Br. J. Surp. Vol. 63 (1976) 44-46
Some observations on gallstones and bile composition D . J U N E S U T O R , A. G . A . C O W l E A N D C. G . C L A R K * SUMMAKY
A series of 3 I consecutive putients undergoing srrrgery for gallstone disease has been studied and the compositiori oJ the gallstones and bile from the gallbladder und common duct determined. As a result of the stone analysis by the X-ray powder di8raction method, the patients were classified according to whether their stones consisted of cholesterol, calcium salts or (I mixture of the two. The mean composition of the common duct bile for the groups with cholesterol and mixed stones was just outside the micellar region of cholesterol solubility. The gullbladder bile .from the cholesterol group of stone-formers was also supersaturated, but the gallblader bile from the group of mixed stone-formers was undersaturated with respect to cholesterol. None of the patients .forming gallstones of calcium salts showed any abnormality in the cholesterol content q f their bile.
DURING the past few years there has been considerable interest in the physicochemical aspects of cholesterol gallstone formation. Stones can only nucleate and grow when bile is supersaturated with respect to the precipitating material, and the purpose of many investigations has been to determine whether the liver, gallbladder or both is (are) involved in the production of a supersaturated bile. There is, however, some disagreement among the various studies. In order to investigate the problem further, a series of 31 patients undergoing surgery for gallstone disease at University College Hospital, London, in 1970 was studied and the composition of the bile was related to the composition of the gallstones.
Patients and methods Thirty-one consecutive unselected patients (26 females and 5 males) undergoing surgery for gallstone disease were studied. The gallstones obtained at operation were washed and blotted dry, placed in formalin solution for 24 hours, then washed and air-dried. The crystalline composition was determined by X-ray diffraction (Sutor and Wooley, 1971) and then used to classify the patients according to whether their gallstones consisted of cholesterol, cholesterol + calcium salts or calcium salts (Sutor and Wooley, 1973). Bile was obtained at operation both from the gallbladder and from the common bile duct, taking care, as far as possible, to avoid contamination of the common duct bile by gallbladder bile. The bile samples were analysed chemically. Cholesterol concentration was determined by a method derived from that of Babson et al. (1962) and Leffler and McDougald ( I 963). To measure phospholipid concentration, the 44
bile was extracted with a 2 : 1 mixture of chloroform and methanol, the extract digested with 5 N sulphuric acid and oxidized with 30 per cent hydrogen peroxide and then phospholipid phosphorus was measured by the method of Fiske and Subbarrow (1925). Total cholate concentration was determined by the method of Irvin et al. (1944). An approximate estimate of total bile salt was obtained by assuming the ratio of total cholate to bile salt remained relatively constant at 1 : 2.5 (Sjovall, 1960). This is, in fact, not strictly the case since in studies where individual bile salts have been measured (Nakayama, 1967; Thistle and Schoenfield, 1971) the ratio varies from 1.7 to 2-95 in patients with cholesterol gallstones and patients without disease of the hepatobiliary tract. Accordingly, these factors were used to give some indication of minimum and maximum values for the bile salt concentrations. Results Sixteen patients had stones consisting of 97-100 per cent crystalline cholesterol and I 1 patients had stones composed of cholesterol + calcium salts. Such salts could be one or several of the following: calcite, aragonite and vaterite (all forms of calcium carbonate), apatite and whitlockite (both calcium phosphates) and calcium palmitate (Sutor and Wooley, 1971). They accounted for 20-72 per cent of the crystalline material of each stone and averaged 43 per cent. Only 4 patients had stones consisting entirely of calcium salts. Table I gives the mean results of the bile analysis in millimoles per litre, and these concentrations converted to percentages of the total millimoles are shown in Fig. 1. The lines represent the range for the bile composition corresponding to minimum and maximum conversion factors of 1 *7-2.95 for obtaining total bile salt from cholate measurement. The continuous line in the bottom left hand corner defines the solubility product of cholesterol in this three-component system (Small et al., 1966; Admirand and Small, 1968). A solution with a composition lying below the solubility product (i.e. in the micellar zone) is undersaturated with respect to cholesterol.0utside this zone a solution is supersaturated and cholesterol either crystallizes out or forms a metastable solution. In the group of cholesterol stone-formers the line representing the mean composition of common duct bile lies just above the solubility product for cholesterol, whereas that for gallbladder bile lies even
* Department of Chemistry, University College, and Department of Surgery, University College Hospital Medical School, London.
