Adverse roles of citric acid and L-histidine in the transition metal-dependent generation of hydroxyl radical at circumneutral pH

Adverse roles of citric acid and L-histidine in the transition metal-dependent generation of hydroxyl radical at circumneutral pH | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Adverse roles of citric acid and L-histidine in the transition metal-dependent generation of hydroxyl radical at circumneutral pH Yoichi Kurokawa, Atsushi Matsuzawa, Hirotaka Ogawa, Yusuke Hirata, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5163186/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Through the present work, we have examined the possible role of L-histidine and citrate in the regulation of iron (II) ion-induced generation of hydroxyl radical (OH*), the most detrimental reactive oxygen species (ROS), via Fenton reaction. We observed that the presence of metal chelators (such as EDTA or citric acid) was necessary for the iron ion-dependent generation of OH*, when assessed with 3-coumarin carboxylic acid (3-CCA) as a probe specific for the radical. We also found that citrate acid as well as EDTA promoted the iron ion-dependent generation of the radical on a dose-dependent manner when they repressed the copper ion-dependent generation of the radical. In contrast, L-histidine promoted the copper ion-dependent generation of the radical at less than equimolar to the ion and repressed it at more than equimolar, whereas L-histidine hardly promoted the iron ion-dependent generation of the radical. Finally, we found that EDTA and citric acid promoted the iron and hydrogen peroxide-induced degradation of bovine serum albumin and repressed the copper and hydrogen peroxide-induced degradation of the protein, suggesting that the contrasting roles of EDTA and citric acid in promoting and repressing the generation of hydroxyl radical, depending on iron ion and copper ion, respectively. Biological sciences/Biochemistry/Metals/Iron Physical sciences/Chemistry/Chemical biology/Metals Fenton reaction hydroxyl radical metal chelator oxidative protein degradation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Iron is the second most abundant metal, and an essential element for many organisms including bacteria, plant and animals. Iron participates in many important roles in metabolic pathways, redox reactions and electron transport chain mechanism, in order to sustain biological homeostasis. On the other hand, excess amount of iron can be highly toxic and lead to life-threatening conditions such as cancer, diabetes, liver disease, cardiovasucular disease and heart failure 1 . Iron participates in the generation of deleterious OH*—arguably the most injurious ROS species in vitro , by the Fenton reaction (Fe(II)-catalyzed H 2 O 2 oxidation process), thus making a toxic feature of iron 2 . The Fenton reaction also has a great role in trace metal and natural organic matter biogeochemistry, its utility in water treatment and its role in oxidative cell degradation and associated human disease 3 . However, the Fenton reaction is effective at acidic pH, not at neutral or basic pH, because iron can form hydroxide with low solubility at high pH conditions and decreases its ability to generate ROS by Fenton reaction. Therefore, the Fenton reaction requires chelators to solubilize otherwise precipitable iron ion at circumneutral pH. EDTA, citric acid or polyhydric organic acids have been reported to enhance the production of OH* 4–8 . The Fenton-like reaction, involving the catalytic interplay of copper ions with hydrogen peroxide 9 , serves as another prototypical system for chemical ROS generation in a manner which does not require a metal chelator. We are interested to find a suitable and biologically relevant chelator which promotes or represses the metal ion-dependent generation of OH* and also to examine the effective concentration of such a chelator as promoter or repressor of the radical generation, in order to examine a possible mechanism for the generation of the radical in vivo . Citric acid is ubiquitous in nature, and has various biological functions in energy production and iron metabolism in animal and plant. In plants, citric acid is present in root exsudates and is responsible for solubilizing the ferric hydroxide in the soils to mobilize the iron to the plant root cells 10 . Bradyrhizobium japonicum has been shown to release citric acid under iron-deficient growth conditions 11 . In animals, citric acid has various essential functions and regulates the homeostasis of iron in vivo as citrate 12 : citric acid has also been proposed to be a constituent of the low molecular weight cytosolic iron pool 13 and non-transferrin-bound iron has been proposed to exist largely as complexes with citric acid in the plasma of iron-overloaded patients 14 . Yet, citric acid is detrimental when present in excess amount: although the anti-oxidative or anti-tumor role of citric acid is reported 15 – 16 , lines of evidence have shown that it is involved in the inflammation responses 1 or the onset of liver-disease like MASLD (metabolic dysfunction-associated steatotic liver disease) 17 – 20 or cancer 12 , 16 . The toxic aspects of citric acid can be in part explained by its ability to promote the generation of OH* 21 . Several reports have been made with citrate-iron complex in generating OH* 2, 22 – 25 , whereas citric acid is reported to repress the copper ion-dependent ROS generation, as scavenger of ROS 26 , 27 . Thus, citric acid has been reported to have dual roles as promoter and repressor for the generation of OH*. Does excess amount of citric acid, a metal chelator, prevent the iron-dependent ROS generation, even when they promote it at low concentration? What is the effective concentration of the ligand to serve as either promoter or repressor of the generation of the radical? According to the reports by Engelmann, metal chelators (EDTA or citric acid) differently affected the electrocatalytic reduction of hydrogen peroxide, depending on the relative concentration of the chelators to that of metal ion: they found that the reduction of hydrogen peroxide was repressed when EDTA was added at a ratio of 5-fold to iron ion, and that it was repressed when citric acid was added at a ratio of more than 50-fold to that of iron 24 . These differences may be in part due to the change of the structure of citrate-iron complex according to the conditions such as pH or temperature in aqueous solution 28 . Compared to the well-studied role of iron (II)-EDTA or citric acid complex on the production of ROS, relatively little is known on the role of other organic acid or L-histidine as iron complex, including the optimum ratio of these acids or histidine /iron in generating ROS, when iron(II)-histidine complex is the most major iron (II) species in the blood plasma of about 15%, followed by iron (II)-citrate complex of about 10% 29 . However, the detailed role of the iron (II)-L-histidine remains unknown. One possible explanation is the ambiguous role of L-histidine in regulating the metal ion-dependent ROS production. Several lines of evidences suggest that L-histidine represses the copper ion-induced generation of ROS generation 30 – 32 by tightly binding copper ion and inhibiting the copper ion-dependent generation of ROS 33 . On the other hand, L-histidine is also reported to induce the ROS production in vivo 34 . However, its detailed mechanism has not been known, including whether the ROS generation depends on metal ion including copper ion. Thus, although L-histidine is reported to have an ambiguous role in the metal ion-dependent ROS production, there have been no report, to our best knowledge, on the information regarding the effective concentration at which the ligand either promote or inhibit the copper or iron ion-dependent ROS generation. Likewise, histamine, decarboxylated L-histidine, is shown to produce oxidative stress or to cause inflammation 35 . 36 and is suggested to suppress the ROS generation 37 . Thus, we are interested to examine whether organic acids, L-histidine or histamine either promote or repress the metal ion-induced generation of ROS, depending on the ratio of ligand/iron. We are interested to examine and compare the effects of organic acids (citric aid, malic acid, succinic acid, tartaric acid and L-histidine) on the iron- or copper ion-induced generation of OH* and also on the degradation of biopolymer. We examine in the present study whether these organic acids promote the Fenton reaction-induced generation of OH* and evaluate the molar ratio of ligand/Fe(II) or Cu(II)- catalyzed oxidation of O 2 or H 2 O 2 ., by using 3-hydroxycoumarin carboxylic acid (3CCA) 38 as a conventional and less time-consuming detector of OH* or H 2 O 2 . We found the possibility that L-histidine or histamine promotes the copper (II) ion-dependent generation of OH*, but not the iron (II) ion-dependent generation of the ROS, which may have a closely relation with onset of some diseases. A new finding on the role of carboxylic acids will be discussed in promoting and suppressing heavy metal ion-dependent ROS generation. Results Fluorescence intensity of 7-hydroxycoumarin-3-carboxylic acid (7OHCCA) differently increased in the presence of both hydrogen peroxide and copper (II) chloride or iron (II) chloride. We first measured the effects of copper (II) chloride or iron (II) chloride on the production of hydroxyl radical when measured with 3CCA, a specific indicator for hydroxyl radical. We incubated 3CCA in the presence of hydrogen peroxide and either copper (II) chloride or iron (II) chloride, and measured the fluorescence intensity of 7OHCCA, 3CCA hydroxylation product. When the control sample (3CCA alone) was incubated and measured for 7OHCCA-specific fluorescence intensity, the fluorescence intensity was hardly detected (Fig. 1 A lane 1; Table S1 A). When 3CCA was treated with hydrogen peroxide and increasing concentration of copper chloride (1–10 µM), the 7OHCCA-specific fluorescence intensity increased significantly according to the concentration of copper chloride: from about 6600 (1 µM) to about 23000 (10 µM) (Fig. 1 A lanes 2–6; Table S1 A ). In contrast, much more concentration of iron (II) chloride was required to reach the comparable level of 7OHCCA intensity in the presence of copper (II) chloride: when 3CCA was incubated with hydrogen peroxide and increasing concentration of iron chloride (100–1000 µM), the 7OHCCA-specific fluorescence intensity increased from about 4000 (100 µM) to about 17000 (1000 µM)(Fig. 1 B lanes 1–6; Table S1 B). A strong, good relationship between the concentration of metal salt and the fluorescent signal intensity of 7OHCCA was observed with copper chloride or iron chloride, and hydrogen peroxide (Fig. 1 C and 1 D). These results suggest that hydroxyl radical is generated in the presence of hydrogen peroxide and either copper chloride or iron chloride, although the latter chemical was much less efficient than the former. Search for organic acids that enhances fluorescence intensity of 7OHCCA incubated in the presence of both hydrogen peroxide and iron (II) chloride. EDTA was known to promote the iron/hydrogen peroxide-induced generation of hydroxyl radical 5 , 6 . Citric acid is also known to promote the generation of the radical in the presence of iron/hydrogen peroxide 11 , 12 . Because both EDTA and citric acid are polyhydric carboxylic acids containing multiple carboxylic groups that may