Hydrogen Peroxide Formation by Reaction of Peroxynitrite with HEPES and Related Tertiary Amines
IMPLICATIONS FOR A GENERAL MECHANISM*

Michael KirschDagger , Elena E. LomonosovaDagger , Hans-Gert Korth§, Reiner Sustmann§, and Herbert de GrootDagger

From the Dagger  Institut für Physiologische Chemie, Universitätsklinikum, Hufelandstrasse 55, D-45122 Essen and § Institut für Organische Chemie, Universität-GH Essen, Universitätsstrasse 5, D-45117 Essen, Germany

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Organic amine-based buffer compounds such as HEPES (Good's buffers) are commonly applied in experimental systems, including those where the biological effects of peroxynitrite are studied. In such studies 3-morpholinosydnonimine N-ethylcarbamide (SIN-1), a compound that simultaneously releases nitric oxide (·NO) and superoxide (Obardot 2), is often used as a source for peroxynitrite. Whereas in mere phosphate buffer H2O2 formation from 1.5 mM SIN-1 was low (~15 µM), incubation of SIN-1 with Good's buffer compounds resulted in continuous H2O2 formation. After 2 h of incubation of 1.5 mM SIN-1 with 20 mM HEPES about 190 µM H2O2 were formed. The same amount of H2O2 could be achieved from 1.5 mM SIN-1 by action of superoxide dismutase in the absence of HEPES. The increased H2O2 level, however, could not be related to a superoxide dismutase or to a NO scavenger activity of HEPES. On the other hand, SIN-1-mediated oxidation of both dihydrorhodamine 123 and deoxyribose as well as peroxynitrite-dependent nitration of p-hydroxyphenylacetic acid were strongly inhibited by 20 mM HEPES. Furthermore, the peroxynitrite scavenger tryptophan significantly reduced H2O2 formation from SIN-1-HEPES interactions. These observations suggest that peroxynitrite is the initiator for the enhanced formation of H2O2. Likewise, authentic peroxynitrite (1 mM) also induced the formation of both Obardot 2 and H2O2 upon addition to HEPES (400 mM)-containing solutions in a pH (4.5-7.5)-dependent manner. In accordance with previous reports it was found that at pH >= 5 oxygen is released in the decay of peroxynitrite. As a consequence, peroxynitrite(1 mM)-induced H2O2 formation (~80 µM at pH 7.5) also occurred under hypoxic conditions. In the presence of bicarbonate/carbon dioxide (20 mM/5%) the production of H2O2 from the reaction of HEPES with peroxynitrite was even further stimulated. Addition of SIN-1 or authentic peroxynitrite to solutions of Good's buffers resulted in the formation of piperazine-derived radical cations as detected by ESR spectroscopy. These findings suggest a mechanism for H2O2 formation in which peroxynitrite (or any strong oxidant derived from it) initially oxidizes the tertiary amine buffer compounds in a one-electron step. Subsequent deprotonation and reaction of the intermediate alpha -amino alkyl radicals with molecular oxygen leads to the formation of Obardot 2, from which H2O2 is produced by dismutation. Hence, HEPES and similar organic buffers should be avoided in studies of oxidative compounds. Furthermore, this mechanism of H2O2 formation must be regarded to be a rather general one for biological systems where sufficiently strong oxidants may interact with various biologically relevant amino-type molecules, such as ATP, creatine, or nucleic acids.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

The term "peroxynitrite" is commonly used to describe the equilibrium mixture of oxoperoxonitrate(1-) (ONOO-) and its conjugated acid, hydrogen oxoperoxonitrate(1-) (peroxynitrous acid, ONOOH). Peroxynitrite is a strong oxidant formed in the diffusion-controlled reaction of superoxide (Obardot 2) and nitric oxide (nitrogen monoxide, ·NO) (k = 3.9-6.7 × 109 M-1 s-1) (1, 2). Peroxynitrite has been suggested to play a major role in many pathological processes like atherosclerosis (3) and stroke (4). The pathological activity of ONOO-/ONOOH is assumed to result from its ability to attack various biological targets, including protein- and non-protein sulfhydryls (5), DNA (6), low density lipoproteins (3), or membrane phospholipids (7). A favored method to generate peroxynitrite for experimental purposes is to use SIN-11 (8). This compound decays in solution in the presence of oxygen with simultaneous release of ·NO and Obardot 2 in a 1:1 stoichiometry (9). Consequently, SIN-1 has been shown to attack many biological targets in nearly the same manner as authentic peroxynitrite (3). The formation of ONOO- from SIN-1 can be suppressed by the enzyme superoxide dismutase (SOD), resulting in the formation of ·NO and hydrogen peroxide (H2O2) as major products (10).

In obvious contradiction to the assumption of a central role of ONOO-/ONOOH in cell injuring processes, almost complete protection from SIN-1 cytotoxicity in experiments with rat liver endothelial cells and Fu5 rat hepatoma cells was provided by catalase but not by SOD (11, 12). Since catalase does not effectively react with peroxynitrite (13), these results strongly suggest a participation of H2O2 in SIN-1-mediated cytotoxicity rather than a participation of ONOO-/ONOOH. Indeed, formation of H2O2 from SIN-1 was observed under certain experimental conditions (12). We (14) have recently demonstrated that the formation of H2O2, and consequently the protection exerted by catalase, decisively depends on the presence of the organic buffer compound HEPES in the incubation medium. In the absence of this "Good's buffer" neither H2O2 was formed nor was catalase protective. The question, however, how HEPES and similar Good's buffers (15, 16) mediate the formation of H2O2 from SIN-1 remained open. The present study aims at the elucidation of the underlying chemical mechanism.

