Light Emission from Fe²⁺-EGTA-H₂O₂ System Depends on the pH of the Reaction Milieu within the Range That May Occur in Cells of the Human Body

Abstract

A Fe²⁺-EGTA(ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid)-H₂O₂ system emits photons, and quenching this chemiluminescence can be used for determination of anti-hydroxyl radical (•OH) activity of various compounds. The generation of •OH and light emission due to oxidative damage to EGTA may depend on the buffer and pH of the reaction milieu. In this study, we evaluated the effect of pH from 6.0 to 7.4 (that may occur in human cells) stabilized with 10 mM phosphate buffer (main intracellular buffer) on a chemiluminescence signal and the ratio of this signal to noise (light emission from medium alone). The highest signal (4698 ± 583 RLU) and signal-to-noise ratio (9.7 ± 1.5) were noted for pH 6.6. Lower and higher pH caused suppression of these variables to 2696 ± 292 RLU, 4.0 ± 0.8 at pH 6.2 and to 3946 ± 558 RLU, 5.0 ± 1.5 at pH 7.4, respectively. The following processes may explain these observations: enhancement and inhibition of •OH production in lower and higher pH; formation of insoluble Fe(OH)₃ at neutral and alkaline environments; augmentation of •OH production by phosphates at weakly acidic and neutral environments; and decreased regeneration of Fe²⁺-EGTA in an acidic environment. Fe²⁺-EGTA-H₂O₂ system in 10 mM phosphate buffer pH 6.6 seems optimal for the determination of anti-•OH activity.

1. Introduction

1.1. Fenton Systems as a Tool for Determination of Anti-(•OH) Activity and Limiting Buffers Interactions

Hydroxyl radicals (•OH) are very reactive and their excessive formation in the human body is involved in numerous pathological processes [1] including rheumatoid arthritis [2], neurodegenerative disorders [3], cancer [4], and atherosclerosis [4,5]. Fenton and Fenton-like reactions are the most important sources of •OH in the human body [4]; however, other processes such as the enhanced formation of peroxynitrite with its subsequent decomposition can contribute to tissue •OH overload [6]. Apart from breaking chemical reactions between H₂O₂ and transition metal ions (mainly Fe²⁺, Fe³⁺ Cu²⁺, Cu⁺) with the specific metal chelators, application of drugs or dietary supplements that can directly react with •OH (•OH scavengers) before their oxidative attack on cellular biomolecules seems to be a possible preventive or therapeutic approach [7]. Compounds being possible candidates for such treatment should be tested in detail in vitro before experiments on laboratory animals and clinical trials. Aqueous systems based on Fenton or Fenton-like reactions are frequently used for in vitro evaluation of the antioxidant activity. Because •OH generation can be significantly influenced by the pH of the reaction milieu [8,9], it is necessary to keep stable hydronium ions (H₃O⁺) activity during the experiment. Changes of pH related, for instance, to the addition of a tested compound may inhibit or enhance •OH generation and lead to false results. Unfortunately, the vast majority of buffering compounds can react with •OH and, in consequence, decrease the signal-to-noise ratio and repeatability of results. For instance, organic buffers such as Tris, Tricine, and Hepes were reported to effectively scavenge •OH radicals [10]. Even bicarbonate and phosphate buffers can react with •OH radicals [11,12,13] and may inhibit •OH reactions with an appropriate probe. It should be pointed out that Fenton’s systems dedicated to the evaluation of anti-•OH activity must resemble in vivo conditions and be relatively simple to facilitate the interpretation of obtained results. Bicarbonate and phosphate buffers are the main buffers in extracellular (e.g., blood) and intracellular fluid [14], respectively. There are also other intracellular buffers such as amino-acids, proteins, and organic acids (mainly carboxylic acids) (e.g., acetic acid, lactic acid, citric acid, succinic acid) that, with their dissociated form, can stabilize pH inside the cell [14]. On the other hand, •OH radicals can effectively oxidize the aforementioned intracellular buffers [15,16]. Moreover, oxidation of some proteins and tryptophane can lead to the formation of excited carbonyl groups with a subsequent photon emission [17,18,19], making results obtained with the Fe²⁺-EGTA-H₂O₂ system almost impossible to interpret. Therefore, phosphate buffer composed of dibasic sodium phosphate and monobasic sodium phosphate seems to be the optimal intracellular buffer that can be used for investigation of the effect of pH changes on UPE of Fe²⁺-EGTA-H₂O₂ system.

