JOURNAL
OF SURGICAL
RESEARCH
Dissolution
19, 47-53 (1975)
of Human
Gallstones:
Bile Salt Plus Lecithin
The Efficacy
and Heparin
of Bile Salt,
Solutions
JAMES TOOULI, M.B., B.S.,’ PAULA JABLONSKI, Ph.D., AND JAMES McK. WATTS, M.B., B.S., F.R.A.C.S. Department of Surgery, Monash University, Prince Henry’s Hospital, Melbourne 3004, Victoria, Australia Submitted for publication
stones were multifaceted and of variable size and weight. They were of uniform shape, and therefore an assumption was made that stone weight was proportional to stone size. Stone weight was used as the parameter determining changes in the stone brought about by immersion in the test solutions.
An uncommon complication following biliary surgery is one or more stones retained in the common bile duct. Two alternative methods of treatment are usually considered: to re-operate for the stones or to withdraw the T-tube and hope that the stone will pass and cause no further problem. A satisfactory noninvasive method for treating this complication was not found until recently when three types of solution have been used for irrigation of the biliary tree: bile salt solutions at different concentrations [9, lo], heparin solutions [3] and saline solutions [2]. This present study examines the effect of various solutions of bile salts, bile salt plus lecithin and heparin in dissolving human cholesterol gallstones. The study was carried out in vitro and the aim was to define the optimal solution for clinical use.
Solutions
The composition of the various solutions tested is shown in Table 1. Only readily available preparations of various bile salts were tested. The solutions were prepared as follows: (a) Sodium taurocholate (TC, KochLight Laboratories). The bile salt was
dissolved in 0.05 it4 phosphate buffer, pH 7.4. (b) Sodium cholate (C, Sigma), and Sodium deoxycholate (DC, Calbiochem ). The
bile acid was dissolved in an equimolar volume of 1 M sodium hydroxide and made up to volume with 0.05 M phosphate buffer, pH 7.4.
MATERIALS Gallstones
Human gallstones were obtained from the gallbladder of two patients with multiple gallstones who had a cholecystectomy. More than 100 stones were obtained from one patient and 30 stones from the other. The stones were rinsed with distilled water and then with methanol and dried in a stream of hot air. Eight of the 100 stones from the first patient and two of the 30 stones from the second patient were assayed for cholesterol content. These stones were composed of 90-100% cholesterol. The ‘TO
whom reprint requests should be addressed.
47 Copyright B 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
December 27. 1974
(c) Sodium chenodeoxycholate (CDC, Weddell Pharmaceuticals). The bile acid was
dissolved in an equimolar volume of 1 M sodium hydroxide and made up to volume with 0.05 M phosphate buffer, pH 7.9. Sodium chenodeoxycholate would not remain in solution at a lower pH. (d) Lecithin. Two sources of lecithin were used: purified soya bean lecithin (Nattermann) and egg lecithin (B.D.H.). The weighed lecithin was dissolved in the test bile-salt solution. (e) Heparin. Sodium heparin (Pularin) was added to normal saline solutions to the
48
JOURNAL OF SURGICAL
RESEARCH VOL. 19, NO. 1, JULY 1975
TABLE 1 Test Solutions Bile salt solutions Sodium deoxycholate 25-200 mM Sodium cholate 50-200 mM Sodium taurocholate 100-200 mM Sodium chenodeoxycholate 100-200 mM Sodium deoxycholate 100 mM, Sodium cholate 100 mM Bile salt plus lecithin solutions Sodium deoxycholate 200 mM, Lecithin 100 mM Sodium cholate 200 mM, Lecithin 100 mM Sodium taurocholate 200 mM, Lecithin 100 mM Sodium chenodeoxycholate 200 mM, Lecithin 100 mM Heparin solutions Sodium heparin 1,000 IU, 2,500 IU, 5,000 IU, 10,000 IU in 30 ml of normal saline Control 0.9 M Sodium chloride (normal saline) 0.05 M Phosphate buffer, pH 7.4
desired concentration. The heparin used contained 0.15% chlorocresol as a preservative. METHODS Stones in Bile Salt, Bile Salt Plus Lecithin and Saline Solutions Thirty milliliters of each solution were placed in 50 ml conical flasks and a single stone of known weight was placed in each flask. The flasks were placed in a constant temperature water bath at 375°C and gently agitated. The stones were removed from the flasks on days 4, 7, 11, 14, 18 and 21 and the solution rinsed from the stones with methanol. The stones were then dried in a stream of hot air for approximately 2 hr and weighed. On days 7, 14 and 21, 1 ml samples of each solution were taken for estimation of cholesterol content in each flask.
comparison, saline and bile-salt solutions were run in parallel, with the same changes in experimental procedure. The stones were weighed on days 4,7, 11 and 14 and samples of solution were taken for cholesterol estimation. These experiments were carried out for 14days. Estimation of Rate of Dissolution In each experiment stones were matched for size so that there was less than 10% variation in dry stone weight. In order to normalize the data the stone weights on each test day were expressed as a percentage of the original weight. These results were averaged and the standard error of the mean and variance were calculated. The Student’s t test was used for comparison of the results, RESULTS E$ect of Normal Saline and Phosphate Bu$er Six stones (84.4- 131 mg) were placed in normal saline and two stones (151.8 and 152 mg) were placed in phosphate buffer. There was no change in weight over 21 days and no detectable cholesterol in the supernatant fluid.
