenic effects of 'NO become more pro- nounced due to shifts in the liquid-gas equilibria of 'NO and 0, and the consequent redirection of 'NO reaction pathways (70).

The role of iron was precisely defined in the systems reported herein, because both XO-dependent lipid oxidation and lipid peroxidation propagation reactions have a critical requirement for metal catalysis (71, 72). Peroxidative processes depend on 'OH production via the Fenton reaction and on the formation of oxidizing iron species such as ferry1 ion (Fe02+), kinetically equivalent to 'OH (73). Thus, iron depletion of reaction systems inhibited XO-induced lipid peroxidation as expected (Figs. 1 and 3). Because 'NO can serve as an iron ligand to form iron- nitrosyl complexes (74), it has been proposed that 'NO will modulate the pro-oxidant effects of iron and other transition metals, thereby limiting their role in the Haber-Weiss-catalyzed formation of 'OH and iron-dependent electron transfer reactions (42). Other investigators have observed that the relative concentration of 'NO influences whether myoglobin-induced lipoprotein oxidation is reduced or enhanced (75). How- ever, ESR analysis showed that the aerobic 0;-containing reaction systems reported herein, which were exposed to lower and sometimes more biological rates of production of 'NO, did not yield detectable iron-nitrosyl complexes. This was not un- expected, because the rate of 'NO reaction with ferrous iron (2 x lo7 " l d , Ref. 76) is significantly slower than for 'NO reaction with either 0, or lipid alkoxy1 and peroxyl radical species (11, 77). I t should be noted that 'NO may also exert prooxidant effects by reducing ferric iron complexes, thus inducing a release of bound iron and indirectly substituting for other reduc- tants in the Haber-Weiss reaction-mediated production of 'OH from H,O, (78).

Nitric oxide reacts rapidly with 'OH (12 = 10 x 10" M-'.s") to form nitrous acid (79). Nitric oxide is expected to have only limited potential as a 'OH scavenger in both aqueous and lipid phases. The potent nonselective reactivity and short diffusion distance of 'OH would mean that there would have to be ex- tremely high and non-biological concentrations of 'NO present to effectively compete with other reaction pathways for 'OH. The 'OH scavengers mannitol and Me,SO afforded minimal inhibition of 0NOO"dependent benzoate hydroxylation to salicylic acid (Table 111). This indicates that at the often submi- cromolar concentrations of 'NO employed in our reaction systems, 'NO would not significantly inhibit the 'OH-like reactivity of ONOO-. Since the concentration of benzoate was 3 orders of magnitude greater than 'NO in these reaction SYS- tems, 'NO would not expected to effectively compete with benzoate as a 'OH scavenger. The partial inhibitory effect of 'NO observed toward benzoate hydroxylation may also be due to reaction of 'NO with the trans-form of ONOO-, critical for expression of the 'OH-like reactivity of ONOO- following proto- nation (80, 81). The experimental conditions where partial inhibition of benzoate hydroxylation by either nitrosothiols or

'NO Regulation of O,IONOO--dependent Lipid Peroxidation 26073

FIG. 6. Proposed reaction mechanisms and structural characteristics of 'NO-dependent lipid oxidation products.

Other *NO-derived products

'NO resulted in often complete inhibition of lipid oxidation. In lipid-containing reactions, fatty acids will also effectively compete with 'NO for reaction with the highly reactive 'OH. In aggregate, our results indicate that the principal mechanism of 'NO inhibition of lipid oxidation is not via scavenging of ONOO- or ONOOH; instead, it occurs by 'NO annihilation of lipid radical species, thus terminating radical chain propagation processes.

The mechanisms of linolenic acid and liposome oxidation induced by reactive oxygen species plus transition metals in- volves formation of hydroxyl ('OH), alkoxyl (LO'), and peroxyl (LOO') radicals as intermediates, with 'OH a poor initiator of oxidation in comparison to LOO' (82). Oxidants that do not readily initiate lipid oxidation in the absence of metals include H,O,, organic hydroperoxides, and, most notably, 0,. It has recently been reported that 'NO reacts with LO' and LOO' at near diffusion-limited rates (for LOO', k = 1.3 x lo9 M - ~ . S - ~ , Ref. 77). These reactions are important to consider, since 'NO will significantly concentrate in lipophilic cell compartments, with a 1ipid:water partition coefficient of 8:l . This further enhances the ability of 'NO to serve as a critical regulator of oxidant- induced membrane or lipoprotein oxidation by terminating radical chain propagation reactions.

