nitroxidation, nitration, and oxidation of a bodipy ...lab.pharm.uic.edu/thatcher/pub/p_5.pdf ·...

ARTICLE IN PRESS

www.elsevier.com/locate/yniox

Nitric Oxide xxx (2006) xxx–xxx

Nitroxidation, nitration, and oxidation of a BODIPY fluorophoreby RNOS and ROS

Adrian C. Nicolescu 1, Qian Li 1, Laurie Brown, Gregory R.J. Thatcher *

Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago,

833 S. Wood St., Chicago, IL 60612-7231, USA

Received 10 September 2005; revised 11 December 2005

Abstract

BODIPY C11 581/591 (BODIPY11) represents a sensitive probe for quantification of relative antioxidant capacity. However, themechanism of BODIPY11 fluorescence decay in the presence of reactive oxygen species (ROS) and reactive nitrogen oxide species(RNOS) requires clarification. Azo-initiators provide a continuous source of peroxyl radicals that in simple, aerobic, homogeneous, buf-fered solution simulate lipid peroxyl radical formation. Inhibition of BODIPY11 fluorescence decay was assayed and quantified for sev-eral families of antioxidants, including phenols, NO donors, and thiols. Fluorescence decay of BODIPY11 in these systems demonstratedsimilar patterns of antioxidant activity to those observed in classical oxygen pressure measurements, and provided a readily appliedquantification of antioxidant capacity and mechanistic information, which was analyzed by measurement of induction periods, initialrates, and net oxidation. LC/MS analysis confirmed that peroxyl radical-induced irreversible fluorescence decay of the BODIPY11 fluo-rophore is due to oxidative cleavage of the activated phenyldiene side chain. The behavior of BODIPY11 towards RNOS was more com-plex, even in these simple systems. Incubation of BODIPY11 with bolus peroxynitrite or a sydnonimine peroxynitrite source produced avariety of novel products, characterized by LC/MS, derived from oxidative cleavage, nitroxidation, and nitration reactions. The ‘‘NOscavenger’’ PTIO reinforced the antioxidant activity of NO, and inhibited BODIPY11 oxidation induced by the sydnonimine. Theseobservations suggest that BODIPY11 is a well-behaved fluorescence probe for peroxidation and antioxidant studies, but that for studyof RNOS even co-application of fluorescence decay with LC/MS measurements requires careful analysis and interpretation.� 2006 Elsevier Inc. All rights reserved.

Keywords: Antioxidant; Fluorescent probe; Nitroxidation; Nitration; Oxidation; Peroxynitrite; ROS; RNOS

Oxidative stress denotes an imbalance between the pro-duction of oxidants and the defense systems of an organism[1]. Reactive oxygen species (ROS) and reactive nitrogenoxide species (RNOS) are important oxidants in vivo.Amongst ROS and RNOS, peroxyl radicals resulting fromlipid peroxidation radical chain reactions, and peroxyni-trite (ONOO�) resulting from the diffusion controlled reac-tion of nitric oxide (NO) and superoxide radical anion, areimportant cellular components of oxidative stress. Peroxylradicals are toxicologically relevant species since they areinvolved not only in the disruption of cell membrane integ-

1089-8603/$ - see front matter � 2006 Elsevier Inc. All rights reserved.

doi:10.1016/j.niox.2006.01.010

* Corresponding author. Fax: +1 312 996 7107.E-mail address: [email protected] (G.R.J. Thatcher).

1 These authors contributed equally to this work.

rity, but also in the oxidation of membrane proteins andthe inactivation of receptors and membrane-boundenzymes. Both peroxynitrite and organic peroxyl radicals(ROO�) are important reactive species likely to play a rolein a number of pathophysiological conditions, such as neu-rodegenerative disorders, aging, reperfusion injury afterischaemia, and atherosclerosis.

Oxidation can be initiated in a number of ways, includ-ing ionizing radiation, chemical reaction, enzyme activity,and through transition metal catalysis. A number of initia-tors have been used to mimic, in vitro, the oxidative stressthat leads to lipid peroxidation in vivo, including: (a) azo-initiators; (b) xanthine oxidase; (c) lipoxygenase; (d) per-oxynitrite; (e) cupric and cuprous ions; and (f) ferric andferrous ions [2–9]. The water- and lipid-soluble thermal

mailto:[email protected]

2 A.C. Nicolescu et al. / Nitric Oxide xxx (2006) xxx–xxx

ARTICLE IN PRESS

radical sources 2,2 0-azobis(2-methylpropionamidine)dihy-drochloride (ABAP)2 and 2,2 0-azobis(2,4-dimethylvalero-nitrile) (AMVN), respectively, provide reliable initiatorsfor peroxidation studies.

Antioxidants, which help cells to cope with oxidativestress by inhibiting damage due to ROS, RNOS, and otherfree radicals, have been linked to disease prevention. Pro-teins, enzymes, and a variety of small molecules manifestantioxidant activity. Lipid-soluble antioxidants, in particu-lar, play a major role in protecting membranes and lipo-proteins. The assessment of classical antioxidant activityrequires measurement of absolute or relative peroxidationrates. Several methods have been used for the quantifica-tion of peroxidation, including the spectrophotometricmonitoring of formation of conjugated dienes [10]; HPLCand GC monitoring of primary (hydroperoxide) and sec-ondary (aldehyde) peroxidation product formation [11];spectrophotometric and HPLC monitoring of thiobarbitu-ric acid adduct formation [2]; monitoring redox-sensitivedyes [12–21]; and measuring oxygen uptake by a peroxi-dizing lipid [3,22].

A suitable method for quantifying the inhibitory effectof antioxidants ideally should offer mechanistic insightsinto the interaction between the antioxidants and the per-oxidizing or oxidizing substrate. One method that meetsthese requirements employs an oxygen pressure transducerfor the measurement of the oxygen uptake during the oxi-dation of lipid substrates [22–24], but this method requiresdedicated instrumentation that is not suitable for parallelprocessing of multiple samples. Spectrofluorometric meth-ods provide for monitoring of lipid peroxidation with highsensitivity and for parallel measurements on multiple sam-ples. The boron dipyrromethene difluoride (BODIPY) fluo-rophore is insensitive to pH variation, is thermally stableand relatively insensitive to air oxidation, and possessesgood spectrofluorometric characteristics (i.e., a high fluo-rescence quantum yield (0.9); absorbance and emissionwavelength maxima >500 nm) [12–21].

A BODIPY fluorescent probe finding increasing usein vitro both in model and cell-based systems is BODIPYC11 581/591 (BODIPY11), which possesses a 4-phenyl-1,3-butadienyl group attached to a conjugated pyrrole system

2 Abbreviations used: ABAP, 2,2 0azobis(2-methylpropionamide)hydro-chloride; AMVN, 2,2 0-azobis(2,4-dimethyl-valeronitrile); AUC, areaunder the curve; BODIPY11, 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid; DCN, 1-decyl nitrite;DEA/NO, diethylamine NONOate; DETA/NO, diethylenetriamineNONOate; DLPC, dilinoleoyl-phosphatidylcholine; FU, fluorescenceunits; GSNO, S-nitrosoglutathione; 5HT, serotonin (5-hydroxytrypta-mine); IAN, i-amyl nitrite; LC/MS, liquid chromatography coupled withelectrospray mass spectroscopy; MeCN, acetonitrile; NONOate,diazeniumdiolate salt; PBS, phosphate-buffered saline; PEN, 2-phenoxy-1-ethyl nitrite; PTIO, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide; ROO�, peroxyl radical; ROS, reactive oxygen species; RNOS,reactive nitrogen oxide species; SDS, sodium dodecyl sulfate; SIN-1,morpholinosydnonimine; SPE/NO, spermine NONOate; a-TcOH, a-tocopherol; Trolox C, 6-hydroxy-2,5,7,8-tetramethylchromane-2-carbox-ylic acid.

susceptible to oxidation. We have used BODIPY11 previ-ously to measure lipid peroxidation induced by azo-initia-tors in phospholipid liposomes [2,6]. In this paper, thereaction kinetics and products of the BODIPY11 probe inresponse to ROS and RNOS generated from azo-initiators,NO, and peroxynitrite are examined. Given the increasingusage of the BODIPY11 fluorescent probe in more complexbiological systems, a better understanding of reactivity andproducts in simple model systems is required. Using a vari-ety of antioxidants, including NO and related species,BODIPY11 was shown to be a simple, useful reporter formeasuring relative antioxidant efficiency, however, in thepresence of RNOS, behavior was more complex, withproducts from oxidation, nitroxidation, and nitration ofthe conjugated diene moiety observed.

