Predrag Ljubuncic, Omar Abu-Salach, Arieh Bomzon, Department of Pharmacology, Ruth and Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel
Correspondence to: Arieh Bomzon, Department of Pharmacology, Ruth and Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Efron Street, Po Box 9649, Haifa 31096, Israel.  bomzon@tx.technion.ac.il
Telephone: +972-4-8295259    Fax: +972-4-8524978
Received: 2004-12-24    Accepted: 2005-01-13

Abstract
AIM: To investigate the ability of ursodeoxycholic acid (UDCA) to scavenge superoxide anion (O₂).

METHODS: We assessed the ability of UDCA to scavenge (O₂) generated by xanthine-xanthine oxidase (X-XO) in a cell-free system and its effect on the rate of O₂-induced ascorbic acid (AA) oxidation in hepatic post-mitochondrial supernatants.

RESULTS: UDCA at a concentration as high as 1 mmol/L did not impair the ability of the X-XO system to generate O₂, but could scavenge O₂ at concentrations of 0.5 and 1 mmol/L, and decrease the rate of AA oxidation at a concentration of 100 mmol/L.

CONCLUSION: UDCA can scavenge O₂, an action that may be beneficial to patients with primary biliary cirrhosis.

ã 2005 The WJG Press and Elsevier Inc. All rights reserved.

Key words: Ursodeoxycholic acid; Superoxide anion; Antioxidant

Ljubuncic P, Abu-Salach O, Bomzon A. Ursodeoxycholic acid and superoxide anion. World J Gastroenterol  2005; 11(31): 4875-4878
http://www.wjgnet.com/1007-9327/11/4875.asp

INTRODUCTION
Ursodeoxycholic acid (UDCA) is a naturally occurring tertiary dihydroxy hydrophilic bile acid used with considerable success in the treatment of primary biliary cirrhosis (PBC)^[1-5]. Traditional mechanisms whereby UDCA is beneficial to the diseased liver center on its ability to block the deleterious actions and encourage the choleresis of toxic bile acids^[6]. We have reported that UDCA has antioxidant properties. This finding is based on our observation that 100 mmol/L UDCA could prevent oxidative activation of cultured macrophages by the pro-oxidant hydrophobic bile acid, deoxycholic acid^[7]. In a follow-up in vivo study, we demonstrated that the therapeutic dose of 15 mg/kg UDCA administered for 24 d, suppresses the augmented extent of lipid peroxidation in the diseased liver of bile duct-ligated (BDL) rats, a widely used animal model of cholestatic liver disease^[8]. Accordingly, we proposed the antioxidant action of UDCA could contribute to its beneficial effect in patients with PBC^[7,8]. This proposal was recently confirmed by Serviddio and his colleagues who reported UDCA administered for 28 d minimized oxidative damage in the diseased liver of BDL rats^[9]. Other studies have established that UDCA can augment the biosynthesis of glutathione (GSH) and thiol-containing proteins in hepatocytes^[10,11] and could scavenge the hydroxyl radical (OH) in a cell-free system^[12]. Collectively, these findings have contributed to our current awareness that UDCA is a binary antioxidant possessing direct and indirect antioxidant properties.
    Superoxide anion (O₂) is a reactive oxygen species (ROS) generated in microsomal and mitochondrial electron transport systems when oxygen is reduced by a single electron. It can also be generated by numerous oxidative enzymes including cytosolic xanthine oxidase (XO) during oxidation of xanthine (X) to uric acid. The generation of O₂ is important, because its biotransformation can lead to formation of hydrogen peroxide through the activity of superoxide dismutase, generation of OH radical in the presence of transition metals such as Fe²⁺ or formation of the reactive peroxynitrite radical by interacting with nitric oxide. Any intervention reducing or preventing excessive generation of O₂ would result in decreased production of reactive oxygen and nitroxy species thereby lowering the extent of oxidative stress.
    Mitsuyoshi et al.^[10], reported that UDCA increases hepatocyte levels of GSH and thiol-containing proteins. In this experiment, they spectrophotometrically measured the rate of oxidation of ascorbic acid (AA) by O₂ and reported that the difference of absorbance before and after the addition of XO was lower in UDCA-treated hepatocytes than in controls. Since bile acids are enzyme inhibitors^[13,14], the difference in absorbance may be due to UDCA inhibiting the ability of the X-XO system to generate O₂. Because Mitsuyoshi et al., did not assess the effect of UDCA on the generation of O₂ by X-XO system in their investigation, we evaluated the ability of UDCA to scavenge O₂ generated in the X-XO system and its effect on the activity of XO.

