Short-term fasting increases biliary calcium and bilirubin

Fasting has been associated clinically with the development of gallbladder sludge and pigment gallstones, both of which are composed primarily of calcium bilirubinate. Although fasting has been demonstrated to increase the cholesterol saturation of bile, its effect on biliary calcium and bilirubin has not been investigated. We, therefore, tested the hypothesis that short-term fasting would increase gallbladder bile calcium and bilirubin levels. Fifteen prairie dogs were studied. Seven animals were not fasted, whereas eight were fasted for 16 hr prior to acute experiments. Gallbladder and hepatic bile samples were obtained and analyzed for calcium, bilirubin, pH, and biliary lipids. Gallbladder bile ionized calcium levels were significantly increased in fasted animals (1.7 +/- 0.2 mM) compared to those in nonfasted animals (1.1 +/- 0.1 mM). Similarly, total calcium (4.3 +/- 0.5 mM vs 2.3 +/- 0.3 mM), total bilirubin (63 +/- 12 microM vs 29 +/- 8 microM), and bilirubin monoglucuronide (58 +/- 10 microM vs 22 +/- 8 microM) were significantly increased in the fasted group. Fasted animals were also noted to have an increased biliary cholesterol saturation index (0.57 +/- 0.04 vs 0.36 +/- 0.03) and decreased biliary pH (6.9 +/- 0.1 vs 7.6 +/- 0.1). These data indicate that in the prairie dog short-term fasting results in significant alterations in gallbladder bile composition. The increased concentrations of gallbladder calcium and bilirubin observed in these experiments may account, in part, for the formation of pigment gallstones and gallbladder sludge seen clinically with prolonged fasting.     

Altered bile composition during cholesterol gallstone formation: cause or effect?

Gallbladder stasis, increased gallbladder absorption, and elevated biliary levels of calcium, hydrogen ion, and bilirubin have been implicated as factors potentially critical to cholesterol crystal precipitation. Previous studies, however, have analyzed bile only when crystals or gallstones have already formed. Therefore, we tested the hypothesis that changes in bile composition are a late effect, occurring only after crystal formation. Adult male prairie dogs were fed a standard nonlithogenic control diet (n = 7) or a lithogenic 1.2% cholesterol diet for 5, 9, or 14 days to cause cholesterol saturation (n = 7), cholesterol monohydrate crystals (n = 7), or gallstones (n = 7). Gallbladder bile was examined microscopically for crystals, and analyzed for ionized calcium, bilirubin, pH, total protein, and biliary lipids. The ratio of gallbladder to hepatic bile radiolabeled cholic acid specific activity (Rsa) was calculated as an index of gallbladder stasis. Cholesterol saturation index was calculated. The results demonstrate that increased gallbladder bile cholesterol saturation and total protein concentration precede cholesterol monohydrate crystal precipitation. However, changes in gallbladder ionized calcium, unconjugated bilirubin, pH, stasis, and absorption were noted only after crystals and gallstones had already formed. These data indicate that alterations in gallbladder bile calcium, bilirubin, pH, stasis, and absorption are not early changes, but occur simultaneously with or after crystal formation. Increased biliary protein, however, which was elevated prior to nucleation, may be an important mediator of cholesterol precipitation in cholesterol-saturated bile.     

Caffeine prevents cholesterol gallstone formation

Methylxanthines are known to inhibit in vitro gallbladder absorption. Increased gallbladder absorption has been observed during formation of cholesterol gallstones. Therefore we tested the hypothesis that caffeine would inhibit in vivo gallbladder absorption and thus prevent formation of cholesterol gallstones. Sixteen adult male prairie dogs received a control nonlithogenic diet, and 16 were fed a diet containing 1.2% cholesterol. Half of the animals in each group received caffeine in their drinking water. Gallbladder and hepatic bile were examined microscopically and analyzed for biliary lipids and electrolytes. The gallbladder/hepatic bile ratios of bile acids and sodium were calculated as indices of gallbladder absorption. All eight animals receiving the 1.2% cholesterol diet formed cholesterol gallstones, whereas none of the eight animals fed the cholesterol diet plus caffeine formed gallstones. The cholesterol saturation index was similar, however, in both groups. In animals fed a control diet, the administration of caffeine significantly increased hepatic bile flow and decreased the gallbladder/hepatic bile ratio for both bile acids (5.4 +/- 0.9 vs 3.6 +/- 0.3; p less than 0.05) and sodium (1.26 +/- 0.03 vs 1.12 +/- 0.03; p less than 0.01). In animals fed the high-cholesterol diet, caffeine significantly decreased the ratios for both bile acids (9.0 +/- 1.6 vs 5.3 +/- 0.6; p less than 0.05) and sodium (1.37 +/- 0.06 vs 1.21 +/- 0.01; p less than 0.05), lowered gallbladder bile protein levels, normalized gallbladder stasis, and lowered serum cholesterol levels. In summary, caffeine prevented formation of cholesterol gallstones in this experimental model. The effect of caffeine may be the result of alterations in multiple biliary parameters including the inhibition of gallbladder absorption.     

