COLLOIDAL PHENOMENA I N GALL STONES BY HARRY B. WEISER AND GEORGE R. GRAY
The importance of colloidal behavior in the formation of gall stones and other concretions has been recognized for a long time. Thus Hippocrates and Galen, the fathers of medicine, attributed such formations to an accumulation of mucus which clung to the organ and served as a nucleus for the stone which subsequently formed. The first experimental evidence which indicated the r61e of colloids in concrement formation was obtained by A. von Heyde who dissolved out the crystalline material from urinary calculi and observed a residual framework. This was recognized quite clearly by Meckel von Helmbach in his book on Microgeologie published in 1856. Thus he writes: “Two basic factors underlie the formation of every true gall or urinary stone; first, the presence of an organic substance, mucus, in which there may be deposition of salts; second, a suitable urinary or gall fluid to serve as the mother liquor for these sediments. The decomposable organic substance. mucus, is unquestionably necessary, because urinary salts and gall substances of themselves can yield only crystalline, pulverulent or granular precipitates and never larger pieces. Stones are formed only when an organic binder is carried down too.” While the presence of an organic binder resulting from inflammatory processes has been definitely established as essential for the formation of certain types of gall and urinary stones,’ it was demonstrated twenty years ago by Aschoffz and by Schade3 that both gall and urinary calculi may form under certain conditions without being accompanied by inflammation. In this paper the colloidal phenomena which are concerned with the formation of gall stones without coexisting inflammation will first be reviewed, after which gall stones formed during inflammation will be considered especially from the point of view of the mechanism of the formation of the concentric rings or layers in such stones. Gall Stones formed without Inflammation When stones are formed in the gall bladder without the coexistence of inflammation there is usually but a single stone composed largely of cholesterol. Such stones are called “pure cholesterol stones” although they usually Pfeiffer: t h Cong. Int. Med., Wiesbaden (1886);Posner: Arch. klin. Med., 5, (188j); 16, (1889); daunyn: “Klinik der Cholelithiasis,” Lei zip (1892); Gilbert and Dominici: Compt. rend. soo. biol., 28, 1033 (1893)‘ Moritz: 14th zona. Int. Med., Wiesbaden (1896); Gilbert: Arch. g6n. de MBd., 2,257 (1896);Gilbert and Fournier: Prease Med., 7,259 (1898); Mignot: Arch. g6n. de MBd., 2, 129,263 (1898);Schreiber:Virchow’sArchiv, 153,147(1898); Cushing: Bull. Johns Hopkins Hosp., 10, 166 (18991. * Aschoff and Bacmeister: “Cholelithiasis” (1909); Kleinschmidt: “Die Harnsteine” (1911). Munch. med. Wochenschr., Nos. I and z (1909);Kolloid-Z., 4, 175, 26 (1909);Kolloidchem. Beihefte, 1, 371 (1910);Alexander’s “Colloid Chemistry,” 2, 801 (1928);cf., also, Boysen: “Uber die Struktur and Pathogenese der Gallensteine” (1909).
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contain small amounts of alkali and calcium cholates, bile pigments, etc. Fig. I shows IL cross section of a stone which is nearly pure cholesterol. The specimon is white, hence is free from bile pigments. A portion of the stone was found to he almost completely soluble in et her, which indicates the absence of alkali or calcium cholates. A prohahlr mechanism of the formation of such "pure cholesterol stones" was suggested by Ord' and was rxtended and formulated by Schade.? The
Fro.
,
Cronx-Section of Pure Cholesterol Stone.