    EXPERIMENTAL PROCEDURES
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Procedures
Results
Discussion
References

Materials-- Catalase from beef liver (EC 1.11.1.6), peroxidase from horseradish (EC 1.11.1.7), cytochrome c from horse heart, and copper-zinc superoxide dismutase from bovine erythrocytes (EC 1.15.1.1) were obtained from Boehringer Mannheim (Mannheim, Germany). Dihydrorhodamine 123 (DHR123) and spermine NONOate were purchased from Molecular Probes (Leiden, The Netherlands). Diethylenetriaminepentaacetic acid (DTPA), manganese dioxide, hydrogen peroxide (H2O2), bovine hemoglobin, HEPES, and other Good's buffers were obtained from Sigma (Deisenhofen, Germany). SIN-1 and its decomposition product, SIN-1C, were generously provided by Dr. K. Schönafinger (Hoechst Marion Roussel, Frankfurt/Main, Germany). Nitrogen 5.0, commercially available mixtures of oxygen 5.0 and nitrogen 5.0 (20.5% O2, 79.5% N2, "synthetic air"), and commercially available mixtures of oxygen 5.0 and nitrogen 5.0 and carbon dioxide 4.6 (20.5% O2, 74.5% N2, 5% CO2) were purchased from Messer-Griessheim (Oberhausen, Germany). Oxoperoxonitrate(1-) (0.62 M) was prepared by isoamyl nitrite-induced nitrosation of hydrogen peroxide (0.12 mol of isoamyl nitrite, 100 ml of H2O2 (1 M) plus DTPA (2 mM)) and purified (e.g. solvent extraction, removal of excess H2O2, N2-purging, storage) as described by Uppu and Pryor (17). Oxyhemoglobin was prepared as described previously (12). All other chemicals were of the highest purity commercially available.

Solutions-- Care was taken to exclude possible contamination by bicarbonate/carbon dioxide. Doubly distilled water was bubbled (2 liters/min) with synthetic air at room temperature for 20 min. This water was used for synthesis of oxoperoxonitrate(1-), NaOH (0.01-0.5 N), and for all other solutions. Potassium phosphate buffer (50 mM) containing DTPA (0.1 mM) was prepared freshly each day. The pH was adjusted to 7.5 at 37 °C, and the solution was again bubbled (2 liters/min) with synthetic air (normoxia) or with nitrogen (hypoxia) or with the carbon dioxide mixture for 20 min. In the case of bubbling with the CO2 mixture, the pH must be readjusted to 7.5. SIN-1 and spermine NONOate solutions were prepared as 100× stock solutions at 4 °C in 50 mM KH2PO4 and 10 mM NaOH, respectively, and used within 15 min. DHR123 200× stock solution was prepared in water-free, nitrogen-purged dimethylformamide and stored in the dark at -20 °C.

Experimental Conditions-- SIN-1 and spermine NONOate (final pH 7.5) were added to 1 ml of phosphate buffer and incubated in 12-well cell culture plates (volume of each well 7 ml, Falcon, Heidelberg, Germany). For the detection of H2O2 with the catalase assay, SIN-1 was added to 10 ml of buffer and incubated in tissue culture dishes (75 ml, Falcon, Heidelberg, Germany). Under normoxic conditions these plates/dishes were placed in an air-tight anaerobic vessel (10 liters). During the first 15 min of each experiment these vessels were flushed (5 liters/min) with synthetic air in a warming incubator (Heraeus, Hanau, Germany). In the presence of HCO3-/CO2 the plates/dishes were placed in an incubator for cell culture (37 °C, humidified atmosphere of 95% authentic air and 5% CO2, Labotect, Göttingen, Germany). The experiments with authentic peroxynitrite were performed in reaction tubes (1.4 ml, Eppendorf, Hamburg, Germany) by using the drop-tube Vortex mixer technique as described by Sampson et al. (18). In brief, 1 ml of buffer (50 mM potassium phosphate, 0.1 mM DTPA, pH 7.5, 37 °C, in few experiments also in the presence of HCO3-/CO2 (20 mM/5%)), containing a known amount of target molecules (Good's buffer, p-HPA), was carefully placed into a reaction tube. A drop of peroxynitrite solution (2 µl, 0.5 M ONOO- in 0.5 N NaOH) was layered over the buffer at the dry wall. The reaction was started by vortexing. Thereby the pH increased to 7.56 ± 0.01. Under hypoxic conditions both the experiments with authentic peroxynitrite and the treatment of the solutions were performed in a glove bag (Roth, Karlsruhe, Germany) under nitrogen.

Determination of H2O2, O2, and Obardot 2-- Hydrogen peroxide was quantified by two techniques. First, H2O2 was determined by the horseradish peroxidase-catalyzed reaction of H2O2 with 4-aminoantipyrine and 3,5-dichloro-2-hydroxybenzenesulfonic acid to a quinoneimine dye that was measured spectrophotometrically at 546 nm (12) (peroxidase assay). Alternatively, H2O2 was quantified by the amount of O2 released upon addition of catalase (1000 units/ml) (catalase assay). O2 was determined polarographically with a Clark-type oxygen electrode (Eschweiler, Kiel, Germany). This electrode was also used to detect the production of O2 from decomposed peroxynitrite.

Superoxide radicals were determined by using the modified ferricytochrome c3+ reduction technique of McCord and Fridovich (19). Peroxynitrite was vortexed to the reaction solution in the absence and presence of various concentrations of HEPES. Cytochrome c (20 µM) or cytochrome c plus SOD (100 units/ml) were added 2 min after peroxynitrite addition. This time lag guaranteed that neither cytochrome c nor SOD reacted directly with peroxynitrite. The resulting mixture was stored for 40 min at 37 °C. The amount of cytochrome c reduction was determined by reading the absorbance at 550 nm (Delta epsilon 550 = 21,000 M-1 s-1) (20). The amount of reduced cytochrome c in the presence of SOD was subtracted from the amount of reduced cytochrome c in the absence of SOD for each HEPES concentration. This difference was used to calculate the amount of trapped Obardot 2.

Capillary Zone Electrophoresis Measurements-- SIN-1 and SIN-1C were quantified on a Beckman P/ACE 5000 apparatus. Separation conditions for SIN-1 and SIN-1C were as follows: fused silica capillary (50-cm effective length, 75 µM internal diameter), hydrodynamic injection for 5 s, temperature 23 °C, voltage 20 kV, normal polarity, UV detection at 280 nm, and 100 mM potassium phosphate, pH 6.3, as electrolyte system.

