Department of Radiochemistry-Biophysics, Niigata College of Pharmacy, Karnishin-ei , Niigata , Japan

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1 Vol. 46, No. 1, September 1998 Pages ENHANCEMENT OF HYDROXYL RADICAL GENERATION IN THE FENTON REACTION BY ALPHA-HYDROXY ACID M. Aktar Ali and Tetsuya Konishi* Department of Radiochemistry-Biophysics, Niigata College of Pharmacy, Karnishin-ei , Niigata , Japan Received June 9, 1998 SUMMARY The effect of various organic acids on hydroxyl radical (, ) generation in the Fenton reaction were examined by the ESR spin trapping technique, where 5,5-dimethyl-1-pyroline-N-nitroxide (DMPO) and a-phenyl-tert- butyl nitrone (PBN) were used as the spin trapping reagents, ct-hydroxy acids such as lactic acid, glycolic acid and 2-hydroxy isobutyric acid were found to markedly enhance 9 generation in the reaction. In contrast, [3-hydroxy acid, ct-keto acid, esters of ct-hydroxy acids, aldehydes and other straight chain organic acids had no such enhancing activity, a-amino acids had also no enhancing effect. The results suggest that the et-hydroxy acid moiety is prerequisite for the enhancement of- generation in the Fenton reaction. Superoxide dismutase did not inhibit the enhancing effect of ct-hydroxy acids whereas catalase completely inhibited the. generation. Thus, ct-hydroxy acids directly enhanced the. generation via the Fenton reaction but not the Haber-Weiss reaction. Possible role of lactic acid manipulating, generation is discussed in relation to the ischemia-reperfusion cell damage. Keywords: ct-hydroxy acid, Fenton reaction, Hydroxyl radical, ESR spin trapping. INTRODUCTION Reactive oxygen species (ROS) such as superoxide radical (02-), hydroxyl radical (. ) and hydrogen peroxide (H202) are constantly produced under physiological conditions in the human body. Several defense systems such as small antioxidants and enzymes balance their physiological levels [1,2]. Among these oxygen species,- is said to be the most harmful in the biosystem because of its high and non-selective reactivities [3]. The major physiological sources of" are the Fenton and Haber-Weiss type reactions [4,5]. Abbreviations: ESR, electron spin resonance; DMPO, 5,5-dimethyl-1-pyroline-N-nitroxide; PBN, a-phenyl-tert -butyl nitrone; MES, [2-(N-morpholino)ethanesulphonic acid- H20]; NADH, nicotinamide adenine dinucleotide (reduced form). *To whom correspondence should be addressed (Fax: ) t /98/130137~) /0 Copyright by Academic Press Australia. All rights of reproduction in any form reserved

