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Annals of Clinical & Laboratory Science 35:37-45 (2005)
© 2005 Association of Clinical Scientists

Free Radical Scavenging, DNA Protection, and Inhibition of Lipid Peroxidation Mediated by Uric Acid

Beth Stinefelt1, Stephen S. Leonard2, Kenneth P. Blemings1, Xianglin Shi2 and Hillar Klandorf1
1 Division of Animal and Veterinary Science, West Virginia University, Morgantown, West Virginia2 Pathology and Physiology Research Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, West Virginia

Address correspondence to Kenneth P. Blemings, Ph.D., West Virginia University, P.O. Box 6108, Morgantown, WV 26506, USA; tel 304 293 2631; fax 304 293 2232; e-mail kbleming{at}wvu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Uric acid (UA) has been proposed to be the dominant antioxidant in birds. The objective of this study was to investigate the quenching effect of varying concentrations of UA, including those found in avian plasma, on specific reactive oxygen species (ROS) and to determine the ability of UA to protect DNA and cellular membranes from ROS-mediated damage. Hydroxyl (•OH) and superoxide (O2•–) radicals were detected by electron spin resonance (ESR) and their presence was reduced following addition of UA (p <0.05) in a concentration-dependent manner. UA inhibited hydroxyl-mediated DNA damage, indicated by the presence of more precise, dense bands of {lambda} Hind III DNA after agarose gel electrophoresis and ethidium bromide staining (p <0.05). Lipid peroxidation of silica-exposed RAW 264.7 cell membranes was diminished (p <0.02) after addition of UA to the cell incubation mixture. These studies demonstrate that UA scavenges hydroxyl and superoxide radicals and protects against DNA damage and lipid peroxidation. These results indicate specific antioxidant protection that UA may afford birds against ROS-mediated damage.

(received 6 October 2004; accepted 21 October 2004)

Keywords: uric acid, oxidative stress, electron spin resonance, lipid peroxidation, DNA oxidation


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Molecular oxygen is required for indispensable mechanisms in aerobic organisms. However, some oxygen and nitrogen metabolites, termed reactive oxygen or nitrogen species, are toxic and the body requires defense mechanisms against these highly reactive molecules. Oxidative stress occurs when reactive oxygen/nitrogen species overwhelm the antioxidant defense system. This is observed as a change in the organism’s redox status that favors a disproportionate increase in reactive species or a decrease in the antioxidant defense [1]. If reactive species are not scavenged by antioxidants, they react with other cellular components [2]. The consequences of such detrimental reactions include lipid peroxidation [3], protein modification [4], and DNA oxidation [5].

The synergistic theory of aging proposes that age-related deterioration of tissues is, in part, related to the accumulation of glycosylation end-products and their interactions with Maillard products and free radicals [6], which indicate and cause oxidative stress and oxidative damage to proteins [7], DNA [8], and lipids [9]. Avian species have a metabolic rate (oxygen consumption) that is approximately 2 to 2.5 times higher than that of mammals of comparable body size [10]. In mammals, mitochondrial electron leak, which is a major source of hydrogen peroxide and superoxide, is estimated to be 1 to 2% of the oxygen consumption rate [11]. Hence, considering avian metabolism and the increased potential for reactive species production, birds should age faster than mammals of comparable size. However, the opposite actually occurs; birds live much longer than mammals of similar size [10].

A combination of oxidative stress-reducing factors characteristic of avian species, when coupled with existing theories of aging, may explain avian longevity. One factor is that birds have a lower rate of heart, brain, kidney, and lung mitochondrial reactive oxygen species production, resulting from a reduced free electron leak relative to mammals of similar body size [12]. Another contributing factor is that birds have higher concentrations of plasma uric acid than mammals of similar size [13–15], which protects tissues from reactive species-mediated damage. Positive correlations have been reported between the maximum life span potential (MLSP) and the uric acid concentrations in brain and plasma per specific metabolic rate [16].

The plasma concentration of uric acid in broiler chickens ranges from 0.2 to 0.8 mM [17,18]. Decreasing uric acid production in birds by approximately 33% causes an increase in oxidative stress, as evidenced by increased accumulation of markers of reactive species mediated tissue damage [19]. Skin pentosidine, an intramolecular tissue glycoxidation product that accumulates in collagen, and breast muscle shear force, which are both markers of aging or oxidative stress, were also increased in birds with low plasma uric acid concentrations [20]. Treating broiler chickens with inosine, a uric acid precursor, increases plasma uric acid concentrations, and is associated with a reduction of oxidative stress and some reactive species mediated markers of aging [18]. These studies are consistent with the birds using uric acid as a defense to prevent premature aging and tissue damage caused by reactive species.

