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2402 J. Agric. Food Chem. 1994, 42, 2402-2406

Oxidation of Ascorbic Acid and Metal Ions As Affected by NaCltStela Hare1

Department of Food Science,ARO, he Volcani Center, P.O. Box 6, Bet Dagan 50250, Israel

Ascorbic acid is one of the biological redu ctants that reduce ferric or cupric ions to ferrous or cuprousforms, inducing in th e presence of oxygen a metal redox cycle which init iates a cascade of reactionsproducing reactive oxygen species. NaCl inhibits ascorbic acid oxidation and the reactions thatproduce 02-, H202, and HO by preventing the interaction of the transition metal (Fe, Cu) withoxygen. If th e transition metal is liganded by a weak ligand such as acetate o r ADP, NaCl preventsthe interaction between oxygen and the transit ion metal. However, if the met al ion is chelated bya strong ligand such as citrate or EDTA, the effect of NaCl on ferrous oxidation is weak ornonexistent. NaCl does not inhibi t the interaction of th e metal ions (Fe, Cu) with H202 and thegeneration of hydroxyl radica ls from preformed H202.Keywords: Oxidation; ascorbic acid; metal ions; Na Cl

INTRODUCTIONSodium and chloride ions ar e present in all living cells

(Mat hews and va n Holde, 1990). Researchers includeNaCl in buffers or solutions t o simulate its concentrationin blood plasma and to provide a n ine rt environmentfor reactions (Minotti and Aust, 1992).

The chloride anion, a weak nucleophile, was found toenhance hemoglobin autoxidation (Wallace et al., 1974).The chloride anion in the presence of H202 is a substratefor chloroperoxidase and myeloperoxidase, producinghypochlorite, as a part of plant and animal defensemechanisms a gai nst microorganisms (Morrison an dSchonbaum, 1976).

Sodium chloride, an important additive in the foodindustry, has been reported to act as a pro-oxidant ofmuscle lipid peroxidation (Lea, 1937; Chang an d Watts,1950; Tappel, 1952; Banks, 1961; Ellis et al., 1968;Powers and Mast, 1980; Kanner and Kinsella, 1983;Kanner et al., 1991)or as an inhibitor of this reaction(Chang and Watt s, 1950; Mabrouk and Dugan, 1960).

Recently, the pro-oxidative effect of NaCl was s tudiedin model systems, using an extract of turkey muscletissue (Kanner et al., 1991)or fish muscle (Osinchak e tal., 1992). Earl ier publications (Mapson, 1941, 1945)showed that NaCl prevents ascorbic acid oxidation bycupric ions, especially at a low pH.

The purpose of this study was to elucidate th e effectof NaCl on autoxidation of ferrous a nd cuprous ions inth e presence of ascorbic acid and severa l iron chelators.

MATERIALS AND METHODSMaterials. Ascorbic acid, sodium chloride, potassiumchloride, and sodium citrate were purchased from Merck(Darmstadt, Germany); ferrous sulfate, Ferrozine, scopoletin,and horseradish peroxidase (HRP) from Sigma Chemical Co.(St. Louis, MO); and ethylenediaminetetraacetic acid disodiumsalt (EDTA) and sodium benzoate from BDH Chemicals Ltd.(Poole, England). Adenosine 5-diphosphate disodium salt(ADP) was obtained from Boehringer (Mannheim, Germany)and ferric sulfate from Riedel-de Haen (Seelze, Holland). Allchemicals used were of AR quality and met ACS requirements.Contribution from th e Agricultural Research Orga-nization, The Volcani Cente r, Bet D agan, Isra el; No.1222-E, 1993 series.

0021-8561 94/1442-2402$04.50/0

Methods. Hydroxyl radical generation in the model sys-tems was determined by benzoate hydroxylation, using amethod developed by Baker and Gebicki (1984). The additionof HO radicals t o benzoate, a t pH 7.3, produces 2-monohy-droxy benzoate, which was separated by HPLC from an RP-18 column by 0.05 M acetate buffer, pH 4.5, and acetonitrile(8:2 v/v) and detected fluorometricallyusing excitation at 300nm and emission at 410 nm. The amount of monohydroxybenzoate produced was calculated from a salicylate standardcurve.

