HPB Surgery, 1996, Vol.10, pp. 73-77
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The Ability of Bile to Scavenge S-uperoxide
Radicals and Pigment Gallstone Formation in
Guinea Pigs*
CONG LIN, TAO SHEN, XIANBO FU and XIAOSI ZHOU
Department of Surgery, Third Teaching Hospital, Beijing Medical University,
Beijing, 100083, P. R. China
(Received 10 February 1994)
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 (/)<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 (/)<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 Ca+ concentrations, the participation of oxygen radicals might be of importance for
pigment gallstone formation induced by bile duct obstruction.
KEY WORDS: Free radical pigment gallstone formation
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, obstruc-
tion 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).
*This work was supported by the National Natural Science
Foundation of China, No. 3880768.
Correspondence to: Dr. X. S. Zhou, Department of Surgery,
Third Teaching Hospital, Beijing Medical University, 49 North
Garden Road, Beijing, 100083, P. R. China.
73
MATERIALS AND METHODS
Reagents
Bilirubin (99%) was purchased from fluka Company
(Switzerland). Xanthine oxidase was purchased from
Dongfend Biochemical Technical Company ofShang-
hai Biochemical Institute (Chinese Academy of Sci-
ences). Xanthine was obtained from Sigma Chemical
Company(USA).
Grouping ofAnimals
52 male guinea pigs (300-700 g body weight) were
divided into three groups. (1) Stricture group (S): 19
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 CONG LIN et al.
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 ob-
served. After 1100 g centrifugation for 15 minutes, the
scavenging rate (SR)4'5 ofbile on exogenous superoxide
radical (O), concentrations ofionized calcium (ICa),
total bilirubin (TBr) and indirect reactive
bilirubin (IBr)6, which represents unconju-
gated 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 compo-
nent 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 allo-
cated in the corresponding group, especially in the C
group (see table).
The Ca2+, TBr and IBr concentration ofgallbladder
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
Ca2+
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 ofC group (p>0.05) (table
andfigure).
Statistical Analyses
For the comparison of gallstone incidence,
exact probability was calculated. The results
ofgallbladder 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
Ourprevious study revealed that the increase ofeither
UCB or Ca2+
concentration would elevate the ion prod-
uct of [UCB] and [Ca2+]. Once the ion product exceed
their conditional solubility product (K'sp), calcium-
bilirubinate would precipitate. This is called
EQUILIBRIUM THEORY of calcium bilirubinate
precipitation and dissolution.
RESULTS
Gallstone Incidence
Table 1 Gallbladder bile components
Three animals in S group died of complete s
CBD ligation on the fourth day after the operation, and ICa
gallstones were found in all of them. In S+V group, uM SD 441
four animals died ahead of schedule and gallstones n 16
were not found in them. Two ofthem died ofcomplete TBr mean 40.8
uM SD 30.3
CBD ligation (four and five days after operation n 16
respectively); the other two did ofrupture ofabdominal IBr mean 12.6
uM SD 8.9
incision on the sixth day. These seven animals were n 16
excluded. SR mean 68.3
The PS incidence of S group (14/16=87.5%) % SD 31.1
and S+V group (5/14-35.7%) were significantly higher n 14
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).
Bile Group t- es
components S+ V C S vs C S+ V vs S
mean 765 720
304
14
69.2
40.3
13
16.9
11.5
13
92.1
8.2
14
281
113 t=3.537 t=0.315
p<0. 01 NS
13.7
8.1 t=2.301 t=2.165
7 p<0. 05 p<0. 05
5.2
3.4 t=2.106 t=1.124
7 p<0. 05 NS
94.4
4.8 t=2.475 t=2.772
9 p<0. 05 p<0. 05
note: C Control group S Stricture group
S+V Stricture plus Vit. E, C group
ICa Ionized calcium TBr Total bilirubin
IBr Unconjugated bilirubin SR Scavenging rate
NS No significance
FREE RADICAL AND PIGMENT GALLSTONE 75
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 Ca2+
con-
centrations in bile. But, the drop ofPS incidence from S
group to S+V group might not be attributed to the
coexistent change oftheUCB and Ca2+
concentrations
in bile, because the decrease ofCa2+
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 signifi-
cantly; when the PS incidence dropped from S group to
S+V group, the SR increased significantly and
approximated to the level ofC group. These facts sug-
gested that in addition to the increase ofUCB and Ca2+
concentrations, the participation offree 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 equilib-
rium ofcalcium bilirubinate might exist, and this is to
be investigated.