Uric acid is an antioxidant because it can inactivate an oxidant by an electron transfer before the oxidant can react with the targeted biological molecule [21]. Evidence suggests that uric acid quenches hydroxyl radical generation by the Fenton reaction [22]. Uric acid is unreactive with diatomic atmospheric oxygen [21]. The ability of uric acid to quench the diatomic superoxide radical is questionable. Although uric acid is unable to protect alcohol dehydrogenase from inactivation when exposed to superoxide [23], uric acid can inhibit superoxide-mediated DNA damage [5].

The objective of this study was to determine the ability of uric acid to scavenge specific free radicals and to protect DNA from oxidation by the hydroxyl radical. The ability of uric acid to protect monocyte cell membranes from lipid peroxidation mediated by reactive species released during an oxidative burst was also determined. The uric acid concentrations utilized in these experiments include and exceed the plasma concentrations of uric acid in birds.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Acknowledgements
 References
 
Free radical measurements.  Electron spin resonance (ESR) spin trapping was used to detect short-lived free radical intermediates using a modified technique [24]. ESR involves the addition-type reaction of a short-lived radical with a spin-trap to form a relatively long-lived free radical product (spin adduct) that can be detected by conventional ESR. The intensity of the signal is used to quantify the amount of free radicals produced in the reaction; hyperfine splittings of the spin-adduct are used to identify the trapped radicals. All ESR measurements were conducted using a Bruker EMX spectrometer (Bruker Instruments, Billerica, MA) and a flat cell assembly. The Acquisit program was used for data acquisition and analysis.

The hydroxyl radical was generated from a Fenton reaction with a final concentration of 0.5 mM iron sulfate (FeSO4), 0.5 mM hydrogen peroxide (H2O2), and 10.0 mM 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin-trap. Uric acid solution was added to the reaction mixture to final concentrations of 0.0, 0.5, 1.5, 2.5, and 3.5 mM. Sodium formate (50 mM) was added to an additional Fenton reaction along with uric acid. The reaction mixtures were brought to a 1 ml volume with phosphate buffered saline (PBS).

The superoxide radical was generated with 3.5 mM xanthine and 4 units xanthine oxidase, with 100 mM DMPO as a spin trap. All reagents were kept on ice to retard decomposition of superoxide radical adducts. Uric acid was added to the reaction mixture to final concentrations of 0.0, 0.125, 0.25, 0.5, and 1.0 mM. The reaction mixtures were brought to a volume of 1 ml with PBS.

The PBS (pH 7.4) was treated with Chelex 100 to remove transition metal ion contaminants. DMPO was purified by charcoal decolorization. All reactions were initiated by mixing in a test tube and each reaction mixture was transferred to a flat cell for ESR measurement. Experiments were performed under ambient laboratory conditions. Signal intensity was quantified by averaging the two individual heights (mm) from the valley to the peak of the two highest signals.

DNA strand breakage assay.  The DNA strand breakage assay was carried out by an established method [25]. Briefly, each reaction mixture contained 10 mg DNA ({lambda} Hind III fragments), 1 mM FeSO4, 10 mM H2O2, and 0.0, 0.5, 1.0, or 2.5 mM uric acid; the volume was brought to 100 µl with PBS. The mixtures were incubated for 20 min in a test tube at 37°C. Five µl of the reaction mixtures were mixed with 2 µl of gel loading solution (0.1 M EDTA, 0.5% sodium dodecyl sulfate, 40% sucrose, and 0.5% bromophenol blue) and loaded into individual wells in a 0.7% agarose gel for electrophoresis at 1 to 2 V/cm in TBE buffer (0.1 M tris; 0.09 M boric acid; 0.001 M EDTA). Gels were stained with Vistra Green (Amersham Biosciences, San Francisco, CA) for 30 min and were photographed under ultraviolet light using a Stratagene Eagle Eye II camera (Stratagene Inc, La Jolla, CA). DNA damage was assessed by quantitative analysis of the first smeared band, which indicates fragmentation resulting from hydroxyl-mediated strand cleavage. Densitometry was completed in the immediate region surrounding the first band in each lane.