bind metal ion, we were interested in examining the effects of series of polyhydric carboxylic acids (EDTA, citric acid, malic acid, tartaric acid and succinic acid) and L-histidine that contain different numbers of carboxylic groups. When iron chloride was incubated alone, the 7-OHCCA fluorescence signal intensity was only about 370 (Fig. 2 lane 2; Table S2). When iron chloride was incubated with hydrogen peroxide, the signal increased to about 15800 (Fig. 2 lane 4; Table S2), which was about half of the sample containing copper chloride and hydrogen peroxide (about 38000) (Fig. 2 lane 15; Table S2). When EDTA was further added with iron and hydrogen peroxide, the signal significantly increased to 80000 − 10000 at 0.1 or 1 mM of EDTA (Fig. 2 lanes 13 and 14; Table S2), suggesting that iron chloride becomes more efficient hydroxyl radical generator than copper chloride in the presence of iron. Similar effect was observed with citric acid at 1.0 mM (Fig. 2 lane 6; Table S2). No significant effects were observed with tartaric acid, malic acid or succinic acid. Opposing effects of EDTA and citric acid in enhancing or decreasing the fluorescence intensity of 7OHCCA incubated in the presence of both hydrogen peroxide and iron (II) chloride or copper (II) chloride. We found that EDTA and citric acid increase the 7-OHCCA derived fluorescent signal that represents the generation of hydroxyl radical in the presence of iron chloride and hydrogen peroxide. We next examined whether these compounds promote the signal in the presence of copper chloride and hydrogen peroxide. When iron chloride and hydrogen peroxide was incubated with 3CCA, the 7-OHCCA fluorescence intensity was about 7000 (Fig. 3 A lane 2; Table S3A), which was less than one fifth of that of the sample containing copper chloride and hydrogen peroxide (about 36000) (Fig. 3 B lane 2; Table S3B). When citric acid was further added with iron and hydrogen peroxide, the signal significantly increased to 8000–10000 at 0.1 or 1 mM of citric acid (Fig. 3 A lanes 3 and 7; Table S3A). EDTA more efficiently increased the signal than citric acid: when EDTA was further added with iron and hydrogen peroxide, the signal significantly increased to 20000–40000 at 0.01–0.1 mM of EDTA (Fig. 3 A lanes 8–12; Table S3A), suggesting that EDTA more efficiently generates hydroxyl radical than citric acid. In contrast, when copper chloride and hydrogen peroxide was incubated in the presence of 3CCA, EDTA and citric acid significantly decreased the 7-OHCCA-derived fluorescence signal intensity: when citric acid was added with copper chloride and hydrogen peroxide, the intensity significantly decreased from 26000 (0.1 mM) to about 3000 (1 mM) of citric acid (Fig. 3 B lanes 3 and 7; Table S3B). EDTA hardly affected the signal intensity from 0.01 to 0.04 mM (Fig. 3 B lanes 8–10), whereas it decreased the signal about to 17000 (0.08 mM) and 1400 (0.1 mM) (Fig. 3 B lanes 11 and 12; Table S3B). We next examined how addition of citric acid or EDTA of more than 1 mM affects the metal ion and hydrogen peroxide-induced 7-OHCCA-derived fluorescence intensity: addition of 1–4 mM citric acid only marginally affected the signal level of the sample containing iron chloride and hydrogen peroxide, whereas addition of 8 mM of the compound decreased the signal level to the that of the control (iron ion and hydrogen peroxide)(Fig. 3 C lanes 4–7; Table S3C), suggesting that the optimum hydroxyl radical-promoting concentration of citric acid is about 1–2 mM and that the generation of the radical is repressed at 8 mM. In contrast, addition of citric acid significantly decreased the signal of the sample containing copper chloride and hydrogen peroxide on a dose-dependent manner, almost to the control level (Fig. 3 D lanes 4–7; Table S3D). Addition of 1 mM EDTA increased the 7-OHCCA derived fluorescence intensity to about 60000 (Fig. 3 C Lane 8; Table S3C). Addition of higher concentration of EDTA decreased the signal about to 40000 at 8 mM (Fig. 3 C lane 11; Table S3C), suggesting that the optimum hydroxyl radical-promoting concentration of EDTA is about 1 mM in the iron chloride and hydrogen peroxide-dependent generation of hydroxyl radical and that EDTA represses generation of the radical at the concentration of higher than 1 mM. In contrast, the 7-OHCCA derived fluorescence intensity remained less than 1000 when EDTA of higher than 1 mM was added to the sample containing copper chloride and hydrogen peroxide, suggesting that EDTA inhibits generation of the radical under the experimental conditions employed. Taken together, these results suggest that citric acid and EDTA promote the iron-hydrogen peroxide-induced generation of hydroxyl radical at the ligand/metal ion mole ratio of less than equimolar to twice-molar (citric acid) and less than 10 times-molar (EDTA) and that they repress the copper-hydrogen peroxide-induced generation of the radical. L-histidine enhances the fluorescence intensity of 7OHCCA incubated in the presence of hydrogen peroxide and copper (II) chloride at concentration lower than that of copper ion, when it hardly affected the fluorescence intensity of 7OHCCA incubated in the presence of iron (II) chloride. We next examined how the addition of L-histidine or histamine, which have imidazole moiety with metal chelating ability, affects the 7-OHCCA-derived fluorescence intensity when iron chloride and hydrogen peroxide was incubated in the presence of 3CCA. When iron chloride was incubated in the presence of hydrogen peroxide, the 7OHCCA-derived fluorescence intensity increased to about 5000 (Fig. 4 A lane 3; Table S4A). When L-histidine or histamine (0.05–0.4 mM) was further added to the sample containing iron chloride and hydrogen peroxide, the 7OHCCA-derived fluorescent hardly increased (Fig. 4 A lanes 4–7, 8–11; Table S4A), which makes a sharp contrast to EDTA that enhanced the iron/hydrogen peroxide-dependent increase of 7OHCCA signal intensity at 0.01-1.0 mM (Fig. 4 A lanes 12–14; Table S4A) and decreased it at 10 mM (Fig. 4 A lane 15; Table S4A). In contrast, when copper chloride was incubated in the presence of hydrogen peroxide, the 7OHCCA-derived fluorescent intensity increased to about 34000 (Fig. 4 B lane 3; Table 4A). The further addition of L-histidine (0.05–0.1 mM) surprisingly increased the 7-OHCCCA derived signal level to about 70000 by about 2-fold (Fig. 4 B lanes 4, 5; Table S4B). Addition of histamine (0.05–0.1 mM) also doubled the 7OHCCA-derived signal level obtained only in the presence of copper chloride and hydrogen peroxide (Fig. 4 B lanes 8–9; Table S4B). Addition of L-histidine or histamine gave a sharp contrast to that of EDTA that did not increase and only marginally affected the 7-OHCCA derived signal at less than equimolar amount copper ion (Fig. 4 B lanes 12, 13; Table S4B), whereas addition of more than 10-times molar of EDTA almost completely repressed the 7OH-CCCA derived signal level (Fig. 4 B lanes 14, 15; Table S4B). These results suggest that L-histidine or histamine promoted the generation of hydroxyl radical that was induced by copper chloride and hydrogen peroxide when present at less than equimolar amount of that of copper ion and that these compounds repressed the generation of the radical when present at more than 4-times. Taken together, these results suggest that L-histidine and histamine effectively promotes the copper ion-induced hydroxyl radical generation and is less effective in promoting the iron ion-induced hydroxyl radical generation. Opposing effects of EDTA and citric acid in enhancing the degradation of BSA incubated in the presence of both of hydrogen peroxide and iron (II) chloride, and in preventing the degradation of BSA incubated in the presence of hydrogen peroxide and copper (II) chloride We finally examined whether the addition of EDTA or citric acid affects the metal-catalyzed degradation of BSA, a model protein. When BSA was incubated alone or in the presence of 5 mM hydrogen peroxide, followed by Native-PAGE analysis, the band of the native-form of BSA remained unaffected (Fig. 5 A lanes 1 and 2). When BSA was incubated in the presence of hydrogen peroxide and 1 mM iron (II) chloride, the native-form BSA band was detected to the same level as the control (BSA alone) (Fig. 5 A lane 3). When BSA was incubated in the presence of hydrogen peroxide and iron chloride, and also 0.1–0.4 mM citric acid, the native BSA band remained intact (Fig. 5 A lanes 4–6). In contrast, when BSA was incubated in the presence of hydrogen peroxide, iron chloride and higher concentration of citric acid, the native BSA band was thin at 0.8 or 1.0 mM citric acid and was hardly detected at 2.0 mM (Fig. 5 A lanes 7–9). A similar result was obtained when BSA was incubated in the presence of the oxidants and also EDTA instead of citric acid: the native BSA band was detected almost unaffected when BSA was incubated in the presence of the oxidants and 0-0.02 mM EDTA (Fig. 5 A lanes 10–12). When BSA was incubated in the presence of higher concentration of EDTA, the native-form BSA band was thin at 0.04 or 0.08 mM EDTA and was hardly detected at 0.1 mM (Fig. 5 A lanes 13–15). To note, the totally different results were obtained when BSA was incubated in the presence of copper (II) chloride instead of iron (II) chloride, and hydrogen peroxide as well as citric acid or EDTA. The native BSA band was detected to the similar level when BSA was incubated in the absence of any oxidant or in the presence of hydrogen peroxide (Fig. 5 B lanes 1 and 2). In contrast, when BSA was incubated in the presence of 0.01 mM copper chloride ad hydrogen peroxide, the native BSA band was not detected (Fig. 5 B lane 3), suggesting that BSA underwent degradation under metal-catalyzed oxidation in the presence of copper chloride and hydrogen peroxide. Upon addition of increased concentration of citric acid together with the oxidants (copper chloride and hydrogen peroxide), the native BSA band was clearly detected at more than 0.4 mM citric acid (Fig. 5 B lanes 6–9), suggesting that BSA escaped from the metal-catalyzed degradation in the presence of citric acid. Likewise, upon addition of EDTA of more than 0.01 mM, the native BSA band clearly detected (Fig. 5 B lanes 11–15), suggesting that EDTA is superior to citric acid in protecting BSA from copper chloride and hydrogen peroxide-induced degradation of BSA. Discussion In the present study, we compared the effects of polyhydric acids (EDTA and citric acid) or L-histidine in iron (II) ion or copper (II) ion and hydrogen peroxide-induced generation of hydroxyl radical and examined the effective concentration at which these ligands either promote or repress the generation of the radical, in order to find a possible physiologically relevant chemical that regulates the generation of ROS in vivo . We will discuss how differently these ligands act against metal ion by forming an ion/ligand complex that either promotes or represses the metal ion-catalyzed generation of hydroxyl radical. First, we found that EDTA with four carboxylic groups in a molecule promoted the iron (II) ion-hydrogen peroxide-induced generation of hydroxyl radical (when present at less than equimolar to iron (II) ion) and that EDTA repressed the copper (II) ion-hydrogen peroxide-induced generation of the radical at the same molar ratio of the ligand/metal ion. Second, we found that among organic acids with multiple carboxylic groups, citric acid with three carboxylic groups in a molecule