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Dietary fish oil inhibits cholesterol monohydrate crystal nucleation and gallstone formation in the prairie dog
Dietary fish oil inhibits cholesterol monohydrate crystal nucleation and gallstone formation in the prairie dog Thomas H. Magnuson, MD, Keith D. LiIlemoe, MD, Rhett C. High, MD, and Henry A. Pitt, MD, Baltimore, Md.
Background. Epidemiologic studies suggest that populations consuming a diet rich in fish oil have lower rates of both atherosclerotic heart disease and gallstones. The mechanisms underlying this inhibitory effect on cholesterol gallstone formation remain unclear. We therefore studied the effect of dietary fish oil on bile composition and cholesterol precipitation in an animal model of gallstone disease. Methods. Adult male prairie dogs were fed a standard control diet (n = 12) or a lithogenic 1.2% cholesterol diet (n = 16). One half of the animals in each group had their diet supplemented with concentrated fish oil. Results. After 14 days animals receiving the cholesterol diet all developed biliary cholesterol monohydrate crystals and gallstones. When fish oil was added to this high cholesterol diet, solid cholesterol crystal precipitation and gallstone formation were completely inhibited. This inhibition of gallstone formation was accompanied by a significant decrease in biliary calcium and total protein concentration. Microscopic cholesterol liquid crystals were evident in the bile of all of the animals fed the cholesterol plus fish oil diet. Dietary fish oil also significantly prolonged cholesterol monohydrate crystal observation time in animals receiving the lithogenic diet. Conclusions. These data suggest that dietary fish oil exerts a potent antilithogenic effect on cholesterol gallstone disease and may induce a stable liquid crystalline phase retarding nucleation. (SuRGF~Y 1995;118:517-23.) From the Departments of Surgery, The Johns Hopkins University School of Medicine, and Johns Hopkins Bayview Medical Center, Baltimore, Md.
EPIDEMIOLOGIC STUDIES SUGGESTTHAT populations consuming a diet rich in fish oil, such as the North American a n d G r e e n l a n d Eskimo, exhibit lower rates o f atherosclerofic h e a r t disease. 1, 2 This beneficial effect o f fish oil has b e e n attributed, in large part, to the high c o n t e n t o f 0~-3 fatty acids present in fish oil. Interestingly, these same populations also a p p e a r to enjoy a relatively low incidence of cholesterol gallstone disease. 3 However, this a p p a r e n t relationship between fish oil c o n s u m p t i o n a n d d i m i n i s h e d cholesterol gallstone disease remains relatively u n e x p l o r e d . Fish oil a n d ~-3 fatty acid administration have b e e n shown to impact various metabolic processes such as prostaglandin biosynthesis, lipid metabolism, a n d hepatic lipoprotein metabolism, which are i m p o r t a n t in the pathogenesis o f atherosclerofic disease. 4-7 Many of these same factors are also Accepted for publication Jan. 10, 1995. Reprint requests: Thomas H. Magnuson, MD, Department of Surgery, Johns Hopkins BayviewMedicalCenter, 4940 Eastern Ave.,Baltimore, MD 21224. Copyright 9 1995 by Mosby-YearBook, Inc. 0039-6060/95/$3.00 + 0 11/56/64381
t h o u g h t to play i m p o r t a n t roles in the d e v e l o p m e n t o f cholesterol gallstones, a n d their alteration may account for fish oil's a p p a r e n t antilithogenic effects. T h e p r e s e n t study was designed to explore the effect o f dietary fish oil o n gallstone pathogenesis a n d bile composition a n d to test the hypothesis that dietary fish oil would prevent the nucleation a n d precipitation o f cholesterol crystals from bile a n d inhibit cholesterol gallstone formation.
MATERIAL A N D M E T H O D S E x p e r i m e n t a l design. Adult male prairie dogs (cy-
nomys ludovicianus) caught in the wild were used in the study. All animals were h o u s e d in a t h e r m o r e g u l a t e d r o o m with a constant l i g h t / d a r k cycle a n d allowed 3 weeks of acclimation to the laboratory e n v i r o n m e n t before study. T h e animals were divided into f o u r experimental groups: (1) control diet, n = 6; (2) control diet s u p p l e m e n t e d with fish oil (FO), n = 6; (3) high cholesterol lithogenic diet (CHOL), n = 8; a n d (4) high cholesterol lithogenic diet s u p p l e m e n t e d with fish oil ( C H O L + FO), n = 8. T h e control diet consisted of a standard trace (0.03%) cholesterol nonlithogenic diet SURGERY
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Table I. Incidence of biliary crystals and cholesterol gallstones Control Fish oil CHOL
Gallstones Cholesterol monohydrate crystals Cholesterol liquid crystals
CHOL + FO
0/6 0/6
0/6 0/6
8/8* 8/8*
0/8 0/8
0/6
6/6J"
0/8
8/8 t
*p< 0.01 versusother dietarygroups. "~p< 0.01 versuscontrol and CHOL. (Purina rat chow; Ralston Purina Co., St. Louis, Mo.). The high cholesterol diet was similar to the control diet but contained 1.2% cholesterol by weight (Teklad test diets; Madison, Wis.). This diet has been used extensively by this laboratorys and others ~ 10 to induce cholesterol gallstones in prairie dogs during a 2- to 3-week period of time. Fish oil was administered by oral gavage to the FO and CHOL + FO groups as a concentrated fish oil preparation (Promega; Parke-Davis, Morris Plains, N.J.) supplying 200 m g / k g / d a y polyunsaturated ~ 3 fatty acids (eicosapentaenoic and docosahexaenoic acid). The animals were maintained on their respective diets for 14 days. At the end of the 14-day feeding period and after an overnight fast, each animal was anesthetized with intramuscular ketamine (100 mg/kg), and a laparotomy was performed. The intact gallbladder was excised, and gallbladder bile was completely aspirated anaerobically, protected from light, and stored on ice. At the termination of the experiment, blood was obtained by cardiac puncture and the animal was killed. Gallstone and crystal analysis. All gallbladders were examined grossly for evidence of cholesterol gallstones. Gallbladder bile and gallbladder mucosa were then examined under a polarizing light microscope for any evidence of cholesterol crystals. Cholesterol monohydrate crystals were identified by their typica! birefringent, notched rhomboidal shape, it Cholesterol liquid crystals were identified as previously described by their birefringent "Maltese cross" configuration) 2 Liquid ,crystals are known to represent fused cholesterol-phospholipid vesicles important in the solubilization of cholesterol in bile. 13 Bile analysis. Fresh anaerobic gallbladder bile was immediately analyzed for pH and ionized calcium. An aliquot was mixed with ascorbic acid (1:1, vohvol) for later bilirubin analysis. The remaining bile was then centrifuged for 5 minutes at 2000 rpm, and the supernatant was stored at -20 ~ for subsequent analysis. After rewarming to room temperature, bile was aria-