Gallstones and bile composition Tahle I : MEAN COMPOSITION OF BILE FROM THE GALLBLADDER AND COMMON BILE DUCT Gallbladder Common bile duct Group Phospholipid Cholesterol Total bile salt Phospholipid Cholesterol Total bile salt Cholesterol stones
20.36
Stones of cholesterol 1 calcium salts
20.96
9.36
20.4 1 Range:
8.70
14.14-24.54 76.20
4.19
52.79-9 1.60 42.98
6.3 I
3.34
9.2X
5.4 I
14.23-24.69 34.10
2.12
23.62-40.09 21.14
Range: Stones or calcium salts
16.03
20.54
Range: Range: 6.19
Range: 29.77-51.67
Range: 14.64-25.4 I
Results given as mmolil. The minimum and maximum values for total bile salts for the ratios 1.7 and 2.95 are also given.
of mixed composition, the common duct bile was also supersaturated with cholesterol, whereas the gallbladder bile was undersaturated. Both the common duct and gallbladder biles from the group of patients with calcium stones showed no theoretical abnormality with respect to the cholesterol concentration. Our results therefore support the early work of Small and Rap0 (1970) and Vlahcevic et al. (1970) that the primary abnormality in cholesterol precipitation is the secretion of a ‘hepatic’ bile supersaturated with cholesterol.
I00
\
I00
80
60
40
20
I00
% bile salts Fig. I. Three-component diagram showing variation in mean values for the different biles using ratios o f 1.7-2.95 to convert measured cholate concentrations to total bile salt. Ch, Cholesterol stone-formers; Ch Ca, cholesterol tcalcium stone-formers; Ca, calcium stone-formers; - - --, common duct bile; . . . . .gallbladder bilc.
+
further from the region of cholesterol solubility. For stones of mixed composition the line representing the mean composition of common duct bile is also just outside the micellar zone, but the line for the gallbladder bile is within this area. The lines for common duct and gallbladder bile giving rise to calcium stones are within the area of cholesterol solubility.
Discussion The classification of gallstones and bile according to the crystalline composition of the former has shown a considerable difference in the composition of pathogenic biles. Cholesterol stone formation appears to be associated with ‘hepatic’ bile, which is supersaturated with respect to this compound, and this bile may become even more unstable in the gallbladder. However, to what extent supersaturation can occur is difficult to estimate since microcrystals are frequently present in gallbladder bile and our samples were not filtered. For the group of patients with stones
Acknowledgements We thank the surgeons at University College Hospital for allowing their patients to be studied, Mrs S. E. Wooley for taking the X-ray photographs and Miss J. Fowler and Miss C. S. Dines for carrying out the bile analysis. We are grateful to the Medical Research Council and the Nuffield Foundation for financial assistance. References A D M I R A N D w. H.
and SMALL D. M. (1968) The physicochemical basis of cholesterol gallstone formation in man. J . Clin. Invest. 47, 1043-1052. RARSON A L., SHAPIRO P. 0.and PHILLIPS G . E. (1962) A new assay for cholesterol and cholesterol esters in serum which is not affected by bilirubin. Clin. Chim. Actu 7, 800-804. FISKE c. H . and SUBBARROW J . (1925) The colorimetric determination of phosphorus. J . B i d . Chem. 66, 375-400. I R V I N J. L., JOHNSON c. G . and KOPALA J. (1944) A photometric method for the determination of cholates in bile and blood. J . Bid. Chrm. 153, 439-457. I.TFFLER H. H . and MCDOUCALI) c. H. (1963) Estimation of cholesterol in serum. Am. J . Clin. Puihol. 39, 31 1-315. NAKAYAMA F. (1967) Quantitative microanalysis of bile. J. Lab. Cliti. Med. 69, 594609. SJOVALL J . (1960) Bile acids in man under normal and pathological conditions. Bile acids and steroids 73. Clin. Chim. Acta 5, 33-41. SMALL D . M., BOURGkS M. and DERVICHIAN D . C. (1966) Ternary and quaternary aqueous systems containing bile salt, lecithin, and cholesterol. Nutrtre (Loud.) 211, 816 818.
45
D. June Sutor et al. and RAPO s. (1970) Source of abnormal bile in patients with cholesterol gallstones. N P W EngI. J . Med. 283, 53-57. SUTOR D. J. and WOOLEY s. E. (1971) A statistical survey of the composition of gallstones in eight countries. Gut 12, 55-64. SUTOR D. J. and WOOLEY s. E. (1973) The nature and incidence of gallstones containing calcium. Gilt 14, 215-220. SMALL D. M.
44
and SCHOENFIELD L. .I (1971) Induced alterations i n composition of bile of persons having cholelithiasis. Gastroenterology 61, 488496. VLAHCEVIC A. R., BELL c. c. jun. and SWELL L. (1970) Significance of the liver in the production of lithogenic bile in man. Gostromtrrology 59, 62-69. THISTLE J. L.
