^•BMPO-OOH Spin-Adduct as a Model for Study of Decomposition of Organic Hydroperoxides and the Effects of Sulfide/Selenite Derivatives. An EPR Spin-Trapping Approach

Abstract

Lipid hydroperoxides play an important role in various pathophysiological processes. Therefore, a simple model for organic hydroperoxides could be helpful to monitor the biologic effects of endogenous and exogenous compounds. The electron paramagnetic resonance (EPR) spin-trapping technique is a useful method to study superoxide (O₂^•−) and hydroxyl radicals. The aim of our work was to use EPR with the spin trap 5-tert-butoxycarbonyl-5-methyl-1-pyrroline-N-oxide (BMPO), which, by trapping O₂^•− produces relatively stable ^•BMPO-OOH spin-adduct, a valuable model for organic hydroperoxides. We used this experimental setup to investigate the effects of selected sulfur/selenium compounds on ^•BMPO-OOH and to evaluate the antioxidant potential of these compounds. Second, using the simulation of time-dependent individual BMPO adducts in the experimental EPR spectra, the ratio of ^•BMPO-OH/^•BMPO-OOH—which is proportional to the transformation/decomposition of ^•BMPO-OOH—was evaluated. The order of potency of the studied compounds to alter ^•BMPO-OOH concentration estimated from the time-dependent ^•BMPO-OH/^•BMPO-OOH ratio was as follows: Na₂S₄ > Na₂S₄/SeO₃²⁻ > H₂S/SeO₃²⁻ > Na₂S₂ ~Na₂S₂/SeO₃²⁻ ~H₂S > SeO₃²⁻ ~SeO₄²⁻ ~control. In conclusion, the presented approach of the EPR measurement of the time-dependent ratio of ^•BMPO-OH/^•BMPO-OOH could be useful to study the impact of compounds to influence the transformation of ^•BMPO-OOH.

1. Introduction

Exogenously added and endogenously produced hydrogen sulfide (H₂S) and polysulfides affect many physiological and pathologic processes [1,2,3,4]. They modulate oxidative stress by reacting with reactive oxygen and nitrogen species [1,5,6,7]. Selenium (Se) is an essential trace element for humans, with multiple and complex effects on health, having antioxidant properties due to its presence in 25 selenoproteins in the form of selenocysteine amino acid [8]. Se compounds and H₂S are present in living organisms and either alone or in combination interact with reactive oxygen species (ROS) [1,9,10,11,12]. In our previous work, we found that products of the sulfide/selenite (H₂S/SeO₃²⁻) interaction scavenge superoxide-derived radicals, cleave plasmid DNA (pDNA) and modulate tonus of isolated rat aorta and blood pressure [13]. However in this work, using the procedure of addition of KO₂ as a source of (O₂^•−) into the mixture of the compounds (H₂S, SeO₃²⁻) with the spin-trapping agent 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide (BMPO) it was not clear which components of the electron paramagnetic resonance (EPR) spectra resulted from the compounds/O₂^•− or compounds/^•BMPO-OOH interactions. To solve this issue, a new experimental strategy for the interaction of the compounds with ^•BMPO-OOH only is herein presented.

Lipid peroxidation is a process under which oxidants such as free radicals attack lipids containing carbon-carbon double bond(s), leading to the radical chain reactions. It has an important role in cell biology and in various pathophysiological processes in which lipid hydroperoxides display a crucial function [14]. Importantly, many pathophysiological states can be regulated by the modulation of lipid peroxidation induced by exogenous compounds. Therefore, simple models of organic hydroperoxide could be useful for studying the effects of various compounds. EPR spin-trapping technique using cyclic nitrone BMPO is a reliable method to study O₂^•− and hydroxyl radicals (HO^•) [15,16,17].

The spin-trapping agent BMPO in aqueous solutions represents a racemic mixture of two enantiomers [S (−) and R (+)], and the previous detailed study evidenced that starting with racemate, as well as with the individual enantiomers, the reaction with O₂^•− resulted in the identical EPR spectrum representing two signals of diastereoisomers at the same ratio (trans and cis with respect to the tert-butoxycarbonyl group) (Scheme 1). The EPR spectra of ^•BMPO-OOH diastereoisomers are characterized with the similar nitrogen splittings and slightly different β-hydrogen splittings [18,19,20]. Analogously, the experimental EPR spectra of ^•BMPO-OH were interpreted considering the superposition of individual signals of two diastereoisomers with the different hyperfine coupling constants [15,21] (Scheme 1).

