Effect of Physiological Concentrations of Vitamin C on the Inhibitation of Hydroxyl Radical Induced Light Emission from Fe²⁺-EGTA-H₂O₂ and Fe³⁺-EGTA-H₂O₂ Systems In Vitro

Abstract

Ascorbic acid (AA) has antioxidant properties. However, in the presence of Fe²⁺/Fe³⁺ ions and H₂O₂, it may behave as a pro-oxidant by accelerating and enhancing the formation of hydroxyl radicals (^•OH). Therefore, in this study we evaluated the effect of AA at concentrations of 1 to 200 µmol/L on ^•OH-induced light emission (at a pH of 7.4 and temperature of 37 °C) from 92.6 µmol/L Fe²⁺—185.2 µmol/L EGTA (ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid)—2.6 mmol/L H₂O₂, and 92.6 µmol/L Fe³⁺—185.2 µmol/L EGTA—2.6 mmol/L H₂O₂ systems. Dehydroascorbic acid (DHAA) at the same range of concentrations served as the reference compound. Light emission was measured with multitube luminometer (AutoLumat Plus LB 953) for 120 s after automatic injection of H₂O₂. AA at concentrations of 1 to 50 µmol/L and of 1 to 75 µmol/L completely inhibited light emission from Fe²⁺-EGTA-H₂O₂ and Fe³⁺-EGTA-H₂O₂, respectively. Concentrations of 100 and 200 µmol/L did not affect chemiluminescence of Fe³⁺-EGTA-H₂O₂ but tended to increase light emission from Fe²⁺-EGTA-H₂O₂. DHAA at concentrations of 1 to 100 µmol/L had no effect on chemiluminescence of both systems. These results indicate that AA at physiological concentrations exhibits strong antioxidant activity in the presence of chelated iron and H₂O₂.

1. Introduction

Reactive oxygen species (ROS) are involved in numerous physiological and pathological processes in the human body [1,2]. ROS include various radicals and oxidants and among them the most reactive species are hydroxyl radicals (^•OH) [1]. Iron (Fe²⁺) dependent reduction of H₂O₂ (Fenton chemistry) is the major source of ^•OH radicals [1,3]. However, other transition metals such as copper, manganese, vanadium [4,5], as well as ionizing radiation [6,7] and peroxynitrite [8,9] could also contribute to generation of ^•OH radicals in vivo. The reaction of Fe²⁺ with H₂O₂ initiates numerous radical and non-radical processes, leading to the formation of ^•OH radicals, Fe³⁺, superoxide radicals (O_2•⁻), and singlet oxygen [10,11,12]. O_2•⁻ can reduce Fe³⁺ to Fe²⁺ and this in turn accelerates the formation of ^•OH radicals [10,11]. Moreover, H₂O₂ can directly react with Fe³⁺ with subsequent creation of Fe²⁺, hydroperoxyl radical (HO₂^•), and H⁺ [11]. Thus, one may say that Fe²⁺ ions are regenerated and catalyze the reduction of H₂O₂ into ^•OH radicals. It is well known that chemical compounds which can effectively reduce Fe³⁺ to Fe²⁺ added to Fenton’s reagent (aqueous solution containing Fe²⁺ and H₂O₂) strongly enhanced ^•OH radicals generation. This was noted for hydroxylamine, ascorbic acid (AA), cysteine, 3-hydroxyanthranilic acid [13,14], and plant phenolics such as gallic acid, phloroglucinol, 3,4-dihydroxyphenylacetic acid, and phloretin [15]. Moreover, AA can undergo autooxidation, generating additional amounts of H₂O₂ [16,17]. Application of Fe²⁺-regenerating compounds appears to be a promising approach for Fenton chemistry in order to develop the effective methods of degradation of hazardous pollutants [13,14]. However, in the case of ascorbic acid (AA), this activity seems to be a double-edged sword. This is because H₂O₂ and free Fe²⁺ and Fe³⁺ ions (labile plasma iron) when present in circulating blood [18,19,20] AA may promote the generation of ^•OH radicals and induction of the peroxidative damage to a variety of biomolecules [20]. It should be pointed out that combinations of iron ions with AA and/or H₂O₂ were widely used for the induction of DNA damage in in vitro studies [21,22,23,24]. On the other hand, this mechanism was proposed as one of those responsible for the killing of cancer cells by high AA concentrations [16,17,25]. Recently, we developed a system composed of Fe²⁺, EGTA (ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid) and H₂O₂, which is a source of ^•OH radicals-induced ultra-weak photon emission (UPE) [26]. ^•OH radicals generated in the reaction of Fe²⁺ with H₂O₂ can attack and cleave the ether bond in an EGTA backbone structure. This leads to the formation of products containing triplet excited carbonyl groups and photons emissions [26]. Some studies indicate that the effect of AA on ^•OH generation by Fenton’s reagent may depend on its concentration and time of addition from the onset of reaction of Fe²⁺ with H₂O₂ [13]. Furthermore, AA itself can react with ^•OH radicals [27,28] while making the chemistry aspects of this process more complicated. Therefore, in this study we examined the effect of AA at a wide range of concentrations (including those present in human plasma) on the UPE of an Fe²⁺-EGTA-H₂O₂ system, as well as the UPE of medium containing Fe³⁺-EGTA-H₂O₂. The activity of AA was compared with that revealed by dehydroascorbic acid (DHAA), a relatively stable product of AA oxidation [13].

