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

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.

All tested concentrations of DHAA had no effect on the UPE of the Fe 2+ -EGTA-H 2 O 2 system (Figure 2A), while the DHAA at concentrations of 1 and 200 µmol/L did not alter the light emission from the control systems, Fe 2+ -H 2 O 2 , Fe 2+ -EGTA-H 2 O, or the medium alone ( Figure 2B).
DHAA at concentrations of 1, 5, 10, 25, and 50 µmol/L had no effect on the UPE of the Fe 3+ -EGTA-H 2 O 2 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 Fe 3+ -H 2 O 2 (p < 0.05) but had no effect on other controls (Fe 3+ -EGTA or medium alone) ( Figure 4B). olecules 2021, 26, x FOR PEER REVIEW 3 of 15 no effect of AA (final concentrations of 1 and 200 µmol/L) on the UPE of incomplete systems (Fe 2+ -H2O2 and Fe 2+ -EGTA -H2O) and medium alone ( Figure 1B). In these cases, the light emissions did not differ from the signal noted for medium alone.  p < 0.05. EGTA-ethylene glycol-bis (β-amino ethyl ether)-N,N,N′,N′-tetra acetic acid, RLU-relative light units.

Discussion
Numerous reactions can occur simultaneously in an Fe 2+ -EGTA-H 2 O 2 system [12][13][14]29,30]. Those leading to the generation of • OH radicals, superoxide radicals (O 2• − ), hydroperoxyl radicals (HO 2 • ), singlet oxygen (O 2 ( 1 ∆g)), and the reduction of Fe 3+ to Fe 2+ are presented below: Hydroperoxyl radicals (HO 2 • ) 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 Hydroxyl radicals generated in an Fe 2+ -EGTA-H 2 O 2 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 Fe 3+ into Fe 2+ ions, which again react with H 2 O 2 and generate • OH radicals. Therefore, the UPE of the Fe 2+ -EGTA-H 2 O 2 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 O 2 and H 2 O 2 [26]. The rates of reactions (7) and (8) are much slower than that of the reaction (1) [13,14]. Therefore, the UPE of Fe 2+ -EGTA-H 2 O 2 was higher than the light emissions from Fe 3+ -EGTA-H 2 O 2 . In both Fe 2+ -EGTA-H 2 O 2 and Fe 3+ -EGTA-H 2 O 2 systems, the iron concentration was 28-times lower than the concentration of H 2 O 2 . Therefore, one may expect that the addition of reducing agent to both systems would increase the light emissions via the regeneration of Fe 2+ ions and enhanced • OH radicals formation.

