Biliary clearance of inert carbohydrates

GASTROENTEROLOGY

Biliary Clearance of hert Expectations and Reality

1988;94:217-28

Carbdhydrates

NICOLA TAVOLONI Department of Medicine, The Polly Annenberg Levee Hematology Center, Mount Sinai School of Medicine of the City University of New York, New York, New York

tiring the last 20 yr, measurement of the biliary clearance of inert carbohydrates has been a conventional procedure in studies of bile secretory physiology and pathophysiology. Molecules like erythritol, mannitol, sucrose, and inulin have received most of the attention, although other solutes, e.g., polyethylene glycols and dextrans of various molecular weights, have also been used. The objectives of these clearance studies have been (a) to separate canalicular secretion from ductular secretion or reabsorption (e.g., to quantitate hepatocytic bile flow) and (b) to determine the integrity of the canalicular membrane permeability or, as more commonly claimed, to define the permeability characteristics of the transjunctional shunt pathway between hepatocytes. Radioactive erythritol and mannitol have been used to serve the first purpose, whereas labeled sucrose and inulin have been used to serve the second. As discussed below, theoretical considerations and a number of experimental observations are consistent with these postulates. Yet, direct supporting evidence has never been obtained and recent findings have questioned the usefulness of these clearance approaches. Over the last few years, our laboratory has been involved in studies aimed at clarifying where and how inert hydrophilic molecules enter the biliary lumen. We have learned from our own work, both from the failures and the more successful attempts. Certainly, we have learned a great deal from the experiences of others, particularly from those studies that have utilized different approaches. Because much new information has been obtained from recent experiments, it seems timely to critically evaluate the state of knowledge of nonelectrolyte biliary permeability and its relevance to the physiology and pathophysiology of bile secretion. This review is not intended as a comprehensive summary because it is our wish to provide a personal, selective, and somewhat speculative discussion. Much excellent work, particularly that which

D

touches upon this subject peripherally, will not be discussed or even mentioned here. We will focus on three major areas: (a] the theory behind the use of these clearance experiments; (b) the experimental data supporting or refuting the usefulness of these approaches; and (6) current thinking about the role of these clearance measurements in the understanding of bile formation and cholestasis. Our discussion will be limited to biliary entry of erythritol, mannitol, sucrose, and inulin. Only when relevant to the interpretation of the biliary clearance of these inert carbohydrates will other solutes receive some attention.

Theoretical Considerations Because the biliary tree is a multistructural and multifunctional apparatus, a fundamental question in biliary physiology is: where along the biliary tract is bile formed? There is indirect but strong evidence that the hepatic parenchyma is the primary site of bile secretion. However, how much of the fluid collected at the common bile duct is hepatocytic in origin is a problem that has lured students of bile formation for more than two decades. Furthermore, as the lining of the biliary lumen is composed of enithelial cells held together by junctional complexes, the question whether bile water reaches the lumen transcellularly or paracellularly, or via both routes, has recently aroused the interest of biliary physiologists. Inasmuch as the molecular events underlying vectorial transport of water across the biliary tree are not known, the answers to these questions cannot simply be obtained from studies of biliary membrane structure and function. Similarly, as all biological membranes are permeable to water, and there are no techniques for distinguishing diffusive from osmotic water movement, kinetic studies 0 1988

