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Lysosomal enzyme activities in gallbladder mucosa of gallstone-free subjects and patients with gallstones
Journal of Hepatology 1996; 25:895-899 Printed in Denmark. All rights reserved Munksgaard. Copenhagen
Copyright © European Association for the Study of the Liver 1996 Journal of Hepatoiogy
ISSN 0168-8278
Lysosomal enzyme activities in gallbladder mucosa of gallstone-free subjects and patients with gallstones Staffan Sahlin l, Hans Glauman 2,/~ke Danielsson 3 and Kurt Einarsson 4 t Department of Surgery, Danderyd Hospital and Departments of 2pathology and Infectious Diseases, and 4Medicine, Huddinge University Hospital, Karolinska lnstitutet, Stockholm, and 3Department of Medicine, University of Umed, Sweden
Background~Aims: Gallstone patients have a reduced cellular lysosome content in the gallbladder mucosa cells compared with gallstone-free subjects. The purpose of the study was to further evaluate the possible role of lysosomes in the pathogenesis of cholesterol gallstone formation in humans. Methods: Lysosomai enzyme activities were assayed in gallbladder mucosa and for comparison in liver specimens of 19 gallstone-free subjects and 24 gallstone patients undergoing cholecystectomy. Results: Gallstone patients had 25-50% lower activities of the lysosomal proteases cathepsin B, D and L in their gallbladder mucosa compared with gallstone-free subjects. The activity of acid phosphatase also tended to be decreased in gallstone
patients. The liver lysosomal enzyme activities were not significantly different between the two groups. Conclusions: The results show that gallstone patients have diminished lysosomal enzyme activities in the gallbladder mucosa, a finding which may be related to decreased intracellular degradation of proteins and/or mucin in the mucosal cells. This may lead to a higher concentration of mucin in gallbladder bile and thus an increased risk of precipitation of cholesterol crystals and gallstone formarion.
CORDING to current concepts, supersaturation of bile is a prerequisite for cholesterol gallstone formation in man (1,2). Other factors are, however, also important. The nucleation process leading to formation of cholesterol crystals seems to be a crucial event in gallstone formation. The nucleation time is thus much shorter in gallbladder bile of gallstone patients compared with gallstone-free subjects (3,4). The nucleation time seems to be shortened by an excess of pronucleating factors and/or by a lack of antinucleating factors. An imbalance between these factors may precipitate crystals (2,5). Several studies in experimental animals have provided evidence for that mucin and glycoproteins may play a role as nucleating factors (6-10). According to several reports, gallbladder bile of gallstone patients has
higher concentrations of mucin and total protein than that of gallstone-free subjects (11-16). This could be caused by either increased production or decreased degradation by the mucosal cells. In a recent study we found that gallstone patients have a markedly reduced cellular lysosome content in the gallbladder mucosa cells compared with gallstone-free controls (17). A reduced lysosome content might be related to a decreased intracellular degradation of proteins and/or mucin leading to higher secretion rate and concentration in the gallbladder bile. It was therefore considered of importance to further evaluate the possible role of the lysosomes in the pathogenesis of cholesterol gallstone formation in humans. The aim of the present study was to assay the activity of some lysosomal enzymes in the gallbladder mucosa of gallstones patients and gallstone-free controls. For comparison, corresponding lysosomal enzyme activities in liver tissue were also determined in surgical biopsies obtained from the same patients.
Received 13 December 1995; revised 21 March; accepted 25 March 1996
Correspondence: Staffan Sahlin, Department of Surgery, Danderyd Hospital, S-182 88 Danderyd, Sweden. Tel.: +46-8-655 5000. Fax: +46-8-622 64 06.
Key words: Cholecystectomy; Cholesterol gallstone; Cholesterol saturation; Liver parenchyma; Mucin.
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from the muscle layer, put into ice-cold homogenizing buffer and rapidly transported to the laboratory.
Materials and Methods Patients The study was carried out in 24 consecutive patients with cholesterol gallstones and 19 gallstone-free subjects undergoing cholecystectomy. A preoperative cholecystography had shown a well functioning gallbladder in all cases. The gallstones were classified as cholesterol stones by laboratory analysis (18,19). Indications for cholecystectomy in the gallstone-free subjects were roentgenographic indication of polyp or adenomyoma of the gallbladder in combination with suspected biliary pain. None of the patients had clinical or laboratory evidence of diabetes mellitus, hyperlipoproteinemia or other diseases affecting the liver, thyroid or kidney function. All patients were of normal weight (Table 1). Informed consent was obtained from each patient before the operation, and the study was approved by the Committee of Ethics at the Karolinska Institute, Stockholm (February 1, 1988).