ESR Measurements-- ESR spectra were recorded at ambient temperature on a Bruker ESP300E X-band spectrometer (Bruker, Rheinstetten, Germany) equipped with a TM110 wide bore cavity. Solutions were prepared from 1 ml of the buffer solution (pH 7.5) containing HEPES or PIPES (both 400 mM) and SIN-1 (10 mM). Alternatively, 200 µl of authentic peroxynitrite (500 mM) and 1.8 ml of the buffer solution (pH 7.5) containing HEPES or PIPES (both 1 M) were vortexed. The reaction solutions were quickly transferred to an aqueous solution quartz cell (Willmad, Buena, NY). The first spectra were run as fast as possible, i.e. within 1 min, and then in 5-min intervals. Recording conditions were as follows: microwave frequency, 9.8 GHz; modulation, 0.04 mT; signal gain, 5 × 105; sweep range, 20 mT; sweep time, 4 min. Spectrum simulation was performed using the WinSim program (21).

Determination of SIN-1-driven Reactions-- The peroxynitrite-forming properties of 25 µM SIN-1 were detected with 50 µM DHR123. Formation of rhodamine 123 (RH123) was quantified spectrophotometrically at 500 nm (epsilon M = 78,000 M-1 cm-1) (22).

Thiobarbituric acid-reactive substances (TBARS) produced from the SIN-1 (1.5 mM)-induced oxidation of deoxyribose (1.5 mM) were detected spectrophotometrically at 532 nm using the TBARS assay (23). A standard calibration curve was prepared from known amounts of malondialdehyde generated by acid hydrolysis of 1,1,3,3-tetramethoxypropane.

Determination of Peroxynitrite-driven Nitration Reactions-- The peroxynitrite (1 mM)-dependent nitration of para-hydroxyphenylacetic acid (p-HPA, 1 mM) was employed. After vortexing, the sample was allowed to stand for 2 min. Then 0.5-1 N NaOH was added (4:1 v/v, final pH 11-11.5), and the formed 3-nitro-4-hydroxyphenylacetic acid (3-NO2-4-HPA) was detected spectrophotometrically at 430 nm (epsilon M = 4400 M-1 cm-1) (17).

Determination of Superoxide Dismutase-like Activity-- SOD-like activity of HEPES was evaluated using both the NADH/Mn2+/EDTA/mercaptoethanol (24) and the xanthine oxidase/xanthine/cytochrome c3+ (19) assay.

Determination of NO Scavenger Activity-- The spermine NONOate (25 µM)-driven reduction of oxyhemoglobin (70 µM) in the absence and presence of HEPES (1-20 mM) was used. Formation of methemoglobin was quantified spectrophotometrically at 578 nm (epsilon M = 12100 M-1 cm-1) (12).

    RESULTS
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Introduction
Procedures
Results
Discussion
References

Peroxynitrite from SIN-1 Decomposition

H2O2 Production from SIN-1 in the Presence of Good's Buffers-- In mere phosphate buffer low levels of H2O2 (15.2 ± 1.8 µM) were found after 2 h of incubation with 1.5 mM SIN-1 (Fig. 1). Addition of 20 mM HEPES to the phosphate buffer largely stimulated H2O2 formation. The H2O2 concentration increased almost linearly within 2 h up to 183.7 ± 1.5 µM. The yield of H2O2 after 2 h of incubation increased with the concentration of HEPES to reach a plateau value of 203.0 ± 12.6 µM H2O2 at about 50 mM HEPES (Fig. 2A). The effect of HEPES on the formation of H2O2 from SIN-1 was half-maximal at 7 ± 1 mM. In all experiments described here 0.1 mM DTPA was present to inhibit the influence of heavy metal contaminations (25). DTPA (0.1 mM) itself did not affect H2O2 formation, neither in the presence nor in the absence of HEPES (data not shown).


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Fig. 1.   Time dependence of hydrogen peroxide formation from SIN-1. SIN-1 (1.5 mM) was incubated in 50 mM potassium phosphate buffer (pH 7.5, 37 °C, 0.1 mM DTPA) in the absence and presence of 20 mM HEPES or 100 units/ml SOD. Aliquots were taken at selected time points, and H2O2 production was quantified using the peroxidase assay. Data are means ± S.D. of three experiments performed in duplicate.


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Fig. 2.   Influence of HEPES and SOD on hydrogen peroxide formation from SIN-1. SIN-1 (1.5 mM) was incubated for 2 h in 50 mM potassium phosphate buffer (pH 7.5, 37 °C, 0.1 mM DTPA). A, in the absence and presence of various concentrations of HEPES (0-100 mM). B, in the absence and presence of various activities of SOD (0-10000 units/ml). After the incubation, H2O2 was quantified using the peroxidase assay. Data are means ± S.D. of three experiments performed in duplicate.

The rate of H2O2 generation in the presence of 20 mM HEPES was comparable to the rate of H2O2 production in the presence of 100 units/ml SOD (Fig. 1). 100 units/ml was already the optimal SOD activity to induce maximal H2O2 production from SIN-1 (Fig. 2B). Both decreasing the SOD activity to 1 unit/ml or increasing it to 10,000 units/ml strongly decreased H2O2 formation, in full agreement with results reported by Gergel et al. (10). Thus, 20 mM HEPES already stimulated H2O2 formation from SIN-1 to the maximum yield that could be obtained by SOD.

Other Good's buffers at concentrations of 20 mM were also able to induce H2O2 formation from SIN-1 (Table I). The highest yield of H2O2 after 2 h was observed with EPPS (~207 µM). HEPES, POPSO, and PIPES induced moderately less H2O2 formation from SIN-1. The non-piperazine-type organic buffer triethanolamine (20 mM) also stimulated some production of H2O2 from SIN-1, about 30% of the maximum amount obtained from HEPES.

                              
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Table I
Hydrogen peroxide production from SIN-1 stimulated by Good's buffers
SIN-1 (1.5 mM) was added to various Good's buffers (20 mM) or SOD (100 units/ml) dissolved in 50 mM potassium phosphate buffer (0.1 mM DTPA, 37 °C, pH 7.5). After a 2-h incubation at 37 °C, H2O2 was determined using the peroxidase assay. Each value represents the mean ± S.D. of three experiments performed in duplicate.