EJTect of Bile-Salt Solutions All bile-salt solutions tested produced dissolution of the gallstones (Table 2). The rates of dissolution varied depending on the bile-salt solution used (Fig. 1). Using stones of comparable weight and solutions of the same concentration, DC was more effective in producing dissolution than C, which was more effective than CDC and TC. These Stones in Heparin Solutions differences became significant after eleven The procedure was similar to that days and increased with time. described in the previous paragraph. A measure of the dissolution rate was also However, heparin denatures in solution, obtained from the cholesterol concentration therefore a number of changes to the of the supernatant solutions. The DC soluprocedure were made. Heparin (the test dose tions contained the highest concentration of in 1 ml of normal saline) was added to each cholesterol followed by C, CDC and TC flask every 24 hr and on days 4,7, 11 and 14 (Fig. 2). The increase in cholesterol conceneach solution was replaced. For purposes of tration paralleled the decreasein stone size.
TOOULI, JABLONSKI
AND WATTS: DISSOLUTION
OF HUMAN
49
GALLSTONES
TABLE 2 Dissolution of Gallstones Immersed in Bile Salt Solutions
Solution 200 mM DC 200 mM C 200 mMCDC 200 mM TC 100 mM DC, 100mMC
No. of stones
Initial weight of stones4 (ms)
8 I 3 4
108.5 (77.0-139.0) 100.7 (74.2-136.7) 103.8 (98.6-112.4) 99.5 (63.0-131.0)
70 f 75 f 79 f 79 f
6
106.7 (73.7-149.5)
Percent original weigh@ I
11
14
18
21
4 3 3 3
49 + 4 59 f 3 68 f 4 70 f 5
28 f 4 44 f 4 58 + 6 61 *6
12* 2 35 +4 53 * 7 55 f 6
3+1 26 -f 4 47+ 8 49 f 8
0 21*4 44 f 4 45 lr 8
75 f 1
59 f 2
37* 4
25 + 4
11 f 4
I+3
P < 0.001 P< 0.001 P< 0.001 P 4 0.05 P < 0.05 N.S. P < 0.05 P < 0.05
P < 0.001 P < 0.001 P< 0.001 P < 0.05 P < 0.02 N.S. P < 0.05 P < 0.05
4
Comparison of results using the Student’s t test DCvsC DC vs CDC DC vs TC C vs CDC CvsTC CDC vs TC DCvsDC+C CvsDC+C
N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S.
P < 0.05 P < 0.001 N.S. P < 0.05 P < 0.01 P < 0.001 P< 0.02 P < 0.01 P < 0.001 N.S. N.S. N.S. N.S. N.S. P < 0.05 N.S. N.S. N.S. N.S. N.S. P < 0.05 N.S. N.S. N.S.
aThe average weight is shown and the range of weights indicated in the bracket. bThe weight on each of the days indicated is expressed as a percentage of the original weight f SEM.
Effect of a Combination of Bile-Salt Solutions The effect on gallstone dissolution of a 200-mM bile-salt solution containing both DC (100 mM) and C (100 mM) was com-
pared with 200 mM solutions of each bile salt alone. The results are shown in Table 2 and Fig. 1. The combination of the two bile salts produced a rate of dissolution of the stones which was between that achieved with the individual bile-salt solutions. 90
80. 70. + :
60.
F
50.
0
14
7
21
Days 0
4
7
11
14
18
21
Days
FIG. 1. The change in weight of comparable human gallstones immersed in different 200 mM bile-salt solutions. The SEM is shown for each point.
FIG. 2. The cholesterol released [(mg cholesterol in solution/original weight of stone) x
UW1)l from human gallstones immersed in different 200 mM bile-salt solutions. TheSEM is shown for each point.
50
OF SURGICAL
JOURNAL
RESEARCH VOL. 19, NO. 1, JULY 1975
TABLE 3 The Effect of Different Concentrations of Bile Salt on Gallstone Dissolution
Solution 200 mM 150 mM 100 mM 75 mM 50 mM 25 mM
DC DC DC DC DC DC
200 mM C 150 mM C 100 mM C 50mMC
No. of stones
Initial weight of stones (mg)
4
8 4 4 4 4 4
108.5 (77.0-139.0) 101.5 (75.2-137.5) 96.2 (67.8-134.4) 98.1 (79.3-124.8) 97.5 (67.2-129.8) 96.8 (68.1-133.8)
70 f 4 79 f 0 84 f 0 88 f 1 92+ 1 96 + 1
49 f 63+ 72+ 802 85 f 93*
8 3 4 3
96.6 (68.0-136.7) 111.9 (77.7-137.0) 96.7 (77.9-118.4) 100.8 (73.7-114.8)
75 k 2 88 f 0 89 f 2 99 f 2
Percent original weight
E&et of Varying the Concentrations of the Bile-Salt Solutions C and DC solutions of varying concentrations were compared to determine their effectiveness (Table 3). The higher the concentration of the bile salt the more rapid was the dissolution (Fig. 3). Hence, for each bilesalt solution the highest concentration employed (i.e., 200 mM) was the most effective concentration. DC-
11
14
18
21
4 1 1 0 1 1
28 + 4 44 t 4 56 t 4 69+ 3 77 k 4 88+ 3
12* 2 30 + 8 48 + 5 64 f 4 74 f 4 87 + 3
3kl 20 f 9 40 f 6 59 +4 70 f 5 86i: 3
0 14 f 8 34 f 8 54 + 6 68 f 6 85* 3
59? 2 79 -r 0 83 i 3 98* 1
43 + 4 68? 3 75 r 4 94+ 1
33 * 4 63 i 4 73 *4 93 + 1
24 * 4 58 *5 70 t 5 92+ 1
18 f 4 5s f 7 68 2 5 91-c 1
7
E$ect of Adding Lecithin to the Bile-Salt Solutions When 100 mM lecithin was added to the test 200 mM bile-salt solutions the difference in dissolving capacity of the different bile salts was reduced (Table 4). Lecithin produced a significant (P < 0.05 after one week, P < 0.001 after 2 wk) acceleration of the dissolution rate of stones placed in TC solutions (Fig. 4). Thus, there was no significant difference in the dissolution of stones placed in solutions of TC plus lecithin and the dissolution of stones in solutions of DC plus lecithin (Fig. 5). There was no difference between the highly unsaturated soya bean and the saturated egg lecithins in their ability to alter the effect of bile-salt solutions. Efect of Heparin Heparin, at concentrations ranging from 1,000 IU/30 ml. of solution to 10,000 IU/30 ml. of solution had no effect in dissolving human gallstones (67.6-91.4 mg) (Table 5). Under the same experimental conditions, four similar stones (54.0-85.4 mg) were completely dissolved in 14 days when placed in 200 mM DC solutions.
