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Cholesterol nucleation and gallstone formation
Journal of Hepatology, 1988; 6:383-387
383
Elsevier HEP 00419
Leader
Cholesterol nucleation and gallstone formation
A l b e r t K. G r o e n , J a n A . G . D r a p e r s a n d G u i d o N . J . T y t g a t Department of Gastroenterology, Aeademic Medical Centre, A msterdam ( The Netherlands)
The first step in the formation of cholesterol gallstones is nucleation of cholesterol monohydrate crystals from bile. Until recently it was generally accepted that the main determinant of crystal formation was the degree to which bile was supersaturated with cholesterol. During the last three years it has become clear that the situation is more complex. Not only has the hitherto-used definition of cholesterol saturation become questionable, but also the existence of biliary proteins that promote or inhibit cholesterol nucleation has been reported. In this mini-review we will discuss these recent developments. The concept of cholesterol saturation index (CSI) was developed by Admirand and Small in 1968 [1]. It was based on experiments with model systems. By mixing cholesterol, phospholipid and bile salts in different proportions the solubility limit of cholesterol was determined. Later Carey and Small [2] refined the experiments by including the total lipid content of the system. It turned out that dilution led to a decrease in cholesterol solubility. The critical tables of cholesterol solubility constructed by Carey [3] are used generally to calculate the saturation index of bile samples. It can be argued that the results of the model bile systems cannot be applied directly to human bile. Carey and Small [2] let their model system evolve to thermodynamic equilibrium, which could
take as long as 14 days. In humans, bile probably almost never has the time to reach equilibrium. Therefore, the rate at which bile reaches equilibrium could be more important for the pathogenesis of gallstone disease than the actual value of the CSI. Indeed, Holan et al. [4] have shown that the nucleation time, defined as the time interval to the appearance of the first cholesterol crystal, is a much better predictor of gallstone disease than the CSI. The factors that determine the metastability of supersaturated bile have been studied intensively in the past few years. An implicit assumption in the treatment of Carey and Small [2] was that cholesterol in bile is solubilised mainly in mixed bile salt-lecithin micelles. In 1983 Somjen and Gilat [5] suggested that for human bile this assumption may not be correct. They studied human bile with quasi-elastic light scattering and found cholesterol to be associated with lecithin in vesicles with a diameter of about 700 A. These results have now been confirmed by several groups [6-11] and it turns out that in gallbladder bile 10-70% of total cholesterol is solubilised in vesicular form [6,9]. In more dilute hepatic bile the proportion of vesicular cholesterol is even larger and can reach 90% [6,7,9,10]. Whether these values for cholesterol distribution are an accurate estimate of the situation in native bile samples is not clear. The methods used so far to iso-
Correspondence: Dr. A.K. Groen, Department of Gastroenterology, F0-116, Academic Medical Centre, Meibergdreef 9, 1105 AZ
Amsterdam, The Netherlands. 0168-8278/88/$03.50 (~) 1988 Elsevier Science Publishers B.V. (Biomedical Division)
384 late the vesicular and micellar phase can introduce shifts in the cholesterol distribution. Reuben et al. [12] and Somjen and Gilat [7] have shown that when using gel-permeation chromatography for the separation the results are strongly influenced by the amount of bile salt present in the elution buffer. Without bile salt all cholesterol is present in the vesicular phase. At high concentrations of bile salt cholesterol shifts to the micellar phase. Probably the experimental artefact will be smallest at concentrations of bile salt around the critical micellar concentration (CMC) and most groups choose this condition in their experiments. Density gradient ultracentrifugation could be a somewhat more reliable procedure to separate the two phases in bile. The dilution of the bile sample is less so that redistribution of cholesterol is perhaps also less when this technique is used. To date, the results of both methods have not been compared rigorously. The importance of the cholesterol/lecithin vesicles in nucleation of cholesterol has been revealed in a landmark study of Halpern et al. [13]. They studied nucleation of cholesterol by time-lapse video enhanced microscopy. In abnormal bile, nucleation of cholesterol was preceded by formation of vesicles large enough to be visualized with this technique. Subsequently the vesicles aggregated and crystals seemed to emerge from these aggregates. These events were observed in biles from both stone patients and normals. However, in biles from stone patients the process was much faster. Lee et al. [9] have confirmed the importance of the vesicles experimentally by showing that crystal formation reduces the amount of cholesterol in the vesicular phase whilst it has no effect on micellar cholesterol. The group of Holzbach [8,14] has studied the dynamic interchange of micellar and vesicular cholesterol extensively in a model bile system. Ultracentrifugation was used to separate the vesicular and micellar phases. As expected, all cholesterol was solubilised in mixed micelles at a saturation index of 1.0. At higher saturation indices cholesterol in the micellar phase remained approximately constant and the extra cholesterol was found in the vesicular phase. Interestingly, the cholesterol/lecithin ratio in the re-