Experimental procedures

All reactions, unless otherwise specified, were performedin 40% (v/v) acetonitrile in 10 mM phosphate-buffered sal-ine (PBS, 50 mM NaCl) pH 7.4 at 37 �C. The PBS stocksolution was stored over Chelex resin to minimize effectsdue to trace transition metals.

Chemicals

All chemicals, unless otherwise stated, were obtainedfrom Sigma (St. Louis, MO), Aldrich Chemicals (Milwau-kee, WI), or BDH (Toronto, Canada). 4,4-Difluoro-5-(-4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (BODIPY11) was obtained from MolecularProbes (Eugene, OR). Peroxynitrite was synthesized by tworoutes: (1) from the reaction of i-amyl nitrite (IAN) withaqueous H2O2 followed by exhaustive washing to removeIAN [6,7]; (2) from the reaction of aqueous NaNO2 withan acidic H2O2 solution with rapid alkaline quenching [8].In both cases, excess H2O2 was removed with MnO2 andUV–Vis spectroscopy was used to quantify peroxynitriteand check for contamination; typically NO2

� and IAN areimpurities in the respective preparations [9]. SIN-1 was syn-thesized by minor adaptation of literature procedures [25].

Oxygen pressure transducer measurements

The dual channel pressure transducer system has beenpreviously described [6,22]. The reaction vessel (10 ml)was shaken in a 37 �C bath fitted with a thermostat. Exper-iments with micelles used 2 ml volume linoleic acid solutionin 0.5 M SDS in 10 mM PBS, pH 7.4. An equal volume of2 ml PBS was used in the reference cell. Reaction was initi-ated by addition of 50 ll ABAP stock solution in 10 mMPBS, pH 7.4. When the rate of oxygen uptake became con-stant, a small volume (10 ll) of Trolox C in 0.5 M SDSsolution was added. After the rate of oxygen uptakereturned to that before the addition of Trolox C, the samevolume of tested compound solution in SDS wasadded and the rate of oxygen uptake monitored. All

A.C. Nicolescu et al. / Nitric Oxide xxx (2006) xxx–xxx 3

ARTICLE IN PRESS

concentrations of substrate, initiator, and inhibitors werecalculated using the micellar reaction volume of2.54 · 10�4 L corresponding to 2.0 ml of 0.5 M SDS [26].For experiments with DLPC liposomes (7.7–10 · 10�5 mol) containing AMVN (5.0–8.6 · 10�6 mol),the liposome emulsion prepared in 10 mM PBS (pH 7.4;2 ml) was added to the sample cell, and antioxidants wereadded as described above. The oxygen uptake was correct-ed for nitrogen and oxygen evolution from the azo-initiatorand peroxyl radical recombination, respectively, and oxy-gen consumption by the initiator.

Fluorescence assay

Fluorometric measurements were performed on a Spec-tramax Gemini XS spectrofluorometer (Molecular Devic-es) using a quartz covered 96-well glass microplate intriplicate (excitation wavelength 540 nm; emission wave-length at 600 nm; cutoff filter at 590 nm). Aliquots (5 ll)of inhibitor and BODIPY11 (200 nM) stock solutions pre-pared in 40% acetonitrile/60% 10 mM PBS were added toeach well (0.2 ml final reaction volume) and incubated at37 �C. The reaction was initiated by addition of small vol-umes (10 ll) of ABAP and SIN-1 stock solution in PBS orperoxynitrite in 10 mM NaOH (final pH of reactions solu-tions was assayed to avoid artifacts due to adjuvantinduced pH changes), and the fluorescence decay of theprobe was monitored to completion. The emission intensityin relative fluorescence units (RFU) was measured withtime, and normalized relative to 100% RFU immediatelyprior to addition of initiator.

LC/MS and LC/MS/MS analyses

All LC/MS and LC/MS/MS analyses were performedusing an Agilent LC/MSD Ion Trap SL mass spectrometerequipped with an Agilent 1100 HPLC system.HPLC method 1: Agilent Zorbax Rx-C8 column(4.6 mm · 250 mm, 5 lm), water containing 0.1% formicacid (A) and methanol (B) as mobile phase, a 1.5 min iso-cratic elution at 1% A increasing to 3% A in 1.5 min fol-lowed by a gradient of 3–20% A over 7 min. HPLCmethod 2: Supelco ODS-2C18 column (4.6 mm · 250 mm,5 lm) eluting with a linear gradient of 10–95% acetonitrileover 60 min, the counter solvent was water containing2.5 mM ammonium acetate. HPLC method 3: SupelcoODS-2C18 column (4.6 mm · 250 mm, 5 lm), water con-taining 0.3% acetic acid (A) and methanol (B) as mobilephase, a 8 min isocratic elution at 50% A increasing to90% A in 12 min then keeping over 10 min. The flow ratewas 0.8 ml/min split to 0.2 ml/min prior to the mass spec-trometer. Method 2 was used for BODIPY derivative anal-ysis; method 3 was used for ferrulic acid.

LC/MS analysis was performed for the reactions ofBODIPY11 (40 lM) with: bolus peroxynitrite (0.5 and1 mM) from either synthetic procedure; SIN-1 (1 mM)incubated for 6 h; or ABAP (20 mM) incubated at 37 �C

for 3 h. The product mixtures from the reactions of BOD-IPY11 with ABAP or peroxynitrite were loaded onto Prep-Sep C18 solid-phase extraction cartridges (0.5 ml), washedwith water to remove inorganic salts, and eluted with meth-anol. The neutral loss of a 20 Da fragment was indicativeof BODIPY11 derivatives and these signals were furtheranalyzed by MS–MS. For the isotope exchange experi-ments, an equal volume of D2O was added to the eluateand the mixture was directly injected into the mass spec-trometer for the quantification of isotope peaks of BOD-IPY11 derivatives. The results were compared withsimulated isotope distributions.

Results

Antioxidants and O2 consumption

Lipid peroxidation by definition involves the consump-tion of oxygen by a readily oxidizable lipid substrate: themeasurement of the oxygen uptake provides quantitativemechanistic information. The pressure transducer methodis very reliable for kinetic measurements, but labor inten-sive. SDS micelles of linoleic acid and multilamellarDLPC liposomes were used as lipid peroxidation models.The well-studied antioxidant Trolox C was used to calcu-late the rate of chain initiation, Ri, employing the expres-sion Ri = n[Inhibitor]/s, where s is the inhibition periodproduced by a known amount of antioxidant and n rep-resents the number of peroxyl radical equivalents termi-nated by one equivalent of antioxidant, which in thecase of Trolox C is 2 [24,27,28] (Fig. 1). A kinetic chainlength (number of substrate molecules oxidized per mol-ecule of radical that initiates the peroxidation chain)greater than three during the inhibition period ensuresthat the antioxidant reacts mainly with lipid peroxyl rad-icals rather than radicals derived directly from the azo-initiator.

For classical antioxidants that give a clear inhibitionperiod, absolute inhibition rate constants (kinh) can bedetermined by linear regression from the slopes of the plotsof the measured oxygen uptake (DO2) during the inhibitionperiod versus �ln (1 � t/s), where the slope is kp[L � H]/kinh, where [L � H] is the concentration of the lipid sub-strate and kp is the propagation rate constant for linoleatein lipid bilayers and micelles (36.1 M�1 s�1 under theexperimental conditions) [29,30]. For Trolox C, studied atvaried concentrations in SDS micelles, kinh was found1.66 ± 0.12 · 104 M�1 s�1 which compares well with the lit-erature [26,31]. In the DLPC liposomal system using thelipid-soluble azo-initiator, AMVN, kinh for Trolox C wasfound to be 3.08 ± 0.63 · 104 M�1 s�1 (Figs. 1A and B).

