High concentrations of antioxidants may not improve defense against oxidative stress

ELSEVIER

Archives of Gerontology and Geriatrics 17 (1993) 189-201

ARCHIVES OF GERONTOLOGY AND G E R I A T R I C S

High concentrations of antioxidants may not improve defense against oxidative stress Guohua Cao, Richard G. Cutler* Molecular Physiology and Genetics Section, Laboratory of Cellular and Molecular Biology, Gerontology Research Center, National Institute on Aging, National Institutes of Health, 4940 Eastern A venue, Baltimore, MD 21224, USA

(Received 7 August 1993; revision received 20 September 1993; accepted 21 September 1993)

Abstract It is often assumed that the oxygen radical defense could be further improved by higher concentration of antioxidants. But this has not been demonstrated over a wide range of concentrations. There are different types of oxygen radicals produced in the body and the antioxidant protection against them may not positively related to their concentrations. We report here that by using H202 with Cu 2+ as an hydroxyl-radical generator in vitro, ascorbic acid shows no oxygen-radical absorbing capacity. We also found that the net hydroxyl-radical absorbing capacity of a water soluble t~-tocopherol analogue (Trolox) and uric acid increases with concentration only when the concentration is lower than the normal value found for a-tocopherol and uric acid in human serum. At higher concentrations, the hydroxyl-radical absorbing capacity of the ct-tocopherol analogue and uric acid decreases. The mechanism involved in the decrease of hydroxyl radical absorbance capacity of Trolox and uric acid at high concentration may be related to their reaction with hydroxyl radicals or other oxygen radicals produced in the presence of both H20 2 and Cu 2÷. This kind of reaction could lead to the formation of additional many Trolox or uric acid radicals at the same time. These results may be important not only in evaluating antioxidant activities of antioxidants in vitro but also in studying the potential efficiency of antioxidants in vivo in affecting oxidative stress status. Key words." Ascorbic acid; a-Tocopherol; Uric acid; Trolox; Oxygen-radical

* Corresponding author. 0167-4943/93/$06.00 © 1993 Elsevier Science Publishers B.V. All rights reserved. SSD1 0167-4943(93)00525-X

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1. Introduction

At present there is major interest in the possibility that free radical-mediated events in biochemical processes may lie at the heart of the etiology and natural history of a number of disorders that are of major concern worldwide, in particular cardiovascular disease and cancer, as well as a number of other disease states. Free radicals are atoms or molecules that have one or more unpaired electrons in their atomic structure and are thus generally highly reactive. It follows, therefore, that the mechanisms that exist for the control of the generation of free radicals and of their proliferation are of central importance in considering the prevention of these disease states. An array of intra- and extra-cellular antioxidants plays a very important role in this control. These antioxidants include enzyme systems such as the cytoplasmic, mitochondrial, and extracellular forms of superoxide dismutase, catalase, and the glutathione system, macromolecules such as ceruloplasmin and transferrin, and an array of small molecules, including glutathione, methionine, ascorbic acid, c~tocopherol, 13-carotene, uric acid and bilirubin. A large and steadily growing body of evidence exists that implies the importance of antioxidants in preventing a number of diseases, in particular cardiovascular disease and certain forms of cancer (Esterbauer, et al., 1991a; Gey et al., 1991; Stahelin et al., 1991a,b; Steinberg et al., 1992;). However, there are no published double-blind, carefully controlled, large-scale studies to establish the value of antioxidant supplementation (Steinberg et al., 1992). Perhaps, an important question need to be addressed first in this kind of study is the dosage of antioxidants supplemented. Can more antioxidant always bring more benefits? One of the difficulties in determining the optimal dosage of an antioxidant supplemented is the quantitation of the antioxidant capacity given by the antioxidant of certain amounts. Recently, our laboratory has developed a method that can quantitate total oxygen-radical absorbance capacity (ORAC) of a sample (Cao et al., 1993) in which we demonstrated that peroxyl radical absorbing capacity of some antioxidants, including ascorbic acid, Trolox (a water soluble a-tocopherol analogue) and uric acid, are linearly correlated with their concentration. However, the peroxyl radical may not be the best model system to assess the ability of a compound to protect against physiologically important free radicals (Halliwell and Gutteridge, 1990). We are therefore trying to use different oxygen radical generators to assess the oxygen radical absorbance capacity of an antioxidant. Hydrogen peroxide and copper are widely distributed in the body. The copper contained in an enzyme like superoxide dismutase can also react with hydrogen peroxide and produce hydroxyl radicals (Hodgson and Fridovich, 1975; Gutteridge and Wilkins, 1983; Sato et al., 1992). Copper plus hydrogen peroxide or copper only is widely used, as well, as an oxidant in the studying of protein and DNA damage (Parthasarathy et al., 1989; Dizdaroglu et al., 1991; Hanna and Mason, 1992), LDL oxidation and atherosclerosis (Esterbauer, et al., 1991b) and, thus, may be more physiologically relevant. In this study we used the H202-Cu 2÷ system as a hydroxyl radical generator and found in in vitro studies that ascorbic acid has no oxygen radical absorbing capacity. The hydroxyl radical absorbing quality of Trolox and uric acid depends upon their concentrations. Their hydroxyl radical absorbance capacity decreases at high