A.K. GROEN et al. sicular phase increased when the CSI of the model system was raised. A similar phenomenon was observed when at a constant CSI and total lipid content the bile salt/lecithin ratio was changed. The vesicular cholesterol/phospholipid ratio increased from 1 to 2 when the bile salt/lecithin ratio was raised from 1.9 to 3, which lies in the range of physiological bile salt/lecithin ratios. A third important variable influencing the vesicular cholesterol/lecithin ratio was the total lipid content of the system. At a constant concentration ratio of cholesterol, lecithin and bile salt there was a positive correlation between the total lipid content and the vesicular cholesterol/lecithin ratio. However, the CSI and hence the amount of cholesterol solubilised in vesicles strongly increased when the total lipid content decreased. The reason for this effect is the shift of bile salt from the micellar to the monomeric phase upon dilution of the system. This will reduce the micellar capacity to solubilise cholesterol. Why is the vesicular cholesterol/lecithin ratio so important? Halpern et al. [8] observed a strong correlation between the cholesterol/lecithin ratio in the vesicles and the nucleation time of model bile. In contrast, there was no correlation between the amount of cholesterol solubilised in vesicles and the nucleation time. For instance, at low total lipid con.centrations up to about 40% of cholesterol was vesicular whilst such a model bile nucleated only very ,_slowly. It is a pity that the group of Holzbach [8,14] did not include human biles in their studies, since it is not yet clear whether the results obtained with the model system can be extrapolated to the human situation. For human bile Lee et al. [9] observed a strong negative correlation (r = -0.89) between total lipid concentration and the percentage of nonmicellar cholesterol, which is in agreement with the data of Halpern et al. [8] in the model system. With gallbladder bile no correlation between the CSI and the amount of cholesterol in the vesicles was found [9]. For hepatic biles and mixtures of hepatic bile and gallbladder bile a weak correlation was observed. Recently Ulloa et al. [10], using density gradient ultracentrifugation, also showed a lack of correlation between the amount of vesicular cholesterol and the saturation index. For both gallbladder bile and hep-
CHOLESTEROL NUCLEATION AND GALLSTONE FORMATION atic bile Lee et al. [9] and Ulloa et al. [10] reported relatively large amounts of vesicular cholesterol at CSI values of close to 1. These results are very important because they indicate that the capacity of mixed micellar cholesterol solubilisation is lower in human bile than in model bile. What is the consequence of these new insights into the dynamics of cholesterol solubilization for understanding the pathogenesis of gallstone disease? We now understand why intrahepatic cholesterol gallstones are so rare. Hepatic bile is often highly supersaturated with cholesterol but the total lipid content of hepatic bile is so low that most of the cholesterol is solubilised in very stable vesicles with a low cholesterol/lecithin ratio. When extrapolating the results of the group of Holzbach [8,14] obtained with model bile to human bile the following events should take place in the gallbladder. Concentration of hepatic bile in the gallbladder reduces the amount of vesicular cholesterol relative to micellar cholesterol but concomitantly the vesicular cholesterol/lecithin ratio increases, which renders the vesicles instable and nucleation can occur. However, there is no significant difference in the lipid composition of biles from healthy subjects as compared to biles from stone patients. Therefore, the situation must be more complex. Since the vesicles seem to play a crucial role in cholesterol nucleation one can expect that either the ~lmount of vesicles or the vesicular cholesterol/lecithin ratio is higher in biles of stone patients. If this is true, and future studies have to substantiate this hypothesis, other factors than the biliary lipids per se must be responsible for these differences. Likely candidates are the factors that have been shown to influence cholesterol nucleation. Two opposing classes of cholesterol nucleation-iniluencing factors have been proposed: nucleation inhibitors and nucleation promotors. In 1984 Holzbach ct al. [15] reported inhibition of nucleation by protein fractions from human gallbladder bile. They chromatographed human bile on a gel-permeation column :rod obtained two protein peaks. Both protein peaks contained nucleation-inhibiting activity. The activity could be destroyed by treatment with proteolytic enzymes. The proteins responsible for the inhibiting ef-