promotes the iron (II) ion-hydrogen peroxide-induced generation of hydroxyl radical at concentration of 20-times or less in molar ratio to copper (II) ion and that it represses the radical generation at higher concentration, when other organic acid with two carboxylic groups (tartaric acid, malic acid, succinic acid) in a molecule neither promoted nor repressed the metal ion-induced generation of hydroxyl radical under the experimental procedures employed. Third, we found that L-histidine or histamine promoted the copper ion and hydrogen peroxide-induced generation of the radical at the concentration less than equimolar to that of copper ion, when these ligands hardly affected the iron ion-dependent generation of the radical. These findings suggest that polyhydric acids (EDTA, citric acid) and L-histidine or histamine have a contrasting effect in regulating the iron- or copper-ion dependent generation of hydroxyl radical: EDTA or citric acid promited the iron ion-dependent generation of the radical and repressed the copper ion-dependent generation of the radical, whereas L-histidine or histamine promoted the copper ion-dependent generation of the radical, depending on the ligand/metal ion ratio. These findings will give a fundamental knowledge on the transition metal ion-induced generation of ROS. We will discuss how differently EDTA, citric acid and L-histidine or histamine regulates the generation of hydroxyl radical and the relevance of our findings. Although EDTA and citric acid was known to promote and repress the hydroxyl radical generation by Fenton reaction in vitro , depending on the molar ratio of metal ion/ligand 2 , the mode of action of the ligands is apparently different: the optimum promoting ratio of the ligand/iron ion was reported to be about 1 for EDTA and citric acid, whereas the repressing ratio of the ligand/iron ion was about 5 for EDTA and about 50:1 for citric acid 24 . Similarly, the optimum ratio of the ligand/iron ion was about 0.5 ~ 1.0 for citric acid 3 . The promoting ratio probably represents that these ligands solubilize iron ion which otherwise tends to form insoluble precipitate at circumneutral pH and is not involved in the ROS generation, and allows redox reaction at an equimolar ratio to that of metal ion by forming a ‘basket complex’ 39 . The difference of the repressing ratio may be ascribed to the difference of the ligand-metal ion structure 40 , as will be discussed below. We found in the present study that repressing ratio of the ligand/iron ion was about 80 for citric acid, when we were not able to determine the accurate ratio (more than 80) for EDTA under the conditions employed (data not shown). The difference of ours and Engelman MD may be ascribed to the difference of the analytical method to measure the hydroxyl radical generation. As described in Introduction, the role of iron (II)-histidine complex in the regulation of ROS generation has been largely unknown, when L-histidine was reported to have dual opposing effects in generation of ROS: L-histidine was reported to repress the copper ion-induced generation of ROS 30 – 32 , and to induce the ROS production in vivo 34 , of which mechanism remains largely unknown. However, a similar dual role of L-histidine has been reported in the nickel ion-dependent generation of the radical: L-histidine was proposed to enhance the generation of hydroxyl radical 41 , 42 and also to scavenge the radical 42 , depending on its concentration. We here discuss the possibility that L-histidine promotes and also represses the ROS generation, on a manner dependent on copper ion. Our present data clearly suggest that L-histidine enhances the copper ion-induced generation of hydroxyl radical at concentration of 2-times or less in molar ratio of the copper ion and that L-histidine represses it at more than 4-fold. Likewise, L-histidine was found to promote the nickel ion-dependent disproportionation of hydrogen peroxide, that is, hydroxyl radical generation at molar ratio below half concentration of nickel ion and to repress it at equimolar concentration 41 . Thus, L-histidine may similarly act against copper ion- or nickel ion-induced generation of the radical, when their effective concentration differed, possibly by the difference of the metal ion-ligand interaction or the experimental procedures employed. Our data also suggest that L-histidine hardly promotes the iron ion-induced generation of the radical. These results are in good accordance with the idea that L-histidine tightly forms a complex with iron ion that does not allow the ROS generation, which is proposed by Zabek-Adamska 33 and that L-histidine can generate ROS when not efficient amount of the ligand is available to chelate copper ion. The same is true with the role of histamine in regulating the generation of the radical. Histamine was reported to produce oxidative stress, to cause inflammation and also to repress the generation of ROS 36 , 37 . Our present data suggests that histamine promotes the copper ion-induced generation of hydroxyl radical at the concentration less than equimolar of the copper ion and that histamine represses it at more than 2-fold, when histamine hardly promotes the iron ion-induced generation of the radical. These results may be that histamine has a dual role in promoting and repressing the metal ion-dependent ROS generation. This idea is in a good accordance with the previous reports: histamine is known to stimulate neutrophil ROS production 35 , 43 , has been known as a cause of neurodegeneration inflammation as well as neuroprotector 44 , 45 . To our best knowledge, there have been no reports on the L-histidine or histamine-induced hydroxyl radical generation which is driven by copper ion and hydrogen peroxide. We believe that these ligands regulate the copper ion-dependent generation of the radical, by a similar mechanism of L-histidine regulating the nickel ion-dependent generation of hydroxyl radical 41 , 42 , depending on its concentration. Further studies are required to reveal whether L-histidine or histamine promotes or represses the generation of ROS, which will give a fundamental knowledge on the possible medical phenomenon such as inflammation or the onset of a disease. As discussed above, our present data suggest that polyhydric acids (EDTA or citric acid) differently behaved toward the metal ion-dependent generation of hydroxyl radical. Let us first consider the difference of the structure of iron ion- or copper ion-complex with a chelator EDTA that either allows or represses a redox reaction for generation of ROS, and also the structure of the metal ion-citric acid complex. Iron occurs in biological systems in the form of ferrous or ferric ions. These ions can form six coordination bonds with ligands that are able to donate electron pair 7 . A metal chelator EDTA is a potential hexadentate ligand and can possibly occupy all coordination sites of iron ion, forming six coordination bonds. However, EDTA has been suggested to be too small to completely encompass all the coordination sites of iron ions. As a result, a seventh coordination site is generated in EDTA, which is occupied by water molecule that is readily exchangeable by oxidant/reductant. The ligand in the “free coordination site” is probably more crucial for the redox reaction that generates ROS by the Fenton reaction itself 39 , 46 , 47 . Thus, EDTA incompletely shields the surface of iron ion and forms an open complex( ‘basket complex’) that allows generating iron ion-catalyzed generation of ROS 39 . In contrast to iron ion, copper ion apparently forms ‘stable complex’ with EDTA 48 that completely shields copper ion and does not allow redox reaction to generate ROS 39 . In a good agreement with this idea, our present data clearly suggest that addition of EDTA completely represses the copper ion-induced ROS generation when EDTA was present at equimolar to that of copper ion, even when EDTA promoted the iron ion-induced generation of the radical at the concentration. These results suggest that EDTA, when present at more than equimolar amount to that of copper ion, does not allow the metal ion-dependent Fenton-like reaction, by tightly binding copper ion, making its contrasting effect to iron ion to promote the ROS generation. The difference may be derived from the difference of structure of metal ion-EDTA complex in which EDTA incompletely shield the surface of iron ion and completely shield that of copper ion, as proposed by Flora et al 39 . This mechanism allows the differential regulation by EDTA toward metal ion-dependent redox reaction. This possibility awaits to be examined in the future. Iron(II)-citrate complex is ubiquitous in many types of organism 12 and has been shown as an important catalyst in generating ROS in vivo 25 , 49 . However, compared to the well-examined iron (II)-EDTA complex, the chemistry of the oligomeric complexes of iron (II)-citrate seems to be complex, which depends on the solution pH value and the mole ratio of iron:citric acid 28 , 50 . When Engelmann MD et al. examined how addition of metal chelators (EDTA or citric acid) affects the electrocatalytic reduction of hydrogen peroxide, they found that addition of EDTA or citric acid similarly increased the current when these ligands were added at a ratio of 1:1 to iron ion, suggesting that EDTA or citric acid enhanced the reduction of hydrogen peroxide, i. e. , the generation of hydroxyl radical 24 . They also found that EDTA represses the Fenton-reaction when present at 10 times higher of iron and that citric acid did not marginally affect the Fenton reaction when present at 10 times higher of iron 24 . We found in the present study that EDTA optimally enhanced the 7-OHCCA–derived fluorescence signal intensity, which represents the hydroxyl radical generation, at 10-fold to the iron ion concentration and that citric acid optimally enhanced the 7-OHCCA-derived signal when present at 10 to 40-fold to the iron concentration. We also found that citric acid decreased the 7-OHCCA-derived signal to the control level (iron ion and hydrogen peroxide only) at 80-fold to the iron concentration, whereas we were not able to determine at which EDTA concentration the 7-OHCCA derived signal falls down to the control level. In contrast, the 7-OHCCA-derived signal fell down to the control level, when EDTA or citric acid was present at 10-times or equimolar to that of copper ion, respectively, in the copper ion and hydrogen peroxide-induced ROS generation. These results suggest that excess amount of EDTA or citric acid allows the Fenton reaction when present at more than 10-times to that of iron ion, which is much broader than that observed by Engelmann MD (equimolar or 10-times of iron ion for EDTA or citric acid, respectively). The difference of Engelmann MD and our data may be derived from that in the experimental procedures. Our data suggest that citric acid represses the iron ion-dependent ROS generation that requires the ligand as much as 80-times and the copper ion-dependent ROS generation that requires only equimolar amount. Although we cannot completely exclude the possibility that citric acid behaves as hydroxyl radical scavenger, these results suggest that citric acid behaves as an efficient chelator to copper ion, not iron ion. This idea is proposed by Martinez A et al . 