The NONOate NO donor, DEA/NO, and the organicnitrites (i-amyl nitrite, IAN; decyl nitrite, DCN; phenoxy-ethyl nitrite, PEN) inhibited oxygen consumption in lipidperoxidation with defined induction periods (Figs. 1Aand B, and Table 1), allowing calculation of inhibitionparameters. A relative antioxidant efficiency (RAE) can

Fig. 1. Induction periods observed for classical antioxidants in azo-initiator induced peroxidation of linoleate and oxidation of BODIPY11. (A) Inhibition ofoxygen uptake by DEA/NO, IAN, and Trolox C in ABAP (40 mM) induced peroxidation of linoleic acid in SDS micelles. Data for DCN and PEN are omittedfor clarity. (B) Inhibition of oxygen uptake by Trolox C in AMVN (7.25 lM) induced lipid peroxidation of DLPC multilamellar liposomes. (C) Inhibition ofABAP-induced fluorescence decay of BODIPY11 by Trolox C in 40% acetonitrile in 10 mM PBS, pH 7.4 at 37 �C; inset: linear dependence of induction periodon concentration of Trolox C. (D) a-Tocopherol (a-TcOH) inhibition of AMVN-induced fluorescence decay of BODIPY11 in acetonitrile at 37 �C; inset: lineardependence of induction period on a-TcOH concentration. Fluorescence units (FU) are normalized to 100% intensity immediately prior to addition ofantioxidant or vehicle, with 0% intensity corresponding to complete reaction. Time courses in triplicate (SD 6 5%).

Table 1Quantitative parameters for inhibition of lipid peroxidation obtained by measurement of oxygen consumption

Inhibitor Concentration (M) s (s) Ri · 108 (Ms�1) Chain length kinh (M�1 s�1) RAE

DEA/NOa 9.16 · 10�5 2460 1.19 6–21 1.20 · 104 0.72DEA/NO 5.60 · 10�8 2430 3.27 4–35 3.15 · 105 7.6IANa 8.03 · 10�3 1920 1.18 12–40 5.27 · 103 0.32DCNa 9.18 · 10�3 3360 1.26 7–23 3.35 · 103 0.20PENa 2.36 · 10�3 1110 1.34 4–40 9.44 · 103 0.57

a In micellar system; otherwise in DLPC liposomal system.


ARTICLE IN PRESS

then be defined as the ratio between the rate constant oftest inhibitor and the rate constant of Trolox C [32].

Antioxidants and BODIPY11 degradation

In simile with the classical oxygen consumption experi-ment (Figs. 1A and B), in the BODIPY11 degradation assaythe antioxidant activity of Trolox C was manifested by aninhibition period, the length of which was linearly propor-tional to antioxidant concentration (Fig. 1C). The experi-

mental radical generation rate (Rg), calculated using theobserved inhibition periods (s) and applying the formulaRg = 2 · [Trolox C]/s, was 3.48 ± 0.62 · 10�8 Ms�1. Thisis in good agreement with the theoretical value(3.39 · 10�8 Ms�1) calculated from the initial concentra-tion of the azo-compound and using the expressionRg = 2ekd[ABAP], where e (0.43) and kd (1.32 · 10�6 s�1

at 37 �C) are the efficacy and the decomposition rate con-stant for ABAP, respectively [23,33]. After the inductionperiod, when the antioxidant has been consumed, the


ARTICLE IN PRESS

fluorescence of BODIPY11 decays with the same rate asthat in the absence of the inhibitor.

The BODIPY11 fluorescence decay induced by thelipophilic azo-initiator AMVN, studied in acetonitrile, wasquenched by a-tocopherol (a-TcOH), with induction periodslinearly dependent on a-TcOH concentration (Fig. 1D). Theexperimental Rg (10.01 ± 2.45 · 10�8 Ms�1) was in goodagreement with the calculated value (9.48 · 10�8 Ms�1)using e (0.095) and kd (4.99 · 10�6 s�1 at 37 �C) for thedecomposition of 0.1 M AMVN [23]. Trolox C (30 lM) alsoinhibited BODIPY11 fluorescence decay initiated by AMVNin acetonitrile (data not shown) and gave an Rg

(11.1 ± 0.6 · 10�8 Ms�1) in agreement with the calculatedvalue.

Pseudo-first-order analysis of the rate of BODIPY11

fluorescence decay showed a linear dependence on ABAPconcentration in aqueous acetonitrile, yielding a second-or-der rate constant approximately 2-fold less than in eggphosphatidylcholine liposomes (1 mg/ml) under similarconditions (Fig. 2). The rate dependence on peroxyl radi-cals rather than ABAP itself is defined by the equationkobs = kb[ROO�]ss, where kb is the second-order rate con-stant for the reaction of BODIPY with the peroxyl radicalsgenerated by ABAP, and [ROO�]ss is the steady-state con-centration of the tertiary peroxyl radicals. The linear decayof the fluorescence indicates that steady-state conditionsare met. [ROO�]ss can be calculated using the equation[ROO�]ss = (Rg/2kt)

1/2, where Rg is the rate of radical gen-eration by ABAP, and 2kt is the second-order rate constantfor the self-recombination reaction of two peroxyl radicals(2ROO� fi non-radical products). Rg depends on the initialconcentration of ABAP and can be determined experimen-tally using the inhibition period method for different con-centrations of a classical antioxidant such as Trolox C.For 30 mM ABAP in 40% acetonitrile/PBS, Rg was3.48 ± 0.62 · 10�8 Ms�1. The rate constant, 2kt, for neutral

Fig. 2. Observed rate constants as a function of ABAP concentration, forfluorescence decay of BODIPY11 in aqueous acetonitrile (solid line) and inDLPC liposomes (dashed line); triplicate data showing SD.

tertiary peroxyl radicals in organic solvents at 30 �C hasbeen reported to range between 0.1 and 60 · 104 M�1s�1

[34]. ABAP produces cationic peroxyl radicals that areanticipated to have slower recombination rates due to elec-trostatic repulsion. Thus, choosing the lower value for 2kt

yields kb = 6.2 · 103 M�1 s�1, which compares to a valueof 6.0 · 103 M�1 s�1 reported for the reaction of BOD-IPY11 with tertiary peroxyl radicals generated fromAMVN in acetonitrile at 37 �C [15].

The NO donor, DEA/NO (t1/2 = 2 min at 37 �C and pH7.4), behaved as an apparent, classical antioxidant produc-ing short induction periods for ABAP-induced BODIPY11

oxidation (Fig. 3A). The dependence of s on DEA/NOconcentration was not linear. This is expected, as the anti-oxidant species is NO, not DEA/NO itself. The calculatednumber of quenched peroxyl radical equivalents (n = [In-hibitor]/(Rgs)) was lower at higher DEA/NO concentration(e.g., 0.41 at 23.7 lM, and 0.15 at 94.0 lM DEA/NO,respectively), because at higher fluxes, the NO in excessof peroxyl radicals is lost through alternative reactions,such as oxidation, or will simply effuse. SPE/NO(t1/2 = 39 min at 37 �C) and DETA/NO (t1/2 = 20 h at37 �C) also showed induction periods for inhibition ofABAP-induced BODIPY11 fluorescence decay at higherconcentrations (Figs. 3B and C): from these data SPE/NO (0.15 mM) was calculated to quench 0.48 peroxylradical equivalents.

Thiols have both antioxidant and prooxidant propertiesand may simply undergo H-atom abstraction or oxidationto terminate chain propagating peroxidation reactions, butthe resulting sulfur–oxygen radicals can propagate radicalchains [2,6,35]. Cysteine inhibited ABAP-induced fluores-cence decay of BODIPY11 with induction times that werenot linear in cysteine concentration (Fig. 4A). The calculat-ed number of quenched peroxyl radical equivalents (n) wasfound to be 0.13. However, BODIPY11 degradation wasonly retarded in the induction period, compatible withBODIPY11 degradation by electrophilic thiyl radicals(which add readily to dienes) and sulfur–oxygen radicalsformed from peroxyl radical scavenging (reactions (3)and (4) lead to loss of BODIPY fluorescence):

R�+ O2!ROO� ð1Þ

ROO�+ CysSH!ROOH + CysS� ð2ÞCysS�+ O2!CysSOO�! propagation ð3Þ

CysS�+ BODIPY–CH@CH–CH@CHR0

!BODIPY–CðSCysÞH–CH@CH–HC�R0 ð4Þ

Human serum albumin (HSA) is a major blood plasmaprotein that contributes to the antioxidant capacity ofblood. At concentrations lower than normally present inhuman blood plasma (i.e., 3–5 g/100 ml or 4.51–7.52 lM),HSA depressed the rate of BODIPY11 fluorescence decayin a concentration-dependent manner (Fig. 4B), acting toretard the BODIPY11 oxidation rate. Only at high concen-tration (2.26 lM) was an induction period observed.