G. Cao, R.G. Cutler~Arch. Gerontol. Geriatr. 17 (1993) 189-201

191

concentrations. These findings may have important implication for the application of antioxidants in basic and clinical research. 2. Materials and methods

2.1. Materials L-Ascorbic acid, uric acid and porphyridium cruentum B-phycoerythrin (B-PE) were purchased from Sigma Chemical Co. (St. Louis, MO). Trolox (6-hydroxy2,5,7,8-tetramethylchroman-2-carboxylic acid) was obtained from Aldrich Chemical Co. (Milwaukee, WI). Chelex 100 resin (200-400 mesh, Na form) was obtained from Bio-Rad Co. (Richmond, CA). Cupric sulfate was obtained from Mallinckrodt Inc. (Paris, KY). Hydrogen peroxide was obtained from Fisher Chemical Co. (Fair Lawn, N J). 2.1. Hydroxyl-radical absorbance capacity assay The hydroxyl radical absorbance capacity was determined for ascorbic acid, Trolox and uric acid at their different final concentrations. The final reaction mixture for the assay contained 3.34 x 10-8 M ~-PE, 9 x 10-6 M Cu 2+ and 0.3% H202 in 7.5 x 10-2 M NaK phosphate buffer, pH 7.0. The buffer had been passed through Chelex 100 resin before it was used in preparing all solutions. A final volume of 2 ml was used in 10-mm wide cuvettes. Into each sample tube, 20 #1 of different concentrations of antioxidant solutions were also added. This gave different final concentrations of a specific antioxidant. Cu 2+ and H202 were used as the hydroxyl radical generator to start the reaction. Once Cu 2+ was added, the reaction mixture was incubated at 37°C. Fluorescence was measured every 5 or 2 min at the emission of 565 nm and excitation of 540 nm using a Perkin-Elmer LS-5 fluorescence spectrophotometer until zero fluorescence occurred. For a standard, 20 #1 of a 1-mM (10 #M in final concentration) Trolox stock solution was assayed during each run. The hydroxyl radical absorbance capacity (ORACoH.) refers to the net protection area under the quenching curve of ~-PE in the presence of an antioxidant. One ORACoH. unit has been assigned the net protection area (S) provided by 1 ml of 10 #M Trolox in final concentration. The ORACoH. value (units) of a sample is calculated as follows (Fig. 1): ORACoH. Value (units/ml)

= (Ssample - SBlank)/(STrolox - SBlank)

Blank: 20/~1 phosphate buffer was added. S refers to the area under the quenching curve of fl-PE. This area is integrated by a computer connected directly to the output of the fluorescence spectrophotometer. The program used by the computer was based on the formula shown in Fig. lB. Here, the coefficient of variation within a run was 4% (n = 6) and from run to run was 6% (n = 4). 3. Results

Hydrogen peroxide with copper ions can damage fl-PE, the indicator protein of

G. Cao, R.G. Cutler~Arch. Gerontol. Geriatr. 17 (1993) 189-201

192

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the assay system, resulting in a decrease in its fluorescence. Hydrogen peroxide alone almost has no damaging effect on/3-PE. The damaging effect of Cu 2÷ alone on riPE is also obvious but much weaker than that of H202 plus Cu 2÷ (Fig. 2A). The damage to fl-PE depended upon the concentration of both H202 and Cu 2÷. However, the effect of the concentration of Cu 2÷ on the quenching of fl-PE is much stronger than that of H202 (Fig. 2B).