26 . Further studies are required to reveal the structure and function of copper-citric acid complex. Methods Chemicals Ethylenediaminetetraacetic acid (EDTA) was purchased from Nakalai Tesuque (Kyoto, Japan). Citric acid, tartaric acid, succinic acid, and malic acid were purchased from Fujifilm Wako Pure Chemical Corp. (Osaka, Japan). Coumarin-3-carboxylic acid (3CCA) was purchased from Tokyo Chemical Industry (Tokyo, Japan). Hydroxyl radical production A molecular probe, coumarin-3-carboxylic acid (3CCA) was used to evaluate generation of hydroxyl radical in aqueous solution, by measuring the fluorescence intensity of 7-hydroxycoumarin − 3-carboxylic acid (7OHCCA), the major hydroxylation product of irradiated 3-CCA, according to the methods reported by Manevich Y et al 38 . Experiments were initiated by addition of FeCl 2 (II) in 50 mM phosphate sodium-150 mM NaCl (pH7.4) buffer containing 1.0 mM hydrogen peroxide and 0–1 mM EDTA or organic acids (citric acid, malic acid, tartaric acid, succinic acid) in black coated 96-well microplates (FluoroNunc 96-Well Microplates, ThermoFisher), using a microplate reader (Synergy4, BioTek, USA). The excitation/emission wavelengths were set 388/450 nm based on previous publications. Oxidative degradation of bovine serum albumin (BSA). In order to assess the oxidation-induced degradation of BSA, BSA was incubated similarly as reported 52 , followed by native-PAGE analysis 53 . Shortly, BSA (1 µg) was incubated in the presence of either 10 µM copper (II) chloride or iron (II) chloride, 5000 µM hydrogen peroxide, in the presence of 0.1- 2.0 mM citric acid or 0.01–0.1 mM EDTA in 50 mM phosphate sodium-150 mM sodium chloride buffer (pH 7.4) at 37 ◦ C for 1h. Portion of the reaction mixture was then applied to native (non-denaturating)-polyacrylamide -gel (10% (w/v)) electrophoresis at a constant current of 20 mA per gel for 100 min using an electrophoresis system (Taitec, Tokyo, Japan), followed by staining with Coomassie Blue R-250. Statistics The data are expressed as mean ± SD. The data were analyzed for statistical significances using Student’s t-test. Difference was assessed with one-side test. The statistical significance was set at p < 0.05. Declarations Author Contributions Y.K. conceived the concept, designed the study which was supervised by A.M., H.O. and H.K. Y. K, Y. H. and T. N wrote the main manuscript text and Y.K. prepared all the figures. All the authors contributed to manuscript preparation and reviewed it. Conflict of Interest Statement We have no conflicts of interest to declare. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Data Availability Statement The datasets used and analyzed during the current study will be available from the corresponding author on reasonable request. Acknowledgements We thank Mrs. Ryunosuke Sato and Osuke Tajima for their technical assistance. References Xia, Y. et al. Ferric citrate-induced colonic mucosal damage associated with oxidative stress, inflammation responses, apoptosis, and the changes of gut microbial composition. Ecotoxicol. Environ. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5163186","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":370425037,"identity":"02adf939-d2a5-4ad0-b09f-94bfbacd4f91","order_by":0,"name":"Yoichi Kurokawa","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA10lEQVRIiWNgGAWjYBACCSBmbAAS/MwHICJgHlFaJNsSSNVicCyBSIdJzsh9+HBGzWF542O8xyQYauwYmGcTsEZaIt3YcMOxw4bbjvGlSTAcS2ZgnHMAvxY5iTQ2yQdstxm33e8xk2BgO8DAOIOACyFa/t2239zGA9Tyjwgt0iAtG9tuJ25gA2phbCNCi2TPM2bDmX3/k2cc40u2SOxL5iHoF4njaYwPe76l2fa38R688eGbnZwhoRBDAjwMDEAn8RjOIFoHSAsIyEsQr2UUjIJRMApGBgAAFP9Aw9E5oV4AAAAASUVORK5CYII=","orcid":"","institution":"Fukui Prefectural University","correspondingAuthor":true,"prefix":"","firstName":"Yoichi","middleName":"","lastName":"Kurokawa","suffix":""},{"id":370425038,"identity":"29624a46-11a3-4c9e-92d0-e79a43c6b47c","order_by":1,"name":"Atsushi Matsuzawa","email":"","orcid":"","institution":"Tohoku University","correspondingAuthor":false,"prefix":"","firstName":"Atsushi","middleName":"","lastName":"Matsuzawa","suffix":""},{"id":370425039,"identity":"52d03ef4-9415-4a51-8e55-124f4be18d47","order_by":2,"name":"Hirotaka Ogawa","email":"","orcid":"","institution":"Nagoya Industrial Science Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Hirotaka","middleName":"","lastName":"Ogawa","suffix":""},{"id":370425041,"identity":"4a2e66b0-f07e-4005-bb2a-3d07fc110ece","order_by":3,"name":"Yusuke Hirata","email":"","orcid":"","institution":"Tohoku University","correspondingAuthor":false,"prefix":"","firstName":"Yusuke","middleName":"","lastName":"Hirata","suffix":""},{"id":370425042,"identity":"90ab730e-87ba-47af-a87d-51004f4a9907","order_by":4,"name":"Takuya Noguchi","email":"","orcid":"","institution":"Tohoku University","correspondingAuthor":false,"prefix":"","firstName":"Takuya","middleName":"","lastName":"Noguchi","suffix":""},{"id":370425043,"identity":"124e4cb2-b41d-4ae6-a73d-7cee3c2bfbf4","order_by":5,"name":"Hiroki Kawashima","email":"","orcid":"","institution":"Nagoya University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Hiroki","middleName":"","lastName":"Kawashima","suffix":""}],"badges":[],"createdAt":"2024-09-27 07:38:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5163186/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5163186/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":67620457,"identity":"e9376c02-8ad4-4c2c-8e3b-6c676f1ab6e7","added_by":"auto","created_at":"2024-10-28 07:02:58","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":907913,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCopper (II) chloride is a more potent hydroxyl radical generator than iron (II) chloride in the presence of hydrogen peroxide.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;(A, B) 3CCA (1.0 mM) was incubated either with 1.0 mM hydrogen peroxide in the presence of indicated concentration of CuCl\u003csub\u003e2\u003c/sub\u003e (A) or FeCl\u003csub\u003e2\u003c/sub\u003e (B) in 50 mM phosphate-150 mM sodium chloride buffer (pH7.4) in 96-well black plate (n=3) at 37℃ for 1 h. 7-hydroxycoumarin carboxylic acid (7OHCCA) -derived fluorescence signal (ex./em. = 388/450 nm) were measured with Synergy H4 Hybrid Microplate Reader (Agilent/BioTek, Sant Clara, USA). Mean value and standard deviations were calculated (n=3).\u003c/p\u003e\n\u003cp\u003e(C, D) The concentration of CuCl\u003csub\u003e2\u003c/sub\u003e (C) or FeCl\u003csub\u003e2\u003c/sub\u003e (D) has a good linearity with the 7OHCCA-derived fluorescence signal intensity. Experiments were repeated at least three times.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5163186/v1/efdfffdf21136bc42fa75075.jpeg"},{"id":67620463,"identity":"cd8062c1-88d5-47de-a65d-21bf2c83e947","added_by":"auto","created_at":"2024-10-28 07:02:58","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":333249,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEDTA and citric acid enhances the iron (II) chloride-dependent generation of hydroxyl radical.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExperiment was performed according to basically the same methods as described in the legends to Fig.1. Shortly, 3CCA (1.0 mM) was incubated either with 1.0 mM hydrogen peroxide in the presence of 0.1 mM CuCl\u003csub\u003e2\u003c/sub\u003e or 1.0 mM FeCl\u003csub\u003e2\u003c/sub\u003e and indicated concentration of organic acids or EDTA in 50 mM phosphate-150 mM sodium chloride buffer (pH7.4) in 96-well black plate (n=3) at 37℃ for 1 h. 7-hydroxy coumarin carboxylic acid-derived fluorescence signal were measured and analyzed similarly with the legends to Fig.1 (†\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.1, * \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003e P \u003c/em\u003e\u0026lt; 0.01, compared to the control (containing hydrogen peroxide and iron chloride).\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5163186/v1/ce3bbfd8f704658a2691990f.jpeg"},{"id":67622092,"identity":"29a844fe-527a-4c49-8ecf-d93b08b28445","added_by":"auto","created_at":"2024-10-28 07:10:58","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":548905,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEDTA or citric acid enhances the iron (II) chloride-dependent generation of hydroxyl radical, and represses the copper (II) chloride-dependent generation of the radical.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExperiment was performed according to basically the same methods as described in the legends to Fig.2, except that the sample contained 1.0 mM hydrogen peroxide, 0.1 mM CuCl\u003csub\u003e2\u003c/sub\u003e (A, C) or FeCl\u003csub\u003e2\u003c/sub\u003e (B, D) and indicated concentration of citric acid or EDTA. Note that the addition of EDTA (0.1 mM) to the sample containing iron chloride and hydrogen peroxide increased the 7-OHCCA fluorescence signal intensity to about 38000, which was a comparable level to that containing copper chloride and hydrogen peroxide and that the addition of higher concentration of the compound decreased the signal intensity of the sample containing iron chloride and hydrogen peroxide. Experiments were performed and analyzed similarly with the legends to Fig.2 (†\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.1, * \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003e P \u003c/em\u003e\u0026lt; 0.01, *** \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001 compared to the respective control (containing hydrogen peroxide and iron chloride or copper chloride)).\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5163186/v1/1651014c99b513748f8a7cee.jpeg"},{"id":67620458,"identity":"9c7719d5-8da8-4626-ae05-fe348e74cf42","added_by":"auto","created_at":"2024-10-28 07:02:58","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":449500,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eL-histidine or histamine enhances the copper (II) chloride-dependent generation of hydroxyl radical, and hardly affects the iron (II) chloride-dependent generation of the radical at concentration that is lower than that of metal ion.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExperiment was performed according to basically the same methods as described in the legends to Fig. 3.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5163186/v1/bd5e48defff9af128e7336c2.jpeg"},{"id":67620462,"identity":"2e0c0bc8-32dd-4e88-885d-33f80c4c01d6","added_by":"auto","created_at":"2024-10-28 07:02:58","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":845240,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEDTA or citric acid enhances the iron (II) chloride-dependent hydrogen peroxide-induced degradation of BSA, and represses the copper (II) chloride-dependent degradation of BSA.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBSA (1.0 mM) was incubated with 1.0 mM hydrogen peroxide in the presence of 0.01 mM CuCl\u003csub\u003e2\u003c/sub\u003e (A) or 1.0 mM FeCl\u003csub\u003e2\u003c/sub\u003e (B) and indicated concentration of citric acid or EDTA in 50 mM phosphate-150 mM sodium chloride buffer (pH7.4) in Eppendorf tubes at 37℃ for 1 h, followed by native-PAGE analysis (20 mA/gel, 100 min) and gel staining and destaining.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5163186/v1/4d85b113d3803758dfc77883.jpeg"},{"id":79067212,"identity":"288b97ea-566e-4d14-821b-c9fbbd53f370","added_by":"auto","created_at":"2025-03-24 04:46:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4002684,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5163186/v1/8ea00166-f2a5-405c-a371-b317d6b5ad15.pdf"},{"id":67622091,"identity":"19db367e-c6d4-49cf-8b96-3956fdc4d76c","added_by":"auto","created_at":"2024-10-28 07:10:58","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":98610,"visible":true,"origin":"","legend":"","description":"","filename":"Sci.Rep2024TableS14Oct.24.docx","url":"https://assets-eu.researchsquare.com/files/rs-5163186/v1/7816be369b54f37ac08857ca.