Experimental procedure The patients were admitted to the surgical ward the day prior to operation and given the regular hospital diet. All operations were performed between 8 and 10 a.m. after an overnight fast. After opening of the abdomen, the gallbladder was completely emptied of bile using a sterile needle and syringe to avoid possible stratification of the bile (20). A wedge biopsy weighing 2--4 g was taken peroperatively from the liver. A small portion of the tissue sample was sent for microscopic examination and the remainder was immediately put into ice-cold homogenizing buffer and rapidly transported to the laboratory. A regular cholecystectomy was then performed. A piece of the gallbladder was subjected to microscopic examination. The remainder was rinsed with ice-cold homogenizing buffer. The mucosa was carefully separated
Analysis of bile Gallbladder bile was examined with polarized light microscopy at 37 °C immediately after operation. Cholesterol monohydrate crystals were identified by birefringence and typical flat rhomboidal shapes. For determination of cholesterol and total phospholipids a portion of the bile samples obtained was immediately extracted with 20 vol of chloroformmethanol 2:1 (vol/vol). Cholesterol was determined by an enzymatic method (21) and phospholipids by the method of Roser et al. (22). The total bile acid concentration in one aliquot of the bile samples was determined using the 3o~-hydroxysteroid dehydrogenase assay (23). Cholesterol saturation was calculated as a percentage of the predicted cholesterol solubility at the respective biliary lipid concentration and composition as described by Carey (24). The nucleation time was determined by the method of Holan et al. (3) with minor modifications (4). The gallbladder content of protein was determined by a Bio-Rad protein assay as described recently (25).
Preparation of a lysosomal enriched fraction from liver and gallbladder mucosa Aliquots of the liver and gallbladder mucosa were minced and homogenized with a loose fitting Teflon pestle in 9 vol of a 50 mM Tris-HC1 buffer pH 7.4, containing 0.3 M sucrose, 10 mM DTT, 10 mM EDTA and 50 mM NaC1. The homogenate was centrifuged at 20000 g for 15 min. The pellet (lysosomal enriched fraction) obtained, containing the lysosomes, was frozen at -20°C until analysis.
Assay of lysosomal enzymes TABLE 1 Clinical data of gallstone patients and gallstone-free subjects undergoing cholecystectomy (mea~+SE) Groups of patients
Cholesterol gallstone
Gallstone-free
n Sex (M/F) Age (years) Body weight (kg) Relative body weight (%)a Plasma cholesterol (mmol/1) Plasma triglycerides (mmol/1)
24 6/18 49 (3) 73 (3) 104 (3) 5.4 (0.2) 1.3 (0.1)
19 5/14 47 (3) 67 (3) 96 (3) 5.8 (0.2) 1.0 (0.1)
a body weight (kg) • Relativebodyweightcalculateda s ~ t ( - ~ -- ]-~ x 100
896
Cathepsin B and L activities were measured as described by Barrett & Kirschke (26) with Z-Arg-Arg-NMec as substrate. Acid phosphatase and cathepsin D activities were assayed as described by Bowers et al. (27). For biochemical assessment of enrichment and purity of the lysosomal enriched fractions from the gallbladder mucosa of gallstone-free and gallstone patients, lysosomal enzymes were measured in the total homogenate and two marker enzymes were analyzed, NADPH-cytochrome c reductase (microsomes) and succinate c reductase (mitochondria). Based on four patients from whom material was available, the enrichment factor (lysosomal enriched
Lysosomal enzymes in gallbladder fraction over homogenate) was 11.5 (gallstone-free) and 13.2 (gallstone) for cathepsin B, and 14.5 (gallstone-free) and 12.3 (gallstone) for cathepsin D. The levels of contamination of microsomes to the lysosomal fractions were 10.7% for gallstone-free and 12.3% for gallstone patients. Corresponding levels for mitochondria were 15.7% (gallstone-free) and 17.1% (gallstone). Thus, there was no significant difference in the quality and purity of the preparations between gallstone patients and controls.
Statistical analysis Data are given as means (standard error of the mean, SEM). Significance of differences was evaluated with Student's t-test or the Mann-Whitney U-test.
TABLE3 Lysosomal enzyme activities in liver tissue and gallbladder specimens from gallstonepatients and gallstone-freesubjects undergoing cholecystectomy. Enzyme activities are given per mg of protein (SEM) and are expressed as ~tmolNMec released per min (cathepsin B and L), nmol tyrosinereleasedper rain (cathepsinD), and I.tmolPi released per min (acid phosphatase).Means+SE Groups of patients Cathepsin B
Cholesterol Gallstone-free gallstone Livertissue 168 (15) 213 (26) Mucosa 31 (4)* 49 (6) Cathepsin D Livertissue 30 (2) 31 (3) Mucosa 23 (2)* 31 (3) Cathepsin L Livertissue 782 (27) 848 (91) Mucosa 84 (16)* 172 (34) Acid Livertissue 1,89 (0,13) 2.09 (0.14) phosphatase Mucosa 1.19 (0.11) 1.29 (0.09) *Significantlydifferentfrom gallstone-free,p<0.05.
Results Light microscopy All gallbladders were routinely examined by a pathologist not involved in the study. Those with gallstones showed evidence of light to moderate chronic inflammation. In the gallstone-free cases the suspected polyps were foldings of epithelium and the adenomyomas were confirmed.