2. Results

The light emission (UPE-ultra weak photon emission) from 92.6 µmol/L Fe²⁺—185.2 µmol/L EGTA—2.6 mmol/L H₂O₂ system was 2306 ± 910 (2052; 71) RLU (n = 11). The UPE from incomplete control systems Fe²⁺-H₂O₂ and Fe²⁺-EGTA was significantly lower (p < 0.05, n = 11) and reached 1022 ± 295 (958; 339) RLU and 675 ± 111 (678; 137) RLU, respectively. These results are in agreement with those previously described [26], which also showed no light emissions from EGTA-H₂O₂ and H₂O₂ alone in comparison to the medium alone.

2.1. Effect of Ascorbic Acid and Dehydroascorbic Acid on Light Emission from Fe²⁺-EGTA-H₂O₂

The addition of AA (final concentrations of 1, 5, 10, 25, and 50 µmol/L) to the Fe²⁺-EGTA-H₂O₂ system completely abolished light emission (Figure 1A). The percent inhibition of UPE for concentrations of AA of 1, 5, 10, 25, and 50 µmol/L reached 102.3 ± 5.9 (99.7; 4.8), 101.7 ± 4.6 (101.7; 5.6), 100.2 ± 5.1 (100.0; 5.3), 100.7 ± 3.1 (99.3; 2.8), and 96.6 ± 2.4 (96.9; 1.0) (p < 0.05), respectively. The inhibition percentage slightly higher than 100 was most probably due to fluctuation of the baseline signal from medium alone. Higher concentrations of AA did not suppress the UPE of Fe²⁺-EGTA-H₂O₂ (Figure 1A). Even at concentrations of 100 µmol/L and 200 µmol/L, a moderate tendency (but not significant) of the enhancement of light emission was noted (Figure 1A). Control experiments showed no effect of AA (final concentrations of 1 and 200 µmol/L) on the UPE of incomplete systems (Fe²⁺-H₂O₂ and Fe²⁺-EGTA-H₂O) and medium alone (Figure 1B). In these cases, the light emissions did not differ from the signal noted for medium alone.

All tested concentrations of DHAA had no effect on the UPE of the Fe²⁺-EGTA-H₂O₂ system (Figure 2A), while the DHAA at concentrations of 1 and 200 µmol/L did not alter the light emission from the control systems, Fe²⁺-H₂O₂, Fe²⁺-EGTA-H₂O, or the medium alone (Figure 2B).

2.2. Effect of Ascorbic Acid and Dehydroascorbic Acid on Light Emission from Fe³⁺-EGTA-H₂O₂

The Fe³⁺-EGTA-H₂O₂ system was a weaker light emitter than the Fe²⁺-EGTA-H₂O₂ system [1531 ± 292, (1493; 443) RLU, n = 8 vs. 2306 ± 910 (2052; 71) RLU, n = 11, p < 0.05]. Addition of AA to the Fe³⁺-EGTA-H₂O₂ system to final concentrations of 1, 5, 10, 25, 50, and 75 µmol/L completely abolished the UPE (Figure 3A) and the percent inhibition of the UPE was 103.1 ± 5.8 (105.1; 3.9), 92.5 ± 22.0 (102.3; 13.2), 102.4 ± 1.7 (102.8; 2.2), 106.5 ± 2.7 (105.3; 1.6), 106.3 ± 3.5 (106.5; 3.2), and 100.5 ± 3.6 (101.4; 4.0), respectively. AA at concentrations of 100 and 200 µmol/L did not significantly change the UPE of the Fe³⁺-EGTA-H₂O₂ system (p > 0.05, Figure 3A). AA at concentrations of 1 and 200 µmol/L decreased the light emission from the Fe³⁺-H₂O₂ (p < 0.05) but had no effect on Fe³⁺-EGTA-H₂O and medium alone (Figure 3B).