Lipid peroxidation assay.  Lipid peroxidation of silica-exposed RAW 264.7 mouse peritoneal monocytes was measured with a colorimetric assay for lipid peroxidation products (Bioxytech MDA-586 kit, Oxis International, Portland, OR). The reaction mixture included 1 x 107 cells, 100 µl of 1 mg/ml Min-U-Sil (crystalline silica) or 5 mg/ml Min-U-Sil, with final concentrations of 0.0, 0.5, 1.0, 2.0, or 2.5 mM uric acid. Reactions were brought to a final volume of 1 ml with PBS. The mixture was incubated 1 hr in a shaking water bath at 37°C. Measurement of lipid peroxidation was based on the reaction of a chromogenic reagent with malondialdehyde (MDA) and 4-hydroxyalkenals after a further incubation at 45°C for 60 min. Standards and reagent blanks were used to generate a standard curve. After cells were removed by centrifugation (500 x g for 5 min), the absorbance of the supernatant was measured at 586 nm.

Statistical analysis.  Data were analyzed by analysis of variance with the PC-SAS general linear models procedure for significant differences among treatment means. In the event of a significant F value, the LSD procedure was used for means comparisons. Differences were considered significant at p 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Acknowledgements
 References
 
Amelioration of hydroxyl and superoxide radicals.  Representative ESR spectra generated from the Fenton reaction using DMPO as a spin trap are shown in Fig. 1Go. This spectrum consists of a 1:2:2:1 quartet with splittings of aH = aN = 14.9 G [26]. Based on these splitting constants the quartet was assigned to a DMPO/•OH adduct. The Fenton reaction was also exposed to sodium formate, which serves as a hydroxyl radical scavenger to confirm the presence of the hydroxyl radical adduct. In this reaction, a new radical is formed that can be trapped by DMPO, which shows a new adduct signal with hyperfine splittings of aH = 15.8 G and aN = 18.8 G that are typical of a DMPO/•CO2– adduct. The intensity of the ESR signal generated from the Fenton reaction without any uric acid serves as the control and is set at 0% inhibition. Adding uric acid to the Fenton reaction was effective in reducing spin adduct signal intensity over 50% with 3.5 mM uric acid (Fig. 1FGo). Table 1Go shows the concentration-dependent effect of uric acid on the ESR signal intensity (r2 = 0.77, p = 0.05). Spectra are shown in Fig. 1Go.



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  		Fig. 1: ESR spectra of hydroxyl radical adducts. Spectra indicate radical adducts created in a PBS buffered system containing 10.0 mM DMPO only (A) and 10.0 mM DMPO, 0.5 mM FeSO4, and 0.5 mM H2O2 with 0.0 mM uric acid (B), 0.5 mM uric acid (C), 1.5 mM uric acid (D), 2.5 mM uric acid (E), 3.5 mM uric acid (F), and 3.5 mM uric acid and 50 mM sodium formate (G). ESR spectrometer settings were: receiver gain, 6.32 x 104; time constant, 20.48 msec; modulation amplitude, 1.00 G; scan time, 20.97 sec; magnetic field 3480 ± 100 G.

 

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  		Table 1. Concentration-dependent effects of uric acid on spectral intensities of the hydroxyl and superoxide ESR signals (see text).
 
Fig. 2Go displays the spin adduct generated by the xanthine and xanthine oxidase system in the absence or presence of uric acid. The superoxide-DMPO adducts produced from this reaction are represented as the hyperfine splittings in Fig. 2Go [26]. A reduction (70%) of the signal is observed when the superoxide generating reaction occurs in 1.0 mM uric acid (Fig. 2EGo). Table 1Go shows the concentration-dependent effect of uric acid on the superoxide adduct intensity (r2 = 0.88, p <0.02) that is observed in the ESR spectra of Fig. 2Go.



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  		Fig. 2. ESR spectra of superoxide radical adducts. Reaction mixtures in a PBS buffered system containing 100 mM DMPO only (A) and 100 mM DMPO, 3.5 mM xanthine, 4 units xanthine oxidase and 0.0 mM uric acid (B) 0.125 mM uric acid (C), 0.25 mM uric acid (D), 0.5 mM uric acid (E), and 1.0 mM uric acid (F). ESR spectrometer settings were: receiver gain, 6.32 x 104; time constant, 20.48 msec; modulation amplitude, 0.50 G; scan time, 20.97 sec; magnetic field 3480 ± 100 G.

