Pro-oxidant activity of vitamin C in drinking water ... - Ãbo Akademi

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Hydrogen peroxide formation in drinking water 1237Table III. Vitamin C induced H 2 O 2 formation in drinking water.Household drinking water (from taps) Bottled waterH 2 O 2 (mM)formationSamplenumberH 2 O 2 (mM)formationSamplenumberH 2 O 2 (mM)formationSamplenumberH 2 O 2 (mM)formationSamplenumberH 2 O 2 (mM)formationSamplenumberH 2 O 2 (mM)formationSamplenumber1 417.3 ^ 17.4 9 219.5 ^ 8.2 17 0 25 282.8 ^ 20.3 33 106.7 ^ 27.4 41 43.7 ^ 12.82 20.1 ^ 13.2 10 92.0 ^ 13.1 18 396.4 ^ 7.1 26 62.6 ^ 3.5 34 294.5 ^ 19.0 42 123.9 ^ 2.53 252.6 ^ 22.9 11 96.4 ^ 20.6 19 408.1 ^ 10.8 27 398.9 ^ 9.2 35 175.9 ^ 8.2 43 97.3 ^ 13.44 85.6 ^ 4.3 12 482.0 ^ 19.0 20 426.7 ^ 16.2 28 361.7 ^ 3.9 36 175.9 ^ 4.9 44 71.2 ^ 42.75 481.7 ^ 13.6 13 115.9 ^ 7.8 21 160.3 ^ 3.8 29 142.8 ^ 8.8 37 356.7 ^ 12.3 45 226.2 ^ 11.76 488.4 ^ 8.8 14 177.6 ^ 32.2 22 476.4 ^ 30.2 30 440.3 ^ 8.7 38 188.9 ^ 6.8 46 44.2 ^ 8.57 488.4 ^ 16.6 15 454.2 ^ 9.1 23 121.4 ^ 5.9 31 252.6 ^ 3.9 39 375.9 ^ 8.6 47 457.8 ^ 12.58 219.2 ^ 7.1 16 326.4 ^ 12.8 24 67.3 ^ 6.9 32 305.1 ^ 7.4 40 135.6 ^ 42.7 48 474.2 ^ 4.4Vitamin C (2 mM) induced H2O2 formation in tap water samples (numbered 1–40) and domestic bottle water samples (numbered 41–48). The values shown are the concentration of H2O2 formedduring a 6-h incubation in dark at room temperature. Data are expressed as means ^ SD of triplicates of one representative experiment out of three conducted.Figure 2. Effect of bicarbonate and iron on ascorbic acid inducedhydrogen peroxide accumulation. Ascorbic acid (2 mM) and variousconcentrations of Fe(III) were added to Milli-Q watersupplemented with 0.1 mg/l Cu(II) and 0 mg/l (†); 25 mg/l (W);50 mg/l (O) and 100 mg/l (K) HCO 3 . The H 2 O 2 concentration wasmeasured in the samples after a 6 h incubation at room temperature.Data points are mean ^ SD of three experiments.(20 times below the amount of copper that is allowedin drinking water (MCL in Europe)). Of the othermetals tested, only Co(II) generated significantamounts of H 2 O 2 (Table I).The ascorbic acid induced H 2 O 2 accumulation incopper supplemented Milli-Q water was significantlyhigher than the H 2 O 2 formation observed earlier incell culture and cell culture medium [37,38].However, addition of ascorbic acid to the Milli-Qwater model system used in our experiments, resultedin a much more acidic milieu (pH 3.5) than the onenormally seen in buffered cell culture medium. Alsothe pH in our drinking water samples were close to orunder the pK al value of ascorbic acid (4.25). Duringmore neutral conditions (pH 6–6.5) that was obtainedwhen magnesium ascorbate, calcium ascorbate orsodium ascorbate were added to Cu(II) supplementedMilli-Q water, less amount of H 2 O 2 was generatedwithin 6 h (data not shown). These results are in betteragreement with the amounts of H 2 O 2 generated in cellcultures and cell culture medium [37,38]. Thisemphasizes the importance of pH in the ability ofascorbic acid to induce H 2 O 2 accumulation in thepresence of Cu(II) ions.In our hands, of the metal ions tested, only Fe(III)was found to strongly affect the ascorbic acid/Cu(II)induced H 2 O 2 formation in Milli-Q water duringacidic conditions (pH 3.5) (Table II). The inhibitionof the H 2 O 2 accumulation could be observed after 2 hincubation, and after 6 h incubation the majority ofthe H 2 O 2 formed had been eliminated from the water