DHAA at concentrations of 1, 5, 10, 25, and 50 µmol/L had no effect on the UPE of the Fe³⁺-EGTA-H₂O₂ system (Figure 4A). For the concentrations of 75 and 100 µmol/L, a slight tendency to increase the light emission was noted, while at the DHAA concentration of 200 µmol/L, a tremendous increase in mean UPE (almost 70-times) was found (Figure 4A). DHAA at concentrations of 1 and 200 µmol/L also increased the light emission from Fe³⁺-H₂O₂ (p < 0.05) but had no effect on other controls (Fe³⁺-EGTA or medium alone) (Figure 4B).

3. Discussion

Numerous reactions can occur simultaneously in an Fe²⁺-EGTA-H₂O₂ system [12,13,14,29,30]. Those leading to the generation of ^•OH radicals, superoxide radicals (O_2•⁻), hydroperoxyl radicals (HO₂^•), singlet oxygen (O₂(¹∆g)), and the reduction of Fe³⁺ to Fe²⁺ are presented below:

Fe²⁺-EGTA + H₂O₂ → Fe³⁺-EGTA + OH⁻ + ^•OH (formation of hydroxyl radicals)

(1)

Fe³⁺-EGTA + H₂O₂ → Fe³⁺OOH⁻-EGTA + H⁺

(2)

Fe³⁺OOH⁻-EGTA + H₂O₂ → FeO²⁺-EGTA + HO₂^• + H₂O

(3)

FeO²⁺-EGTA + H₂O₂ → Fe³⁺-EGTA + HO₂^• + OH⁻

(4)

HO₂^• → H⁺ + O_2•⁻ (formation of superoxide radicals)

(5)

Fe³⁺-EGTA + O_2•⁻ → Fe²⁺-EGTA + O₂

(6)

Fe³⁺-EGTA + H₂O₂ → Fe²⁺-EGTA + HO₂^•

(7)

Fe³⁺-EGTA + HO₂^• → Fe²⁺-EGTA + O₂ + H⁺.

(8)

Hydroperoxyl radicals (HO₂^•) are generated in reactions (3), (4), and (7).

Reduced iron formed in reactions (6), (7), and (8) can enter reaction 1 to enhance the generation of hydroxyl radicals

O_2•⁻ + ^•OH + H⁺ → H₂O₂ + O₂(¹∆g) formation of singlet oxygen

(9)

2O_2•⁻ + 2H⁺ → H₂O₂ + O₂(¹∆g) formation of singlet oxygen.

(10)

Hydroxyl radicals generated in an Fe²⁺-EGTA-H₂O₂ system can cleave one of the ether bonds in the backbone structure of EGTA, leading to formation of products with a triplet excited carbonyl group responsible for light emission [26]. O_2•⁻ radicals can reduce Fe³⁺ into Fe²⁺ ions, which again react with H₂O₂ and generate ^•OH radicals. Therefore, the UPE of the Fe²⁺-EGTA-H₂O₂ system was strongly inhibited by scavengers of ^•OH radicals (dimethylsulfoxide and mannitol) and partially by superoxide dismutase, which very rapidly catalyzes the dismutation of O_2•⁻ radicals into O₂ and H₂O₂ [26]. The rates of reactions (7) and (8) are much slower than that of the reaction (1) [13,14]. Therefore, the UPE of Fe²⁺-EGTA-H₂O₂ was higher than the light emissions from Fe³⁺-EGTA-H₂O₂. In both Fe²⁺-EGTA-H₂O₂ and Fe³⁺-EGTA-H₂O₂ systems, the iron concentration was 28-times lower than the concentration of H₂O₂. Therefore, one may expect that the addition of reducing agent to both systems would increase the light emissions via the regeneration of Fe²⁺ ions and enhanced ^•OH radicals formation.

