by Harwood Academic Publishers Printed in Malaysia The Ability of Bile to Scavenge S-uperoxide Radicals and Pigment Gallstone Formation in

After partial ligation of the common bile duct (CBD) of guinea pigs, 14 of 16 animals developed pigment gallstones within one week (S group). Intraperitoneal injection of Vit. E and C, each 10 mg/kg daily from 3 days before CBD ligation to one week after the operation (S+V group), decreased the gallstone incidence to 5/14 (exact probability<0.01). The gallstone incidence in the control group, that only received laparotomy without ligation of the CBD, was 0/15. Biochemical analysis of the gallbladder bile showed that stricture of the CBD was associated with a significant increase in levels of unconjugated bilirubin (UCB) and Ca2+ (p<0.05 and <0.01). Simultaneously the scavenging rate (SR) of superoxide radical in bile significantly decreased (p<0.05). Comparing S+V group with S group, the effect of Vit. E and C on the concentrations of UCB and Ca2+ in bile was not significant (both p>0.05), but Vit. E and C normalized the SR, and the difference between S group and S+V group was significant (p<0.05). These results suggested that Vit. E and C, known as antioxidants, enhanced the ability to scavenge oxygen radical in S+V group; and that in addition to the increases of UCB and Ca2+ concentrations, the participation of oxygen radicals might be of importance for pigment gallstone formation induced by bile duct obstruction.


INTRODUCTION
Free radical signals have been detected in pigment gallstones (PS) in vivo under anoxic condition, and the intensities of the signals correlated positively with the amounts of calcium bilirubinate1. However, obstruction ofthe biliary tract could induce an increase ofboth free radical reaction and formation of PS3. These results suggested that oxygen radicals might be related to the formation ofPS. The aim ofthis experiment is to study the relation between active oxygen radicals and PS formation using a guinea pig model, in which PS were induced by partial ligation of common bile duct (CBD). * animals, their CBD were partially ligated3. Normal saline was intraperitoneally administered as placebo from 3 days before the operation to 7 days after the operation. (2) Stricture plus Vitamin E, C group (S+V): 18 animals, the operation was the same as that 74 of S group, but the normal saline was substituted with vitamin E and C, each 10 mg per Kg body weight, injected intraperitoneally once a day. (3) Control group (C), 15 animals, laparotomy without ligation of CBD was performed, and the use of the placebo is the same as S group.

Gallstone Incidence and Bile Analyses
The guinea pigs were sacrificed 7 days after the operation.Having fasted for 12 hours, animals were anesthetized by intraperitoneal injection of sodium pentobarbital (30 mg/Kg body weight). Laparotomy was performed. Gallbladder bile was collected and gallstones in the biliary tree were observed. After 1100 g centrifugation for 15 minutes, the scavenging rate (SR) 4'5 ofbile on exogenous superoxide radical (O), concentrations of ionized calcium (ICa), total bilirubin (TBr) and indirect reactive bilirubin (IBr)6, which represents unconjugated bilirubin (UCB), in the supernatant were determined.

Gallbladder t?ile Components
In some animals, their gallbladder bile was too small to satisfy the need ofbiochemical analysis. In this case, bile specimens from two individuals in a same group were mixed up and used, even so, some mixed bile specimens were still not enough for all the bile component items. Therefore, the numbers of bile specimen, "n" in the table for each bile component, were not the same and often less than the numbers of animals allocated in the corresponding group, especially in the C group (see table).
The Ca2+, TBr and IBr concentration of gallbladder bile in S group were significantly higher than those ofC group (p<0.01, 0.05, and 0.05 respectively); and the SR decreased significantly (p<0.05). In S+V group, the Ca 2+ and IBr concentrations were not significantly different from those of S group (/)>0.05 both), and the concentration of TBr was even higher (/)<0.05). But, SR rose significantly from the level of S group (p<0.05), and closed to that of C group (p>0.05) (table   andfigure).
Statistical Analyses For the comparison of gallstone incidence, exact probability was calculated. The results of gallbladder bile components was expressed as mean + standard deviation, and grouped Student's t-test was used. P values less than 0.05 were accepted as significant.