1238P. J. Jansson et al.sample (Table II, Figure 1). The shape of the ascorbicacid/Cu(II) induced H 2 O 2 curve, obtained in thepresence of ferric iron, strongly indicated that ametabolite from ascorbic acid might be involved in theH 2 O 2 decomposition reaction. Consistent with thisassumption, we found that oxalic acid, together withFe(III) induced a prompt inhibition of ascorbicacid/Cu(II) induced H 2 O 2 formation in Milli-Qwater (Figure 1). The reason why we used oxalicacid was because we have previously demonstratedthat oxalic acid is one of the degradation productswhen ascorbic acid is oxidatively decomposed inCu(II) contaminated drinking water [36].Oxalic acid also decreased the ascorbic acid/Cu(II)induced H 2 O 2 formation in Milli-Q water, implyingdirect interaction between Cu(II) and oxalic acid. Themechanism for the oxalic acid mediated inhibitoryeffect on ascorbic acid/copper induced H 2 O 2 formationin Milli-Q water might be due to its ability tointerfere with copper redox-cycling. This is becauseoxalic acid is known to have affinity for both Fe(III)and Cu(II), even at very low pH. Alternatively,copper/oxalic acid could catalyze H 2 O 2 to hydroxylradicals. However, we could not detect any ascorbicacid induced hydroxyl radical formation when oxalicacid was added to copper supplemented water (datanot shown), implying reduced copper redox-activitywhen oxalic acid was present. Interestingly, it has beensuggested that oxalic acid could act as an antioxidantin some systems, because oxalic acid reduces the rateof ascorbic acid oxidation in the presence of H 2 O 2 andCu(II) [39].The ability of Fe(III) and oxalic to regulate ascorbicacid/Cu(II)-induced H 2 O 2 formation during acidicconditions as shown in our experiments could be ofimportance in vivo when acidic conditions prevail.This could emphasize the importance of vitamin C,oxalic acid and iron during certain conditions such asthe inflammatory process. Thus, when Cu(II) ionsand Vitamin C are present during acidic conditions,the presence or absence of free redox-active iron andoxalic acid will determine the amount of H 2 O 2 thatwill be accumulated. In particular, during conditionswhere catalase is not present or not functioningproperly e.g. in the presence of ascorbic acid andcopper [40,41], the iron/oxalic acid complex could beof importance in regulating H 2 O 2 toxicity duringacidic conditions in various biological systems in vivo.The vitamin C induced accumulation of H 2 O 2could also be demonstrated in 40 household tap watersamples and 8 domestic bottled water samples. In25% of the drinking water samples tested, over400 mM of H 2 O 2 was formed during the 6 hincubation and some drinking waters generated closeto 500 mMofH 2 O 2 within 6 h (Table III). The kineticsfor the H 2 O 2 formation in these drinking waters wasrelative fast, reaching close to 300 mM H 2 O 2 in only2 h (data not shown). The ascorbic acid inducedhydrogen peroxide formation was particularly evidentin the poorly buffered copper contaminated drinkingwater samples. When bicarbonate was added to Milli-Q water supplemented with copper, the hydrogenperoxide accumulation was much lower (Figure 2).During these conditions the hydrogen peroxide isconverted to hydroxyl radicals [32].Hydrogen peroxide has been shown to have strongantibacterial effects [42]. One might speculate,whether the ascorbic acid/Cu(II)-induced H 2 O 2formation and the ability of Fe(III) and oxalic acidto regulate H 2 O 2 formation, as demonstrated here indrinking water, could be involved in controlling thesurvival of pathogenic bacteria in the humangastrointestinal tract. On the other hand, our resultsdemonstrating that vitamin C induces H 2 O 2 formationin drinking water could, in fact, result inincreased oxidative stress in vivo. In particular, in theabsence of Fe (III) ions, ascorbic acid/Cu(II)-inducedH 2 O 2 formation in drinking water, as demonstratedhere, could strongly enhance the total H 2 O 2 load andH 2 O 2 induced oxidative stress in some individuals.In conclusion, our results show that copper, but notiron, can support vitamin C induced H 2 O 2 formationin weakly buffered drinking water. Moreover, we showthat iron, together with ascorbic acid-derived oxalicacid prevents ascorbic acid/Cu(II) induced H 2 O 2accumulation. Our results strongly indicate that theiron oxalic acid complex might function as animportant regulator of ascorbic acid/copper-inducedH 2 O 2 formation and copper mediated tissue damage.The iron/oxalic acid induced inhibition of H 2 O 2formation, as demonstrated here in drinking waters,might explain the mechanism for the protective effectof iron against ascorbic acid (vitamin C) induced celltoxicity in cell cultures.AcknowledgementsThis work was supported by Magnus Ehrnroothfoundation, Paulon Säätiö, K. Albin JohanssonsStiftelse, Stiftelsen för Åbo Akademi, Academy ofFinland and Svenska Kulturfonden.References[1] Stahl W, Sies H. Antioxidant defense: Vitamins E and C andcarotenoids. Diabetes 1997;46:S14–S18.