THE COMPOSITION OF HUMAN GALLBLADDER BILE AND ITS ...

THE COMPOSITION OF HUMAN GALLBLADDER BILE AND ITSRELATIONSHIP TO CHOLELITHIASIS

John G. Reinhold, … , L. Kraeer Ferguson, Ambrose Hunsberger Jr.

J Clin Invest. 1937;16(3):367-382. https://doi.org/10.1172/JCI100864.

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THE COMPOSITIONOF HUMANGALLBLADDERBILE AND ITSRELATIONSHIP TO CHOLELITHIASIS

By JOHN G. REINHOLD, L. KRAEERFERGUSONAND AMBROSEHUNSBERGER,JR.(From the Biochemical Division, Laboratories and the Surgical Services, Philadelphia General Hospital; the Sur-

gical Outpatient Department, University of Pennsylvania Hospital; and the Department of PhysiologicalChemistry, School of Medicine, University of Pennsylvania, Philadelphia)

(Received for publication December 17, 1936)

Chemical investigations of the activity of thegallbladder have aided notably in clarifying theetiology of gallstones. Detailed reviews havebeen published by Lichtwitz (1) and Ivy (45).Particularly important is the striking alterationof gallbladder function caused by infection of orinjury to the mucosa demonstrated in the dog bythe experiments of Drury, Rous and McMaster;Ravdin, Johnston and Riegel; and of Andrewsand associates. It has been shown by these work-ers that changes in the composition of bile ex-posed to the action of pathologically altered gall-bladders tended to diminish the effectiveness ofthe bile as a solvent for cholesterol. Thus, con-siderable experimental evidence has been pre-sented to connect the development of gallstoneswith infection of or injury to the gallbladdermucosa. The most convincing support has beenderived from experiments done upon dogs, al-though data have been published from the labora-tories of Ravdin and of Andrews showing thatsimilar processes appear to operate in the humangallbladder. Species differences, however, neces-sitate caution in drawing close analogy betweenobservations made in the presence of experimentalgallbladder injury of dogs and in human cholecyst-itis. As has been pointed out, human bile differsin composition from dog bile, and also, the re-sponse to disease and injury of the biliary tractin man is not quite the same as that of the dogunder experimental conditions. Human bile isrich in cholesterol as compared with dog bile. Itdiffers in type and amount of bile acid. Chole-cystitis in the dog causes the gallbladder contentsto become more alkaline, but in man the oppositeeffect is generally observed. Therefore, morecomplete investigation of additional human mate-rial is desirable to establish clearly the conse-quences of cholecystitis and its r6le in cholelithia-sis, as well as to correlate more closely suchobservations with the studies on animals. The

results of a chemical study of human gallbladderbile are reported in this paper. The explanationof gallstone formation provided by the work ofRavdin, Johnston, Riegel and associates and ofAndrews, $choenheimer and Hrdina is supportedby our data.

PROCEDURE

Bile was aspirated directly from the gallbladder intoa syringe by the surgeon in the course of operation.Precautions were taken to avoid exposure to air or lossof carbon dioxide. Preceding the operation, the usualsaline enemas and morphine were given. Some of thepatients received glucose intravenously in addition. Gen-erally, the patients had fasted 12 to 15 hours beforeoperation, occasionally somewhat longer.

Determinations of pH were completed within 1 to 2hours after collection of the specimen. Other tests alsowere started with a minimum of delay. Occasionalspecimens could not be analysed promptly; pH deter-minations were then omitted. Analytical findings werenot influenced appreciably by delays of several hours.The specimens were centrifuged and the precipitate, ifany, examined for the presence of crystals or otherformed elements. Measured amounts of centrifugedbile were added to warm absolute ethyl alcohol, heatedto boiling, centrifuged, decanted, and the residue ex-tracted twice with alcohol. The alcohol solution andwashings were combined and diluted to volume in avolumetric flask. Aliquots were used for the determina-tion of cholic acid, phosphorus and cholesterol. Theabsence of cholesterol esters from bile and the solventaction of the bile acids and phospholipid permit the useof alcohol alone as an extractive solvent for cholesterol.The latter was estimated by a modification of theAutenrieth and Funk method (2). Bile saponified twohours with 25 per cent potassium hydroxide was ex-tracted with ether in a continuous extractor of the typedescribed by Quick (3). Cholic acid was determined bythe modification of the Gregory-Pascoe procedure de-veloped by Reinhold and Wilson (4), using the pre-cautions recommended for analysis of human bile. Tur-bidity resulting from the presence of desoxycholic acidwas removed by addition of alcohol after color develop-ment as described in the method. Phospholipid valuesrepresent total phosphorus of the alcohol soluble com-ponerrts of bile.

367

JOHN G. REINHOLD, L. KRAEERFERGUSONAND AMBROSEHUNSBERGER,JR.