Chelatable iron ions were assayed by using ferric chloridefor standard calibration. Iron ions were determined by amethod developed by Stookey (1970) using Ferrozine reagent.Ferrozine forms with ferrous ions a complex with a highextinction coefficient of E562 = 2.79 x l o 4M- cm-l.Ascorbic acid oxidation by ferric ions, with or withoutchelators, was monitored by its absorbance at 265 nm.Oxygen absorption in a reaction system containing 0.05 M

acetate, pH 7, was determined using an oxygen monitor(Yellow Springs Instrument Co., Model 53 ) with a Clarkelectrode. The aqueous solutions of ferrous or cuprous ionswere prepared by passing nitrogen free of oxygen through thedistilled water and through the solutions. Anaerobic oxidationwas done by passing a continuous stream of nitrogen throughthe solutions. The solubility of oxygen in distilled water at25 C and 1 atm is 8.26 mg/L, while in a 0.5 M NaCl solutionit is 6.75 mgL. A linear interpolation was used for calibrationvalues. The solubility of oxygen in each sample was takeninto account during calculations.Generation of HzOz was determined by the HRP-dependentreaction of HzOz with the fluorescent dye scopoletin anddetected by fluorescence measurements (excitation 350 nm,emission 460 nm) (Loschen and Flohe, 1971). A decrease influorescence intensity demonstrates oxidation of scopoletinby

HzOz-activated HRP.During the reactions and analysis the pH was taken intoaccount and corrected.RESULTS AND DISCUSSION

Ascorbic acid is one of the biological red ucta nts thatmay reduce ferric or cupric ions to ferrous or cuprousforms and by this induce in the presence of molecularoxygen a metal redox cycle which initi ates a cascadeof react ions producing superoxide anion radica l (02*-),Hz02, and hydroxyl radicals (H O) (Barber, 1966; Scarpaet al., 1983; Kanner et al., 1986, 1987; Miller a nd Aust,1989;Miller e t al., 1990; Minotti and Aust, 1992). Thiscascade of reactions, which in the end produces the0 1994 American Chemical Society

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Effect of NaCl on Ascorbic Acid Oxidation J. Agric. Food Chem., Vol. 42, No, 11, 1994 2403Fe3++ AH, * e2++ AH' + H+ (1)

Ferrous ions interact with oxygen, producing superoxideand ferric ions (Kann er et al., 1986). Superoxide couldfurther interact with ferrous ion, producing H202 (Co-hen and Sinet, 1980; Aust et al., 1990; Miller et al.,1990) by th e following reactions:

Fe2' + 0, - e3++ 0;-Fe2++ 0;- ?!. e3++ H20 ,

(2 )(3 )

Po cum .

>6

5

4

3

z

1

0TIME (mln)

Figure 1. Effect of NaCl on the generation of hydroxylradicals by ascorbic acid and metal ions, detected by benzoatehydroxylation: (W ) 13pM FeCl3; ( 0 ) p M CuSO4;(0)3 pMFeCl3 + 0 .5 M NaC1; (0 )5 p M CuSO4 + 0.5 M NaC1. Thereaction was performed in 0.05M acetate, pH 7, at 37 "C with200 p M ascorbic acid.hydroxyl radical, known to be the biologically mostoxidizing compound, was found to be inhibit ed by NaCl(Figure 1). Oxidation of ascorbic acid estimated byoxygen absorption (Figure 2a) or spectrophotometricallyat 265 nm (Figure 2b) (Buettner, 1986,1988;Aronovitchet al., 1987)was inhib ited by NaC1. Tounderstand thisinhibition, several of the reactions th at participate inth e oxygen meta l redox cycle cascade were determined .

The first reaction in this system is the chelation ofiron by ascorbic acid and the reduction of the ferric toferrous ion reaction (eq 11,which was estimated by theFerrozine method (Stookey, 1970). This reaction wasaffected only partially by NaCl(-lO%) at pH 7.0 (resultsnot shown).

.0 0

B O

60

40

20

00 5TIME (min)

The reactants and products of these proposed reac-tions were determined by (a ) oxygen absorption (Figures3 and 41,(b) accumulation of HzO2 (Figure 51, and ( c )oxidation of ferrous ion (Figure 6). The methods usedto estimate reactions 2 and 3 showed that NaCl inhibitsthese reactions, most probably by preventing the inter -action of the transi tion meta l with oxygen. If th etransiti on metal is liganded by a weak ligand such asacetate o r ADP, NaCl prevents the interaction betweenoxygen and the transition metal (Figures 3 and 4).However, if the metal ion is chelated by a s tron g ligandsuch a s citrate or EDTA, the effect of NaCl on ferrousoxidation is weak o r nonexistent.It was also assumed that NaCl could affect thereaction of ferrous ion with H202.