SOD-like Activity-- Due to the fact that in the presence of HEPES H2O2 was formed at virtually the same rate as could maximally be achieved by SOD (Fig. 1 and Fig. 2, A and B), we first assumed that HEPES might accelerate the dismutation of Obardot 2. However, we were not able to detect any SOD-like activity of HEPES neither with a NADH/Mn2+/EDTA/mercaptoethanol assay nor with a xanthine oxidase/xanthine/cytochrome c3+ assay (data not shown). This result is in agreement with the work of Weiss et al. (26) who found that 60 mM HEPES does not inhibit Obardot 2-mediated cytochrome c3+ reduction.

·NO Scavenger Properties of HEPES-- An alternative explanation for the HEPES-dependent increase of H2O2 formation from SIN-1 was that the buffer compound might act as a ·NO scavenger, by this means preventing peroxynitrite formation and thus allowing the released Obardot 2 to dismutate to H2O2. As the release of nitric oxide from spermine NONOate is not affected by oxygen (27), we used the spermine NONOate-driven reduction of oxyhemoglobin to verify ·NO scavenger properties of HEPES. However, HEPES (1-20 mM) did not inhibit methemoglobin generation (data not shown).

Involvement of Peroxynitrite-- Since the foregoing experiments showed that the HEPES-dependent H2O2 formation from SIN-1 is neither mediated by a SOD-like activity of HEPES nor by ·NO-HEPES interactions, we concluded that reaction between peroxynitrite and HEPES should be responsible for the stimulation of H2O2 generation.

We (14) and others (28) have found that HEPES and SOD influence the decomposition of SIN-1. To exclude possible artifacts by an altered decomposition of this compound in the presence of HEPES or SOD, we performed the subsequent experiments as end point determinations. DHR123 is oxidized by peroxynitrite to RH123 in the presence of DTPA but neither by Obardot 2 nor ·NO alone (29), and TBARS are formed from deoxyribose only by very strong oxidants like hydroxyl radicals (30) and peroxynitrite (31). The influence of HEPES (20 mM) and SOD (100 units/ml) on the SIN-1-driven oxidation of both DHR123 and deoxyribose is shown in Fig. 3. After 3 h of incubation 25 µM SIN-1 was completely converted into SIN-1C as monitored by capillary electrophoresis. At that time SIN-1 (25 µM) had oxidized DHR123 (50 µM) with formation of 11.4 ± 0.2 µM RH123, i.e. with an efficiency of about 46%. In agreement, two groups have reported that authentic peroxynitrite oxidizes DHR123 with an efficiency close to 44% (32) or 50% (29). RH123 formation decreased to 5.6 ± 0.6 µM in the presence of HEPES (20 mM) and to 3.7 ± 0.2 µM in the presence of SOD (100 units/ml), indicating that both compounds were able to either scavenge peroxynitrite or to prevent its formation. The solubility of DHR123 in phosphate buffer is too low to allow concentrations higher than 50 µM. For that reason the SIN-1 concentration was limited to 25 µM. To corroborate the reactivity of SIN-1 in the presence of HEPES, we also studied the oxidation of deoxyribose. After 5 h of incubation of 1.5 mM SIN-1 with 1.5 mM deoxyribose, i.e. after 1.5 mM SIN-1 was completely decomposed, 3.0 ± 0.2 µM TBARS were found. This result agrees fairly well with data of Hogg et al. (23) who reported the formation of about 3 µM TBARS after 5 h incubation of 1 mM SIN-1 and 1 mM deoxyribose. In contrast to the findings of Hogg et al. (23), the presence of SOD (100 units/ml) did not inhibit the oxidation of deoxyribose. Other reports support our results. Yim et al. (33) observed a Cu,Zn-SOD-mediated production of hydroxyl radicals from H2O2, and SOD model compounds induced the formation of TBARS from deoxyribose in the presence of H2O2 (34). The addition of HEPES (20 mM) to the buffer solution reduced the production of TBARS to 1.0 ± 0.1 µM. The strong inhibition of both TBARS and RH123 formation (Fig. 3) indicated that peroxynitrite produced from SIN-1 should have reacted with HEPES.


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Fig. 3.   Effect of HEPES and SOD on the SIN-1-induced oxidation of DHR123 and deoxyribose. SIN-1 and DHR123 (25 µM/50 µM) or SIN-1 and deoxyribose (both 1.5 mM) were incubated for 3 or 5 h, respectively, in 50 mM potassium phosphate buffer (pH 7.5, 0.1 mM DTPA, 37 °C) in the absence and presence of HEPES (20 mM) or SOD (100 units/ml). RH123 was quantified by reading the absorbance at 500 nm and deoxyribose oxidation by determining TBARS formation. Data are means ± S.D. of three experiments performed in duplicate and expressed in percent relative to control values. Formation of 100% product indicated 11.4 ± 0.2 µM RH123 and 3.0 ± 0.2 µM TBARS, respectively.

Influence of Tryptophan and Bicarbonate/Carbon Dioxide on H2O2 Formation-- To support the foregoing conclusion further, we attempted to scavenge peroxynitrite with tryptophan, which has been reported to scavenge peroxynitrite with a rate constant of k = 184 M-1 s-1 (35) but should not react with ·NO or Obardot 2 at significant rates. Accordingly, tryptophan (10 mM) inhibited the HEPES (5 and 10 mM)-stimulated H2O2 production from SIN-1 by about 50% (Fig. 4A). The inhibitory effect of tryptophan decreased with increasing concentrations of HEPES and was abolished at 50 mM HEPES.


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Fig. 4.   Effect of tryptophan on the formation of hydrogen peroxide by SIN-1. SIN-1 (1.5 mM) was incubated for 5 h in 50 mM potassium phosphate buffer (pH 7.5, 0.1 mM DTPA, 37 °C) in the absence and presence of tryptophan (10 mM) and various concentrations of HEPES (0-50 mM). A, in HCO3-/CO2-free solutions. B, in the presence of HCO3-/CO2 (20 mM/5%). H2O2 production was quantified using the catalase assay. Data are means ± S.D. of three experiments performed in duplicate.