Some observations on gallstones and bile composition D . J U N E S U T O R , A. G . A . C O W l E A N D C. G . C L A R K * SUMMAKY
A series of 3 I consecutive putients undergoing srrrgery for gallstone disease has been studied and the compositiori oJ the gallstones and bile from the gallbladder und common duct determined. As a result of the stone analysis by the X-ray powder di8raction method, the patients were classified according to whether their stones consisted of cholesterol, calcium salts or (I mixture of the two. The mean composition of the common duct bile for the groups with cholesterol and mixed stones was just outside the micellar region of cholesterol solubility. The gullbladder bile .from the cholesterol group of stone-formers was also supersaturated, but the gallblader bile from the group of mixed stone-formers was undersaturated with respect to cholesterol. None of the patients .forming gallstones of calcium salts showed any abnormality in the cholesterol content q f their bile.
DURING the past few years there has been considerable interest in the physicochemical aspects of cholesterol gallstone formation. Stones can only nucleate and grow when bile is supersaturated with respect to the precipitating material, and the purpose of many investigations has been to determine whether the liver, gallbladder or both is (are) involved in the production of a supersaturated bile. There is, however, some disagreement among the various studies. In order to investigate the problem further, a series of 31 patients undergoing surgery for gallstone disease at University College Hospital, London, in 1970 was studied and the composition of the bile was related to the composition of the gallstones.
Patients and methods Thirty-one consecutive unselected patients (26 females and 5 males) undergoing surgery for gallstone disease were studied. The gallstones obtained at operation were washed and blotted dry, placed in formalin solution for 24 hours, then washed and air-dried. The crystalline composition was determined by X-ray diffraction (Sutor and Wooley, 1971) and then used to classify the patients according to whether their gallstones consisted of cholesterol, cholesterol + calcium salts or calcium salts (Sutor and Wooley, 1973). Bile was obtained at operation both from the gallbladder and from the common bile duct, taking care, as far as possible, to avoid contamination of the common duct bile by gallbladder bile. The bile samples were analysed chemically. Cholesterol concentration was determined by a method derived from that of Babson et al. (1962) and Leffler and McDougald ( I 963). To measure phospholipid concentration, the 44
bile was extracted with a 2 : 1 mixture of chloroform and methanol, the extract digested with 5 N sulphuric acid and oxidized with 30 per cent hydrogen peroxide and then phospholipid phosphorus was measured by the method of Fiske and Subbarrow (1925). Total cholate concentration was determined by the method of Irvin et al. (1944). An approximate estimate of total bile salt was obtained by assuming the ratio of total cholate to bile salt remained relatively constant at 1 : 2.5 (Sjovall, 1960). This is, in fact, not strictly the case since in studies where individual bile salts have been measured (Nakayama, 1967; Thistle and Schoenfield, 1971) the ratio varies from 1.7 to 2-95 in patients with cholesterol gallstones and patients without disease of the hepatobiliary tract. Accordingly, these factors were used to give some indication of minimum and maximum values for the bile salt concentrations. Results Sixteen patients had stones consisting of 97-100 per cent crystalline cholesterol and I 1 patients had stones composed of cholesterol + calcium salts. Such salts could be one or several of the following: calcite, aragonite and vaterite (all forms of calcium carbonate), apatite and whitlockite (both calcium phosphates) and calcium palmitate (Sutor and Wooley, 1971). They accounted for 20-72 per cent of the crystalline material of each stone and averaged 43 per cent. Only 4 patients had stones consisting entirely of calcium salts. Table I gives the mean results of the bile analysis in millimoles per litre, and these concentrations converted to percentages of the total millimoles are shown in Fig. 1. The lines represent the range for the bile composition corresponding to minimum and maximum conversion factors of 1 *7-2.95 for obtaining total bile salt from cholate measurement. The continuous line in the bottom left hand corner defines the solubility product of cholesterol in this three-component system (Small et al., 1966; Admirand and Small, 1968). A solution with a composition lying below the solubility product (i.e. in the micellar zone) is undersaturated with respect to cholesterol.0utside this zone a solution is supersaturated and cholesterol either crystallizes out or forms a metastable solution. In the group of cholesterol stone-formers the line representing the mean composition of common duct bile lies just above the solubility product for cholesterol, whereas that for gallbladder bile lies even
* Department of Chemistry, University College, and Department of Surgery, University College Hospital Medical School, London.