The effects of amiloride on biliary calcium and cholesterol gallstone formation.

Recent studies indicate that gallbladder absorption increases during the early stages of experimentally-induced cholesterol gallstone formation. The purpose of the present study was to ascertain whether pharmacologic inhibition of gallbladder ion transport and absorption reduces the incidence of experimentally-induced cholesterol gallstones. Prairie dogs were fed either a control chow or a 1.2% cholesterol-enriched chow for 15 days. One group of cholesterol-fed animals received saline via an orogastric tube; another group received amiloride, a drug known to inhibit in vitro ion transport in the prairie dog gallbladder. The incidence of gallstones in cholesterol-fed animals was reduced from 83% to 13% (p less than 0.025) when the animals were treated with amiloride; this occurred despite a cholesterol-saturation index comparable to that observed in gallstone animals. Additionally, although biliary calcium decreased in the gallbladder, hepatic bile did not in the amiloride-treated animals. These data provide further evidence that altered gallbladder absorption and increased biliary calcium are important factors in the pathogenesis of cholesterol gallstones.     

Triglyceride and cholesterol content in bile, blood, and gallbladder wall

Cholesterol gallstone disease is frequent in Chile compared with other countries, as is cholesterolosis of the gallbladder. The purpose of this study was to determine any differences in bile composition and cholesterol content in the gallbladder wall and serum of patients compared with findings in patients with gallstones and control subjects. In control subjects, cholesterol content of the gallbladder wall was determined in autopsy material. Patients with cholesterolosis had bile composition similar to that of patients with gallstones, with supersaturation of cholesterol in the bile. Also, these patients had increased levels of cholesterol in the gallbladder wall but normal serum cholesterol levels. These findings suggest that altered bile composition occurs in patients with cholesterolosis and gallstones.     

Effect of dietary ethanol on gallbladder absorption and cholesterol gallstone formation in the prairie dog

Dietary ethanol has been reported to protect against cholesterol gallstone formation. Because enhanced gallbladder absorption of water is important in cholesterol cholelithiasis, we examined the hypothesis that ethanol acts by inhibiting the absorptive function of the gallbladder. Eighteen adult male prairie dogs were fed a lithogenic liquid diet containing 0.4% cholesterol. Half of the animals received 30% of total calories as ethanol, whereas their pair-fed controls received equicaloric amounts of maltose-dextrin. After 3 months, the gallbladders were inspected for gallstones and crystals, and gallbladder and hepatic bile were analyzed. Cholesterol stones and crystals were present in all nine controls. None of the alcohol-fed animals had stones, but four had cholesterol crystals. Gallbladder cholesterol, phospholipids, and total calcium were significantly decreased in alcohol-fed animals. In both gallbladder and hepatic bile, the cholesterol saturation index was significantly lower in alcohol-fed animals, as was the ratio of trihydroxy to dihydroxy bile salts. The ethanol-supplemented diet produced a significant decrease in the absorption of water by the gallbladder as indicated by changes in the gallbladder bile to hepatic bile ratios of the total bile salt concentration (7.29 +/- 1.25 versus 3.84 +/- 0.56; p less than 0.05) and the total calcium (3.37 +/- 0.24 versus 2.43 +/- 0.29; p less than 0.05). These findings indicate that the protective effect of ethanol may be related to its ability both to inhibit gallbladder absorption of water and to alter the composition of biliary lipids.     