Fe2++ H20, - e3++HO'+ HO- (4)This reaction was demonstrated by measuring thedecrease of ferrous ion and hydroxylation of benzoateto hydroxylated compounds. The results demonstrateno effects of NaCl on these reactions (Figure 7) (resultsof benzoate hydroxylation not shown).The results in Figure 7 demonstrate a very rapidoxidation of ferrous ion (200pM) in the presence of H202(5 0 PM) with o r without NaCl (0.5MI. The stoichiom-

I

0 1 5 30 45TIME (min)Figure 2. Inhibition by NaCl of ascorbic acid oxidation in the presence of metal ions: (a, left) reactions determined by oxygenabsorption; (b, right) reactions determined spectrophotometrically at 265 nm; ( 0 ) pM CuS04; W) 13 pM FeCl3; ( 0 )CuS04o rFeC13 + 0.5 M NaC1. The reaction was performed in 0.05 M acetate, pH 7 , at 25 "C and with 200 pM ascorbic acid.

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2404 J. Agric. Food Chem., Vol . 42, No. 11, 1994 Harel

CONTROLS +NaCl (0.5M)

0v .--0 1 2 3 4 5

l-Lhm (min)Figure 3. Inhibition by NaCl of oxygen consumption during the oxidation of ferrous ion: (a , left) (E!)control (250 pM FeS04 in0.05 M acetate, pH 7 ) ;(0)ontrol + 330pM EDTA; (A ) control + 330 pM sodium citrate; ( 0 ) ontrol + 330 pM ADP; v) ontrol+ 330 y M ascorbic acid; (b, right) in the presence of 0.5 M NaC1.etry indicates tha t it is consistent with reactions 4 and5 which show oxidat ion of ferro us ion by H202 and HO'.

Fe2++ HO'- e3++ HO- ( 5 )At the end of both reactions 4 and 5, the remaining

ferrous ion was oxidized by oxygen and this reaction wasfound, as previously, to be affected by NaC1.Determination of ferrous oxidation by the Ferrozinemethod is much more sensitive tha n oxygen absorptionin thi s model system. One of the reasons is because forevery 1 mol of oxygen, 4 mol of ferrous ion is oxidizedby OZ'-, H202, and HO ' produced dur ing reactions 2-5.

Fe2++ 0, - ,'- + Fe3+Fez++ Oz*--Hz02 Fe3'Fe2++ H202 HO'+ Fe3'Fez++ HO'- O- + Fe3+

The inhibition of ferrous ion oxidation by NaCl s tem sfrom the interaction of th e chloride anion with th e ironion. Different chloride sal ts, such a s CaC12, MgC12, andLiC1, affect th e react ion very simi larly ( resu lts notshown).The anionic ligands interfere with chloride ions andprevent the interac tion between metal ions and oxygen.If the metal is liganded t o relatively strong chelatorssuch a s EDTA o r citrate, the effect of the chloride ionis very poor o r only moderate (Figure 3) .It was found that NaCl could affect the stability ofoxyhemoglobin and accelerate decomposition of theoxyhemoglobin to methemoglobin (Wallace et al., 1974).It seems th at NaCl prevents o r disturbs the interactionbetween iron o r iron-heme an d oxygen in a simila rmanner.Citrate, phosphate, and EDTA ions increase ironreactivity with oxygen to produce active oxygen species(Miller et al., 1990). Chelation of iron to different

10 0 1

0 4 I0 0 5 1

NaCl CONC. M)Figure 4. Effect of NaCl concentration on oxygen consump-tion during oxidation of ferrous ion. The reaction was per-formed with 250 yM FeS04 and 330yM sodium citrate in 0.05M acetate, pH 7 , at 25 "C .compounds affects iron reactivity by changing i ts redoxpotential a nd also by changing its potential to intera ctwith oxygen, HzO2, o r ligands. If iron is liganded byall of its six coordination positions, as occurs withcytochrome c, ligation of oxygen is prevented butinteraction of iron with H202 o r hydroperoxides is not(Harel et al., 1988).In blood and tissues the concentration of NaCl mayreduce autoxidation of ferrous or cuprous ions ligandedby weak chelators such as ADP or ascorbic acid, therebypreventing th e autoxidation of several important reduc-ing compounds and the formation of oxygen reactivespecies. These effects ar e also relevant to several foods.However, since NaCl does not affect the reactivity offerrous ions with previously formed H202 or hydroper-oxides, it may tu rn t he reactivity of metal ions to thesepathways a nd thereby contribute to and affect (invivoo r in situ) the generation of hydroperoxides and lipidfree radicals.The incre ase of lipid peroxidation by NaCl in severa lmodel systems and in situ in muscle foods was at-