In the presence of bile acid salts alcohol appears to dis-solve completely bile phospholipid, although Mathews(5) states that alcohol precipitates cephalin ordinarily.Analytical methods that are not specifically mentionedare those listed by Reinhold and Wilson (6). Bile wasashed preliminary to determination of cations. To avoidpossibility of error due to the high concentration ofsolids, water was added to the sample taken for thechloride determination, as suggested by Sunderman andWilliams (7). Analytical results have been calculatedon the basis of the concentration per liter of bile.

Attempts to study changes in the bilirubin content ofbile of diseased gallbladders were hampered by thedifficulties of determining this substance quantitatively.When possible, direct comparison of the diluted bilewith solutions of potassium .bichromate was made. Thelatter were standardized with the aid of solutions ofpure bilirubin (0.025 per cent potassium bichromate wasequivalent to 0.38 mgm. per cent bilirubin). Obviously,the method was not applicable when oxidation of the bilepigment had occurred.

The specimens analysed were derived prin-cipally from pathological gallbladders showingevidence of inflammatory disease. In addition,specimens from gallbladders showing no distinctpathological changes, as well as specimens fromgallbladders presenting cholesterosis or neoplasticdisease, have been examined.

To demonstrate the cumulative nature of thepathological changes in cholecystitis, data are ar-ranged in Tables I to III according to the absenceor presence and the severity of cholecystitis.Table I includes data for a group of controlspecimens. In Tables II and III are shown fig-ures for specimens taken from gallbladders ex-hibiting moderate or marked evidence of disease,respectively. When possible, classification hasbeen made upon histological evidence. When thegallbladder was not removed or examined, theconclusions of the surgeon concerning the con-dition of the gallbladder were accepted. Histo-logically, thickening of the walls, extent of fibro-sis, number and quality of villi, and presence anddegree of inflammatory reaction and cellular in-filtration have been given the most weight. Tab-ulated according to these criteria, fair homogene-ity is observed in each group, while significantdifferences between the groups likewise becomeapparent. An alternative grouping, employed inan earlier study (Reinhold and Ferguson (8))and considered in connection with the classifica-tion of the present material, depended on the

presence of calculi in the gallbladder or of bothcalculi and obstruction. Actually, the differencein distribution of specimens according to the twoplans was not great, since only 5 of 35 gallblad-ders showing evidence of inflammatory changeswere without calculi, and only 2 failed to showcrystals of cholesterol.

The calculi encountered were almost all of thecholesterol calcium pigment type or of the choles-terol type. It was not always possible for theauthors to examine the stones removed. Theanalytical findings of Ray (9) and of Riegel,Ravdin, Johnston and Morrison (47) indicatethat there is less difference in composition be-tween various types of calculi than had beensupposed.

Composition of bile from gallbladders normal inappearance

Table I includes observations made upon bilefrom gallbladders without perceptible pathologicallesions or with slight changes. Laparotomy wasundertaken either for exploration or because ofdisease not involving the biliary tract, althoughgallbladder disease was suspected in several pa-tients due to the association of certain charac-teristic symptoms with failure to visualize thegallbladder by the cholecystographic technic ofKirklin (51). The group is heterogeneous, andthere exists a distinct possibility that abnormali-ties of bile secretion and of gallbladder functioninfluenced the composition of some specimens.Bile from normal living individuals, however, israrely available. The specimens have been ar-ranged according to cholate concentrations in de-scending order, since it is reasonably certain thatability to concentrate bile salts is an especially sig-nificant indicator of functional capacity. Thus,specimens in the upper half of the table are mostnearly normal. It is evident that figures forsome specimens in the lower portion overlap datafor specimens from patients suffering from chole-cystic diseases. Graham and Mackey (50) havepointed out difficulties of confirming at times byhistological methods the presence of gallbladderabnormalities. It is possible or even probablethat gallbladder disease existed unrecognized inthis group. Adhesions involving the gallbladderexisted in 6 patients. Crystals, either of choles-

368

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terol or bile pigment, were found in 5 specimens,althouglh always in small numbers. Neither crys-tals, adhesions, nor failure of visualization byx-ray, could be correlated with distinctive changesin composition of the bile specimens. How-ever, Johnston et al. (10), and Riegel and co-workers (47), by repeated testing, frequentlywith larger amounts of dye, have succeeded indemonstrating chemical differences between speci-mens secured from gallbladders exhibiting normalresponse to cholecystography from those with-out calculi that failed to do so. The specimensranking near the top in Table I varied onlyslightly in regard to calcium, chloride and bilesalt concentrations from two samples of normalhuman gallbladder bile analysed for these con-

stituents by Johnston, Ravdin, Riegel and Allison(10). Concentrations of bile acids in those speci-mens we consider most nearly normal (the first7 in Table I) range far above all except twovalues reported by Andrews ( 11 ) for " normal "

gallbladder bile. It is doubtful whether any ofthe specimens labeled normal by this author shouldactually be so considered.