lyzed for total protein, 14 cholesterol, 15 total bile acid, 16 and phospholipid concentration. ]7 The cholesterol saturation index (CSI) of bile was calculated as described by Kuroki et al.lS Bile was also analyzed for unconjugated bilirubin according to the technique of Spivak and Carey 19 by use of high-pressure liquid chromatography. Prairie dog serum was analyzed for total cholesterol concentration. Crystal observation lime. To determine the effect of fish oil on cholesterol crystal observation time, a second study was designed. In this study five prairie dogs were fed the high (1.2%) cholesterol diet, and five animals received the same diet but supplemented with fish oil (900 m g / k g / d a y to-3 fatty acids). The animals received their respective diets for 9 days, at which point shortterm terminal experiments were performed. Gallbladder bile was aspirated into prewarmed syringes and individually filtered through 0.22 micron filters to achieve isotropic, crystal free bile as previously described for human bile nucleation studies. 2~ The individual bile samples were then incubated at 37 ~ C and examined at 12-hour intervals under a polarizing light microscope for evidence of cholesterol crystal nucleation. Crystal observation time was defined as the point when cholesterol monohydrate crystal(s) precipitated from solution and could clearly be identified microscopically. Statistics. Data are presented as the mean +_ the standard error of the mean. Statistical analysis was performed with analysis of variance or Fisher's exact test where appropriate. Nucleation time data were compared by using the Mann-Whitney U test. RESULTS
All animals gained weight during the experimental period and appeared healthy. Food intake was monitored daily, and all four groups of animals consumed similar amounts of food and water. Gallstone and biliary crystal analysis. The incidence of gallstones and biliary crystals in the experimental groups is shown in Table I. As expected, none of the animals in the control group or the fish oil alone group developed gallstones. In contrast, all of the animals fed the high cholesterol diet developed grossly evident cholesterol gallstones. When animals were fed the same lithogenic diet but supplemented with fish oil (CHOL +FO), the incidence of gallstones dropped dramatically to zero (p < 0.01). In parallel with the incidence of gallstones in the various groups, cholesterol monohydrate crystals were present on the microscopic examination of gallbladder bile in only the cholesterol-fed group. Examination of gallbladder bile in the cholesterol plus fish oil group failed to reveal any evidence of cholesterol monohydrate crystal precipitation. Interestingly, this same group
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Table II. Gallbladder bile composition Control
pH Ionized calcium (mmol/L) Unconjugated bilirubin (pmol/L) Cholesterol (mmol/L) Phospholipids (mmol/L) Total bile acids (mmol/L) CSI
6.93 _+ 0.08 1.7 _+ 0.1 7.9 _+ 1.1 4.2 -+ 0.4 16.3 _+ 1.6 154 _+9 0.60 _+ 0.03
Fish oil
6.95 2.0 8.9 3.7 16.2 160 0.52
-+ 0.08 _+ 0.2 -+ 1.0 _+ 0.3 -+ 1.4 +_ 9 -+ 0.03
CHOL
6.57 3.3 19.7 14.8 39.4 209 0.94
-+ 0.16" + 0.2t _+ 4.0* -+ 1.2" _+ 3.9* + 13" -4-0.07*
CHOL + FO
6.86 2.5 16.8 13.2 32.6 191 1.03
+ 0.07 -+ 0.2 -+ 2.6* -+ 0.4* -+ 2.6* _+ 8* -+ 0.06*
*p < 0.005 versus c o n t r o l a n d fish oil. t P < 0.005 versus all o t h e r groups.
( C H O L + FO) exhibited an a b u n d a n c e o f cholesterol liquid crystals on microscopic examination. These birefringent, Maltese cross liquid crystals were also n o t e d in gallbladder bile of animals fed the fish oil diet without high dietary cholesterol. N o n e o f the control o r the cholesterol-fed animals h a d evidence o f cholesterol liquid crystal formation. Gallbladder bile analysis. Analysis of gallbladder bile for pH, ionized calcium, u n c o n j u g a t e d bilirubin, a n d biliary lipids is p r e s e n t e d in Table II. As previously described, 8 biliary p H was significantly decreased a n d ionized calcium was increased in prairie dogs fed the lithogenic cholesterol diet. T h e addition o f fish oil to the control diet or to the high cholesterol diet resulted in no significant changes in biliary pH. Similarly, unconjugated bilirubin levels were increased in the cholesterolfed group a n d unaffected when fish oil was added. Dietary fish oil did, however, significantly prevent the increase in gallbladder bile ionized calcium i n d u c e d by the high cholesterol diet. Gallbladder biliary lipid concentrations a n d CSI for the four dietary groups are also shown in T a b l e II. Biliary cholesterol, phospholipid, a n d total bile acid concentrations were all increased with the cholesterol diet. The addition offish oil to this diet h a d no significant effect on the concentrations o f these biliary lipids. As expected, the CSI of gallbladder bile in the control g r o u p was well below 1.0, the limit o f saturation. T h e gallbladd e r CSI o f the fish oil g r o u p (0.52 - 0.03) showed little change c o m p a r e d with the control g r o u p (0.60 _+ 0.03). T h e addition o f 1.2% cholesterol to the control diet increased the gallbladder CSI to the p o i n t o f cholesterol saturation, consistent with the macroscopic evidence o f cholesterol precipitation a n d gallstones in this group. T h e CSI o f the cholesterol plus fish oil g r o u p (1.03 +- 0.06) was no different from that of the cholesterol-fed g r o u p (0.94 _+ 0.07), despite the fact that no cholesterol precipitation was observed in the animals fed cholesterol plus fish oil. Gallbladder bile total protein levels are r e p r e s e n t e d in Fig. 1. No difference was n o t e d between the control
(0.79 -+ 0.07 m g / m l ) a n d fish oil g r o u p (0.80 -- 0.04 m g / m l ) . T h e addition o f dietary cholesterol resulted in a greater than fourfold increase in biliary p r o t e i n concentration (3.41 + 0.24 m g / m l ) when c o m p a r e d with controls (p < 0.01). This increase in biliary p r o t e i n was diminished in the C H O L + FO animals, resulting in levels significantly less (2.1 -+ 0.21 m g / m l ) than animals fed the same high cholesterol diet but without fish oil (p< 0.01). Serum cholesterol. Serum cholesterol concentrations were m e a s u r e d in the four dietary groups. The high cholesterol diet resulted in a significant increase in serum cholesterol (508 +- 50 m g / d l ) when c o m p a r e d with controls (116 +- 6 m g / d l ; p < 0 . 0 1 ) . A similar increase in cholesterol concentration was n o t e d in the C H O L + FO g r o u p (459 +- 83 m g / d l ) . This level was significantly increased c o m p a r e d with both controls (p< 0.05) a n d the F O group (118 +- 5 m g / d l ; p < 0.05) a n d was n o t statistically different from the C H O L group. Crystal observation time. Gallbladder bile cholesterol crystal observation times were d e t e r m i n e d in five animals receiving the high cholesterol diet a n d five animals receiving the same cholesterol diet b u t supplem e n t e d with fish oil. Cholesterol crystal nucleation has b e e n previously shown to be a critical step in cholesterol precipitation from saturated bile. 21 A m o r e rapid time to crystal f o r m a t i o n correlates with decreased stability with respect to biliary cholesterol solubility a n d enh a n c e d tendency toward precipitation. As p r e s e n t e d in Fig. 2, the cholesterol-fed g r o u p exhibited a r a p i d biliary crystal observation time of 1.4 +- 0.4 days. T h e addition o f fish oil to this diet significantly p r o l o n g e d crystal observation time nearly fourfold to 5.4 +- 1.6 days (p < 0.05).