The effects of ethinylestradiol,a glucose diet and streptozotocin induced diabetes mellitus on gallstone formation and biliary lipid composition in the hamster

Possible risk factors for gallstone formation were examined and the concentrations of biliary lipids and each bile acid in the hepatic and gallbladder bile of hamsters were quantified. Forty female golden Syrian hamsters were divided into 4 groups according to diet; Group I, given control chow, Group II, given an ethinylestradiol and cholesterol supplemented diet, Group III, given a glucose rich diet without induced diabetes mellitus, and Group IV, given a glucose rich diet with diabetes mellitus induced by streptozotocin injection. The formation of cholesterol crystals but not gallstones was induced in Group II associated with a significantly decreased total bile acid concentration in the gallbladder bile but not in the hepatic bile. The formation of cholesterol gallstones and crystals with significantly higher concentrations of cholesterol and phospholipid was observed in Group III, while neither the formation of gallstones nor lithogenicity was enhanced by diabetes mellitus. However, a quite different lithogenicity was evident between the hepatic and gallbladder bile of the Group IV animals. These results suggest that neither the consumption of oral contraceptives nor diabetes mellitus induces gallstone formation, but that these factors can be responsible for dysfunction of the gallbladder.     

Stasis before gallstone formation: Altered gallbladder compliance or cystic duct resistance?

Gallbladder stasis occurs before gallstone formation and provides the link between the hepatic secretion of supersaturated bile and cholesterol cholelithiasis. We recently observed that cystic duct resistance increases while sphincter of Oddi resistance is unchanged in the presence of lithogenic bile without gallstones. Whether alterations in gallbladder function also lead to gallbladder stasis has been unclear. Therefore, we tested the hypothesis that before gallstone formation, stasis results from increased cystic duct resistance and altered gallbladder compliance. Adult, male prairie dogs were fed either a trace cholesterol (control) or a 0.4 percent cholesterol-enriched diet. Cystic duct resistance increased but gallbladder compliance was unchanged before gallstone formation. A significant correlation (p < 0.001) was found between the lithogenic index and cystic duct resistance in pregallstone animals. We conclude that increased resistance to flow across the cystic duct, and not altered gallbladder compliance, is etiologically related to bile stasis, an important event in gallstone formation.     

Bilirubin monoglucuronide promotes cholesterol gallstone formation

Recent evidence suggests that cholesterol (Ch) solubility in bile is determined by a complex interaction of mixed micelles and lecithin-cholesterol vesicles. Bilirubin monoglucuronide (BMG), which binds to bile salts and incorporates into mixed micelles, may displace cholesterol from micelles into vesicles, thus favoring cholesterol monohydrate crystal precipitation. Therefore, we designed an experiment to test the hypothesis that BMG may enhance cholesterol gallstone formation without inducing cholesterol supersaturation. For 8 weeks, 28 adult male prairie dogs were fed either a control, nonlithogenic diet (0.03% Ch), a high carbohydrate diet (CHO) which has no cholesterol but increases hepatic bilirubin secretion, or the same CHO diet plus 0.03% Ch. Cholecystectomy was then performed, and bile was examined microscopically for stones or crystals and analyzed for BMG and biliary lipids. Cholesterol saturation index was calculated. Cholesterol gallstones were found in none of the control animals and in 13% of the CHO-fed animals. However, the addition of trace cholesterol to the CHO diet resulted in an 88% incidence of cholesterol gallstones (P less than 0.001 vs control, P less than 0.01 vs CHO, respectively). Gallbladder bile was unsaturated with cholesterol in all groups. (control = 0.65 +/- 0.05, CHO = 0.46 +/- 0.05, CHO + 0.03% Ch = 0.70 +/- 0.03). CHO feeding alone or with trace cholesterol significantly elevated gallbladder bilirubin monoglucuronide, phospholipid, and cholesterol concentrations when compared to controls. These data suggest that in the prairie dog a high carbohydrate diet with only trace amounts of cholesterol increases bilirubin monoglucuronide in gallbladder bile and causes cholesterol gallstone formation without inducing cholesterol supersaturation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hyodeoxycholic acid: a new approach to gallstone prevention

Hyodeoxycholic acid and its isomer, 6 beta-hyodeoxycholic acid, when added to a lithogenic diet prevented the formation of cholesterol gallstones and crystals in prairie dogs. This beneficial effect occurred in the presence of bile supersaturated with cholesterol. Hyodeoxycholic acid abolished the feedback inhibition of hepatic hydroxymethylglutaryl coenzyme A reductase activity, the rate-limiting enzyme of cholesterol synthesis, and prevented elevations in serum and liver cholesterol observed in animals fed a 0.4 percent cholesterol diet. The gallbladder bile of the animals fed hyodeoxycholic acid and 6 beta-hyodeoxycholic acid contained abundant liquid crystals. This suggests that these bile acids prevented the transition of cholesterol from its liquid crystalline phase to solid crystals and stones.     