DISCUSSION
Our previous study revealed that the increase ofeither UCB or Ca 2+ concentration would elevate the ion product of [UCB] and [Ca2+]. Once the ion product exceed their conditional solubility product (K'sp), calciumbilirubinate would precipitate. This is called EQUILIBRIUM THEORY of calcium bilirubinate precipitation and dissolution. than that of C group (0/15, p<0.01 and p<0.05 respectively); but the PS incidence of S/V group was significantly lower than that of S group (p<0.01) (see Figure). Accordingly, the rise of PS incidence from C group to S group in this study might be explained by the coincident significant elevation of UCB and Ca 2+ concentrations in bile. But, the drop ofPS incidence from S group to S+V group might not be attributed to the coexistent change ofthe UCB and Ca 2+ concentrations in bile, because the decrease of Ca 2+ concentration was not significant; furthermore, the UCB concentration of S+V group even was slightly higher than S group. On the other hand, it is striking that when the PS incidence rose from C group to S group, the SR decreased significantly; when the PS incidence dropped from S group to S+V group, the SR increased significantly and approximated to the level of C group. These facts suggested that in addition to the increase of UCB and Ca 2+ concentrations, the participation of free radical might be an essential factor in the PS formation. But, within the S+V group between animals who developed PS and those who did not, the difference in SR (89.0+8.8, n=5 vs 93.8+7.8, n=9) was not significant (p>0.05). This suggested that factors other than SR and the equilibrium of calcium bilirubinate might exist, and this is to be investigated.

Gallstone Incidence
The necessity of free radicals in PS formation does not belittle the importance of equilibrium theory. Because, the experiments in vitro of our colleagues revealed that there were three effects of free radical on the interaction ofcalcium ion and bilirubin in the presence of sodium cholate (1) accelerating the speed of precipitation; (2) decreasing the K'sp; and (3) enlarging the size of precipitated calcium bilirubinate parti-cles. All ofthese effects promoted the precipitation and polymerization of calcium bilirubinate. Their findings in vitro and the results form this experiment in vivo supported each other. The findings also revealed that free radical changed the equilibrium condition under given concentrations of [UCB] and [Ca2+], such as the reaction speed, the K'sp, and the particle size ofprecipitate. The basis ofthe free radical effectiveness is the precipitation and dissolution equilibrium itself.
In addition, oxygen radicals may promote the formation of gallstones by stimulating glycoprotein secretion The conditions for the stricture group and control group in Xu's experiment were the same as those of S group and C group in this experiment respectively. He had determined the content of glycoprotein of gallbladder bile. The glycoprotein content of stricture group was significantly higher than that ofcontrol group3. The important role of glycoprotein in the formation of both pigment and cholesterol stones is well known. Consequently, the PS incidence of S+V group being lower than that of S group might be related to inhibiting the glycoprotein secretion by scavenging oxygen radicals. Figurel The relationship between pigment gallstone formation and gallbladder bile components with bile duct obstruction.
Comparison of the gallstone incidence, the scavenging rate (SR), and the concentrations of total bilirubin (TBr), indirect reactive bilirubin-(IBr), and ionized calcium (ICa), of gallbladder bile in control (C), stricture (S), and stricture plus Vit. E, C (S+V) groups. exogenous O in bile decreased, the UCB and Ca 2+ concentrations of gallbladder bile increased. 2. Administration of Vit. E, C to the animal models recovered the scavenging rate of the gallbladder bile on exogenous O to the level of control; in spite of that UCB and Ca 2+ concentrations remained high, the incidence of PS decreased significantly. This showed that in addition to the concentrations ofUCB and Ca>, free radical is another essential factor in PS formation. 3. Although administration ofVit. E and Vit. C was capable of decreasing the incidence of pigment gallstone by enhancing the ability of scavenging oxygen radicals in bile, it was not capable to preventing pigment gallstone formation thoroughly.