385
fect have as yet not been identified. Good candidates are apolipoproteins AI and All. Kibe et al. [16] showed that these lipoproteins are capable of inhibiting nucleation in a model bile system. Apolipoproteins B and C and other proteins such as albumin and immunoglobulins had no effect. The presence of apolipoprotein AI and All has been demonstrated in human bile [17]. However, there was no significant difference in the content of these proteins in bile from stone patients as compared to normal bile. Furthermore, to our knowledge the proteins have not yet been isolated from human bile. Hence it is not yet certain whether the biliary apolipoproteins are capable of inhibiting nucleation. The question remains as to why biles from stone patients nucleate so much faster than biles from normals. Since apolipoproteins AI and All are present in both abnormal and normal bile this could and perhaps must be due to the presence of factors that promote nucleation. Two types of these factors have been proposed. In 1983 Burnstein et al. [18] described a heat-labile factor with a molecular weight of less than 300 000. Recently we have isolated a nucleation-promoting factor from bile that binds to concanavalin A-Sepharose [19]. Concanavalin A is specific for glucose and mannose [20]. Therefore this nucleation promotor must be a glucose- or mannose-containing glycoprotein. Promoting activity of this factor was resistant to treatment with proteolytic enzymes. Concanavalin A-binding nucleation promotor was heat-labile and had an apparent molecular weight of 130 000 (Groen et al., unpublished observations). It could be identical to the factor proposed by Burnstein et al. [18]. The group of LaMont [21] suggested that biliary mucin is responsible for the nucleation-promoting effect. They reported the presence of mucin in the nucleus of cholesterol gallstones [22] and were also able to demonstrate activation of nucleation of supersaturated model bile by mucin [21}. The ability of biliary mucin to promote nucleation was confirmed by Gallinger et al. [23], who observed significant shortening of the nucleation time of human bile at a mucin concentration of 40 Mg/ml. However, Gallinger et al. [23] also showed that extraction of mucin from bile by ultrafiltration failed to influence the nucleation time of the
386 bile sample. Furthermore, Harvey et al. [24] did not observe a significant difference in the mucin content of normal bile and bile from stone patients. In contrast, Lee and Nicholls [25] did find a lower mucin content of normal bile. Both groups used the same methodology to determine the mucin content of the bile samples but the values obtained by Harvey et ai. [24] are a factor of three lower than the values of Lee and Nicholls [25]. The reason for the discrepancy is not clear. Both groups have also analysed the amino acid and carbohydrate content of mucin derived from stone patients and controls. No significant difference was observed. It has not yet been studied whether there are differences in the nucleation-promoting potency of mucin from the bile of stone patients as compared to mucin from normal bile. Such differences have been reported for biliary protein fractions from which mucin had been removed. Gallinger et al. [26] isolated biliary proteins and added an aliquot of 250 /zg back to isotropic bile derived from control patients. Protein from stone patient bile shortened nucleation time significantly more than protein of control bile. A disadvantage of mixing experiments with human bile is that no conclusions can be drawn about the level at which the proteins exert their effect. Nucleation in human bile is a complex interplay of cholesterol/phospholipid vesicles, nucleation inhibitors and nucleation promotors. Added protein can interact with any of these factors. Recently we have developed a semi-quantitative assay of nucleation-influencing activity by mixing human gallbladder bile with model bile [27]. The method is based on the fact that nucleation-promoting activity in bile from stone patients is often present in excess. Serial dilutions of human gallbladder bile were added to a supersaturated model bile and the highest dilution that still significantly shortened the nucleation time of the model bile was called Nucleation Promoting Activity Titer (NPAT). Since the bile is always diluted more than 20 times, the lipid content of the diluted bile sample no longer influences the nucleation time of the model bile. The NPAT of control biles was significantly lower than the N P A T of biles from stone patients. However, there was considerable overlap due to abnormal biles with a low NPAT.