2 Over production of. is known to give rise to oxidative damages on biological constituents such as DNA, protein, lipid and carbohydrates, leading to various physiological disorders such as cancer, rheumatoid, aging and postischemic repeffusion injury [6-12]. Thus, it is of interest to study the manipulating mechanism of. generation by biological constituents in the Fenton reaction. A large number of studies have reported on Fenton or Haber-Weiss mediated 9 generation and its scavenging, or on the preventing mechanism of- induced cell damages, but few have reported on the enhanced manipulation of- generation. Catechols are one example which enhances. generation in Fenton system [13]. Recently, we found an ingredient in a chlorella strain, T-l, which enhanced- generation in the Fenton reaction. The chemical structure identified was lactic acid [ 14]. Lactic acid is physiologically produced in the cell, especially under anaerobic condition. It is thus possible that the lactic acid mediated enhancement of- generation in the Fenton reaction might play a significant role in cell damaging processes especially under ischemia-repeffusion condition. We studied here a series of acids for their manipulating activity of. generation in the Fenton reaction and found that the ct-hydroxy acid structure is responsible for the enhancing activity of-. MATERIALS AND METHODS Materials. Lactic acid, glycolic acid, 2-hydroxyisobutyric acid, acetic acid, propionic acid, pyruvic acid, 3-hydroxybutyric acid, methyl lactate and all other organic acids and their derivatives were purchased from Sigma Chemical Co. Ltd. (USA). Spin trapping reagents 5,5- dimethyl-1-pyroline-n-nitroxide (DMPO) and ~x-phenyl-tert-butyl nitrone (PBN) were from LABOTEC Co. Ltd. (Japan). o-phenanthroline, was purchased from Wako Chemical Co. Ltd. (Japan). Hydrogen peroxide (31% w/v) was from Mitsubishi Gas Co. Ltd. (Japan). FeSO4and FeCI3, superoxide dismutase (SOD) and catalase were purchased from Sigma Chemical Co Ltd (USA). All other chemicals used were commercial products of the highest reagent grade available. Metal free re-distilled water was used as the solvent for all reagents. ESR spin trapping assay. Enhancing effects of a series of acids on. generation in the Fenton reaction were studied using a spin trapping ESR method. The reaction was carried out in 300 Ixl aqueous solution, in a microtube, containing 15 mm of H202, 0.2 mm of FeSO4, 300 mm of spin trapping reagents (DMPO or PBN) and an aliquote of investigated samples. In the control, metal free H20 was substituted for the sample. The Fenton reaction was initiated by H202 addition, then 30 od of the reaction mixture was placed in an ESR fiat cell. Hydroxyl radical adduct of DMPO (DMPO-) and PBN (PBN-) were recorded at 3 and 5 min, respectively, after the addition of H202. ESR spectra were recorded by JEOL JES-TE 200 ESR spectrometer (X-Band Microwave Unit). The spectrometer settings were as follows: microwave power, 8 mw; microwave frequency, 9.20 GHz; modulation amplitude, 0. lmt; time constant, 0.03 sec; sweep time, 30 sec; center fields, 332.6/322.6 rot. Ferric ion reduction. Ferric ion reduction by a series of acids was determined by Fe(II)-o-phenanthroline complex formation assay [15]. Reaction mixtures containing 0.2 mm FeCI3, 10 mm o-phenanthroline and 1.5 mm acid sample were allowed to stand for 140 min at room temperature. Then, Fe(II)-o-phenanthroline complex formed was determined by 512 nm absorption. 138

3 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL RESULTS Enhancement of. generation in the Fenton reaction by a-hydroxy acids Typical ESR spectra obtained after all the reagents were mixed up are shown in Fig. 1 and 2. Both spin trapping reagents, DMPO and PBN, give rise to typical 9 adduct (DMPO- and PBN- ) signals (an = 1.49 mt, an = 1.49 mt for DMPO and an = 1.53 mt, an = 0.25 m T for PB N) [ 16, 17]. The spectral pattern was not changed even if the order of reagent addition was changed, that is, the reaction was initiated by adding FeSO4 instead of H202. Both the DMPO- and PBN- signals were markedly enhanced when lactic acid was present in the reaction mixture. In the absence of either hydrogen peroxide or ferrous sulphate, no spin adduct signal was observed (data not shown). The lactic acid dependent enhancement of- generation is not a simple ph effect, because hydrochloric acid addition instead of lactic acid did not enhance the DMPO- signal. The lactic acid dependent enhancement of DMPO- formation was also observed in 5 mm MES (ph 6.2), although the enhancing magnitude was far smaller than that observed in distilled water because of the, scavenging effect of the buffer (data not shown), further confirming that the acidification of the reaction mixture was not the primary cause of the enhancing effect. To examine whether superoxide radicals (02-) are involved in the enhancement of DMPO- formation, superoxide dismutase (SOD) was added to the reaction mixture. The signal intensity of DMPO- was not decreased at all with 0.6 U/Ixl SOD both in the absence or presence of lactic acid A,1" D.r 4"- B 4" C -4" 4"----- Fig. 1. Lactic acid dependent enhancement of DMPO- formation in Fenton reaction. Conditions for the Fenton reaction and ESR spectrum measurement are described in the method section. (A) Control Fenton reaction (without lactic acid), (B) 0.2 mm lactic acid, (C) 0.5 mm lactic acid, (D) I mm lactic acid, (E) 2 mm lactic acid. 139