0
4
7
11
14
18
a
Days
FIG. 3. The effect of varying concentrations of sodium deoxycholate on human gallstone dissolution. The concentration in millimoles of solution is indicated. The SEM is shown for each point.
DISCUSSION The aim of this study was to investigate a number of solutions that had been claimed to dissolve gallstones and to find the optimal solution for dissolving gallstones in the bil-
TOOULI.
JABLONSKI
Dissolution
Solution
AND
WATTS:
DISSOLUTION
TABLE 4 of Gallstones Immersed in Bile Salt Plus Lecithin
No. of stones
Initial weight of stones hd
4
3
128.4 (83.6-206.5)
75+10
58~13
4
124.1 (75.0-171.6)
82+3
6
137.7 (65.4-137.7)
4
112.9 (94.0-147.8)
200 mM DC, 100mMLec 200 mM C, 100mMLec 200 mM TC, 100mMLec 200 mM CDC, lOOmfI4Lec
Comparison DC vs DC/Let C vs C/Let TC vs TC/Lec CDC vs CDC/Lec DC/Let vs C/Let DC/Let vs TC/Lec C/Let vs TC/Lec CDC/Lec vs DC/Let CDC/Lec vs C/Let CDC/Lec vs TC/Lec
OF HUMAN
51
GALLSTONES
Solutions
Percent original weight 7
11
14
18
21
4Ok16
31 f 16
19~ 16
15 k 14
68+4
54 + 5
46 + 6
36 + 6
33 f- 6
72*4
59+5
42 f 6
34 + 5
19+ 5
12 t 5
78+1
68r3
55 + 4
50*
43+ 3
35 + 4
N.S. N.S. P < 0.001 N.S. N.S. N.S. N.S. N.S. N.S. P < 0.02
N.S. N.S. P < 0.001 N.S. N.S. N.S. P < 0.05 N.S. N.S. P < 0.02
3
of results using the Student’s t test N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S.
FIG. 4. The dissolution of stones immersed in a TC solution is compared with TC solution containing 100 mM lecithin (L). The average initial weight and range of the six stones immersed in TC solution was 125.3 (63.0179.0) mg. and that of the six stones immersed in TC + L solution was 137.7 (65.4-137.7) mg. The SEM is shown for each point.
N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S.
N.S. N.S. P c: 0.05 N.S. N.S. N.S. N.S. N.S. N.S. N.S.
N.S. N.S. P < 0.02 N.S. N.S. N.S. N.S. N.S. N.S. N.S.
FIG. 5. The effect on dissolution of gallstones of the addition of lecithin (L) to 200 mM bile-salt solutions. The SEM have not been plotted for the purpose of clarity but are shown on Table 4.
52
JOURNAL OF SURGICAL
RESEARCH VOL. 19, NO. I, JULY 1975
TABLE 5 The Effect of Heparin, Normal Saline and Sodium Deoxycholate on Dissolution of Gallstones
Solution Heparin 10,000 IU/30 5,000 W/30 2,500 IU/30 1,000 IU/30 Normal saline 200 mM DC
ml ml ml ml
No. of stones
Initial weight of stones (mg)
4 4 4 4 4 4
77.8 (67.6-88.0) 80.8 (73.1-86.4) 81.9 (72.8-90.5) 79.9 (64.2-91.4) 74.6 (60.9-84.4) 70.2 (54.0-85.4)
iary ducts by irrigation. The study has revealed that bile salts dissolve human gallstones, different bile salts produce different rates of dissolution and the addition of lecithin tends to eliminate these differences. Heparin at the concentrations tested does not dissolve or fragment human gallstones. Dissolution of human gallstones by increasing the bile-salt concentration around the stone was predicted by Admirand and Small [1] and has been confirmed on numerous occasions. Way, Admirand and Dunphy [lo] infused 100 mM sodium cholate solutions into the biliary tree of patients with stones retained in the common bile duct and were successful in dissolving the stones in 12 of 22 patients. Toouli, Jablonski and Watts [9] using 200 mM sodium cholate solutions in a similar manner were successful in dissolving the stones in 13 of 16 patients. The greater success of the latter study may be attributed to the higher concentration of the bile-salt solution used. Sodium deoxycholate solution (200 mA4) was found to be the most effective solution tested. However, the clinical use of sodium deoxycholate is limited due to production of intestinal side-effects such as diarrhoea [6]. Sodium cholate also produces diarrhoea but to a lesser degree, such that it can be prevented by oral administration of cholestyramine [9]. A 1:1 mixture of sodium cholate and deoxycholate was shown to be more effective than a cholate solution and not as effective as a deoxycholate solution in vitro. Such a combination may eliminate the
Percent original weight 4 102 f 103 * 103 * 103 k 102 * 61 t
I 1 1 1 1 1 3
loo+ 100 f 100 f 100 f 100 f 33*
11 1 1 1 1 1 5
100 t 1 lOOA 1 100 f 1 100 f 1 loo* 1 6*3
14 100 f 100 * 100 i: 100 f 100 f 0
1 1 1 1 1
incidence of unwanted side effects due to deoxycholate whilst at the same time provide a more effective solution than sodium cholate. The addition of lecithin to sodium taurocholate solutions enhanced its capacity for dissolving human gallstones and reduced the differences in dissolution rate of the other bile salts examined. Small [8] showed that lecithin enhanced the dissolving capacity of bile salts for cholesterol but he predicted that some retardation of gallstone dissolution would occur. Higuchi et al. [5] showed a retardation in dissolution of cholesterol gallstones when lecithin was added to solutions of sodium cholate and sodium taurocholate. The stones used by Higuchi were artificially manufactured cholesterol pellets and the retardation was attributed to an increase in the interfacial barrier around the pellet which retarded the dissolution of cholesterol. Our results do not agree with those of Higuchi et al. [5] but agree with the findings of Hegardt and Dam [4] who found that as increasing concentrations of lecithin were added to different bile-salt solutions, the differences that existed in their capacity for dissolving cholesterol were eliminated. From our study a 2: 1 combination of sodium taurocholate plus lecithin is as effective as a 100 mM deoxycholate plus 100 mM cholate solution in dissolving gallstones. The intestinal side effects of sodium taurocholate are prevented by oral administration of cholestyramine therefore a trial of its application in dissolving stones retained in the biliary tree is indicated.