A.K. GROEN et al. Interestingly these biles all had a high CSI. Our resuits for the first time provided evidence for the hypothesis advanced by Burnstein et al. [18] that there are different groups of stone patients: patients with a very high saturation index, and patients with a low saturation index but a high content of nucleationpromoting factors. In its present form our method does not discriminate between mucin and non-mucin pronucleators. For patients with a high N P A T one could envisage a further subdivision in those that have a high mucin content and those with a high amount of non-mucin promotor. The mechanism by which nucleation-promoting factors affect cholesterol crystal formation is still illunderstood. Mucin has been reported to contain hydrophobic domains which could serve as a nidus for nucleation [28]. It induces nucleation in fractions of isolated vesicles and thus probably interacts directly with vesicles [29]. Concanavalin A-binding promotor destabilises micelles and increases the amount of vesicular cholesterol and phospholipid [30]. This could be the explanation for the high concentrations of vesicular cholesterol in human bile with a low CSI reported by Lee et al. [9] and Ulloa et al. [10]. The mechanism underlying this phenomenon has not yet been elucidated. In summary, these data indicate that nucleationinfluencing factors play a crucial role in the pathogenesis of gallstoiae disease. The balance between nucleation-inhibiting and -promoting factors will determine whether or not cholesterol crystals are formed. So far the data suggest that the concentration of promoting factors is most variable and may be more important for gallstone disease. The controversy over the relative importance of mucin and the 'low molecular weight nucleation promotor' will hopefully be resolved in the near future. Attention can then be directed to the development of ways to manipulate the concentration or activity of the nucleation-influencing factors.
Acknowledgements We are indebted to Drs. Peter Jansen and Frans
CHOLESTEROL NUCLEATION AND GALLSTONE FORMATION
H o e k f o r critically r e a d i n g t h e m a n u s c r i p t a n d to
387
manuscript.
Mrs. T r u u s K l e b a c h for h e r h e l p in p r e p a r i n g t h e
References 1 Admirand WH, Small DM. The physico-chemical basis of cholesterol gallstone formation in man. J Clin Invest 1968; 47: 1043-1052. 2 Carey MC, Small DM. The physical chemistry of cholesterol solubility in bile. Relationship to gallstone formation and dissolution in man. J Clin Invest 1978; 61: 998-1026. 3 Carey MC. Critical tables for calculating the cholesterol saturation of native bile. J Lipid Res 1978; 19: 945-955. 4 Holan KR, Holzbach RT, Hermann RE, et al. Nucleation time: a key factor in the pathogenesis of cholesterol gallstone disease. Gastroenterology 1979; 77:611-617. 5 Somjen G J, Gilat T. A non-micellar mode of cholesterol transport in bile. FEBS Lett 1983; 156: 265-268. 6 Pattinson NR. Solubilisation of cholesterol in human bile. FEBS Lett 1985; 181: 339-342. 7 Somjen G J, Gilat T. Contribution of vesicular and micellar carriers to cholesterol transport in human bile. J Lipid Res 1985; 26: 699-704. 8 Halpern Z, Dudley MA, Lynn MP, et al. Vesicle aggregation in model systems of supersaturated bile: relation to crystal nucleation and lipid composition of the vesicular phase. J Lipid Res 1986; 27: 295-306. 9 Lee SP, Park HZ, Madani H, Kaler EW. Partial characterization of a nonmicellar system of cholesterol solubilization in bile. Am J Physiol 1987; 252: G374-G384. 10 Ulloa N, Garrido J, Nervi F. Ultracentrifugal isolation of vesicular carriers of biliary cholesterol in native human and rat bile. Hepatology 1987; 7: 235-244. 11 Pattinson NR, Chapman BA. Distribution of biliary cholesterol between mixed micelles and nonmicelles in relation to fasting and feeding in humans. Gastroenterology 1986; 91: 697-702. 12 Reuben A, Howell KE, Boyer JL. Effects of taurocholate on the size of mixed micelles and their associations with pigment and protein in rat bile. J Lipid Res 1982; 23: 1039-1052. 13 Halpern Z, Dudley MA, Kibe A, et al. Rapid vesicle formation and aggregation in abnormal human biles. Gastroenterology 1986; 90: 875-885. 14 Kibe A, Dudley MA, Halpern Z, et al. Factors influencing cholesterol monohydrate crystal nucleation time in model systems of supersatui'ated bile. J Lipid Res 1985; 26: 1102-1111. 15 Holzbach RT, Kibe A, Thiel E, et al. Biliary proteins. Unique inhibitors of cholesterol crystal nucleation in human gallbladder bile. J Clin Invest 1984; 73: 35-45. 16 Kibe A, Holzbach RT, LaRusso NF, Mao ST. Inhibition of