All specimens listed in Table I contained highconcentrations of solid material. This is largelybile acid and phospholipid, if the two figures forthe latter may be considered representative. De-termined bile acid, glycocholate and taurocholate,often accounted for more than half the totalsolids. The high concentrations of these sub-stances are to be contrasted with the far lowerconcentrations found when the gallbladder mu-

cosa is perceptibly injured. Owing to the factthat the modified Pettenkofer reaction employedfor the determination of bile acids is specific forthe cholic acids (Reinhold and Wilson (4) ), otherbile acids that occur in human bile are includedin the undetermined anion.' Desoxycholic acidappears to be most important quantitatively, ac-

cording to Wieland and Reverey (12) and Doubi-let (13). Chenodesoxycholic acid (12) and litho-cholic acid (14) have been isolated from humanbile and from gallstones, although data are notavailable relative to their concentration. It is to

1 Doubilet (13) has justly pointed out that data forhuman bile based upon this method alone are incomplete.In the present study this objection is avoided becausechanges in the undetermined anion offer means fordetecting gross changes in other bile acids.

be expected that differences between normal andpathological specimens resembling those seen forthe cholic acids exist also for the undeterminedbile acids. High figures for undetermined anionin the normal and moderately pathological speci-mens do indeed support this conclusion althoughthe contrast is not as clearly defined. That thereis a change in the character of bile acids secretedunder certain abnormal conditions is suggested bythe work of Greene, Walters and Fredrickson(15), of Ravdin, Johnston, Riegel and Wright(16), and of Breusch and Johnston (17). Doubi-let (13) recently has demonstrated such a quali-tative alteration. Doubtless the high undeter-mined anion concentration, frequently observedin our series in conjunction with relatively lowcholate, is the result of a similar change in thetype of bile acid secreted by the liver.

Chloride concentrations in specimens fromgallbladders without demonstrable pathologicalchanges were considerably less than the concen-trations of organic anions. The relatively lowchloride figures may be contrasted with the highvalues observed in pathological material describedin the next section. Similar differences have beenobserved by Johnston et al. (10). The concen-tration of chloride varies inversely with that ofbile acid and compensates changes in the latter.Bicarbonate undergoes somewhat similar altera-tions, and it also may replace or be replaced bybile acid. Bicarbonate rarely exceeds chloride inconcentration and ordinarily it is considerablyless. While high bicarbonate has occasionallybeen found associated with diminished chlorideconcentrations, generally both are increased orlowered together.

The concentration of cation in bile likewise isgoverned to a considerable extent by the level ofbile acid; consequently, sodium and other cationsas well are relatively high in the specimens ofthis group. Sodium is by far the most importantof the cations so far as concentration is con-cerned, with calcium, magnesium and potassiumranking in the order mentioned when consideredin terms of milli-equivalents per liter. Mag-nesium concentrations in bile frequently outweighthose of calcium in contrast to serum where mag-nesium approximates only one-half of the calciumconcentration. Furthermore, the divalent cations

372


of bile constitute a considerably larger fractionof the total base than in serum. Concentrationsof all cations of bile normally exceed by consid-erable amounts the concentrations in serum, whilethe total cation concentration is -consistently above250 m.eq. per liter of water as compared withithe average concentration in serum of 167 m.eq.Despite this discrepancy, it has been demonstratedrepeatedly that bile and serum are iso-osmotic(see Brand (48), Strauss (49), Ravdin, Johns-ton, Riegel and Wright (16), and Gilman andCowgill (18)). The anomaly is a manifestationof the abnormal behavior of ions of low molecularweight in the presence of large ions. Hammar-sten (19) has observed that in the presence ofbile acids, as well as of other compounds of highmolecular weight, osmotic activities of smallerions are diminished.

The reaction of bile from gallbladders showingno evidence of disease was found in an earlierinvestigation by two of the writers (Reinhold andFerguson (8) ) to vary between pH 7.10 and 7.30.Although it was suggested that greater variationwas to be expected, in the present series 3 of 5similar specimens fell within these limits. The 2remaining specimens were somewhat more acid inreaction, the pH being 6.90 and 6.95. The rangeof values for specimens of this class must bebroadened to include the latter. Low pH wasassociated with low concentrations of bicarbonate.

Comparison of pH values reported for dog andhuman bile shows that gallbladder bile of dogs hasbeen found to be somewhat more acid in reaction(Okada (20) ; Drury, McMaster and Rous (21);unpublished observations of the writers) thanhuman gallbladder bile (Drury, McMaster andRous (21); Reinhold and Ferguson (8); An-drews (11)). Undoubtedly, differences in thenature and concentration of the species-predomi-nant bile acids are responsible. Human bile con-tains principally the relatively weak glycine-con-jugated cholic and desoxycholic acids. Bile acidof the dog, on the other hand, consists primarilyof the strong taurine-conjugated cholic acid. Ithas been shown that the pH of dog bile is gov-erned by the concentration of taurocholic acid(Reinhold and Wilson (6)). In human bile,because of the relatively weak character of thebile acids, such a relationship is not clearly dem-

onstrated. Interpretation of the role of bile acidin the regulation of the reaction of human bile iscomplicated by the appreciable concentration ofsuch acids included in the undetermined anion.