3.1. Effect of Ascorbic Acid and Dehydroascorbic Acid on the Light Emission from Fe²⁺-EGTA-H₂O₂

AA is a powerful reducer of Fe³⁺ ions [13,14]. Its ability to reduce Fe³⁺ ions into Fe²⁺ ions is stronger than that of uric acid, bilirubin, Trolox, and numerous plant phenolics such as ferulic acid, catechin, gallic acid, and quercetin [31,32]. Therefore, we expected that the addition of ascorbic acid to an Fe²⁺-EGTA-H₂O₂ system would enhance its UPE. Surprisingly, AA at concentrations of 1 to 50 µmol/L completely abolished the light emissions. Only higher AA concentrations of 100 and 200 µmol/L tended to increase the UPE of Fe²⁺-EGTA-H₂O₂ but this effect was not significant. AA can react with various reactive oxygen species such as H₂O₂, O_2•⁻, O₂(¹∆g), and especially ^•OH radicals [33,34,35]. In our previous experiments, sodium azide as a scavenger of O₂(¹∆g) did not suppress the light emission from Fe²⁺-EGTA-H₂O₂ and the contribution of O2_•⁻ to this phenomenon was relatively low [26]. Moreover, the concentration of H₂O₂ was many times higher than that of AA, Fe²⁺, and EGTA in the Fe²⁺-EGTA-H₂O₂ system. Therefore, the plausible reaction of AA with H₂O₂ and O_2•⁻ seems to not be responsible for the quenching of the UPE. Thus, the reaction of AA with ^•OH radicals could have a crucial effect on the UPE of the Fe²⁺-EGTA-H₂O₂ system. The reaction of AA with ^•OH radical leads to the formation of ascorbate radical and H₂O. At a physiological pH, the reaction of disproportionation of two molecules of ascorbate radicals is thermodynamically favored. This is a complex process and involves dimerization of an ascorbate radical, internal electron transfer, and hydrolysis of temporal dimer, and results in the formation of one molecule of DHAA and AA [35,36] which can again react with an ^•OH radical. The rate of the reaction of AA with Fe³⁺, which promotes ^•OH radicals generation, was affected by the pH of the solution and at the pH higher than 6, the rate was slow [37]. Therefore, under conditions of our experiments (pH = 7.4), the reaction of AA with ^•OH radicals dominates and protects molecules of EGTA from oxidative attack and generation of end-products, with triplet excited carbonyl groups responsible for light emission. These may explain the strong inhibitory effect of AA at concentrations of 1 to 50 µmol/L on the UPE of Fe²⁺-EGTA-H₂O₂. However, AA at higher concentrations of 75 to 200 µmol/L did not inhibit the UPE of Fe²⁺-EGTA-H₂O₂. This suggests that under those conditions, there is a relative balance between ^•OH radicals generation and their scavenging by AA and thus the activity of ^•OH radicals is similar in Fe²⁺-EGTA-H₂O₂ with and without high concentrations of AA. AA has chelating activity and was reported to form complexes with Fe²⁺ and Fe³⁺ ions [33,38]. Moreover, the formation of AA-Fe³⁺ complexes is necessary for AA- induced reduction of Fe³⁺ to Fe²⁺ [37,38]. EGTA is a chelating agent which complexes Fe²⁺ and Fe³⁺ ions [39]. In experiments with concentrations of AA of 75 to 200 µmol/L (close to concentration of EGTA of 185.2 µmol/L), there is a substantial possibility of formation of AA-Fe³⁺ complexes. Moreover, it cannot be excluded that under these conditions, mixed chelate complexes of EGTA-AA-Fe³⁺ could be formed. This is supported by the description of Fe³⁺- deferiprone-AA complexes (deferiprone is an iron chelator indicated for the treatment of iron overload) in medium of pH = 7.4 in vitro [33]. Thus, at concentrations of AA of 75 to 200 µmol/L, considerable augmentation of Fe²⁺ ions regeneration can occur. Therefore, the intensities of two reactions: AA- induced scavenging of ^•OH radicals and AA- induced Fe²⁺ regeneration, are comparable and these explain why higher AA concentrations did not inhibit the UPE of the Fe²⁺-EGTA-H₂O₂ system. On the other hand, low concentrations of AA (1 to 50 µmol/L) could not form sufficient amounts of redox active complexes with Fe³⁺ due to an excess of EGTA. These outcomes additionally explain the strong inhibitory effect of low AA concentrations on light emission from the Fe²⁺-EGTA-H₂O₂ system. Figure 5 summarizes the mechanism of inhibitory effect of AA (concentrations of 1 to 50 µmol/L) on the ^•OH radicals-induced UPE of the Fe²⁺-EGTA-H₂O₂ system. Although DHAA can react with H₂O₂ and ^•OH radicals [40], no effect of DHAA on UPE of Fe²⁺-EGTA-H₂O₂ was noted. DHAA is the product of two-electron oxidation of AA [36]. Therefore, it is a much weaker electron donor than AA and the involvement of DHAA in redox reactions after an addition to Fe²⁺-EGTA-H₂O₂ was many times lower than that in the case of AA. Hence, DHA did not alter the light emission from the Fe²⁺-EGTA-H₂O₂ system.