cholesterol crystal formation by apolipoproteins A-I and AII in model systems of supersaturated bile; implications for gallstone pathogenesis in man. Science 1984; 225: 514-516. 17 Sewell RB, Mao ST, Kawamoto T, LaRusso NF. Apolipoproteins of high, low and very low density in human bile. J Lipid Res 1983; 42: 391-401. 18 Burnstein JM, Ilson RG, Petrunka CN, Strasberg SM. Evidence for a potent nucleation factor in gallbladder bile of patients with cholesterol gallstones. Gastroenterology 1983; 85: 801-807. 19 Groen AK, Stout JPJ, Drapers JAG, et al. Cholesterol influencing activity in T-tube bile. Hepatology 1988; 2: in press. 20 Baenziger JU, Fiete D. Structural determinants of concanavalin A specificity for oligosaccharides. J Biol Chem 1979; 254: 2400-2407. 21 Levy PF, Smith BF, LaMont JT. Human gallbladder mucin accelerates in vitro nucleation of cholesterol in artificial bile. Gastroenterology 1984; 87: 270-275. 22 Smith BF, LaMont JH. Identification of gallbladder mucinbilirubin complex in human cholesterol gallstone matrix. Effects of reducing agents on in vitro dissolution of matrix and intact gallstones. J Clin Invest 1985; 76: 439-446. 23 Gallinger S, Taylor RD, Harvey PRC, Strasberg SM. Effect of mucous glycoprotein on nucleation time of human bile. Gastroenterology 1985; 89: 648-658. 24 Harvey PRC, Rupar CA, Gallinger S, et al. Quantitative and. qualitative comparison of gallbladder mucous glycoprotein from patients with and without gallstones. Gut 1986; 27: 374-381. 25 Lee SP, Nicholls JF. Nature and composition of biliary sludge. Gastroenterology 1986; 90: 677-686. 26 Gallinger S, Harvey PRC, Petrunka CN, et al. Biliary proteins and the nucleation defect in cholesterol cholelithiasis. Gastroenterology 1987; 92: 867-875. 27 Drapers JAG, Groen AK, Stout JPJ, et al. Quantification of cholesterol nucleation influencing.activity in human gallbladder bile. Clin Chim Acta 1987; 165: 295-302. 28 LaMont JT, Smith BF, Moore JRL. Role of gallbladder mucin in pathophysiology of gallstones. Hepatology 1984; 4: 51S-56S. 29 Lee TJ, Smith BF. Gallbladder mucin accelerates cholesterol crystal nucleation from cholesterol enriched vesicles in model bile. Gastroenterology 1987; 92: 1748. 30 Groen AK, Ottenhoff R, Noordam C, et al. Cholesterol nucleation promoting activity shifts cholesterol and phospholipid from the micellar to the vesicular phase in model bile. Gastroenterology 1987; 92: 1737.
383
Elsevier HEP 00419
Leader
Cholesterol nucleation and gallstone formation
A l b e r t K. G r o e n , J a n A . G . D r a p e r s a n d G u i d o N . J . T y t g a t Department of Gastroenterology, Aeademic Medical Centre, A msterdam ( The Netherlands)
The first step in the formation of cholesterol gallstones is nucleation of cholesterol monohydrate crystals from bile. Until recently it was generally accepted that the main determinant of crystal formation was the degree to which bile was supersaturated with cholesterol. During the last three years it has become clear that the situation is more complex. Not only has the hitherto-used definition of cholesterol saturation become questionable, but also the existence of biliary proteins that promote or inhibit cholesterol nucleation has been reported. In this mini-review we will discuss these recent developments. The concept of cholesterol saturation index (CSI) was developed by Admirand and Small in 1968 [1]. It was based on experiments with model systems. By mixing cholesterol, phospholipid and bile salts in different proportions the solubility limit of cholesterol was determined. Later Carey and Small [2] refined the experiments by including the total lipid content of the system. It turned out that dilution led to a decrease in cholesterol solubility. The critical tables of cholesterol solubility constructed by Carey [3] are used generally to calculate the saturation index of bile samples. It can be argued that the results of the model bile systems cannot be applied directly to human bile. Carey and Small [2] let their model system evolve to thermodynamic equilibrium, which could
take as long as 14 days. In humans, bile probably almost never has the time to reach equilibrium. Therefore, the rate at which bile reaches equilibrium could be more important for the pathogenesis of gallstone disease than the actual value of the CSI. Indeed, Holan et al. [4] have shown that the nucleation time, defined as the time interval to the appearance of the first cholesterol crystal, is a much better predictor of gallstone disease than the CSI. The factors that determine the metastability of supersaturated bile have been studied intensively in the past few years. An implicit assumption in the treatment of Carey and Small [2] was that cholesterol in bile is solubilised mainly in mixed bile salt-lecithin micelles. In 1983 Somjen and Gilat [5] suggested that for human bile this assumption may not be correct. They studied human bile with quasi-elastic light scattering and found cholesterol to be associated with lecithin in vesicles with a diameter of about 700 A. These results have now been confirmed by several groups [6-11] and it turns out that in gallbladder bile 10-70% of total cholesterol is solubilised in vesicular form [6,9]. In more dilute hepatic bile the proportion of vesicular cholesterol is even larger and can reach 90% [6,7,9,10]. Whether these values for cholesterol distribution are an accurate estimate of the situation in native bile samples is not clear. The methods used so far to iso-
