Pitfalls of peroxynitrite determination by luminescentprobe in diabetic rat aorta

Andras Nemeth • Krisztian Stadler • Judit Jakus •

Tamas Vidoczy

Received: 15 November 2011 / Accepted: 20 January 2012 / Published online: 9 February 2012

� Akademiai Kiado, Budapest, Hungary 2012

Abstract Pitfalls of peroxynitrite (ONOO-) formation in diabetic rat aorta on

luminol-induced chemiluminescence (LCL) are investigated based on a detailed

reaction mechanism in a case where 1.0 9 10-7 M s-1 superoxide formation rate

and nitric oxide (•NO) formation were measured by electron paramagnetic reso-

nance, while ONOO- formation by LCL. Modeling ONOO- formation at equi-

molar reactant ratio at pH 7.4 and 37 �C predicts 2.0 nM ONOO- and

2.1 9 10-6 M steady-state •NO concentrations, which are both biologically rele-

vant. Comparison of steady-state concentrations to those obtained by modeling the

LCL intensity at pH 10 shows that ONOO- concentration increases with 10% while

peroxynitrous acid (ONOOH) concentration decreases complying with the pH shift.

Evaluation of steady-state reaction rates reveals that the contribution of CO3•-

radicals to the formation of luminol radicals is 76%, that of •NO2 is 24%, consid-

erable, but that of •OH radicals negligible. The contribution of additional superoxide

formation by autoxidation of luminol is 13%, not negligible, but that of ONOOH

homolysis is negligible. The •NO2 is predominantly formed from the decomposition

of the ONOO-–carbon dioxide adduct and only 0.5% directly from •NO oxidized

by molecular oxygen. But the contribution of the latter pathway depends strongly on

the •NO and superoxide formation rate ratio, at a ratio of 2:1, it would increase to

A. Nemeth (&) � K. Stadler � J. Jakus

Institute of Biomolecular Chemistry, Chemical Research Center, Hungarian Academy of Sciences,

Pusztaszeri ut 59-67, Budapest 1025, Hungary

e-mail: anemeth@chemres.hu

Present Address:K. Stadler

Oxidative Stress and Disease Laboratory, Pennington Biomedical Research Center, Baton Rouge,

LA 70808, USA

T. Vidoczy

Institute of Structural Chemistry, Chemical Research Center, Hungarian Academy of Sciences,

Pusztaszeri ut 59-67, Budapest 1025, Hungary

123

Reac Kinet Mech Cat (2012) 106:1–10

DOI 10.1007/s11144-012-0427-3

14%. The measured time interval of the initial increase of LCL intensity complies

with the time needed luminol aorta outside and inside concentrations in the sample

to be equalized by diffusion, the 7 9 10-3 s-1 rate constant obtained by modeling

enabled to estimate 5 9 10-7 cm2 s-1 as the diffusion coefficient of luminol in the

diabetic rat aorta.

Keywords Peroxynitrite � Luminol � Chemiluminescence � Reaction mechanism �Modeling

Introduction

Luminol (LH,5-amino-2,3-dihydro-1,4-phthalazinedione) induced chemilumines-

cence (LCL) is one of the most frequently applied methodologies for peroxynitrite

(ONOO-) detection [1, 2]. It has a high selectivity with a detection limit

1 9 10-9 M min-1 for ONOO- fluxes by photon counting [2] and 1 9 10-11 M

for ONOO- concentration by applying a membrane sampler [3]. Luminol is

endosomal membrane permeable [4] and its initial oxidation step, converting

luminol to luminol radical cannot be achieved by superoxide (O2•-) [5].

The LCL probe in biological systems, however, cannot directly asses the

formation of a particular reactive intermediate, since different reactive species can

lead to the formation of the light-emitting species. An early study by Faulkner et al.

[6] argued that in the presence of molecular oxygen, the univalently oxidized

luminol itself can be a source of superoxide formation. Recently, a comprehensive

review advanced by Wardman [7] on the pitfalls of the luminol probe for reactive

oxygen and nitrogen species called attention to the impact of nitric dioxide and

concluded that quantification of mechanistic pathways would be required for the

rational use of the probe.

In accordance with this recommendation, the goal of the present study was to

address (i) the effect of pH shift, a technique used to intensify LCL intensity, (ii) the

implications of additional superoxide formation, (iii) the competition between nitric

dioxide (•NO2), carbonate radical anion (CO3•-) and hydroxyl radical (•OH) in the

production of luminol radicals, (iv) the contribution of peroxynitrous acid

(ONOOH) homolysis, and (v) that of nitric oxide (•NO) oxidation to the formation

of •NO2 by modeling based on a detailed reaction mechanism.