1.2. Properties of Fe²⁺-EGTA-H₂O₂ System Generating Ultra-Weak Chemiluminescence

We developed a system composed of Fe²⁺, EGTA (ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid), and H₂O₂, which generates •OH radicals (Fenton reaction) [20]. •OH radicals can attack and cleave the ether bond in an EGTA backbone structure, leading to the formation of products containing triplet excited carbonyl groups and subsequent ultra-weak photon emissions (UPE) [20]. Measurement of photon emanation (UPE within a defined time) with a sensitive luminometer could be a measure of •OH radicals production [20]. By using this system, we were able to determine pro-oxidant (enhancing •OH production) or anti-oxidant (inhibiting •OH activity) properties of various plant polyphenols at concentrations within the range 5 µmol/L to 50 µmol/L [21] and ascorbic acid [22]. These experiments were performed in phosphate-buffered saline pH = 7.4 [20,21,22] containing 137 mmol/L NaCl, 2.7 mmol/L KCl, and 10 mmol/L phosphate. This relatively high concentration of Cl- can suppress •OH radicals’ activity before their reaction with EGTA, leading to a decrease in photon emanations and low chemiluminescence signal. Apart from the triplet excited carbonyl groups, the decay of singled oxygen (O₂ (1Δg)) that is formed by a Fenton reagent (Fe²⁺, H₂O₂) could be a source of emitted photons from the Fe²⁺-EGTA-H₂O₂ system [14,23]. These photons have three characteristic bands of emission at 1270 nm, 703 nm, and 634 nm [20]. The spectral range of the luminometer used in our experiments was from 380 nm to 630 nm. Therefore, it cannot be ruled out that wavelength photons of 634 nm may contribute to some extent to UPE of Fe²⁺-EGTA-H₂O₂ system. Although we excluded indirectly the significant contribution of these photons to UPE of Fe²⁺-EGTA-H₂O₂ system with the use of sodium azide an O₂ (1Δg) scavenger [20], it would be better to complete these experiments and analyze a UPE signal from a system that specifically generated O₂ (1Δg). If the luminometer is sensitive to 634 nm photons, it will lead to over- or underestimation of •OH scavenging activity of the tested compound, depending on the simultaneous reactivity with O₂ (1Δg).

1.3. Aims of the Study

To better characterize the Fe²⁺-EGTA-H₂O₂ system as a tool for measurement of anti-•OH activity, we therefore evaluated the effect of a medium composed of 10 mmol/L phosphate buffer of pH ranging from 6.0 to 7.4 on UPE, ΔUPE (increment in UPE calculated as the difference between UPE and noise), and the UPE signal-to-noise ratio (light emission from medium alone). Moreover, we analyzed the effect of the system generating O₂ (1Δg) composed of H₂O₂ and sodium hypochlorite (NaOCl) on a UPE signal as well as O₂ (1Δg) decay-dependent photon emission from Fe²⁺-H₂O₂ recorded by a luminometer with a photomultiplier spectrum from 380 nm to 630 nm.

We found that the decay of O₂ (1Δg) did not significantly affect the UPE of the Fe²⁺-EGTA-H₂O₂ system and that pH = 6.6 of reaction milieu results in maximal signal-to-noise ratio being optimal for in vitro determination of anti-•OH activities of various compounds.

2. Results

2.1. Effect of Singlet Oxygen (O₂ (1Δg)) Generating System (H₂O₂-NaOCl) on the Luminescence Signal Recorded by Luminometer with Photomultiplier Spectrum from 380 nm to 630 nm

The baseline signal (UPE of medium alone-PB pH = 6.8 with injected NaCl) was 609 ± 60 (630; 107) RLU. The addition of H₂O₂ or NaOCl alone did not change UPE 628 ± 74 (651; 120) RLU and 651 ± 80 (695; 133) RLU, respectively. Light emission from the O2 (1Δg) generating system (H₂O₂-NaOCl) reached 926 ± 245 (878; 218) RLU and the median value was 1.37-times higher than that of the baseline (

Table 1. Singlet oxygen (O₂ (1Δg))-dependent chemiluminescence signal [RLU] recorded by a luminometer with a photomultiplier spectrum from 380 nm to 630 nm.