Correspondence: Dr. A.K. Groen, Department of Gastroenterology, F0-116, Academic Medical Centre, Meibergdreef 9, 1105 AZ
Amsterdam, The Netherlands. 0168-8278/88/$03.50 (~) 1988 Elsevier Science Publishers B.V. (Biomedical Division)
384 late the vesicular and micellar phase can introduce shifts in the cholesterol distribution. Reuben et al. [12] and Somjen and Gilat [7] have shown that when using gel-permeation chromatography for the separation the results are strongly influenced by the amount of bile salt present in the elution buffer. Without bile salt all cholesterol is present in the vesicular phase. At high concentrations of bile salt cholesterol shifts to the micellar phase. Probably the experimental artefact will be smallest at concentrations of bile salt around the critical micellar concentration (CMC) and most groups choose this condition in their experiments. Density gradient ultracentrifugation could be a somewhat more reliable procedure to separate the two phases in bile. The dilution of the bile sample is less so that redistribution of cholesterol is perhaps also less when this technique is used. To date, the results of both methods have not been compared rigorously. The importance of the cholesterol/lecithin vesicles in nucleation of cholesterol has been revealed in a landmark study of Halpern et al. [13]. They studied nucleation of cholesterol by time-lapse video enhanced microscopy. In abnormal bile, nucleation of cholesterol was preceded by formation of vesicles large enough to be visualized with this technique. Subsequently the vesicles aggregated and crystals seemed to emerge from these aggregates. These events were observed in biles from both stone patients and normals. However, in biles from stone patients the process was much faster. Lee et al. [9] have confirmed the importance of the vesicles experimentally by showing that crystal formation reduces the amount of cholesterol in the vesicular phase whilst it has no effect on micellar cholesterol. The group of Holzbach [8,14] has studied the dynamic interchange of micellar and vesicular cholesterol extensively in a model bile system. Ultracentrifugation was used to separate the vesicular and micellar phases. As expected, all cholesterol was solubilised in mixed micelles at a saturation index of 1.0. At higher saturation indices cholesterol in the micellar phase remained approximately constant and the extra cholesterol was found in the vesicular phase. Interestingly, the cholesterol/lecithin ratio in the re-
A.K. GROEN et al. sicular phase increased when the CSI of the model system was raised. A similar phenomenon was observed when at a constant CSI and total lipid content the bile salt/lecithin ratio was changed. The vesicular cholesterol/phospholipid ratio increased from 1 to 2 when the bile salt/lecithin ratio was raised from 1.9 to 3, which lies in the range of physiological bile salt/lecithin ratios. A third important variable influencing the vesicular cholesterol/lecithin ratio was the total lipid content of the system. At a constant concentration ratio of cholesterol, lecithin and bile salt there was a positive correlation between the total lipid content and the vesicular cholesterol/lecithin ratio. However, the CSI and hence the amount of cholesterol solubilised in vesicles strongly increased when the total lipid content decreased. The reason for this effect is the shift of bile salt from the micellar to the monomeric phase upon dilution of the system. This will reduce the micellar capacity to solubilise cholesterol. Why is the vesicular cholesterol/lecithin ratio so important? Halpern et al. [8] observed a strong correlation between the cholesterol/lecithin ratio in the vesicles and the nucleation time of model bile. In contrast, there was no correlation between the amount of cholesterol solubilised in vesicles and the nucleation time. For instance, at low total lipid con.centrations up to about 40% of cholesterol was vesicular whilst such a model bile nucleated only very ,_slowly. It is a pity that the group of Holzbach [8,14] did not include human biles in their studies, since it is not yet clear whether the results obtained with the model system can be extrapolated to the human situation. For human bile Lee et al. [9] observed a strong negative correlation (r = -0.89) between total lipid concentration and the percentage of nonmicellar cholesterol, which is in agreement with the data of Halpern et al. [8] in the model system. With gallbladder bile no correlation between the CSI and the amount of cholesterol in the vesicles was found [9]. For hepatic biles and mixtures of hepatic bile and gallbladder bile a weak correlation was observed. Recently Ulloa et al. [10], using density gradient ultracentrifugation, also showed a lack of correlation between the amount of vesicular cholesterol and the saturation index. For both gallbladder bile and hep-