3.2. Effect of Ascorbic Acid and Dehydroascorbic Acid on the Light Emission from Fe³⁺-EGTA-H₂O₂

As was stated before, the Fe³⁺-EGTA-H₂O₂ system was a weaker light emitter than the Fe²⁺-EGTA-H₂O₂ one. ^•OH radicals initiating the light emission from Fe³⁺-EGTA-H₂O₂ system are formed in the reaction (1), which occurs as a result of the reaction (7). AA at concentrations of 1 to 75 µmol/L inhibited the UPE of the Fe³⁺-EGTA-H₂O₂ system through direct scavenging of ^•OH radicals. Due to a medium pH of 7.4 and excess of EGTA, these low concentrations of AA could not effectively reduce Fe³⁺ ions to Fe²⁺ ions, therefore the inhibition of light emission was complete. However, at higher concentrations of AA (100 and 200 µmol/L), the process of Fe²⁺ ions formation was enhanced and resulted in higher generation of ^•OH radicals. Thus, AA at concentrations of 100 and 200 µmol/L did not alter the light emission from the Fe³⁺ EGTA-H₂O₂ system due to a dynamic balance between ^•OH radicals scavenging and the promotion of ^•OH radicals generation caused by this vitamin. Because DHAA is a weaker electron donor than AA [36], this compound at concentrations of 1 to 100 µmol/L had no significant effect on the UPE of the Fe³⁺-H₂O₂-EGTA system. However, the concentration of DHAA of 200 µmol/L tremendously (by about 70-times) increased photons emission from the Fe³⁺-H₂O₂-EGTA system. DHAA was reported to react with H₂O₂ through the formation of 4-O-oxalyl-threonate and 3-O-oxalyl-threonate as the main products, small amounts of cyclic oxalyl-threonate, 2-keto-L-xylonate, and threonic acid, and trace amounts of oxalic acid while oxidation of DHAA by ^•OH radicals generated by Fenton’s reagent (Fe²⁺-EDTA-H₂O₂) produced mainly oxalic acid and both isomers of oxalyl threonate and small amounts of threonic acid [40]. Because DHAA at a concentration of 200 µmol/L had no effect on light emission from Fe²⁺-EGTA-H₂O₂ and Fe²⁺-H₂O₂ as well as Fe³⁺-EGTA-H₂O₂ generated substantially less ^•OH radicals than Fe²⁺-EGTA-H₂O₂, one may conclude that reactions leading to formation of cyclic oxalyl-threonate and 2-keto-L-xylonate may be involved in very strong augmentation of the UPE of the Fe³⁺-H₂O₂-EGTA system. DHAA also augmented the light emission from Fe³⁺-H₂O₂ by about 2.5-times, having no effect on this process in medium containing Fe³⁺ and EGTA. This suggests that EGTA is not necessary for moderate augmentation of UPE by DHAA oxidized in the presence of Fe³⁺ and H₂O₂. On the other hand, the combination of EGTA or its derivatives formed after ^•OH- induced oxidative attack with cyclic oxalyl-threonate or 2-keto-L-xylonate may result in chemical reactions which efficiently generate light and strongly augment the UPE of the Fe³⁺-H₂O₂-EGTA system. However, confirmation of these hypothetical mechanisms requires further studies.

3.3. Relevance to Human Physiology

It is believed that plasma concentrations of H₂O₂ range from 1 to 5 µmol/L in healthy subjects. However, in the course of certain diseases, the levels of H₂O₂ in plasma can increase up to 50 µmol/L [18]. The plasma concentration of iron complexed with low molecular weight compounds is about 1 µmol/L in healthy subjects while in patients with hemochromatosis, this can reach 10 µmol/L [41]. The median concentrations of AA and DHAA in plasma of healthy subjects were around 61.4 µmol/L and 2.3 µmol/L [42], however in critically ill patients (sepsis, major-organ failure, severe accidental injury), they decreased to 9.0 µmol/L and 1.4 µmol/L, respectively [42]. Therefore, the studied concentrations of AA and DHAA included the concentration ranges which can occur in healthy subjects and those with a strong inflammatory response. Because oxygen pressure in arterial blood ranges from 75 mmHg to 100 mmHg in healthy subjects [43] and O₂ is involved in final reactions, leading to the formation of a product with triplet excited carbonyl groups [26], we did not use deaerated solutions in our experiments. In addition, AA was stable in undeaerated phosphate buffers of pH = 7.2 and 7.8 for at least 50 min [44]. Thus, unspecific decompositions of AA could not have had any influence on the results of our experiments.