Therefore, the aim of our work was to use EPR with BMPO—which in the presence of O₂^•− forms a relatively stable ^•BMPO-OOH and can serve as a model for organic hydroperoxide—to study the effects of Na₂S_n (n = 1, 2, 4) and Na₂SeO_n (n = 3, 4) on their own or their mixture Na₂S_n/Na₂SeO_n. This approach enables the comparison of the relative potential of the investigated compounds to affect the ROOH bond and to eliminate radicals formed during its decomposition. Since 10% DMSO (v/v) is used in the studied system, the procedure is useful for evaluating antioxidant potency of compounds insoluble in water.

2. Materials and Methods

2.1. Chemicals

Stock solutions of sodium selenite (Na₂SeO₃, 10 or 40 mmol L⁻¹, Sigma-Aldrich 214485, Saint Louis, MO, USA) and sodium selenate (Na₂SeO₄, 10 mmol L⁻¹, Sigma-Aldrich S0882, Saint Louis, MO, USA) were prepared freshly in deionized H₂O, stored at 23 °C and used within 5 h. Na₂SeO₃ dissociates in solution to yield mostly H₂SeO₃ at acidic pH, HSeO₃⁻ at neutral pH and SeO₃²⁻ at alkaline pH. For simplicity, the terms SeO₃²⁻ and SeO₄²⁻ are employed as representative expression to encompass the total mixture of different (de)protonation states. Spin-trapping agent 5-tert-butoxycarbonyl-5-methyl-1-pyrroline-N-oxide (BMPO, 100 mmol L⁻¹, DoJindo B568-10, Munich, Germany) was dissolved in deionized H₂O, stored at −80 °C and used after thawing. Na₂S (100 mmol L⁻¹) as a source of H₂S and polysulfides, sodium disulfide (Na₂S₂, 10 mmol L⁻¹) and sodium tetrasulfide (Na₂S₄, 10 mmol L⁻¹) (Dojindo SB01, SB02 and SB04, respectively, Munich, Germany) were prepared in argon-bubbled deionized H₂O, aliquoted, stored at −80 °C and thawed just before use [5]. Na₂S dissociates in aqueous solution and reacts with H⁺ to yield H₂S, HS⁻ and a trace of S²⁻. We use the term H₂S to describe the total mixture of H₂S, HS⁻ and S²⁻ forms. Similarly, Na₂S₂ and Na₂S₄ dissociate in aqueous solution yielding S_n²⁻, HS_n⁻ and traces of H₂S_n (n = 2 and 4). For simplicity, we use the terms Na₂S₂ and Na₂S₄. For EPR samples, buffer consisting of 50 mmol L⁻¹ sodium phosphate (pH 7.4, 37 °C) and 100 µmol L⁻¹ diethylenetriaminepentaacetic acid (DTPA) was used. Saturated KO₂/DMSO solution was prepared by the addition of powdered KO₂ (Sigma-Aldrich 278904, Steinheim, Germany) into the anhydrous DMSO (1.42 mg mL⁻¹; 23 ± 1 °C; theoretically 20 mmol L⁻¹ KO₂), vortexed for 2 min, sonicated for 20 s and let for 1 h to settle down the undissolved KO₂ powder. When an aliquot of KO₂/DMSO was taken from the bottom part of the stock solution and added into the phosphate buffer, the spectral intensity of ^•BMPO-OOH was 2- to 4-fold higher than that of the aliquot taken from the upper part [5,13]. For EPR study we used the aliquots of the saturated stock KO₂/DMSO obtained from the upper part of the stock solution. The intensity of ^•BMPO-OOH EPR spectra was reproducible when the KO₂/DMSO stock solution was incubated at 23 ± 1 °C and used within ~4 h.