Carbon dioxide tensions of gallbladder bilefrequently are very high. Calculation was madefrom the data for pH and carbon dioxide con-centration by the Henderson-Hasselbalch equa-tion according to Peters and Van Slyke (22).Values of the same magnitude have been estab-lished for urine by Sendroy, Seelig and VanSlyke (23), while contents of intestinal loopsalso may have high carbon dioxide tensions ac-cording to deBeer, Johnston and Wilson (24).It would appear that carbon dioxide tensions ofsecretions may differ appreciably from those ofblood.

In normal bile, total cation concentrations con-sistently exceed the concentrations of total deter-mined anion. The undetermined anion, as alreadyindicated, is made up chiefly of bile acids otherthan cholic acid, although certain phospholipidsmay bind a portion of the base represented inthis fraction. Inorganic phosphorus, if present,exists in amounts insignificant by comparison withother anions. None was found in dog bile (Rein-hold and Wilson (6) ). The presence of ap-preciable concentrations of inorganic phosphorusin bile has been reported; however, this mayhave originated by hydrolysis of phospholipid.Riegel et al. (47) found little phosphate inhuman biliary calculi.

Aronsohn and Andrews (25) have attemptedto explain the acidification of bile in the gallblad-der as a consequence of increased concentrationof phosphorus and protein. Their conclusion,based on an increase in total phosphorus in gall-bladder bile as compared with fistula bile, is notvalid since the total phosphorus consists almostentirely of phospholipid. The latter, accordingto Hammarsten (26), is chiefly lecithin. As leci-thin does not exert appreciably acidic propertiesat the pH of gallbladder bile (Fischgold andChain (27); Chain and Kemp (28) ; Jukes (29) ),it is not permissible to assign to the phosphorusof bile the acidifying action observed. Likewise,it appears improbable that bile protein would pos-sess the powerful acidifying action implied byAronsohn and Andrews. As already noted, there

373


is considerable evidence showing that bile acidconcentration is the chief factor regulating thepH of bile, and that acidification of gallbladderbile is primarily a result of the large increase inbile acid concentration as hepatic bile is con-centrated in the normal gallbladder.

Cholesterol in these specimens from gallblad-ders without pathological changes varied in con-centration between 140 and 1010 mgm. per cent.The highest value cited was also the highest inthe entire series. The patient suffered fromcystic lymphangioma of the peritoneum. Despitethe elevated concentration of bile acids, choles-terol crystals were detected in this specimen.Also, in two other specimens where cholesterolconcentrations were not elevated, a few choles-terol crystals were seen although bile acid con-centrations were high. It must be conceded,however, that crystals could not be found in theabundance that is ordinarily characteristic of bilefrom pathological gallbladders. The importanceof the ratio between bile acid and cholesterol asa factor governing the solubility of cholesterolhas been stressed by Newman (30), Andrews,Schoenheimer and Hrdina (31), and Johnstonet al. (10). Such a relationship between cholateand cholesterol may be seen in the present series.It is noteworthy that only two specimens of thisnormal group had a cholate: cholesterol ratio ofless than 10. The high undetermined anion ofthese non-conforming specimens suggests that theratio of total bile acid to cholesterol would beconsiderably higher.2

2 Other substances in bile beside cholesterol may reactto give color with the Liebermann-Burchard reagents.Thus Wright (32) found the colorimetrically deter-mined values for cholesterol in dog bile to be higher by20 per cent than the results of determination by digitoninprecipitation. Wieland and Reverey (12) report thatanthropodesoxycholic acid gives a weak Liebermann-Burchard reaction. While the term cholesterol has beenused in the text and tables, it should be regarded as anexpression of total non-saponifiable material giving theLiebermann-Burchard reaction. For the purposes of thepresent paper, there is reason to believe that the"cholesterol " values approximate cholesterol concentra-tions without gross inaccuracy. Comparisons withgravimetric digitonin determinations showed that colori-metric estimations did not vary more than 10 per centfrom the digitonin values.

The composition of bile from diseasedgallbladders

Distinct changes from the normal compositionof bile accompanied even slight pathological al-terations of the gallbladder mucosa. The effectsof cholecystitis of mild or moderate degree areshown in Table II. Gallstones were present in16 of the 18 gallbladders from which these speci-mens were removed, while all of the bile speci-mens contained cholesterol crystals. Obstructionof the cystic duct was found at operation in 9 ofthe patients represented in this group.