TOOULI,
JABLONSKI
AND
WATTS:
DISSOLUTION
Heparin was shown to have no effect in dissolving human cholesterol gallstones in vitro. Ostrowitz and Gardner [7] found the negative zeta potentials of bile increased when heparin was added. They suggested that the bile salt-lecithin-cholesterol micelles might be dispersed in solutions of this type so that if intact stones were placed in this milieu, fragmentation would occur. The evidence that this occurred was the disappearance of retained common bile duct stones in 15 out of 19 patients treated by irrigation with heparinized saline [3]. It is probable that in those patients factors other than dissolution or fragmentation were responsible for success. Removal of stones of up to 1 cm in diameter, by an irrigation technique using saline without heparin has been described by Catt et al. [2]. The infusion technique used is similar to that of Gardner [3] which suggests that the effect noted might occur by simple flushing of the calculi into the duodenum. SUMMARY We have studied a number of solutions that have been claimed to dissolve human gallstones in order to determine the best solution for treating, by irrigation, stones retained in the biliary tree. It has been shown that heparin solutions are ineffective in dissolving or fragmenting gallstones and that the most effective bile-salt solution is a 200 mM deoxycholate solution. However, deoxycholate has a number of intestinal sideeffects which prevent its clinical use. 200 mM sodium cholate is the next most effective bile-salt solution and its side-effects are prevented by simultaneous administration of cholestyramine. A combination of deoxycholate and cholate has been shown to be more effective than a cholate solution and due to the smaller concentration of deoxycholate may lack the side-effects of the 200 mM deoxycholate solution. These differences in dissolving capacity between the different bile
OF HUMAN
GALLSTONES
53
salts are eliminated when lecithin is added to the solution, and this study has shown that a 200 mM taurocholate plus 100 mM lecithin solution is as effective in dissolving gallstones as a 100 mM deoxycholate plus 100 mM cholate solution. A careful clinical trial of both the combination of deoxycholate plus cholate and taurocholate plus lecithin solutions is indicated. ACKNOWLEDGMENTS The secretarial assistance of Miss E. Holloway is gratefully acknowledged. This study was supported by a grant from the National Health and Medical Research Council and Dr. J. Toouli is the recipient of an N. H. & M. R. C. Medical Postgraduate Scholarship.
REFERENCES I. Admirand, W., and Small, D. M. The physiological basis of cholesterol gallstone formation in man. J. Clin. Invest. 47,1043, 1968. 2. Catt, P. B., Hogg, D. F., Clunie, G. J. A., and Hardie, 1. R. Retained biliary calculi: Removal by a simple nonoperative technique. Ann. Surg. 180,247, 1974. 3. Gardner,
B. Experiences with the use of intracholcdochal heparinized saline for the treatment of retained common duct stones. Ann. Surg. 177,240, 1973.
4. Hegardt, F. G., and Dam, H. The solubility of cholesterol in aqueous solutions of bile salts and lecithin. Z. Emiihrungswiss. 10,223, 197 1. 5. Higuchi, W. I., Prakongpan, S., Surpuriya, V., and Young F. Cholesterol dissolution rate in micellar bile acid solutions: Retarding effect of added lecithin. Science 178,633, 1972. 6. Low-Beer, T., Schneider, R., and Dobbins, W. Morphological changes of the small intestinal mucosa of guinea-pig and hamster following incubation in vitro and perfusion in viva with unconjugated bile salts. Gut 11,486, 1970. 7. Ostrowitz, A., and Gardner, B. Studies of bile as a suspending medium and its relationship to gallstone formation. Surgery 68,329, 1970. 8. Small, D. M. The formation ofgallstones. Adv. Int. Med. 16,243, 1970. 9. Toouli, J., Jablonski, P., and Watts, J. McK. Dissolution of stones in the common bile duct with bilesalt solutions. Aust. N.Z.J. Surg. 44:336, 1974. 10. Way, L., Admirand W., and Dunphy, E. Management of choledocholithiasis. Ann. Surg. 176,347, 1972.