(x
hi
latter demonstrated that when a z to 6 per cent solution of cholate is saturated with cholesterol a t approximately 80" and the solution cooled mpidly, a cloud of formless white flocs of cholesterol separates out; if the cooling is quite slow relat.ively large crysrals are obtained. On thc other hand, if the hot cholatecholesterol solution is shaken with a drop of olive oil, gall fat, or petroleum oil, and then cooled, the cholesterol separates in myelin-like drops with t,hr oil phase. These drops, which at. first are transparent soft hodies, conleacc and undergo cryst,allization with the separation of fat, giving radially cryst,alline spheres of almost. pure cholestcrol. A similar phenomenon apparently takes place in stasis of the bile. This secretion which a t first possesses a slight alkaline reaction consists of a mixture of alkali cholates, bile pigments, eholesterin, lecithin, fats and soaps, as well as electrolytos which are widely distributed in the body fluids. The cholesterol is colloidally dispelaed in the bile fluid by t,he protect,ive action of the alkali cholates. Now in stasis of the hilc, it has been demonstrated by the surgeon
* "The Influenee of
Colloids on Crystalline Form and Gohenion" (r879). lKolloidehem. Beihefte. 1, 37s (rsioi.
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HARRY B. WEISER AND GEORUE R . GRAY
and pathologist that the amount of cholates diminishes gradually owing to resorption and autolysis, leaving a fluid which may be clear and almost cholate free. This gradual disappearance of cholates leaves a supersaturated solution of cholesterol which separates out.]. But owing to the presence of fat and soaps in the fluid, the separation is in the form of drops which coalesce and subsequently crystallize initiating “pure cholesterol” gall stones. From the above it would appear that stasis of the bile may in itself lead to the formation of gall stones especially if there is an impairment of liver function giving a bile deficient in cholates. It is probable that there are other contributing causes not associated with inflammation, which favor the separation of the cholesterol, in such a condition that stones result. Thus Thudichum2 points out that a stagnant and infected bile will decompose giving an acid reaction. This would convert the cholates into insoluble cholic acids which would allow the cholesterol to precipitate. In this connection Wrada,3 Neilson and Meyer‘ and Rous, McMaster and Drury6 found that bile taken from the gall bladder was slightly acid in a large proportion of cases while that taken from the hepatic duct was nearly always somewhat alkaline and usually markedly so. Whatever may be the cause of the normal change in pH in the gall bladder, it occurred to Petersene and Miller that gall stones might be formed by altering definitely the pH value of the bile. A distinctly acid bile was obtained in a number of experimental animals by grafting a pedicled flap of the stomach to the gall bladder wall and an alkaline bile by anastomosis of the gall bladder to a portion of the duodenum. The bile from gall bladders containing the duodenal transplants was regularly alkaline and was more viscid than normal bile; but no evidence of stone formation was observed in six months. The gall bladders having the gastric transplants always yielded an acid bile. No definite concretions were obtained but the gall bladder wall appeared reddened and congested, with cholesterin particles scattered over the entire surface. This condition is what is known clinically as “strawberry gall bladder,” recognized as a forerunner of gall stones. These observations of Petersen disprove the contention of Porges’ that free, unneutralized acid will not remain in contact with the mucous membrane of the gall bladder and the claim of Bacmeister* that the addition of small amounts of acid to the bile is unsuitable for bringing about the precipitation of cholesterol. The hypothesis that the destruction of cholates by bacterial action contributes to the formation of gall stones as claimed by E w e r and Heirovosky8 and Bondi and HesslO on the basis of observations in vitro, is open to the Cf. Oliver: J. Lab. Clin. Med., 8, 242 (1923). Virchow’s Archiv, 156, 384 (1899). J. Physiol., 50, 114 (1915). 4 J. Infect. Dis., 28, 5 1 1 (1921). J. Exp. Medicine, 39, ( I ) 77; (2) 97; (3) 403 (1924). e Unpublished paper. Kolloid-Z., 5, 301 (1909). * Mtinch. med. Wwhenschr., Nos. 5, 6, and 7 (1908). Arch. klin. Chu., 86, 6og, 643 (1908). lo Wiener klin. Wwhenschr., 21, 271 (1908).