Because HCO3-/CO2 accelerates nitration reactions (e.g. nitration of tryptophan (35)) but inhibits strongly the "hydroxyl radical-like" activity of ONOOH (36), we studied the HEPES-dependent H2O2 formation from SIN-1 in the presence of HCO3-/CO2 (20 mM, 5%) (Fig. 4B). In the presence of HEPES concentrations typically used in experimental systems (10 and 20 mM), the addition of HCO3-/CO2 slightly enhanced the production of H2O2 compared with the bicarbonate-free situation. A maximum value of about 220 µM H2O2 was reached at 20 mM HEPES. Contrary to the bicarbonate-free system, however, higher HEPES concentrations again diminished the H2O2 level. In the presence of HCO3-/CO2, tryptophan (10 mM) inhibited the HEPES (5, 10, and 20 mM)-mediated H2O2 production from SIN-1 somewhat more pronounced than in its absence (Fig. 4B). These results favor the view that peroxynitrite-derived oxidants, e.g. bicarbonate radical, nitrogen dioxide, nitryl cation, that have been postulated to be produced in the presence of carbon dioxide (37) also might be able to induce H2O2 production from HEPES.

Authentic Peroxynitrite

H2O2 Production from Peroxynitrite in the Presence of HEPES-- To address peroxynitrite-HEPES interactions more directly, we also employed solutions of authentic peroxynitrite. As expected, reaction of authentic ONOO- (1 mM) with HEPES (0-400 mM) also resulted in the formation of H2O2 (Fig. 5). In the absence of HEPES only 6.2 ± 0.4 µM H2O2 was found, reflecting the hydrogen peroxide base level (0.65 mol %) of our peroxynitrite stock solution. In the presence of a typical HEPES concentration (20 mM) the yield of H2O2 was about 8-fold higher. A limiting value of 89.9 ± 3.3 µM H2O2 at pH 7.5 was reached again at a concentration of 400 mM HEPES. The addition of 20 mM HCO3-, 5% CO2 did not alter the base level of H2O2 (6.1 ± 0.7 µM H2O2) but increased the yield of hydrogen peroxide by about a factor of 14 in the presence of 20 mM HEPES. A maximum value of 95.4 ± 1.9 µM H2O2 was found at 100 mM HEPES. Further increase of the HEPES concentration to 400 mM diminished the yield of H2O2 to 71.6 ± 3.7 µM. These results are in line with the data obtained from SIN-1 (Fig. 4B).


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Fig. 5.   Peroxynitrite-dependent H2O2 production: influence of HEPES. Peroxynitrite (1 mM) was added by vortexing to various concentrations of HEPES in 50 mM potassium phosphate buffer (0.1 mM DTPA, 37 °C, pH 7.5) in the absence and presence of HCO3-/CO2 (20 mM/5%). After the addition of peroxynitrite, the samples were stored for 40 min in a warming or in a cell culture incubator. H2O2 was quantified using the peroxidase assay. Each value represents the mean ± S.D. of three experiments performed in duplicate.

With regard to the short lifetime of peroxynitrite (t1/2 approx  1 s at 37 °C) H2O2 production by action of authentic peroxynitrite was expected to be complete within a few seconds, provided that no chain reaction would be initiated. Accordingly, about 88% of the maximum (i.e. after 40 min) yield of H2O2 was found already 10 s after mixing of 400 mM HEPES with 1 mM peroxynitrite (Fig. 6). Further growth to the plateau value of 90 µM H2O2 occurred much slower, within 40 min.


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Fig. 6.   Time evolution of H2O2 production from peroxynitrite-HEPES interaction under hypoxic and normoxic conditions. Peroxynitrite (1 mM) was vortexed to HEPES (400 mM) containing potassium phosphate buffer solution (50 mM, 0.1 mM DTPA, 37 °C, pH 7.5) under hypoxic or normoxic conditions. Aliquots were taken at selected time points, and H2O2 production was quantified using the peroxidase assay. Each value represents the mean ± S.D. of three experiments performed in duplicate.

Oxygen Dependence of H2O2 Formation-- According to our proposed mechanism (see "Discussion") atmospheric oxygen is transformed to H2O2; hence, H2O2 production should be critically dependent on the oxygen concentration. However, the yield of H2O2 was found to be largely independent on the oxygen level (Fig. 6). Somewhat surprisingly, even under hypoxic conditions about 88% of the maximum yield of H2O2 was produced within 10 s. Only the minor (12%) long term production of H2O2 appeared to be dependent on the oxygen level.

Oxygen and H2O2 Formation under Hypoxic Conditions-- The apparent independence of H2O2 production on the level of dissolved oxygen implied that either peroxynitrite is directly converted to H2O2 or that a substantial amount of oxygen is released in the decay of peroxynitrite. In fact, oxygen release from peroxynitrite has been reported previously (7, 38, 39). To check on this, we measured the release of O2 from peroxynitrite in the presence and absence of HEPES (400 mM) (Table II). In the absence of HEPES we observed at pH 7.5 the formation of oxygen, in agreement with the above report (39). In mere phosphate buffer an oxygen production of 175 ± 7.9 µM was measured within 10 s after addition of peroxynitrite (1 mM). However, in the presence of DPTA (0.1 mM) the maximum yield of oxygen dropped to 132.8 ± 7.8 µM, in agreement with data published by Radi et al. (7). In any case, oxygen is produced at a level sufficient to account for the observed yield of H2O2 (78.9 ± 2.9 µM). On the other hand, release of oxygen from peroxynitrite was abolished in the presence of 400 mM HEPES. In mere phosphate buffer at pH 5 the release of oxygen was reduced by about 90% (17.0 ± 3.9 µM). Consequently, H2O2 production was strongly suppressed to 11.8 ± 0.9 µM in the presence of HEPES under these hypoxic conditions (Table II). By way of contrast, under normoxic conditions (see Fig. 10) peroxynitrite stimulated formation of H2O2 at this pH to about 50 µM. Thus, atmospheric oxygen and/or oxygen donated from peroxynitrite is necessary for H2O2 production from peroxynitrite-HEPES interactions.