Severe cholecystitis, with widespread changesin the mucosa, caused alterations in the composi-tion of the contents of the gallbladders similar tothose observed in moderate gallbladder disease,though far more pronounced. Data for 15 speci-mens of this character are presented in Table III.Here, likewise, the incidence of calculi was high.However, two specimens contained no crystals,nor were calculi found in the gallbladders. Par-tial or complete occlusion of the bile ducts causedby calculi existed in 10 of the 14 patients.Changes from the normal were most marked inthe presence of obstruction. The group was farfrom homogeneous in respect to appearance ofthe specimens, which varied from essentially nor-mal pigmentation to practically pigment-free"white bile."

The lowered concentration of total solids tobe seen even in the presence of moderate injuryto the gallbladder (Table II) reflects a markedimpairment of the ability to retain and concentratematerial reaching the gallbladder in the hepaticbile. Severe damage to the gallbladder mucosadepressed the solids content of the bile to agreater extent (Table III). Particularly strikingchanges were associated with obstruction of thecystic and common bile ducts. Estimation of theprotein contained in certain of these specimensshowed increased concentrations in both groupsas compared with normal specimens. Thus theloss of ability to concentrate bile as measured bydeterminations of total solids is obscured to someextent by elevated protein, and was greater thanthe figures for total solids would indicate.

Striking reductions in concentration of bileacids in bile in the presence of gallbladder diseasehave been reported (10, 11, 30, 31, 47). Our

374


analyses likewise showed that cholic acid concen-trations were lowered consistently in specimenscomprising this group. The loss of bile acid ex-plains the major portion of the decrease in solids.Whereas cholate in normal specimens exceeds 150m.eq. per liter, the average concentration in thepresence of gallbladder disease was reduced to34 m.eq. per liter (Table II). Under similarconditions, undetermined anion concentrationswere generally below the normal level althoughnot as much so as cholate. It seems probablethat undetermined bile acids behaved not unlikethe cholic acids. Even lower concentrations ofbile acids were observed in bile from badly dis-eased gallbladders (Table III), and in a fewspecimens, particularly in the presence of ob-struction and empyema, only traces of cholate

the gallbladder, equivalent quantities of inorganicanions, largely chloride, replace the organic elec-trolyte so lost. Excessive concentrations of chlo-ride are observed in the presence of marked edemaof the gallbladder mucosa when the chloride con-centration of the contents often exceeds that ofserum. Riegel, Ravdin, Johnston and Morrison(33) have reported high chloride concentrationin hydrops fluid. The importance of increasedchloride as an indicator of gallbladder dysfunc-tion has been stressed by these workers (10).

Bicarbonate, like chloride, may replace bile acidthat diffuses from the injured gallbladder. Incertain of the specimens represented in TablesII, III, IV and V, bicarbonate is increased incomparison with the normal while a few genu-inely high values are seen. However, in the most

TABLE IV

Cholesterosis t

Operative findrings L i:3 Remarkis 0 .

Q 00 Q~~. 0 CE 0 C)~ ~

m.eq. m.e. m.eq. m.eq. m.eq. m.eq. m.eq. m.eq. mum.per per lie per literperr liter per liter per liter per liter per liter mM. m. perof bil of bile of bile of bile of bil of bil of bde of bil cent

49 Cholsterods b, c Cs 63.1 32.1 23 3.9 240 3.828 Choleterosis c Cr 8.1 22.3 82 36 177 4.0 11.8 9.8 6154 Cholesterosis Gangre-

nous pancreatitis c Yellow 8, Cr 12.2 15.1 38.6 76 137 9.3 192 16.1

t For legend, see Table I.

could be detected while other bile acids likewiseappeared to have vanished. Heightened permea-bility of the gallbladder mucosa which ceases toact as a barrier to bile salts and perhaps to othersubstances of high molecular weight undoubtedlyis foremost among the factors leading to suchchanges. There is little doubt that the loweredconcentration of bile acid diminishes the solventaction of the bile for lipids, and leads in turn tocrystallization of cholesterol.

High chloride concentrations were associatedconsistently with the low bile acid concentrationstypical of cholecystitis. Normally, as the con-centration of bile acid increases, chloride diffusesfrom the gallbladder, thus maintaining osmoticequilibrium. However, in the presence of mod-erate cholecystitis the relatively high concentrationof chloride present in hepatic bile not only re-mains undiminished, but, as bile acid diffuses from

severely damaged gallbladders bicarbonate is moreoften decreased. It appears that the abundantsecretion of bicarbonate in the dog described byRavdin, Johnston, Austin and Riegel (34) as aconsequence of injury to the mucosa is not theprevailing response to chronic cholecystitis in thehuman. Perhaps it represents an early phase inthe response to injury, although the occurrenceof calculi composed of calcium carbonate impliesa persisting increase in bile pH.