Fig. 4. Effects of different antioxidants on ABAP-induced fluorescence decay of BODIPY11 in 40% acetonitrile in 10 mM PBS, pH 7.4, at 37 �C: (A)cysteine; (B) human serum albumin (HAS); (C) serotonin (5HT); (D) S-nitrosoglutathione (SNOG or GSNO); (E) i-amyl nitrite (IAN). Fluorescence units(FU) are normalized to 100% intensity immediately prior to addition of antioxidant or vehicle, with 0% intensity corresponding to complete reaction. Timecourses in triplicate (SD 6 5%).

Fig. 3. Effects of NONOates on ABAP-induced fluorescence decay of BODIPY11 in 40% acetonitrile in 10 mM PBS, pH 7.4, at 37 �C: (A) DEA/NO; (B)SPE/NO; (C) DETA/NO. Fluorescence units (FU) are normalized to 100% intensity immediately prior to addition of antioxidant or vehicle, with 0%intensity corresponding to complete reaction. Time courses in triplicate (SD 6 5%).


ARTICLE IN PRESS

Serotonin (5HT), an important neurotransmitter, isexpected to be a classical phenolic chain-breaking antioxi-dant, inhibiting BODIPY11 oxidation. 5HT is able to forman N-stabilized phenoxyl radical, but 5HT was observedonly to retard the rate of BODIPY11 oxidation, albeit ina concentration-dependent manner (Fig. 4C).

In contrast to NONOates, which cleanly and spontane-ously generate NO in neutral aqueous solution, S-nitrosoand O-nitroso compounds did not show an induction peri-od in inhibition of BODIPY11 oxidation. It has beenreported that nitrosothiols may act as effective antioxidants

in vivo; S-nitrosoglutathione (GSNO) was reported to be a100-fold more potent antioxidant than its thiol parent[36,37]. In azo-induced BOPIPY11 oxidation, GSNO wasless effective than cysteine in that an induction periodwas not observed (Fig. 4D). The formation of antioxidantNO from a nitrosothiol by homolysis is a 1e� reductionrequiring catalysis by e� donors including transition metalions or photolysis. In the present experiments, the workingexcitation wavelength (540 nm) and the use of Chelex C toremove adventitious transition metal cations reduce NOrelease and antioxidant activity of GSNO. In simile with


ARTICLE IN PRESS

the nitrosothiol, IAN also retarded BODIPY11 oxidation,albeit with much lower potency than GSNO, compatiblewith the slow hydrolysis and minor flux of NO producedby organic nitrites (Fig. 4E) [6].

Quantification of antioxidant activity using BODIPY11

The inhibitors used in the peroxyl radical trapping fluo-rescence assay are categorized in two groups: (i) inhibitorsthat show definite inhibition periods, and (ii) inhibitorsthat depress the rate of fluorescence decay without showinginhibition periods. Three semi-quantitative methods wereused to quantify the antioxidant effect on ABAP-inducedfluorescence decay of BODIPY11.

(1) Defined induction periods. The constant rate of per-oxyl radical generation in the reaction mixture usingan azo-initiator can be measured using Trolox C,which is known to trap two mole equivalents of per-oxyl radicals (Rg = 2[Trolox]/sTrolox). The inhibitorycapacity is found by multiplying the rate of peroxylradical generation by the induction period shownby antioxidants (Rgs), which leads to a molar antiox-idant capacity (ACs = Rgs/[inhibitor]). In the aque-ous acetonitrile system, the rate of peroxyl radicalgeneration from ABAP (30 mM) was found to be3.70 ± 0.19 · 10�8 Ms�1, thus for DEA/NO, SPE/NO, and cysteine, ACs can be calculated. For cys-teine, ACs = 0.153 ± 0.025 · 10�3. For NONOates,ACs varies with concentration, since the antioxidantis not the NONOate itself, but NO: the highest valuesfrom the collected data are 0.55 · 10�3 and0.24 · 10�3 for SPE/NO and DEA/NO, respectively.

(2) Retardation of BODIPY decay. For antioxidantsthat do not show an inhibition period, the ratio of ini-tial rates for inhibited (v0,inh) and uninhibited, control(v0) reactions yields a measure of relative antioxidantcapacity (ACv = 1 � v0,inh/v0). The ACv data

Fig. 5. Antioxidant capacity from AUC measurements of BODIPY fluorescencTrolox C and 5HT); dashed lines (first-order fits for HSA, cysteine, GSNO); dthat of cysteine residues.

can be plotted to estimate IC50 values forinhibition of peroxidation by antioxidants, yieldingIC50 = 0.2 ± 0.15, 5 ± 2, 80 lM, 4, 0.1, and 2 mMfor HSA, serotonin, GSNO, IAN, SPE/NO, andDETA/NO, respectively.

(3) Net protection or area-under-the-curve (AUC). TheAUC method has been used in a number of studiesand approximates the net protection against oxidationwith contributions from both chain-breaking antioxi-dant and retardation activity. The net antioxidantcapacity (ACAUC = (AUCinh � AUCun)/AUCun) iscalculated from the area under the curve of the BOD-IPY11 fluorescence decay in the presence (AUCinh)and absence (AUCun) of an antioxidant and is depen-dent on antioxidant concentration (Fig. 5). In thisanalysis, the phenolic antioxidants Trolox C and5HT show a linear dependence upon concentration,despite the fact that one is acting as a classical chain-breaking antioxidant and the other as an oxidationretardant. Interestingly, 5HT has a higher antioxidantcapacity than Trolox C by this measure. ACAUC valuescalculated for the thiol antioxidants, cysteine andHSA, showed saturation behavior compatible withthe influence of chain propagating and prooxidantactivity at higher concentrations. The optimumNONOate chain-breaking antioxidant would releasea flux of NO greater than the flux of peroxyl radicals(�40 nM�1) and maintain this flux over the course ofthe experiment. Consequently, the short half life ofDEA/NO is insufficient to maintain such a flux andtherefore saturation behavior is observed.

Peroxynitrite and BODIPY11 degradation

BODIPY11 reaction in phosphatidylcholine liposomeswith bolus peroxynitrite led to fluorescence decay(Fig. 6A). Similar observations were made in homogeneous

e decay (ACAUC = (AUCinh � AUCun)/AUCun). Solid lines (linear fits forotted lines (first-order fits for NONOates). For HSA, the concentration is

Fig. 6. Time course of BODIPY11 fluorescence decay in response to peroxynitrite. (A) Repetitive bolus addition (1.31 mM) to egg phospholipid liposomesin PBS. (B) Repetitive bolus addition (shown by arrows) of peroxynitrite (1.61 mM) to methanol (s) and 40% acetonitrile in 10 mM PBS, pH 7.4 (n).(C) SIN-1 effect on BODIPY11 fluorescence in 40% acetonitrile in 10 mM PBS, pH 7.4. Fluorescence units (FU) are normalized to 100% intensityimmediately prior to initial addition of peroxynitrite or SIN-1; time courses in triplicate.