’
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object,ion that even under the extreme conditions chosen by them, there still remained enough cholates to keep the cholesterol dispersed.’ As a result of chemical-pathological investigations, Chauffard* observed that stone formation in the absence of inflammation is frequently accompanied by a pathological condition which results in an increase in the cholesterol content of the blood. I n such cases the primary cause is a disturbance in the metabolism. Since cholesterol is colloidally dispersed in the bile, Porges3 attempts to explain the precipitation by assuming the coagulating action of substances on the sol. His suggestion follows from analogy with the behavior of hydrosols of lecithin and cholesterol toward electrolytes. The difficulty is that one must postulate the presence in the bile of an amount of coagulating electrolyte considerably larger than is likely to be present. While all of the above factors may contribute to the formation of gall stones in the absence of inflammation, it seems probable that sufficiently marked stasis of the bile to allow of resorption of the cholates is adequate in itself to cause stone formation provided the conditions are favorable for the separation of the cholesterol in myelin-like drops which coalesce and subsequently undergo crystallization. This would seem to be particularly true if the bile were deficient in cholates or especially rich in cholesterol as a result of impaired liver function. I n addition to the pure cholesterol stones, pure pigment stones‘ and pure calcium carbonate stones5 are formed under certain conditions without apparent inflammation. Peel6 analyzed some pigment stones of this kind and found them to be entirely free from cholesterol. They contained some free pigment but were largely calcium and copper bilirubinate. The following description of these stones indicates that like pure cholesterol stones, they are formed as a result of the dropwise separation of the insoluble calcium salts Of the bile pigments: “They vary in size from that of a pin-head to a cherry seed and possess a fine granular surface such that they appear as if they were built up of several small globules. I n a fresh condition they are somewhat hard; on drying, the hardness increases enormously. I n cross section they appear uniform throughout. They show neither radial nor concentric layers nor a distinguishable central core.” The formation of the pure calcium pigment stones is probably favored by a disturbed metabolism which gives a bile relatively rich in pigments and in calcium and possibly copper salts.’ It is probably true that this condition alone without inflammation would not cause the calcium pigment to precipitate as a concrement unless the initial separation was in the form of drops. Porges: Kolloid-Z., 5 , 301 (1909). Lepons sur le lithiase hiliare” (1914). 3Kolloid-Z., 5 , 301 (1909); Porges and Xeuhauer: Biochem. Z., 7, I j2 (1907). Boysen: Uber die Struktur und Pathogenese der Gallensteine” (1909; Bacmeister: ErRebn. inn. Med., 11, I (1913). -5 Halpert: Arch. Path., 6, 630 (1928). Z. physiol. Chem., 167, 269 (1927). Peel: 2. physiol. Chem., 167, 274 (1927). ?
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HARRY B. WEISER AND GEORQE R. GRAY
Gall Stones of Inilammatory Oriffin
An inflammation in the bile ducts or the gall bladder introduces into the bile irreversibly precipitating hydrophilic colloids such as serum albumin, globulin, and fibrin. If cholesterol or calcium bile pigments separate in the presence of such colloids, there is mutual adsorption with the result that the whole is united into a coherent mass giving what is sometimes termed a colloid-crystalline stone as distinct from the pure crystalline stone considered in the last section. Since gall stones are usually of inflammatory origin, it follows that colloid-crystalline stones are the type most commonly found in the gall bladder. As has been pointed out, the formation of stones in the absence of inflammation is probably preceded by the separation of the stone-forming material in a drop-like form. A similar condition may obtain when inflammation is present but in the latter case dropwise separation is not essential since the colloidal material resulting from the inflammatory process may bind the mass firmly into a concrement. Indeed, Schadel produced stones artificially by allowing coagulation of fibrin to occur in solution in which freshly precipitated, sediment-like discrete crystals were suspended. The importance of the colloidal binding material in concrement formation is shown by dissolving out first one constituent and then the other from a colloid-crystalline stone. If the crystalline material is removed with a suitable solvent, a firm coherent skeleton of colloidal matter remains which shows the details of structure of the stone. On the other hand, if the albuminous