Under conditions where linolenic acid emulsions oxidized by X0 or ONOO--derived reactive species showed inhibition of lipid peroxidation by 'NO (Tables I and 11), mass spectral analysis of oxidation products showed formation of novel nitrogen- containing lipid adducts (Table IV, Figs. 4, A and B , and 5, A and B ) , which were not detectable in the absence of 'NO

sources. These nitrogen-containing products have been tenta- tively identified as nitritolinolenate (molecular mass = 323), nitrosoperoxolinolenate (molecular mass = 339), hydroxylni- trosoperoxolinolenate (molecular mass = 355), and hydroperoxonitrosoperoxolinolenate (molecular mass = 371). Nitrosyla- tion of unsaturated lipids is analogous to oxygenation reactions proposed for linolenic acid (82). I t is possible that lipid reaction or decomposition pathways for ONOO- involve production of low concentrations of 'NO or 'NO,, since minor amounts of the molecular mass 323 and 339 nitrogen-containing oxidized lipid species were detected in ONOO--oxidized linolenate. The scheme in Fig. 6 shows a proposed mechanism for the reaction of 'NO with oxidizing lipids. Further analysis of preparations of the 371 molecular mass product is required to define the potential extent and time course of formation of the alkyl nitrate derivative of linolenic acid, since this ion has the same molecular mass as the hydroperoxonitrosoperoxolinolenate derivative. Similarly, the molecular mass 322 product will require further characterization to differentiate nitritolinolenate from the nitrolinolenate derivative. It has been proposed that nitro- sation of hydroperoxides with N,O, yields a nitrosoperoxo- derivative which would decompose via 0-0 bond homolysis to give an alkoxyV'N0, caged radical pair and ultimately yield an alkyl nitrate derivative (83).

From this product analysis, we conclude that dissolved 'NO gas and 'NO generated from decomposition of either SNAP or GSNO inhibits both X0 and 0NOO"dependent lipid oxidation by direct reaction with the lipid alkoxyl and peroxyl radical products formed by both X 0 and ONOO- during lipid peroxi-

26074 'NO Regulation of Oil 0NOO"dependent Lipid Peroxidation

dation, thus terminating lipid radical chain propagation reactions. These novel lipid oxidation adducts are in part organic peroxynitrites and would be expected to occur in vivo, where diverse inflammatory and pathological processes give rise to similar combinations of reactive species. Because of their in- stability (Table IV) and potential to decompose through radical or oxidative pathways, the nitrogen-containing lipid oxidation products may also serve as potent inflammatory mediators or as intermediates in cytotoxic processes. For this reason, the inhibitory effect of 'NO toward lipid peroxidative processes may not fully support terming this property an antioxidant event. While net yields of lipid peroxidation products were less in the presence of higher concentrations of 'NO, an effective antioxidant would not only have to react rapidly with oxidizing species, but also form less reactive or toxic species. This may not be the case when reactive nitrogen-containing lipid oxidation products are formed, because undefined cytotoxic reactions of these species may occur. With this qualification, it is still intriguing to note that the rate constant for 'NO reaction with peroxyl radicals is more than 2 x lo3 times greater than for a-tocopherol reaction with peroxyl radicals ( k = 2.5 x lo6 M-%', Ref. 771, with tissue concentrations and lipophilicity of 'NO and a-tocopherol not that dissimilar. From this, one might specu- late that 'NO may under some conditions predominate over a-tocopherol as an oxidant-protective molecule in tissues.

From the data reported herein, we conclude that (a) the relative rates of production and steady state concentrations of 0; and 'NO, ( b ) the cellular and anatomical sites of production of 0, and 'NO, and (c) the dominant operative mechanisms of oxidant damage in tissues at the time of 0, and 'NO production profoundly influence expression of the differential oxidant injury-enhancing and protective effects of 'NO. Critical events mediated by 'NO will include formation of potent secondary oxidants such as ONOO-, redirection of 0,-mediated cytotoxic reactions to other oxidative pathways, and 'NO annihilation of other radical species (while at the same time forming additional secondary reactive products). Full understanding of the physiological roles of 'NO, coupled with detailed insight into 'NO regulation of oxygen radical-dependent reactions, should yield a more rational basis for the use of 'NO donors and in- hibitors of 'NO synthases for therapeutic purposes.

Acknowledgments-We appreciate helpful discussions with Drs. Joseph Beckman, John Crow, Wim Koppenol, Patricia Bounds, and Victor Darley-Usmar.

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