Gallstones and bile composition Tahle I : MEAN COMPOSITION OF BILE FROM THE GALLBLADDER AND COMMON BILE DUCT Gallbladder Common bile duct Group Phospholipid Cholesterol Total bile salt Phospholipid Cholesterol Total bile salt Cholesterol stones
20.36
Stones of cholesterol 1 calcium salts
20.96
9.36
20.4 1 Range:
8.70
14.14-24.54 76.20
4.19
52.79-9 1.60 42.98
6.3 I
3.34
9.2X
5.4 I
14.23-24.69 34.10
2.12
23.62-40.09 21.14
Range: Stones or calcium salts
16.03
20.54
Range: Range: 6.19
Range: 29.77-51.67
Range: 14.64-25.4 I
Results given as mmolil. The minimum and maximum values for total bile salts for the ratios 1.7 and 2.95 are also given.
of mixed composition, the common duct bile was also supersaturated with cholesterol, whereas the gallbladder bile was undersaturated. Both the common duct and gallbladder biles from the group of patients with calcium stones showed no theoretical abnormality with respect to the cholesterol concentration. Our results therefore support the early work of Small and Rap0 (1970) and Vlahcevic et al. (1970) that the primary abnormality in cholesterol precipitation is the secretion of a ‘hepatic’ bile supersaturated with cholesterol.
I00
\
I00
80
60
40
20
I00
% bile salts Fig. I. Three-component diagram showing variation in mean values for the different biles using ratios o f 1.7-2.95 to convert measured cholate concentrations to total bile salt. Ch, Cholesterol stone-formers; Ch Ca, cholesterol tcalcium stone-formers; Ca, calcium stone-formers; - - --, common duct bile; . . . . .gallbladder bilc.
+
further from the region of cholesterol solubility. For stones of mixed composition the line representing the mean composition of common duct bile is also just outside the micellar zone, but the line for the gallbladder bile is within this area. The lines for common duct and gallbladder bile giving rise to calcium stones are within the area of cholesterol solubility.
Discussion The classification of gallstones and bile according to the crystalline composition of the former has shown a considerable difference in the composition of pathogenic biles. Cholesterol stone formation appears to be associated with ‘hepatic’ bile, which is supersaturated with respect to this compound, and this bile may become even more unstable in the gallbladder. However, to what extent supersaturation can occur is difficult to estimate since microcrystals are frequently present in gallbladder bile and our samples were not filtered. For the group of patients with stones
Acknowledgements We thank the surgeons at University College Hospital for allowing their patients to be studied, Mrs S. E. Wooley for taking the X-ray photographs and Miss J. Fowler and Miss C. S. Dines for carrying out the bile analysis. We are grateful to the Medical Research Council and the Nuffield Foundation for financial assistance. References A D M I R A N D w. H.
and SMALL D. M. (1968) The physicochemical basis of cholesterol gallstone formation in man. J . Clin. Invest. 47, 1043-1052. RARSON A L., SHAPIRO P. 0.and PHILLIPS G . E. (1962) A new assay for cholesterol and cholesterol esters in serum which is not affected by bilirubin. Clin. Chim. Actu 7, 800-804. FISKE c. H . and SUBBARROW J . (1925) The colorimetric determination of phosphorus. J . B i d . Chem. 66, 375-400. I R V I N J. L., JOHNSON c. G . and KOPALA J. (1944) A photometric method for the determination of cholates in bile and blood. J . Bid. Chrm. 153, 439-457. I.TFFLER H. H . and MCDOUCALI) c. H. (1963) Estimation of cholesterol in serum. Am. J . Clin. Puihol. 39, 31 1-315. NAKAYAMA F. (1967) Quantitative microanalysis of bile. J. Lab. Cliti. Med. 69, 594609. SJOVALL J . (1960) Bile acids in man under normal and pathological conditions. Bile acids and steroids 73. Clin. Chim. Acta 5, 33-41. SMALL D . M., BOURGkS M. and DERVICHIAN D . C. (1966) Ternary and quaternary aqueous systems containing bile salt, lecithin, and cholesterol. Nutrtre (Loud.) 211, 816 818.
45
D. June Sutor et al. and RAPO s. (1970) Source of abnormal bile in patients with cholesterol gallstones. N P W EngI. J . Med. 283, 53-57. SUTOR D. J. and WOOLEY s. E. (1971) A statistical survey of the composition of gallstones in eight countries. Gut 12, 55-64. SUTOR D. J. and WOOLEY s. E. (1973) The nature and incidence of gallstones containing calcium. Gilt 14, 215-220. SMALL D. M.
44
and SCHOENFIELD L. .I (1971) Induced alterations i n composition of bile of persons having cholelithiasis. Gastroenterology 61, 488496. VLAHCEVIC A. R., BELL c. c. jun. and SWELL L. (1970) Significance of the liver in the production of lithogenic bile in man. Gostromtrrology 59, 62-69. THISTLE J. L.