 
Reduction of DNA fragmentation.  Hydroxyl radicals generated by the Fenton reaction are capable of oxidizing DNA and causing strand breaks, resulting in DNA fragmentation. After gel electrophoresis of DNA exposed to hydroxyl radicals, the fragmented DNA looks smeared throughout the lane due to the different migration rates of the various fragment sizes (Fig. 3Go). DNA exposed to the Fenton reaction is protected from hydroxyl radical-mediated fragmentation when incubated with uric acid, as observed by more precise bands with reduced smearing (r2 = 0.97, p <0.02). In Fig. 3Go, lanes 2 through 5 represent DNA exposed to the Fenton reaction and uric acid at specified concentrations. The % density of the first band, scaled to the first band in lane 1, declines as the concentration of uric acid decreases. Therefore, uric acid protects DNA from hydroxyl-mediated fragmentation in a concentration-dependent manner, as shown by a density reduction and visually less precise bands with more smearing as concentrations of uric acid decrease.



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  		Fig. 3. Agarose gel electrophoresis of {lambda} Hind III DNA fragments. Incubation mixtures include 10 mg DNA only (lane 1), and 10 mg DNA, 1 mM FeSO4, 10 mM H2O2 and 0.0 mM uric acid (lane 2), 0.5 mM uric acid (lane 3), 1.0 mM uric acid (lane 4), and 2.5 mM uric acid (lane 5). % density = top band density/control band density x 100. Control band density is set at 100%.

 
Lipid peroxidation.  Min-U-Sil is a stimulant of oxidative burst in phagocytes. Incubation of RAW 264.7 mouse peritoneal monocytes with Min-U-Sil causes an oxidative burst that damages cell membranes, as evidenced by lipid peroxidation products. Incubation of the cells with uric acid reduced lipid peroxidation (p <0.02) in a concentration-dependent manner (Fig. 4Go). However, lipid peroxidation increased when reaction mixtures were incubated with 2.5 mM uric acid (p < 0.05), perhaps due to generation of a potent radical from interacting reactive species (see Discussion).



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  		Fig. 4. Effect of uric acid on lipid peroxidation of RAW 264.7 mouse perintoneal monocytes. Different letters represent significant differences within a Min-U-Sil level, p < 0.05. Values represent averages between 3 replicates ± SEM. Regression of lipid peroxidation with uric acid concentrations resulted in r2 = 0.67 and p = 0.018 for 1 mg/mL Min-U-Sil and r2 = 0.79, p = 0.020 for 5 mg/mL Min-U-Sil.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Acknowledgements
 References
 
The range of uric acid concentrations used in the present study includes and exceeds the plasma concentrations of uric acid in broiler chickens. The concentration-dependent decrease in the hydroxyl radical adduct ESR signal indicates that uric acid can scavenge the hydroxyl radical. Therefore, birds and other species with high uric acid concentrations may have an enhanced defense against the highly reactive, nonspecific hydroxyl radical. This enhanced ability to scavenge hydroxyl radicals may contribute to the reduction in markers of reactive species-mediated tissue injury observed in broiler chickens with elevated plasma uric acid concentrations [18]. Hydroxyl radicals are highly unstable, reacting nonspecifically with substrates. The efficacy of uric acid to scavenge these radicals is crucial for broiler health and longevity. Consequently, birds with low uric acid concentrations may be more vulnerable to hydroxyl-mediated tissue damage and should exhibit an increase in the age-related markers of oxidative stress.

Results from the superoxide experiments indicate that the superoxide radical can be scavenged by uric acid concentrations from 0.0 to 1.0 mM. Superoxide is a free radical produced from electron leak in mitochondria, and its rate of production there is inversely related to mammalian MLSP [27]. The ability to scavenge superoxide at its production site is crucial for the protection of mitochondrial components. Considering the average plasma uric acid concentration of a bird, it is likely that the mitochondrial uric acid concentration falls within the range utilized in the present experiment. Therefore, uric acid scavenging of the superoxide radical in mitochondria is likely to occur, which, in addition to the superoxide scavenging enzymes already in place, may afford the bird additional free radical protection and help to extend its longevity.

There is conflicting evidence regarding the ability of uric acid to protect cellular components from superoxide-mediated oxidation. By examining the chemistry of this reaction, it appears that uric acid can scavenge the superoxide radical only if secondary generation of the hydroxyl radical can occur [28]. In other studies, uric acid was able to protect DNA from superoxide-mediated damage generated by a xanthine oxidase system [5]. The ability of uric acid to react with the superoxide radical may be attributed to the reactivity of the urate radical with superoxide. Generation of the urate radical can only occur if uric acid is oxidized by certain reactive species such as the hydroxyl radical, hypochlorous acid, or peroxynitrite [29], which are the short-lived reactive species that uric acid has been found to be most effective at scavenging [30].