The most important finding was that AA at concentrations of 5 to 50 µmol/L which can occur in human plasma suppressed the ^•OH radicals-induced light emission from both systems: Fe²⁺-EGTA-H₂O₂ and Fe³⁺-EGTA-H₂O₂. Moreover, the concentration of AA of 75 µmol/L inhibited the UPE of the Fe³⁺-EGTA-H₂O₂ but had no significant effect on that of Fe²⁺-EGTA-H₂O₂.These suggest that under physiological conditions, the antioxidant activity (scavenging of ^•OH radicals) of AA prevails over its plausible pro-oxidant activity related to the reduction of Fe³⁺ to Fe²⁺ ions. It should be pointed out that even higher concentrations of AA of 100 and 200 µmol/L did not significantly alter the UPE of Fe²⁺-EGTA-H₂O₂ and Fe³⁺-EGTA-H₂O₂ systems. However, circulating blood plasma containing H₂O₂ and iron complexed with low molecular weight chelating compounds is much more complex medium than our in vitro model. Recent clinical studies showing that intravenous administration of AA in a single dose of 750 mg or 7500 mg for six days did not increase oxidative stress markers (plasma concentrations of thiobarbituric acid reactive substances and urinary 8-oxoguanosine) in healthy subjects [45] support our observations.

Circulating blood has a pH of around 7.4 and a temperature of 37 °C. However, locally at the place of inflammation and also in certain solid tumors, the tissue environment could be acidic with a pH ranging from 5.7 to 7.0 [46]. This may predispose towards the reduction of Fe³⁺ to Fe²⁺ by AA and the enhanced generation of ^•OH radicals. Therefore, pro-oxidant activity of ascorbate cannot be excluded under such circumstances.

3.4. Limitations of the Study

The UPE was measured with a luminometer equipped with a photon counter sensitive to photons emitted in the 380 nm–630 nm range. We proposed a mechanism of light emission by Fe²⁺-EGTA-H₂O₂ which involves an ^•OH- induced cleavage of the ether bond in the backbone chain of EGTA molecule, its further degradation and formation of another radical, and triplet excited carbonyl groups [26]. Triplet excited carbonyl groups emit photons with a spectral range of 350 nm to 550 nm [47]. The human body spontaneously emits light, mostly within the wavelength range of 420 nm to 570 nm [48]. This suggests the occurrence of other sources of UPE in body fluids than triplet excited carbonyl groups. Therefore, a lack of spectral analysis of the UPE of Fe²⁺-EGTA-H₂O₂ and Fe³⁺-EGTA-H₂O₂ with and without studied compounds could be recognized as the limitation of our study. Unfortunately, there were no technical capabilities to use any cut-off filters for spectral analysis in AutoLumat Plus LB 953. Spectral analysis of the UPE would be especially helpful for an explanation of 200 µmol/L DHAA-induced enhancement of light emission form Fe³⁺-EGTA-H₂O₂. If the emission spectra of Fe³⁺-EGTA-H₂O₂ and Fe³⁺-EGTA-DHAA-H₂O₂ would be similar, one may conclude that this enhancement of the UPE is the consequence of increased formation of triplet excited carbonyl groups. However, from the physiological and clinical point of view, this is not important because concentrations of DHAA of 200 µmol/L could not occur in human plasma, even after intravenous administration of high doses of AA [45]. On the other hand, it cannot be excluded that strong light emission from Fe³⁺-EGTA-DHAA-H₂O₂ may be used for the determination of anti-oxidant properties of other compounds. Therefore, further experiments to elucidate the mechanism of DHAA-induced augmentation of the UPE of Fe³⁺-EGTA-H₂O₂ are worth conducting.

