Lithogenic Diet Research Articles

Cholelithiasis in mice: Effects of different chemicals upon formation and prevention of gallstones

Prevention of gallstones induced in mice by 1% cholesterol and 0.5% cholic acid (lithogenic diet) for 8 weeks was obtained by simultaneously feeding sulfaguanidine (1.5%) and dodecyl sodium sulfate (0.5–1.0%) along with the lithogenic diet. Citrus pectin (3%), egg lecithin (1%), n-octyl alcohol (1%), neomycin sulfate (0.2–0.5%) and l-ascorbic acid (5%) added to the lithogenic diet did not prevent gallstone formation. The condition of the liver, fatty or normal, in the experiment could not be correlated with the stone formation. Lower serum and liver cholesterol levels and an elevation of lecithin concentrations in serum was noticed in mice fed the sulfaguanidine and dodecyl sodium sulfate diet.

A scanning electron microscopic study on the formation of cholesterol stones.

Sediments from the bile of mice fed a lithogenic diet, and of patients with cholesterol stones were observed under a scanning electron microscope. In the murine bile, single cholesterol crystals appeared at 1 week after the start of lithogenic diet. At 2 weeks, clusters of a few cholesterol crystals were seen. They gradually grew into clusters of many cholesterol crystals in 6 weeks, and finally into cholesterol stones at 20 weeks. In the sediment of the bile from cholesterol stone patients, single cholesterol crystals and clusters of various sizes were observed. From these results, the process of cholesterol stone formation in the bile of man and mouse was presumed as follows: Single cholesterol crystals appear as the first step, then clusters of crystals are formed by the process of new crystal growth on the surface of older crystals or by the coagulation of crystals by cohesive force, and finally cholesterol stones are formed through a repetition of these processes.

The effect of phenobarbital on cholesterol gallstones in hamsters.

This experimental study was undertaken to follow the influence of phenobarbital on bile chemistry and gallstone formation. Phenobarbital (400 mg/kg/day) was administered to golden hamsters receiving a diet known to induce cholesterol gallstones. After a 28-day period none of the control animals had gallstones whereas five of 12 animals with added phenobarbital developed stones. Ten of 19 animals on the lithogenic diet formed gallstones but when phenobarbital was added gallstones occurred in 16 of 19 hamsters. The administration of phenobarbital resulted in the production of bile which was relatively more saturated with cholesterol, the bile salt + phospholipid: cholesterol of 24.8 +/- 12.7 in control animals altering to 9.1 +/- 6.2 in animals receiving phenobarbital. Hepatic bilirubin uridine diphosphate-glucuronyl transferase activity was not increased in animals on phenobarbital. It was concluded that, in the hamster at least, there is no indication that large doses of phenobarbital will reduce the potential for gallstone formation.

Alterations in biliary lipids of mice during dehydrocholic acid feeding

Mice were fed a lithogenic diet consisting of Purina chow and 0.5% dehydrocholic acid (DHA group). Controls received Purina chow. Every 2 wk for 20 wk animals were killed, and biliary phospholipid, cholesterol, and bile salt concentrations were determined, as well as the extent of gallstone formation. With time there was a gradual, significant decline in the concentration and the relative composition of phospholipid in both groups compared with initial values. There was a significant increase in biliary cholesterol concentration and relative amount in the DHA group compared with the control. No significant differences were found in the relative amounts of bile salt or phospholipid between the two groups. Feeding DHA resulted in an increased concentration of bile salts and the sum of measured lipid compared with controls. After 8 wk, gallstones were found in approximately 60% of autopsied animals and correlated with increased cholesterol concentration. Our data support the hypothesis that there is a component of cholesterol secretion that may not be bile salt- or phospholipid-dependent. Our data also suggest that biliary phospholipid secretion decreases with age.

Biliary Excretion of Bile Acids, Lecithin, and Cholesterol in Hamsters with Gallstones

Young hamsters were fed an essential fatty acid-deficient diet. The incidence of cholesterol gallstones was 68%, 100% when 0.1% chenodeoxycholic acid was added, 40% with 2% ethyl linoleate, and zero with 0.1% hyodeoxycholic acid. Relative hepatic bile concentrations of cholesterol averaged 8.8 moles % in the lithogenic group and 3.6 moles % in controls. Both cholesterol to lecithin and lecithin to bile acid ratios were increased (means 0.573 and 0.216 compared with 0.280 and 0.164 in controls). Hyodeoxycholic acid caused extremely high concentrations of cholesterol and lecithin (25.8 and 21.3 moles % compared with 3.6 and 13.5 in controls). During intravenous sodium glycocholate infusion at both 0.02 and 0.2 μmole per min the lithogenic group had high lecithin to bile acid ratios (0.179 and 0.115 versus 0.102 and 0.069), but the highest ratios occurred after hyodeoxycholic acid (0.468 and 0.246). Cholesterol output in response to bile acid secretion was also increased in the lithogenic group and markedly increased after hyodeoxycholic acid. Acute infusion of sodium glycohyodeoxycholate in chow-fed animals caused a much greater increase in lipid output than infusion of glycocholate. The lithogenic diet appears to: (1) increase the availability of cholesterol, and (2) increase the amount of lecithin carried into the bile by bile acid secretion. Hyodeoxycholic acid causes extremely high biliary lipid output (possibly by a toxic effect on membranes), and thus may prevent solid gallstone formation by causing cholesterol to be excreted as a liquid crystal or gel.

Liver morphology and gallstone formation in hamsters and mice treated with chenodeoxycholic acid.