Effect of Ascorbic Acid and Dehydroascorbic Acid on the Light Emission from
AA is a powerful reducer of Fe 3+ ions [13,14]. Its ability to reduce Fe 3+ ions into Fe 2+ 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 Fe 2+ -EGTA-H 2 O 2 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 Fe 2+ -EGTA-H 2 O 2 but this effect was not significant. AA can react with various reactive oxygen species such as H 2 O 2 , O 2• − , O 2 ( 1 ∆g), and especially • OH radicals [33][34][35]. In our previous experiments, sodium azide as a scavenger of O 2 ( 1 ∆g) did not suppress the light emission from Fe 2+ -EGTA-H 2 O 2 and the contribution of O2 • − to this phenomenon was relatively low [26]. Moreover, the concentration of H 2 O 2 was many times higher than that of AA, Fe 2+ , and EGTA in the Fe 2+ -EGTA-H 2 O 2 system. Therefore, the plausible reaction of AA with H 2 O 2 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 Fe 2+ -EGTA-H 2 O 2 system. The reaction of AA with • OH radical leads to the formation of ascorbate radical and H 2 O. 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 Fe 3+ , 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 Fe 2+ -EGTA-H 2 O 2 . However, AA at higher concentrations of 75 to 200 µmol/L did not inhibit the UPE of Fe 2+ -EGTA-H 2 O 2 . 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 Fe 2+ -EGTA-H 2 O 2 with and without high concentrations of AA. AA has chelating activity and was reported to form complexes with Fe 2+ and Fe 3+ ions [33,38]. Moreover, the formation of AA-Fe 3+ complexes is necessary for AA-induced reduction of Fe 3+ to Fe 2+ [37,38]. EGTA is a chelating agent which complexes Fe 2+ and Fe 3+ 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-Fe 3+ complexes. Moreover, it cannot be excluded that under these conditions, mixed chelate complexes of EGTA-AA-Fe 3+ could be formed. This is supported by the description of Fe 3+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 Fe 2+ ions regeneration can occur. Therefore, the intensities of two reactions: AA-induced scavenging of • OH radicals and AA-induced Fe 2+ regeneration, are comparable and these explain why higher AA concentrations did not inhibit the UPE of the Fe 2+ -EGTA-H 2 O 2 system. On the other hand, low concentrations of AA (1 to 50 µmol/L) could not form sufficient amounts of redox active complexes with Fe 3+ due to an excess of EGTA. These outcomes additionally explain the strong inhibitory effect of low AA concentrations on light emission from the Fe 2+ -EGTA-H 2 O 2 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 Fe 2+ -EGTA-H 2 O 2 system. Although DHAA can react with H 2 O 2 and • OH radicals [40], no effect of DHAA on UPE of Fe 2+ -EGTA-H 2 O 2 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 Fe 2+ -EGTA-H 2 O 2 was many times lower than that in the case of AA. Hence, DHA did not alter the light emission from the Fe 2+ -EGTA-H 2 O 2 system. s 2021, 26, x FOR PEER REVIEW 9 of 15 Figure 5. Postulated mechanism of inhibitory effect of ascorbic acid (AA) on the • OH radical-induced ultraweak photon emission (UPE) of the Fe 2+ -EGTA-H2O2 system in medium of pH = 7.4. • OH radicals generated in the reaction of Fe 2+ with H2O2 (1) attack one of the two ether bonds in the backbone structure of EGTA, leading to its cleavage and formation of other radicals that results in the creation of one product with a triplet excited carbonyl group (R-CH = O *). Electronic transitions from the triplet excited state to the ground state are accompanied by the photon emission (λν). AA can effectively react with • OH radicals (2) through the formation of an ascorbate radical (AA • ). This protects the ether bonds of EGTA from oxidative attack and completely inhibits light emission from the Fe 2+ -EGTA-H2O2 system. Two molecules of AA • undergo disproportionation reaction (3) with the formation of one molecule of dehydroascorbic acid (DHAA) and one of AA, which again can react with • OH radicals. AA can reduce Fe 3+ to Fe 2+ (4) and therefore, enhances • OH radicals generation. However, under conditions of pH = 7.4 and excess of EGTA as chelating agent, this process has low intensity. This pathway of Fe 2+ regeneration is enhanced for higher concentrations of AA (75 to 200 µmol/L) and therefore, they do not inhibit the UPE of the Fe 2+ -EGTA-H2O2 system. For more details, please refer to [26,35,36].

Effect of Ascorbic Acid and Dehydroascorbic Acid on the Light Emission from Fe 3+ -EGTA-H2O2
As was stated before, the Fe 3+ -EGTA-H2O2 system was a weaker light emitter than the Fe 2+ -EGTA-H2O2 one. • OH radicals initiating the light emission from Fe 3+ -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 Fe 3+ -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 Fe 3+ ions to Fe 2+ ions, therefore the  (3) with the formation of one molecule of dehydroascorbic acid (DHAA) and one of AA, which again can react with • OH radicals. AA can reduce Fe 3+ to Fe 2+ (4) and therefore, enhances • OH radicals generation. However, under conditions of pH = 7.4 and excess of EGTA as chelating agent, this process has low intensity. This pathway of Fe 2+ regeneration is enhanced for higher concentrations of AA (75 to 200 µmol/L) and therefore, they do not inhibit the UPE of the Fe 2+ -EGTA-H 2 O 2 system. For more details, please refer to [26,35,36].

