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Electrolyte Excretion in BileBy HENRY 0. WHEELER, M.D., OSWALDo L. RAMOs, M.D., AND

ROBERT T. WHITLOCK, M.D.

The high concentration of certain test substances in both urine and bile suggests thatsimilarities exist between the biliary and renal tubular functions. Evidence from dogswith permanent duodenal fistulas indicates that the rate of elaboration of bile dependsprimarily on the rate of secretion of bile salts. The variations in flow and compositionof hepatic bile seem to result from the addition of a fluid whieh is similar in somerespects to pancreatic juice.

IN COMMON with other extrarenal secre-tory structures, the biliary tract appar-

ently depends upon energy-consuming cellu-lar processes rather than upon a hydrostaticfiltration system for the production of itscharacteristic effluent. Nevertheless, there ap-pear to be certain rather fundamental similar-ities between biliary and renal tubular func-tion. These similarities become apparent whenone considers the number of compounds whichare secreted in high concentrations in bothurine and bile. Examples of such compoundsare phenolsulfonphthalein,l fluorescein,2 p-aminohippurate,3 penieillin,3 and phlorhizin.4

It is noteworthy that among the substanceswhich are most actively secreted into the bileare included the most potent known choler-eties. The correlation between active secretionand choleretic potency may well have im-portant implications with regard to the gen-eral nature of bile formation. Outstandingcholeretic substances are the natural bilesalts and their synthetic derivatives, eincho-phen5 and, to a lesser extent, the phthaleindyes such as Bromsulphalein, phenol red andbromeresol green.1 All of these, it should benoted, are organic acids. Of these cholereticcompounds, the natural bile salts are, ofcourse, normally present in abundance andare therefore of the greatest physiologic in-terest.The 2 major bile acids in the dog are the di-

and tri-hydroxy cholanic acids, deoxycholicand cholic acid; of the 2, the latter is themore abundant. These bile acids represent the

From the Department of Medicine, College ofPhysicians and Surgeons, Columbia University, NeNiYork, N. Y.

major end-product of cholesterol metabo-lism.6' 7 In the dog and in other carnivoresthey are conjugated with taurine. Because ofthe free sulfonic acid group of taurine, theresulting conjugates are completely dissoci-ated and highly soluble in the physiologic pHrange, having a pK of about 1.5. These 2 bilesalts, which can be measured accurately by themethod of Mosbach et al.,8 will be referred tohereafter simply as " taurocholate."

In the intact animal, the bile salts undergoextensive enterohepatic recirculation so that,as shown in figure 1, approximately 85 to 90per cent of the bile salt in the bile at anygiven time represents material previously ex-creted and reabsorbed from the bowel.9 Theremainder represents new bile salt synthesizedby the liver. The phenomenon of recirculationwas described as early as 1870 by Schiff, andin the course of his experiments he also ob-served that bile itself, when introduced intothe duodenum, was the most potent availablestimulant for increased bile production.10 Itwill be obvious, then, that interruption of thisenterohepatic cycle must have major physio-logic consequences which have to be consid-ered in the planning and interpretation of anystudy in which bile is removed from the ex-perimental subject and not replaced.Time does not permit a review of the many

ingenious technies which have been developedfor repeated collections of bile from un-anesthetized animals. The particular device wehave employed is a duodenal cannula develop-ed by Thomas."' This apparatus, which is heldin the duodenum by a hard rubber flange,permits the creation of a permanent duodenalfistula opening directly over the ampulla of

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ELECTROLYTE EXCRETION IN BILE

Vater. Between studies it is kept stoppered,but during an experiment it is opened, and asmall ureteral catheter is inserted directlythrough the ampulla of Vater and advancedwell into the common duct. Except for thefact that the animals are cholecystectomized,this technic leaves the biliary tract in normalcondition between studies and free of per-manent foreign bodies. All of the studieswhich will be considered in this paper wereconducted on 4 such dogs. In each study, thedog was held in the upright position by meansof a sling. Bile was collected either by gravityor with the assistance of gentle suction pro-vided by a tuberculin syringe.When bile is collected continuously over a