Hamsters fed a lithogenic diet showed a significantly stronger tendency to gallstone formation if the diet was supplemented with 0.5 per cent chenodeoxycholic acid. In hamsters and in mice the addition of chenodeoxycholic acid to the diet resulted in a rapid and drastic shift towards this bile acid in the animals' bile. Mice were fed normal chow with supplements of 1 per cent cholesterol, 1 per cent cholesterol plus 0.5 per cent cholic acid and 1 per cent cholesterol plus 0.5 per cent chenodeoxycholic acid respectively. After four months, slight fatty degeneration with occasional cholesterol crystals were observed in the livers of the animals of the first group. In the second group there was marked fatty degeneration with a massive accumulation of cholesterol crystals. In the third group, the liver histology was essentially normal. Gallstones were present in the second group and absent in the other two. In a regression experiment, hamsters were first fed chow supplemented with cholesterol and cholic acid. After one month they were divided into two groups which were fed chow supplemented with 0.5 per cent chenodeoxycholic acid and unsupplemented chow respectively. When the animals were sacrificed 40 days later the massive accumulation of cholesterol crystals, induced by the cholesterol cholic acid containing diet, was still present in all animals fed chow. In the hamsters fed chow supplemented with chenodeoxycholic acid there were far less such crystals. The conclusion is that with chow as the basic diet a chenodeoxycholic acid supplement aids the hamster in freeing itself from excess cholesterol. On the other hand, inflammatory‐ changes of the liver were present in many of the chenodeoxyholic acid treated hamsters while they were missing in the controls. The same experiment was repeated with a gallstone provoking diet as the basic diet during the regression period. With this basic diet, chenodeoxycholic acid did not have any effect on the accumulation of cholesterol crystals in the liver. It did, however, result in the development of inflammatory changes in the liver, comparable to those observed in animals fed lithocholic acid. The results of these experiments perhaps suggest that the basic diet may be an important factor also in the clinical trials with chenodeoxycholic acid which are now carried out. They also stress the importance of careful control of liver function and morphology during these trials.

Acute renal ischemia and experimental stone formation (author's transl)

The influence of an acute renal ischemia on experimental stone formation has been proved in 60 rabbits. In the ischemically damaged kidney there was no additional or increased stone formation. A temporary renal ischemia has no additional influence on urinary stone formation in the case of lithogenic diet or damage.

Cholesterol metabolism in rabbits with oleic acid-induced cholelithiasis.

SummaryCholesterol metabolism has been studied in rabbits with bile acid gallstones induced by feeding a high oleic acid diet. Lithogenic rabbits showed hypercholesterolemia, compared with control rabbits, which was accompanied by a striking increase in the liver cholesterol concentration. The lithogenic diet contained no supplementary cholesterol and hepatic cholesterol synthesis was markedly inhibited. Since intestinal cholesterol synthesis was unchanged in lithogenic rabbits, it is suggested that the increase in plasma and liver cholesterol concentrations was due to increased absorption of cholesterol by the presence of 15% oleic acid in the lithogenic diet. The significance of the raised plasma and liver cholesterol concentrations in relation to the 2-3 fold increase in cholestanol concentration in the livers of lithogenic rabbits remains to be elucidated.The authors are grateful to Mr. A. Shaw for technical assistance.

Biliary Mucous Substances in Dihydrocholesterol-Induced Cholelithiasis

Mucous substances have been studied in dihydrocholesterol-induced cholelithiasis by measuring hexosamines in gallbladders, gallstones, and bile and by the use of light and electron microscopy of the gallbladder mucosa. This approach permitted the study of mucous substances prior to and during gallstone formation. In response to the lithogenic diet, the gallbladder epithelial cells secreted mucous substances in increased amounts before gallstones were formed. The enhanced secretion continued during the period of gallstone growth. The stimulus for enhanced secretion appeared to be contained in the bile because increased secretion did not occur if pathological bile was not allowed to enter the gallbladder. The findings support the hypothesis that mucous substances may play a role in gallstone formation.

Gallstone formation in the conventional mouse: The role of bacteria

Aerobic and anaerobic cultures of the gallbladder wall and bile of conventional mice on a lithogenic diet did not show growth. It is unlikely that bacterial cholecystitis or a bacterial nidus has any role in gallstone formation in the mouse.

Gallstone formation in the germ-free mouse

Higher serum cholesterol levels developed in germ-free mice fed a lithogenic diet than in conventional mice, but gallstones developed more slowly than in conventional mice fed the same diet. Cholecystitis occurs in the absence of bacteria as a result of a high cholesterol diet.

Dietary production and dissolution of cholesterol gallstones in the mouse.

Groups of animals fed lithogenic diets were sacrificed at intervals, with serum and bladder bile obtained. Bile was analyzed for bile acids, lecithin, free fatty acids, and cholesterol concentrations, serum for cholesterol and free fatty acids. Gall bladders were also examined for histological change. The chemical response to the diets was a prompt increment in serum and bile cholesterol concentrations. After an initial rise, bile acid content of bile decreased, resulting in a fall in bile acid-to-cholesterol ratios to the point of cholesterol precipitation. Stones once formed dissolved when animals harboring stones were fed Purina laboratory chow. Histological changes appeared in the gall bladder after 2 weeks on the diets and were progressive.

Alimentary production of gallstones in hamsters. 6. Disappearance of cholesterol stones by treatment with a non-lithogenic diet.

Summary.As shown earlier by us, gallstones consisting of cholesterol and/or pigments can be produced and prevented in hamsters by dietary means.It has now been shown that cholesterol stones and cholesterol from amorphous stones developed in the gallbladder of hamsters by a lithogenic diet can be dissolved in the bile by changing the diet to a non‐lithogenic type.