Leo Hrdina

Liver Autolysis in Vivo.

The experimental work of Mason 1 called our attention to the extremely toxic reaction produced by the implantation of liver into the abdominal cavity of animals. Ellis and Dragstedt 2 showed that death of these animals was not strictly due to toxemia but there was a constant finding of large numbers of organisms of the B. welchii group in the abdominal cavity and drew attention to the fact that this B. welchii organism was a normal inhabitant of the livers of dogs. They implanted fetal liver into the abdomens of dogs and also adult livers which had been previously autoclaved and found that no toxic reaction ensued. Our experiments were undertaken with a view to establishing the mechanism of death in peritonitis due to liver autolysis. It was found earlier in the course of these experiments that if the liver was ground and allowed to autolyse and then sterilized that its implantation into the abdomen caused the same picture of an overwhelming B. welchii peritonitis as when the infected liver had been used. The seeming contradiction in the work of Ellis and Dragstedt is probably due to the fact that in their experiments the hard cooked mass of liver had less exposed surface and did not undergo autolysis so promptly. There is, therefore, some mechanism at work by which the ordinary flora of the intestine as represented by the Welch bacilli are allowed to make their way through the intestinal wall and infect these masses of autolysed liver which are shown to have been previously sterile by culture. The following attempts were made to isolate the toxic factor in this reaction. 100 gm. specimens of liver were ground and incubated for 24 hours and then extracted with large amounts of water.

The etiology of gall-stones. 1. Chemical factors.

The problem of gall stone formation is the problem of the precipitation of cholesterol out of the bile. An explanation is only-possible when we know how the cholesterol is held in solution in the bile. The bile differs from all the other body fluids in that it contains large amounts of bile salts which, for a long time, have been known to have the power to hold many ordinarily insoluble substances in solution. Long ago Wieland and Sorge 1 showed that desoxycholic acid, a split product of the glyco- and taurodesoxy-cholic acids occurring in bile formed with various substances, insoluble in water, a soluble addition compound. We have investigated the question as to whether one certain bile acid or all bile acids are responsible for the solubility of the cholesterol in the bile. We have found that when bile acids are precipitated from human gall bladder or fistula bile by FeCl3, lead acetate by acidification, or by any other means there is a simultaneous precipitation of cholesterol. This can easily be proved by dissolving the bile acid fraction in alcohol and adding digitonin, which brings down a precipitate of cholesterol-digitonid. The complex of bile acids and cholesterol is readily soluble in water and may be purified by various methods without lessening the cholesterol content. These substances have about the same cholesterol content, 6-9%, without regard to the form of bile acids from which they are made. These same products may be prepared from pure bile acids. We have made these complexes from cholic, desoxycholic acid, glycocholic and taurocholic acids. The properties of these substances are very similar. The bond between the cholesterol and the bile acid is a very loose one and even in the preparation there is very often a slight loss of cholesterol.

Effect of Prolonged Stasis on the Bile Salt-Cholesterol Ratio

In our studies of the relative concentration of cholesterol in the bile and bile salts which hold it in solution, the element of time in which the bile lies in the gall bladder is important. If prolonged stasis produces any differential absorption it would profoundly influence the likelihood of cholesterol precipitation. As shown in previous papers 1 , 2 the ligature of the cystic duct in a dog brings about marked infection in the gall bladder but common duct ligature, although it usually results in the presence of bacteria in the bile, only occasionally causes a marked cholecystitis. In the series of experiments reported below pathological studies of the gall bladders are reserved for a later paper and only the chemical investigations are included here. Fifty-one dogs were used and the common ducts ligated and the animals sacrificed or died at various periods. It can be seen from these data that there is no differential absorption of either bile salts or cholesterol. Prolonged stasis does not cause any change in the bile salt-cholesterol ratio, and hence cannot be considered as a factor in gall stone formation per se. Other points to be noted are the wide fluctuations in both the bile salts and cholesterol but the tendency of the ratio between the 2 to remain at a fairly definite level. Of 51 operated dogs 25 died and 26 were killed. In the 2 series the average cholesterol content was the same but in the dogs who died the average bile salt content was 25% lower. This observation is of importance as it throws considerable doubt on chemical examination of post mortem material whether human or experimental in studies of this sort.