CHOLESTEROL NUCLEATION AND GALLSTONE FORMATION atic bile Lee et al. [9] and Ulloa et al. [10] reported relatively large amounts of vesicular cholesterol at CSI values of close to 1. These results are very important because they indicate that the capacity of mixed micellar cholesterol solubilisation is lower in human bile than in model bile. What is the consequence of these new insights into the dynamics of cholesterol solubilization for understanding the pathogenesis of gallstone disease? We now understand why intrahepatic cholesterol gallstones are so rare. Hepatic bile is often highly supersaturated with cholesterol but the total lipid content of hepatic bile is so low that most of the cholesterol is solubilised in very stable vesicles with a low cholesterol/lecithin ratio. When extrapolating the results of the group of Holzbach [8,14] obtained with model bile to human bile the following events should take place in the gallbladder. Concentration of hepatic bile in the gallbladder reduces the amount of vesicular cholesterol relative to micellar cholesterol but concomitantly the vesicular cholesterol/lecithin ratio increases, which renders the vesicles instable and nucleation can occur. However, there is no significant difference in the lipid composition of biles from healthy subjects as compared to biles from stone patients. Therefore, the situation must be more complex. Since the vesicles seem to play a crucial role in cholesterol nucleation one can expect that either the ~lmount of vesicles or the vesicular cholesterol/lecithin ratio is higher in biles of stone patients. If this is true, and future studies have to substantiate this hypothesis, other factors than the biliary lipids per se must be responsible for these differences. Likely candidates are the factors that have been shown to influence cholesterol nucleation. Two opposing classes of cholesterol nucleation-iniluencing factors have been proposed: nucleation inhibitors and nucleation promotors. In 1984 Holzbach ct al. [15] reported inhibition of nucleation by protein fractions from human gallbladder bile. They chromatographed human bile on a gel-permeation column :rod obtained two protein peaks. Both protein peaks contained nucleation-inhibiting activity. The activity could be destroyed by treatment with proteolytic enzymes. The proteins responsible for the inhibiting ef-
385
fect have as yet not been identified. Good candidates are apolipoproteins AI and All. Kibe et al. [16] showed that these lipoproteins are capable of inhibiting nucleation in a model bile system. Apolipoproteins B and C and other proteins such as albumin and immunoglobulins had no effect. The presence of apolipoprotein AI and All has been demonstrated in human bile [17]. However, there was no significant difference in the content of these proteins in bile from stone patients as compared to normal bile. Furthermore, to our knowledge the proteins have not yet been isolated from human bile. Hence it is not yet certain whether the biliary apolipoproteins are capable of inhibiting nucleation. The question remains as to why biles from stone patients nucleate so much faster than biles from normals. Since apolipoproteins AI and All are present in both abnormal and normal bile this could and perhaps must be due to the presence of factors that promote nucleation. Two types of these factors have been proposed. In 1983 Burnstein et al. [18] described a heat-labile factor with a molecular weight of less than 300 000. Recently we have isolated a nucleation-promoting factor from bile that binds to concanavalin A-Sepharose [19]. Concanavalin A is specific for glucose and mannose [20]. Therefore this nucleation promotor must be a glucose- or mannose-containing glycoprotein. Promoting activity of this factor was resistant to treatment with proteolytic enzymes. Concanavalin A-binding nucleation promotor was heat-labile and had an apparent molecular weight of 130 000 (Groen et al., unpublished observations). It could be identical to the factor proposed by Burnstein et al. [18]. The group of LaMont [21] suggested that biliary mucin is responsible for the nucleation-promoting effect. They reported the presence of mucin in the nucleus of cholesterol gallstones [22] and were also able to demonstrate activation of nucleation of supersaturated model bile by mucin [21}. The ability of biliary mucin to promote nucleation was confirmed by Gallinger et al. [23], who observed significant shortening of the nucleation time of human bile at a mucin concentration of 40 Mg/ml. However, Gallinger et al. [23] also showed that extraction of mucin from bile by ultrafiltration failed to influence the nucleation time of the