ARTICLE IN PRESS

solutions of 40% MeCN/PBS and in methanol (Fig. 6B).The diminished BODIPY degradation in methanol waslikely the result of the well-known radical scavenging prop-erty of alcohols. The fast decomposition of peroxynitrite atneutral pH leads to the production of oxidizing radicalsthat can add to BODIPY or more rapidly undergo radi-cal–radical reactions followed by hydrolysis to stableNOx anions. The short lifetime of peroxynitrite and theabsence of a substrate able to propagate a chain reactionlead to the observed incomplete oxidation of BODIPY11

by bolus peroxynitrite (Figs. 6A and B).The sydnonimine SIN-1 undergoes base-assisted ring-

opening to generate an intermediate that will release asteady, but non-linear flux of NO with concomitant1e� transfer to an oxidant, usually O2. Thus the simulta-neous generation of NOþO2

�� in a 1:1 stoichiometryleads to SIN-1 acting as a peroxynitrite donor, exceptin cases where other oxidants interfere with electrontransfer. In aqueous acetonitrile, the decay of BODIPY11

fluorescence was observed to follow the expected kinetictime course for peroxynitrite release from SIN-1(Fig. 6C).

Fig. 7. Effect of NO donors and NO scavengers on ABAP- or SIN-1-inducedABAP induction; (B) SPE/NO effect on SIN-1 induction; (C) PTIO effect on Simmediately prior to addition of antioxidant or vehicle, with 0% intensity cor

NO scavengers and BODIPY11 degradation

To further explore and compare the response of BOD-IPY11 to ROS and RNOS and the influence of antioxi-dants, the effect of NONOate NO donors was assayed incombination with the putative NO trap nitronyl nitroxide,2-(phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide(PTIO). In ABAP-initiated oxidation, the induction periodof SPE/NO was prolonged by the addition of increasingconcentrations of PTIO, thus PTIO did not serve to trapthe antioxidant NO released from SPE/NO (Fig. 7A). IfPTIO had behaved as a selective trap for NO, the expecta-tion would be for diminution of the antioxidant effect ofthe NO donor in ABAP-induced oxidation. SPE/NO,alone, was seen to inhibit the oxidation of BODIPY11 bySIN-1 with an induction period at higher concentration(Fig. 7B). In addition, PTIO, alone, efficiently inhibitedthe decay of BODIPY11 fluorescence induced by SIN-1(Fig. 7C). The effects of the NO donor and of PTIO onthe reaction of SIN-1 with BODIPY11 are anticipated.The maximum flux of ROS and RNOS from peroxynitritewill occur at a stoichiometry of NO=O2

�� of 1:1; simplistically,

BODIPY11 fluorescence decay: (A) PTIO effect on SPE/NO inhibition ofIN-1 induction. Fluorescence units (FU) are normalized to 100% intensityresponding to complete reaction. Time courses in triplicate (SD 6 5%).


ARTICLE IN PRESS

at other stoichiometries, ROS and RNOS will be trappedby the excess NO or O2

��. The observations with PTIOin ABAP-induced oxidation show that PTIO does not actspecifically as an NO trap.

LC/MS analysis

BODIPY11 and its reaction products can beidentified in LC/MS/MS analysis by the loss of a char-acteristic HF fragment (20 Da). Reaction of the water-soluble azo-initiator ABAP (20 mM) with BODIPY11

(40 lM) in 40% MeCN in 10 mM PBS, pH 7.4, gaveas major product the carboxylic acid (m/z

Fig. 8. LC/MS analysis of BODIPY-containing products (undergoing neutral(A) bolus peroxynitrite (1 mM) (inset: 0.5 mM peroxynitrite) in 10 mM PBS,after 6 h in 10 mM PBS, pH 7.4; (C) ABAP (20 mM) in 40% acetonitrile inproducts from reaction of BODIPY11 with bolus peroxynitrite, m/z [M � H]�

[M � H]� = 419) resulting from oxidative cleavage ofthe phenyldiene moiety (Fig. 8C). This product waspreviously reported in a 24-h incubation of ABAP inabsolute ethanol to be the major product of reaction(>90% by relative intensity) [16]. The minor productof ABAP-induced oxidation, resulting from olefinicbond cleavage adjacent to the phenyl group (m/z445), was not observed in this study, although severalsmall signals (<5%) in the chromatograms were notinvestigated in detail. The small difference in observa-tions is compatible with the differences in solvent (eth-anol is an H atom donor solvent) and reactionsconditions employed in these experiments.

loss of HF) from reaction solutions of BODIPY11 (40 lM) at 37 �C with:pH 7.4; (B) peroxynitrite produced by decomposition of SIN-1 (0.1 mM)10 mM PBS, pH 7.4. (D) and (E) H/D isotope exchange experiment for= 419 (top), 519 (down), showing single label incorporation.


ARTICLE IN PRESS

Reaction of BODIPY11 with bolus peroxynitrite (1 mM)in the aqueous acetonitrile solvent system generated amuch greater range of products, including the product ofoxidative cleavage (m/z [M � H]� = 419) (Fig. 8A). Fourcompounds were characterized by LC/MS/MS analysis(in addition to the oxidative cleavage product), corre-sponding to substitution of an OH and/or NO2 radicalfor H: negative ion analysis indicated substitution productscontaining HO (m/z 519), HO + NO2 (m/z 564), andHO + 2NO2 (m/z 609) (Figs. 8 and 9). The four signalsobserved corresponded to compounds containing BOD-IPY11, all fragmenting to undergo loss of HF (�20 Da).All three nitration products underwent loss of a neutralfragment (m/z 47; corresponding to HNO2). Similarly,nitrolinoleic acid derivatives have been reported to losean HNO2 fragment in MS analysis [38]: collectively theseresults support the nitration of BODIPY11 at an olefinicrather than an aromatic site.

In aerobic solution, SIN-1 undergoes base catalyzed ring-opening to an intermediate that further decomposes torelease a low flux of peroxynitrite, which was observed todegrade the BODIPY11 fluorophore. Two products werecharacterized by LC/MS/MS analysis of a 6 h incubationof SIN-1 with BODIPY11 in buffered aqueous CH3CN, iden-tified as the aldehyde oxidative cleavage products of BOD-IPY11 (m/z 403) (Fig. 8A). In accord with the observations

Fig. 9. BODIPY11 oxidation, nitroxidation, and nitration products: (A) ox(B) epoxidation of BODIPY diene by peroxynitrous acid; (C) radical addition oaddition of NO2, capture of O2, and subsequent peroxide decomposition; (E)addition of peroxynitrous acid to BODIPY diene, followed by elimination of nitalternative isomers compatible with MS data are possible. Negative ion m/z v

on ABAP-induced oxidative cleavage, above, and a litera-ture report [16], selectivity was observed for cleavage of theolefinic bond closest to the pyrrole group of BODIPY.

Isotope distributions were compatible with structuralassignments in all MS spectra. In order to distinguishbetween product isomers, product solutions were incubatedin the presence of D2O, leading to isotope exchange with acid-ic protons. Isotope exchange with some species (e.g., m/z 419)gave no change in the negative ion mode MS spectra, whereasisotope exchange with others (e.g., m/z 419, 519, 564, and609) gave addition of one mass unit with isotope patternsconfirming deuterium incorporation (Figs. 8D and E).

Model reactions were carried out with cinnamic acid,5-phenyl-2,4-pentadienoic acid, 1,4-diphenyl-1,3-butadi-ene, and ferrulic acid in admixture with bolus peroxynitrite(0.5 and 1 mM). Only ferrulic acid underwent reactionunder these conditions, as assessed by LC/MS analysis.Ferrulic acid almost quantitatively gave a product withm/z 239 in negative ion mode (Scheme 1), which fragment-ed with loss of oxygen (data not shown).

Discussion

Fluorescent probes and dyes are widely used in bothantioxidant and NO research, providing ready quantifica-tion and visualization of phenomena in vitro and in some

idative cleavage of BODIPY diene by oxyradicals produced by ABAP;f NO2 to BODIPY diene and reconjugation with loss of nitrite; (D) radicalHO� radical addition and oxidation of BODIPY diene; (F) electrophilic

rous acid. These structures are representative of products and mechanisms,alues are given with the agent that yields the product in parentheses.

Scheme 1.


ARTICLE IN PRESS

cases in vivo. BODIPY-based fluorophore probes are find-ing increasing use in cell culture [16]. A number of fluores-cence reagents are available for monitoring lipidperoxidation, for example, fluorescein derivatives, parina-ric acid and, more recently, boron dipyrromethene difluo-ride (BODIPY) reagents. The use of several fluorescentdyes has been criticized because of sensitivity to factorssuch as light and pH, suboptimal fluorescence properties,and reversible redox properties whereby the probe is notinert.