DISCUSSION T h e p r e s e n t study suggests that dietary fish oil may exert a p o t e n t antilithogenic effect d u r i n g cholesterol gallstone formation. In the cholesterol-fed prairie dog, a widely accepted animal m o d e l o f cholesterol gallstone disease, fish oil s u p p l e m e n t a t i o n resulted in a signifi-
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4 7 6
3
~5
p<0.0s
,
3 O 1
,O 1 0
-
-
CONTROL
FISHOIL
CHOL
CHOL+FO
Fig. 1. Gallbladder bile total protein concentration in animals fed standard diet (CONTROL), fish oil supplemented diet (FISH OIL), high cholesterol diet (CHOL), and high cholesterol diet with fish oil (CHOL +FO).
cant inhibition of both cholesterol monohydrate crystal precipitation and gallstone formation and nearly a fourfold prolongation of in vitro cholesterol crystal observation time. These changes were accompanied by decreased gallbladder bile concentrations of both ionized calcium and protein. Dietary fish oil had no effect on the concentration of biliary lipids or the CSI of gallbladder bile. Fish oil did, however, induce the formation of apparently stable phospholipid-cholesterol vesicles as evidenced by the microscopic appearance of liquid crystals in gallbladder bile. Epidemiologic studies have suggested that populations consuming diets rich in fish oil and ~-3 fatty acids may be relatively protected from cholesterol gallstone disease. 3 In a preliminary report we first noted that fish oil had potential antilithogenic effects in an animal model of gallstone disease. 22 Several other investigators have since addressed this relationship, and their results support the theory that fish oil may impact cholesterol gallstone pathogenesis. In the African green monkey, Scobey et al.23 reported a 45% decrease in gallstone formation in animals whose diets were supplemented with fish oil. Similarly, Booker et al.lO noted a significant decrease in the incidence of biliary cholesterol crystals in prairie dogs fed a high cholesterol lithogenic diet supplemented with fish oil. More recent studies in the hamster ~4 and mouse 25 also suggest that cholesterol gallstone formation is inhibited when dietary fish oil is added to a normally lithogenic experimental diet. A number of human studies have shown conflicting results. Ben" et al.26 studied patients with known gallstones who were given concentrated dietary fish oil supplements. They noted no changes in the incidence of cholesterol monohydrate crystal or in cholesterol nucleation time. A similar study byJanowitz et al.27 found that feeding of fish oil accelerated cholesterol nucleation time, theoretically favoring cholesterol precipita-
CHOLESTEROL
CHOLESTEROL + FISH OIL
Fig. 9. Gallbladder bile crystal observation time in animals fed high cholesterol diet without and with fish oil. don. Cautious interpretation of these clinical studies is indicated, however, because many of the patients had preexisting stones that may have served as a continuing nidus for nucleation-precipitation despite dietary-induced biliary changes. Furthermore, bile samples were also obtained from an endoscopic, transduodenal approach and therefore may be invalid reflections of the tree gallbladder environment. 28 We have recently studied patients with cholesterol gallstones treated before operation for 2 weeks with fish oil and compared their cholesterol crystal nucleation times with patients with gallstones who were not receiving fish oil and a nongallstone control group. 29 At cholecystectomy, bile was completely aspirated from the patient's gallbladder, and cholesterol nucleation time was determined. Nucleation times were significantly prolonged in patients with gallstones receiving fish oil when compared with patients with untreated gallstones and were no different from crystal observation times in the control nongallstone group. A variety of mechanisms exist that may explain fish oil's inhibitory effect on gallstone development. Altered hepatic cholesterol and lipoprotein metabolism have previously been noted with fish oil consumption and have been thought to be responsible, in part, for fish oil's apparent protective role in atherosclerotic cardiovascular disease. 4, 6 Such changes in cholesterol metabolism could impact biliary cholesterol and, thus, gallstone formation, tn the present study, however, we noted no changes in concentrations of serum cholesterol or gallbladder bile cholesterol or biliary CSI in the animals fed fish oil that might explain the observed decrease in cholesterol precipitation. This finding concurs with previous studies showing no significant changes in biliary cholesterol saturation with feeding of fish 0i1.23, 24, 29 Others, however, have noted that cholesterol saturation is decreased with fish oil, primarily because of a relative increase in phospholipid secretion into bile.aO, ~6,27 Conversely, in a rat model biliary cholesterol
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concentrations were noted to increase as serum cholesterol concentrations decreased when fish oil was added to the diet. 3~ Cholesterol precipitation in bile is governed by a wide variety of factors i n addition to the relative concentrations of cholesterol, phospholipids, and bile acids, the traditional determinants of CSI. Such factors may influence cholesterol precipitation in bile by enhancing the relative solubility of cholesterol in solution or by preventing a supersaturated solution from reaching thermodynamic equilibrium. More than 20 years ago, when cholesterol was thought to be exclusively solubilized by mixed micelles, Olszewski and Holzbach 12 first noted the presence of cholesterol liquid crystals in human gallbladder bile and postulated that they served to "inhibit the precipitation of excess cholesterol to solid crystalline form by delaying its nucleation." We now know that cholesterol is transported and solubilized not only by mixed micelles but also by cholesterol-phospholipid vesicles. 13 These vesicles in mulfilamellar form represent liquid crystals, and cholesterol nucleation and precipitation into solid crystals proceed from this liquid crystalline phase. 31 In the present experiment fish oil supplementation blocked this phase transition and induced relatively stable cholesterol-phospholipid vesicles as manifested by the microscopic appearance of liquid crystals and prolonged crystal observation time. Although not directly determined in this study, dietary fish oil has been shown in human beings z6 and animals 1~ 23 to alter the composition of phospholipids in bile and result in species highly enriched at the sn-2 position with o)-3 fatty acids. Such a change in phospholipid composition might theoretically alter vesicular stability and the cholesterol carrying capacity of bile and potentially account for the observed inhibition of cholesterol precipitation. Indeed, Cohen and Carey3z and Booker et al. 33 have demonstrated that altering phospholipid composition by changing fatty acyl length and unsaturation can significantly impact cholesterol solubility in bile and the partitioning of cholesterol between vesicles and micelles. In a preliminary report we have recently noted that fish oil administered to patients with gallstones results in the preferential incorporation of n-3 fatty acids into micellar as opposed to vesicular phospholipids. 34 Interestingly, ursodeoxycholic acid, when given orally for gallstone prevention or dissolution, has also been noted to prolong cholesterol crystal observation time and induce the formation of stable liquid crystals similar to those seen in this experiment. 35, 36 In the present study cholesterol gallstone formation was accompanied by a significant increase in gallbladder bile protein concentration. Many investigators have focused on biliary proteins and their role as potential factors influencing cholesterol crystal nucleation, z~ 37 Lee
Magnuson et al.