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Effect of NaCl on Ascorbic Acid Oxidation J. Agric. Food Chem., Vol. 42, No. 11, 1994 24051 0 -

PON

vN

3 0 5 -

02NaC l0 1 2 3 4 5 6

TIMB (mln)Figure 5. Effect of NaCl on ferrous ion oxidation and thegeneration of HzOz. The reaction was performed in 250 pMFeS04 in 0.05 M acetate, pH 7, at 25 C with and without 0.5M NaC1: ( 0 ) ontrol; (0).5 M NaCl.

25 0

2 00

9 15 0vr)

100

50

00 10 20 30 40

TlMB (mln)Figure 6. Effect of NaCl (0.5 M) on ferrous ion oxidation asdetermined by the ferrozine method. The reaction was per-formed in 0.05 M acetate, pH 7, a t 25 C: (0)50 pM FeS04;( 0 ) 50 pM FeS04 + 330 pM ADP.tributed to the possible effect of NaCl to elevate theamoun t of free iron in tissues (Kan ner et al., 1991). Onthe basis of our recent results we suggest that, inaddition, NaCl ma y play a role in lipid peroxidation byshifting ferrous ions from interaction with oxygen toreaction with previously formed Hz02 o r hydroperoxidesand decomposing these compounds to free radicals,accelerating in particular the peroxidation process.In conclusion, the cascade reactions generated dur ingthe inte raction of ascorbic acid with ferric or cupric ionsin the presence of oxygen are prevented by NaC1.Sodium chloride prevents only partly th e interac tion ofthe metal ions with ascorbic acid but completely pre-vents the interaction of the metal with oxygen and bythis the formation of superoxide, HzOz, and hydroxylradicals. In this way the oxidation and autoxidation ofascorbic acid and the reduced metals by oxygen arepreven ted by NaC1. The effect of NaCl is significant ifthe metal ion is chelated by weak chelators such asacetate, ADP, o r ascorbic acid, only moderate withcitrate, and nonexistent with strong chelators suchEDTA.

I\ +NaCIN+ 1002 / I \

0 10 L O 30 40TlMB (mln)

0

Figure 7. Effect of NaCl (0.5 M) on ferrous ion oxidation byHzOz, as determined by the ferrozine method. The reactionwas performed in 0.05 M acetate, pH 7: (0)00 pM FeS04;( 0 ) 00 pM FeS04 + 50 pM HzOz.ACKNOWLEDGMENT

I thank Professor Joseph Kanner for useful advice,critical comments, and discussion during th e course ofthis study. This work was supported by the U.S.-IsraelBinational Agricultural Research and DevelopmentFund (BARD) (Project VS-1525-88).LITERATURE CITEDAronovitch,J. ;Godinger, D.; Samuni, A.; Czapski, G. Ascorbicacid oxidation and DNA scission catalyzed by iron andcopper chelates. Free Radical Res. Commun. 1987,2,41.Aust, S. D.; Miller, D. M.; Samokyszyn, V. M. Iron redoxreactions and lipid peroxidation. Methods Enzymol . 1990,186, 457.Baker, M. S.; Gebicki,J. M. The effect of pH on the conversionof superoxide t o hydroxyl free radicals. Arch. Biochem.Biophys. 1984,234, 58.Banks, A. The role of cytochrome c in the autoxidation ofunsaturated fatty acids. Chem. Ind. (London)1961,2,40.Barber, A. B. Lipid peroxidation in rat tissue homogenates:Interaction of iron and ascorbic acid as the normal catalyticmechanism. Lipids 1966, , 146.Buettner, G. R. Ascorbate autoxidation in the presence of ironand copper chelates. Free Radical Res. Commun. 1986, ,349.Buettner, G. R. In the absence of catalytic metals ascorbatedoes not autoxidize at pH 7: ascorbate as a test for catalyticmetals. J . Biochem. Biophys. Methods 1988, 6, 27.Chang, I.; Watts, B. M. Some effects of salt and moisture onrancidity in fats. Food Res. 1950, 5, 313.Cohen, G.; Sinet, P. M. In Development in Biochemistry;Bannister, J. V., Hill, H. A. O., Eds.; Elseviermorth-Holland: New York, 1980; Vol. 11A, pp 27-37.Ellis, R. ; Currie, G. T.; Thornton, F. E.; Bollinger, N. C.;Gaddis, A. M. Carbonyls in oxidizing fat. 11. The effect ofthe prooxidant activity of sodium chloride in pork tissue.J . Food Sci. 1968, 3, 13.Harel, S.; alan, M. A.; Kanner, J. Iron release from metmyo-globin, methaemoglobin and cytochrome c by a systemgenerating hydrogen peroxide. Free Radical Res. Commun.1988, ,11. Carotenedestruction and oxygen evolution in a system containinglactoperoxidase, hydrogen peroxide and halides. Lipids1983, 8, 198.Kanner, J. ; Harel, S.; Hazan, B. Muscle membranal lipidperoxidation by an iron redox cycle system: initiation byoxy radicals and site-specific mechanism. J . Agric. FoodChem. 1986,34,06 .