skeleton is dissolved out by antiformin, there is complete disintegration, nothing remaining but a slimy mass of minute crystals.2 Since the relative amounts of the various constituents which make up gall stones of inflammatory origin may vary widely, it is obvious that the composition, appearance, and physical properties of the stone will show almost infinite variation, In a stone in which there is a relatively small amount of hydrophilic colloid, the soft irregular masses consisting largely of cholesterol, undergo solution and recrystallization, ultimately giving a radial structure similar to that in the pure cholesterol stone. Adsorption of the bile pigments or their precipitation as the calcium salts stains the stone to a greater or lesser degree depending on the relative amount of pigment adsorbed or thrown down. If the amount of hydrophilic colloids present in the stone is relatively large, crystal growth is inhibited and the formation of long crystals is prevented. Since the hydrophilic colloids lose water and shrink with time, stones containing a large amount of such colloids may become sufficiently friable that they fall to pieces, In other cases the ageing may make radial rifts leading out from the center leaving an irregular hollow space which fills with liquid. Miinch. med. Wochenschr., Nos. I and z (1909). Schade: Med. Klinik, No. 15 ( 1 9 1 1 ) . 8 The so-called calcium salt of the bile pigments is probably an adsorption complex of indefinite composition. 1
2
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The most common form of gall stones are the colloid-cholesterol-calcium pigment stones that are characterized by the presence of concentric rings varying in color. Such concretions are termed layered stones or “common gall stones.” I n these stones the kernel and the surrounding ring structure are readily distinguished. The former consists largely of albumin, calcium, bilirubin and cholesterol without the presence of a definite ring structure while in the latter, there are a number of colored rings in crystals of cholesterol. Gall stones of inflammatory origin seldom or never occur singly. In rare instances there may be but two or three stones but, as a rule, there is a much larger number, 100 or more in some cases. Because of the pressure of the gall bladder the stones are seldom spherical but are faceted and usually of widely varying shapes. I n certain cases the shape may be more or less uniform. In a collection obtained from one gall bladder there were 50 stones each of which was an almost perfect pyramid with rounded edges. The cause of the concentric rings in the common gall stones is usually attributed to layering as a result of variations in conditions which produce alternate layers of cholesterol and pigment. Schadel attributes the formation of a layered rather than a radiating structure to the effect of pressure which flattens out the crystals of cholesterol. This does not account for the beautiful regular formation of alternate dark and light rings. Moreover, in most stones of this type there is a distinct evidence of radiating structure and in many cases this is quite marked as shown in the photographs of the cross-section of stones shown in Figs. z and 3. The appearance of the concentric rings suggcsts to the colloid chemist that they may originate as a result of the rhythmic banding first described by Liesegang as a result of his observations of the formation of rings of silver chromate when silver nitrate is allowed to diffuse into a gelatin jelly containing dichromate. The possibility that the rings in gall stones are not layers but rhythmic bands has been taken seriously by few people. Schade? dismisses the suggestion promptly by contending that for the formation of Liesegang rings as a primary process, the diffusion must take place in a jelly which has no points of resistance to diffusion. This, he points out, does not obtain in gall stones owing to the presence of crystalline cholesterol irregularly included in the framework of hydrophilic colloids. Moreover] he rules out the formation of Liesegang rings as a secondary process, since this would give very irregular lines, which do not occur. Sweet3takes the position that the concentric rings are due to the Liesegang phenomenon. He gets around the difficulty confronting Schade by postulating that the cholesterol forms a gel containing calcium into which the bile pigments such as bilirubin can diffuse giving rhythmic bands of calcium bilirubin just as silver nitrate diffuses into dichromate-gelatin jelly and gives rhythmic bands of silver chromate. ~
Alexander’s “Colloid Chemistry,” 2, 817 (1928). Alexander’s “Colloid Chemistry,” 2, 830 (1928). Colloid Symposium Annual, 8,249 (1930).
H A R R Y B. WElSEH A X D GEORGE It. GHAV
COLLoiDAL PHENOMENA 1N GALL STONES
Fie. 3 Cros-Sect,ion of Nstuisl Gall Stoee. ( X
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10).