BODIPY11 contains a C11 saturated fatty acid chain inaddition to a conjugated phenyldiene moiety at the fifthposition in the boron dipyrromethene ring, which providesthe molecule with (a) an extended conjugated chromophorethat absorbs light of low energy (kmax = 582 nm,e = 140,000 M�1 cm�1 in methanol) and (b) a functionalgroup potentially sensitive to oxidants such as lipid peroxylradicals.

BODIPY11 fluorescence decay was earlier suggested tobe sensitive to alkoxyl, peroxyl, and hydroxyl radicals,but not to superoxide, NO, or hydroperoxides [39],although reaction with peroxynitrite was subsequentlyreported [16,40]. The fluorescence of BODIPY11 reagentsthat lack a conjugated diene is insensitive to oxidizingagents. Furthermore, diene oxidation yields a product thatexhibits green fluorescence, similar to that of unsubstitutedboron dipyrromethene difluoride ring [16]. The reactions ofROS and RNOS with BODIPY11, that lead to loss of fluo-rescence, must occur at the phenyldiene moiety owing tothe insensitivity of the parent BODIPY core. WhereasROS are expected to oxidize the diene group, RNOS mayreact to produce a variety of products. The potential utilityof the BODIPY11 probe in quantifying oxidation inresponse to ROS and RNOS in biological systems requiresa fuller understanding of probe reactivity in simple modelsystems. Accordingly, (a) the BODIPY11 probe was bench-marked using standard antioxidants and an azo-initiatorsource of peroxyl radicals, and comparison made with oxy-gen-uptake data that are direct measures of antioxidantactivities under controlled oxygen pressure; (b) the reactiv-ity of BODIPY11 towards RNOS was studied; and (c) theinfluence of NO-related antioxidants on the reactions ofROS and RNOS with BODIPY11 was examined.

In our experiments, the rate of BODIPY11 fluorescencedecay was dependent on the concentration of azo-initiatorsboth in a homogenous aqueous solution and in phosphati-dylcholine liposomes (Fig. 2). The choice of a bufferedaqueous acetonitrile solution provides a medium withoutinherent antioxidant capacity (in contrast to alcohols) inwhich a variety of lipophilic antioxidants (and their oxida-tion products) are soluble. The observed linear dependenceon azo-initiator concentration suggests a reaction mecha-nism in which the addition of the peroxyl radical is ratedetermining:

R–N@N–RðABAPÞ

!kd2R� þN2

R� þO2 ��!k>109 M�1 s�1

ROO�

ROO� þ BODIPY��!kBODIPY

ROO� BODIPY�

ROO� þ inhibitor�!kinh

products

2ROO��!2kt

termination productsþO2

Azo-initiated decay of BODIPY11 fluorescence followsfirst-order kinetics both in the presence, and in the absenceof classical chain-breaking antioxidants in simile with theO2 consumption measurements (Fig. 1), indicatingsteady-state kinetics. Comparison of O2 consumption withBODIPY11 decay demonstrates similar behavior for theseclassical antioxidants, which compete for peroxyl radicals,regardless of whether the system contains polyunsaturatedfatty acid liposomes or is a simple homogenous solution.

Depending on the rates of peroxyl radical reaction withthe antioxidant inhibitor and BODIPY11, respectively, theprofile of fluorescence decay will reflect one of three scenar-ios: (i) when the rate of peroxyl radical trapping by theinhibitor is much greater than with BODIPY11 (kinh�kBODIPY), the rate of BODIPY11 fluorescence decay will

be zero order until the inhibitor is consumed; (ii) whenthe rate of peroxyl radical reaction with BODIPY11 ismuch greater than that with the inhibitor (kBODIPY� kinh),the rate of BODIPY11 fluorescence decay will be first orderand the same as the uninhibited reaction, and (iii) when therate of peroxyl radical reaction with the inhibitor is compa-rable to that with BODIPY11 (kBODIPY � kinh), the rate of


ARTICLE IN PRESS

BODIPY fluorescence decay will be first order and retard-ed compared to the uninhibited reaction. All these types ofbehavior, predicted for antioxidants that scavenge peroxylradicals, are observed in this study (Figs. 1, 3, and 4).Moreover, the decay profiles are compatible with the anti-oxidant mechanisms, elucidated by other methods, for avariety of inhibitors, including NO donors (for which theflux of NO is the determining factor), thiols (which alsopossess prooxidant activity), and chain-breaking phenolicantioxidants. The measurement of BODIPY11 decay inaqueous acetonitrile may be used reliably to quantify rela-tive antioxidant activity and to classify antioxidant mecha-nisms (Fig. 5).

The antioxidant activity of NO has been well studied [2–4,41]. The antioxidant potency of NONOates is quantita-tively related to the rate of NO release, with a lipid radicalchain termination stoichiometry of 0.4–0.5 mol of lipid per-oxyl radicals per mole of NO [2]. The nitronyl nitroxide,PTIO, and its derivative, cPTIO, are often used as ‘‘selec-tive’’ NO scavengers [42], and therefore the effect of PTIOon NO antioxidant and SIN-1 prooxidant activity was ofinterest. PTIO has frequently been employed to trap NOand thence to yield the prooxidant NO2, which wouldreverse the antioxidant effect of NO. In this work, PTIOdid not act as an NO scavenger in co-incubation withSPE/NO and BODIPY11, but instead enhanced the antiox-idant activity of NO towards azo-induced BODIPY11 oxi-dation, compatible with the known antioxidant activity ofnitronyl nitroxides (Fig. 7). In reaction with SIN-1, PTIOhas previously been proposed to inhibit superoxide forma-tion, thus enhancing NO release, although other mecha-nisms are now favored [43,44]. In this study, both PTIOand SPE/NO inhibited SIN-1-induced oxidation of BOD-IPY11, which is explained by inhibition of peroxynitrite for-mation and scavenging of peroxynitrite and its radicalproducts.

The major product derived from peroxyl radical addi-tion to BODIPY was identified by LC/MS analysis as thecarboxylate resulting from oxidative cleavage of the olefin-ic bond adjacent to the pyrrole ring (Figs. 8C and 9) . Theidentification of this major product is in accord with a pre-vious study in which under different reaction conditions,the minor product of oxidative cleavage of the alternateolefinic bond was also observed [16].

In contrast to the azo-initiated, peroxyl radical-mediatedfluorescence decay of BODIPY11, reaction with bolus per-oxynitrite elicited a more complex profile of fluorescencedecay and of BODIPY11 products (Figs. 6 and 8). The timecourse of BODIPY11 decay elicited by bolus peroxynitriteconfirmed that in a simple homogeneous solution and inthe absence of a polyunsaturated lipid substrate, a chainpropagation mechanism is not supported (Fig. 6). In aero-bic, homogeneous solutions, one decomposition pathwayof peroxynitrite will yield NO2 and an oxy-radical (eitherCO3��; or HO� in the absence of CO2). Whereas, the 1,4-di-

ene moiety of fatty acids readily undergoes H-atom abstrac-tion, the 1,3-diene of BODIPY11 can only undergo radical

addition. Thus, the predicted initial products of reactionof BODIPY11 with peroxynitrite are those of addition ofNO2 and of the oxy-radicals; decomposition to final prod-ucts is driven by reconjugation either by abstraction of Hor H+, yielding radical substitution products. An alterna-tive pathway that might lead to some of the same productsis electrophilic addition of peroxynitrous acid to BODIPY11

(Fig. 9). Interestingly, simple phenyldiene model com-pounds were seen to be unreactive towards bolus peroxyni-trite, showing that the phenyldiene moiety of BODIPY11 isof enhanced reactivity towards oxidation.

The olefinic cleavage product was not the major productobserved in reactions with peroxynitrite. The reactionproducts of BODIPY11 with bolus peroxynitrite were iden-tified by LC/MS as those derived from addition of an oxy-gen atom and of NO2, as well as the oxidative cleavageproduct also observed in BODIPY11 peroxidation (Figs.8 and 9). One candidate structure for the O-additionproduct (m/z 519) is an oxirane, which is a typical productof olefinic oxidation (Fig. 9B). LC/MS and H/D isotopelabeling studies were required to rule out the oxirane infavour of the alternative conjugated enol as the majorproduct of BODIPY nitroxidation.