G. Cao, R.G. Cutler~Arch. GerontoL Geriatr. 17 (1993) 189-201

193

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Fig. 2. The decay in ~-PE emission on incubation with different oxidants. A (a) H202 (0.3%) plus Cu 2÷ (9 × l0 -6 M); (b) H202 (0.3%), Cu 2+ (9 x 10 -6 M) and antioxidant (Trolox, 4 ttM); (c) Cu E÷ (9 x 10 -6 M) only; (d) H202 (0.3%) only; and (e) N a K phosphate buffer only. B (a) 1.8 x l0 -5 M Cu z+ + 0.3% H202; (b) 1.8 x 10 -5 M Cu 2+ + 0.03% H202; (c) 1.8 × 10 -6 M Cu 2+ + 0.3% H202; (d) 1.8 x 10 -6 M Cu 2+ + 0.03% H202. /~-PE was 3.34 x l0 -8 M throughout and the reactions were performed in 2 ml 0.075 M N a K phosphate buffer (pH 7.0) at 37°C.

The assay system chosen for the determination of the hydroxyl radical absorbance capacity of an antioxidant was one that needs relatively lower concentration of copper ions and that can also totally destroy/~-PE within 20-30 min in the absence of any antioxidants. Thus, the assay for an antioxidant sample can usually be finished within 60 min. Ascorbic acid showed no net hydroxyl radical absorbance capacity in the presence of H20 z and Cu 2÷ in this assay system when its final concentration was between 0.1

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G. Cao, R.G. Cutler~Arch. Gerontol. Geriatr. 17 (1993) 189-201

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Fig. 3. The hydroxyl radical absorbance capacity of ascorbic acid. A The kinetics of/3-PE quenching with different concentrations of ascorbic acid. B The O R A C o H . value of ascorbic acid at its different concentrations (10-1000 #M): y = -0.3919 log x + 0.3796, n = 7, r = -0.9939, P = 0,00001. The reaction mixture contains also 3.34 × 10 -8 M /3-PE, 0.3% H202 and 9 × 10 -6 M Cu 2+. The reactions were performed in 2 ml 0.075 M N a K phosphate buffer (pH 7.0) at 37°C.

/~M and 10/~M in the incubation mixture. When the final concentration of ascorbic acid was more than 10/~M in the incubation mixture, it acted as a powerful oxidation stimulator. This result is shown in Fig. 3. The hydroxyl radical absorbance capacity of Trolox increased with its final concentration in the incubation mixture from 0.1 to 20 ~M and decreased from 20 to 400 #M. Trolox acted as an oxidation stimulator rather than an antioxidant when its final concentration was higher than 1000 tzM. The maximum hydroxyl radical absorbance capacity of this water-soluble oe-tocopherol analogue was obtained with a final concentration range from 10 to 40/~M in the incubation mixture (Fig. 4). The hydroxyl radical absorbance capacity of uric acid increased in the concentration range of 0.1-100 t~M in the final incubation mixture and decreased in the range of 100-1000 t~M (uric acid was found to be insoluble at a concentration above 1000

195

G. Cao, R.G. Cutler/Arch. Gerontol. Geriatr. 17 (1993) 189-201

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#M at 37°C). The maximum hydroxyl radical absorbance capacity of uric acid was obtained from 40 to 250 t~M (Fig. 5). The concentration-dependent changes in the hydroxyl radical absorbance quality of ascorbic acid, Trolox and uric acid were not related to the direct interaction between B-PE and these antioxidants. When different concentrations of ascorbic acid (1, 10, 100 and 1000/zM, respectively), Trolox (1, 10, 100, 1000 and 4000 #M, respectively) and uric acid (1, 10, 100 and 1000/~M, respectively) were individually incubated with/3-PE, they all gave an exactly same/~-PE quenching curve as curve e in Fig. 2A, i.e. as that produced by incubating/3-PE itself in the absence of any other reagents. When only H202 or Cu 2+ instead of both H202 and Cu 2+ was used as an oxidant, Trolox also showed a similar concentration-dependent change in its antioxidant quality. The protection produced by Trolox for B-PE against H202 or Cu 2+ increases with its concentration from 0 to 10/zM and decreases from 10 to 4000 #M

196

G. Cao, R.G. Cutler / Arch. Gerontol. Geriatr. 17 (1993) 189-201

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(Fig. 6A). However, the protection produced by uric acid for B-PE against H202 or Cu 2+ increases with its concentration within the whole range from 0 to 1000 #M (Fig. 6B). However, both the protection produced by Trolox (at low concentration) or uric acid for B-PE against H202 or Cu 2+ and the damage to ~-PE enhanced by Trolox (at high concentration) are very small, if these results are compared with those obtained by using both H202 and Cu 2+ as a hydroxyl radical generator. Ascorbic acid strongly enhanced the oxidant capacity of H202 or Cu 2+ used separately, showing a concentration-dependent increase. This is shown in Fig. 6C. 4. Discussion H 2 0 2 with copper ions has been shown to cause oxidative damage in biological systems in vitro, such as oxidation of protein amino acid residues (Gutteridge and