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Adverse roles of citric acid and L-histidine in the transition metal-dependent generation of hydroxyl radical at circumneutral pH","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIron is the second most abundant metal, and an essential element for many organisms including bacteria, plant and animals. Iron participates in many important roles in metabolic pathways, redox reactions and electron transport chain mechanism, in order to sustain biological homeostasis. On the other hand, excess amount of iron can be highly toxic and lead to life-threatening conditions such as cancer, diabetes, liver disease, cardiovasucular disease and heart failure\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIron participates in the generation of deleterious OH*\u0026mdash;arguably the most injurious ROS species \u003cem\u003ein vitro\u003c/em\u003e, by the Fenton reaction (Fe(II)-catalyzed H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e oxidation process), thus making a toxic feature of iron\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The Fenton reaction also has a great role in trace metal and natural organic matter biogeochemistry, its utility in water treatment and its role in oxidative cell degradation and associated human disease\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. However, the Fenton reaction is effective at acidic pH, not at neutral or basic pH, because iron can form hydroxide with low solubility at high pH conditions and decreases its ability to generate ROS by Fenton reaction. Therefore, the Fenton reaction requires chelators to solubilize otherwise precipitable iron ion at circumneutral pH. EDTA, citric acid or polyhydric organic acids have been reported to enhance the production of OH* \u003csup\u003e4\u0026ndash;8\u003c/sup\u003e. The Fenton-like reaction, involving the catalytic interplay of copper ions with hydrogen peroxide \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, serves as another prototypical system for chemical ROS generation in a manner which does not require a metal chelator. We are interested to find a suitable and biologically relevant chelator which promotes or represses the metal ion-dependent generation of OH* and also to examine the effective concentration of such a chelator as promoter or repressor of the radical generation, in order to examine a possible mechanism for the generation of the radical \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eCitric acid is ubiquitous in nature, and has various biological functions in energy production and iron metabolism in animal and plant. In plants, citric acid is present in root exsudates and is responsible for solubilizing the ferric hydroxide in the soils to mobilize the iron to the plant root cells\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eBradyrhizobium japonicum\u003c/em\u003e has been shown to release citric acid under iron-deficient growth conditions\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. In animals, citric acid has various essential functions and regulates the homeostasis of iron \u003cem\u003ein vivo\u003c/em\u003e as citrate\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e : citric acid has also been proposed to be a constituent of the low molecular weight cytosolic iron pool\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e and non-transferrin-bound iron has been proposed to exist largely as complexes with citric acid in the plasma of iron-overloaded patients\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Yet, citric acid is detrimental when present in excess amount: although the anti-oxidative or anti-tumor role of citric acid is reported\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, lines of evidence have shown that it is involved in the inflammation responses\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e or the onset of liver-disease like MASLD (metabolic dysfunction-associated steatotic liver disease) \u003csup\u003e\u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e or cancer \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. The toxic aspects of citric acid can be in part explained by its ability to promote the generation of OH* \u003csup\u003e21\u003c/sup\u003e. Several reports have been made with citrate-iron complex in generating OH* \u003csup\u003e2, \u003cspan additionalcitationids=\"CR23 CR24\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, whereas citric acid is reported to repress the copper ion-dependent ROS generation, as scavenger of ROS\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Thus, citric acid has been reported to have dual roles as promoter and repressor for the generation of OH*.\u003c/p\u003e \u003cp\u003eDoes excess amount of citric acid, a metal chelator, prevent the iron-dependent ROS generation, even when they promote it at low concentration? What is the effective concentration of the ligand to serve as either promoter or repressor of the generation of the radical? According to the reports by Engelmann, metal chelators (EDTA or citric acid) differently affected the electrocatalytic reduction of hydrogen peroxide, depending on the relative concentration of the chelators to that of metal ion: they found that the reduction of hydrogen peroxide was repressed when EDTA was added at a ratio of 5-fold to iron ion, and that it was repressed when citric acid was added at a ratio of more than 50-fold to that of iron\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. These differences may be in part due to the change of the structure of citrate-iron complex according to the conditions such as pH or temperature in aqueous solution\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCompared to the well-studied role of iron (II)-EDTA or citric acid complex on the production of ROS, relatively little is known on the role of other organic acid or L-histidine as iron complex, including the optimum ratio of these acids or histidine /iron in generating ROS, when iron(II)-histidine complex is the most major iron (II) species in the blood plasma of about 15%, followed by iron (II)-citrate complex of about 10% \u003csup\u003e29\u003c/sup\u003e. However, the detailed role of the iron (II)-L-histidine remains unknown. One possible explanation is the ambiguous role of L-histidine in regulating the metal ion-dependent ROS production. Several lines of evidences suggest that L-histidine represses the copper ion-induced generation of ROS generation\u003csup\u003e\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e by \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003etightly binding copper ion\u003c/span\u003e and inhibiting the copper ion-dependent generation of ROS\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. On the other hand, L-histidine is also reported to induce the ROS production \u003cem\u003ein vivo\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. However, its detailed mechanism has not been known, including whether the ROS generation depends on metal ion including copper ion. Thus, although L-histidine is reported to have an ambiguous role in the metal ion-dependent ROS production, there have been no report, to our best knowledge, on the information regarding the effective concentration at which the ligand either promote or inhibit the copper or iron ion-dependent ROS generation. Likewise, histamine, decarboxylated L-histidine, is shown to produce oxidative stress or to cause inflammation\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e and is suggested to suppress the ROS generation\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Thus, we are interested to examine whether organic acids, L-histidine or histamine either promote or repress the metal ion-induced generation of ROS, depending on the ratio of ligand/iron.\u003c/p\u003e \u003cp\u003eWe are interested to examine and compare the effects of organic acids (citric aid, malic acid, succinic acid, tartaric acid and L-histidine) on the iron- or copper ion-induced generation of OH* and also on the degradation of biopolymer. We examine in the present study whether these organic acids promote the Fenton reaction-induced generation of OH* and evaluate the molar ratio of ligand/Fe(II) or Cu(II)- catalyzed oxidation of O\u003csub\u003e2\u003c/sub\u003e or H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e., by using 3-hydroxycoumarin carboxylic acid (3CCA)\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e as a conventional and less time-consuming detector of OH* or H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. We found the possibility that L-histidine or histamine promotes the copper (II) ion-dependent generation of OH*, but not the iron (II) ion-dependent generation of the ROS, which may have a closely relation with onset of some diseases. A new finding on the role of carboxylic acids will be discussed in promoting and suppressing heavy metal ion-dependent ROS generation.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cem\u003eFluorescence intensity of 7-hydroxycoumarin-3-carboxylic acid (7OHCCA) differently increased in the presence of both hydrogen peroxide and copper (II) chloride or iron (II) chloride.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eWe first measured the effects of copper (II) chloride or iron (II) chloride on the production of hydroxyl radical when measured with 3CCA, a specific indicator for hydroxyl radical. We incubated 3CCA in the presence of hydrogen peroxide and either copper (II) chloride or iron (II) chloride, and measured the fluorescence intensity of 7OHCCA, 3CCA hydroxylation product. When the control sample (3CCA alone) was incubated and measured for 7OHCCA-specific fluorescence intensity, the fluorescence intensity was hardly detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e1\u003c/span\u003eA lane 1; Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). When 3CCA was treated with hydrogen peroxide and increasing concentration of copper chloride (1\u0026ndash;10 \u0026micro;M), the 7OHCCA-specific fluorescence intensity increased significantly according to the concentration of copper chloride: from about 6600 (1 \u0026micro;M) to about 23000 (10 \u0026micro;M) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e1\u003c/span\u003eA lanes 2\u0026ndash;6; Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA ). In contrast, much more concentration of iron (II) chloride was required to reach the comparable level of 7OHCCA intensity in the presence of copper (II) chloride: when 3CCA was incubated with hydrogen peroxide and increasing concentration of iron chloride (100\u0026ndash;1000 \u0026micro;M), the 7OHCCA-specific fluorescence intensity increased from about 4000 (100 \u0026micro;M) to about 17000 (1000 \u0026micro;M)(Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e1\u003c/span\u003eB lanes 1\u0026ndash;6; Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). A strong, good relationship between the concentration of metal salt and the fluorescent signal intensity of 7OHCCA was observed with copper chloride or iron chloride, and hydrogen peroxide (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). These results suggest that hydroxyl radical is generated in the presence of hydrogen peroxide and either copper chloride or iron chloride, although the latter chemical was much less efficient than the former.