period of hours, there is progressive diminu-tion in bile flow because of the interruption ofenterohepatic circulation of bile salts. Figure2 illustrates tLis phenomenon and also certaingeneral features of bile composition withwhich we shall be concerned. At the top isshown bile flow in ml./min., in the middle isthe pH, relative to a line drawn at 7.4, at thebottom is shown the electrolyte composition ofindividual bile specimens. In each block dia-gram the major cations, sodium and potas-sium, are on the left and the anions, tauro-cholate, bicarbonate and chloride, are on theright. The specimen of bile labeled CD istypical of bile removed from the common ductat the moinent of catheterization. We shallshortly examine this type of bile in more de-tail. It should be noted here, however, that,in contrast to subsequent samples of flowinghepatic bile, the concentration of taurocholatein "common duct bile" is very high, the totalionic concentration is high, the pH is low,and there is very little bicarbonate or chloride.Over the course of 2 to 3 hours there is pro-gressive diminution in bile flow and also intaurocholate concentration. Thus, in this typeof experiment the output of all solutes, butparticularly the output of bile salt, diminishesas expected. Not only is the electrolyte com-position of bile difficult to interpret underthese circumstances, but actually it is oftendifficult even to obtain bile in sufficient quan-tity for analysis. It was found that this "un-steady" state can be averted by the constantCirculation, Volume XXI, May 1960

Excretion1 5 °/a

Figure 1Enterohepatic circulation of bile salts. About 85to 90 per cent of the bile salt excreted into theduodenmum is reabsorbed and returned to the liverby way of the portal vein.

intravenous replacement of bile salt. Beforeproceeding to the results which were obtainedby using this technic, let us examine moreclosely the composition of "common ductbile. "

The specimen on the left of figure 3 is typi-cal of many specimens of "common duct bile"obtained upon catheterization of fasting dogs.Presumably it represents bile which wasformed over a period of several hours prior tocatheterization and held in the duct systemby the normal action of the sphincter of Oddi.In the center is shown the composition ofcanine gallbladder bile, and on the right, forcontrast, that of canine plasma. There is ob-viously a striking similarity between " com-

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WHEELER, RAMOS, WHITLOCK

cc/min CrG C20.1 _ -u

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120 ..

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BILE FL OW

12 Na+ rD Taurocholatem K + E HC03

fS CI-.~~~~~~: ..

,,~~~~~~~~~~~~~~~~: ,.1..-...:.:..-............. ........

114 mOsm

j I(ILI -A

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Figure 2Bile flow and composition during continuous collectio7z. Bile withdraawn from the commonduct immediately after catheterization (CD) has a high concentration of taurocholateand low concentrations of chloride and bicarbonate. During continuous collection, with-out bile salt replacement, the flow of bile and the concentration of taurocholate diminishprogressively. Composition of plasma is showvn at left for comparison.

pH 6.2 pH 6.5 El Na+* K<+ m

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-7.~ ~ ~ ~ ~ ~

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200

150 :- :: : ::::300m Osny <g

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"COMMON GALL PLASMA

DUCT BLADDER

BILE BILE

Figure 3

Comnparison of typical fa ting canine "'commnon duct" and gallbladder bile. Bile retained

in the bile ducets by the sphincter of Oddi is similar to ga.llbladder bile in that it has a

high concentration of taurocholate, lowl concenztrations of chloride and bicarbonate and

a low p11. Note that the total ionic conlcentration is highJ, although osmnolality is equal

to that of pla,sma (at right). (Republished by permission of the Journal of Clinical

Investigation^.'6)


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991ELECTROLYTE EXCRETION IN BILE

TAUROCHOL ATE*c-/i 13AM/min /V0.20

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Figure 4Bile composition and flow during constant intravenous jifusion of taurocholate. The rateof bile salt excretion is constant, but there are spontaneous variations in flow and electro-lyte composition. Highest flows are accompanied principally by increased pH and con-centration of bicarbonate.

TAUROCHOLA TE1/3,iM/min IV

cc /mine0.30 BILE Scretn

0.20 K FLOWIOuI0.10

08 _

EZ2 Na+ rJ Taurocholate- K+ EE HC03

E CI-

7

2 00 250MINUTES

Figure 5Effect of secretin on the flow and the composition of bile. Intra!renous administration ofsecretin causes a marked choleresis and a very high pH and bicarbonate concentration.