MATERIALS AND METHODS
Chemicals and reagents
All chemicals and reagents of the highest purity were purchased from the Sigma Chemical Co. (St. Louis, MO, USA), except for UDCA that was purchased as its sodium salt from Megapharm Ltd, the Israeli agent of Calbiochem-Novabiochem Corporation.

Preparation of hepatic post-mitochondrial supernatants
The livers were harvested from healthy rats used as "healthy untreated controls" in institute-approved animal-based investigations. The harvested livers were washed in normal ice-cold saline, weighed and then cut into small pieces using scissors before their homogenization in 100 mmol/L ice-cold phosphate buffer, pH 7.4 at 4 ℃ with a Potter-Elvehjem glass homogenizer. The crude liver homogenates were centrifuged (1 000 r/min×10 min) at 4 ℃. The resultant supernatants were then centrifuged at 10 000 g×10 min at 4 ℃ to pellet mitochondria and the supernatant was collected. The protein content in the supernatants was determined by the method of Lowry et al.^[15].

Analytical procedures
Ability of UDCA to scavenge superoxide anion  Using the xanthine-xanthine oxidase (X-XO) reaction to generate O₂-^[16], we evaluated the ability of 0.01-1 mmol/L UDCA to scavenge O₂ by the nitroblue tetrazolium (NBT) reduction assay^[17]. The reaction mixture contained 100 mmol/L Na₂EDTA, 40 mmol/L X, and 40 mmol/L NBT in 10 mmol/L phosphate buffer pH 7.4. The reaction was started by adding 10 mU/mL XO and its rate was continuously monitored spectrophotometrically at A₅₆₀ nm for 15 min at 25 ℃ in the absence and presence of different concentrations of UDCA. The specificity of the reaction was confirmed by 300 mU/mL superoxide dismutase. The ability of UDCA to scavenge O₂, as expressed as percentage of inhibition of NBT reduction in UDCA-present samples compared to NBT reduction in UDCA-absent samples. The experiment was repeated between 9 and 11 times at each UDCA concentration.

Effect of UDCA on activity of xanthine oxidase
Compounds interacting with XO could affect the kinetics of reaction of oxidation of xanthine to uric acid^[18]. Accordingly, we assessed the effect of 0.01-1 mmol/L UDCA on XO activity by spectrophotometrically monitoring the rate of uric acid formation at A₂₉₅ nm for 3 min at 25 ℃ in the absence and presence of UDCA^[19]. The rate of uric acid formation was compared in the absence and presence of UDCA. The specificity of the reaction was confirmed by 100 mg/mL allopurinol. The experiment was repeated nine times at each UDCA concentration.

Effect of UDCA on hepatic antioxidant capacity
The effect of 100 mmol/L UDCA on hepatic antioxidant capacity was determined by monitoring the rate of oxidation of AA by the O₂ generated by the X-XO according to the original method of Nishikimi^[20] with modifications described by Mitsuyoshi et al.^[10]. Liver supernatants were incubated for 120 min at 37 ℃ in shaking water bath in the absence and presence of 100 mmol/L UDCA. Upon completion of the incubation, the change in the rate of oxidation of AA was monitored in a quartz cuvette containing 1 mL reaction mixture. The post-mitochondrial liver supernatant contained 100 mmol/L X, 100 mmol/L EDTA, 22 mg/mL catalase, 100 mmol/L AA and 0.1 mol/L phosphate buffer pH 7.4. The assay reaction was commenced by adding 10 mU/mL XO. The reaction was monitored spectrophotometrically at A₂₅₀ nm for 10 min at 25 ℃. The differences in absorbance between UDCA-absent and UDCA-added samples were compared. The results were expressed as the reaction rate of AA oxidation (△ absorbance/mg protein). The experiment was repeated thrice.

Experimental design and statistical analysis
The data were analyzed using a two-tailed t-test. The sample size for each experiment was determined by power analysis arbitrarily set between 80-90% in order to detect the effect at the 5% significance level using StatmateTM version 1 (GraphPad Software Inc., San Diego, CA, USA). All figures were prepared using PrismTM version 3.02 (GraphPad Software Inc., San Diego, CA, USA). All data were expressed as mean±SD.