Products of olefinic nitration were identified (m/z 564and 609), but the expected simple mononitration product(m/z 549) was not observed (Fig. 9). The simple mononitra-tion product would have reduced reactivity towards furtherradical addition, which suggests that BODIPY11 nitroxida-tion precedes nitration or that NO2 olefinic addition isreversible. This is also in accord with the observed productdistributions at different peroxynitrite concentrations(Fig. 8). There are a number of potential pathways to theenol nitroxidation product (m/z 519), including epoxida-tion/ring-opening and addition of NO2 (or CO3

��) fol-lowed by rearrangement (Fig. 9). Given reasonablealternative pathways, there is no need to invoke simplehydroxyl radical olefinic addition; hydroxyl radical addi-tion is not probable given the known very rapid trappingof peroxynitrite by CO2 [45]. Nitroxidation and nitrationof BODIPY11 leads to substitution products that apparent-ly possess the same extended conjugation as the parent,however, their enolate tautomers have broken conjugation,compatible with loss of fluorescence at higher wavelengthin these products relative to BODIPY11 itself [43].

The usefulness of bolus peroxynitrite is problematiceven in simple model systems because of the very high localconcentrations and the influence of mixing. Alternatively,sydnonimines (almost exclusively SIN-1) have been usedas a continuous source of peroxynitrite. BODIPY11 wassensitive to SIN-1 showing a decay time course compatiblewith the known pathway of decomposition of SIN-1 to per-oxynitrite, but the BODIPY11 products identified by LC/MS were not the same as those seen in reactions with bolusperoxynitrite (Figs. 6C and 8). SIN-1 treatment of BOD-IPY11 gave oxidative olefinic cleavage in simile with BOD-IPY11 peroxidation, however, BODIPY11-derived aldehydeproducts were observed for SIN-1, reflecting the relatively


ARTICLE IN PRESS

weak oxidizing environment compared to that produced bythe azo-initiator. Observation of differences in reactionproducts from treatment with either bolus peroxynitriteor SIN-1 is not uncommon, for example, SIN-1 does notsupport significant tyrosine nitration in contrast to bolusperoxynitrite. Drummen et al. reported the detection ofthe oxidative cleavage product and the BODIPY11-derivedenol in cell culture treated with bolus peroxynitrite, but didnot detect nitration products, suggesting that NO2 is rapid-ly scavenged by molecules such as glutathione [16].

In summary, the present study analyzed mechanisms ofBODIPY11 fluorescence decay in simple, aerobic, bufferedsolution, and the reaction products of BODIPY11 withROS and RNOS (peroxyl radicals and peroxynitrite)involved in biologically relevant oxidative processes. Per-oxyl radical-induced fluorescence decay of BODIPY11 inthis system demonstrated similar patterns of antioxidantactivity to those observed in classical oxygen pressure mea-surements in micelles and liposomes, and provided both areadily applied quantitation of antioxidant capacity andmechanistic information.

No less than six novel nitroxidation and nitration prod-ucts from the reaction of BODIPY11 with bolus peroxyni-trite and a peroxynitrite donor (SIN-1) were identified byLC/MS/MS. The identity of the previously reportednitroxidation product (m/z 519) as the conjugated enolwas unambiguously established by H/D isotope exchange.The behavior of BODIPY11 towards RNOS is more com-plex, even in the simple systems used in this study, yieldinga variety of oxidative cleavage and substitution productsderived from nitroxidation and nitration reactions. Thediverse BODIPY-derived products, dependent upon theidentity of the ROS and RNOS, may allow the use ofBODIPY11, in combination with LC/MS, to identify thedifferent ROS and RNOS in biological systems, but thecomplexity of the product distribution must be consideredin any analysis. Similarly, the observed behavior of thenitronyl nitroxide PTIO as an antioxidant rather than asan NO scavenger emphasizes the need for caution in anal-ysis of more complex biological systems.

Acknowledgments

This work was supported in part both by NSERC Grant245617-01 and NIH Grant CA 102590. Professor RossBarclay is acknowledged for assistance with oxygen pres-sure measurements.

References

[1] B. Halliwell, J.M.C. Gutteridge, Free Radicals in Biology andMedicine, third ed., Oxford University Press, New York, 1999.

[2] A.C. Nicolescu, S.I. Zavorin, N.J. Turro, J.N. Reynolds, G.R.Thatcher, Inhibition of lipid peroxidation in synaptosomes andliposomes by nitrates and nitrites, Chem. Res. Toxicol. 15 (2002) 985–998.

[3] V.B. O’Donnell, P.H. Chumley, N. Hogg, A. Bloodsworth, V.M.Darley-Usmar, B.A. Freeman, Nitric oxide inhibition of lipid

peroxidation: kinetics of reaction with lipid peroxyl radicals andcomparison with alpha-tocopherol, Biochemistry 36 (1997) 15216–15223.

[4] H. Rubbo, R. Radi, M. Trujillo, R. Telleri, B. Kalyanaraman, S.Barnes, M. Kirk, B.A. Freeman, Nitric oxide regulation of superoxideand peroxynitrite-dependent lipid peroxidation. Formation of novelnitrogen-containing oxidized lipid derivatives, J. Biol. Chem. 269(1994) 26066–26075.

[5] S.R. Thomas, M.J. Davies, R. Stocker, Oxidation and antioxidationof human low-density lipoprotein and plasma exposed to 3-morpho-linosydnonimine and reagent peroxynitrite, Chem. Res. Toxicol. 11(1998) 484–494.

[6] A.C. Nicolescu, J.N. Reynolds, L.R. Barclay, G.R. Thatcher, Organicnitrites and NO: inhibition of lipid peroxidation and radical reactions,Chem. Res. Toxicol. 17 (2004) 185–196.

[7] R.M. Uppu, W.A. Pryor, Synthesis of peroxynitrite in a two-phasesystem using isoamyl nitrite and hydrogen peroxide, Anal. Biochem.236 (1996) 242–249.

[8] J.S. Beckman, T.W. Beckman, J. Chen, P.A. Marshall, B.A. Freeman,Apparent hydroxyl radical production by peroxynitrite: implicationsfor endothelial injury from nitric oxide and superoxide, Proc. Natl.Acad. Sci. USA 87 (1990) 1620–1624.

[9] M.N. Hughes, H.G. Niklin, The Chemistry of pernitrites. Part 1.Kinetics of decomposition of pernitrous acid,, J. Chem. Soc. A 12(1968) 450–452.

[10] F.P. Corongiu, S. Banni, Detection of conjugated dienes by secondderivative ultraviolet spectrophotometry, Methods Enzymol. 233(1994) 303–310.

[11] J. Liu, H.C. Yeo, S.J. Doniger, B.N. Ames, Assay of aldehydes fromlipid peroxidation: gas chromatography-mass spectrometry comparedto thiobarbituric acid, Anal. Biochem. 245 (1997) 161–166.

[12] Y.M. Naguib, A fluorometric method for measurement of peroxylradical scavenging activities of lipophilic antioxidants, Anal. Bio-chem. 265 (1998) 290–298.

[13] Y.M. Naguib, A fluorometric method for measurement of oxygenradical-scavenging activity of water-soluble antioxidants, Anal. Bio-chem. 284 (2000) 93–98.

[14] G. Aldini, K.J. Yeum, R.M. Russell, N.I. Krinsky, A method tomeasure the oxidizability of both the aqueous and lipid compartmentsof plasma, Free Radic. Biol. Med. 31 (2001) 1043–1050.

[15] Y. Yoshida, S. Shimakawa, N. Itoh, E. Niki, Action of DCFH andBODIPY as a probe for radical oxidation in hydrophilic andlipophilic domain, Free Radic. Res. 37 (2003) 861–872.

[16] G.P. Drummen, B.M. Gadella, J.A. Post, J.F. Brouwers, Massspectrometric characterization of the oxidation of the fluorescent lipidperoxidation reporter molecule C11-BODIPY(581/591), Free Radic.Biol. Med. 36 (2004) 1635–1644.