OF SURGICAL
RESEARCH
Dissolution
19, 47-53 (1975)
of Human
Gallstones:
Bile Salt Plus Lecithin
The Efficacy
and Heparin
of Bile Salt,
Solutions
JAMES TOOULI, M.B., B.S.,’ PAULA JABLONSKI, Ph.D., AND JAMES McK. WATTS, M.B., B.S., F.R.A.C.S. Department of Surgery, Monash University, Prince Henry’s Hospital, Melbourne 3004, Victoria, Australia Submitted for publication
stones were multifaceted and of variable size and weight. They were of uniform shape, and therefore an assumption was made that stone weight was proportional to stone size. Stone weight was used as the parameter determining changes in the stone brought about by immersion in the test solutions.
An uncommon complication following biliary surgery is one or more stones retained in the common bile duct. Two alternative methods of treatment are usually considered: to re-operate for the stones or to withdraw the T-tube and hope that the stone will pass and cause no further problem. A satisfactory noninvasive method for treating this complication was not found until recently when three types of solution have been used for irrigation of the biliary tree: bile salt solutions at different concentrations [9, lo], heparin solutions [3] and saline solutions [2]. This present study examines the effect of various solutions of bile salts, bile salt plus lecithin and heparin in dissolving human cholesterol gallstones. The study was carried out in vitro and the aim was to define the optimal solution for clinical use.
Solutions
The composition of the various solutions tested is shown in Table 1. Only readily available preparations of various bile salts were tested. The solutions were prepared as follows: (a) Sodium taurocholate (TC, KochLight Laboratories). The bile salt was
dissolved in 0.05 it4 phosphate buffer, pH 7.4. (b) Sodium cholate (C, Sigma), and Sodium deoxycholate (DC, Calbiochem ). The
bile acid was dissolved in an equimolar volume of 1 M sodium hydroxide and made up to volume with 0.05 M phosphate buffer, pH 7.4.
MATERIALS Gallstones
Human gallstones were obtained from the gallbladder of two patients with multiple gallstones who had a cholecystectomy. More than 100 stones were obtained from one patient and 30 stones from the other. The stones were rinsed with distilled water and then with methanol and dried in a stream of hot air. Eight of the 100 stones from the first patient and two of the 30 stones from the second patient were assayed for cholesterol content. These stones were composed of 90-100% cholesterol. The ‘TO
whom reprint requests should be addressed.
47 Copyright B 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
December 27. 1974
(c) Sodium chenodeoxycholate (CDC, Weddell Pharmaceuticals). The bile acid was
dissolved in an equimolar volume of 1 M sodium hydroxide and made up to volume with 0.05 M phosphate buffer, pH 7.9. Sodium chenodeoxycholate would not remain in solution at a lower pH. (d) Lecithin. Two sources of lecithin were used: purified soya bean lecithin (Nattermann) and egg lecithin (B.D.H.). The weighed lecithin was dissolved in the test bile-salt solution. (e) Heparin. Sodium heparin (Pularin) was added to normal saline solutions to the
48
JOURNAL OF SURGICAL
RESEARCH VOL. 19, NO. 1, JULY 1975
TABLE 1 Test Solutions Bile salt solutions Sodium deoxycholate 25-200 mM Sodium cholate 50-200 mM Sodium taurocholate 100-200 mM Sodium chenodeoxycholate 100-200 mM Sodium deoxycholate 100 mM, Sodium cholate 100 mM Bile salt plus lecithin solutions Sodium deoxycholate 200 mM, Lecithin 100 mM Sodium cholate 200 mM, Lecithin 100 mM Sodium taurocholate 200 mM, Lecithin 100 mM Sodium chenodeoxycholate 200 mM, Lecithin 100 mM Heparin solutions Sodium heparin 1,000 IU, 2,500 IU, 5,000 IU, 10,000 IU in 30 ml of normal saline Control 0.9 M Sodium chloride (normal saline) 0.05 M Phosphate buffer, pH 7.4
desired concentration. The heparin used contained 0.15% chlorocresol as a preservative. METHODS Stones in Bile Salt, Bile Salt Plus Lecithin and Saline Solutions Thirty milliliters of each solution were placed in 50 ml conical flasks and a single stone of known weight was placed in each flask. The flasks were placed in a constant temperature water bath at 375°C and gently agitated. The stones were removed from the flasks on days 4, 7, 11, 14, 18 and 21 and the solution rinsed from the stones with methanol. The stones were then dried in a stream of hot air for approximately 2 hr and weighed. On days 7, 14 and 21, 1 ml samples of each solution were taken for estimation of cholesterol content in each flask.
comparison, saline and bile-salt solutions were run in parallel, with the same changes in experimental procedure. The stones were weighed on days 4,7, 11 and 14 and samples of solution were taken for cholesterol estimation. These experiments were carried out for 14days. Estimation of Rate of Dissolution In each experiment stones were matched for size so that there was less than 10% variation in dry stone weight. In order to normalize the data the stone weights on each test day were expressed as a percentage of the original weight. These results were averaged and the standard error of the mean and variance were calculated. The Student’s t test was used for comparison of the results, RESULTS E$ect of Normal Saline and Phosphate Bu$er Six stones (84.4- 131 mg) were placed in normal saline and two stones (151.8 and 152 mg) were placed in phosphate buffer. There was no change in weight over 21 days and no detectable cholesterol in the supernatant fluid.