The necessity of free radicals in PS formation does
not belittle the importance of equilibrium theory. Be-
cause, the experiments in vitro of our colleagues
revealed that there were three effects offree radical on
the interaction ofcalcium ion and bilirubin in the pres-
ence of sodium cholate (1) accelerating the speed of
precipitation; (2) decreasing the K'sp; and (3) enlarg-
ing the size of precipitated calcium bilirubinate parti-
cles. All ofthese effects promoted the precipitation and
polymerization ofcalcium 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 condi-
tion 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 effective-
ness is the precipitation and dissolution equilibrium
itself.
In addition, oxygen radicals may promote the for-
mation of gallstones by stimulating glycoprotein se-
cretion The conditions for the stricture group and
control group in Xu's experiment were the same as
those ofS group and C group in this experiment respec-
tively. He had determined the content of glycoprotein
of gallbladder bile. The glycoprotein content of stric-
ture group was significantly higher than that ofcontrol
group3. The important role of glycoprotein in the for-
mation 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.
CONCLUSION
1. The incomplete obstruction of guinea pig com-
mon bile ducts resulted in high pigment gallstone inci-
dence. At the same time, the ability of scavenging
1oo
i// "k" // iiiii!iii!i!i!i!
INCIDENCE(%) TBr(uM) IBr(uM) ICa(lO"M)
IC S 8S+V
S vs C or S+V vs S * p<O.05 ,. p<O.01
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.
76 CONG LIN et al.
exogenous O in bile decreased, the UCB and Ca2+
concentrations of gallbladder bile increased.
2. Administration ofVit. E, C to the animal models
recovered the scavenging rate of the gallbladder bile
on exogenous O to the level ofcontrol; in spite ofthat
UCB and Ca2+
concentrations remained high, the inci-
dence 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 gall-
stone by enhancing the ability of scavenging oxygen
radicals in bile, it was not capable to preventing pig-
ment gallstone formation thoroughly.
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COMMENTARY
Pigment gallstones are of two major types, black and
brown stones. These two pigment types stones are
morphologically, compositionally and clinically
distinct1,2.
Black pigment gallstones forms usually within
the gallbladder although they can migrate to the bile
duct.They are associated with heamolytic anaemia,
cirrhosis, and alcoholism. The incidence ofblack gall-
stone disease increases with age2. The pathogenesis of
black stones is probably related to nonbacterial,
nonenzymatic hydrolysis ofbilirubin conjugates, espe-
cially the monoconjugates3.
De novo common bile duct stones are likely to be
brown pigment stones. The presence ofajuxtapapillary
duodenal diverticulum is an important associated risk
factor for formation ofbrown pigment stones probably
because ofthe change in the bacterialflora in the region
of the papilla4. Brown pigment stones are formed with
stasis or infection in the biliary system1. Maki postu-
lated that such stones form following hydrolysis ofbili-
rubin diglucuronide by bacterial glucuronidase to
form glucuronic acid and bilirubin5. Unlike bilirubin
diglucuronide, which is freely water soluble, unconju-
gated bilirubin is almost insoluble in water and may
precipitate by combining with calcium to form calcium
bilirubinate1. Both bacteria and tissues contain
gltcuronidases, and although the pH optimal for the
enzymes are different, the pH ofthe microenvironment
in which the hydrolysis occurs is unknown and may
allow either or both types of enzymes to act1. Similarly
bothbacteria and tissue containphospholipases and the
fatty acids present in the stone are very likely the product
of either bacterial or tissue catalysis or hydrolysis of
biliary lecithin8. Recent data indicate that a variety of
calcium salts offatty acids are present in brown stones.
Palmitate is only one ofthese salts2.
This study attempts to show that oxygen radicals
might be related to the formation of pigment stones
in animals withpartial bile duct occlusion in addition to
the increases in unconjugated bilirubin and calcium
concentrations. The administration of intraperitoneal
vitamins E, C, known as antioxidants, is capable of
decreasing the incidence ofpigment stones but not com-
pletely preventing its formation, suggesting that free
radicals may play a role in pigment stone formation.
W.Y. Lau, F.R.A.C.S.
A.K.C. Li, F.R.A.C.S.
Department ofSurgery
The Chinese University ofHong Kong
Prince ofWales Hospital
Shatin, New Territories, Hong Kong
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FREE RADICAL AND PIGMENT GALLSTONE 77
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