The excretion of bile salt is unaffected. (Republished by permission of the Journalof Clinical Investigation.16)


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WHEELER, RAMOS. WHTIjTOCK

rA UROCHOL A rE/3,vM/min /V

Introduodenal HCIF 0.05N 7.7cc/min+

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= CI-

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100 150 200 2 50 30CMINUTES

Figure 6Effect of intraduodenal infusion of hydrochloric acid. The choleresis and changes in

electrolyte composition are similar to those produced by exogenous secretin and are

probably attributable to release of endogenous secretin.

mon duct bile"' and gallbladder bile in that,so far as anions are concerned, each bile ispractically a pure solution of bile salt. Itseems quite probable that, at least in chole-cystectomized dogs, the common duct and itsmajor branches serve in effect as a gallbladderin concentrating the bile salts by removal ofother solutes and water.The osmolality of bile, according to all re-

ports and under all the circumstances we shalldiscuss, is very close to that of plasma (thatis, about 300 mOsm./Kg.). In all bile speci-mens, however, and particularly in those ofthe type illustrated in figure 3, the total ionicconcentration (sum of anions plus cations)is much greater than 300 mEq./L. Thismarked discrepancy between ionic concentra-tion and osmolality can be attributed to thefact that tauroeholate, like many other sur-

face-active substances, forms large multipolaraggregates, or micelles, when its concentratioinexceeds a critical value known as the "micellepoint." The "micelle point" of pure tauro-

cholate, as determined by Pethica and Schul-man,12 is about 0.007 M, which is much lowerthan the concentration of taurocholate in bile.Thus, the taurocholate ion itself is virtuallyinactive osmotically. Its osmotic significanceis, in effect, attributable solely to the cationwhich must accompany it to preserve electro-neutrality. Consistent with this view is thefinding that, regardless of taurocholate con-

centration, virtually all the osmotic activityof any bile specimen may be accounted for as

the sum of sodium, potassium, chloride andbicarbonate.To return to the composition of flowing

hepatic bile, figure 4 illustrates a typical studyin which sodium taurocholate was infused in-travenously at a constant rate of 14.5 ,LM/min. In this and similar studies, such a con-

stant infusion resulted in stabilization oftaurocholate excretion at a constant rate ap-

proximately equal to the rate of infusion.There was no progressive diminution in bileflow. Nevertheless, as shown in figure 4, sig-


BILE FLOW

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nificant fluctuations in bile flow often oc-curred. There was a reciprocal relationshipbetween bile flow and taurocholate concentra-tion, consistent with the constant output ofthis constituenit. There were also eharacteris-tic changes in other electrolytes. As flow in-creased, the chloride concentration increasedslightly, but there was a more striking in-crease in bicarbonate concentration and a cor-respondilng rise in pH.When 100 units of the intestinal hormone,

secretin, was adminiistered intravenously (fig.5), a nmarked increase in bile flow occurred,exceeding the highest spontaneous flows ob-served in the same animal. The excretion rateof taurocholate was unchanged, but the secre-tin choleresis was accompanied by a veryhigh concentration of bicarbonate (reachingvalues as great as 60 mEq./L.) and pH (to ashigh as 7.8).The intraduodenal infusion of hydrochloric

acid is known to stimulate the release of en-dogenous secretin.'3 As shown in figure 6, thismaneuver resulted in a change similar to thatproduced by exogenous secretin. There wasan impressive choleresis accompanied, onceagain, by a high bicarbonate concentratiorLand pH. As with exogenous secretin, thesechanges occurred in the face of a constant rateof taurocholate secretion.

In each animal there was a reproduciblerelationship between bile composition and bileflow as shown in figure 7. Whenever bile flowinereased, whether spontaneously or underthe influence of secretin (on the extremeright), there was a marked increase in pH andconcentration of bicarbonate. The concentra-tion of chloride also increased, but this changewas small compared to the increment in bicar-bonate. In contrast to these changes, the en-tirely different effect of intravenous acetazole-amide is also shown in figure 7. This agent, ina dose of 60 mg./Kg., resulted in a choleresiswhich was characterized by a relatively highchloride concentration and a comparativelylow bicarbonate concentration and pH.

All of the studies to which we have alludedwere conducted with the rate of secretion oftaurocholate arbitrarily fixed at about 15Circulation, Volume XXI, May 1960

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rigure 7Relationship between bile flow and compositionduring constant infusion of taurocholate. At higherrates of flow, the concentrations of bicarbonateand chloride and the p11 were increased. Theincrement in bicarbonate was greater than thatin chloride. Highest flows were observed afterintravenous administration of secretin or intra-duodenal administration of hydrochloric acid.Acetazoleamide produced a choleresis in whichchloride was the predominating anion. (Repub-lished by permission of the Journal of ClinicalInvestigation.16)

,uM/min. It should be mentioned, however,that we have observed a very similar qualita-tive relationship between bile compositionand flow at a taurocholate secretion rate ofabout 40 ptM/min.-although, of course, theabsolute values of bile flow were much higherthan those shown here. It is also worth notingthat the arbitrary rate of taurocholate secre-tion employed in the present studies repre-sents only about one-tenth of the maximal rateat which the liver is apparently capable ofsecreting this bile salt. The transport maxi-

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9WHEELER, RAMOS, WHITLOCK

TAUROCHOLATEFRACT ION

BILE ELECTROLYTEFRACTION

a

R

.