The results of the DNA protection assay indicate that uric acid protects DNA against hydroxyl mediated damage generated by Fenton chemistry. These results agree with observations that uric acid can protect DNA from damage mediated by other free-radical generating systems [5,31]. The ability of uric acid to protect DNA can be attributed to its ability to scavenge hydroxyl radicals before the radicals oxidize DNA. Although the intranuclear uric acid concentration of avian cells is unknown, presumably the uric acid found in the nucleus can protect DNA from oxidation by the hydroxyl radical. Mitochondrial DNA is the most vulnerable to oxidation due to its proximity to the mitochondrial membrane and lack of protective histones [32,33]. Therefore the ability of uric acid to scavenge superoxide to prevent DNA oxidation is most significant at this location. Uric acid may contribute to the repair of mitochondrial DNA as it has been found to restore guanine from the guanyl radical generated by pulse radiolysis [21]. The quantity of oxidized DNA, measured as 8-hydroxy-2'-deoxyguanosine (8-oxoG) in heart mitochondria, is higher in rats than in pigeons [34] and correlates negatively with lifespan across mammalian species [35]. Mitochondrial 8-oxoG increases with age in various rat [36] and human tissues [37], which is consistent with a link between reactive species production and aging. In birds, the effect of both a lower rate of reactive oxygen species production in mitochondria and a higher concentration of plasma uric acid may be the reduction of mitochondrial DNA oxidation and subsequent retardation of the aging process.

Uric acid protects erythrocyte ghosts from lipid peroxidation induced by oxo-heme oxidants [38] and t-butylhydroperoxide [3]. In the present experiment, RAW 264.7 cells were exposed to silica to stimulate an oxidative burst of reactive species. Reactive species released during oxidative burst include superoxide, hydrogen peroxide, and the hydroxyl radical, each of which has the ability to damage cellular membranes, causing lipid peroxidation [39]. Unsaturated fatty acids in tissue are more sensitive to peroxidation than saturated fatty acids and this sensitivity increases as the number of double bonds increases. It would seem advantageous to have relatively lower polyunsaturated fatty acids to reduce sensitivity to lipid peroxidation. Birds seem to have developed a non-antioxidant defense against lipid peroxidation. Compared to rats, pigeons have a lower abundance of unsaturated fatty acids in heart and liver mitochondria, and, as expected, pigeons are more resistant to lipid peroxidation [40,41]. This increased resistance to peroxidation presumably contributes to avian longevity. Also contributing to the reduced lipid peroxidation in avian cells may be a higher concentration of uric acid compared to that of other short-lived mammals.

In the present experiment, uric acid reduced lipid peroxidation caused by reactive species released upon silica-induced oxidative burst. The observed increase in lipid peroxidation in 2.5 mM uric acid is consistent with a report that uric acid may have a stimulatory effect on lipid peroxidation at higher concentrations [42]. This may occur by free radical production from uric acid decomposition products, such as the urate radical, that can further react with reactive nitrogen species to create a strong reducing agent. The 2.5 mM concentration of uric acid that resulted in increased lipid peroxidation exceeds the range of plasma uric acid found in birds. Therefore, the conditions that favor a pro-oxidative action of uric acid are unlikely to occur in vivo.

Results of the present study indicate that uric acid has a concentration-dependent effect on scavenging hydroxyl radicals and superoxide and inhibits oxidation of DNA and of lipids in cellular membranes. The ability of uric acid to prevent reactive species oxidation of biological components gives it a crucial antioxidant role in vivo, slowing the accumulation of reactive species-mediated markers of tissue injury. Consistent with the synergistic theory of aging, uric acid is important as an antioxidant for its contribution to deceleration of aging, which is supported by positive correlation between uric acid concentrations and MLSP. A reduced mitochondrial reactive oxygen species production, a lower proportion of polyunsaturated fatty acids, and a higher plasma uric acid concentration may act in combination to reduce age-related tissue damage caused by reactive species and contribute to avian longevity.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
References
 
This research was supported by the West Virginia Agriculture and Forestry Experiment Station (H393). This is paper 2899 of the West Virginia Agriculture and Forestry Experiment Station.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

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