Experimental

Most of the experimental data are from our already reported work [8]. Three-week-

old male Wistar rats were kept under standard conditions then randomly divided

into diabetic and age-matched control groups containing 6–8 animals for each

experiment. To induce diabetes, animals were anesthetized and STZ was injected

via the vena femoralis. Induction of diabetes was confirmed by measuring the

urinary glucose level 3 days later. 1, 2, 3, and 7 weeks after the onset of diabetes,

control and diabetic rats were exsanguinated under anesthesia.

2 A. Nemeth et al.

123

To determine the rate (spin clearance) of in vivo formation of oxygen-centered

free radicals in the aorta, rats received the spin probe 3-carbamoyl-proxyl

(c-PROXYL), 56 mg kg-1 i.p. at 5, 10 and 15 minutes before sacrificing. Then,

organ samples were prepared in liquid nitrogen and electron paramagnetic

resonance (EPR) signal intensities were registered. Co-administration of the

superoxide dismutase (SOD) enzyme with c-PROXYL revealed that the majority of

the formed oxygen-centered free radicals was superoxide. The accumulation and

decay of c-PROXYL were measured between 5 and 30 min and registered EPR

signal intensities from six diabetic aorta pieces were evaluated by double integration

using an EPR analysis program. Intensities in arbitrary units were calibrated with

2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO), a stable free radical.

Electron paramagnetic resonance measurements and spin trapping procedures

were carried out with an X-Band computer-controlled spectrometer constructed by

Magnettech GmbH (Berlin, Germany). Approximately 100 mg of tissue samples

were frozen into a rod-shaped form and spectra of the samples were recorded at

77 K using a quartz finger-Dewar filled up with liquid nitrogen. Instrument settings

were 100 kHz modulation frequency, 0.7050 mT modulation amplitude, 18 mW

microwave power, 1 min scan time and 20.63 mT field sweep. For the evaluation of

the EPR signals, Mn/MnO as an internal standard was used.

A luminol-dependent chemiluminescence method originally described by Zweier

et al. [9] and modified by Brovkovych et al. [10] was used to measure ex vivo

ONOO--derived radical generation in the rat aorta tissue (each sample weighing

8–10 mg) placed in 2 mL Hanks buffer solution (pH 7.4), and starting photon

counting immediately after luminol addition. In another series of measurements,

1 mL alkaline sodium bicarbonate-luminol solution was added to the sample (final

concentrations were 400 mM of luminol, 50 mM of bicarbonate.

Method of modeling and reaction mechanism

First, the fate of ONOO- was modeled by a CO2-only consumption of ONOO-

yielding the biologically maximum ONOO- concentration at pH 7 and 37 �C [11].

The NO formation rate, taking into account that in biological systems ONOO-

related effects are generally observed at equimolar reactant rates of ONOO-

formation was set to be equal to that measured for superoxide. Then, modeled

steady-state concentrations were compared to those obtained by modeling LCL

intensity at pH 10 when luminol and bicarbonate solutions were added to the sample

and reactions of luminol appended to the mechanism. Finally, contribution analysis

of the steady-state reaction rates was carried out to assess the impact of pitfalls on

LCL intensity.

The reaction mechanism compiled from the literature is shown in Table 1. Their

rate constants are at pH 7.4 and 37 �C, when not indicated otherwise. If data were

not available under these conditions, we used the closest obtainable value. Reactions

(1–17) consist of the reduced set of ONOO- formation and decomposition based on

the consideration that in a biological environment the reactions of ONOO- with

CO2 outcompetes other decay process [14]. In order to account for the impact of

Pitfalls of peroxynitrite determination by luminescent probe 3

123

Table 1 Reaction mechanism

No. Reaction k or pK Reference

1 •NO ? O2•- ? ONOO- 1.6 9 1010 M-1 s-1 [25]

2 ONOO- ? CO2 ? ONOOCO2- 3.0 9 104 M-1 s-1 [26]

3 ONOOCO2- ? •NO2 ? CO3

•- 6.5 9 108 s-1 [27]

4 ONOOCO2- ? NO3

- ? CO2 1.25 9 109 s-1 [27]

5 2O2•- (?2H?) ? O2 ? H2O2 2.0 9 105 M-1 s-1 [28]

6 O2•- ? CO3

•- ? O2 ? CO32- 6.5 9 108 M-1 s-1 [29]