RESULTS
Ability of UDCA to scavenge O 2 and effect of UDCA on activity of xanthine oxidase
Figure 1A summarizes the experiments to establish whether UDCA could scavenge O₂ using the NBT reduction assay. UDCA (10 mmol/L and 100 mmol/L) had no effect on NBT reduction (Figures 1 A1 and A2). Higher concentrations of UDCA (500 mmol/L and 1 mmol/L) slowed the rate of NBT reduction by 11% and 16% respectively (Figure 1A3 and A4). In order to eliminate the possibility that UDCA suppressed the rate of conversion of X to uric acid to account for this O₂ scavenging ability, we also measured the effects of the identical concentrations of UDCA on XO activity. None of the UDCA concentrations affected the rate of formation of uric acid (Figure 1B). Overall, these experiments demonstrated UDCA could scavenge O₂ without affecting the activity of XO.

Figure 1  (PDF) Ability of UDCA to scavenge O₂– ( A) and effect of UDCA on activity of xanthine oxidase ( B) in a cell-free system. ¹P<0.02, ^bP<0.01 vs control.

Effect of UDCA on rate of oxidation of ascorbic acid
UDCA (100 mmol/L) significantly (P<0.002) decreased the rate of AA oxidation (Figure 2).

Figure 2  (PDF) Effect of 100 mmol/L UDCA on rate of oxidation of AA by O₂– generated by X-XO in post-mitochondrial hepatic supernatants. ¹P<0.002 vs control.

DISCUSSION
The aim of the present study was to assess the ability of UDCA to scavenge O₂ and its effects on the activity of XO in a cell-free system and post-mitochondrial supernatants prepared from rat livers. In the cell-free experiments, we used UDCA concentrations not greater than 1 mmol/L in order to avoid the problems when extrapolating data to cell-containing systems in which millimolar concentrations of bile acids solubilize membranes to form micellar poly-aggregates (critical micellar concentration)^[21-23]. On the other hand, we used 100 mmol/L UDCA in the experiments conducted in post-mitochondrial supernatants because this is the concentration used by Mitsuyoshi et al.^[10], and that found in the plasma of patients treated with UDCA^[24]. Using NBT reduction in the cell-free X-XO system to evaluate the ability of UDCA to scavenge O₂, we found UDCA could scavenge O₂ at concentrations of 0.5 and 1 mmol/L, respectively. Given the knowledge that bile acids can inhibit enzyme activity^[13,14], we checked the possibility that suppression of the activity of XO accounted for ability of UDCA to scavenge O₂. We found that UDCA at concentrations as high as 1 mmol/L did not inhibit the rate of conversion of X to uric acid. After establishing that UDCA could not impair the ability of the X-XO system to generate O₂, we then evaluated the effect of 100 mmol/L UDCA on the rate of O₂-induced AA oxidation in hepatic post-mitochondrial supernatants. We found that UDCA decreased the rate of AA oxidation. This result agrees with that of Mitsuyoshi et al.^[10].
    Is the scavenging of O₂ by UDCA therapeutically relevant? Free-radical-induced peroxidation of phospholipids has been implicated in the pathogenesis of the formation of cholesterol gallstones^[25-27]. In addition, Liu and Hu demonstrated that O₂ can attack the bilirubin molecule to generate cytotoxic bilirubin radicals. ROS can also be generated in bile because hydrophobic bile acids are pro-oxidants^[28,29]. It was reported that the concentration of UDCA in bile may reach as high as 29 mmol/L in individuals given 750 mg UDCA daily for 2-3 wk^[30]. In our experiments, UDCA scavenged O₂ at the concentrations of 0.5 and 1 mmol/L suggesting that the ability of UDCA to scavenge O₂ may be beneficial in bile.
    Lapenna et al.^[12], reported that UDCA is an efficient OH scavenger. We have confirmed their finding and established the second order rate constant (k₂) for the reaction of UDCA with OH in the D-ribose oxidation assay exceeded the rate constants of other OH radical scavengers, such as mannitol or dimethylsulfoxide (Ljubuncic, Abu-Salach and Bomzon, unpublished results). In conclusion, UDCA is a direct scavenger of superoxide anion and hydroxyl radicals.

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