2.2. EPR Study of the BMPO-Adducts

A modified protocol from our previous study was used [13]. First, ^•BMPO-OOH was prepared by the addition of the saturated KO₂/DMSO solution (10% v/v DMSO/final buffer) into the BMPO (30 mmol L⁻¹ final concentration, 37 °C) diluted in the phosphate buffer. The BMPO/KO₂ sample was mixed for 10 s and then the studied compounds, H₂S_n, SeO_n²⁻ or H₂S_n/SeO_n²⁻ mixtures, were added. The sample was mixed again for 5 s and transferred to a standard cavity aqueous EPR flat cell (WG 808-Q, Wilmad-LabGlass, Vineland, NJ, USA). The first EPR spectrum was recorded 100 s after the addition of the compounds. The sets of the individual EPR spectra of the BMPO spin-adducts were recorded as 15 sequential scans, each 42 s, with a total acquisition time of 11 min. Each experiment was repeated at least twice. EPR spectra of the BMPO spin-adducts were measured on a Bruker EMX spectrometer (Rheinstetten, Germany), X-band ~9.4 GHz, 335.15 mT central field, 8 mT scan range, 20 mW microwave power, 0.1 mT modulation amplitude, 42 s sweep time, 20.48 ms time constant and 20.48 ms conversion time at 37 °C. To compare the relative potency of the compounds to decrease overall trapped radical concentration, a second integral of the total EPR spectra intensity of the BMPO-adducts was evaluated. To obtain the ^•BMPO-OH/^•BMPO-OOH radical proportion, the simulated spectra ratio was calculated using EasySpin program working on MatLab platform [22].

2.3. Plasmid DNA Cleavage

A pDNA cleavage assay with the use of pBR322 plasmid (New England BioLabs, Inc., N3033 L, Ipswich, MA, USA) was performed as reported previously [13]. In this assay, all samples contained 0.2 μg pDNA in the sodium phosphate buffer (25 mmol L⁻¹ sodium phosphate, 50 μmol L⁻¹ DTPA, pH 7.4). FeCl₂ (150 µmol L⁻¹) was used in control experiments. Powdered KO₂ was dissolved by 4 mmol L⁻¹ BMPO in the buffer containing 10% DMSO (v/v), vortexed for 10 s and 10 µL of the mixture was added into 10 µL solution of pDNA. The resulting mixtures were incubated for 30 min at 37 °C. After incubation, the reaction mixtures were subjected to 0.6% agarose gel electrophoresis. Integrated densities of all pBR322 forms in each lane were quantified using the Image Studio analysis software (LI-COR Biotechnology, Bad Homburg, Germany) to estimate pDNA-cleavage efficiency.

3. Results

3.1. ^•BMPO-OOH as a Model Hydroperoxide and the Effects of Na₂S_n/Na₂SeO_n

KO₂ in DMSO dissociates to K⁺ and relatively stable O₂^•−, but its solubility in DMSO is extremely low [23,24]. EPR spectra after the addition of KO₂ into the BMPO-buffered solution showed signals of two conformers of the ^•BMPO-OOH adduct, as a result of O₂^•− trapping by BMPO (Figure 1a1–a3). The ^•BMPO-OOH signal was relatively stable and slowly decreased (t_1/2 ~23 min) in accordance with [15,16,18]. However, when KO₂ was added into the buffer first, followed by the addition of BMPO 10 s later no radical was trapped by BMPO, i.e., the EPR spectrum was not observed (Figure 1b1–b3). The results confirmed a short life time of O₂^•− in water solution (t_1/2 ~1–10 µs). Therefore, we used the interaction of O₂^•− with BMPO, producing the ^•BMPO-OOH spin-adduct, as a model of organic hydroperoxides to study the effects of Na₂S_n/Na₂SeO_n. The following sequence of the compounds was used to prepare the sample: KO₂ was added into BMPO-buffered solution, resulting in the formation of ^•BMPO-OOH and then the studied compounds were added 10 s later.

Information that can be obtained from the EPR spectra after the interaction of ^•BMPO-OOH with compounds added into the system is as follows: First, it is an effect of compounds on the total integral EPR intensity of the BMPO-adducts obtained as a second integral of EPR spectra. Second, from the simulation of individual BMPO-adducts of the experimental EPR spectra it may be possible to obtain the changes in their relative concentrations (^•BMPO-OOH and ^•BMPO-OH), thereby reflecting the effect of compounds.