Composition of bile The gallbladder bile contained less total lipid in gallstone patients than in gallstone-free patients (Table 2). Gallbladder bile was significantly more saturated with cholesterol in the gallstone patients compared with the gallstone-free subjects. Seventy-nine percent of the gallstone patients displayed cholesterol crystals in their gallbladder bile compared with none of the gallstone-free subjects. The gallstone patients had
TABLE2 Lipid composition, cholesterol saturation, occurrence of cholesterol monohydrate crystals, nucleation time and protein concentrationin gallbladder bile of gallstone patients and gallstone-free subjects undergoingcholecystectomy(means+SE) Groups of Cholesterol Gallstonepatients gallstone free Cholesterol (mmol/l) 12.1 (1.1) 14.6 (1.7) Cholesterol (tool%) 7.2 (0.5) 5.9 (0.5) Bile acids (mol%) 69.6 (1.4) 70.5 (1.0) Phospholipids (mol%) 23.1 (1.0) 23.6 (1.0) Lipid concentration(g/dl) 8.4 (0.7)* 13.3 (1.0) Cholesterol saturation(%) 103 (6)* 78 (7) Crystals, no of patients (+/-) 19/5 0/19 Nucleationtime (days) 2 (1-11) 20 (1-35) Proteinconcentration(mg/ml) 8.3 (1.8) 6.8 (0.9) * Significantlydifferentfrom gallstone-free,p<0.01, *p<0.001 # Significantlydifferentfrom gallstone-free,p<0.0001. Mann Whitney U-test
a significantly shorter gallbladder bile nucleation time than the gallstone-free controls. Only one of the gallstone-free subjects had a short nucleation time (1 day). The protein concentration tended to be higher in the gallstone patients compared with the gallstonefree subjects (8.3 vs. 6.8 mg/ml) but the difference did not reach statistical significance. The protein concentration averaged 9.0 (2.2) mg/ml in the gallstone patients with cholesterol crystals and 6.2 (3.6) mg/ml in those without crystals.
Lysosomal enzyme activities The purity of the lysosomal enriched fractions was similar for gallstone patients and gallstone-free subjects (see Methods section). The activities of cathepsin B, D and L were significantly reduced by 25-50% in the gallbladder mucosa of gallstone patients compared with gallstone-free controls (Table 3). The activity of acid phosphatase was not significantly different between the two groups of patients. There was no difference in hepatic lysosome enzyme activity between gallstone patients and gallstone-free controls.
Discussion A major finding of the present study was that the activities of the lysosomal enzymes cathepsin B, D and L were 25-50% lower in the lysosomal enriched fraction of gallbladder mucosa from gallstone patients compared with gallstone-free subjects. The activity of acid phosphatase also tended to be lower in the gallstone patients. Corresponding enzyme activities in the liver specimens of the gallstone patients and the gallstone-free controls were not significantly different. The results are in good agreement
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with those obtained in a previous ultrastructural study from our group (17). Patients with gallstones were found to have a markedly reduced cellular lysosomal volume density (around 70%) in the gallbladder epithelial cells compared with gallstone-free subjects. Kouroumalis et al. (28,29) have previously reported significantly decreased levels of lysosomal enzyme activities - beta-glucuronidase, acid phosphatase and neutral esterases - in the gallbladder wall of gallstone patients, especially those with chronic cholecystitis. They speculated that the reduced levels might play a significant role in the development of cholecystitis. However, our gallstone patients showed evidence of only slight chronic inflammation and no marked cholecystitis. The mechanism of the decreased levels of lysosomal enzyme activities in the gallbladder mucosa of the gallstone patients is not apparent. The difference between gallstone patients and gallstone-free controls could hardly be due to methodological problems, since the quality and purity of the lysosome enriched fractions from the gallbladder mucosa of gallstonefree and gallstone patients were similar. Furthermore, as mentioned above, a previous ultrastructural study has shown markedly reduced cellular lysosomal volume density in the gallbladder epithelial cells of gallstone patients compared with gallstone-free subjects. Theoretically, genetic differences between gallstone patients and normal subjects might explain the decreased lysosomal enzyme activity in cholesterol gallstone patients, even though there are no studies available to suggest that individuals with low lysosomal enzyme activities in the gallbladder are prone to develop gallstones. Alternatively, the decreased lysosomal enzyme activities in gallstone patients might be a mucosal response to a cytotoxic effect of lithogenic bile, cholesterol crystals and/or gallstones. What, then, could be the clinical significance of reduced levels of lysosomal enzymes in the gallbladder mucosa? Some research groups have recently reported that gallstone patients have higher concentrations of total protein in the gallbladder bile compared with gallstone-free subjects (13,16,30-32). We also found a tendency to higher concentrations of protein in the gallstone patients compared with the gallstone-free subjects, although the difference did not reach statistical significance. Higher concentrations of total protein were found in gallbladder bile samples with cholesterol crystals than in samples without crystals. The protein concentration correlated with the nucleation time in gallstone patients. The hepatic bile protein concentrations, however, were similar in gallstone patients and gallstone-free sub898
jects, indicating that the origin of the increased protein concentration of the gallbladder bile in the gallstone patients would be the gallbladder p e r se. Mucosal inflammation of the gallbladder is common in cholesterol gallstone disease. In the present study, the gallbladders of the gallstone patients showed evidence of light to moderate inflammation. Inflammatory changes of the gallbladder may cause leakage and an increased flux of plasma proteins into the bile. Another explanation of the increased protein concentration of the gallbladder bile of the gallstone patients could be a decreased capacity to degrade protein in the gallbladder. The findings of reduced lysosomal content and concomitant reduced enzyme activity may reflect decreased ability for protein degradation in the gallbladder wall. An alternative explanation could be that lysosomes of the gallbladder mucosa are normally involved in intraceUular degradation of mucin granules. A reduced content of these organelles and their enzyme activities would thus reflect a defect in the elimination of mucin granules, which then are released into the gallbladder lumen, and may act as a nidus for cholesterol crystal formation. Kouroumalis et al. (28) presented a third explanation that lysosomal enzymes are released together with mucin granules into the gallbladder lumen, where they hydrolyze conjugated bilirubin into unconjugated bilirubin. The unconjugated bilirubin can easily precipitate as calcium bilirnbinate, forming a nidus for gallstones. In conclusion, the present study has shown that gallstone patients have diminished lysosomal enzyme activities in the gallbladder mucosa. This finding may be related to decreased intracellular degradation of proteins and/or mucin in the mucosal cells, which may lead to increased concentration of mucin in gallbladder bile and thus an increased risk of precipitation of cholesterol crystals and gallstone formation.