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et al.9 demonstrated that aspirin, an inhibitor of prostaglandin biosynthesis, was able to block gallbladder protein hypersecrefion and thereby prevent gallstones in cholesterol-fed prairie dogs. Recent studies suggest that dietary fish oil may also inhibit gallbladder mucosal prostaglandin synthesis by substituting ~0-3fatty acids for arachidonic acid, the phospholipid fatty acyl species that is the usual substrate for prostaglandin production. 1~ An alteration in gallbladder prostaglandin synthesis and the resultant inhibition of glycoprotein production might explain the drop in protein concentration we observed in the cholesterol plus fish oil dietary group. Failure to secrete a potential pronucleating protein may have prevented cholesterol precipitation. However, we have been unable to demonstrate any qualitative changes in biliary protein composition in prairie dogs fed the fish oil diet. 38 Alternatively, increased protein production by the gallbladder epithelium may have been a secondary phenomenon such as an inflammatory response to the presence of cholesterol crystals and stones. In the group fed fish oil the diminished rise in protein could then be explained by the failure of gallstones to form and, therefore, the elimination of the inflammatory stimulus. Clearly, further studies are needed to determine the exact sequence of events with regard to fish oil's effect on prostaglandin metabolism, gallbladder protein production, and cholesterol precipitation. Biliary pH, 39' 40 calcium,41 and bilirubin 42 have all been implicated as modulators of cholesterol precipitation a n d gallstone formation, but fish oil's effect on these factors is unknown. In the present study gallstone formation in the cholesterol-fed group was associated with a significant decrease in biliary pH and increase in unconjugated bilirubin levels. Fish oil, however, had no effect on these changes despite inhibiting gallstone development. Consistent with previous studies, 43 ionized calcium level was also elevated in the cholesterol-fed gallstone group. Fish oil supplementation inhibited this increase. Calcium has been noted to he an important structural component in the core of cholesterol gallstones, 44 perhaps serving as a nidus for cholesterol crystal precipitation or growth. Calcium has also been reported to accelerate biliary cholesterol crystal formation by inducing the aggregation of unstable cholesterol carrying vesicles.45' 46 The mechanism whereby fish oil decreased biliary calcium level in this study is unclear. Enhanced bile concentration caused by increased gallbladder water absorption or altered biliary motility has previously been noted to elevate calcium levels. 4~ 47 It is unlikely, however, that fish oil affected gallbladder absorption or motility because the concentration of bile acids, a relatively unabsorbed constituent of bile and therefore an index of bile concentration, was similar in
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the cholesterol-fed group and the group fed cholesterol p l u s f i s h oil. I n s u m m a r y , t h e p r e s e n t s t u d y s h o w s t h a t d i e t a r y fish oil p r e v e n t s c h o l e s t e r o l crystal p r e c i p i t a t i o n a n d gallstone formation in the cholesterol-fed prairie dog. This antilithogenic effect may be due to a relative enhancem e n t i n t h e stability o f b i l i a r y p h o s p h o l i p i d - c h o l e s t e r o l vesicles as m a n i f e s t e d b y t h e o b s e r v e d p r e s e n c e o f c h o l e s t e r o l l i q u i d crystals a n d p r o l o n g e d c h o l e s t e r o l crystal o b s e r v a t i o n t i m e s . T h e a p p l i c a b i l i t y o f this m o d e l
to
h u m a n g a l l s t o n e d i s e a s e r e m a i n s to b e d e t e r m i n e d , b u t fish oil m a y p r o v e b e n e f i c i a l i n p o p u l a t i o n s a t h i g h risk f o r g a l l s t o n e f o r m a t i o n o r r e c u r r e n c e s u c h as t h o s e after extracorporeal
shock wave lithotripsy or during
rapid weight reduction.
REFERENCES 1. Bang HO, DyerbergJ, Sinclair HM. The composition of the Eskimo food in northwestern Greenland. Am J Clin Nutr 1980; 33:2657451. 2. Kromhout D, Bosschieter EB, de Lezenne Covlander C. The inverse relationship between fish consumption and 20-year mortality from coronary heart disease. N EnglJ Med 1985;312:1205-9. 3. Schaefer O. When the Eskimo comes to town. Nutr Today 1971;Nov/Dec:8-16. 4. Leaf A, Weber PC. Cardiovascular effects of n-3 fatty acids. N Engl J Med 1988;318:549-57. 5. Field FJ, Albright EJ, Mathur SN. Effect of dietary n-3 fatty acids on HMG-CoA reductase and ACAT activities in liver and intestine of the rabbit. J Lipid Res 1987;28:50-8. 6. Phillipson BE, Rothbrock DW, Connor WE, Harris ES, Illingworth DR. Reduction of plasma lipids, lipoproteins and apoproteins by dietary fish oils in patients with hypertriglyceridemia. N EnglJ Med 1985;312:121045. 7. Smit MJ, Temmerman AM, Wolters H, Kuipers F, Beynen AC, Vonk RJ. Dietary fish oil-induced changes in intrahepatic cholesterol transport and bile acid synthesis in rats. J Clin Invest 1991;88:943-51. 8. Lillemoe KD, Magnnson TH, High RC, Peoples GE, Pitt HA. Caffeine prevents cholesterol gallstone formation. SURGERY 1989;106:400-7. 9. Lee SP, Carey MC, LamontJT. Aspirin prevention of cholesterol gallstone formation in prairie dogs. Science 1981;211:1429-31. 10. Booker ML, Scott TE, LaMorte WW. Effects of dietary fish oil on biliary phospholipids and prostaglandin synthesis in the cholesterol-fed prairie dog. Lipids 1990;25:27-31. 11. Juniper KJr, Burson EN. Biliary tract studies. II. The significance of biliary crystals. Gastroenterology 1957;32:175-211. 12. Olszewski MF, Holzbach RT. Liquid crystals in htmaan bile. Nature 1973;242:336-7. 13. Somjen GJ, Gilat T. A non-micellar mode of cholesterol transport in human bile. FEBS Lett 1983;156:265-8. 14. Bradford MM. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248-54. 15. Roschlau P, Bernt E, Grnber W. Cholesterol and esterified cholesterol. In: Methods of enzymatic analysis. New York: Academic Press, Inc, 1974:1890-93. 16. Talalay P. Enzymatic analysis of steroid hormones. In: Glick D, ed. Methods of biochemical analysis. New York: Interscience Publishers, Inc, 1960:119-43. 17. Dryer RL, Tammes AR, Routh JI. The determination of phos-