The presence of SeO₃²⁻ (25 µmol L⁻¹) did not influence the rate of ^•BMPO-OOH decomposition (Figure 1c1). From the similar shape of the first five cumulative spectra (Figure 1c2) and the last five accumulated spectra (Figure 1c3), it is assumed that there was no interaction between SeO₃²⁻ and ^•BMPO-OOH. The addition of Na₂S (25 µmol L⁻¹; H₂S) slightly increased the rate of ^•BMPO-OOH decay (Figure 1d1), as an indication of scavenging/interaction of the BMPO-adducts by/with H₂S. Since the shape of the last five accumulated spectra (Figure 1d3) was different from the first five spectra (Figure 1d2), it can be concluded that H₂S affects the ^•BMPO-OOH spin-adduct. The details on the interaction are described in the next section. The H₂S/SeO₃²⁻ (25/25 µmol L⁻¹/µmol L⁻¹) mixture decreased ^•BMPO-OOH concentration during 100 s after the sample preparation (before EPR measurement started) and later the BMPO-adducts concentration was approximately constant (Figure 1e1). However, spectra were different (Figure 1e2,e3) from control ^•BMPO-OOH (Figure 1a2,a3), indicating interaction of H₂S/SeO₃²⁻ with ^•BMPO-OOH. Similar effect was detected when polysulfide Na₂S₂ (25 µmol L⁻¹) and the Na₂S₂/SeO₃²⁻ (25/25 µmol L⁻¹/µmol L⁻¹) mixture was used (Figure 1f1–f3,g1–g3). The effects of polysulfide Na₂S₄ (25 µmol L⁻¹) was the most pronounced, it diminished ^•BMPO-OOH concentration during 100 s, before EPR measurement started (Figure 1h1–h3). However, its effect to scavenge the BMPO-adducts decreased when the Na₂S₄/SeO₃²⁻ (25/25 µmol L⁻¹/µmol L⁻¹) mixture was used (Figure 1i1). The spectra (Figure 1i2,i3) revealed pronounced interaction with ^•BMPO-OOH.

When SeO₄²⁻ was used instead of SeO₃²⁻, it did not influence the effects of Na₂S, Na₂S₂ and Na₂S₄, indicating no formation of redox active products of the Na₂S_n/SeO₄²⁻ interaction (Figure 2).

3.2. Simulation of BMPO-Adducts Spectra in the Presence of Na₂S_n/Na₂SeO₃

The studied compounds changed shapes of EPR spectra of the BMPO-adducts, indicating the superposition of signals corresponding to the generation of individual BMPO-adducts. Therefore, we analyzed all accumulated spectra by simulation. Figure 3 shows representative examples of the simulation of the sixth to tenth accumulated spectra only. The results showed that the best fit was obtained when the hyperfine coupling constants for two conformers of ^•BMPO-OOH and two of BMPO-hydroxyl radical (^•BMPO-OH) adducts, along with those of BMPO-adduct with carbon-centered radical (^•BMPO-CR) were inserted in spin-Hamiltonian calculations. The simulated spectra shown in Figure 3 were calculated using the hyperfine coupling constants elucidated from the experimental spectra (

Table 1. Hyperfine coupling constants of the BMPO spin-adducts elucidated from the simulations of experimental spectra measured in the buffer solutions containing KO₂ and 10% DMSO (v/v). ^•BMPO-OOH and ^•BMPO-OH were simulated considering presence of two conformers [15,18,19].

BMPO-Adduct	aN, mT	aHβ, mT	aHγ, mT
•BMPO-OH(1)	1.423 ± 0.011	1.541 ± 0.014	0.078 ± 0.011
•BMPO-OH(2)	1.365 ± 0.038	1.248 ± 0.036	0.073 ± 0.015
•BMPO-OOH(1)	1.339 ± 0.002	1.186 ± 0.007	–
•BMPO-OOH(2)	1.334 ± 0.003	0.958 ± 0.007	–
•BMPO-CR	1.528	2.221	–

).