[17] G.P. Drummen, M. Makkinje, A.J. Verkleij, J.A. Op den Kamp, J.A.Post, Attenuation of lipid peroxidation by antioxidants in rat-1fibroblasts: comparison of the lipid peroxidation reporter moleculescis-parinaric acid and C11-BODIPY(581/591) in a biological setting,Biochim. Biophys. Acta 1636 (2004) 136–150.

[18] Y. Christova, P.S. James, R. Jones, Lipid diffusion in sperm plasmamembranes exposed to peroxidative injury from oxygen free radicals,Mol. Reprod. Dev. 68 (2004) 365–372.

[19] J.P. Fabisiak, Y.Y. Tyurina, V.A. Tyurin, J.S. Lazo, V.E. Kagan,Random versus selective membrane phospholipid oxidation inapoptosis: role of phosphatidylserine, Biochemistry 37 (1998)13781–13790.

[20] J.F. Brouwers, B.M. Gadella, In situ detection and localization oflipid peroxidation in individual bovine sperm cells, Free Radic. Biol.Med. 35 (2003) 1382–1391.

[21] J.W. Borst, N.V. Visser, O. Kouptsova, A.J. Visser, Oxidation ofunsaturated phospholipids in membrane bilayer mixtures is accom-panied by membrane fluidity changes, Biochim. Biophys. Acta 1487(2000) 61–73.

[22] D.D.M. Wayner, G.W. Burton, Measurements of individual antiox-idants and radical-trapping activity, in: J. Miquel (Ed.), Handbook of


ARTICLE IN PRESS

Free Radcals and Antioxidants in Biomedicine, CRC Press, BocaRaton, FL, pp. 223–232.

[23] L.R. Barclay, M.R. Vinqvist, Membrane peroxidation: inhibitingeffects of water-soluble antioxidants on phospholipids of differentcharge types, Free Radic. Biol. Med. 16 (1994) 779–788.

[24] L.R.C. Barclay, K.U. Ingold, Autoxidation of biological molecules. 2.The autoxidation of a model membrane. A comparison of theautoxidation of egg lecithin phosphatidylcholine in water and inchlorobenzene, J. Am. Chem. Soc. 103 (1981) 6478–6485.

[25] K. Masuda, Y. Imashiro, T. Kaneko, Mesoionic compounds. I.Synthesis of 3-dialkylamino-sydnonimines, Chem. Pharm. Bull. 18(1970) 128–132.

[26] L.R.C. Barclay, K.A. Baskin, S.J. Locke, T.D. Schaefer, Benzophe-none-photosensitized autoxidation of linoleate in solution and sodi-um dodecyl sulfate micelles, Can. J. Chem. 65 (1987) 2529–2540.

[27] G.W. Burton, K.U. Ingold, Autoxidation of biological molecules. 1.The antioxidant activity of vitamin E and related chain-breakingphenolic antioxidants in vitro, J. Am. Chem. Soc. 103 (1981) 6472–6477.

[28] T. Doba, G.W. Burton, K.U. Ingold, Antioxidant and co-antioxidantactivity of vitamin C. The effect of vitamin C, either alone or in thepresence of vitamin E or a water-soluble vitamin E analogue, uponthe peroxidation of aqueous multilamellar phospholipid liposomes,Biochim. Biophys. Acta 835 (1985) 298–303.

[29] L.R.C. Barclay, S.J. Locke, J.M. MacNeil, J. VanKessel, Quantitativestudies of the autoxidation of linoleate monomers sequestered inphosphatidylcholine bilayer. Absolute rate constants in bilayer, Can.J. Chem. 63 (1985) 2633–2638.

[30] L.R.C. Barclay, K.A. Baskin, D. Kong, S.J. Locke, Autoxidation ofmodel membrane. The kinetics and mechanism of autoxidation ofmixed phospholipid bilayer, Can. J. Chem. 65 (1987) 2541–2549.

[31] L. Castle, M.J. Perkins, Inhibition kinetics of chain-breaking phenolicantioxidants in SDS micelles. Evidence that intermicellar diffusionrates may be rate-limiting for hydrophobic inhibitors such as a-tocopherol, J. Am Chem. Soc. 108 (1986) 6381–6382.

[32] W.A. Pryor, J.A. Cornicelli, L.J. Devall, B. Tait, B.K. Trivedi, D.T.Witiak, M. Wu, A rapid screening test to determine the antioxidantpotencies of natural and synthetic antioxidants, J. Org. Chem. 58(1993) 3521–3532.

[33] L.R. Barclay, F. Antunes, Y. Egawa, K.L. McAllister, K. Mukai, T.Nishi, M.R. Vinqvist, The efficiency of antioxidants delivered byliposomal transfer, Biochim. Biophys. Acta 1328 (1997) 1–12.

[34] K.U. Ingold, Peroxy radicals, Acc. Chem. Res. 2 (1969) 1–9.[35] H. Moini, L. Packer, N.E. Saris, Antioxidant and prooxidant

activities of alpha-lipoic acid and dihydrolipoic acid, Toxicol. Appl.Pharmacol. 182 (2002) 84–90.

[36] P. Rauhala, K.P. Mohanakumar, I. Sziraki, A.M. Lin, C.C. Chiueh,S-nitrosothiols and nitric oxide, but not sodium nitroprusside, protectnigrostriatal dopamine neurons against iron-induced oxidative stressin vivo, Synapse 23 (1996) 58–60.

[37] P. Rauhala, A.M. Lin, C.C. Chiueh, Neuroprotection by S-nitroso-glutathione of brain dopamine neurons from oxidative stress, FASEBJ. 12 (1998) 165–173.

[38] V.B. O’Donnell, J.P. Eiserich, P.H. Chumley, M.J. Jablonsky, N.R.Krishna, M. Kirk, S. Barnes, V.M. Darley-Usmar, B.A. Freeman,Nitration of unsaturated fatty acids by nitric oxide-derived reactivenitrogen species peroxynitrite, nitrous acid, nitrogen dioxide, andnitronium ion, Chem. Res. Toxicol. 12 (1999) 83–92.

[39] E.H. Pap, G.P. Drummen, V.J. Winter, T.W. Kooij, P. Rijken, K.W.Wirtz, J.A. Op den Kamp, W.J. Hage, J.A. Post, Ratio-fluorescencemicroscopy of lipid oxidation in living cells using C11-BODIPY(581/591), FEBS Lett. 453 (1999) 278–282.

[40] G.P. Drummen, L.C. van Liebergen, J.A. Op den Kamp, J.A. Post,C11-BODIPY(581/591), an oxidation-sensitive fluorescent lipid per-oxidation probe: (micro)spectroscopic characterization and validationof methodology, Free Radic. Biol. Med. 33 (2002) 473–490.

[41] S.P. Goss, N. Hogg, B. Kalyanaraman, The antioxidant effect ofspermine NONOate in human low-density lipoprotein, Chem. Res.Toxicol. 8 (1995) 800–806.

[42] A. Law, S. Gauthier, R. Quirion, Neuroprotective and neurorescuingeffects of isoform-specific nitric oxide synthase inhibitors, nitric oxidescavenger, and antioxidant against beta-amyloid toxicity, Br. J.Pharmacol. 133 (2001) 1114–1124.

[43] S. Goldstein, A. Russo, A. Samuni, Reactions of PTIO and carboxy-PTIO with NO, NO2, and O2

��, J. Biol. Chem. 278 (2003) 50949–50955.

[44] R.J. Singh, N. Hogg, J. Joseph, E. Konorev, B. Kalyanaraman, Theperoxynitrite generator, SIN-1, becomes a nitric oxide donor in thepresence of electron acceptors, Arch. Biochem. Biophys. 361 (1999)331–339.

[45] S. Goldstein, G. Czapski, J. Lind, G. Merenyi, Carbonate radical ionis the only observable intermediate in the reaction of peroxynitritewith CO2, Chem. Res. Toxicol. 14 (2001) 1273–1276.

nitroxidation, nitration, and oxidation of a bodipy ...lab.pharm.uic.edu/thatcher/pub/p_5.pdf ·...

Documents