Department of Medical Imaging Techniques, Medical University of Lodz, Lindleya 6, 90-131 Lodz, Poland.
Radiation Protection, University Hospital No. 2, Medical University of Lodz, Zeromskiego 113, 90-549 Lodz, Poland.
Molecules. 2024 Aug 25;29(17):4014. doi: 10.3390/molecules29174014.
A Fe-EGTA(ethylene glycol-bis (β-aminoethyl ether)-,,','-tetraacetic acid)-HO 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) at neutral and alkaline environments; augmentation of •OH production by phosphates at weakly acidic and neutral environments; and decreased regeneration of Fe-EGTA in an acidic environment. Fe-EGTA-HO system in 10 mM phosphate buffer pH 6.6 seems optimal for the determination of anti-•OH activity.
Fe-EGTA（乙二醇双（β-氨基乙基醚）-N，N，N'，N'-四乙酸）-HO 体系会发射光子，而淬灭这种化学发光可用于测定各种化合物的抗羟自由基（•OH）活性。EGTA 的氧化损伤导致•OH 的产生和光发射可能取决于反应介质的缓冲液和 pH 值。在本研究中，我们评估了 pH 值从 6.0 到 7.4（可能发生在人类细胞中）、用 10 mM 磷酸盐缓冲液（主要细胞内缓冲液）稳定时对化学发光信号及其与噪声的比值（培养基单独产生的光发射）的影响。在 pH 值为 6.6 时，观察到最高的信号（4698 ± 583 RLU）和信号噪声比（9.7 ± 1.5）。较低和较高的 pH 值导致这些变量分别降至 2696 ± 292 RLU、4.0 ± 0.8（pH 值为 6.2）和 3946 ± 558 RLU、5.0 ± 1.5（pH 值为 7.4）。以下过程可能解释了这些观察结果：在较低和较高的 pH 值下，•OH 的产生增强和抑制；在中性和碱性环境下形成不溶性 Fe（OH）；在弱酸性和中性环境下，磷酸盐会增加•OH 的产生；在酸性环境中，Fe-EGTA 的再生减少。在 10 mM 磷酸盐缓冲液 pH 值为 6.6 的 Fe-EGTA-HO 体系似乎是测定抗•OH 活性的最佳选择。