                              
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Table II
Peroxynitrite-induced formation of oxygen and hydrogen peroxide under hypoxic conditions
Peroxynitrite (1 mM) was added under hypoxic conditions in the absence and presence of HEPES (400 mM) or DTPA (0.1 mM) in 50 mM potassium phosphate buffer (37 °C, pH 7.5 and 5). Oxygen was measured polarographically with a Clark-type electrode. In another set of experiments, H2O2 was quantified after an incubation of 40 min using the peroxidase assay. Each value represents the mean ± S.D. of six experiments.

Influence of HEPES on Peroxynitrite-mediated Nitration of p-Hydroxyphenylacetic Acid-- Peroxynitrite (1 mM)-mediated nitration of p-HPA (1 mM) yielded 79.6 ± 1.5 µM 3-NO2-4-HPA in the absence of HCO3-/CO2 (20 mM/5%) and 189.2 ± 2.8 µM in its presence (Table III). These yields (8 and 19%) are virtually identical to those found for nitration of tyrosine (~6-7 and 19%) at the same pH (40). In the presence of SOD (100 units/ml) the yields of 3-NO2-4-HPA increased slightly by about 10%, in line with reports by Beckman et al. (41). The addition of HEPES strongly decreased the peroxynitrite-mediated nitration of p-HPA. At 20 mM HEPES formation of 3-NO2-4-HPA was reduced by 73% in the absence of HCO3-/CO2 and by 64% in its presence. Increasing the HEPES concentration to 400 mM further inhibited 3-NO2-4-HPA formation under both conditions by approximately 90%. The effect of HEPES on the 3-NO2-4-HPA formation from peroxynitrite in HCO3-/CO2-free solution was half-maximal at 6 ± 1 mM, in excellent agreement with the half-maximally stimulation of H2O2 production (see above). In the presence of HCO3-/CO2 the effect of HEPES on the half-maximal yield of H2O2 formation was almost unchanged (5 ± 1 mM), whereas nitration of p-HPA was half-maximal at 9 ± 1 mM. These results suggest that HEPES has been attacked by the same reactive intermediates that are responsible for the nitration of p-HPA.

                              
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Table III
Effect of HEPES and superoxide dismutase on the peroxynitrite-induced nitration of para-hydroxyphenylacetic acid
Peroxynitrite (1 mM) was vortexed to para-hydroxyphenylacetic acid (p-HPA, 1 mM) in the absence and presence of HCO3-/CO2 (20 mM/5%), HEPES (0-400 mM), or SOD (0-100 units/ml) in 50 mM potassium phosphate buffer (0.1 mM DTPA, 37 °C, pH 7.5). After a 5-min incubation at 37 °C, the formation of 3-(NO2)-4-HPA was determined by reading the absorbance at 430 nm. Each value represents the mean ± S.D. of four experiments performed in duplicate.

Radicals from Good's Buffers-- In previous studies (42, 43) fairly persistent ESR spectra have been observed from piperazine buffer compounds by action of various oxidants like Fe(II)/H2O2 or polymeric iron/O2. The spectra were reasonably attributed to the radical cations expected to be generated by one-electron oxidation of the piperazine compounds (42), although the assignment appeared tentative as no detailed analysis of the spectra was performed. We therefore checked, by ESR spectroscopy, the ability of peroxynitrite to mediate radical formation from HEPES and PIPES, both by SIN-1 (10 mM) and by authentic peroxynitrite (50 mM) solutions. After mixing of the reactants by vortexing, we observed "instantaneously" (i.e. within 1 min) weak, multi-line ESR spectra (shown for authentic peroxynitrite in Fig. 7). In some experiments a 2-4-fold increase of the signal intensity was observed during the run. Both the HEPES- and PIPES-derived spectra were virtually identical to those reported in the literature (42, 43). In agreement with the previous observations the signals decayed with half-lifes of about 10-15 min at 20 °C. The spectrum obtained from HEPES was too weak to be evaluated in detail, but the PIPES-derived spectrum was sufficiently intense to be completely analyzed. Elaborate analysis by spectral simulation revealed that the ESR spectrum must be interpreted as a superposition of the two radical cations R1 and R2 (Fig. 8).


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Fig. 7.   ESR spectrum of radical cations from oxidation of PIPES with peroxynitrite. ESR spectrum of radical cations R1 and R2 recorded 4 min after oxidation of PIPES (900 mM) with peroxynitrite (50 mM), pH 7.5, at 20 °C. Top, experimental ESR spectrum. Bottom, simulated ESR spectrum as a 3:1 superposition of R1 and R2. ESR parameters: R1: [a(N) = 0.722 (2N), a(H) = 0.698 (4H), a(H) = 0.522 (4H), a(H) = 0.493 (4H), a(H) = 0.048 (4H) mT; g = 2.0036]; R2: [a(N) = 0.725 (2N), a(H) = 0.695 (4H), a(H) = 0.509 (4H), a(H) = 0.500 (4H), a(H) = 0.035 (4H) mT; g = 2.0036]. The simulated spectrum fits the experimental one with a correlation coefficient of r = 0.999.


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Fig. 8.   Structure of the radical cations from Good's buffers.

The presence of at least two radicals was further indicated by slight temporal changes of the overall spectral shape, indicating somewhat different lifetimes of both species. A similar superposition has been observed previously for HEPES-derived ESR spectra (43). The presence of a small amount of a possible further oxidized species, R3, cannot be completely excluded. The identification of R1, R2 as radical cations unambiguously follows from their ESR spectral properties, that is the even number of interacting nuclei, the g factor, and the magnitude of the corresponding hyperfine splittings, all of which are strikingly similar to the data known for other piperazine and dihydropiperazine radical cations in organic solvents (44-46). Thus, both SIN-1-generated and authentic peroxynitrite are able to oxidize Good's buffers by one-electron processes.