EJTect of Bile-Salt Solutions All bile-salt solutions tested produced dissolution of the gallstones (Table 2). The rates of dissolution varied depending on the bile-salt solution used (Fig. 1). Using stones of comparable weight and solutions of the same concentration, DC was more effective in producing dissolution than C, which was more effective than CDC and TC. These Stones in Heparin Solutions differences became significant after eleven The procedure was similar to that days and increased with time. described in the previous paragraph. A measure of the dissolution rate was also However, heparin denatures in solution, obtained from the cholesterol concentration therefore a number of changes to the of the supernatant solutions. The DC soluprocedure were made. Heparin (the test dose tions contained the highest concentration of in 1 ml of normal saline) was added to each cholesterol followed by C, CDC and TC flask every 24 hr and on days 4,7, 11 and 14 (Fig. 2). The increase in cholesterol conceneach solution was replaced. For purposes of tration paralleled the decreasein stone size.
TOOULI, JABLONSKI
AND WATTS: DISSOLUTION
OF HUMAN
49
GALLSTONES
TABLE 2 Dissolution of Gallstones Immersed in Bile Salt Solutions
Solution 200 mM DC 200 mM C 200 mMCDC 200 mM TC 100 mM DC, 100mMC
No. of stones
Initial weight of stones4 (ms)
8 I 3 4
108.5 (77.0-139.0) 100.7 (74.2-136.7) 103.8 (98.6-112.4) 99.5 (63.0-131.0)
70 f 75 f 79 f 79 f
6
106.7 (73.7-149.5)
Percent original weigh@ I
11
14
18
21
4 3 3 3
49 + 4 59 f 3 68 f 4 70 f 5
28 f 4 44 f 4 58 + 6 61 *6
12* 2 35 +4 53 * 7 55 f 6
3+1 26 -f 4 47+ 8 49 f 8
0 21*4 44 f 4 45 lr 8
75 f 1
59 f 2
37* 4
25 + 4
11 f 4
I+3
P < 0.001 P< 0.001 P< 0.001 P 4 0.05 P < 0.05 N.S. P < 0.05 P < 0.05
P < 0.001 P < 0.001 P< 0.001 P < 0.05 P < 0.02 N.S. P < 0.05 P < 0.05
4
Comparison of results using the Student’s t test DCvsC DC vs CDC DC vs TC C vs CDC CvsTC CDC vs TC DCvsDC+C CvsDC+C
N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S.
P < 0.05 P < 0.001 N.S. P < 0.05 P < 0.01 P < 0.001 P< 0.02 P < 0.01 P < 0.001 N.S. N.S. N.S. N.S. N.S. P < 0.05 N.S. N.S. N.S. N.S. N.S. P < 0.05 N.S. N.S. N.S.
aThe average weight is shown and the range of weights indicated in the bracket. bThe weight on each of the days indicated is expressed as a percentage of the original weight f SEM.
Effect of a Combination of Bile-Salt Solutions The effect on gallstone dissolution of a 200-mM bile-salt solution containing both DC (100 mM) and C (100 mM) was com-
pared with 200 mM solutions of each bile salt alone. The results are shown in Table 2 and Fig. 1. The combination of the two bile salts produced a rate of dissolution of the stones which was between that achieved with the individual bile-salt solutions. 90
80. 70. + :
60.
F
50.
0
14
7
21
Days 0
4
7
11
14
18
21
Days
FIG. 1. The change in weight of comparable human gallstones immersed in different 200 mM bile-salt solutions. The SEM is shown for each point.
FIG. 2. The cholesterol released [(mg cholesterol in solution/original weight of stone) x
UW1)l from human gallstones immersed in different 200 mM bile-salt solutions. TheSEM is shown for each point.
50
OF SURGICAL
JOURNAL
RESEARCH VOL. 19, NO. 1, JULY 1975
TABLE 3 The Effect of Different Concentrations of Bile Salt on Gallstone Dissolution
Solution 200 mM 150 mM 100 mM 75 mM 50 mM 25 mM
DC DC DC DC DC DC
200 mM C 150 mM C 100 mM C 50mMC
No. of stones
Initial weight of stones (mg)
4
8 4 4 4 4 4
108.5 (77.0-139.0) 101.5 (75.2-137.5) 96.2 (67.8-134.4) 98.1 (79.3-124.8) 97.5 (67.2-129.8) 96.8 (68.1-133.8)
70 f 4 79 f 0 84 f 0 88 f 1 92+ 1 96 + 1
49 f 63+ 72+ 802 85 f 93*
8 3 4 3
96.6 (68.0-136.7) 111.9 (77.7-137.0) 96.7 (77.9-118.4) 100.8 (73.7-114.8)
75 k 2 88 f 0 89 f 2 99 f 2
Percent original weight
E&et of Varying the Concentrations of the Bile-Salt Solutions C and DC solutions of varying concentrations were compared to determine their effectiveness (Table 3). The higher the concentration of the bile salt the more rapid was the dissolution (Fig. 3). Hence, for each bilesalt solution the highest concentration employed (i.e., 200 mM) was the most effective concentration. DC-
11
14
18
21
4 1 1 0 1 1
28 + 4 44 t 4 56 t 4 69+ 3 77 k 4 88+ 3
12* 2 30 + 8 48 + 5 64 f 4 74 f 4 87 + 3
3kl 20 f 9 40 f 6 59 +4 70 f 5 86i: 3
0 14 f 8 34 f 8 54 + 6 68 f 6 85* 3
59? 2 79 -r 0 83 i 3 98* 1
43 + 4 68? 3 75 r 4 94+ 1
33 * 4 63 i 4 73 *4 93 + 1
24 * 4 58 *5 70 t 5 92+ 1
18 f 4 5s f 7 68 2 5 91-c 1
7
E$ect of Adding Lecithin to the Bile-Salt Solutions When 100 mM lecithin was added to the test 200 mM bile-salt solutions the difference in dissolving capacity of the different bile salts was reduced (Table 4). Lecithin produced a significant (P < 0.05 after one week, P < 0.001 after 2 wk) acceleration of the dissolution rate of stones placed in TC solutions (Fig. 4). Thus, there was no significant difference in the dissolution of stones placed in solutions of TC plus lecithin and the dissolution of stones in solutions of DC plus lecithin (Fig. 5). There was no difference between the highly unsaturated soya bean and the saturated egg lecithins in their ability to alter the effect of bile-salt solutions. Efect of Heparin Heparin, at concentrations ranging from 1,000 IU/30 ml. of solution to 10,000 IU/30 ml. of solution had no effect in dissolving human gallstones (67.6-91.4 mg) (Table 5). Under the same experimental conditions, four similar stones (54.0-85.4 mg) were completely dissolved in 14 days when placed in 200 mM DC solutions.