\u003c/p\u003e \u003cp\u003e \u003cem\u003eSearch for organic acids that enhances fluorescence intensity of 7OHCCA incubated in the presence of both hydrogen peroxide and iron (II) chloride.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eEDTA was known to promote the iron/hydrogen peroxide-induced generation of hydroxyl radical\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Citric acid is also known to promote the generation of the radical in the presence of iron/hydrogen peroxide\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Because both EDTA and citric acid are polyhydric carboxylic acids containing multiple carboxylic groups that may bind metal ion, we were interested in examining the effects of series of polyhydric carboxylic acids (EDTA, citric acid, malic acid, tartaric acid and succinic acid) and L-histidine that contain different numbers of carboxylic groups.\u003c/p\u003e \u003cp\u003eWhen iron chloride was incubated alone, the 7-OHCCA fluorescence signal intensity was only about 370 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003e lane 2; Table S2). When iron chloride was incubated with hydrogen peroxide, the signal increased to about 15800 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003e lane 4; Table S2), which was about half of the sample containing copper chloride and hydrogen peroxide (about 38000) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003e lane 15; Table S2). When EDTA was further added with iron and hydrogen peroxide, the signal significantly increased to 80000\u0026thinsp;\u0026minus;\u0026thinsp;10000 at 0.1 or 1 mM of EDTA (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003e lanes 13 and 14; Table S2), suggesting that iron chloride becomes more efficient hydroxyl radical generator than copper chloride in the presence of iron. Similar effect was observed with citric acid at 1.0 mM (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003e lane 6; Table S2). No significant effects were observed with tartaric acid, malic acid or succinic acid.\u003c/p\u003e \u003cp\u003e \u003cem\u003eOpposing effects of EDTA and citric acid in enhancing or decreasing the fluorescence intensity of 7OHCCA incubated in the presence of both hydrogen peroxide and iron (II) chloride or copper (II) chloride.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eWe found that EDTA and citric acid increase the 7-OHCCA derived fluorescent signal that represents the generation of hydroxyl radical in the presence of iron chloride and hydrogen peroxide. We next examined whether these compounds promote the signal in the presence of copper chloride and hydrogen peroxide.\u003c/p\u003e \u003cp\u003eWhen iron chloride and hydrogen peroxide was incubated with 3CCA, the 7-OHCCA fluorescence intensity was about 7000 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e3\u003c/span\u003eA lane 2; Table S3A), which was less than one fifth of that of the sample containing copper chloride and hydrogen peroxide (about 36000) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e3\u003c/span\u003eB lane 2; Table S3B). When citric acid was further added with iron and hydrogen peroxide, the signal significantly increased to 8000\u0026ndash;10000 at 0.1 or 1 mM of citric acid (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e3\u003c/span\u003eA lanes 3 and 7; Table S3A). EDTA more efficiently increased the signal than citric acid: when EDTA was further added with iron and hydrogen peroxide, the signal significantly increased to 20000\u0026ndash;40000 at 0.01\u0026ndash;0.1 mM of EDTA (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e3\u003c/span\u003eA lanes 8\u0026ndash;12; Table S3A), suggesting that EDTA more efficiently generates hydroxyl radical than citric acid. In contrast, when copper chloride and hydrogen peroxide was incubated in the presence of 3CCA, EDTA and citric acid significantly decreased the 7-OHCCA-derived fluorescence signal intensity: when citric acid was added with copper chloride and hydrogen peroxide, the intensity significantly decreased from 26000 (0.1 mM) to about 3000 (1 mM) of citric acid (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e3\u003c/span\u003eB lanes 3 and 7; Table S3B). EDTA hardly affected the signal intensity from 0.01 to 0.04 mM (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e3\u003c/span\u003eB lanes 8\u0026ndash;10), whereas it decreased the signal about to 17000 (0.08 mM) and 1400 (0.1 mM) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e3\u003c/span\u003eB lanes 11 and 12; Table S3B).\u003c/p\u003e \u003cp\u003eWe next examined how addition of citric acid or EDTA of more than 1 mM affects the metal ion and hydrogen peroxide-induced 7-OHCCA-derived fluorescence intensity: addition of 1\u0026ndash;4 mM citric acid only marginally affected the signal level of the sample containing iron chloride and hydrogen peroxide, whereas addition of 8 mM of the compound decreased the signal level to the that of the control (iron ion and hydrogen peroxide)(Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e3\u003c/span\u003eC lanes 4\u0026ndash;7; Table S3C), suggesting that the optimum hydroxyl radical-promoting concentration of citric acid is about 1\u0026ndash;2 mM and that the generation of the radical is repressed at 8 mM. In contrast, addition of citric acid significantly decreased the signal of the sample containing copper chloride and hydrogen peroxide on a dose-dependent manner, almost to the control level (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e3\u003c/span\u003eD lanes 4\u0026ndash;7; Table S3D). Addition of 1 mM EDTA increased the 7-OHCCA derived fluorescence intensity to about 60000 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e3\u003c/span\u003eC Lane 8; Table S3C). Addition of higher concentration of EDTA decreased the signal about to 40000 at 8 mM (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e3\u003c/span\u003eC lane 11; Table S3C), suggesting that the optimum hydroxyl radical-promoting concentration of EDTA is about 1 mM in the iron chloride and hydrogen peroxide-dependent generation of hydroxyl radical and that EDTA represses generation of the radical at the concentration of higher than 1 mM. In contrast, the 7-OHCCA derived fluorescence intensity remained less than 1000 when EDTA of higher than 1 mM was added to the sample containing copper chloride and hydrogen peroxide, suggesting that EDTA inhibits generation of the radical under the experimental conditions employed. Taken together, these results suggest that citric acid and EDTA promote the iron-hydrogen peroxide-induced generation of hydroxyl radical at the ligand/metal ion mole ratio of less than equimolar to twice-molar (citric acid) and less than 10 times-molar (EDTA) and that they repress the copper-hydrogen peroxide-induced generation of the radical.\u003c/p\u003e \u003cp\u003e \u003cem\u003eL-histidine enhances the fluorescence intensity of 7OHCCA incubated in the presence of hydrogen peroxide and copper (II) chloride at concentration lower than that of copper ion, when it hardly affected the fluorescence intensity of 7OHCCA incubated in the presence of iron (II) chloride.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eWe next examined how the addition of L-histidine or histamine, which have imidazole moiety with metal chelating ability, affects the 7-OHCCA-derived fluorescence intensity when iron chloride and hydrogen peroxide was incubated in the presence of 3CCA. When iron chloride was incubated in the presence of hydrogen peroxide, the 7OHCCA-derived fluorescence intensity increased to about 5000 (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e4\u003c/span\u003eA lane 3; Table S4A). When L-histidine or histamine (0.05\u0026ndash;0.4 mM) was further added to the sample containing iron chloride and hydrogen peroxide, the 7OHCCA-derived fluorescent hardly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e4\u003c/span\u003eA lanes 4\u0026ndash;7, 8\u0026ndash;11; Table S4A), which makes a sharp contrast to EDTA that enhanced the iron/hydrogen peroxide-dependent increase of 7OHCCA signal intensity at 0.01-1.0 mM (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e4\u003c/span\u003eA lanes 12\u0026ndash;14; Table S4A) and decreased it at 10 mM (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e4\u003c/span\u003eA lane 15; Table S4A).\u003c/p\u003e \u003cp\u003eIn contrast, when copper chloride was incubated in the presence of hydrogen peroxide, the 7OHCCA-derived fluorescent intensity increased to about 34000 (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e4\u003c/span\u003eB lane 3; Table\u0026nbsp;4A). The further addition of L-histidine (0.05\u0026ndash;0.1 mM) surprisingly increased the 7-OHCCCA derived signal level to about 70000 by about 2-fold (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e4\u003c/span\u003eB lanes 4, 5; Table S4B). Addition of histamine (0.05\u0026ndash;0.1 mM) also doubled the 7OHCCA-derived signal level obtained only in the presence of copper chloride and hydrogen peroxide (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e4\u003c/span\u003eB lanes 8\u0026ndash;9; Table S4B). Addition of L-histidine or histamine gave a sharp contrast to that of EDTA that did not increase and only marginally affected the 7-OHCCA derived signal at less than equimolar amount copper ion (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e4\u003c/span\u003eB lanes 12, 13; Table S4B), whereas addition of more than 10-times molar of EDTA almost completely repressed the 7OH-CCCA derived signal level (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e4\u003c/span\u003eB lanes 14, 15; Table S4B). These results suggest that L-histidine or histamine promoted the generation of hydroxyl radical that was induced by copper chloride and hydrogen peroxide when present at less than equimolar amount of that of copper ion and that these compounds repressed the generation of the radical when present at more than 4-times. Taken together, these results suggest that L-histidine and histamine effectively promotes the copper ion-induced hydroxyl radical generation and is less effective in promoting the iron ion-induced hydroxyl radical generation.\u003c/p\u003e \u003cp\u003e \u003cem\u003eOpposing effects of EDTA and citric acid in enhancing the degradation of BSA incubated in the presence of both of hydrogen peroxide and iron (II) chloride, and in preventing the degradation of BSA incubated in the presence of hydrogen peroxide and copper (II) chloride\u003c/em\u003e \u003c/p\u003e \u003cp\u003eWe finally examined whether the addition of EDTA or citric acid affects the metal-catalyzed degradation of BSA, a model protein. When BSA was incubated alone or in the presence of 5 mM hydrogen peroxide, followed by Native-PAGE analysis, the band of the native-form of BSA remained unaffected (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA lanes 1 and 2). When BSA was incubated in the presence of hydrogen peroxide and 1 mM iron (II) chloride, the native-form BSA band was detected to the same level as the control (BSA alone) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA lane 3). When BSA was incubated in the presence of hydrogen peroxide and iron chloride, and also 0.1\u0026ndash;0.4 mM citric acid, the native BSA band remained intact (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA lanes 4\u0026ndash;6). In contrast, when BSA was incubated in the presence of hydrogen peroxide, iron chloride and higher concentration of citric acid, the native BSA band was thin at 0.8 or 1.0 mM citric acid and was hardly detected at 2.0 mM (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA lanes 7\u0026ndash;9). A similar result was obtained when BSA was incubated in the presence of the oxidants and also EDTA instead of citric acid: the native BSA band was detected almost unaffected when BSA was incubated in the presence of the oxidants and 0-0.02 mM EDTA (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA lanes 10\u0026ndash;12). When BSA was incubated in the presence of higher concentration of EDTA, the native-form BSA band was thin at 0.04 or 0.08 mM EDTA and was hardly detected at 0.1 mM (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA lanes 13\u0026ndash;15).