Obardot 2 and H2O2 Formation from HEPES Radicals-- Based on the ESR observations we suspected that the piperazine-derived radical cations may react with O2 to generate Obardot 2 which subsequently dismutates to H2O2 and O2 (see "Discussion"). However, as shown above, about 88% of H2O2 production is already complete within the lifetime of peroxynitrite, whereas the ESR-detected piperazine-type radicals are much more long-lived (t1/2 approx 10-15 min). Thus, only the additional (12%) long term increase of H2O2 concentration should refer to the reaction of the "ESR observable" fraction of the HEPES-derived radical cations with atmospheric oxygen. Consequently, Obardot 2 formation in the same concentration range was expected solely by action of the HEPES-derived radical(s), i.e. without interference by residual peroxynitrite.

In the absence of HEPES the detectable amount of Obardot 2 was low (<= 0.1 µM Obardot 2) but increased with increasing HEPES concentration (Fig. 9). A maximum amount of 2.5 ± 0.3 µM Obardot 2 was formed in the presence 400 mM HEPES. Thus, attack of authentic peroxynitrite on HEPES indeed generates Obardot 2. The fact that only micromolar amounts of Obardot 2 were detected agrees with the low concentrations of the HEPES-derived radical cations as deduced from the ESR signal intensities.


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Fig. 9.   Peroxynitrite-dependent Obardot 2 production. Influence of HEPES. Peroxynitrite (1 mM) was added by vortexing to various concentrations of HEPES in 50 mM potassium phosphate buffer (0.1 mM DTPA, 37 °C, pH 7.5). After 2 min of incubation the samples were divided in 2 aliquots. In 1 aliquot cytochrome c3+ (20 µM) was added, and in the other aliquot cytochrome c3+ plus SOD (100 units/ml) was added. The 2 aliquots were stored for 40 min in a warming incubator. Cytochrome c2+ was detected by reading the absorbance at 550 nm. For each HEPES concentration the difference A550aliquot 1 - A550aliquot 2 was used to calculate the trapped amounts of Obardot 2. Each value represents the mean ± S.D. of three experiments performed in duplicate.

pH dependence of H2O2 Formation-- Only the amino unprotonated forms of HEPES, PIPES, etc. are amenable to one-electron oxidation to yield radicals R1-R3 (Fig. 8). Thus, H2O2 formation was expected to be pH-dependent. In the absence of HEPES a constant, low amount of H2O2 (~7 µM) was found within the pH range 4-8.5 after vortexing of peroxynitrite (1 mM) solutions (Fig. 10). At pH 4 no change of H2O2 level was detected in the presence of HEPES (400 mM). However, with increasing pH H2O2 formation strongly increased to reach a maximum value of about 90 µM at pH 7.5. Further increase of the pH again diminished the production of H2O2. The same pH dependence on H2O2 formation, with maxima at pH 7.5, was also observed for EPPS and MOPS (data not shown). The pH-dependent half-maximal production of H2O2 correlated well with the pKa of the respective buffer compound (Table IV). In agreement with the proposal that the unprotonated forms of the tertiary amines are oxidized by peroxynitrite, the quaternary amino compound tetramethylammonium chloride (400 mM) did not stimulate H2O2 formation (Table IV).


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Fig. 10.   Influence of pH on hydrogen peroxide formation from peroxynitrite-HEPES interactions. Peroxynitrite (1 mM) was added by vortexing to 50 mM potassium phosphate buffer (0.1 mM DTPA, 37 °C) solutions at various pH values (4-8.5) in the absence and presence of 400 mM HEPES. After the addition of peroxynitrite, the samples were stored for 40 min in a warming incubator. H2O2 was quantified using the peroxidase assay. Data are means ± S.D. of three experiments performed in duplicate.

                              
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Table IV
Influence of pH on hydrogen peroxide production from peroxynitrite and various amines
Peroxynitrite (1 mM) was vortexed to various amines (400 mM) in 50 mM potassium phosphate buffer (0.1 mM DTPA, 37 °C) solutions. Maximal H2O2 formation from peroxynitrite-amine interactions were observed at pH 7.5. For pH-dependent half-maximal formation of H2O2 from peroxynitrite-amine interactions, pH was decreased stepwise (Delta pH 0.5) from 7.5 to 4.5. After the addition of peroxynitrite, the samples were stored for 40 min in a warming incubator. H2O2 was quantified using the peroxidase assay. Data are means ± S.D. of three experiments performed in duplicate.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Reactions between authentic or in situ generated peroxynitrite and HEPES or PIPES result in the formation of HEPES- and PIPES-derived radical cation species (R1 and R2) (Fig. 8) as shown by ESR spectroscopy (Fig. 7). A reasonable mechanism for the formation of R1 and R2 is displayed in Fig. 11, where [Ox] stands for the action of any sufficiently strong oxidant in the system, e.g. peroxynitrite and peroxynitrite-derived oxidants formed in the absence and presence of carbon dioxide, without specifying the actual reactive species.


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Fig. 11.   Radical cation formation from Good's buffers by one-electron oxidants. In the first step one-electron oxidation of the piperazine compounds produces the amine radical cation R1 which subsequently undergoes alpha -deprotonation, a common decay path for such types of radical cations (47, 58). The putative alpha -aminoalkyl radical R4 certainly is too short-lived to be observed under the conditions of the ESR experiment (see below). Further oxidation of R4 to yield R2 also is feasible, in particular in the presence of O2 (47, 58). A further reaction of R2 to give R3 could not be verified by the ESR spectra but cannot be ruled out.

Fig. 11 also provides the starting point for the interpretation of Obardot 2 formation in our system. It is common knowledge that carbon-centered radicals react rapidly (k approx 2 × 109 M-1 s-1) with molecular oxygen to give peroxyl radicals (47, 48). However, as shown by von Sonntag's group (reviewed in Ref. 47) in the case of electron-rich alkyl radicals, i.e. those carrying electron-donating substituents, the corresponding peroxyl radicals are highly unstable, decomposing rapidly (k approx 104-109 M-1 s-1) to a cationic species and Obardot 2 (47, 49, 50). In fact, this property of electron-rich alkyl radicals has recently been used as the basis for the development of a thermal superoxide source (51).