0
4
7
11
14
18
a
Days
FIG. 3. The effect of varying concentrations of sodium deoxycholate on human gallstone dissolution. The concentration in millimoles of solution is indicated. The SEM is shown for each point.
DISCUSSION The aim of this study was to investigate a number of solutions that had been claimed to dissolve gallstones and to find the optimal solution for dissolving gallstones in the bil-
TOOULI.
JABLONSKI
Dissolution
Solution
AND
WATTS:
DISSOLUTION
TABLE 4 of Gallstones Immersed in Bile Salt Plus Lecithin
No. of stones
Initial weight of stones hd
4
3
128.4 (83.6-206.5)
75+10
58~13
4
124.1 (75.0-171.6)
82+3
6
137.7 (65.4-137.7)
4
112.9 (94.0-147.8)
200 mM DC, 100mMLec 200 mM C, 100mMLec 200 mM TC, 100mMLec 200 mM CDC, lOOmfI4Lec
Comparison DC vs DC/Let C vs C/Let TC vs TC/Lec CDC vs CDC/Lec DC/Let vs C/Let DC/Let vs TC/Lec C/Let vs TC/Lec CDC/Lec vs DC/Let CDC/Lec vs C/Let CDC/Lec vs TC/Lec
OF HUMAN
51
GALLSTONES
Solutions
Percent original weight 7
11
14
18
21
4Ok16
31 f 16
19~ 16
15 k 14
68+4
54 + 5
46 + 6
36 + 6
33 f- 6
72*4
59+5
42 f 6
34 + 5
19+ 5
12 t 5
78+1
68r3
55 + 4
50*
43+ 3
35 + 4
N.S. N.S. P < 0.001 N.S. N.S. N.S. N.S. N.S. N.S. P < 0.02
N.S. N.S. P < 0.001 N.S. N.S. N.S. P < 0.05 N.S. N.S. P < 0.02
3
of results using the Student’s t test N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S.
FIG. 4. The dissolution of stones immersed in a TC solution is compared with TC solution containing 100 mM lecithin (L). The average initial weight and range of the six stones immersed in TC solution was 125.3 (63.0179.0) mg. and that of the six stones immersed in TC + L solution was 137.7 (65.4-137.7) mg. The SEM is shown for each point.
N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S.
N.S. N.S. P c: 0.05 N.S. N.S. N.S. N.S. N.S. N.S. N.S.
N.S. N.S. P < 0.02 N.S. N.S. N.S. N.S. N.S. N.S. N.S.
FIG. 5. The effect on dissolution of gallstones of the addition of lecithin (L) to 200 mM bile-salt solutions. The SEM have not been plotted for the purpose of clarity but are shown on Table 4.
52
JOURNAL OF SURGICAL
RESEARCH VOL. 19, NO. I, JULY 1975
TABLE 5 The Effect of Heparin, Normal Saline and Sodium Deoxycholate on Dissolution of Gallstones
Solution Heparin 10,000 IU/30 5,000 W/30 2,500 IU/30 1,000 IU/30 Normal saline 200 mM DC
ml ml ml ml
No. of stones
Initial weight of stones (mg)
4 4 4 4 4 4
77.8 (67.6-88.0) 80.8 (73.1-86.4) 81.9 (72.8-90.5) 79.9 (64.2-91.4) 74.6 (60.9-84.4) 70.2 (54.0-85.4)
iary ducts by irrigation. The study has revealed that bile salts dissolve human gallstones, different bile salts produce different rates of dissolution and the addition of lecithin tends to eliminate these differences. Heparin at the concentrations tested does not dissolve or fragment human gallstones. Dissolution of human gallstones by increasing the bile-salt concentration around the stone was predicted by Admirand and Small [1] and has been confirmed on numerous occasions. Way, Admirand and Dunphy [lo] infused 100 mM sodium cholate solutions into the biliary tree of patients with stones retained in the common bile duct and were successful in dissolving the stones in 12 of 22 patients. Toouli, Jablonski and Watts [9] using 200 mM sodium cholate solutions in a similar manner were successful in dissolving the stones in 13 of 16 patients. The greater success of the latter study may be attributed to the higher concentration of the bile-salt solution used. Sodium deoxycholate solution (200 mA4) was found to be the most effective solution tested. However, the clinical use of sodium deoxycholate is limited due to production of intestinal side-effects such as diarrhoea [6]. Sodium cholate also produces diarrhoea but to a lesser degree, such that it can be prevented by oral administration of cholestyramine [9]. A 1:1 mixture of sodium cholate and deoxycholate was shown to be more effective than a cholate solution and not as effective as a deoxycholate solution in vitro. Such a combination may eliminate the
Percent original weight 4 102 f 103 * 103 * 103 k 102 * 61 t
I 1 1 1 1 1 3
loo+ 100 f 100 f 100 f 100 f 33*
11 1 1 1 1 1 5
100 t 1 lOOA 1 100 f 1 100 f 1 loo* 1 6*3
14 100 f 100 * 100 i: 100 f 100 f 0
1 1 1 1 1
incidence of unwanted side effects due to deoxycholate whilst at the same time provide a more effective solution than sodium cholate. The addition of lecithin to sodium taurocholate solutions enhanced its capacity for dissolving human gallstones and reduced the differences in dissolution rate of the other bile salts examined. Small [8] showed that lecithin enhanced the dissolving capacity of bile salts for cholesterol but he predicted that some retardation of gallstone dissolution would occur. Higuchi et al. [5] showed a retardation in dissolution of cholesterol gallstones when lecithin was added to solutions of sodium cholate and sodium taurocholate. The stones used by Higuchi were artificially manufactured cholesterol pellets and the retardation was attributed to an increase in the interfacial barrier around the pellet which retarded the dissolution of cholesterol. Our results do not agree with those of Higuchi et al. [5] but agree with the findings of Hegardt and Dam [4] who found that as increasing concentrations of lecithin were added to different bile-salt solutions, the differences that existed in their capacity for dissolving cholesterol were eliminated. From our study a 2: 1 combination of sodium taurocholate plus lecithin is as effective as a 100 mM deoxycholate plus 100 mM cholate solution in dissolving gallstones. The intestinal side effects of sodium taurocholate are prevented by oral administration of cholestyramine therefore a trial of its application in dissolving stones retained in the biliary tree is indicated.