\u003c/p\u003e \u003cp\u003eTo note, the totally different results were obtained when BSA was incubated in the presence of copper (II) chloride instead of iron (II) chloride, and hydrogen peroxide as well as citric acid or EDTA. The native BSA band was detected to the similar level when BSA was incubated in the absence of any oxidant or in the presence of hydrogen peroxide (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB lanes 1 and 2). In contrast, when BSA was incubated in the presence of 0.01 mM copper chloride ad hydrogen peroxide, the native BSA band was not detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB lane 3), suggesting that BSA underwent degradation under metal-catalyzed oxidation in the presence of copper chloride and hydrogen peroxide. Upon addition of increased concentration of citric acid together with the oxidants (copper chloride and hydrogen peroxide), the native BSA band was clearly detected at more than 0.4 mM citric acid (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB lanes 6\u0026ndash;9), suggesting that BSA escaped from the metal-catalyzed degradation in the presence of citric acid. Likewise, upon addition of EDTA of more than 0.01 mM, the native BSA band clearly detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB lanes 11\u0026ndash;15), suggesting that EDTA is superior to citric acid in protecting BSA from copper chloride and hydrogen peroxide-induced degradation of BSA.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present study, we compared the effects of polyhydric acids (EDTA and citric acid) or L-histidine in iron (II) ion or copper (II) ion and hydrogen peroxide-induced generation of hydroxyl radical and examined the effective concentration at which these ligands either promote or repress the generation of the radical, in order to find a possible physiologically relevant chemical that regulates the generation of ROS \u003cem\u003ein vivo\u003c/em\u003e. We will discuss how differently these ligands act against metal ion by forming an ion/ligand complex that either promotes or represses the metal ion-catalyzed generation of hydroxyl radical. First, we found that EDTA with four carboxylic groups in a molecule promoted the iron (II) ion-hydrogen peroxide-induced generation of hydroxyl radical (when present at less than equimolar to iron (II) ion) and that EDTA repressed the copper (II) ion-hydrogen peroxide-induced generation of the radical at the same molar ratio of the ligand/metal ion. Second, we found that among organic acids with multiple carboxylic groups, citric acid with three carboxylic groups in a molecule promotes the iron (II) ion-hydrogen peroxide-induced generation of hydroxyl radical at concentration of 20-times or less in molar ratio to copper (II) ion and that it represses the radical generation at higher concentration, when other organic acid with two carboxylic groups (tartaric acid, malic acid, succinic acid) in a molecule neither promoted nor repressed the metal ion-induced generation of hydroxyl radical under the experimental procedures employed. Third, we found that L-histidine or histamine promoted the copper ion and hydrogen peroxide-induced generation of the radical at the concentration less than equimolar to that of copper ion, when these ligands hardly affected the iron ion-dependent generation of the radical. These findings suggest that polyhydric acids (EDTA, citric acid) and L-histidine or histamine have a contrasting effect in regulating the iron- or copper-ion dependent generation of hydroxyl radical: EDTA or citric acid promited the iron ion-dependent generation of the radical and repressed the copper ion-dependent generation of the radical, whereas L-histidine or histamine promoted the copper ion-dependent generation of the radical, depending on the ligand/metal ion ratio. These findings will give a fundamental knowledge on the transition metal ion-induced generation of ROS. We will discuss how differently EDTA, citric acid and L-histidine or histamine regulates the generation of hydroxyl radical and the relevance of our findings.\u003c/p\u003e \u003cp\u003eAlthough EDTA and citric acid was known to promote and repress the hydroxyl radical generation by Fenton reaction \u003cem\u003ein vitro\u003c/em\u003e, depending on the molar ratio of metal ion/ligand\u003csup\u003e2\u003c/sup\u003e, the mode of action of the ligands is apparently different: the optimum promoting ratio of the ligand/iron ion was reported to be about 1 for EDTA and citric acid, whereas the repressing ratio of the ligand/iron ion was about 5 for EDTA and about 50:1 for citric acid\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Similarly, the optimum ratio of the ligand/iron ion was about 0.5\u0026thinsp;~\u0026thinsp;1.0 for citric acid\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. The promoting ratio probably represents that these ligands solubilize iron ion which otherwise tends to form insoluble precipitate at circumneutral pH and is not involved in the ROS generation, and allows redox reaction at an equimolar ratio to that of metal ion by forming a \u0026lsquo;basket complex\u0026rsquo; \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. The difference of the repressing ratio may be ascribed to the difference of the ligand-metal ion structure\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, as will be discussed below. We found in the present study that repressing ratio of the ligand/iron ion was about 80 for citric acid, when we were not able to determine the accurate ratio (more than 80) for EDTA under the conditions employed (data not shown). The difference of ours and Engelman MD may be ascribed to the difference of the analytical method to measure the hydroxyl radical generation.\u003c/p\u003e \u003cp\u003eAs described in Introduction, the role of iron (II)-histidine complex in the regulation of ROS generation has been largely unknown, when L-histidine was reported to have dual opposing effects in generation of ROS: L-histidine was reported to repress the copper ion-induced generation of ROS\u003csup\u003e\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, and to induce the ROS production \u003cem\u003ein vivo\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, of which mechanism remains largely unknown. However, a similar dual role of L-histidine has been reported in the nickel ion-dependent generation of the radical: L-histidine was proposed to enhance the generation of hydroxyl radical\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e and also to scavenge the radical\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, depending on its concentration. We here discuss the possibility that L-histidine promotes and also represses the ROS generation, on a manner dependent on copper ion. Our present data clearly suggest that L-histidine enhances the copper ion-induced generation of hydroxyl radical at concentration of 2-times or less in molar ratio of the copper ion and that L-histidine represses it at more than 4-fold. Likewise, L-histidine was found to promote the nickel ion-dependent disproportionation of hydrogen peroxide, that is, hydroxyl radical generation at molar ratio below half concentration of nickel ion and to repress it at equimolar concentration\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Thus, L-histidine may similarly act against copper ion- or nickel ion-induced generation of the radical, when their effective concentration differed, possibly by the difference of the metal ion-ligand interaction or the experimental procedures employed. Our data also suggest that L-histidine hardly promotes the iron ion-induced generation of the radical. These results are in good accordance with the idea that L-histidine tightly forms a complex with iron ion that does not allow the ROS generation, which is proposed by Zabek-Adamska\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e and that L-histidine can generate ROS when not efficient amount of the ligand is available to chelate copper ion.\u003c/p\u003e \u003cp\u003eThe same is true with the role of histamine in regulating the generation of the radical. Histamine was reported to produce oxidative stress, to cause inflammation and also to repress the generation of ROS\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Our present data suggests that histamine promotes the copper ion-induced generation of hydroxyl radical at the concentration less than equimolar of the copper ion and that histamine represses it at more than 2-fold, when histamine hardly promotes the iron ion-induced generation of the radical. These results may be that histamine has a dual role in promoting and repressing the metal ion-dependent ROS generation. This idea is in a good accordance with the previous reports: histamine is known to stimulate neutrophil ROS production\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, has been known as a cause of neurodegeneration inflammation as well as neuroprotector\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo our best knowledge, there have been no reports on the L-histidine or histamine-induced hydroxyl radical generation which is driven by copper ion and hydrogen peroxide. We believe that these ligands regulate the copper ion-dependent generation of the radical, by a similar mechanism of L-histidine regulating the nickel ion-dependent generation of hydroxyl radical\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, depending on its concentration. Further studies are required to reveal whether L-histidine or histamine promotes or represses the generation of ROS, which will give a fundamental knowledge on the possible medical phenomenon such as inflammation or the onset of a disease.\u003c/p\u003e \u003cp\u003eAs discussed above, our present data suggest that polyhydric acids (EDTA or citric acid) differently behaved toward the metal ion-dependent generation of hydroxyl radical. Let us first consider the difference of the structure of iron ion- or copper ion-complex with a chelator EDTA that either allows or represses a redox reaction for generation of ROS, and also the structure of the metal ion-citric acid complex.