[2] Frei B. On the role of vitamin C and other antioxidants inatherogenesis and vascular dysfunction. Proc Soc Exp BiolMed 1999;222:196–204.[3] Padayatty SJ, Katz A, Wang Y, Eck P, Kwon O, Lee JH, ChenS, Corpe C, Dutta A, Dutta SK, Levine M. Vitamin C as anantioxidant: Evaluation of its role in disease prevention. J AmColl Nutr 2003;22:18–35.[4] Block G. Vitamin C and cancer prevention: The epidemiologicevidence. Am J Clin Nutr 1991;53:270S–282S.
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Pro-oxidant activity of vitamin C i
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Supervised byDocent Tommy Nordströ
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ContentsCONTENTSLIST OF ORIGINAL PU
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List of original publicationsLIST O
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AcknowledgementsACKNOWLEDGEMENTSThi
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AbbreviationsABBREVIATIONSAsc …
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Review of the literatureREVIEW OF T
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Review of the literatureSince vitam
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Review of the literaturestill added
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Review of the literatureantioxidant
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Review of the literatureThe α-toco
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Review of the literatureCopper, wil
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Review of the literatureOH • + H
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Review of the literatureFormation o
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Review of the literature3.2. The ro
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Review of the literaturecopper conc
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Experimental proceduresEXPERIMENTAL
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Experimental procedures2.2. Measure
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Experimental procedurestetrahydrate
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ResultsRESULTS1. Vitamin C induces
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Results3. Oxidative decomposition o
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Resultsdifferent water samples vari
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DiscussionDISCUSSIONNowadays, ascor
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DiscussionCu 2+ + Asc → Cu + + As
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Discussion3. Iron inhibits vitamin
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Discussionperoxide might have an im
Page 54 and 55: ConclusionsCONCLUSIONSThe main focu
Page 56 and 57: ReferencesREFERENCES1. Arrigoni O,
Page 58 and 59: References34. Padayatty SJ, Katz A,
Page 60 and 61: References66. Sies H, Stahl W, Sund
Page 62 and 63: References95. Halliwell B. Role of
Page 64 and 65: References127. Park S, Han SS, Park
Page 66 and 67: References157. Critchley MM, Cromar
Page 68 and 69: References185. Liao CH, Kang SF, Wu
Page 70 and 71: References214. Orr CW. Studies on a
Page 72: References243. Miller C, Kennington
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Pro-oxidant activity of vitamin C in drinking water ... - Ãbo Akademi