phorus and phosphatase with N-phenyl-p-phenylenediamine. J Bit Chem 1956;255:177-83. 18. Kuroki S, Cohen BI, Carey MC, Mosbach EH. Rapid computation with the personal computer of the percent cholesterol saturation of bile samples. J Lipid Res 1986;27:44245. 19. Spivak W, Carey MC. Reverse-phase HPLC separation, quantification, and preparation of bilirubin and its conjugates from native bile. Biochem J 1985;225:787-805. 20. Holzbach RT, Kibe A, Thiel E, Howell JH, Marsh M, Hermann RE. Biliary proteins: unique inhibitors of cholesterol crystal nucleation in human gallbladder bile. J Clin Invest 1984;73:3545. 21. Holan KR, Holzbach RT, Hermann RE, Cooperman AM, Claffey WT. Nucleation time: a key factor in the pathogenesis of cholesterol gallstone disease. Gastroenterology 1979;77:611-7. 22. Magnnson TH, Lillemoe KD, High RC, Pitt HA. Fish oil prevents cholesterol crystal nucleation and gallstone formation [Abstract]. Gastroenterology 1989;96:A625. 23. Scobey MW, Johnson FL, ParksJS, Rudell LL. Dietary fish oil effects on biliary lipid secretion and cholesterol gallstone formarion in the African Green monkey. Hepatology 1991;14:67984. 24. Cohen BI, Mnsbach EH, Ayyad N, Miki S, McSherry CK. Dietary fat a n d fatty acids modulate cholesterol cholelithiasis in the hamster. Lipids 1992;27:526-32. 25. Banerjee B, Singh E. Fish oil prevents cholesterol crystal formation and development of gallstones in mice [Abstract]. Gut 1989; 30:A1459. 26. Berr F, Holl J, Jungst D, et al. Dietary n-3 polyunsaturated fatty acids decrease biliary cholesterol saturation in gallstone disease. Hepatology 1992;16:960-7. 27.Janowitz P, Swobodnik W, Weschler JG, Janowitz A, Saal D, Ditshuneit H. Fish oil, enriched with polyunsaturated fatty acids of the omega-3 type accelerates the nucleation time in healthy subjects. Klinische Wochenschrift 1991;69:289-93. 28. Tera H. Stratification of human gallbladder bile in vivo, Acta Chir Scand 1960;256(S):1-85. 29. Tierney S, Ahrendt SA, Talbot KF, et al. Fish oil reduces biliary cholesterol and prolongs nucleation of human gallbladder bile [Abstract]. Gastroenterology 1993;104:380A. 30. Balasubramaniam SB, Simons LA, Chang S, HickieJB. Reduction in plasma cholesterol and increase in biliary cholesterol by a diet rich in n-3 fatty acids in the rat. J Lipid Res 1985;26:684-9. 31. Carey MC. Pathogenesis of gallstones. AmJ Surg 1993;165:410-9. 32. Cohen DE, Carey MC. Acyl chain unsaturation modulates distribution of lecithin molecular species between mixed micelles and vesicles in model bile: implications for particle structure and metastable cholesterol solubilities. J Lipid Res 1991;32:1291-302. 33. Booker ML, LaMorte WW, Ahrendt SA, Lillemoe KD, Pitt HA. Distribution of phosphatidylcholine molecular species between mixed micelles and phospholipid-cholesterol vesicles in human gallbladder bile: dependence of acyl chain length and unsaturation. J Lipid Res 1992;33:1485-92. 34. Tierney S, Ahrendt SA, Booker ML, et al. Fish oil fatty acids are preferentially incorporated into micelles [Abstract]. Gastroen-' terology 1994;106:362A. 35. Park YH, Igimi H, Carey MC. Dissolution of human cholesterol gallstones in simulated chenodeoxycholate-rich and ursodeoxycholate-rich biles. Gastroenterology 1984;87:150-8. 36. Igimi H, Asakawa S, Watanabe D, Shirmura H. Liquid crystal formarion in ursodeoxycholic-rich human gallbladder bile. Gastroenterology Jpn 1983;18:93-7. 37. MarksJW, Bonorris GG, Albers G, Schoenfield LJ. The sequence of biliary events preceding the formation of gallstones in humans. Gastroenterology 1992;103:566-70. 38. Magnuson TH, D'Souza P, Fox-Talbot K, Lillemoe KD, Pitt HA.
Surgery Volume 118, Number 3 The influence of dietary fish oil on biliary nonmucin glycoproteins [Abstract]. Hepatology 1989;10:734A. 39. Magnuson TH, Lillemoe KD, Zarkin BA, Pitt HA. Patients with gallstones acidify bile normally. Dig Dis Sci 1992;37:1517-22. 40. Shiffman ML, Sugerman HJ, Moore EW. Human gallbladder mucosal function: effect of concentration and acidification of bile on cholesterol and calcium solubility. Gastroenterology 1990;99:1452-9. 41. Moore EW. Biliary calcium and gallstone formation. Hepatology 1990;12:206S-18S. 42. Ostrow JD. Unconjugated bilirubin and cholesterol gallstone formation. Hepatology 1990;12:219S-26S. 43. Strichartz SD, Abedin MZ, Abdou MS, et al. Increased biliary calcium in cholesterol gallstone formation. Am J Surg 1988; 155:131-7.
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44. Kaufman HS, Magnuson TH, Pitt HA, Frasca P, Lillemoe KD. The distribution of calcium salt precipitates in the core, periphery and shell of cholesterol, black pigment and brown, pigment gallstones. Hepatology 1994;19:1124-32. 45. Berenson MM, Cardinal JR. Calcium accelerates cholesterol phase transitions in analog bile. Experientia 1985;41:1328-30. 46. Kibe A, Dudley MA, Halpern Z, Lynn MP, Breuer AC, Holzbach RT. Factors affecting cholesterol monohydrate crystal nucleation time in model systems of supersaturated bile. J Lipid Res 1985;26:1102-11. 47. Magnuson TH, Ahrendt SA, Lillemoe KD, Kaufman HS, Watt PC, Pitt HA. Short-term fasdng increases gallbladder calcium and bilirubin. J Surg Res 1991;50:524-34.
Background. Epidemiologic studies suggest that populations consuming a diet rich in fish oil have lower rates of both atherosclerotic heart disease and gallstones. The mechanisms underlying this inhibitory effect on cholesterol gallstone formation remain unclear. We therefore studied the effect of dietary fish oil on bile composition and cholesterol precipitation in an animal model of gallstone disease. Methods. Adult male prairie dogs were fed a standard control diet (n = 12) or a lithogenic 1.2% cholesterol diet (n = 16). One half of the animals in each group had their diet supplemented with concentrated fish oil. Results. After 14 days animals receiving the cholesterol diet all developed biliary cholesterol monohydrate crystals and gallstones. When fish oil was added to this high cholesterol diet, solid cholesterol crystal precipitation and gallstone formation were completely inhibited. This inhibition of gallstone formation was accompanied by a significant decrease in biliary calcium and total protein concentration. Microscopic cholesterol liquid crystals were evident in the bile of all of the animals fed the cholesterol plus fish oil diet. Dietary fish oil also significantly prolonged cholesterol monohydrate crystal observation time in animals receiving the lithogenic diet. Conclusions. These data suggest that dietary fish oil exerts a potent antilithogenic effect on cholesterol gallstone disease and may induce a stable liquid crystalline phase retarding nucleation. (SuRGF~Y 1995;118:517-23.) From the Departments of Surgery, The Johns Hopkins University School of Medicine, and Johns Hopkins Bayview Medical Center, Baltimore, Md.
EPIDEMIOLOGIC STUDIES SUGGESTTHAT populations consuming a diet rich in fish oil, such as the North American a n d G r e e n l a n d Eskimo, exhibit lower rates o f atherosclerofic h e a r t disease. 1, 2 This beneficial effect o f fish oil has b e e n attributed, in large part, to the high c o n t e n t o f 0~-3 fatty acids present in fish oil. Interestingly, these same populations also a p p e a r to enjoy a relatively low incidence of cholesterol gallstone disease. 3 However, this a p p a r e n t relationship between fish oil c o n s u m p t i o n a n d d i m i n i s h e d cholesterol gallstone disease remains relatively u n e x p l o r e d . Fish oil a n d ~-3 fatty acid administration have b e e n shown to impact various metabolic processes such as prostaglandin biosynthesis, lipid metabolism, a n d hepatic lipoprotein metabolism, which are i m p o r t a n t in the pathogenesis o f atherosclerofic disease. 4-7 Many of these same factors are also Accepted for publication Jan. 10, 1995. Reprint requests: Thomas H. Magnuson, MD, Department of Surgery, Johns Hopkins BayviewMedicalCenter, 4940 Eastern Ave.,Baltimore, MD 21224. Copyright 9 1995 by Mosby-YearBook, Inc. 0039-6060/95/$3.00 + 0 11/56/64381
t h o u g h t to play i m p o r t a n t roles in the d e v e l o p m e n t o f cholesterol gallstones, a n d their alteration may account for fish oil's a p p a r e n t antilithogenic effects. T h e p r e s e n t study was designed to explore the effect o f dietary fish oil o n gallstone pathogenesis a n d bile composition a n d to test the hypothesis that dietary fish oil would prevent the nucleation a n d precipitation o f cholesterol crystals from bile a n d inhibit cholesterol gallstone formation.