7 •NO ? CO3•- ? NO2

- ? CO2 3.5 9 109 M-1 s-1 [29]

8 2•NO ? O2 ? 2•NO2 2.9 9 106 M-2 s-1 [30]

9 •NO ? •NO2 ? N2O3 1.1 9 109 M-1 s-1 [29]

10 N2O3 ? •NO ? •NO2 8.4 9 104 s-1 [29]

11 N2O3 (?H2O) ? 2NO2- ? 2H? 5.22 9 104 s-1 [30, 32]

12 •NO2 ? O2•- ? O2NOO- 4.5 9 109 M-1 s-1 [33]

13 O2NOO- ? •NO2 ? O2•- 1.1 s-1 [33]

14 O2NOO- ? NO2- ? O2 1.3 s-1 [33]

15 2•NO2 ? N2O4 4.5 9 108 M-1 s-1 [29]

16 N2O4 ? 2•NO2 6.9 9 103 s-1 [29]

17 N2O4 (?H2O) ? NO2- ? NO3

- ? 2H? 1.0 9 103 s-1 [31]

18 ONOOH ? •NO2 ? •OH 0.35 s-1 [35]

19 •NO2 ? •OH ? ONOOH 4.5 9 109 M-1 s-1 [35]

20 •OH ? •OH ? H2O2 5.5 9 109 M-1 s-1 [34, 36]

21 •NO2 ? •OH ? NO3- ? H? 4.5 9 109 M-1 s-1 [34]

22 O2•- ? SOD ? SODred

- ? O2 2.0 9 109 M-1 s-1 [40]

23 O2•- ? SODred (?2H?)?H2O2 ? SOD- 2.0 9 109 M-1 s-1 [40]

24 Tyrosine ? CO3•- ? tyr-O• ? HCO3

- 4.5 9 107 M-1 s-1 [29]

25 Tyrosine?•NO2 ? tyr-O• ? NO2- ? H? 3.2 9 105 M-1 s-1 [41]

26 Tyr-O• ? •NO2 ? 3-nitrotyrosine 1.3 9 109 M-1 s-1 [31, 41, 42]

27 Tyr-O• ? O2•- ? P1 1.5 9 109 M-1 s-1 [43]

28 Tyr-O• ? •NO ? Tyr-ONO 1.0 9 109 M-1 s-1 [31, 44]

29 Tyr-ONO ? Tyr-O• ? •NO 1.0 9 103 s-1 [31, 44]

30 Tyr-ONO ? P2 1.8 9 10-1 s-1 [31]

31 2Tyr-O• ? 3,3-dityrosine 2.25 9 108 M-1 s-1 [44]

32 ONOOH $ ONOO- ? H? 6.6 [46]

33 CO2 ? H2O $ H? ? HCO3- 6.1 [37, 38]

34 HCO3- $ H?? CO3

2- 10.3 [37, 38]

35 LHins- ? CO3

•- ? LH• ? CO32- 9.0 9 108 M-1 s-1 [7]

36 LHins- ? •NO2 ? LH• ? NO2

- 1.0 9 106 M-1 s-1 [7]

37 LHins- ? •OH ? LH• ? -OH 9.0 9 109 M-1 s-1 [7]

38 LH• ? O2•- ? light ? APht (pH 10) 2.0 9 105 M-1 s-1 [46]

39 LH• ? O2 ? Lox ? O2•- 6.0 9 102 M-1 s-1 [39]

40 LHouts ? LHins 7 9 10-3 s-1 This work

The symbol tyr-O• indicates the tyrosyl radical, P1 substituted indole-2-carboxylic acids [23], P2 3-ni-trotyrosine or hydrolysis products [47]. APht is ground state aminophthalate [12], Lox is 5-aminophthal-azine-1,4-dione [47], subscripts outs and ins denote luminol sample outside and inside, respectively

4 A. Nemeth et al.

123

•OH radicals on the production of luminol radicals, its production by homolysis of

ONOOH and corresponding termination reactions were added, reactions (18–21).

The impact of the biological environment on ONOO- formation and decay is much

more complex and its reaction mechanism is less elaborated [12, 13]. A mechanism

was recently suggested to model tyrosine nitration by superoxide and •NO fluxes in

biological systems as well as a mechanism for the role of SOD [14]. These reactions

(22–31) were assumed for the present case as well. SOD is a powerful superoxide

scavenger found practically uniformly distributed within the cell, operating as a

drain of excess O2•-. Species derived from tyrosine are not quantitatively important

since they represent a tiny fraction of ONOO- consumed, but their importance lies

in their stability and the use as the trail of ONOO- in vivo. Efficient ONOO-

scavengers, such as peroxiredoxin, some thiol proteins, glutathione peroxidase, and

several heme proteins could not be taken into account because their concentrations

were not known in the investigated system. Reactions (32–34) set the constraints for

the equilibrium between ONOO- and ONOOH, and the carbonates.