G. Cao, R.G. Cutler~Arch. Gerontol. Geriatr. 17 (1993) 189-201

197

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Fig. 6. Protection of (A) Trolox, (B) uric acid and (C) ascorbic acid for 19-PE against the attacking of H202 or Cu 2+. n , H202; +, Cu 2+. The protection for/9-PE against the attacking of H202 or Cu 2+ was expressed by using arbitrary units. The arbitrary units = (Ssampie - SBlank)/SBlank. S was calculated accoro ding to the formula showed in Fig. 1. #-PE was 3.34 × 10- a M, H 2 0 2 was 0.3%, Cu 2+ was 9 × IG- 6 M. The reactions were performed in 2 ml 0.075 M N a K phosphate buffer (pH 7.0) at 37°C. The incubation time was about 30-32 rain. However, in these experiments that used only H20 2 or only Cu 2+ as an oxidant it was difficult to drive the reaction to completion, as either H 2 0 2 or Cu 2+ was a much weaker oxidant compared with H20 2 plus Cu 2+ (Fig. 2A).

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G. Cao. R.G. Cuth, r/Arch. Gerontol. Geriatr. 17 (1993) 189-201

Wilkins, 1983;), hemolysis (Aaseth et al., 1984), lipid peroxidation (Chan et al., 1982) and DNA base modification and cleavage (Dizdaroglu et al., 1991; Tkeshelashvili et al., 1991). This H202-Cu 2+ system-induced oxidative damage is generally attributed to the formation of hydroxyl radical (Rowley and Halliwell, 1983; Reed and Douglas, 1989; Sagripanti and Kraemer, 1989; Dizdaroglu et al., 1991: Hanna and Mason, 1992; Sato et al., 1992), although other oxygen radicals are also produced in the system (Hanna and Mason, 1992). The hydroxyl radical is the most harmful oxygen radical produced in the body. The generation of hydroxyl radicals and the subsequent damage to macromolecules in the widely used H202-Cu 2+ system can be accounted for by the following sequence of reactions (Gutteridge and Wilkins, 1983; Hanna and Mason, 1992): CU 2+ + H202 -- Cu + + HO2" + H + 2HO2" -- 202" - + 2H + -- H202 + 02 Cu 2+ + O 2" - __ CU + + 02 Target-Cu + complex + H202 ~ target-Cu2+-OH • + O H Target-Cu2+-OH. -- damaged target + Cu 2+ where the target in this assay is fl-PE. In this system both H202 and Cu 2+ can be reproduced during the reaction process. The continued production of hydroxyl radicals is assured by the continued reduction of Cu 2+ to Cu ÷. The copper concentration used in the assay is lower than that in human serum but higher than that in human urea. Ascorbic acid has no hydroxyl radical absorbance capacity at low concentration and acts as a strong oxidation stimulator at a concentration higher than 10 taM in the assay system. This is because the ascorbate-Cu 2÷ system can also produce hydroxyl radicals (Glazer, 1988). Ascorbic acid is a very strong reductant in terms of its ability in reducing Cu 2+ to Cu +. Ascorbate and fl-PE were also used for the detection of hydroxyl radical scavengers (Glazer, 1988). The weakness of the method in this report (Glazer, 1988) is the difficulty in quantifying results. The hydroxyl radical absorbance capacity of Trolox increases with its concentration at low concentrations and decreases at high concentrations. The maximum hydroxyl radical absorbance capacity of this water soluble ot-tocopherol analogue was obtained within a final concentration range from 10 to 40 taM in the incubation mixture. Furthermore, the hydroxyl radical absorbance capacity of Trolox at 4 taM or 100 taM is still about 90% of its maximum value. This is interesting because (i) the concentration range from 10 to 40 taM is just the normal range found for atocopherol in human plasma, and (ii) Trolox and ot-tocopherol have the same ring portion which is responsible for their oxygen-radical absorbing quality. Numerous surveys on human populations who did not take ot-tocopherol supplements showed the mean value of plasma tocopherol determined from 36 different studies to be around 21 taM, with a range of 8-28 tzM (Chan, 1989). A recent study on 1373 cancer-free Finnish men and women also showed that the mean value of serum ottocopherol was 20 taM among men and 24 taM among women (Knekt et al., 1988). Trolox and c~-tocopherol have the same peroxyl-radical absorbance capacity on a