Although but little change in pH of bile ac-companies moderate gallbladder disease, a shifttoward more acid reactions commonly is asso-ciated with marked injury to the walls of thisorgan. In this respect, data obtained in the pres-ent study confirm the earlier observations of thewriters.

Decreased pH and bicarbonate under these cir-cumstances provide an interesting contrast to the

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changes observed in the gallbladder bile of dogsfollowing the development of cholecystitis.Drury, McMaster and Rous (21), and Johnstonet al. (10) have shown that injury to the mucosaof the gallbladder causes the pH and bicarbonateof dog's gallbladder bile to increase. Again, thedifferences in the chemical properties of the pre-dominating bile acids in human and dog bile aidin explaining the inconsistency. Salts of theweakly acidic glycine-conjugated acids of humanbile on undergoing hydrolysis would impart aslightly alkaline reaction to solutions. As alreadymentioned, the slightly alkaline reaction of normalhuman gallbladder bile is a consequence of thisproperty. On the other hand, when glycocholicacid diffuses from the diseased gallbladder andis replaced principally by chloride, the reactionwill tend to become less alkaline. In contrast,the strong taurine-conjugated bile acids of dogbile normally displace practically all of the bicar-bonate (and chloride) as concentration proceedsin the gallbladder so that ordinarily the reactionis distinctly acid. Under these circumstances lossof bile acid from the gallbladder of the dog inthe presence of cholecystitis, particularly if ac-companied by secretion of bicarbonate (see Rav-din et al. (34)), will cause the marked increasein bile pH observed in this species. Impairedbuffering capacity facilitates changes in reactionthat occur in injured gallbladders.

The lowered pH of the contents of severelydamaged gallbladders indicates that acidic con-stituents are present in significant concentrations.These are not cholic acids, free or conjugated,since low cholate is found in this class of speci-mens. Appreciable amounts of undeterminedanion are observed. Its identity has not beenestablished. That it is not phospholipid is shownby simultaneous occurrence of very low bicar-bonate with the smallest phospholipid concentra-tions of the entire series.

Diffusion of bile acid out of the diseased gall-bladder is only partially compensated by the risein chloride (with or without a rise of bicarbonate).There is invariably a simultaneous and roughlyproportional decrease in concentration of totalbase. While quantitatively smaller than the al-teration of the chloride, it is nevertheless of suffi-cient size to bring about marked reduction of

the total cation concentration of the pathologicalspecimens, as well as that of individual cations.Sodium, being the principal cation, accounts forthe major portion of the change. However, cal-cium concentrations do not change to the sameextent as a concentration of other cations.

The presence of considerable amounts of cal-cium as a constituent of many gallstones arousesinterest in the concentration of this ion in bileof diseased gallbladders. Marked irregularity isshown in the response of calcium concentration ofbile to gallbladder disease. Alterations that havebeen observed are most frequently in the direc-tion of lower values. Occasional high concen-trations were found in the contents of certainbadly diseased gallbladders. Calcium concentra-tion in normal bile varies directly as the concen-tration of bile acids, and no doubt a relationshipexists analogous to that between serum calciumand serum protein. Thus high concentrations ofbile acid would exert a protective action tendingto keep calcium in solution by diminishing ioniza-tion. On the contrary, low bile acid levels wouldfavor precipitation of calcium, thus contributingto a widespread occurrence of calcium in calculi.Inouye and Ryuichi (35) found that sodiumtaurocholate prevented precipitation of calciumalthough glycocholate was ineffective.

In disease, the changes in bile calcium are com-plex. Quite probably high calcium concentra-tions in the presence of severe cholecystitis are tobe attributed to exudation or transudation. Thisexplanation is supported by the simultaneous in-crease in the protein of the bile shown by un-published observations of the writers. Riegel etal. (33) refer to the process by which calciumis increased as secretion. Phemister, Day andHastings (36) have presented interesting exam-ples of extreme accumulation of calcium in thegallbladder.

In cholecystitis, concentrations of bile magne-sium are diminished. The calcium-magnesiumratio of the gallbladder contents ordinarily is low-ered in comparison with that observed in normalspecimens, approaching more nearly the ratio thatcharacterizes blood serum or transudates. Po-tassium, like sodium, also approaches values com-monly found in serum.

Contrary to expectations, cholesterol concen-

377


trations are lower in our specimens from patho-logical gallbladders, as judged by averages, thanin the normal material. Differences becomegreater as the severity of injury to the mucosaincreases, until the average cholesterol of GroupIV (marked cholecystitis) is about half that ofthe normal. Between these extremes fall theaverages of the intermediate groups. Individualvariations within all groups are large. The lowfigures undoubtedly reflect a decreased capacityof the bile for holding cholesterol in solution.