Acknowledgements The skillful technical assistance of Ms Ingela Arvidsson, Ms Lisbet Benthin, Ms Danuta Cosliani and Ms Helena Jansson is gratefully acknowledged. This study was supported by grants from the Swedish Medical Research Council (03X-4793). References 1. Hofmann AF. Pathogenesis of cholesterol gallstones. J Clin Gastroenterol 1988; 10 (suppl 2): SI-11. 2. Lamont JT, Carey MC. Cholesterol gallstone formation. 2. Pathobiology and pathomechanics. Progr Liver Dis 1992; 10: 165-91.
Lysosomal enzymes in gallbladder 3. Holan KR, Holzbach RT, Hermann RE, Cooperman AM, Claffey WJ. Nucleating time: a key factor in pathogenesis of cholesterol gallstone disease. Gastroenterology 1979; 77: 611-7. 4. Sahlin S, Ahlberg J, Angelin B, Reihnrr E, Einarsson K. Nucleation time of gallbladder bile in gallstone patients: influence of bile acid treatment. Gut 1991; 32: 1554-7. 5. Holzbach RT. Recent progress in understanding cholesterol crystal nucleation as a precursor to human gallstone formation. Hepatology 1986; 6: 1403-6. 6. Doty JE, Pitt HA, Kuchenbecker SL, Porter-Fink V, DenBesten LW. Role of gallbladder mucus in the pathogenesis of cholesterol gallstones. Am J Surg 1983; 145: 54--61. 7. Zac RA, Frenkiel PG, Marks JW, Bonorris GG, Allen A, Schoenfield LJ. Cyclic nucleotides and glycoproteins during formation of cholesterol gallstones in prairie dogs. Gastroenterology 1984; 87: 263-9. 8. LaMont JT, Smith BF, Moore JRL. Role of gallbladder mucin in pathophysiology of gallstones. Hepatology 1984; 4: $51-6. 9. LaMorte WW, Booker ML, Scott TE, Williams LF Jr. Increases in gallbladder prostaglandin synthesis before the formarion of cholesterol gallstones. Surgery 1985; 98:445-51. 10. Li YF, Moody FG, Weisbrodt NW, Zalewsky CA, Coelho JCU, Senninger ND. Gallbladder contractility and mucus secretion after cholesterol feeding in the prairie dog. Surgery 1986; 100: 90(0-4. 11. Bouchier IAD, Cooperband SR, E1 Kodsi BM. Mucous substances and viscosity of normal and pathological human bile. Gastroenterology 1965; 49: 343-53. 12. Lee SP, Lira TH, Scott AJ. Carbohydrate moieties of glycoproteins in human hepatic and gallbladder bile, gallbladder mucosa and gall stones. Clin Sci 1979; 56: 533-8. 13. Gallinger S, Harvey PRC, Petrunka CN, Ilson RG, Strasberg SM. Biliary proteins and the nucleation defect in cholesterol cholelithiasis. Gastroenterology 1987; 92: 867-75. 14. Sahlin S, Danielsson Angelin B, Reihnrr E, Henriksson R, Einarsson K. Mucin in gallbladder bile of gall stone patients: influence of treatment with chenodeoxycholic acid and ursodeoxycholic acid. Gut 1988; 29: 1506--10. 15. Mingrone G, Greco AV, Finotti E, Passi S. Free fatty acids: a stimulus for mucin hypersecretion in cholesterol gallstone biles. Biochim Biophys Acta 1988; 958: 52-9. 16. Swobodnik W, Wenk H, Janowitz P, Hagert N, Kratzer W, Berghold J, Zhang Y, Bittner R, Schusdiziarra V, Ott R, Kuhn K, Classen M. Total biliary protein, mucus glycoproteins, cyclic-AMP, and apolipoproteins in the gallbladder bile of patients with cholesterol gallstones and stone-free controls. Scand J Gastroenterol 1991; 26: 771-8.