The effects of ethinylestradiol, a glucose diet and streptozotocin induced diabetes mellitus on gallstone formation and biliary lipid composition in the hamster

Possible risk factors for gallstone formation were examined and the concentrations of biliary lipids and each bile acid in the hepatic and gallbladder bile of hamsters were quantified. Forty female golden Syrian hamsters were divided into 4 groups according to diet; Group I, given control chow, Group II, given an ethinylestradiol and cholesterol supplemented diet, Group III, given a glucose rich diet without induced diabetes mellitus, and Group IV, given a glucose rich diet with diabetes mellitus induced by streptozotocin injection. The formation of cholesterol crystals but not gallstones was induced in Group II associated with a significantly decreased total bile acid concentration in the gallbladder bile but not in the hepatic bile. The formation of cholesterol gallstones and crystals with significantly higher concentrations of cholesterol and phospholipid was observed in Group III, while neither the formation of gallstones nor lithogenicity was enhanced by diabetes mellitus. However, a quite different lithogenicity was evident between the hepatic and gallbladder bile of the Group IV animals. These results suggest that neither the consumption of oral contraceptives nor diabetes mellitus induces gallstone formation, but that these factors can be responsible for dysfunction of the gallbladder.     

Ileal resection-induced gallstones: altered bilirubin or cholesterol metabolism?

Ileal resection has been shown to increase the risk of cholelithiasis. Earlier studies in humans suggested that ileal resection increases the cholesterol saturation index. Recent data from patients on long-term parenteral nutrition and from animals, however, have suggested that ileal resection predisposes to pigment gallstone formation. We therefore tested the hypothesis that ileal resection alters bile calcium and bilirubin metabolism without affecting the cholesterol saturation index. Adult male prairie dogs underwent either sham laparotomy (eight prairie dogs) or ileal resection (16 prairie dogs). All animals were fed a trace cholesterol (nonlithogenic) diet before and for 4 weeks after operation. Pigment gallstones were present in 44% of the ileal-resected animals and in none of the sham animals (p less than 0.05). Calcium bilirubinate crystals were present in 94% of the ileal-resected animals and in none of the sham animals (p less than 0.01). Gallbladder bile calcium (25.6 +/- 2.4 versus 17.2 +/- 1.1 mg/dl; p less than 0.05) and total bilirubin (29.3 +/- 4.0 versus 9.4 +/- 1.8 mg/dl; p less than 0.01) concentrations were significantly greater in ileal-resected animals. The cholesterol saturation index of gallbladder bile, however, was no different in ileal-resected (0.53 +/- 0.04) and in sham-operated animals (0.50 +/- 0.04). Although initial studies suggested that the cholesterol saturation index of hepatic bile was increased after ileal resection, a second set of experiments demonstrated that this phenomenon resulted from washout of bile salts that were already in extremely low concentrations in hepatic bile. We conclude that alterations in bilirubin, but not cholesterol, metabolism result in pigment gallstone formation after ileal resection.     