U. 15m /min

CG-

0.1OmI/mi n

Figure 8Hypothetic fractions of bile. The concentra,tions and flows are based on the assumptionthat each solution is isosmotic with respect to plasma. When the output of taurocholateis constant, all variations in bile flow and composition can be attributed to changes in

flow and composition of the "electrolyte fraction." (Republished by permission of theJournal of Clinical Investigation.16)

mum for taurocholate is well over 100 [M/min. in dogs of this size.One way of explaining the observed varia-

tions in bile flow and composition would beto postulate that bile is formed by the admix-ture of a number of solutions which differfrom one another in comnposition and mode ofproduction. With this thought in mind, we

have arbitrarily elected to regard each bilespecimen as a mixture of 2 hypothetic isosmot-ic solutions as shown in figure 8. On the leftis a pure solution of taurocholate which-be-cause of the associative properties of thetaurocholate ion-would be isosmotic at a con-

centration of about 300 muM/L. On the rightis a solution of chloride and bicarbonatewhich we shall call the "electrolyte fraction ";the sum of these ions would be equal to about150 mEq./L. for an isosmotic solution. Thus,on the basis of the osmotic behavior of allthese constituents, it is possible to calculate,for each bile specimen, the flow and composi-

tion of these 2 hypothetic constituents. Underthe conditions we have emnployed, the outputof the "tauroeholate fraction" is maintainedat a constant rate. Hence, the changes in bileflow and composition must be attributed tochanges in the output and composition of the"electrolyte fraction," and we shall thereforeexamine these changes.When one examines the relationship be-

tween composition and output in the "elec-trolyte fraction" (fig. 9), it is apparent thatincreasing output is accompanied by recipro-

cal changes in chloride and bicarbonate con-

centration. At the very highest rates of flow-after secretin administration-the concentra-tion of bicarbonate achieves its highest valueof about 75 mEq./L. and chloride concentra-tion reaches a minimum at about the same

level. This figure bears a striking resemblanceto illustrations of the behavior of pancreaticsecretion14 although, of course, much higherconcentrations of bicarbonate and lower con-


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centrations of chloride have been observed inpancreatic juice under maximal secretin stim-ulation. Note again the fact that acetazoleam-ide administration results in high concentra-tions of chloride and low concentrations ofbicarbonate.The results of these studies are consistent

with the view that at least 2 processes are in-volved in the elaboration and modification ofbile. First, it is obvious that the rate of bileproduction is profoundly affected by the rateof secretion of a number of substanees ofwhich the bile salts are of the greatest physi-ologic importance. In spite of the variationsnoted there is, in fact, a rough proportionalitybetween total bile flow and rate of bile saltsecretion. Also, as noted earlier, bile flow be-comes almost vanishingly small in the animalwhich is acutely deprived of recirculating bilesalt unless the bile salt is replaced by anotherroute. It would seem entirely reasonable topostulate that the priluary event in bile for-mation is the active secretion of bile salts and,to a lesser extent, of certain other substanees.The addition of water and many diffusibleconstituents could then occur passively alongthe resulting osmotic and electrochemical gra-dients. This viewpoint has been enunciated ina recent review by Sperber'5 with which ourdata are wholly in accord.The second process involves the modifica-

tion of the final composition of bile by theaddition-at an unknown site in the biliarytract-of a solution which is similar in manyrespects to panereatic juice. This bicarbonate-rich fluid appears to be responsible for thespontaneous variations in bile flow which oc-cur in spite of the constant rate of secretionof bile salts, and its output is maximal fol-lowing stimulation by exogenous or endoge-nous secretin.

Finally, the possibility of reabsorptive mech-anisms in the bile ducts must not be over-looked, although at present the only evidenceof the existence of such mechanisms is thatwhich can be inferred from the similar com-position of gallbladder bile and bile restingin the common duct.Circulation, Volume XXI, May 1960

120

10

E90

a0Rx- 80

70

60

020°0

z

0

l0 I

K Dog NormaTAUROCHOLATE145A,M/m inm V

CHLORIDE

4%.

B8CARBONATE

.