MATERIAL A N D M E T H O D S E x p e r i m e n t a l design. Adult male prairie dogs (cy-
nomys ludovicianus) caught in the wild were used in the study. All animals were h o u s e d in a t h e r m o r e g u l a t e d r o o m with a constant l i g h t / d a r k cycle a n d allowed 3 weeks of acclimation to the laboratory e n v i r o n m e n t before study. T h e animals were divided into f o u r experimental groups: (1) control diet, n = 6; (2) control diet s u p p l e m e n t e d with fish oil (FO), n = 6; (3) high cholesterol lithogenic diet (CHOL), n = 8; a n d (4) high cholesterol lithogenic diet s u p p l e m e n t e d with fish oil ( C H O L + FO), n = 8. T h e control diet consisted of a standard trace (0.03%) cholesterol nonlithogenic diet SURGERY
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Table I. Incidence of biliary crystals and cholesterol gallstones Control Fish oil CHOL
Gallstones Cholesterol monohydrate crystals Cholesterol liquid crystals
CHOL + FO
0/6 0/6
0/6 0/6
8/8* 8/8*
0/8 0/8
0/6
6/6J"
0/8
8/8 t
*p< 0.01 versusother dietarygroups. "~p< 0.01 versuscontrol and CHOL. (Purina rat chow; Ralston Purina Co., St. Louis, Mo.). The high cholesterol diet was similar to the control diet but contained 1.2% cholesterol by weight (Teklad test diets; Madison, Wis.). This diet has been used extensively by this laboratorys and others ~ 10 to induce cholesterol gallstones in prairie dogs during a 2- to 3-week period of time. Fish oil was administered by oral gavage to the FO and CHOL + FO groups as a concentrated fish oil preparation (Promega; Parke-Davis, Morris Plains, N.J.) supplying 200 m g / k g / d a y polyunsaturated ~ 3 fatty acids (eicosapentaenoic and docosahexaenoic acid). The animals were maintained on their respective diets for 14 days. At the end of the 14-day feeding period and after an overnight fast, each animal was anesthetized with intramuscular ketamine (100 mg/kg), and a laparotomy was performed. The intact gallbladder was excised, and gallbladder bile was completely aspirated anaerobically, protected from light, and stored on ice. At the termination of the experiment, blood was obtained by cardiac puncture and the animal was killed. Gallstone and crystal analysis. All gallbladders were examined grossly for evidence of cholesterol gallstones. Gallbladder bile and gallbladder mucosa were then examined under a polarizing light microscope for any evidence of cholesterol crystals. Cholesterol monohydrate crystals were identified by their typica! birefringent, notched rhomboidal shape, it Cholesterol liquid crystals were identified as previously described by their birefringent "Maltese cross" configuration) 2 Liquid ,crystals are known to represent fused cholesterol-phospholipid vesicles important in the solubilization of cholesterol in bile. 13 Bile analysis. Fresh anaerobic gallbladder bile was immediately analyzed for pH and ionized calcium. An aliquot was mixed with ascorbic acid (1:1, vohvol) for later bilirubin analysis. The remaining bile was then centrifuged for 5 minutes at 2000 rpm, and the supernatant was stored at -20 ~ for subsequent analysis. After rewarming to room temperature, bile was aria-
lyzed for total protein, 14 cholesterol, 15 total bile acid, 16 and phospholipid concentration. ]7 The cholesterol saturation index (CSI) of bile was calculated as described by Kuroki et al.lS Bile was also analyzed for unconjugated bilirubin according to the technique of Spivak and Carey 19 by use of high-pressure liquid chromatography. Prairie dog serum was analyzed for total cholesterol concentration. Crystal observation lime. To determine the effect of fish oil on cholesterol crystal observation time, a second study was designed. In this study five prairie dogs were fed the high (1.2%) cholesterol diet, and five animals received the same diet but supplemented with fish oil (900 m g / k g / d a y to-3 fatty acids). The animals received their respective diets for 9 days, at which point shortterm terminal experiments were performed. Gallbladder bile was aspirated into prewarmed syringes and individually filtered through 0.22 micron filters to achieve isotropic, crystal free bile as previously described for human bile nucleation studies. 2~ The individual bile samples were then incubated at 37 ~ C and examined at 12-hour intervals under a polarizing light microscope for evidence of cholesterol crystal nucleation. Crystal observation time was defined as the point when cholesterol monohydrate crystal(s) precipitated from solution and could clearly be identified microscopically. Statistics. Data are presented as the mean +_ the standard error of the mean. Statistical analysis was performed with analysis of variance or Fisher's exact test where appropriate. Nucleation time data were compared by using the Mann-Whitney U test. RESULTS
All animals gained weight during the experimental period and appeared healthy. Food intake was monitored daily, and all four groups of animals consumed similar amounts of food and water. Gallstone and biliary crystal analysis. The incidence of gallstones and biliary crystals in the experimental groups is shown in Table I. As expected, none of the animals in the control group or the fish oil alone group developed gallstones. In contrast, all of the animals fed the high cholesterol diet developed grossly evident cholesterol gallstones. When animals were fed the same lithogenic diet but supplemented with fish oil (CHOL +FO), the incidence of gallstones dropped dramatically to zero (p < 0.01). In parallel with the incidence of gallstones in the various groups, cholesterol monohydrate crystals were present on the microscopic examination of gallbladder bile in only the cholesterol-fed group. Examination of gallbladder bile in the cholesterol plus fish oil group failed to reveal any evidence of cholesterol monohydrate crystal precipitation. Interestingly, this same group
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Table II. Gallbladder bile composition Control
pH Ionized calcium (mmol/L) Unconjugated bilirubin (pmol/L) Cholesterol (mmol/L) Phospholipids (mmol/L) Total bile acids (mmol/L) CSI
6.93 _+ 0.08 1.7 _+ 0.1 7.9 _+ 1.1 4.2 -+ 0.4 16.3 _+ 1.6 154 _+9 0.60 _+ 0.03
Fish oil
6.95 2.0 8.9 3.7 16.2 160 0.52
-+ 0.08 _+ 0.2 -+ 1.0 _+ 0.3 -+ 1.4 +_ 9 -+ 0.03
CHOL
6.57 3.3 19.7 14.8 39.4 209 0.94
-+ 0.16" + 0.2t _+ 4.0* -+ 1.2" _+ 3.9* + 13" -4-0.07*
CHOL + FO
6.86 2.5 16.8 13.2 32.6 191 1.03
+ 0.07 -+ 0.2 -+ 2.6* -+ 0.4* -+ 2.6* _+ 8* -+ 0.06*
*p < 0.005 versus c o n t r o l a n d fish oil. t P < 0.005 versus all o t h e r groups.