The role of cystic duct resistance in the pathogenesis of cholesterol gallstones

Several recent clinical and laboratory observations suggest that impaired gallbladder emptying is important in the pathogenesis of cholesterol cholelithiasis. However, the exact mechanism by which gallbladder stasis occurs in the majority of patients who form gallstones has not been clear. We tested the hypothesis that impaired gallbladder emptying antedates cholelithiasis and results from increased resistance to bile flow. Using the prairie dog gallstone model, resistance to flow through the cystic duct (CD) and sphincter of Oddi (SO) was measured in control and cholesterol-fed animals. Prairie dogs were fed either a control (trace cholesterol) or a 0.4% cholesterol-enriched diet known to induce gallstones in 6 weeks. Resistance across the CD and SO was measured at 4 weeks (pregallstone) and 16 weeks (gallstone). Resistance was measured by infusing lactated Ringer's solution through the CD and SO at four separate flow rates while gallbladder and distal common bile duct pressures were recorded. Resistance to flow through the cystic duct increased prior to gallstone formation and continued to increase during the 16 weeks of cholesterol feeding. In comparison, sphincter of Oddi resistance remained normal despite chronic exposure to lithogenic bile and formation of stones within the gallbladder. The increased cystic duct resistance observed prior to gallstone formation provides a mechanism for diminished gallbladder emptying and suggests an etiological role for increased cystic duct resistance in the pathogenesis of cholesterol gallstones.     

In vitro nucleation factors--mucus glycoprotein and proteins: changes and mechanisms in the process of sludge and gallstone formation]

Both mucus glycoprotein (MGP) and proteins are in vitro nucleation factors. Biliary sludge proved to be a precursor of gallstones. In this study, the MGP in gallbladder bile (GB) and the RNA content of the membrane-bound polyribosome in gallbladder epithelium were significantly higher in the sludge group than that in gallstone and control group. These facts demonstrated the increased synthesis and secretion of glycoproteins in the prestone stage. The RNA content of free polyribosome in the sludge group was also significantly greater than that in the control group so that in prestone stage there was an increase of cell proliferation in the gallbladder epithelium. Patients with sludge and cholesterol gallstones had significantly higher protein concentrations in GB than those without gallstones or with pigment stones. The gallbladder-hepatic bile ratio of proteins was significantly less than that of cholesterol, and there was a highly positive correlation between glycoproteins and proteins in GB. Therefore, the only reasonable explanation for the higher protein content in these two kinds of patients was that the mucus selectively inhibited protein absorption in the gallbladder. The increases in MGP and protein content in GB during the embryonic stage of gallstones might play a significant role in the formation of both cholesterol and pigment gallstones.     

The role of dietary iron in pigment gallstone formation

Recent studies suggest that dietary factors may be responsible for the increasing incidence of pigment gallstones. Although iron deficiency alters the activities of several hepatic enzymes, its effects on biliary lipid metabolism are not known. The aim of this study was to define the role of dietary iron in pigment gallstone formation. Three groups of prairie dogs were maintained for 2 months on either a control chow (iron-198 ppm), a high-carbohydrate diet with normal iron levels (CHO group; iron-220 ppm), or a high-carbohydrate, iron-deficient (iron-56 ppm) diet (CHO-FeD group). Serum analysis confirmed iron deficiency in the CHO-FeD group. The CHO animals had a significant (p less than 0.01) increase in hepatic bile phospholipids, while CHO-FeD animals had increased (p less than 0.01) concentrations of phospholipids and cholesterol as compared with controls. Similar findings were noted in gallbladder bile with the addition of increased calcium levels in both carbohydrate groups. Calcium bilirubinate crystals and stones were found in only 17% of CHO animals, as compared with 67% of CHO-FeD animals. These data indicate that consumption of diets rich in carbohydrates but deficient in iron alters hepatic metabolism of cholesterol and may be an important etiologic factor in pigment gallstone formation. Iron supplementation may prevent pigment gallstones in certain high-risk groups.     

Some observations on gallstones and bile composition.

A series of 31 consecutive patients undergoing surgery for gallstone disease has been studied and the composition of the gallstones and bile from the gallbladder and common duct determined. As a result of the stone analysis by the X-ray powder diffraction method, the patients were classified according to whether their stones consisted of cholesterol, calcium salts or a mixture of the two. The mean composition of the common duct bile for the groups with cholesterol and mixed stones was just outside the micellar region of cholesterol solubility. The gallbladder bile from the cholesterol group of stone-formers was also supersaturated, but the gallblader bile from the group of mixed stone-formers was undersaturated with respect to cholesterol. None of the patients forming gallstones of calcium salts showed any abnormality in the cholesterol content of their bile.     