No.	Sample	pH of Reaction Milieu (10 mmol/L PB) pH = 6.8	pH of Reaction Milieu (10 mmol/L PB) pH = 6.6
1.	Complete system H2O2-NaOCl	926 ± 245 (878; 218) *	809 ± 124 (827; 105) *,†
2.	Complete system with O2 (1Δg) scavenger-H2O2-NaN3-NaOCl	1073 ± 105 (1102; 96)	930 ± 93 (974; 161)
3.	Incomplete system I H2O2-NaCl	628 ± 74 (651; 120)	641 ± 78 (653; 129)
4.	Incomplete system II H2O-NaOCl	651 ± 80 (695; 133)	654 ± 85 (669; 131)
5.	H2O-NaN3-NaCl	640 ± 46 (648; 50)	636 ± 54 (643; 84)
6.	H2O-NaCl	609 ± 60 (630; 107)	661 ± 104 (659; 101)
7.	NaCl	640 ± 47 (649; 65)	635 ± 56 (653; 121)

Total light emission was measured for 2 min just after the automatic injection of 100 μL of NaOCl or NaCl solution. Final sample volume 1080 μL. Results are expressed as mean and standard deviation and (median; interquartile range) of RLU. The final concentrations of H₂O₂, NaOCl, NaCL, and NaN₃ were 2.6 mmol/L, 2.6 mmol/L, 2.6 mmol/L, and 18.5 mmol/L. *—vs. corresponding values obtained for samples No 3, 4, 5, 6, and 7—p < 0.05. †—vs. corresponding value for sample No 2—p < 0.05.

). NaN₃ did not suppress UPE of H₂O₂–NaOCl (1073 ± 105 (1102; 96) RLU). Similar results were obtained for the second series of experiments with PB pH = 6.6. H₂O₂–NaOCl increased median RLU 1.28 times (Table 1). However, surprisingly, light emission from H₂O₂-NaOCl-NaN₃ was higher (p < 0.05) than that of H₂O₂-NaOCl.

2.2. Effect of pH of Reaction Milieu on Light Emission from Fe²⁺-EGTA-H₂O₂ System

Figure 1 shows the effect of pH of reaction milieu (changes from 6.0 to 7.4) on the ratios of UPE and ΔUPE to noise. They were highest (p < 0.05) at pH 6.6 and were 9.7 ± 1.5 (9.3; 2.3) and 8.7 ± 1.5 (8.3; 2.3) (Figure 1). The UPE of the Fe²⁺-H₂O₂ system was small and ranged between 620 ± 70 RLU (at pH 6.6) and 826 ± 76 RLU (at pH 7.2) as well as the ratios of UPE of the Fe²⁺-H₂O₂ system to baseline were low at the whole studied pH range and did not exceed 1.3 (Figure 1). Similarly, UPE and ΔUPE of the Fe²⁺-EGTA-H₂O₂ system revealed maximal values for pH 6.6 and 6.8 (p < 0.05) 4698 ± 583 RLU (4557; 1062), 4207 ± 586 RLU (4006; 1069) and 4651 ± 410 RLU (4756; 651), 4004 ± 387 RLU (4144; 605), respectively. For higher pH, they gradually decreased (p < 0.05) and reached 3946 ± 558 RLU (3745; 465) and 3023 ± 658 RLU (2983; 544) at pH = 7.4. Lowering pH (more acidic environment) resulted in suppression of UPE and ΔUPE to values of 2696 ± 292 RLU (2674; 345) and 2001 ± 340 (1991; 198) RLU at pH 6.2 (p < 0.05) (Figure S1). More details on UPE and ratios of UPE to noise of the Fe²⁺-EGTA-H₂O₂ system and controls are shown in Tables S1 and S2 and Figure S1.