TOOULI,
JABLONSKI
AND
WATTS:
DISSOLUTION
Heparin was shown to have no effect in dissolving human cholesterol gallstones in vitro. Ostrowitz and Gardner [7] found the negative zeta potentials of bile increased when heparin was added. They suggested that the bile salt-lecithin-cholesterol micelles might be dispersed in solutions of this type so that if intact stones were placed in this milieu, fragmentation would occur. The evidence that this occurred was the disappearance of retained common bile duct stones in 15 out of 19 patients treated by irrigation with heparinized saline [3]. It is probable that in those patients factors other than dissolution or fragmentation were responsible for success. Removal of stones of up to 1 cm in diameter, by an irrigation technique using saline without heparin has been described by Catt et al. [2]. The infusion technique used is similar to that of Gardner [3] which suggests that the effect noted might occur by simple flushing of the calculi into the duodenum. SUMMARY We have studied a number of solutions that have been claimed to dissolve human gallstones in order to determine the best solution for treating, by irrigation, stones retained in the biliary tree. It has been shown that heparin solutions are ineffective in dissolving or fragmenting gallstones and that the most effective bile-salt solution is a 200 mM deoxycholate solution. However, deoxycholate has a number of intestinal sideeffects which prevent its clinical use. 200 mM sodium cholate is the next most effective bile-salt solution and its side-effects are prevented by simultaneous administration of cholestyramine. A combination of deoxycholate and cholate has been shown to be more effective than a cholate solution and due to the smaller concentration of deoxycholate may lack the side-effects of the 200 mM deoxycholate solution. These differences in dissolving capacity between the different bile
OF HUMAN
GALLSTONES
53
salts are eliminated when lecithin is added to the solution, and this study has shown that a 200 mM taurocholate plus 100 mM lecithin solution is as effective in dissolving gallstones as a 100 mM deoxycholate plus 100 mM cholate solution. A careful clinical trial of both the combination of deoxycholate plus cholate and taurocholate plus lecithin solutions is indicated. ACKNOWLEDGMENTS The secretarial assistance of Miss E. Holloway is gratefully acknowledged. This study was supported by a grant from the National Health and Medical Research Council and Dr. J. Toouli is the recipient of an N. H. & M. R. C. Medical Postgraduate Scholarship.
REFERENCES I. Admirand, W., and Small, D. M. The physiological basis of cholesterol gallstone formation in man. J. Clin. Invest. 47,1043, 1968. 2. Catt, P. B., Hogg, D. F., Clunie, G. J. A., and Hardie, 1. R. Retained biliary calculi: Removal by a simple nonoperative technique. Ann. Surg. 180,247, 1974. 3. Gardner,
B. Experiences with the use of intracholcdochal heparinized saline for the treatment of retained common duct stones. Ann. Surg. 177,240, 1973.
4. Hegardt, F. G., and Dam, H. The solubility of cholesterol in aqueous solutions of bile salts and lecithin. Z. Emiihrungswiss. 10,223, 197 1. 5. Higuchi, W. I., Prakongpan, S., Surpuriya, V., and Young F. Cholesterol dissolution rate in micellar bile acid solutions: Retarding effect of added lecithin. Science 178,633, 1972. 6. Low-Beer, T., Schneider, R., and Dobbins, W. Morphological changes of the small intestinal mucosa of guinea-pig and hamster following incubation in vitro and perfusion in viva with unconjugated bile salts. Gut 11,486, 1970. 7. Ostrowitz, A., and Gardner, B. Studies of bile as a suspending medium and its relationship to gallstone formation. Surgery 68,329, 1970. 8. Small, D. M. The formation ofgallstones. Adv. Int. Med. 16,243, 1970. 9. Toouli, J., Jablonski, P., and Watts, J. McK. Dissolution of stones in the common bile duct with bilesalt solutions. Aust. N.Z.J. Surg. 44:336, 1974. 10. Way, L., Admirand W., and Dunphy, E. Management of choledocholithiasis. Ann. Surg. 176,347, 1972.