A consideration of ihe p l l stone-bile system reveals, however, a marked difference between it and the gelatin-electrolyte system of Liesegang. In thr first place, the gall stone is probably not a true jelly at any stage of its histoiy, as assumed hy Sweet. And if it did possess u true jelly structure like fhat, of gelatin, rhythmic bands could not form as a result of diffusion s i n e the hilc pigments are in colloidal solution and so diffuse but little if at all. Rchade's objection t o the handing theory on the ground that rhythmic precipitation takes place only in a jelly, does not hold since the phenomenon manifests itself not only by diffusion into jellies hut also into celatively non-unifonn amorphous and crystalline masses. Accordingly, it is a h g e t h e r possible 1,hat t,hr precipitation of crystalline cholesterol in the presrncc of hydrophilic colloids will, under certain conditions, give a NUUS into which the colloidal pigment can diffuse and hy interacting with IiNIe or ot,her calcium salt carried down with the cholesterol, precipitate colored rhyihmic hands. In other words, the very fact that, cholcstcrol is definitely crystalline may he the determining factor in producing rhythmic hands therein sincc a ma&s of small crystals would allow t,he interdiffusion of the colloidal bile pigment,s. If this is a true statement of the case it, should he possible to simulate the conditions sufficiently closely that rhythmic hands of calcium bile pigrnont will be formed in a m i i s of precipitated cholesterol containing lime. This has actually been accomplished as will he described in the following experiments.
Experimental Rhythmic Bands of Ay,CrO* in Chofeslmol. To show that rhythmic hunding will take place in a mass of cholesterol crystals, the following experiments were carried out: One gram of gelatin was dissolved in io0 cc. of water con-
2 94
HARRY B . WEISER AND GEORGE R. QRAY
taining 0.1gram of K2Cr04 and heated to 70'. Into this solution was poured rapidly 2 5 cc. of a hot alcoholic solution of cholesterol containing z grams of the pure compound. The cholesterol precipitated immediately in the form of minute crystals. These were matted firmly and uniformly in the bottom of a test tube by centrifuging and the supernatant solution was poured off. The precipitate was then covered with an 8 percent solution of AgN03. Cpon standing, the familiar rhythmic bands of Ag2CrO4were formed. The above procedure was repeated keeping all the factors constant except the amount of gelatin in the solution into which the alcoholic cholesterol was poured. The gelatin solutions contained 0.75, 0.50, 0 . 2 5 and 0.05 gram, respectively, in IOO cc. In every case rhythmic bands were formed. With decreasing amounts of gelatin the bands were broader, less distinct and further apart. When no gelatin was used, bands were not formed, but crystals of Ag2Cr04were scattered irregularly throughout the mass. Portions of the precipitate thrown down in the presence of a small amount of gelatin when placed on a watch glass and surrounded with silver nitrate gave rhythmic rings similar to those obtained by diffusion in gelatin. Since bands of silver chromate in cholesterol are formed in the presence of such minute amounts of gelatin, it is obvious that the rhythmic precipitation is not taking place in a gelatin jelly. The gelatin merely serves to inhibit the growth of the cholesterol crystals and so to give a mass of minute crystals in which the diffusion phenomenon can take place under such conditions that rhythmic bands or rings result. Rhythmic Bands of Calcium Bile Pigment in Cholesterol. Solutions of bile pigment were prepared in the following way. Twenty-five grams of finely powdered human gall stones were extracted in a Soxhlet tube with zoo cc. of ether for two days to remove cholesterol and fat. The residue was dried, washed with hot water, then with I O percent acetic acid and finally with water. This residue was dried and ground in small portions with z N NaOH and the filtered solution was used in the experiments. When a portion of the highly colored solution was subjected to dialysis in a cellophane bag, the color did not diffuse showing that it was in the colloidal state just as it is in the bile fluid. The addition of a dilute solution of calcium nitrate did not result in immediate precipitation of the calcium-pigment complex but upon standing, a precipitate settled out which varied in color from reddish brown to olive green, depending upon the degree of oxidation of the pigment. Although the colloidal bile pigments do not diffuse through gelatinous membranes, it seemed not unlikely, in the light of the above experiments with Ag2Cr04,that a suitably precipitated mass of cholesterol crystals containing calcium would serve as a medium into which the colloidal pigments would diffuse to form rhythmic bands, thus simulating the process which probably takes place in nature. This hypothesis was confirmed by the following method of procedure: Into 50 cc. of a solution containing 0.5 gram of gelatin and 0.5 gram of calcium nitrate a t 70' was poured 12.5 cc. of a hot alcoholic solution of cholesterol containing I gram of the pure compound. After prolonged centrifuging the supernatant solution was removed from the mass of precipitate and
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HARRY B. WEISER A N D GEORGE R . G R A 1
FIG.5 Synthetic Cholesterol-Calrium Pigment Stones shoring how t,he Shape of the Stone influences the Form of the Rhythmic Bands. ( x 10).