17. Sahlin S, Ahlberg J, Einarsson K, Henriksson R, Danielsson A. Quantitative ultrastructural studies of gallbladder epithelium in gallstone-free subjects and patients with gallstones. Gut 1990; 31: 100-5. 18. Trotman BW, Ostrow JD, Soloway RD. Pigment v cholesterol cholelithiasis: comparison of stone and bile composition. Am J Dig Dis 1974; 19: 585-90. 19. Whiting MJ, Down RH, Watts JMcK. Biliary crystals and granules, the cholesterol saturation index, and the prediction of gallstone type. Surg Gastroenterol 1982; 1: 17-21. 20. Tera H. Stratification of human gallbladder bile in vivo. Acta Chit Scand 1960; Suppl 256: 4-65. 21. Roda A, Festi D, Sama C, Mazzeli G, Aldini R, Roda E, et al. Enzymatic determination of cholesterol in bile. Clin Chim Acta 1975; 64: 337--41. 22. Rouser G, Sidney F, Akira Y. Two dimensional thin-layer chromatography separation of polar lipids and determination of phospholipids by phosphorus analysis of spots. Lipids 1975; 5: 494---6. 23. Fausa O, Skhlhegg BA. Quantitative determination of bile acids and their conjugates using thin-layer chromatography and purified 3-~-hydroxysteroid dehydrogenase. Scand J Gastroent 1974; 9: 249-54. 24. Carey MC. Critical tables for calculating the cholesterol saturation of native bile. J Lipid Res 1978; 19: 945-55. 25. Sahlin S. Total protein content in human gallbladder bile: relation to cholesterol gallstone disease and effects of bile acid and aspirin treatment. Eur J Surg 1996; 162: 463-9. 26. Barrett AJ, Kirschke H. Cathepsin B, cathepsin H and cathepsin L. Meth Enzymol 1981; 80: 535-81. 27. Bowers WE, Finkelstaedt JT, de Duve C. Lysosomes in lymphoid tissue. 1. The measurement of hydrolytic activities in whole homogenates. J Cell Biol 1967; 32: 325-41. 28. Kouroumalis E, Hopwood D, Ross PE, Milne G, Bouchier IAD. Gallbladder epithelial acid hydolases in human cholecystis. J Pathol 1983; 139: 179-91. 29. Kouroumalis E, Hopwood D, Ross PE, Bouchier lAD. Human gallbladder epithelium; non-specific esterases in cholecystitis. J Pathol 1984; 142: 151-9. 30. Strasberg SM, Toth JL, Gallinger S, Harvey PRC. High protein and total lipid concentration are associated with reduced metastability o; bile in an early stage of cholesterol gallstone formation. Gastroenterology 1990; 98: 739-46. 31. Yu L, Guan-Zang L. Qualitative and quantitative comparison of gallbladder protein from patients with and without cholesterol gallstones. Dig Dis Sci 1990; 35: 47-9. 32. Jungst D, Lang T, von Ritter C, Paumgartner G. Role of high total protein in gallbladder bile in the formation of cholesterol gallstones. Gastroenterology 1991; 100:1724-9.
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Copyright © European Association for the Study of the Liver 1996 Journal of Hepatoiogy
ISSN 0168-8278
Lysosomal enzyme activities in gallbladder mucosa of gallstone-free subjects and patients with gallstones Staffan Sahlin l, Hans Glauman 2,/~ke Danielsson 3 and Kurt Einarsson 4 t Department of Surgery, Danderyd Hospital and Departments of 2pathology and Infectious Diseases, and 4Medicine, Huddinge University Hospital, Karolinska lnstitutet, Stockholm, and 3Department of Medicine, University of Umed, Sweden
Background~Aims: Gallstone patients have a reduced cellular lysosome content in the gallbladder mucosa cells compared with gallstone-free subjects. The purpose of the study was to further evaluate the possible role of lysosomes in the pathogenesis of cholesterol gallstone formation in humans. Methods: Lysosomai enzyme activities were assayed in gallbladder mucosa and for comparison in liver specimens of 19 gallstone-free subjects and 24 gallstone patients undergoing cholecystectomy. Results: Gallstone patients had 25-50% lower activities of the lysosomal proteases cathepsin B, D and L in their gallbladder mucosa compared with gallstone-free subjects. The activity of acid phosphatase also tended to be decreased in gallstone
patients. The liver lysosomal enzyme activities were not significantly different between the two groups. Conclusions: The results show that gallstone patients have diminished lysosomal enzyme activities in the gallbladder mucosa, a finding which may be related to decreased intracellular degradation of proteins and/or mucin in the mucosal cells. This may lead to a higher concentration of mucin in gallbladder bile and thus an increased risk of precipitation of cholesterol crystals and gallstone formarion.