By analogy, we propose that a similar mechanism is operative in the production of Obardot 2 from Good's buffers by attack of authentic or SIN-1-generated peroxynitrite (Fig. 12). Thus, it would appear that the rate of Obardot 2 production is governed by the first two steps (electron transfer and deprotonation) of the reaction sequence. On the other hand, R4 may also be generated directly from the parent compound by hydrogen abstraction by suitably reactive radicals X·, e.g. hydroxyl or peroxyl radicals. In line with this assumption, formation of R1 and R4 has been postulated in the autoxidation of DNA/Cu2+/H2O2 systems in the presence of HEPES and PIPES (52).


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Fig. 12.   Proposed mechanism of hydrogen peroxide formation from piperazine-based buffer compounds and peroxynitrite or other oxidants. As already shown in Fig. 11 the reaction sequence is initiated by one-electron oxidation of the piperazine compound by peroxynitrite. The alpha -aminoalkyl radical R4 formed by proton loss from the initial radical cation R1 would react at a close to diffusion-controlled rate with oxygen. Although not detected by ESR, radical R1 may likewise undergo deprotonation at the side chains to give exocyclic alpha -amino radicals R4', which would react with O2 in the same manner. Rapid fragmentation of the so-formed peroxyl radicals R5, R5' produces Obardot 2 and the cationic species 6. The latter would be rapidly trapped by reaction with water or other nucleophiles (47) and/or further oxidized to the radical cation R2. Dismutation of Obardot 2 finally leads to H2O2.

Further consequences arise from the foregoing. First, Obardot 2/H2O2 production is expected to be pH-dependent, decreasing with decreasing pH and reflecting the respective pKa of the organic buffer compound. This hypothesis was confirmed by the data of Fig. 10 and Table IV, in agreement with the requirement of an unshared electron pair at nitrogen for the oxidation process. Second, the efficiency of Obardot 2/H2O2 production among the various buffer compounds should be somehow affected by the structure and properties of the side chains attached to the piperazine rings. As amino-derived radical cations are strong electrophiles and are normally rapidly quenched in aqueous solution (47, 53, 54), one might hypothesize that the negatively charged sulfonic acid side chain(s) exert some "protective" effect on the cationic radical center in R1, thereby inhibiting the rate of deprotonation. This would agree with the observations that the monosulfonylated piperazines HEPES and EPPS gave higher yields of H2O2 (Tables I and IV) than the bis-sulfonylated POPSO and PIPES, but, vice versa, the HEPES-derived radical showed a much lower ESR signal intensity (steady-state concentration) than the PIPES-derived one.

There is no contradiction in the fact that the ESR spectra of radical cations R1 and R2 have been detected although their reaction with oxygen is extremely fast. What we detected by ESR is just the small "excess" amount of R1 and R2 after all of the dissolved oxygen has been consumed. This explains the sometimes observed short term growth of the ESR signals immediately after mixing of the reactants.

The results presented here clearly demonstrate that the interaction of peroxynitrite and piperazine-type buffers, regardless of the presence of HCO3-/CO2, may lead to serious consequences concerning the investigation of peroxynitrite-driven actions under physiological conditions, e.g. necrosis, apoptosis, inhibition of enzymes, formation of metabolites, etc. There are several reports in the literature in which similar effects of the interaction of HEPES with strong oxidants other than peroxynitrite are mentioned, although no satisfying explanation has been given. For example, vanadyl induces hemolysis of vitamin E-deficient erythrocytes in HEPES buffer but not in phosphate buffer (55); HEPES stimulates hydroxyl radical generation significantly in the presence of both copper ions and H2O2 (56), and HEPES promotes also hypochlorous acid-induced oxidation of ferrocyanide very efficiently (57). In conclusion, it must be emphasized that Obardot 2/H2O2 formation according to the above mechanism (Fig. 12) is not restricted to peroxynitrite as an oxidant and not to piperazine buffer compounds as targets but should be regarded as a general pathway for compounds from which electron-rich alkyl radicals (preferably alpha -amino or alpha ,alpha -dialkoxyl radicals) can be generated. Accordingly, H2O2 formation in the range of 75 µM has been found in the reaction of peroxynitrite (1 mM) with other tertiary amines (400 mM), viz. triethylamine and triethanolamine.2 Generally speaking, any oxidant strong enough to oxidize certain tertiary amines in a one-electron step would be able to initiate the above reaction sequence. Furthermore, any other reaction, preferably hydrogen abstraction, that generates alpha -amino alkyl or alpha ,alpha -dialkoxyl radicals would induce the same process. Because tertiary amine groups as targets for strong oxidants are present in a variety of biological molecules, e.g. in ATP, creatine, or nucleic acids, we propose that Fig. 12 for Obardot 2/H2O2 production must be considered to be a general mechanism under in vivo conditions. This certainly sheds a new light on a number of studies aimed at the pathophysiological effects of various oxidative species, as actually hydrogen peroxide might have been the true operating agent.

    ACKNOWLEDGEMENT

We thank Angela Wensing for excellent technical assistance.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Institut für Physiologische Chemie, Universitätsklinikum Essen, Hufelandstrasse 55, D-45122 Essen, Germany. Tel.: +201/723-4101; Fax: +201/723-5943.

1 The abbreviations used are: SIN-1, 3-morpholinosydnonimine N-ethylcarbamide; SOD, superoxide dismutase; EPPS, N-2-hydroxyethylpiperazine-N'-3-propanesulfonic acid; PIPES, piperazine-N,N'-bis(2-ethanesulfonic acid); POPSO, piperazine-N,N'-bis(2-hydroxypropanesulfonic acid); MOPS, 3-(N-morpholino)propanesulfonic acid; DHR123, dihydrorhodamine 123; RH123, rhodamine 123; TBARS, thiobarbituric acid-reactive substances; 3-NO2-4-HPA, 3-nitro-4-hydroxyphenylacetic acid; p-HPA, 4-hydroxyphenylacetic acid; DTPA, diethylenetriaminepentaacetic acid; mT, millitesla.

2 M. Kirsch, H. G. Korth, R. Sustmann, and H. de Groot, unpublished observations.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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