3. Discussion

3.1. Contribution of 634 nm Photons Derived from Singlet Oxygen Decay to Maximal Signal Generated by the Fe²⁺-EGTA-H₂O₂ System

Light emission from Fe²⁺-EGTA-H₂O₂ was tested in PB with pH from 6.0 to 7.4. The maximal signal and optimal signal-to-noise ratio were observed at pH 6.6 and 6.8. In the majority of cells, the intracellular pH is within the range of 6.7 to 7.2 or even lower in some cellular organelles such as lysosomes where it reached 4.5–5.0 [24]. Therefore, our findings are relevant to conditions inside the cells where H₂O₂ can leak from mitochondria and is involved in intracellular signaling [25] and also reacts with iron to form •OH radicals [26]. The lowest UPE-to-noise ratio was 4.0 ± 0.8 at pH 6.2. In the case of the system generating O₂ (1Δg) the UPE increased only by 1.3- and 1.4 times at pH 6.6 and 6.8 in comparison to noise (PB with the addition of NaCl). Moreover, the ratio of UPE of Fe²⁺-H₂O₂ system to noise (which depends on the formation of O₂ (1Δg) did not exceed 1.3 within the pH range from 6.0 to 7.4. Therefore, the contribution of 634 nm photons from decay of O₂ (1Δg) to maximal UPE is low and could not be responsible for bias during estimation of anti-•OH activity of tested compounds using the Fe²⁺-EGTA-H₂O₂ system and light measurement with luminometer AutoLumat Plus LB 953. NaN₃ is a frequently used scavenger of O₂ (1Δg) especially generated by H₂O₂-NaClO system because it does not react with H₂O₂ and NaOCl [27]. NaN₃ also quenched O₂ (1Δg) dependent light emission from acetonitrile- H₂O₂ in alkaline environment [28]. We used a concentration of NaN₃ comparable to that applied in the afore-mentioned experiments and therefore it is difficult to explain no inhibitory effect of this compound on the chemiluminescence of H₂O₂-NaOCl system. Thermal decomposition of NaN₃ is accompanied by photon emanation [29] and may contribute to increased light emission from H₂O₂-NaN₃-NaOCl samples. On the other hand, UPE of H₂O-NaN₃-NaCl did not differ from that of H₂O-NaCl or NaCl alone making this explanation unlikely.

3.2. Effect of pH of Phosphate Buffer on Light Emission from the Fe²⁺-EGTA-H₂O₂ System

There are two reactions responsible for the generation of •OH radicals in the Fe²⁺-EGTA-H₂O₂ system.

Fe²⁺-EGTA + H₂O₂ → Fe³⁺-EGTA + OH⁻ + •OH

(1)

Fe³⁺-EGTA + O₂^•- → Fe²⁺-EGTA + O₂

(2)

Chemical reaction (2) leads to regeneration of Fe²⁺-EGTA complex that can enter reaction (1) to increase •OH generation, oxidative damage to EGTA and formation of excited carbonyl groups with subsequent photon emission [20].

Two other reactions can be also involved in Fe²⁺ regeneration [30].

H₂O₂ + Fe³⁺-EGTA → HO₂^• + H⁺ + Fe²⁺-EGTA

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

The ratio of H₂O₂ to Fe²⁺ in our Fe²⁺-EGTA-H₂O₂ system was around 28. Thus, the availability of Fe²⁺ is the limiting factor for •OH formation. Therefore, the reduction of Fe³⁺-EGTA to Fe²⁺-EGTA described by reaction (2) has an important contribution to total •OH radicals generation and light emission. An increase in pH above 6.8 was accompanied by a lower UPE of the Fe²⁺-EGTA-H₂O₂ system. Such conditions facilitate the formation of insoluble Fe(OH)₃. Although the concentration of EGTA was 2-fold higher than that of iron ions, Fe³⁺ could be grabbed from the complex with EGTA and precipitated as Fe(OH)₃, thus limiting Fe²⁺ regeneration. Five ferric hydrolysis products (Fe³⁺, FeOH₂⁺, FeO₂⁻, FeO₂H and FeO⁺) can contribute to the total content of Fe³⁺ in aqueous solution shoving the complexity of chemical reactions involving Fe²⁺ and Fe³⁺ ions. The aqueous solubility of these products decreases across the pH range from 5.0 to 8.0 [31,32]. Especially Fe³⁺ solubility decreases from 10⁻⁸ mol/L, 7 × 10⁻⁹ mol/L to about 5 × 10⁻⁹ mol/L at pH 6.0, 6.8 and 7.0, respectively [31]. In our Fe²⁺-EGTA-H₂O₂ system, the concentration of Fe²⁺ ions was 92.6 μmol/L (mostly chelated with EGTA) and the concentration of H₂O₂ was 28- times higher than that of Fe²⁺. Assuming carefully that after injection of H₂O₂ to Fe²⁺-EGTA, only 10% of Fe²⁺ ions would be oxidized to Fe³⁺ over 2 min observation, the concentration of Fe³⁺ (9.26 × 10⁻⁶ mol/L) can be substantially higher than its solubility at pH 6.8 and 7.0 and result in Fe³⁺ precipitation and decrease in UPE of Fe²⁺-EGTA-H₂O₂.