Kanner, J.; Kinsella, E. J. Lipid Deterioration:

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2406 J Agric. Food Chem., Vol. 42, No. 11, 1994Kanner, J. ; German, J. B.; Kinsella, J. E. Initiation of lipidperoxidation in biological systems. Crit. Rev. Food Sci.Nutr. 1987,25, 317.Kanner, J.;Harel, S.; Jaffe, R. Lipid peroxidation of musclefood as affected by NaC1. J . Agric. Food Chem. 1991, 39,1017.Lea, C. H . The influence of tissue oxidases on rancidity.Oxidation of the fat of bacon. J . SOC. hem. Ind., London1937,56, 3761.Loschen, G.; Flohe, L. Respiratory chain linked H202 produc-tion in pigeon heart mitochondria. FEBS Lett. 1971, 18,

261.Mabrouk, A. F.; Dugan, L. R., Jr . A kinetic study of theautoxidation of methyl linoleate and linoleic acid emulsionsin the presence of sodium chloride. J . Am . Oi l Chem. SOC.1960, 37, 486.Mapson, L. W. The influence of halides on the oxidation ofascorbic acid. Biochem. J . 1941,35, 1332.Mapson, L. W. Influence of halides on the oxidation of ascorbicacid. 2. Action of C1- on the cupric-cuprous system.Biochem. J . 1945, 39, 228.Mathews, K. C.; van Holde, E. K. Biochemistry; Benjamin/Cummings: Redwood City, CA, 1990; pp 9-10,Miller, D. M.; Aust, S. D. Studies of ascorbate dependent, ironcatalyzed lipid peroxidation. Arch. Biochem. Biophys. 1989,271, 113.Miller, D. M.; Buettner, G. R.; Aust, D. S. Transition metalsas catalysts of autoxidation reactions. Free Radicals Biol.Med. 1990, 8 , 95.

HarelMinotti, G.; Aust, S. D. Redox cycling of iron and lipidperoxidation. Lipids 1992, 27, 219.Morrison, M.; Schonbaum, G. R. Peroxidase-catalyzed halo-genation. Annu . Rev. Biochem. 1976,45, 861.Osinchak, E. J. ;Hultin, 0.H.; Zajicek, T. 0.;Kelleher, D. S.;Huang, C. H. Effect of NaCl on catalysis of lipid oxidationby the soluble fraction of fish muscle. Free Radicals Biol.M e d . 1992, 12, 35.Powers, J. M.; Mast, M. G. Quality differences in simulatedkosher and conventionally-processed chicken. J . Food Sci.1980, 45, 760.Scarpa, M.; Stevanado, R.; Viglino,P.; Rigo, P. Superoxide ionas active intermediate in the autoxidation of ascorbate bymolecular oxygen. J . Biol. Chem. 1983,258, 6695.Stookey, L. L. Ferrozine, a new spectrophotometric reagentfor iron. Anal. Chem. 1970, 4 2 , 779.Tappel, A. L. Linoleate oxidation catalyzed by hog muscle andadipose tissue extract. Food Res. 1962, 17, 550.Wallace, W. J.; Maxwell, J. C.; Caughey, W. S. The mecha-nisms of hemoglobin autoxidation. Evidence for proton-assisted nucleophilic displacement of superoxide by anions.Biochem. Biophys. Res. Commun. 1974, 57, 1104.Received fo r review December 1, 1993. Revised manuscriptreceived April 14, 1994. Accepted August 8, 1994.@

@ Abstract published in Advance ACS Abstracts, Sep-tember 15, 1994.