These results are in agreement with those ofNewman (30), Spanner and Bauman (37), An-drews et al. (31) and Riegel et al. (47) in show-ing that the underlying cause of the crystalliza-tion of cholesterol is not a primary excess of thissubstance, but rather a defect of the solvent. Theobvious lowering in the ratio of cholic acid tocholesterol in pathological specimens, as comparedwith normals, provides a quantitative measure ofthis change. Our ratios are lower than those ofcertain other workers who employed the less spe-cific determination of hydrolyzed amino-nitrogenfor analysis of bile acids. In general, however,the results are similar. Despite the high choles-terol concentrations of many normal specimens,crystals of cholesterol were seldom encounteredin such specimens. On the other hand, it ap-pears that crystals and calculi rarely are absentfrom the contents of diseased gallbladders andusually are present in great abundance despite thelowered concentration of dissolved cholesterol.

Bilirubin solubility, like that of cholesterol,must depend upon the lipid-bile acid relationshipsince gallbladder bile usually lacks the alkalinityrequisite to maintain bilirubin in aqueous solution.Furthermore, the activity of calcium undoubtedlyincreases as bile acid concentrations decrease.Thus loss of bile acid and lipid with the furtherlikelihood of an increase in calcium concentrationand activity may readily bring about precipitationof bilirubin. Possibly, because of the limitationsof the method, it has not been possible to relatethe occurrence of gallstones with the incidence ofeither high or low concentrations of bilirubin.The concentration of bilirubin in bile, as deter-mined by the procedure described, varied widelyboth in the normal and in the several groups ofpathological specimens. However, low concen-

trations were characteristic of the specimens fromthe most severely damaged gallbladders, as onemight expect on the basis of physical factors.Not infrequently, crystals of bile pigment, theso-called calcium-bilirubin crystals, were presentin specimens of all groups. Their presence ap-peared to be independent of concentration ofbilirubin, calcium or hydrogen ions. The closecorrelation with the occurrence of cholecystitisthat was found to exist in the case of cholesterolcrystals was not observed.

The investigations of iFurth and Scholl (38)have again demonstrated the importance of phos-pholipid in the presence of bile acid as a solventfor cholesterol. Since bile is rich in lecithin andperhaps other phospholipids as well, these sub-stances undoubtedly exert an important influenceon the properties of bile as a solvent. An at-tempt has been made to determine the approxi-mate concentration of phospholipid in the variousspecimens for the purpose of discovering whethercholecystitis led to any significant alterations inthese substances. It appears that, like cholesterol,lipid phosphorus tends to remain in the damagedgallbladder while bile acid diffuses away. How-ever, being comparatively unstable and perhapsmore readily soluble than cholesterol, these sub-stances would not persist as concretions as choles-terol does.

Protection of the gallbladder against the dele-terious effects of bile salts is undoubtedly animportant function of the lipids of bile. Aninjurious action of aqueous bile acid solutionson the gallbladder mucosa has been described byRiegel, Ravdin and Johnston (39), and Aronsohnand Andrews (25). Riegel and associates .havealso described a protective action of cholesterolagainst bile salts, while Ishii (40) also has founda similar protective action of phospholipid againstbile salts.

Cholesterosis. No characteristic chemical com-position of bile could be related to the presenceof cholesterosis (strawberry gallbladder). Whilechanges from the normal exist (Table IV), thesewere in general similar to those observed in chole-cystitis of corresponding severity. The bilecholesterol was not elevated.

Neoplasm. The composition of the contents ofthe gallbladder in the presence of malignant

378


growths involving the biliary tract is shown inTable V. Obstruction of the cystic or commonbile ducts existed as well in all of these patients.Despite the absence in most cases of conspicuousgross or microscopic changes in the gallbladder,the composition of its contents was profoundlyaltered. Such a finding suggests that obstructionmay alter the composition as well as the secretionof bile. It is known that suppression of bilesecretion follows obstruction of the common bileduct. Contrasting with the remainder of thegroup is Specimen 60, where malignancy and ob-struction of possibly as long as 5 months' durationhad no effect other than marked inspissation ofthe bile.

Hepatic cyst. An hepatic cyst was encounteredin Patient 11. Fluid was aspirated from the cyst,and, after removal of leukocytes by centrifuga-tion, was analysed by the methods used for bile.The specimen was colorless but distinctly opal-escent. Cultures were negative. The specimencontained 7.89 per cent solids, 118.1 m.eq. perliter chloride, 1.6 m.eq. bicarbonate; the pH was5.86. The resemblance of these figures to thosefound for the contents of severely damaged gall-bladders is at once evident.