Altered biliary prostaglandins and cholesterol gallstones: an in vivo study

Recent investigations suggest that biliary prostaglandin metabolism is altered during cholesterol gallstone formation. Most of the available data, however, has been obtained from in vitro studies. The purpose of the present study was to define the effects of cholesterol gallstone formation on in vivo biliary prostaglandin metabolism. Male prairie dogs were fed either a control chow for 21 days or a 1.2% cholesterol-enriched chow for 14-21 days. Cholecystectomy was performed and gallbladder tissue and bile were collected for analysis of prostaglandin concentrations using radioimmunoassay techniques. Gallbladder bile was examined for the presence of crystals and stones. No control animals but all cholesterol-fed animals developed either cholesterol crystals or gallstones (P less than 0.001). Concentrations of prostaglandin E2, prostaglandin F2 (PGF2 alpha), and the stable metabolic products of prostacyclin and thromboxane A2, 6-keto-PGF1 alpha and thromboxane B2 (TXB2), respectively, were decreased 60-85% in the gallbladder tissue of animals with crystals and gallstones compared to controls. Additionally, gallstone containing animals and those with crystals demonstrated a significant increase in the gallbladder bile concentrations of PGF2 alpha, 6-keto-PGF1 alpha, and TXB2. These findings lend support to previously reported in vitro studies suggesting that prostaglandin synthesis increases at an early stage of experimentally induced cholesterol gallstone formation.     

Reversal of pigment gallstone disease in a canine model

Unlike dietary-induced cholesterol gallstones, which may disappear spontaneously when the lithogenic diet is withdrawn, little is known about the natural history of pigment gallstones. We examined whether pigment gallstone disease, which can be uniformly induced in the dog by six weeks of a methionine-deficient diet, can be reversed by return to normal diet. As previously reported, all dogs develop pigment gallstones as well as significant increases in biliary total calcium, free ionized calcium, and cholesterol concentrations after six weeks of a lithogenic diet. These changes are accompanied by a significant increase in the concentration of unconjugated bile salts in bile. In addition, histologic changes in the gallbladder wall occur that are consistent with a moderate degree of chronic cholecystitis. This study clearly demonstrates that return to a normal diet for six weeks allows bile composition to normalize, gallstones to disappear in 50% of dogs, and gallbladder histologic changes to return toward normal. Thus, it would appear that pigment gallstone disease in this model may be reversible, at least early during its course. Although the relevance of these findings to pigment gallstones in humans must be established, the potential for nonoperative treatment of pigment gallstones should not be discounted.     

Oral calcium promotes pigment gallstone formation

Dietary calcium supplementation has been recommended for prevention of osteoporosis and has become a standard component of most "health food" diets. Biliary calcium has been recognized to play a central role in the formation of pigment gallstones. We have recently demonstrated that 5 days of oral calcium supplementation significantly increases biliary calcium in the prairie dog (K. D. Lillemoe, T. H. Magnuson, G. E. Peoples, et al., Gastroenterology 94: A563, 1988). We hypothesized, therefore, that long-term oral calcium supplementation would promote pigment gallstone formation. Sixteen adult male prairie dogs were maintained on a standard nonlithogenic diet. Eight animals received calcium supplementation (2.5 x control levels) in their water, while the remaining eight animals served as controls. After 8 weeks, cholecystectomy was performed, and the common bile duct was cannulated. Bile was examined microscopically and analyzed for ionized calcium, bilirubin, glycoprotein, and biliary lipids. The cholesterol saturation index (CSI) was calculated. Pigment stones and calcium bilirubinate sludge were present in all animals receiving calcium supplementation. Only one control animal had evidence of pigment stones (P less than 0.001). Biochemical analysis of gallbladder bile demonstrated a significant increase in total bilirubin and bilirubin monoglucuronide (P less than 0.01) as well as bile glycoprotein content (P less than 0.05) after oral calcium supplementation. Gallbladder bile ionized calcium was also increased although not significantly. These data suggest that oral calcium supplementation promotes gallbladder sludge and pigment gallstone formation in the prairie dog. This observation raises concern that oral calcium supplementation, especially in the older female population, may enhance gallstone formation.     

Bile acid alterations in patients with cholesterol gallstones

Study of individual bile acid ratios in eight patients with cholesterol gallstones in functioning gallbladders and lithogenic bile leads to the conclusion that the colonic phase of the EHC is altered in these patients, compared to six control subjects. Whether or not the changes observed are of a primary nature, or are merely secondary to the gallstones causing minimal disturbance of gallbladder storage function is not apparent from this investigation.