\u003c/p\u003e \u003cp\u003eIron occurs in biological systems in the form of ferrous or ferric ions. These ions can form six coordination bonds with ligands that are able to donate electron pair\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. A metal chelator EDTA is a potential hexadentate ligand and can possibly occupy all coordination sites of iron ion, forming six coordination bonds. However, EDTA has been suggested to be too small to completely encompass all the coordination sites of iron ions. As a result, a seventh coordination site is generated in EDTA, which is occupied by water molecule that is readily exchangeable by oxidant/reductant. The ligand in the \u0026ldquo;free coordination site\u0026rdquo; is probably more crucial for the redox reaction that generates ROS by the Fenton reaction itself \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Thus, EDTA incompletely shields the surface of iron ion and forms an open complex( \u0026lsquo;basket complex\u0026rsquo;) that allows generating iron ion-catalyzed generation of ROS\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn contrast to iron ion, copper ion apparently forms \u0026lsquo;stable complex\u0026rsquo; with EDTA\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e that completely shields copper ion and does not allow redox reaction to generate ROS\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. In a good agreement with this idea, our present data clearly suggest that addition of EDTA completely represses the copper ion-induced ROS generation when EDTA was present at equimolar to that of copper ion, even when EDTA promoted the iron ion-induced generation of the radical at the concentration. These results suggest that EDTA, when present at more than equimolar amount to that of copper ion, does not allow the metal ion-dependent Fenton-like reaction, by tightly binding copper ion, making its contrasting effect to iron ion to promote the ROS generation. The difference may be derived from the difference of structure of metal ion-EDTA complex in which EDTA incompletely shield the surface of iron ion and completely shield that of copper ion, as proposed by Flora \u003cem\u003eet al\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. This mechanism allows the differential regulation by EDTA toward metal ion-dependent redox reaction. This possibility awaits to be examined in the future.\u003c/p\u003e \u003cp\u003eIron(II)-citrate complex is ubiquitous in many types of organism\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e and has been shown as an important catalyst in generating ROS \u003cem\u003ein vivo\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. However, compared to the well-examined iron (II)-EDTA complex, the chemistry of the oligomeric complexes of iron (II)-citrate seems to be complex, which depends on the solution pH value and the mole ratio of iron:citric acid\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. When Engelmann MD \u003cem\u003eet al.\u003c/em\u003e examined how addition of metal chelators (EDTA or citric acid) affects the electrocatalytic reduction of hydrogen peroxide, they found that addition of EDTA or citric acid similarly increased the current when these ligands were added at a ratio of 1:1 to iron ion, suggesting that EDTA or citric acid enhanced the reduction of hydrogen peroxide, \u003cem\u003ei. e.\u003c/em\u003e, the generation of hydroxyl radical\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. They also found that EDTA represses the Fenton-reaction when present at 10 times higher of iron and that citric acid did not marginally affect the Fenton reaction when present at 10 times higher of iron\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. We found in the present study that EDTA optimally enhanced the 7-OHCCA\u0026ndash;derived fluorescence signal intensity, which represents the hydroxyl radical generation, at 10-fold to the iron ion concentration and that citric acid optimally enhanced the 7-OHCCA-derived signal when present at 10 to 40-fold to the iron concentration. We also found that citric acid decreased the 7-OHCCA-derived signal to the control level (iron ion and hydrogen peroxide only) at 80-fold to the iron concentration, whereas we were not able to determine at which EDTA concentration the 7-OHCCA derived signal falls down to the control level. In contrast, the 7-OHCCA-derived signal fell down to the control level, when EDTA or citric acid was present at 10-times or equimolar to that of copper ion, respectively, in the copper ion and hydrogen peroxide-induced ROS generation. These results suggest that excess amount of EDTA or citric acid allows the Fenton reaction when present at more than 10-times to that of iron ion, which is much broader than that observed by Engelmann MD (equimolar or 10-times of iron ion for EDTA or citric acid, respectively). The difference of Engelmann MD and our data may be derived from that in the experimental procedures.\u003c/p\u003e \u003cp\u003eOur data suggest that citric acid represses the iron ion-dependent ROS generation that requires the ligand as much as 80-times and the copper ion-dependent ROS generation that requires only equimolar amount. Although we cannot completely exclude the possibility that citric acid behaves as hydroxyl radical scavenger, these results suggest that citric acid behaves as an efficient chelator to copper ion, not iron ion. This idea is proposed by Martinez A \u003cem\u003eet al\u003c/em\u003e.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Further studies are required to reveal the structure and function of copper-citric acid complex.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eChemicals\u003c/h2\u003e \u003cp\u003eEthylenediaminetetraacetic acid (EDTA) was purchased from Nakalai Tesuque (Kyoto, Japan). Citric acid, tartaric acid, succinic acid, and malic acid were purchased from Fujifilm Wako Pure Chemical Corp. (Osaka, Japan). Coumarin-3-carboxylic acid (3CCA) was purchased from Tokyo Chemical Industry (Tokyo, Japan).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHydroxyl radical production\u003c/h3\u003e\n\u003cp\u003eA molecular probe, coumarin-3-carboxylic acid (3CCA) was used to evaluate generation of hydroxyl radical in aqueous solution, by measuring the fluorescence intensity of 7-hydroxycoumarin \u0026minus;\u0026thinsp;3-carboxylic acid (7OHCCA), the major hydroxylation product of irradiated 3-CCA, according to the methods reported by Manevich Y \u003cem\u003eet al\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eExperiments were initiated by addition of FeCl\u003csub\u003e2\u003c/sub\u003e (II) in 50 mM phosphate sodium-150 mM NaCl (pH7.4) buffer containing 1.0 mM hydrogen peroxide and 0\u0026ndash;1 mM EDTA or organic acids (citric acid, malic acid, tartaric acid, succinic acid) in black coated 96-well microplates (FluoroNunc 96-Well Microplates, ThermoFisher), using a microplate reader (Synergy4, BioTek, USA). The excitation/emission wavelengths were set 388/450 nm based on previous publications.\u003c/p\u003e \u003cp\u003e \u003cem\u003eOxidative degradation of bovine serum albumin (BSA).\u003c/em\u003e \u003c/p\u003e \u003cp\u003eIn order to assess the oxidation-induced degradation of BSA, BSA was incubated similarly as reported \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e, followed by native-PAGE analysis\u003csup\u003e53\u003c/sup\u003e. Shortly, BSA (1 \u0026micro;g) was incubated in the presence of either 10 \u0026micro;M copper (II) chloride or iron (II) chloride, 5000 \u0026micro;M hydrogen peroxide, in the presence of 0.1- 2.0 mM citric acid or 0.01\u0026ndash;0.1 mM EDTA in 50 mM phosphate sodium-150 mM sodium chloride buffer (pH 7.4) at 37 \u003csup\u003e◦\u003c/sup\u003eC for 1h. Portion of the reaction mixture was then applied to native (non-denaturating)-polyacrylamide -gel (10% (w/v)) electrophoresis at a constant current of 20 mA per gel for 100 min using an electrophoresis system (Taitec, Tokyo, Japan), followed by staining with Coomassie Blue R-250.\u003c/p\u003e\n\u003ch3\u003eStatistics\u003c/h3\u003e\n\u003cp\u003eThe data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. The data were analyzed for statistical significances using Student\u0026rsquo;s t-test. Difference was assessed with one-side test. The statistical significance was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.K. conceived the concept, designed the study which was supervised by A.M., H.O. and H.K. Y. K, Y. H. and T. N wrote the main manuscript text and Y.K. prepared all the figures. All the authors contributed to manuscript preparation and reviewed it.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe have no conflicts of interest to declare.\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and analyzed during the current study will be available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Mrs. Ryunosuke Sato and Osuke Tajima for their technical assistance.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eXia, Y. et al. 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Rep.\u003c/em\u003e \u003cb\u003e7\u003c/b\u003e, 40744. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/srep40744\u003c/span\u003e\u003cspan address=\"10.1038/srep40744\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2017).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Fenton reaction, hydroxyl radical, metal chelator, oxidative protein degradation","lastPublishedDoi":"10.21203/rs.3.rs-5163186/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5163186/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThrough the present work, we have examined the possible role of L-histidine and citrate in the regulation of iron (II) ion-induced generation of hydroxyl radical (OH*), the most detrimental reactive oxygen species (ROS), via Fenton reaction. We observed that the presence of metal chelators (such as EDTA or citric acid) was necessary for the iron ion-dependent generation of OH*, when assessed with 3-coumarin carboxylic acid (3-CCA) as a probe specific for the radical. We also found that citrate acid as well as EDTA promoted the iron ion-dependent generation of the radical on a dose-dependent manner when they repressed the copper ion-dependent generation of the radical. In contrast, L-histidine promoted the copper ion-dependent generation of the radical at less than equimolar to the ion and repressed it at more than equimolar, whereas L-histidine hardly promoted the iron ion-dependent generation of the radical. Finally, we found that EDTA and citric acid promoted the iron and hydrogen peroxide-induced degradation of bovine serum albumin and repressed the copper and hydrogen peroxide-induced degradation of the protein, suggesting that the contrasting roles of EDTA and citric acid in promoting and repressing the generation of hydroxyl radical, depending on iron ion and copper ion, respectively.\u003c/p\u003e","manuscriptTitle":"Adverse roles of citric acid and L-histidine in the transition metal-dependent generation of hydroxyl radical at circumneutral pH","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-28 07:02:53","doi":"10.21203/rs.3.rs-5163186/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5936ebdb-9c1f-403b-a80d-fcf5cc74ed38","owner":[],"postedDate":"October 28th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":39427908,"name":"Biological sciences/Biochemistry/Metals/Iron"},{"id":39427909,"name":"Physical sciences/Chemistry/Chemical biology/Metals"}],"tags":[],"updatedAt":"2025-03-24T04:38:26+00:00","versionOfRecord":[],"versionCreatedAt":"2024-10-28 07:02:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5163186","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5163186","identity":"rs-5163186","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}