4 A ~ 4"-- B 4"- Fig. 2. Lactic acid dependent enhancement of PBN- formation in Fenton reaction. Conditions for the Fenton reaction and ESR spectrum measurement are described in material and method section. (A) Control Fenton reaction (without lactic acid), (B) 0.1 mm lactic acid, (C) 0.2 mm lactic acid, (D) 0.4 mm lactic acid. (Fig. 3). On the other hand, ethanol (1.8 M) decreased the DMPO- signal to approximately 55 %, instead, CHaCH radical was formed (Fig. 3). Thus, the signals observed here reflect the. generated primarily in the Fenton reaction. Indeed, the DMPO- was disappeared when catalase (45 U/O.I) was added to the reaction mixture both in the presence and absence of lactic acid (Fig. 3). The experiments described above strongly indicate that lactic acid directly enhances 9 generation in the Fenton system, and not via superoxide radical generation. The concentration dependencies of lactic acid, ferrous sulphate and hydrogen peroxide were determined. The DMPO- signal increased with increased concentration of lactic acid (Fig. 4). The signal also increased with increasing concentrations of ferrous sulphate and hydrogen peroxide (data not shown). Neither Cu 1+ nor Co 2+ were effective for the enhancement (data not shown), suggesting that iron is primarily involved in the enhancing mechanism of. generation. To analyze the acid structure and the activity relationship, a series of organic acids were further studied for their. enhancing activities (Table 1). ct-hydroxy acids such as glycolic acid and 2-hydroxy isobutyric acid enhanced the" generation to the same extent as lactic acid. Simple acids such as acetic acid, propionic acid and isobutyric acid had no enhancing effect. Methyl lactate, the ester of lactic acid, significantly decreased the enhancing activity of lactic acid. Both acetaldehyde and propionaldehyde were ineffective. 3-Hydroxy butyric acid, a 13-hydroxy acid, did not enhance. 140

5 ~ 150 o, 100 ~ 50 I:1 o 0 "~ 0 ~ o 9 _-1 "~ o + ~ + + Fig. 3. Effect of super oxide dismutase (SOD), catalase and ethanol on DMPO- enhancement by lactic acid in Fenton reaction. Reaction condition is given in material and method section. DMPO- spectrum was recorded at 3 min after the reaction was initiated by I-I202 addition. Final concentrations of lactic acid, SOD, catalase and Et were 1 rnm, 0.6U/ttl, 45U/ttl and 1.8 M respectively. generation. Pyruvic acid, the keto form of lactic acid, also did not enhance the reaction. In contrast, []- hydroxy pyruvic acid, which can be tautomerized to form (x-hydroxy enol form, effectively enhanced the 9 generation. Further, (x-amino acids such as alanine and serine did not show any enhancing effect. These results were confirmed using PBN as a spin trap (data not shown). It is thus clearly indicated that the (x-hydroxy acid structure is prerequisite for the enhancement of" generation in the Fenton reaction. DISCUSSION In the present study, we found that the ct-hydroxy acids such as lactic acid, glycolic acid and 2-hydroxyisobutyric acid enhance. generation in the Fenton reaction. This finding is interesting because (x-hydroxy acids are commonly produced as a cellular component under 141

6 BIOGHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL 20 t5 e~0 0 d I, I, I, I, I Lactic acid (mm) Fig. 4. Dose effect of lactic acid on DMPO- formation in Fenton reaction. Reaction condition is given in material and method section. DMPO- spectrum was recorded at 3 min after the reaction was initiated by H202 addition. physiological conditions, and it is generally accepted that the Fenton mediated, generation is the major cause of various physiological disorders such as cancer, rheumatoid, diabetes and ischemia-reperfusion [ Cellular redox imbalances in ischemic tissues are one of the fundamental factors contributing to the complications of this pathological disorder[18]. In ischemic tissues, impaired mitrocondrial oxidation of NADH to NAD + because of oxygen depletion results in an increased intracellular NADH/NAD + ratio [19]. The increased cellular NADH/NAD + ratio causes so-called reductive stress which in turn inhibits several major metabolic processes such as glycolysis and fatty acid oxidation [20]. This condition accelerates the intracellular formation of ROS leading to further cell injury [21]. At the same time, the increased NADH/NAD + ratio promotes the release of cellular iron (Fe 3+) from ferritin and elevates free Fe 2+ level [22]. Thus, the Fenton reaction is accelerated to produce excess intracellular -. Lactic acid also accumulates in these cells by anaerobic glycolysis. It is therefore plausible that lactic acid may play an important role in the pathogenesis of ischemic or post ischemic dysfunction via its enhancing effect on, generation [23] as found here. Although acidification due to accumulation of lactic acid was suggested as one of the causes of ischemia-reperfusion damage in the tissues [23-251, our finding strongly indicates that 142