Moreover, the reaction rate (1) depends on pH with maximal intensity at pH around 3 [33]. This may additionally elucidate the suppression of UPE when pH increased from 7.0 to 7.4. On the other hand, mean signal suppression in neutral (decrease by 1.19 times at pH 7.0) and alkaline environments (decrease by 1.24 times at pH 7.2) was not so great, taking into consideration the observation that, under these conditions, high-valent oxoiron [13,34] species are the main product of Fenton reaction. This is probably due to the presence of phosphate buffer (phosphates), which can augment •OH radicals formation even in moderate alkaline solutions [13].

Surprisingly, lowering the pH of the reaction milieu from 6.6 to 6.0 did not increase the UPE of the Fe²⁺-EGTA-H₂O₂ system. Quite the contrary, light emission decreased and reached the lowest values at pH 6.2. It should be pointed out that three radicals •OH, O₂-, and O₂ (1Δg) are produced in Fenton’s system [33]. The reactions leading to the production of these radicals can compete with each other and increased generation of one radical may affect the intensity of the remaining two reactions [33]. Thus increased generation of •OH radicals in a more acidic environment (reaction 1) may suppress the production of O₂-radicals involved in the regeneration of Fe²⁺ ions and finally suppress the total yield of •OH radicals. It should be pointed out that •OH radicals can react with phosphate [13] which is a potential limitation in the use of phosphate buffer to stabilize the pH of the Fenton system reaction milieu. On the other hand, the kinetics of phosphate reactions with •OH radicals is significantly lower than that of the vast majority of organic compounds [13] including other buffers such as Tris and Hepes. Moreover, phosphate buffer plays a major role in maintaining the acid-base balance inside cells [35]. The intracellular concentration of free phosphates (H₂PO⁴⁻ and HPO⁴⁻) is within the range of 0.5 mmol/L to 5 mmol/L [27]. In addition, concentration of labile organophosphates (e.g., phosphocreatine) is up to 20 times higher [36]. What is more, high UPE-to-noise (mean 9.7) and ΔUPE-to noise (mean 8.7) ratios at pH 6.6 suggest that scavenging of •OH radicals by phosphates did not substantially suppress light emission from the Fe²⁺-EGTA-H₂O₂ system which could be used for evaluation of anti-•OH radicals activity of various organic compound. Previously we tested four increasing concentrations of the Fe²⁺-EGTA-H₂O₂ system as an emitter of light under a stable ratio of Fe²⁺ to EGTA to H₂O₂ molar concentrations [20]. The optimal UPE was found for 92.6 µmol/L Fe²⁺-185.2 µmol/L EGTA-2.6 mmol/L H₂O₂ system [20]. Moreover, concentrations of 92.6 µmol/L Fe²⁺ and 2.6 mmol/L H₂O₂ correspond to some extent to the values that occur in vivo. The plasma levels of H₂O₂ and Fe complexed with low molecular weight compounds, can reach 50 µmol/L and 10 µmol/L in certain diseases [37,38]. It is believed that H₂O₂ concentrations can be even higher in a close neighborhood of activated inflammatory cells such as polymorphonuclear leukocytes and macrophages [39]. Additionally, the subcellular iron concentration calculated per average neuron of a brain can reach around 0.6 mM/L [40].