CORDING to current concepts, supersaturation of bile is a prerequisite for cholesterol gallstone formation in man (1,2). Other factors are, however, also important. The nucleation process leading to formation of cholesterol crystals seems to be a crucial event in gallstone formation. The nucleation time is thus much shorter in gallbladder bile of gallstone patients compared with gallstone-free subjects (3,4). The nucleation time seems to be shortened by an excess of pronucleating factors and/or by a lack of antinucleating factors. An imbalance between these factors may precipitate crystals (2,5). Several studies in experimental animals have provided evidence for that mucin and glycoproteins may play a role as nucleating factors (6-10). According to several reports, gallbladder bile of gallstone patients has
higher concentrations of mucin and total protein than that of gallstone-free subjects (11-16). This could be caused by either increased production or decreased degradation by the mucosal cells. In a recent study we found that gallstone patients have a markedly reduced cellular lysosome content in the gallbladder mucosa cells compared with gallstone-free controls (17). A reduced lysosome content might be related to a decreased intracellular degradation of proteins and/or mucin leading to higher secretion rate and concentration in the gallbladder bile. It was therefore considered of importance to further evaluate the possible role of the lysosomes in the pathogenesis of cholesterol gallstone formation in humans. The aim of the present study was to assay the activity of some lysosomal enzymes in the gallbladder mucosa of gallstones patients and gallstone-free controls. For comparison, corresponding lysosomal enzyme activities in liver tissue were also determined in surgical biopsies obtained from the same patients.
Received 13 December 1995; revised 21 March; accepted 25 March 1996
Correspondence: Staffan Sahlin, Department of Surgery, Danderyd Hospital, S-182 88 Danderyd, Sweden. Tel.: +46-8-655 5000. Fax: +46-8-622 64 06.
Key words: Cholecystectomy; Cholesterol gallstone; Cholesterol saturation; Liver parenchyma; Mucin.
895
S. Sahlin et al.
from the muscle layer, put into ice-cold homogenizing buffer and rapidly transported to the laboratory.
Materials and Methods Patients The study was carried out in 24 consecutive patients with cholesterol gallstones and 19 gallstone-free subjects undergoing cholecystectomy. A preoperative cholecystography had shown a well functioning gallbladder in all cases. The gallstones were classified as cholesterol stones by laboratory analysis (18,19). Indications for cholecystectomy in the gallstone-free subjects were roentgenographic indication of polyp or adenomyoma of the gallbladder in combination with suspected biliary pain. None of the patients had clinical or laboratory evidence of diabetes mellitus, hyperlipoproteinemia or other diseases affecting the liver, thyroid or kidney function. All patients were of normal weight (Table 1). Informed consent was obtained from each patient before the operation, and the study was approved by the Committee of Ethics at the Karolinska Institute, Stockholm (February 1, 1988).
Experimental procedure The patients were admitted to the surgical ward the day prior to operation and given the regular hospital diet. All operations were performed between 8 and 10 a.m. after an overnight fast. After opening of the abdomen, the gallbladder was completely emptied of bile using a sterile needle and syringe to avoid possible stratification of the bile (20). A wedge biopsy weighing 2--4 g was taken peroperatively from the liver. A small portion of the tissue sample was sent for microscopic examination and the remainder was immediately put into ice-cold homogenizing buffer and rapidly transported to the laboratory. A regular cholecystectomy was then performed. A piece of the gallbladder was subjected to microscopic examination. The remainder was rinsed with ice-cold homogenizing buffer. The mucosa was carefully separated
Analysis of bile Gallbladder bile was examined with polarized light microscopy at 37 °C immediately after operation. Cholesterol monohydrate crystals were identified by birefringence and typical flat rhomboidal shapes. For determination of cholesterol and total phospholipids a portion of the bile samples obtained was immediately extracted with 20 vol of chloroformmethanol 2:1 (vol/vol). Cholesterol was determined by an enzymatic method (21) and phospholipids by the method of Roser et al. (22). The total bile acid concentration in one aliquot of the bile samples was determined using the 3o~-hydroxysteroid dehydrogenase assay (23). Cholesterol saturation was calculated as a percentage of the predicted cholesterol solubility at the respective biliary lipid concentration and composition as described by Carey (24). The nucleation time was determined by the method of Holan et al. (3) with minor modifications (4). The gallbladder content of protein was determined by a Bio-Rad protein assay as described recently (25).
Preparation of a lysosomal enriched fraction from liver and gallbladder mucosa Aliquots of the liver and gallbladder mucosa were minced and homogenized with a loose fitting Teflon pestle in 9 vol of a 50 mM Tris-HC1 buffer pH 7.4, containing 0.3 M sucrose, 10 mM DTT, 10 mM EDTA and 50 mM NaC1. The homogenate was centrifuged at 20000 g for 15 min. The pellet (lysosomal enriched fraction) obtained, containing the lysosomes, was frozen at -20°C until analysis.