For simplicity, the relative concentration of two conformers ^•BMPO-OH(1) and ^•BMPO-OH(2) were summed up and described as ^•BMPO-OH. Analogously, ^•BMPO-OOH(1) and ^•BMPO-OOH(2) were summed up and described as ^•BMPO-OOH. The time dependence of such evaluated BMPO-adducts EPR integral intensity is shown in Figure 4. In this figure, the absolute integral of individual BMPO-adducts is depended besides sample composition also on time. For example, the first to fifth accumulated spectra of control integral intensity of ^•BMPO-OOH spectra (circle; measured 1.7–5.2 min after sample preparation; see Figure 4a tick 1–5) has value 100 (r.u.) and the integrals of ^•BMPO-OH (triangle) and ^•BMPO-CR (cross) components of the sample are close to zero. The same control sample is measured 5.2–8.7 min after sample preparation (sixth to tenth accumulated spectra; see Figure 4a tick 6–10) and shows decrease to 73.5 (r.u.) for ^•BMPO-OOH component, whereas integral of ^•BMPO-OH is close to zero and ^•BMPO-CR increased to 4.2.

In controls and samples with SeO₃²⁻, similar concentration of radicals and a slow decay of the ^•BMPO-OOH component was seen, indicating no interaction of SeO₃²⁻ with ^•BMPO-OOH (Figure 4a,b). The addition of Na₂S increased the rate of ^•BMPO-OOH decay (Figure 4c), whereas slightly increased the concentration of ^•BMPO-OH. The decay was several times pronounced when the H₂S/SeO₃²⁻ mixture was used (Figure 4d). Na₂S₂ and Na₂S₄ alone and their mixture with SeO₃²⁻ significantly decreased the number of radicals and increased the rate of ^•BMPO-OOH decay (Figure 4e–h), whereas slightly increased concentration of ^•BMPO-OH with exception of Na₂S₄, where radical concentration was close to zero (Figure 4g). It is noticed that spin adducts concentration in the case of Na₂S₄/SeO₃²⁻ was noticeably higher compared to Na₂S₄ alone (Figure 4g vs. Figure 4h). The total integral EPR intensity of all components (Figure 4) revealed that the order of potential to transform/scavenge the BMPO-adducts was Na₂S₄ > Na₂S₂ > H₂S (Figure 4c,e,g) and H₂S/SeO₃²⁻ > H₂S (Figure 4c,d), Na₂S₂ ~Na₂S₂/SeO₃²⁻ (Figure 4e,f), but Na₂S₄ > Na₂S₄/SeO₃²⁻ (Figure 4g,h).

In order to compare the relative ratio of components of the BMPO-adducts the sum of the radicals at each time was normalized to 100% (Figure 5). In controls and samples with SeO₃²⁻, the major ^•BMPO-OOH component was relatively constant over the time with minor ^•BMPO-CR component, indicating no decomposition of ^•BMPO-OOH by SeO₃²⁻ (Figure 5a,b). H₂S and the H₂S/SeO₃²⁻ mixture decreased ^•BMPO-OOH component and increased ^•BMPO-OH over the time (Figure 5c,d), suggesting time-dependent transformation/decomposition of the model hydroperoxide ^•BMPO-OOH to ^•BMPO-OH. Similar qualitative results were observed when Na₂S₂ alone or in the combination with SeO₃²⁻ was used (Figure 5e,f). Na₂S₄ alone significantly decreased ^•BMPO-OOH and increased the ^•BMPO-OH component (Figure 5g). The Na₂S₄/SeO₃²⁻ mixture had similar, but slightly lower effects compared to Na₂S₄ (Figure 5h). The order of the compounds potency to cleave ^•BMPO-OOH, estimated from the time-dependent ^•BMPO-OH/^•BMPO-OOH ratio was: Na₂S₄ > Na₂S₄/SeO₃²⁻ > H₂S/SeO₃²⁻ > Na₂S₂ ~Na₂S₂/SeO₃²⁻ ~H₂S > SeO₃²⁻ ~control.

3.3. Cleavage of Plasmid DNA

Since Na₂S interacted with ^•BMPO-OOH (Figure 4 and Figure 5), it was of interest to know whether radical species, which can damage DNA, were formed during the interaction. The procedure for EPR experiments was modified to observe pDNA cleavage. In the control experiment, Fe²⁺ (150 µmol L⁻¹) cleaved pDNA. However, when the reaction buffer contained 10% (v/v) DMSO, Fe²⁺ did not cleave pDNA. Cleaving of pDNA was observed neither in the presence of BMPO/KO₂ mixture in buffer with 10% (v/v) DMSO, nor when Na₂S was added into BMPO/KO₂/DMSO (Figure 6). From the experiments with Fe²⁺ it is evident that DMSO interfered with the assay. This is supported also by information that DMSO is a scavenger of HO^• radicals [13,25]. Therefore, we did not precede with the pDNA experiments.