The oxidation potential of luminol was also thoroughly investigated, reactions

(35–39). The initial step of oxidation is the interaction between LH- and radicals

CO3•-, •OH, and •NO2 producing LH• radicals. Here we note that the rate constant

of the latter interaction is known only from personal communications of Merenyi as

already referred to in [7], estimated to be at least 106 M-1 s-1. Next, addition of

O2•- produces a rather unstable endoperoxide, which decomposes very fast into

excited aminophthalate and nitrogen, the former leading subsequently to the

emission of chemiluminescence. Luminol radicals can react with oxygen to generate

O2•-, where Lox is 5-aminophthalazine-1,4-dione.

The effect of luminol diffusion, reaction (40) will be discussed in the next

chapter. KINAL software was used to model the reaction mechanism and fit to

measured data to extract the contribution from the reactions of interest [15].

Results and discussion

A fixed parameter of the kinetic analysis was the experimentally determined

1.0 9 10-7 M s-1 superoxide formation rate. The initial concentration for oxygen

was taken as 1.0 9 10-5 M, for total carbon dioxide (CO2 ? HCO3-) as

1.4 9 10-3 M, for tyrosine as 1.0 9 10-3 M, these being the usual values in

plasma as already referred to in [14], 5.5 9 10-6 M for SOD the average of its

biological relevant range in the present case as earlier modeled by us [16],

4 9 10-4 M for luminol and 50 mM for bicarbonate.

Complying with the experiments ONOO- formation and decay was first modeled

at pH 7.4 and 37 �C, conditions at which the superoxide formation rate was

measured. For the reason discussed above, the rate of •NO formation was set to

1.0 9 10-7 M s-1 the measured rate of superoxide formation. The predicted

2.0 9 10-9 M ONOO- and 2.1 9 10-6 M •NO steady-state concentrations are in

biologically relevant ranges as already referred to in [11]. Steady-state concentra-

tions and reaction rates obtained were compared to those obtained by modeling LCL

at pH 10 when luminol and bicarbonate solutions were added to the sample.

Pitfalls of peroxynitrite determination by luminescent probe 5

123

In this modeling, however, we were confronted with the problem that the

measured LCL intensity showed an expressed time dependence approaching a

maximum after a time lag of approximately 20 min (vide measured curve in Fig. 1).

This observation was seemingly in contradiction with the steady-state condition

prevailing in the sample. We assumed that the discrepancy could be the

consequence of the experimental procedure, and hypothesized that this time

dependence was related to the time required for the luminol concentration to reach

its equilibrium within the sample (since CO2 diffuses much faster, there was no need

to take its diffusion into consideration by a similar approach). Therefore, the

reaction mechanism was amended by reaction (40) where indices outs and ins

denote luminol concentrations aorta outside and inside in the sample. Since the

measured LCL intensity was available only in arbitrary units (cps) the computed

rate of luminol radical formation which is directly related to the rate of light

producing reaction was normed to the maximum cps value measured. By the trial

and error method, an acceptable fit between the time dependence of measured and

computed curves was obtained at k40 = 7 9 10-3 s-1 (Fig. 1). The descending part

of the measured curve following the maximum was possibly due to deteriorating

cell functioning in our in vitro system. The hypothesis was also confirmed by

plotting inner and outer luminol concentrations against time (Fig. 2), illustrating

that they became equal within the recorded time lag.

In Table 2, modeled changes of steady-state concentrations are summarized. The

concentrations of both O2•- and •NO are significantly affected, but their product, i.e.,

the rate of ONOO- formation in less measure. The increase of ONOO-

concentration was 10%, the ONOOH concentration decreased complying with the

pH shift while their sum remained unchanged. Modeling indicated a shift from

NO2- to NO3

- accumulation. The consumption of tyrosine was negligible.