DISCUSSION

Diminished concentration of bile acid, as typi-fied by cholic acid, proves to be an almost in-variable accompaniment of injury to the gall-bladder mucosa. This close association providesevidence of increased permeability of the wall ofthe gallbladder to bile acid, while loss by diffusionbest explains the low bile acid concentrations.However, in the presence of injury to the hepaticparenchyma (Doubilet (13); Andrews, Hrdinaand Dostal (41)), in pregnancy (Riegel, Ravdin,Morrison and Potter (42)) or obstruction ofthe bile ducts (Goff, Hrdina and Andrews (43);McMaster, Broun and Rous (44); Greene, Wal-ters and Fredrickson (15)), alteration or sup-pression of bile acid secretion mray be additionalfactors. Cholesterol and, presumably, phospho-lipid, fail to diffuse from the damaged gallbladderas does bile acid. The failure of phospholipidto form deposits like those of cholesterol may beexplained by differences in stability and solubility.The evidence provided by Andrews, Schoen-

heimer, and Hrdina (31) and by Ravdin, Riegel,Johnston, and Morrison (52) to explain forma-tion of calculi in the gallbladder receives addi-tional confirmation from our data.

Other changes in chemical composition of bilein cholecystitis may in a large measure be re-ferred to a primary loss of bile acid. Chlorideor, less frequently, bicarbonate replaces the anionlost by diffusion of bile acid from the gallbladder.Thus the concentrations of these ions are in-creased. Diminished total solids and decreasedconcentration of total base likewise are directlyrelated to the decline in bile acid concentration.

An end result of changes that occur in thecontents of the gallbladder with an inert or ab-normally functioning mucosa is replacement ofthe hydrolyzed salts of weak acids, i.e., sodiumglycocholate and glycodesoxycholate, by sodiumchloride, with a shift of pH from the slightlyalkaline reaction normally observed toward moreacid reactions. The distinctly acid reactions ofmany specimens from badly damaged gallbladdersare unexplained, however, although loss of buf-fering capacity facilitates the change. Increasedalkalinity, at times observed, results from secre-tion of bicarbonate under similar circumstances.Weiser and Gray (46) and others suggest as onemechanism for gallstone formation, a shift inpH from alkaline to acid with resultant precipi-tation of cholate. Actually, bile acid concentra-tion governs the pH of bile. It is our belief thatchanges in pH are incidental to more fundamentalchanges in the constitution of bile, and thatchange of pH is of secondary importance as acausative factor in formation of cholesterol ormixed stones. Despite the primary r6le in chole-lithiasis assigned to changes in pH by many work-ers, no suggestion has been offered by them as tohow the hypothetical change in the reaction ofbile giving rise to gallstone formation is to bebrought about. Because of the acid reaction ofmany specimens of gallbladder bile, it is quiteunlikely that soaps are present in significantamounts.

Calcium usually is lowered in the contents ofdiseased gallbladders, although this response toinjury is variable. Ocasional noteworthy excep-tions with striking increases have been observed.While commonly lowered in comparison with val-

379


ues found in normal specimens, calcium concen-trations remain higher than those of blood serum.It is significant that the concentration of bile cal-cium is lowered less in proportion to the normalthan is the concentration of bile acid. Since bileacids undoubtedly aid in maintaining calcium(and bilirubin) in solution, the ubiquitous occur-rence of calcium in gallstones would follow asa result of this altered relationship between bileacids and calcium. An additional factor that mayfavor precipitation of calcium is secretion of bi-carbonate by the gallbladder. Although a typicalresponse in dogs, our data suggest that it is infre-quently encountered in humans. The behaviorof magnesium differs from that of calcium, andmagnesium concentrations are not maintained athigh levels in the presence of gallbladder disease.

Sterile fluid found in an hepatic cyst closelyresembled in composition the fluid collected fromgallbladders where obliteration of functioningmucosa or complete long-standing obstruction ofbile ducts had rendered the organ incapable offunction.

Cholesterol crystals in the gallbladder contentsquite consistently accompanied cholecystitis. Thedetection of such crystals in specimens of biletherefore provides good evidence for the exist-ence of cholecystitis. Such evidence is not nec-

essarily conclusive for occasionally crystals are

found in the absence of pathological changes ofthe gallbladder. However, our data support theapplication of this test for diagnosis of cho-lecystitis, provided representative samples of gall-bladder bile are secured for examination. Aclose correlation existing between the ratio ofcholic acid to cholesterol and the incidence ofcholesterol crystals or concretions reported by pre-vious workers has been confirmed.

SUMMARY

Severe cholecystitis brings about serious de-fects in gallbladder physiology that result inmarked alteration in chemical composition of gall-bladder bile. Changes that have been observedin the presence of gallbladder disease are suf-ficiently marked (qualitatively and quantitatively)to lead to calculus formation. Cholesterol varieswidely in concentration in the control specimensas well as in those specimens from pathological

gallbladders. Low values for cholesterol werefound in the presence of obstruction of the bileducts. Similarly, concentrations of bile pigmentbecame lower progressively with increasing injuryto the gallbladder.

The writers were aided by George R. Kingsley andHiram F. Snider, who assisted in tests of the accuracyof the procedure employed for cholesterol determinationand who also performed most of the cholesterol analyses.We are also indebted to Doctors E. L. Eliason, I. S.Ravdin, G. P. Mueller and H. L. Owen for specimens.

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