4. Materials and Methods

4.1. Chemicals and Solutions

All chemicals were of analytical grade. Sodium phosphate monobasic monohydrate (NaH₂PO₄·H₂O), sodium phosphate dibasic heptahydrate (Na₂HPO₄·7H₂O), iron (II) sulfate heptahydrate (FeSO₄·7H₂O), hydrochloric acid (HCl), sodium hydroxide (NaOH), sodium azide (NaN₃), sodium hypochlorite (NaOCl), sodium chloride (NaCl), 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 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 FeSO₄, 20 mmol/L NaN₃ and 28 mmol/L NaCl were prepared before the assay. Thirty-percent solution of H₂O₂ was diluted with water to a final concentration of 28 mmol/L (working H₂O₂ solution) and the concentration was confirmed by the measurement of the absorbance at 240 nm using a molar extinction coefficient of 43.6/mol cm [41]. The stock solution of EGTA (100 mmol/L) was prepared in 10 mmol/L phosphate buffer pH = 8.0 and stored at room temperature in the dark for no longer than 3 months. A working solution of 10 mmol/L EGTA was obtained by the dilution of EGTA stock solution with water.

4.2. Effect of pH of Reaction Milieu on the Light Emission by Fe²⁺-EGTA-H₂O₂ System

Chemical reactions of 92.6 μmol/L Fe²⁺-185.2 μmol/L EGTA-2.6 mmol/L H₂O₂ system leading to UPE were carried out in 10 mmol/L phosphate buffers with pH ranging from 6.0 to 7.4 (pH = 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, and 7.4). UPE was measured 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 signal. An amount of 10 mmol/L phosphate buffers with aforementioned pH were prepared following the prescription of AAT Bioquest (https://www.aatbio.com/resources/buffer-preparations-and-recipes/phosphate-buffer-ph-5-8-to-7-4, accessed on 19 August 2024). For instance, 10 mmol/L phosphate buffer (PB) pH = 6.0 was prepared by addition of 36.704 mg of Na₂HPO₄·7H₂O and 0.119 mg of NaH₂PO₄·H₂O to 80 mL of water, pH was adjusted to 6.0 with HCl and then distilled water was added to the final volume of 100 mL. Other buffers were done in the same way and amounts of Na₂HPO₄·7H₂O and NaH₂PO₄·H₂O were taken from AAT Bioquest page and pH was adjusted to the desired value with HCl or NaOH solutions. 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 PB (pH = 6.0). Then, 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/L H₂O₂ solution was added by an automatic dispenser and the total light emission (expressed in RLU) was measured for 120 s. The final concentrations of FeSO₄, EGTA, and H₂O₂ in the reaction mixture were 92.6 µmol/L, 185.2 µmol/L, and 2.6 mmol/L, respectively. For experiments with other buffers (pH = 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, and 7.4), the procedure was the same. Controls included: incomplete system I (Fe²⁺-H₂O₂ in PB); incomplete system II (EGTA-H₂O₂ in PB); H₂O₂ alone in PB; Fe²⁺ and EGTA in PB without H₂O₂; and medium alone (

Table 2. Design of experiments on the effect of pH of reaction milieu on light emissions by the Fe²⁺-EGTA-H₂O₂ system.

No.	Sample	Working Solutions Added to Luminometer Tube (µL)
		A-PB	B-EGTA	C-FeSO4	D-H2O	E-H2O
1.	Complete system	940	20	20	100	0
2.	Incomplete system I	960	0	20	100	0
3.	Incomplete system II	960	20	0	100	0
4.	H2O2 alone	980	0	0	100	0
5.	Fe2+ + EGTA without H2O2	940	20	20	0	100
6.	Medium alone	980	0	0	0	100

Working solutions were mixed in alphabetical order. (A)—10 mM phosphate buffer (PB) with increasing pH from 6.0 to 7.4 (pH = 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, and 7.4); (B)—10 mmol/L aqueous solution of ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), and (C)—5 mmol/L solution of FeSO₄. Then, after gentle mixing the tube was placed into a luminometer chain, incubated for 10 min at 37 °C and then (D)—28 mmol/L aqueous solution of H₂O₂ or (E)—H₂O was automatically injected with dispenser and total light emission was measured for 2 min.

).