GeSpontaneous f/ow,a /ntraduodenol HGC03 InarGvenous secretinVT tntravenous acetazolamide

lIl

0.25D 0005 0.10 0 15 0 20OUTPUT OF ELECTROLYTE FRACTION-ml/min

Figure 9Composition of "electrolyte fraction" duringchazges in its output. A reciprocal change inbicarbonate and chloride, similar to that observedin pancreatic juice, is observed as output increases.After the administration of acetazoleamide, theconcentration of chloride is high and the coceen-tration of bicarbonate is low. (Republished bypermission of the Journal of Clinical Investiga-tion.'6)

References1. SPERBER, I.: Biliary excretion and choleretic ef-

fect of somiie phenolsulfonephthaleins. Actaphysiol. scandinav. 42: Suppl. 145, 129, 1957.

2. HANZON, V.: Liver cell secretion under normaland pathologic conditions studied by fluores-cence microscopy on living rats. Acta physiol.scandinav. 28: Suppl. 101, 1, 1952.

3. CooK, D. L., LAWLER, C. A., CALVIN, L. D., ANDGREEN, D. M.: MIechanisnis of bile formation.Am. J. Physiol. 171: 62, 1952.

4. JENNER, F. A., AND SAIYTH, D. H.: Excretion ofphlorrhizin by the liver. J. Physiol. 137: 18P,1957.

5. BERMAN, A. L., SNAPP, E. P., ATKINSON, A. J.,AND IVY, A. C.: Effect of cinchophen on bileformation. J. Lab. & Clin. Med. 28: 682, 1943.

6. BLOCH, K., BERG, B. N., AND RITTENBERG, D.:Biological conversion of cholesterol to cholicacid. J. Biol. Cheai. 149: 511, 1943.

7. SIPERSTEIN 2M. D., AND MURRAY, A. W.: Choles-terol metabolism in man. J. Clin. Invest. 34:1449, 1955.

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8. MOSBACH, E. H., KALINSKY, H. J., HALPERN,E., AND KENDALL, F. E.: Determination ofdeoxycholic and cholic acids in bile. Arch.Biochem. 51: 402, 1954.

9. SCHMIDT, C. R., BEAZELL, J. M., BERMAN, A. L.,IVY, A. C., AND ATKINSON, A. J.: Studies onthe secretion of bile. Am. J. Physiol. 126:120, 1939.

10. SCHIFF, M.: Gallenbildung, abhaingig von derAufsauguing der Gallenstoffe. Pfluigers Arch.ges. Physiol. 3: 598, 1870.

11. THOMAS, J. E.: An improved cannula for gastricand intestinal fistulas. Proe. Soc. Exper. Biol.& Med. 46: 260, 1941.

12. PETHICA, B. A., AND SCHULMAN, J. H.: Haemoly-tic and surface activity of sodium taurocho-late. Nature 170: 117, 1952.

13. THOMAS, J. E., AND CRIDER, J. 0.: A quantitativestudy of acid in the intestine as a stimulusfor the pancreas. Am. J. Physiol. 131: 349,1940.

14. HART. W. M., AND THOMAS, J. E.: Bicarbonateaiid chloride of pancreatic juice secreted inlresponse to various stimuli. Gastroenterology4: 409, 1945.

15. SPERBER, I.: Secretion of organic anions in theformation of urine and bile. Pharmacol. Rev.11: 109, 1959.

16. WHEELER, H. O., AND RAMOS, 0. L.: Determi-nants of the flow and composition of bile inthe unanesthetized dog during constant infu-sions of sodium taurocholate. J. Clin. Invest.39: 161, 1960.

The Origin of LifeAt first there were the simple solutions of organic substances, whose behavior was

governed by the properties of their component atoms and the arrangement of thoseatoms in the molecular structure. But gradually as a result of growth and increasedcomplexity of the new molecules new properties have come into being and a new colloid-chemical order was imposed upon the more simple organic chemical relations. Thesenewer properties were determined by the spatial arrangement and mutual relationshipof the molecules. Even this configuration of organic matter was still insufficient to giverise to primary living things. For this, the colloidal systems in the process of theirevolution had to acquire properties of a still higher order, which would permit the attain-ment of the next and more advanced phase in the organization of matter. In this processbiological orderliness already comes into prominence. Competitive speed of growth,struggle for existence and, finally, natural selection determined such a form of materialorganization which is characteristic of living things of the present time.-A. I. Oparin.The Origin of Life. Translated with annotations by S. Morgulis. Ed. 2. New York, DoverPublications, 1953, pp. 250-251.


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HENRY O. WHEELER, OSWALDO L. RAMOS and ROBERT T. WHITLOCKElectrolyte Excretion in Bile

Print ISSN: 0009-7322. Online ISSN: 1524-4539 Copyright © 1960 American Heart Association, Inc. All rights reserved.

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