Etiology of Gall Stones. III. Bile Salt-Cholesterol Ratio in Human Gall Stone Cases.∗

If, as has been suggested in a previous paper, cholesterol stones are formed in the human gall bladder because there were not enough bile salts present to hold the cholesterol in solution, one would expect to find in human gall bladders with cholesterol stones a lesser quantity of bile salts. The accompanying table amply confirms this expectation and corroborates the findings of Newman. 1 Newmans studies were all upon autopsy material, and for that reason might have been questioned. The first series here given are all on fresh surgical specimens and hence not open to the criticism of agonal changes. As controls in these studies it is of course impossible to obtain bile from normal human gall bladders, and the autopsy findings of Newman must serve. In his series the controls from normal human gall bladders gave a bile acid-cholesterol ratio of from 10 to 24, and his lowest figures of bile acid content of normal gall bladders were nearly double our highest one in a stone containing gall bladder. As a further control the figures cited by Hammarsten on 2 cases of sudden accidental death are interesting. His analyses showed ratios of 10 and 40 respectively and the total bile acids were 8.2% and 8.7%, approximately 16 times as high as the average of our operative cases. It is further very interesting to note that in several of our specimens the bile contained a large amount of cholesterol crystals and those cases had little or no bile acid in the bile. This was true not only in 2 cases with little or no pigment in the bile as a result of long cystic duct obstruction, but in another bile which appeared absolutely normal grossly except for the presence of cholesterol crystals and on chemical examination proved to contain but 42 mg.% of bile salts.

Is Cholesterol Excreted by the Gall Bladder Mucosa

Conclusions The normal gall bladder mucosa secretes calcium in large amounts and cholesterol in negligible amounts.

The Etiology of Gall Stones. II. Role of the Gall Bladder.

In the preceding paper it was shown that the greater content of cholesterol in human bile than in that of animals was associated with the formation of a water soluble complex with bile salts. By this means more cholesterin is held in solution. It is obvious therefore that the maintenance of the cholesterol in solution is dependent on the concentration of bile salts in the bile and that if it falls below a certain level the cholesterol will be precipitated. The following studies of the differential absorption rate of bile acids and cholesterol seem to show that the gall bladder under certain conditions may play the major part in gall stone formation. It will be seen that under normal conditions the gall bladder mucosa in its action in concentrating the bile is able to absorb water, of course, rapidly, and the bile salts and cholesterol, if they are absorbed at all, are absorbed at the same rate. In other words the cholesterol content of the bile rises no faster if as fast as the bile salt content, so that there is a wide margin of safety, and no danger that there will not be enough bile salts to hold the cholesterol in solution. When the gall bladder is diseased a diametrically opposite action takes place. In this series, the cystic duct was ligated which, as we have shown, 1 leads at once to the infection of the gall bladder. Such a gall bladder with the added insult of the insertion of a needle and ligature of the needle hole looks grossly thickened and diseased at the end of 24-48 hours. In some of the experiments it was also traumatized by scraping its mucosa. In this series of experiments an entirely opposite differential absorption took place.

Effect of Stasis on the Calcium Content of the Bile.

When bile is stored in the gall bladder its solid constituents undergo a 6 to 8 fold concentration due to the absorption of water. This should produce very high calcium levels theoretically, unless some of the calcium was also absorbed. The solution of this problem is of interest on account of the calcium content of gall stones. Dogs were anesthetized with ether, the abdomen opened and the cystic duct clamped immediately to prevent emptying of the gall bladder. Then the common duct was opened, cannulae inserted and sufficient liver bile collected for analysis. In 5 experiments the average content of the liver bile on fasting animals was 14.6 mg. per 100 cc. while the simultaneous gall bladder bile contained 52.2 mg. per 100 cc. This is a concentration of about three and a half times, considerably less than the total concentration of the bile, indicating that some calcium as well as water had been absorbed. The average concentration of normal cystic duct bile in 10 control electrocuted fasting animals was 47.7 mg. In 11 animals in whom the cystic duct had been ligated for varying periods the average calcium content was 39.2 mg. per 100 cc. indicating that a progressive absorption of calcium was taking place. These figures do not truly represent the amount of absorption as the total volume of the gall bladders was always much less after cystic duct ligation. This of course could not be measured quantitatively but it was grossly quite apparent. Jaundice had no effect on the procedure. When common duct ligation as well as cystic was done the average for the series was 41.6 mg. per 100 cc. Common duct ligation alone leaving the cystic duct open gave an average of 39.9 mg. per 100 cc. for the cystic bile.