Assay of lysosomal enzymes TABLE 1 Clinical data of gallstone patients and gallstone-free subjects undergoing cholecystectomy (mea~+SE) Groups of patients
Cholesterol gallstone
Gallstone-free
n Sex (M/F) Age (years) Body weight (kg) Relative body weight (%)a Plasma cholesterol (mmol/1) Plasma triglycerides (mmol/1)
24 6/18 49 (3) 73 (3) 104 (3) 5.4 (0.2) 1.3 (0.1)
19 5/14 47 (3) 67 (3) 96 (3) 5.8 (0.2) 1.0 (0.1)
a body weight (kg) • Relativebodyweightcalculateda s ~ t ( - ~ -- ]-~ x 100
896
Cathepsin B and L activities were measured as described by Barrett & Kirschke (26) with Z-Arg-Arg-NMec as substrate. Acid phosphatase and cathepsin D activities were assayed as described by Bowers et al. (27). For biochemical assessment of enrichment and purity of the lysosomal enriched fractions from the gallbladder mucosa of gallstone-free and gallstone patients, lysosomal enzymes were measured in the total homogenate and two marker enzymes were analyzed, NADPH-cytochrome c reductase (microsomes) and succinate c reductase (mitochondria). Based on four patients from whom material was available, the enrichment factor (lysosomal enriched
Lysosomal enzymes in gallbladder fraction over homogenate) was 11.5 (gallstone-free) and 13.2 (gallstone) for cathepsin B, and 14.5 (gallstone-free) and 12.3 (gallstone) for cathepsin D. The levels of contamination of microsomes to the lysosomal fractions were 10.7% for gallstone-free and 12.3% for gallstone patients. Corresponding levels for mitochondria were 15.7% (gallstone-free) and 17.1% (gallstone). Thus, there was no significant difference in the quality and purity of the preparations between gallstone patients and controls.
Statistical analysis Data are given as means (standard error of the mean, SEM). Significance of differences was evaluated with Student's t-test or the Mann-Whitney U-test.
TABLE3 Lysosomal enzyme activities in liver tissue and gallbladder specimens from gallstonepatients and gallstone-freesubjects undergoing cholecystectomy. Enzyme activities are given per mg of protein (SEM) and are expressed as ~tmolNMec released per min (cathepsin B and L), nmol tyrosinereleasedper rain (cathepsinD), and I.tmolPi released per min (acid phosphatase).Means+SE Groups of patients Cathepsin B
Cholesterol Gallstone-free gallstone Livertissue 168 (15) 213 (26) Mucosa 31 (4)* 49 (6) Cathepsin D Livertissue 30 (2) 31 (3) Mucosa 23 (2)* 31 (3) Cathepsin L Livertissue 782 (27) 848 (91) Mucosa 84 (16)* 172 (34) Acid Livertissue 1,89 (0,13) 2.09 (0.14) phosphatase Mucosa 1.19 (0.11) 1.29 (0.09) *Significantlydifferentfrom gallstone-free,p<0.05.
Results Light microscopy All gallbladders were routinely examined by a pathologist not involved in the study. Those with gallstones showed evidence of light to moderate chronic inflammation. In the gallstone-free cases the suspected polyps were foldings of epithelium and the adenomyomas were confirmed.
Composition of bile The gallbladder bile contained less total lipid in gallstone patients than in gallstone-free patients (Table 2). Gallbladder bile was significantly more saturated with cholesterol in the gallstone patients compared with the gallstone-free subjects. Seventy-nine percent of the gallstone patients displayed cholesterol crystals in their gallbladder bile compared with none of the gallstone-free subjects. The gallstone patients had
TABLE2 Lipid composition, cholesterol saturation, occurrence of cholesterol monohydrate crystals, nucleation time and protein concentrationin gallbladder bile of gallstone patients and gallstone-free subjects undergoingcholecystectomy(means+SE) Groups of Cholesterol Gallstonepatients gallstone free Cholesterol (mmol/l) 12.1 (1.1) 14.6 (1.7) Cholesterol (tool%) 7.2 (0.5) 5.9 (0.5) Bile acids (mol%) 69.6 (1.4) 70.5 (1.0) Phospholipids (mol%) 23.1 (1.0) 23.6 (1.0) Lipid concentration(g/dl) 8.4 (0.7)* 13.3 (1.0) Cholesterol saturation(%) 103 (6)* 78 (7) Crystals, no of patients (+/-) 19/5 0/19 Nucleationtime (days) 2 (1-11) 20 (1-35) Proteinconcentration(mg/ml) 8.3 (1.8) 6.8 (0.9) * Significantlydifferentfrom gallstone-free,p<0.01, *p<0.001 # Significantlydifferentfrom gallstone-free,p<0.0001. Mann Whitney U-test
a significantly shorter gallbladder bile nucleation time than the gallstone-free controls. Only one of the gallstone-free subjects had a short nucleation time (1 day). The protein concentration tended to be higher in the gallstone patients compared with the gallstonefree subjects (8.3 vs. 6.8 mg/ml) but the difference did not reach statistical significance. The protein concentration averaged 9.0 (2.2) mg/ml in the gallstone patients with cholesterol crystals and 6.2 (3.6) mg/ml in those without crystals.