4. Materials and Methods

4.1. Reagents

All chemicals were of analytical grade. Iron (II) sulfate heptahydrate (FeSO₄ × 7H₂O), iron (III) chloride hexahydrate (FeCl₃ × 6H₂O), sodium L-ascorbate (AA), L-dehydroascorbic acid (DHAA), and ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) were purchased from Sigma-Aldrich Chemicals (St. Louis, MO, USA.) H₂O₂ 30% solution (w/w) was from Chempur (Piekary Slaskie, Poland). Sterile phosphate buffered saline (PBS, pH 7.4, without Ca²⁺ and Mg²⁺) was obtained from Biomed (Lublin, Poland). Sterile deionized pyrogen-free water (freshly prepared, resistance >18 MW/cm, HPLC H₂O Purification System, USF Elga, Buckinghamshire, UK) was used throughout the study. Working aqueous solutions of 5 mmol/L of FeSO₄ and 5 mmol/L of FeCl₃ were prepared before the assay. A working solution of 28 mmol/L of H₂O₂ was also prepared before the assay by dilution of 30% of H₂O₂ solution and the concentration was confirmed by the measurement of absorbance at 240 nm using a molar extinction coefficient of 43.6 mol⁻¹cm⁻¹ [49]. A stock solution of EGTA (100 mmol/L) was prepared in PBS with pH adjusted to 8.0 with 5 mol/L of NaOH and was stored at room temperature in the dark for no longer than 3 months. Ten mmol/L of EGTA working solution was obtained by appropriate dilution of EGTA stock solution with water before the assay. AA and DHAA solutions in PBS (7.2 mmol/L) and their 2-, 2.7-, 4-, 8-, 20-, 40-, and 200-times dilutions were prepared freshly before the assay.

4.2. System Generating Light and Measurement of Light Emission

For light generation, we used 92.6 µmol/L Fe²⁺—185.2 µmol/L EGTA—2.6 mmol/L H₂O₂ system, as previously described [26]. This system generates the UPE, which depends mainly on ^•OH radicals. ^•OH generated in the course of reaction of Fe²⁺ with H₂O₂ can oxidatively attack and cleave ether bonds in EGTA molecule, which leads to formation of triplet excited carbonyl groups and light emission [26]. Briefly, 20 µL of 10 mmol/L EGTA solution was added to the tube (Lumi Vial Tube, 5 mL, 12 × 75 mm, Berthold Technologies, Bad Wildbad, Germany) containing 940 µL of PBS. Afterwards, 20 µL of 5 mmol/L solution of FeSO₄ was added and after gentle mixing, the tube was placed in the luminometer chain and incubated for 10 min in the dark at 37 °C. Then, 100 µL of 28 mmol H₂O₂ solution was added by an automatic dispenser and the total light emission (expressed in RLU—relative light units) was measured for 120 s with a multitube luminometer (AutoLumat Plus LB 953, Berthold, Germany) equipped with a Peltier-cooled photon counter (spectral range from 380 to 630 nm) to ensure high sensitivity and low and stable background noise signals.

4.3. Effect of Ascorbic Acid and Dehydroascorbic Acid on Light Emission from Fe²⁺-EGTA-H₂O₂ System

In order to determine the effect of AA on the UPE of 92.6 µmol/L Fe²⁺—185.2 µmol/L EGTA—2.6 mmol/L H₂O₂ system, 30 µL of working solution of AA in PBS or its appropriate dilutions were added to the luminometer tube containing EGTA and FeSO₄ in PBS and incubated for 10 min at 37 °C in the dark and then 100 µL of H₂O₂ solution was injected and the total light emission was measured for 2 min. The final concentrations of AA in the reaction mixture were 1, 5, 10, 25, 50, 75, 100, and 200 µmol/L, respectively. Controls included: Fe²⁺-EGTA-H₂O₂ in PBS without AA, incomplete system Fe²⁺-H₂O₂ with and without AA, Fe²⁺-EGTA with and without AA, AA alone in PBS, and medium alone. The final concentrations of AA in the controls were 1 and 200 µmol/L. The same procedures were executed when the effect of DHAA on the UPE of 92.6 µmol/L Fe²⁺—185.2 µmol/L EGTA—2.6 mmol/L H₂O₂ was studied. The design of these experiments is shown in

Table 1. Design of experiments on the effect of ascorbic acid and dehydroascorbic acid on light emissions from the Fe²⁺-EGTA-H₂O₂ system.