Effect of Ascorbic Acid and Dehydroascorbic Acid on the Light Emission from Fe 3+ -EGTA-H 2 O 2
As was stated before, the Fe 3+ -EGTA-H 2 O 2 system was a weaker light emitter than the Fe 2+ -EGTA-H 2 O 2 one. • OH radicals initiating the light emission from Fe 3+ -EGTA-H 2 O 2 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 Fe 3+ -EGTA-H 2 O 2 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 Fe 3+ ions to Fe 2+ ions, therefore the inhibition of light emission was complete. However, at higher concentrations of AA (100 and 200 µmol/L), the process of Fe 2+ 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 Fe 3+ EGTA-H 2 O 2 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 Fe 3+ -H 2 O 2 -EGTA system. However, the concentration of DHAA of 200 µmol/L tremendously (by about 70-times) increased photons emission from the Fe 3+ -H 2 O 2 -EGTA system. DHAA was reported to react with H 2 O 2 through the formation of 4-O-oxalyl-threonate and 3-O-oxalylthreonate 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 (Fe 2+ -EDTA-H 2 O 2 ) 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 Fe 2+ -EGTA-H 2 O 2 and Fe 2+ -H 2 O 2 as well as Fe 3+ -EGTA-H 2 O 2 generated substantially less • OH radicals than Fe 2+ -EGTA-H 2 O 2 , one may conclude that reactions leading to formation of cyclic oxalylthreonate and 2-keto-L-xylonate may be involved in very strong augmentation of the UPE of the Fe 3+ -H 2 O 2 -EGTA system. DHAA also augmented the light emission from Fe 3+ -H 2 O 2 by about 2.5-times, having no effect on this process in medium containing Fe 3+ and EGTA. This suggests that EGTA is not necessary for moderate augmentation of UPE by DHAA oxidized in the presence of Fe 3+ and H 2 O 2 . 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 Fe 3+ -H 2 O 2 -EGTA system. However, confirmation of these hypothetical mechanisms requires further studies.

Relevance to Human Physiology
It is believed that plasma concentrations of H 2 O 2 range from 1 to 5 µmol/L in healthy subjects. However, in the course of certain diseases, the levels of H 2 O 2 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 O 2 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: Fe 2+ -EGTA-H 2 O 2 and Fe 3+ -EGTA-H 2 O 2 . Moreover, the concentration of AA of 75 µmol/L inhibited the UPE of the Fe 3+ -EGTA-H 2 O 2 but had no significant effect on that of Fe 2+ -EGTA-H 2 O 2 .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 Fe 3+ to Fe 2+ 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 Fe 2+ -EGTA-H 2 O 2 and Fe 3+ -EGTA-H 2 O 2 systems. However, circulating blood plasma containing H 2 O 2 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 Fe 3+ to Fe 2+ by AA and the enhanced generation of • OH radicals. Therefore, pro-oxidant activity of ascorbate cannot be excluded under such circumstances.

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 Fe 2+ -EGTA-H 2 O 2 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 Fe 2+ -EGTA-H 2 O 2 and Fe 3+ -EGTA-H 2 O 2 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

Reagents
All chemicals were of analytical grade. Iron (II) sulfate heptahydrate (FeSO 4 × 7H 2 O), iron (III) chloride hexahydrate (FeCl 3 × 6H 2 O), 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.) H 2 O 2 30% solution (w/w) was from Chempur (Piekary Slaskie, Poland). Sterile phosphate buffered saline (PBS, pH 7.4, without Ca 2+ and Mg 2+ ) was obtained from Biomed (Lublin, Poland). Sterile deionized pyrogen-free water (freshly prepared, resistance >18 MW/cm, HPLC H 2 O Purification System, USF Elga, Buckinghamshire, UK) was used throughout the study. Working aqueous solutions of 5 mmol/L of FeSO 4 and 5 mmol/L of FeCl 3 were prepared before the assay. A working solution of 28 mmol/L of H 2 O 2 was also prepared before the assay by dilution of 30% of H 2 O 2 solution and the concentration was confirmed by the measurement of absorbance at 240 nm using a molar extinction coefficient of 43.6 mol −1 cm −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.

System Generating Light and Measurement of Light Emission
For light generation, we used 92.6 µmol/L Fe 2+ -185.2 µmol/L EGTA-2.6 mmol/L H 2 O 2 system, as previously described [26]. This system generates the UPE, which depends mainly on • OH radicals. • OH generated in the course of reaction of Fe 2+ with H 2 O 2 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 FeSO 4 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 H 2 O 2 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.

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 Fe 2+ -EGTA-H 2 O 2 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, Fe 3+ -EGTA-H 2 O 2 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.

Conclusions
Ascorbic acid within the concentration range of 1 to 50 µmol/L very effectively inhibited • OH-induced light emission from Fe 2+ -EGTA-H 2 O 2 and Fe 3+ -EGTA-H 2 O 2 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.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.