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HARRY B. WEISER AND GEORGE R. GRAY
portions of the latter were placed on a microscope slide and surrounded by the solution of bile pigments. The specimens were placed in a desiccator containing an ammonia solution to prevent their drying out and to minimize the oxidation of bilirubin to biliverdin. After standing over night rhythmic rings resulted as shown by the photographs reproduced in Figs. 4 and 5 . In Fig. 5 the effect of the shape of the mass on the form of the bands is clearly shown. In order to secure a plane surface to photograph, in some cases a cover glass was pressed down gently on top of the specimen flattening out the high places. A comparison of the synthetic preparations with the natural gall stones reveals a marked similarity in appearance which indicates that rhythmic banding plays a rBle in the formation of the natural stones just as it does in the synthetic ones. The objections may be raised that there is no gelatin in natural gall stones. The answer is that the action of gelatin is not specific. It is only necessary that a protective colloid be present to inhibit the growth of the cholesterol crystals and to serve as a kind of binder. Albumin and fibrin which do occur in natural gall stones may be substituted for gelatin ip the synthesis of banded stones: The specimens shown in Fig. 6 were prepared in the same way as those in Figs. 4 and 5 using instead of gelatin, 50 cc. of water in which was peptized 0.I to 0 . 2 gram of fibrin. Conclusions r om a survey of the conditions which result in the formation of the socalled “layered” stones and the experimental results given in the previous section, it would appear that the formation of such concretions is favored or initiated by inflammation, which yields irreversibly precipitated protein materials, such as fibrin and albumin. Pathologic changes bring about the precipitation of the cholesterol, carrying calcium with it, the nature of the precipitate depending upon the amount of hydrophilic colloid present. Into this mass the colloidally dispersed bile pigments diffuse and are precipitated in the form of rhythmic bands. The structure and arrangement of the bands is influenced by the shape of the mass, its density due to the pressure of other stones, and by variations in the composition of the bile fluid. After the bands are formed the structure may be invaded by radial crystallization of the cholesterol, cracks may develop, further deposition of the cholesterol may occur, or the stone may undergo alteration in other ways, producing the wide variety of forms which are found in gall bladder disease. summary
The following is a, brief summary of the results of this paper: I . Rhythmic rings of calcium-bile pigment and of silver chromate were obtained in a mass of cholesterol crystals precipitated in the presence of a small amount of hydrophilic colloid material such as gelatin, albumin, and fibrin.
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z . The concentric rings in the “common gall stones” of inflammatory origin are not the result of the deposition of alternate light and dark colored layers but are a manifestation of the Liesegang phenomenon. 3 . The colloidal bile pigments diffuse into a mass of cholesterol crystals, hydrophilic colloids and lime and the calcium-bile pigment complex is deposited in concentric bands. The term “layered stone” as applied to the “common gall stone” is a misnomer. 4. Concentric bands are not formed in either natural or synthetic cholesterole stones in the absence of hydrophilic colloids. 5 . The structure and arrangement of the bands in both synthetic and natural gall stones is influenced by the density and shape of the mass, the nature and amount of hydrophilic colloid present and the composition of the bile fluid. 6. A survey has been given of the rele of colloidal behavlor in the formation of the so-called “pure cholesterol” and “pure pigment” stones resulting without inflammation and of the mixed colloid-crystalline stones of inflammatory origin. The Rice Instilute, Houston, Texas.