No.	Sample	Volumes of Working Solutions Added to Luminometer Tube [µL]
		A	B	C	D	E	F	G
		PBS	EGTA	FeSO4	AA	DHAA	H2O2	H2O
1	Complete system	940	20	20	-	-	100	-
2	Complete system + AA	910	20	20	30	-	100	-
3	Complete system + DHAA	910	20	20	-	30	100	-
4	Incomplete system	960	-	20	-	-	100	-
5 *	Incomplete system + AA	930	-	20	30	-	100
6 *	Incomplete system + DHAA	930	-	20	-	30	100	-
Additional controls
7	Fe2+-EGTA without H2O2	940	20	20	-	-	-	100
8 *	Fe2+-EGTA without H2O2 +AA	910	20	20	30	-	-	100
9 *	Fe2+-EGTA without H2O2 +DHAA	910	20	20	-	30	-	100
10 *	AA alone	950	-	-	30	-	-	100
11 *	DHAA alone	950	-	-	-	30	-	100
12	Medium alone	980	-	-	-	-	-	100

Working solutions were added to the luminometer tube in alphabetical order: A—sterile phosphate buffered saline (PBS) (pH = 7.4) without divalent cations; B—10 mmol/L aqueous solution of EGTA: C—5 mmol/L aqueous solution of FeSO₄; D—solution of AA in PBS (final concentration of AA in the reaction mixture ranged from 1 to 200 µmol/L, eight concentrations); E—solution of DHAA in PBS (final concentration of DHAA in the reaction mixture ranged from 1 to 200 µmol/L). Then after gentle mixing the tube was placed into luminometer chain, incubated for 10 min at 37 °C, and then 28 mmol/L H₂O₂ (F) or water (G) was automatically injected by dispenser and the total light emission was measured for 2 min. *—in control experiments, two concentrations (1 and 200 µmol/L) of AA or DHA were tested.

. In each series of experiments (repeated at least 4 times), eight concentrations of AA or DHAA were tested. The inhibitory effect of AA or DHAA on the light emission was expressed as a percent inhibition (%I) calculated according to the formula: %I = [(A − B)/(A − C)] × 100% where A, B, and C are the total light emission from Fe²⁺-EGTA-H₂O₂, Fe²⁺-EGTA-studied compound (AA or DHAA)-H₂O₂, and medium (H₂O injected into PBS), respectively. In the case of augmentation of the UPE, the percent enhancement (%E) was calculated as follows: %E = [(B − A)/(A − C)] × 100%. In additional experiments, the effect of AA and DHAA on light emission from 92.6 µmol/L Fe³⁺—185.2 µmol/L EGTA—2.6 mmol/L H₂O₂ was examined. The design of these experiments was the same as in Table 1, except for the addition of 20 µL of working solution of FeCl₃ instead of 20 µL of working solution of Fe₂SO₄.

4.4. Statistical Analysis

Results (total light emission, % inhibition or % enhancement of light emission) were expressed as mean (standard deviation) and median and interquartile range (IQR). The comparisons between the UPE of the Fe²⁺-EGTA-H₂O₂ system and the light emission from corresponding samples of a modified system (e.g., an incomplete system, system with the addition of AA or DHAA, Fe³⁺-EGTA-H₂O₂ with and without addition of AA or DHAA, and medium alone) were analyzed with the independent-samples (unpaired) t-test or Mann-Whitney U test depending on the data distribution, which was tested with the Kolmogorov–Smirnov–Liliefors test. The Brown–Forsythe test for analysis of the equality of the group variances was used prior to the application of the unpaired t-test and if variances were unequal, then the Welch’s t-test was used instead of the standard t-test. The comparisons of % inhibition or % enhancement of light emission caused by AA and DHAA were analyzed in the same way. A p-value < 0.05 was considered significant.

5. Conclusions

Ascorbic acid within the concentration range of 1 to 50 µmol/L very effectively inhibited ^•OH-induced light emission from Fe²⁺-EGTA-H₂O₂ and Fe³⁺-EGTA-H₂O₂ systems in vitro. Higher concentrations of 75 to 200 µmol/L did not significantly enhance the UPE of both modified Fenton systems. Because studied concentrations of AA involved those present in human plasma, one may conclude that AA can act as an antioxidant in the presence of iron complexed with low molecular weight compounds in circulating blood. Dehydroascorbic acid within the range of physiological concentrations of 1 to 5 µmol/L had no effect on the intensity of ^•OH- induced reaction, resulting in the light emission. Although these results were obtained from in vitro experiments, they strongly suggest the low risk of pro-oxidant activity of AA in healthy subjects.