4. Discussion

In our previous study, when KO₂ as a source of O₂^•− was added into the mixture of H₂S/BMPO or H₂S/SeO₃²⁻/BMPO, the concentration of trapped radicals changed and a superposition of several individual BMPO-adducts was detected [13]. It was not clear which components of the spectra resulted from the compounds/O₂^•− or from the compounds/^•BMPO-OOH interaction. Therefore, in the present work we used a different approach, which allowed us to study interaction of the compounds with a model of organic hydroperoxide ^•BMPO-OOH. This approach is based on the short lifetime of O₂^•− in water solution and relatively stable ^•BMPO-OOH to which the studied compounds were added.

This approach allowed the study of the effects of compounds on decomposition/transformation of the organic hydroperoxide ^•BMPO-OOH to the ratio of ^•BMPO-OH/^•BMPO-OOH, which was detected by EPR. To elaborate this, we performed a detailed simulation of the experimental EPR spectra obtaining the hyperfine coupling constants of the individual BMPO-adducts under these experimental conditions, along with their relative concentrations. From the comparison of the experimental and the simulated EPR spectra (Figure 3), it can be concluded that the BMPO-adducts spectra were very well simulated by the hyperfine coupling constants (Table 1), based on the presence of two conformers, ^•BMPO-OH(1) and ^•BMPO-OH(2), ^•BMPO-OOH(1) and ^•BMPO-OOH(2) and ^•BMPO-CR. The hyperfine coupling constants were clearly indicated from the spectra simulations (Table 1) and were comparable to the published BMPO-adducts. The relative intensity of ^•BMPO-OOH decreased slowly over time and was comparable, but not identical, to the reported values under physiological conditions without DMSO [15,16,18]. It is likely that the ^•BMPO-CR component resulted from the trapping carbon-centered radical, originating from DMSO. From the time dependence of the integral EPR intensity (Figure 4 and Figure 5) it was possible to evaluate the time-dependent effects of the compounds on the total BMPO-adduct concentration and the ^•BMPO-OH/^•BMPO-OOH ratio, which is suggested to be proportional to the organic hydroperoxide ^•BMPO-OOH transformation/decomposition. The presence of SeO₃²⁻ potentiated the decrease of integral intensity of spin-adducts induced by Na₂S, had no significant effect on Na₂S₂ potency, but decreased the potency of Na₂S₄ (Figure 4). These results indicate that the interaction of SeO₃²⁻ with Na₂S_n, which leads to the formation of redox radical species significantly depends on the number of S atoms.

The order of ability to decrease the total integral intensity of the BMPO-adducts (Figure 4) of H₂S/SeO₃²⁻ > H₂S or Na₂S₄ > H₂S was similar to the order when KO₂ was added to the mixtures of the BMPO/compounds or DEPMPO/compounds [5,13]. This indicates that in the case when KO₂ was added to the mixtures of spin trap/compounds, the compounds affected mostly ^•BMPO-OOH or ^•DEPMPO-OH radicals.

A comparison of the time-dependent ratio of ^•BMPO-OH/^•BMPO-OOH (Figure 5) revealed that the increase of the ^•BMPO-OH spectral component was at the expense of the ^•BMPO-OOH component, supporting the concept of decomposition/transformation of organic hydroperoxide. Interactions of H₂S and polysulfides with radical species are complex [4,7] and more studies are needed to explain the most profound potential of Na₂S₄ > Na₂S₂ or effects of SeO₃²⁻ to decrease the potency of Na₂S₄ to cleave ^•BMPO-OOH.

In conclusion, ^•BMPO-OOH was demonstrated to be a helpful model of organic hydroperoxide. The presented approach of EPR spectra measurement and analysis of the time-dependent ratio of the ^•BMPO-OH/^•BMPO-OOH spin-adducts utilizing the spectra simulation could be useful to study potential of compounds to transform/decompose ^•BMPO-OOH. Using this approach, the impact of sulfide derivatives (Na₂S_n) alone or in the combination with SeO₃²⁻ to transform/decompose ^•BMPO-OOH was detected and compared. Since 10% DMSO (v/v) is used in the studied system, the procedure is useful for evaluating antioxidant potency of compounds insoluble in water.