0 500 1000 1500 2000 2500 3000

time, s

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

LCL

inte

nsity

, cps

(LH

. xO2.-

xk37

) no

rmed

to c

ps

calculated

measured

k39= 7.0x10-3 s-1

Fig. 1 Evaluation of luminol diffusion rate constant in rat aorta: fitting the time dependence of the rate ofLH• formation (normed to cps) to that of measured LCL (in cps)

6 A. Nemeth et al.

123

In order to asses the effect of pitfalls on LCL intensity, which is directly related

to the rate of LH• radical formation, contribution analysis was carried out. This

revealed that the contribution of luminol autooxidation to O2•- formation was 13%,

not negligible but that of ONOOH homolysis negligible. The contribution of CO3•-

radicals to LH• radical formation was 76%, that of •NO2 24%, a considerable impact

on LCL intensity, while that of •OH radicals negligible.

With regard to the pivotal role of •NO2, the contribution of pathways to its

formation were also evaluated. In the present case, •NO2 was predominantly formed

via the decomposition of the ONOOCO2- adduct and only 0.5% directly from • NO

0 400 800 1200time, s

0E-01

1E-04

2E-04

3E-04

4E-04

5E-04

conc

entr

atio

n, M

LH-ins

LH-outs

Fig. 2 Evaluation of the time interval of initial increase in LCL intensity: modeled luminolconcentrations outside and inside of a rat aorta sample in function of time

Table 2 Impact of luminol

and pH shift on steady-state

concentrations

pH 7.4 pH 10

M M Change, %

LH- 4.0 9 10-4

O2•- 2.4 9 10-12 4.2 9 10-12 75

•NO 2.1 9 10-6 1.3 9 10-6 -38

ONOO- 2.0 9 10-9 2.2 9 10-9 10

ONOOH 4.9 9 10-10 1.4 9 10-12 -100

ONOO- ? ONOOH 2.5 9 10-9 2.5 9 10-9 &0•NO2 2.3 9 10-11 2.4 9 10-11 4

CO3•- 5.2 9 10-13 8.0 9 10-14 -85

•OH 1.2 9 10-10 1.4 9 10-19 &-100

NO3- 5.6 9 10-5 6.4 9 10-5 14

NO2- 4.1 9 10-5 2.7 9 10-5 -34

Tyrosine 1.0 9 10-3 1.0 9 10-3 &0

3-Nitrotyrosine 2.2 9 10-7 1.4 9 10-7 -36

Tyr-ONO 1.6 9 10-8 6.5 9 10-9 -59

Pitfalls of peroxynitrite determination by luminescent probe 7

123

oxidized by molecular oxygen. It follows, however, from the reaction mechanism,

that the contribution of the latter pathway is strongly related to the •NO–superoxide

formation rate ratio. A computational exercise showed that 15% of •NO2 would be

formed through this bypass way at 2:1 •NO–superoxide formation rate ratio. The

reaction graph of corresponding reaction pathways and contributions are illustrated

in Fig. 3.

As an additional result, from the diffusion rate constant k40, an approximate value

5 9 10-7 cm2 s-1 as bulk diffusion coefficient of luminol in the diabetic rat aorta

was calculated by a method already referred to in [14]. The diffusion coefficient of

luminol in 0.1 M NaOH solution is 6.6 9 10-6 cm2 s-1 [17, 18]. In biological

systems, such data are not known to us, but values were reported for •NO in

liposomes and for red blood cells to be half [19–21], and in rat aorta wall to be

nearly fourfold less than in water [22]. The calculated value is less than that

suggested by this analogy. The difference might be attributed to the harmful effect

of diabetes. The literature supports this explanation as diabetes mellitus [23] and

related hypertension [24] lead to increased rat aorta rigidity.

Conclusions

Modeling rendered useful information on possible pitfalls of ONOO- determination

by LCL. It confirmed that neither the impact of •NO2 nor the contribution of the

autoxidation of luminol producing additional superoxide formation can be

neglected. On the other hand, frequently discussed pitfalls, such as the impact of

ONOO−

ONOOCO2−

ONOOH

O2

O2•−

CO2

•NO

85%

15%

•NO2

~0%

NO2-

NO3-

Fig. 3 Contribution of reactionpathways to the formation andconsumption of •NO2 radicalsmodeled at nitric oxide–superoxide formation rate ratio2:1 and pH 10, in percentage

8 A. Nemeth et al.

123

ONOOH hydrolysis on •NO2 formation and the impact of •OH radical on LCL

intensity, seem to be minor in the present case. It also demonstrated that the pH shift

affected the steady-state concentrations. It is, however, recognized that doubts can

arise regarding the oversimplifications we were forced to apply, but which at the

same time emphasize an authentic need for new and better data obtained in

biological systems.

Acknowledgment Helpful discussion with Professor I Papai on the carbonate equilibrium was

gratefully acknowledged.

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