386 bile sample. Furthermore, Harvey et al. [24] did not observe a significant difference in the mucin content of normal bile and bile from stone patients. In contrast, Lee and Nicholls [25] did find a lower mucin content of normal bile. Both groups used the same methodology to determine the mucin content of the bile samples but the values obtained by Harvey et ai. [24] are a factor of three lower than the values of Lee and Nicholls [25]. The reason for the discrepancy is not clear. Both groups have also analysed the amino acid and carbohydrate content of mucin derived from stone patients and controls. No significant difference was observed. It has not yet been studied whether there are differences in the nucleation-promoting potency of mucin from the bile of stone patients as compared to mucin from normal bile. Such differences have been reported for biliary protein fractions from which mucin had been removed. Gallinger et al. [26] isolated biliary proteins and added an aliquot of 250 /zg back to isotropic bile derived from control patients. Protein from stone patient bile shortened nucleation time significantly more than protein of control bile. A disadvantage of mixing experiments with human bile is that no conclusions can be drawn about the level at which the proteins exert their effect. Nucleation in human bile is a complex interplay of cholesterol/phospholipid vesicles, nucleation inhibitors and nucleation promotors. Added protein can interact with any of these factors. Recently we have developed a semi-quantitative assay of nucleation-influencing activity by mixing human gallbladder bile with model bile [27]. The method is based on the fact that nucleation-promoting activity in bile from stone patients is often present in excess. Serial dilutions of human gallbladder bile were added to a supersaturated model bile and the highest dilution that still significantly shortened the nucleation time of the model bile was called Nucleation Promoting Activity Titer (NPAT). Since the bile is always diluted more than 20 times, the lipid content of the diluted bile sample no longer influences the nucleation time of the model bile. The NPAT of control biles was significantly lower than the N P A T of biles from stone patients. However, there was considerable overlap due to abnormal biles with a low NPAT.
A.K. GROEN et al. Interestingly these biles all had a high CSI. Our resuits for the first time provided evidence for the hypothesis advanced by Burnstein et al. [18] that there are different groups of stone patients: patients with a very high saturation index, and patients with a low saturation index but a high content of nucleationpromoting factors. In its present form our method does not discriminate between mucin and non-mucin pronucleators. For patients with a high N P A T one could envisage a further subdivision in those that have a high mucin content and those with a high amount of non-mucin promotor. The mechanism by which nucleation-promoting factors affect cholesterol crystal formation is still illunderstood. Mucin has been reported to contain hydrophobic domains which could serve as a nidus for nucleation [28]. It induces nucleation in fractions of isolated vesicles and thus probably interacts directly with vesicles [29]. Concanavalin A-binding promotor destabilises micelles and increases the amount of vesicular cholesterol and phospholipid [30]. This could be the explanation for the high concentrations of vesicular cholesterol in human bile with a low CSI reported by Lee et al. [9] and Ulloa et al. [10]. The mechanism underlying this phenomenon has not yet been elucidated. In summary, these data indicate that nucleationinfluencing factors play a crucial role in the pathogenesis of gallstoiae disease. The balance between nucleation-inhibiting and -promoting factors will determine whether or not cholesterol crystals are formed. So far the data suggest that the concentration of promoting factors is most variable and may be more important for gallstone disease. The controversy over the relative importance of mucin and the 'low molecular weight nucleation promotor' will hopefully be resolved in the near future. Attention can then be directed to the development of ways to manipulate the concentration or activity of the nucleation-influencing factors.
Acknowledgements We are indebted to Drs. Peter Jansen and Frans
CHOLESTEROL NUCLEATION AND GALLSTONE FORMATION
H o e k f o r critically r e a d i n g t h e m a n u s c r i p t a n d to
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manuscript.
Mrs. T r u u s K l e b a c h for h e r h e l p in p r e p a r i n g t h e
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