7 Tab. 1. Effect of various acids on DMPO- formation and on ferric ion reduction. Reaction conditions for DMPO- formation and ferric ion reduction were given in material and method section. The value are the mean of three determinations. Compound Structure tgmpo- formation (relative % of control) Fe~/Fe ~" reduction (relative % of control) None (control) -- I00 I00 Lactic acid CH3-CH-CO Propionic acid CH3-CH2-C(X)H Methyl lactate CH3-CH-COOCI- ~ , Isobutyric acid CH3_Ctt_CO Hydroxy CH~ isobutyric acid CHrC-CO, hydroxybutyric acid CHr,CH-CI~-CO Pyruvic acid CH3-C-CO H 0 13-Hydroxypyruvic acid CH2-C-CO 298 I I I I O Acetic acid CH3-COO H Glycolic acid H~C-CO Acetaldehyde CH 3-CH 10"7 78 II o Propionaldehyde CHr CI-I2-CH, O Alanine CH~-CH-CO NH~ Serine,CHz-CH-CO NH 2 143

8 the lactic acid mediated enhancement of. generation rather than the ph effect is more critical in the propagation of oxidative damages occurring under ischemia-reperfusion condition. In order to determine the enhancing mechanism of. generation by ct-hydroxy acid, the lacic acid mediated reduction of ferric ions to ferrous was studied by using Fe(II)-ophenanthroline complex formation assay [15]. Lactic acid indeed reduced Fe 3 but the reaction rate was too slow to explain the enhanced production of. by Fea+/Fe 2+ recycling (data not shown). Further the Fe 3+ reducibilities of various acids are not quantitatively related to their enhancing activities as shown in Table 1. Especially pyruvate did not enhance the DMPO- formation but strongly reduced Fe 3+. Thus the ct-hydroxy acid mediated Fe3+/Fe 2+ recycling may not be the major cause of the. enhancing mechanism. It is more plausible that a certain stable intermediates are formed by the interaction of Fe3*/lactate with H202 which promotes- generation, since two different spin probes for 9 with different stability of their adducts gave the identical results in the lactic acid effect. Further studies are in progress to clarify the mechanism. ACKNOWLEDGMENT The authors thank Dr. S. Matsugo at Toyama University and also Dr. L. Packer in UCB for their helpful discussion. REFERENCES , 10. Aruoma, O. I. (1996) Free radicals in biology and medicine 5, Halliwell, B.; Gutteridge, J. M. C. (1989) Free radicals in biology and medicine Oxford: Clarendon Press. Franzini, E.; Sellak, H.; Hokin, J.; Pasquier, C. (1993) Biochim. Biophs. Acta 1203, Goldstein, S.; Meyerstein, D.; Czapski, G. (1993) Free Radic. Biol. Med. 15, Koppenol, W. H. (1993) FreeRadic. Biol. Med. 15, Halliwell, B.; Aruoma, O. I. (1993) DNA and free radicals, pp Ellis Horwood. Roberto Bolli.; Mohamed O. Jeroudi; Bharat, S. Patel; Coit, M. Dubose; Edward, K. Lat. (1989) Proc. Natl. Acad. Sci. 86, Halliwell, B.; Cross, C. E.; Gutteridge, J. M. C. (1992) J. Lab. Clin. Med. 1 19, Cerutti, P. A. (1994) Lancet 344, Ames, B. N.; Cathcart, R.; Schwiers, E.; Hochstein, P. (1981) Proc. Natl. Acad. Sci. USA 7 8,

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