( C H O L + FO) exhibited an a b u n d a n c e o f cholesterol liquid crystals on microscopic examination. These birefringent, Maltese cross liquid crystals were also n o t e d in gallbladder bile of animals fed the fish oil diet without high dietary cholesterol. N o n e o f the control o r the cholesterol-fed animals h a d evidence o f cholesterol liquid crystal formation. Gallbladder bile analysis. Analysis of gallbladder bile for pH, ionized calcium, u n c o n j u g a t e d bilirubin, a n d biliary lipids is p r e s e n t e d in Table II. As previously described, 8 biliary p H was significantly decreased a n d ionized calcium was increased in prairie dogs fed the lithogenic cholesterol diet. T h e addition o f fish oil to the control diet or to the high cholesterol diet resulted in no significant changes in biliary pH. Similarly, unconjugated bilirubin levels were increased in the cholesterolfed group a n d unaffected when fish oil was added. Dietary fish oil did, however, significantly prevent the increase in gallbladder bile ionized calcium i n d u c e d by the high cholesterol diet. Gallbladder biliary lipid concentrations a n d CSI for the four dietary groups are also shown in T a b l e II. Biliary cholesterol, phospholipid, a n d total bile acid concentrations were all increased with the cholesterol diet. The addition offish oil to this diet h a d no significant effect on the concentrations o f these biliary lipids. As expected, the CSI of gallbladder bile in the control g r o u p was well below 1.0, the limit o f saturation. T h e gallbladd e r CSI o f the fish oil g r o u p (0.52 - 0.03) showed little change c o m p a r e d with the control g r o u p (0.60 _+ 0.03). T h e addition o f 1.2% cholesterol to the control diet increased the gallbladder CSI to the p o i n t o f cholesterol saturation, consistent with the macroscopic evidence o f cholesterol precipitation a n d gallstones in this group. T h e CSI o f the cholesterol plus fish oil g r o u p (1.03 +- 0.06) was no different from that of the cholesterol-fed g r o u p (0.94 _+ 0.07), despite the fact that no cholesterol precipitation was observed in the animals fed cholesterol plus fish oil. Gallbladder bile total protein levels are r e p r e s e n t e d in Fig. 1. No difference was n o t e d between the control
(0.79 -+ 0.07 m g / m l ) a n d fish oil g r o u p (0.80 -- 0.04 m g / m l ) . T h e addition o f dietary cholesterol resulted in a greater than fourfold increase in biliary p r o t e i n concentration (3.41 + 0.24 m g / m l ) when c o m p a r e d with controls (p < 0.01). This increase in biliary p r o t e i n was diminished in the C H O L + FO animals, resulting in levels significantly less (2.1 -+ 0.21 m g / m l ) than animals fed the same high cholesterol diet but without fish oil (p< 0.01). Serum cholesterol. Serum cholesterol concentrations were m e a s u r e d in the four dietary groups. The high cholesterol diet resulted in a significant increase in serum cholesterol (508 +- 50 m g / d l ) when c o m p a r e d with controls (116 +- 6 m g / d l ; p < 0 . 0 1 ) . A similar increase in cholesterol concentration was n o t e d in the C H O L + FO g r o u p (459 +- 83 m g / d l ) . This level was significantly increased c o m p a r e d with both controls (p< 0.05) a n d the F O group (118 +- 5 m g / d l ; p < 0.05) a n d was n o t statistically different from the C H O L group. Crystal observation time. Gallbladder bile cholesterol crystal observation times were d e t e r m i n e d in five animals receiving the high cholesterol diet a n d five animals receiving the same cholesterol diet b u t supplem e n t e d with fish oil. Cholesterol crystal nucleation has b e e n previously shown to be a critical step in cholesterol precipitation from saturated bile. 21 A m o r e rapid time to crystal f o r m a t i o n correlates with decreased stability with respect to biliary cholesterol solubility a n d enh a n c e d tendency toward precipitation. As p r e s e n t e d in Fig. 2, the cholesterol-fed g r o u p exhibited a r a p i d biliary crystal observation time of 1.4 +- 0.4 days. T h e addition o f fish oil to this diet significantly p r o l o n g e d crystal observation time nearly fourfold to 5.4 +- 1.6 days (p < 0.05).
DISCUSSION T h e p r e s e n t study suggests that dietary fish oil may exert a p o t e n t antilithogenic effect d u r i n g cholesterol gallstone formation. In the cholesterol-fed prairie dog, a widely accepted animal m o d e l o f cholesterol gallstone disease, fish oil s u p p l e m e n t a t i o n resulted in a signifi-
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4 7 6
3
~5
p<0.0s
,
3 O 1
,O 1 0
-
-
CONTROL
FISHOIL
CHOL
CHOL+FO
Fig. 1. Gallbladder bile total protein concentration in animals fed standard diet (CONTROL), fish oil supplemented diet (FISH OIL), high cholesterol diet (CHOL), and high cholesterol diet with fish oil (CHOL +FO).
cant inhibition of both cholesterol monohydrate crystal precipitation and gallstone formation and nearly a fourfold prolongation of in vitro cholesterol crystal observation time. These changes were accompanied by decreased gallbladder bile concentrations of both ionized calcium and protein. Dietary fish oil had no effect on the concentration of biliary lipids or the CSI of gallbladder bile. Fish oil did, however, induce the formation of apparently stable phospholipid-cholesterol vesicles as evidenced by the microscopic appearance of liquid crystals in gallbladder bile. Epidemiologic studies have suggested that populations consuming diets rich in fish oil and ~-3 fatty acids may be relatively protected from cholesterol gallstone disease. 3 In a preliminary report we first noted that fish oil had potential antilithogenic effects in an animal model of gallstone disease. 22 Several other investigators have since addressed this relationship, and their results support the theory that fish oil may impact cholesterol gallstone pathogenesis. In the African green monkey, Scobey et al.23 reported a 45% decrease in gallstone formation in animals whose diets were supplemented with fish oil. Similarly, Booker et al.lO noted a significant decrease in the incidence of biliary cholesterol crystals in prairie dogs fed a high cholesterol lithogenic diet supplemented with fish oil. More recent studies in the hamster ~4 and mouse 25 also suggest that cholesterol gallstone formation is inhibited when dietary fish oil is added to a normally lithogenic experimental diet. A number of human studies have shown conflicting results. Ben" et al.26 studied patients with known gallstones who were given concentrated dietary fish oil supplements. They noted no changes in the incidence of cholesterol monohydrate crystal or in cholesterol nucleation time. A similar study byJanowitz et al.27 found that feeding of fish oil accelerated cholesterol nucleation time, theoretically favoring cholesterol precipita-
CHOLESTEROL
CHOLESTEROL + FISH OIL
Fig. 9. Gallbladder bile crystal observation time in animals fed high cholesterol diet without and with fish oil. don. Cautious interpretation of these clinical studies is indicated, however, because many of the patients had preexisting stones that may have served as a continuing nidus for nucleation-precipitation despite dietary-induced biliary changes. Furthermore, bile samples were also obtained from an endoscopic, transduodenal approach and therefore may be invalid reflections of the tree gallbladder environment. 28 We have recently studied patients with cholesterol gallstones treated before operation for 2 weeks with fish oil and compared their cholesterol crystal nucleation times with patients with gallstones who were not receiving fish oil and a nongallstone control group. 29 At cholecystectomy, bile was completely aspirated from the patient's gallbladder, and cholesterol nucleation time was determined. Nucleation times were significantly prolonged in patients with gallstones receiving fish oil when compared with patients with untreated gallstones and were no different from crystal observation times in the control nongallstone group. A variety of mechanisms exist that may explain fish oil's inhibitory effect on gallstone development. Altered hepatic cholesterol and lipoprotein metabolism have previously been noted with fish oil consumption and have been thought to be responsible, in part, for fish oil's apparent protective role in atherosclerotic cardiovascular disease. 4, 6 Such changes in cholesterol metabolism could impact biliary cholesterol and, thus, gallstone formation, tn the present study, however, we noted no changes in concentrations of serum cholesterol or gallbladder bile cholesterol or biliary CSI in the animals fed fish oil that might explain the observed decrease in cholesterol precipitation. This finding concurs with previous studies showing no significant changes in biliary cholesterol saturation with feeding of fish 0i1.23, 24, 29 Others, however, have noted that cholesterol saturation is decreased with fish oil, primarily because of a relative increase in phospholipid secretion into bile.aO, ~6,27 Conversely, in a rat model biliary cholesterol