Lysosomal enzyme activities The purity of the lysosomal enriched fractions was similar for gallstone patients and gallstone-free subjects (see Methods section). The activities of cathepsin B, D and L were significantly reduced by 25-50% in the gallbladder mucosa of gallstone patients compared with gallstone-free controls (Table 3). The activity of acid phosphatase was not significantly different between the two groups of patients. There was no difference in hepatic lysosome enzyme activity between gallstone patients and gallstone-free controls.
Discussion A major finding of the present study was that the activities of the lysosomal enzymes cathepsin B, D and L were 25-50% lower in the lysosomal enriched fraction of gallbladder mucosa from gallstone patients compared with gallstone-free subjects. The activity of acid phosphatase also tended to be lower in the gallstone patients. Corresponding enzyme activities in the liver specimens of the gallstone patients and the gallstone-free controls were not significantly different. The results are in good agreement
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with those obtained in a previous ultrastructural study from our group (17). Patients with gallstones were found to have a markedly reduced cellular lysosomal volume density (around 70%) in the gallbladder epithelial cells compared with gallstone-free subjects. Kouroumalis et al. (28,29) have previously reported significantly decreased levels of lysosomal enzyme activities - beta-glucuronidase, acid phosphatase and neutral esterases - in the gallbladder wall of gallstone patients, especially those with chronic cholecystitis. They speculated that the reduced levels might play a significant role in the development of cholecystitis. However, our gallstone patients showed evidence of only slight chronic inflammation and no marked cholecystitis. The mechanism of the decreased levels of lysosomal enzyme activities in the gallbladder mucosa of the gallstone patients is not apparent. The difference between gallstone patients and gallstone-free controls could hardly be due to methodological problems, since the quality and purity of the lysosome enriched fractions from the gallbladder mucosa of gallstonefree and gallstone patients were similar. Furthermore, as mentioned above, a previous ultrastructural study has shown markedly reduced cellular lysosomal volume density in the gallbladder epithelial cells of gallstone patients compared with gallstone-free subjects. Theoretically, genetic differences between gallstone patients and normal subjects might explain the decreased lysosomal enzyme activity in cholesterol gallstone patients, even though there are no studies available to suggest that individuals with low lysosomal enzyme activities in the gallbladder are prone to develop gallstones. Alternatively, the decreased lysosomal enzyme activities in gallstone patients might be a mucosal response to a cytotoxic effect of lithogenic bile, cholesterol crystals and/or gallstones. What, then, could be the clinical significance of reduced levels of lysosomal enzymes in the gallbladder mucosa? Some research groups have recently reported that gallstone patients have higher concentrations of total protein in the gallbladder bile compared with gallstone-free subjects (13,16,30-32). We also found a tendency to higher concentrations of protein in the gallstone patients compared with the gallstone-free subjects, although the difference did not reach statistical significance. Higher concentrations of total protein were found in gallbladder bile samples with cholesterol crystals than in samples without crystals. The protein concentration correlated with the nucleation time in gallstone patients. The hepatic bile protein concentrations, however, were similar in gallstone patients and gallstone-free sub898
jects, indicating that the origin of the increased protein concentration of the gallbladder bile in the gallstone patients would be the gallbladder p e r se. Mucosal inflammation of the gallbladder is common in cholesterol gallstone disease. In the present study, the gallbladders of the gallstone patients showed evidence of light to moderate inflammation. Inflammatory changes of the gallbladder may cause leakage and an increased flux of plasma proteins into the bile. Another explanation of the increased protein concentration of the gallbladder bile of the gallstone patients could be a decreased capacity to degrade protein in the gallbladder. The findings of reduced lysosomal content and concomitant reduced enzyme activity may reflect decreased ability for protein degradation in the gallbladder wall. An alternative explanation could be that lysosomes of the gallbladder mucosa are normally involved in intraceUular degradation of mucin granules. A reduced content of these organelles and their enzyme activities would thus reflect a defect in the elimination of mucin granules, which then are released into the gallbladder lumen, and may act as a nidus for cholesterol crystal formation. Kouroumalis et al. (28) presented a third explanation that lysosomal enzymes are released together with mucin granules into the gallbladder lumen, where they hydrolyze conjugated bilirubin into unconjugated bilirubin. The unconjugated bilirubin can easily precipitate as calcium bilirnbinate, forming a nidus for gallstones. In conclusion, the present study has shown that gallstone patients have diminished lysosomal enzyme activities in the gallbladder mucosa. This finding may be related to decreased intracellular degradation of proteins and/or mucin in the mucosal cells, which may lead to increased concentration of mucin in gallbladder bile and thus an increased risk of precipitation of cholesterol crystals and gallstone formation.
Acknowledgements The skillful technical assistance of Ms Ingela Arvidsson, Ms Lisbet Benthin, Ms Danuta Cosliani and Ms Helena Jansson is gratefully acknowledged. This study was supported by grants from the Swedish Medical Research Council (03X-4793). References 1. Hofmann AF. Pathogenesis of cholesterol gallstones. J Clin Gastroenterol 1988; 10 (suppl 2): SI-11. 2. Lamont JT, Carey MC. Cholesterol gallstone formation. 2. Pathobiology and pathomechanics. Progr Liver Dis 1992; 10: 165-91.
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