4.3. Effect of H₂O₂–NaOCl System on the Luminescence Signal Recorded by Luminometer with Photomultiplier Spectrum from 380 nm to 630 nm

During chemical reactions, three radicals are generated in the Fenton system [33]. One of them is O₂ (1Δg) that after decay may emit photons with three bands (634 nm, 703 nm, and 1270 nm). The band of 634 nm is close to the upper border of the luminometer spectrum and may affect •OH—dependent UPE signal giving false positive results in our experiments. However, production of O₂ (1Δg), in Fenton systems is much less intensive than generation of •OH radicals [42,43,44]. Therefore, to study the effect of O₂ (1Δg) on the chemiluminescence signal we choose H₂O₂-NaOCl system a very effective generator of O₂ (1Δg) [45]. The pH of chemical reaction environments 6.6 and 6.8 was chosen based on the results of experiments on effect of pH of reaction milieu on the light emission by Fe²⁺-EGTA-H₂O₂ system. UPE and ΔUPE (UPE minus baseline) reached the highest values at pH 6.6 and 6.8. To estimate this plausible effect, 100 µL of 28 mmol/L H₂O₂ solution was added to the tube containing 880 μL of 10 mmol/L PB pH = 6.8 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 aqueous solution of 28 mmol/L NaOCl was added using an automatic dispenser and the total light emission (expressed in RLU) was measured for 120 s. It should be pointed out that NaOCl solution was prepared in ice-cold water and kept in an ice bath throughout the whole experiment. The final concentrations of H₂O₂ and NaOCL were 2.6 mmol/L. The design of these experiments and control systems are shown in

Table 3. Design of experiments on the effect of singlet oxygen (O2 (1Δg)) generating system (H₂O₂-NaOCl) on the luminescence signal recorded by luminometer with photomultiplier spectrum from 380 nm to 630 nm.

No.	Sample	Volumes of Working Solutions Added to Luminometer Tube (μL)
		A-PB	B-H2O2	C-H2O	D-NaN3	E-NaOCl	F-NaCl
1.	Complete system	880	100	0	0	100	0
2.	Incomplete system I	880	0	100	0	100	0
3.	Complete system + NaN3	780	100	0	100	100	0
Additional control
4.	Incomplete system II	880	100	0	0	0	100
5.	Medium alone	880	0	100	0	0	100
6.	Medium + NaN3	780	0	100	100	0	100
7.	PB alone	980	0	0	0	0	100

Working solutions were mixed in alphabetical order. (A)—10 mM phosphate buffer (PB) (pH = 6.8); (B)—28 mmol/L solution of H₂O₂, (C)—distilled water, (D)—20 mmol/L NaN₃—an O2 (1Δg) scavenger, then after gentle mixing, the tube was placed into a luminometer chain, incubated for 10 min at 37 °C, and then (E)—28 mmol/L aqueous solution of NaOCl or (F)—28 mmol/L NaCl (additional controls) was automatically injected with dispenser and total light emission was measured for 2 min.

.

4.4. Statistical Analyses

Results obtained from 9 series of separate experiments are expressed as means (standard deviations) and medians and interquartile ranges (IQR) of relative light units (RLU). The following parameters were recorded and calculated: UPE (ultra-weak photons emission)—total light emission within the first two minutes after the addition of H₂O₂ or H₂O and NaOCl or NaCl, increment in UPE (ΔUPE = UPE of a given system—UPE of buffer alone (noise), and the ratio of UPE (or ΔUPE) of a given system to noise when pH of reaction milieu increased from 6.0 to 7.4. The comparisons between the UPE and ΔUPE and their ratios to noise observed in different pH of reaction milieu 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, the Welch’s t-test was used instead of the standard t-test. A p-value < 0.05 was considered significant.

5. Conclusions

We found that •OH radicals-induced light emission from the Fe²⁺-EGTA-H₂O₂ system is highest at pH 6.6 stabilized with 10 mmol/L phosphate buffer. Both increase in pH within the range of 6.8 to 7.4 and decrease from 6.4 to 6.0, resulting in suppression of UPE and a decrease in the UPE-to-noise ratio. The following processes summarized in Figure 2 may be responsible for this phenomenon;

• Enhancement and inhibition of •OH production in lower and higher pH, respectively.
• Formation of insoluble and non-reactive Fe(OH)₃ at neutral and alkaline environment.
• Enhancement of •OH production by phosphates at weakly acidic and neutral environments.
• Suppression of O₂^•-production in acidic environment with decreased intensity of Fe²⁺-EGTA complex regeneration.

Phosphates are the main intracellular buffer and the pH range investigated in our study occurs in human body cells. Therefore, the Fe²⁺-EGTA-H₂O₂ system with pH 6.6 stabilized with PB resembles intracellular conditions and seems optimal for the determination of anti-•OH activity of the variety of water-soluble organic compounds.