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concentrations were noted to increase as serum cholesterol concentrations decreased when fish oil was added to the diet. 3~ Cholesterol precipitation in bile is governed by a wide variety of factors i n addition to the relative concentrations of cholesterol, phospholipids, and bile acids, the traditional determinants of CSI. Such factors may influence cholesterol precipitation in bile by enhancing the relative solubility of cholesterol in solution or by preventing a supersaturated solution from reaching thermodynamic equilibrium. More than 20 years ago, when cholesterol was thought to be exclusively solubilized by mixed micelles, Olszewski and Holzbach 12 first noted the presence of cholesterol liquid crystals in human gallbladder bile and postulated that they served to "inhibit the precipitation of excess cholesterol to solid crystalline form by delaying its nucleation." We now know that cholesterol is transported and solubilized not only by mixed micelles but also by cholesterol-phospholipid vesicles. 13 These vesicles in mulfilamellar form represent liquid crystals, and cholesterol nucleation and precipitation into solid crystals proceed from this liquid crystalline phase. 31 In the present experiment fish oil supplementation blocked this phase transition and induced relatively stable cholesterol-phospholipid vesicles as manifested by the microscopic appearance of liquid crystals and prolonged crystal observation time. Although not directly determined in this study, dietary fish oil has been shown in human beings z6 and animals 1~ 23 to alter the composition of phospholipids in bile and result in species highly enriched at the sn-2 position with o)-3 fatty acids. Such a change in phospholipid composition might theoretically alter vesicular stability and the cholesterol carrying capacity of bile and potentially account for the observed inhibition of cholesterol precipitation. Indeed, Cohen and Carey3z and Booker et al. 33 have demonstrated that altering phospholipid composition by changing fatty acyl length and unsaturation can significantly impact cholesterol solubility in bile and the partitioning of cholesterol between vesicles and micelles. In a preliminary report we have recently noted that fish oil administered to patients with gallstones results in the preferential incorporation of n-3 fatty acids into micellar as opposed to vesicular phospholipids. 34 Interestingly, ursodeoxycholic acid, when given orally for gallstone prevention or dissolution, has also been noted to prolong cholesterol crystal observation time and induce the formation of stable liquid crystals similar to those seen in this experiment. 35, 36 In the present study cholesterol gallstone formation was accompanied by a significant increase in gallbladder bile protein concentration. Many investigators have focused on biliary proteins and their role as potential factors influencing cholesterol crystal nucleation, z~ 37 Lee
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et al.9 demonstrated that aspirin, an inhibitor of prostaglandin biosynthesis, was able to block gallbladder protein hypersecrefion and thereby prevent gallstones in cholesterol-fed prairie dogs. Recent studies suggest that dietary fish oil may also inhibit gallbladder mucosal prostaglandin synthesis by substituting ~0-3fatty acids for arachidonic acid, the phospholipid fatty acyl species that is the usual substrate for prostaglandin production. 1~ An alteration in gallbladder prostaglandin synthesis and the resultant inhibition of glycoprotein production might explain the drop in protein concentration we observed in the cholesterol plus fish oil dietary group. Failure to secrete a potential pronucleating protein may have prevented cholesterol precipitation. However, we have been unable to demonstrate any qualitative changes in biliary protein composition in prairie dogs fed the fish oil diet. 38 Alternatively, increased protein production by the gallbladder epithelium may have been a secondary phenomenon such as an inflammatory response to the presence of cholesterol crystals and stones. In the group fed fish oil the diminished rise in protein could then be explained by the failure of gallstones to form and, therefore, the elimination of the inflammatory stimulus. Clearly, further studies are needed to determine the exact sequence of events with regard to fish oil's effect on prostaglandin metabolism, gallbladder protein production, and cholesterol precipitation. Biliary pH, 39' 40 calcium,41 and bilirubin 42 have all been implicated as modulators of cholesterol precipitation a n d gallstone formation, but fish oil's effect on these factors is unknown. In the present study gallstone formation in the cholesterol-fed group was associated with a significant decrease in biliary pH and increase in unconjugated bilirubin levels. Fish oil, however, had no effect on these changes despite inhibiting gallstone development. Consistent with previous studies, 43 ionized calcium level was also elevated in the cholesterol-fed gallstone group. Fish oil supplementation inhibited this increase. Calcium has been noted to he an important structural component in the core of cholesterol gallstones, 44 perhaps serving as a nidus for cholesterol crystal precipitation or growth. Calcium has also been reported to accelerate biliary cholesterol crystal formation by inducing the aggregation of unstable cholesterol carrying vesicles.45' 46 The mechanism whereby fish oil decreased biliary calcium level in this study is unclear. Enhanced bile concentration caused by increased gallbladder water absorption or altered biliary motility has previously been noted to elevate calcium levels. 4~ 47 It is unlikely, however, that fish oil affected gallbladder absorption or motility because the concentration of bile acids, a relatively unabsorbed constituent of bile and therefore an index of bile concentration, was similar in
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the cholesterol-fed group and the group fed cholesterol p l u s f i s h oil. I n s u m m a r y , t h e p r e s e n t s t u d y s h o w s t h a t d i e t a r y fish oil p r e v e n t s c h o l e s t e r o l crystal p r e c i p i t a t i o n a n d gallstone formation in the cholesterol-fed prairie dog. This antilithogenic effect may be due to a relative enhancem e n t i n t h e stability o f b i l i a r y p h o s p h o l i p i d - c h o l e s t e r o l vesicles as m a n i f e s t e d b y t h e o b s e r v e d p r e s e n c e o f c h o l e s t e r o l l i q u i d crystals a n d p r o l o n g e d c h o l e s t e r o l crystal o b s e r v a t i o n t i m e s . T h e a p p l i c a b i l i t y o f this m o d e l
to
h u m a n g a l l s t o n e d i s e a s e r e m a i n s to b e d e t e r m i n e d , b u t fish oil m a y p r o v e b e n e f i c i a l i n p o p u l a t i o n s a t h i g h risk f o r g a l l s t o n e f o r m a t i o n o r r e c u r r e n c e s u c h as t h o s e after extracorporeal
shock wave lithotripsy or during
rapid weight reduction.
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