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molar basis because of their same functional structure (Cao et al., 1993). Both Trolox and ot-tocopherol trap two peroxyl radicals per molecule (Burton et al., 1983;). Here we did not measure the hydroxyl radical absorbance capacity of ottocopherol of different concentrations because of the difficulty in dissolving this antioxidant at high concentration. However, the effective concentration of ottocopherol in a membrane or in the serum attached to an LDL may be higher than the normal t~-tocopherol range found in human serum. But the small decrease (about 10%) of hydroxyl radical absorbance capacity of the a-tocopherol analogue at 100 #M could explain the effectiveness of oL-tocopherol at a concentration several times higher than its normal serum value. The hydroxyl radical absorbance capacity of uric acid also increases with its concentration at low concentrations and decreases at high concentrations. The maximum hydroxyl radical absorbance capacity of uric acid was obtained from 40 to 250 #M, which is almost the normal serum concentration found for this antioxidant in healthy humans. The normal serum uric acid concentration range in human was found to be 2-6 mg/100 ml or 119-357 #M (Lehninger, 1982). The hydroxyl radical absorbance capacity of both Trolox and uric acid decreases at high concentration. In fact, Trolox acts as an oxidant in stead of an antioxidant when its concentration is higher than 1 mM in this assay. This suggests that the human species may have evolved an optimal serum concentration of uric acid, and perhaps also a-tocopherol, to reduce oxygen radicals produced by H202 with copper ions. Any more uric acid may have harmful effects. It is well known that a high serum concentration of uric acid beyond the normal range has been related to the formation of gout in humans. Our experiment results also showed that the concentration-dependent changes in the hydroxyl radical absorbance quality of Trolox and uric acid were not related to the direct interaction between fl-PE and these antioxidants. Trolox may react with H202 or Cu 2+ and enhance their damage to/3-PE. However, even in the presence of high concentration of Trolox, the damage to/3-PE by H202 or Cu 2+ is very limited if it is compared with that to/3-PE by both H202 and Cu 2÷. A more important fact is that the protection produced by uric acid for/3-PE against H202 or Cu 2+ increased with its concentration within any concentration ranges that we could obtain. This means that the antioxidant quality of uric acid is not concentration-dependent when only H202 or Cu 2÷ instead of both H202 and Cu 2+ was used as an oxidant. So, the mechanism involved in the decrease of hydroxyl radical absorbance capacity of Trolox and uric acid at high concentrations may be related to their reaction with hydroxyl radicals or other oxygen radicals produced in the presence of both H202 and Cu 2÷ and thus leading to the formation of too many Trolox or uric acid radicals at the same time. It is known that uric acid radicals themselves are also capable of bring significant damage to macromolecules (Kittridge and Willson, 1984), although these radicals are weak in terms of their ability to react with other molecules. Our results indicated the potential danger of ascorbic acid in the presence of n202 or/and Cu E÷ although it was also reported that dehydro-L-ascorbic acid can protect human low density lipoprotein against Cu2÷-induced oxidative modification (Retsky et al., 1993). In the human body, dehydro-L-ascorbic acid could be reduced quickly to L-ascorbic acid. Considering these points, it may not always be true that more antioxidant in the

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serum w o u l d p r o p o r t i o n a t e l y increase a n t i o x i d a n t capacity. The benefits b r o u g h t by an a n t i o x i d a n t a p p e a r to be o b t a i n e d only within a certain range. T r o l o x is being widely a p p l i e d in b o t h basic a n d clinical studies (Bolkenius et al., 1991; W u et al., 1991; Roveri et al., 1992; Le et al., 1992). The effects o f T r o l o x in different studies could be different because o f the different dosages used. W u et al. (1991) have atready r e p o r t e d that a l t h o u g h in cultured rat h e p a t o c y t e s 0.5 to 16 mmoi/1 o f T r o l o x was observed to p r o l o n g the survival o f cells exposed to o x y r a d i c a l s generated with xanthine o x i d a s e - h y p o x a n t h i n e , the o p t i m a l T r o l o x dosage was only between 1 mmol/1 a n d 2 mmol/1 ( W u et al., 1991). Large dosages, especially o f intravenous a n d / o r i n t r a m u s c u l a r injections o f a single a n t i o x i d a n t like ascorbic acid and T r o l o x itself, m a y even cause h a r m f u l effects. I f true for other a n t i o x i d a n t supplements, these findings w o u l d have m a j o r i m p l i c a t i o n s as well for r e c o m m e n d a t i o n s o f dietary supplements o f a n t i o x i d a n t s b e y o n d n o r m a l ranges.

5. Acknowledgement This w o r k is s u p p o r t e d in p a r t by the Paul G l e n n F o u n d a t i o n for M e d i c a l Research.

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