A Method for Estimation of Both Bile Salts and Cholesterol in Small Amounts of Bile

In experimental and human studies on the chemistry of the bile, it often happens that only very small amounts of bile are available in the gall bladder, as the contents of the diseased viscus are frequently very scanty. It is in these very ones that analysis of the bile may be the most important. The following method enables one to determine both the bile acids and cholesterol in a single cubic centimeter of bile. Following the suggestion of Dr. F. C. Koch the marked affinity of petroleum ether for bile salts was utilized. The principle is that petroleum ether has the power of making a quantitative separation of the bile acids and cholesterol in alcoholic solutions. The procedure is as follows: One cc. of bile is diluted with 6 cc. of alcohol, brought to a boil and filtered to deproteinize. The filtrate is then shaken in a separatory funnel with 30 cc. of petroleum ether and extracted 3 times. The alcoholic fraction is then warmed to drive out the petroleum ether and used for the bile acid determination. The ether fraction (about 100 cc.) containing the cholesterol is placed in an ordinary 37° incubator and will evaporate to dryness in 30 to 45 minutes, as its boiling point is 25° to 65°C. It is then dissolved in chloroform, made up to 5 cc. and used for an ordinary colorimetric cholesterol estimation. The pigments remain in the alcoholic fraction. Thirty-five samples of bile, normal and abnormal from dogs and humans have been studied for controls of this technique. In no case could any test for bile acid be obtained in the ether fraction or for cholesterol in the alcohol fraction. In 2 cases difficulty in separating the layers in the funnel occurred. This in each case was overcome by the addition of 2 drops of water.

Experimental Hepatogenous Cholecystitis.

Gall bladder bile in dogs cultured in our laboratory has been shown by Rewbridge 1 to be almost invariably sterile. In a series of experiments in which biliary stasis had been present for varying periods of time B. welchii and lesser numbers of cocci and B. coli were grown from the bile in 76% of cases. Biliary stasis had been produced not only by ligating the cystic duct, care being taken to avoid the cystic artery, but also by common duct ligation and by both procedures. In most of these experiments not only were these bacteria present but marked evidences of cholecystitis were found. Gross specimens of these gall bladders revealed the interesting fact that the infection undoubtedly made its way into the gall bladder not through the bile or the blood stream but by direct extension from the liver. This could be seen by killing the dogs in various stages of the process. In the early stages (1-4 days), the gall bladders presented the picture of a cholecystitis only on the hepatic surfaces. The free peritoneal side at this period is thin, normal in appearance both grossly and histologically. The hepatic surface is thickened, often to 10 times normal and its wall shows edema and infiltration of leucocytes and round cells. Later the inflammation extends around the entire circumference of the viscus. The mucosa generally remains quite normal even in the later stages when the other layers of the wall show marked inflammation. The work of Dragstedt 2 and later of Andrews 3 on the rich anaerobic flora of the liver has shown that these organisms are constantly present and it now becomes evident that in stasis they may make their way into the gall bladder in large numbers.

Effect of Pancreatic Juice on Absorptive Mechanism of the Gall Bladder

Wolfer 1 showed that pancreatic juice injected into the gall bladder of the dog caused marked histologic changes in the wall of the viscus. The possibility of this being the cause of cholecystitis clinically was thoroughly discussed by him. The secretory pressure of the pancreas is much higher than that of the liver 2 and in case of blockage of the ampulla by stone, anatomic arrangements are such in a high percentage of human subjects that the gall bladder might fill with pancreatic secretions. 3 , 4 Our studies on 19 dogs were aimed to determine if the presence of pancreatic juice would affect the permeability of the gall bladder to the constituents of the bile, especially the bile acids. The pancreatic juice (kindly furnished by Dr. L. R. Dragstedt) was activated and contained about 1000 units. It was injected directly into the gall bladder by needle puncture in some cases, in others, through a ureteral catheter up the common and cystic duct. Four cc. were used. The duration of the experiments was 4 days. The results were as follows: 1. Pathological changes of mild degree were evident in the gall bladder wall in most cases. These, however, were not marked. The susceptibility of the dogs gall bladder to slight stasis and infective influences, as we have previously noted 5 is so marked that it would be hazardous to assert that the changes found were not due to the manipulations. 2. In some experiments the juice was Berkefeld filtered. In these experiments the pathological and chemical changes were much less than from the unfiltered juice. 3. The average cholesterol content of the bile was not reduced, but the concentration of bile acids was reduced to less than one-third of the values found in control experiments.