by the American

Gastroenterological

0016-5085/88/$3.50

Associiition

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GASTROENTEROLOGY

of tritiated water flux across the biliary system would not provide interpretable information. Hence, alternative strategies are required to identify the sites and routes of transhepatic water flow. One of the simplest approaches is to study the biliary entry of molecules that behave kinetically like water, and whose movement across the biliary membranes can easily be traced. Such molecules must be highly soluble in water and virtually insoluble in membrane lipids, be uncharged, and must not chemically react with molecules in plasma and tissues, particularly those in the liver, and with constituents of bile. Labeled erythritol, mannitol, sucrose, and inulin fulfill these requirements. They are highly soluble in aqueous systems and very poorly soluble in lipids (l--3), are uncharged, are not concentrated in tissues (1,4-IO), and enter bile by passive mechanisms (4-7,11-13). Some investigators have suggested that radioactive sucrose is degraded after its administration to live animals (8). However, contrary results have been reported (1,4, l4,15) and evidence is now available to indicate that erythritol, mannitol, sucrose, and inulin are not readily and significantly metabolized by mammalian cells and distribute passively and exclusively among plasma, interstitial water, and body cavity fluids. If they permeate the cell plasma membrane (e.g., erythritol and mannitol), they will also equilibrate with intracellular water. Based on these physicochemical and biological characteristics and on the concept that water crosses biological membranes via an aqueous pathway, conventionally referred to as water channels or pores, entry of these inert hydrophilic molecules into bile can be confined to such a polar route. Consequently, the driving forces for the transfer of these solutes from plasma into bile can only be the chemical potential or osmotic pressure, or both. The chemical potential, or concentration gradient, will allow the solute to diffuse, whereas osmotic pressure will drive it into the lumen by means of bulk water flow. Using the principles of irreversible thermodynamics, solute as described by Kedem and Katchalsky (16), biliary entry us,, mobs) can thus be defined by the following equation: Js, =

Pb.

Ab

(c,

-

cb)

+

Jv (1 - a) c,,

(11

where Pb (cm/s) is the diffusive permeability coefficient for the solute in question, Ab (cm’) is the surface area, Cb and C, are the solute concentrations (mol/cm3) in bile and plasma, respectively, o is the reflection coefficient, JV (cm3/s) is water (bile) flow, and C, = (Cb - C,)/ln (C&r& The first expression on the right-hand side of Equation (1)defines solute entry by simple diffusion (Fick’s law), whereas the second defines permeation by solvent drag. C, (av-

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erage concentration between plasma and bile) is included instead of C, because Equation (1)is not distributed in space and applies solely to a thin homogeneous membrane. If Equation (1) is integrated over the membrane thickness, as described by Patlak et al. (li’), and rearranged, we obtain

Equation (2) is similar to that derived by Forker (11) and Wheeler et al. (S), and describes solute entry into the biliary lumen by diffusion or convection, or both. It holds only under the conditions that (a) solute movement is restricted to the water pore pathway, (b) the biliary tree is composed of a single homogeneous membrane, and (c) Jy is the same throughout the system. In reality, assumptions b and c are not correct. Indeed, it is because of the multifunctional and multistructural nature of the biliary apparatus that the biliary clearance of these inert carbohydrates is measured as a means to identify the sites and routes of transhepatic water flow. Accordingly, the following considerations are in order before Equation (Zj can be applied to estimate hepatocytic bile flow and the permeability of the canalicular membrane. Measurement

of Canalicular

Bile Flow

The biliary excretion of an inert carbohydrate will provide a quantitative assessment of hepatocytic bile flow if two conditions are met. First, this molecule must permeate the bile canaliculus, but not the biliary ductules and ducts. This means that CTfor such a solute in the distal epithelial structures must be 1,and that solute diffusion through the lipids of the bile ductules and ducts must be insignificant. Accordingly, Equation (2) can now be applied to define solute entry exclusively at the canalicular level. Second, (Tfor the canalicular membrane must be 0; that is, the solute must move freely between plasma and canalicular bile. Under these conditions, Equation (2) becomes Js, = C, Iv c9

(3)

in which Js, is solute flux into the bile canaliculus, and JV,is canalicular bile flow. Because such a solute is assumed not to permeate the distal epithelial structures, Jss,= Js, and Jsb = IV . cb. Thus, Jvc = Iv

cb/c,.

(41

Equation (4) indicates that canalicular bile flow can be estimated by simply measuring total bile flow and the bile-to-plasma concentration ratio of the hepatocytic fluid marker. Clearly, inasmuch as canalicular bile must transit through the biliary ductules and

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ducts before it can be collected at the common bile duct cannula, Equation (4) holds only under steady state conditions; that is, C&r must be constant for a given rate of Jy. From inspecting Equation (4), it is evident that, if Cb < C,, canalicular bile flow is smaller than total bile flow. Thus, the difference between Jy and Jyc provides an estimate of net ductular secretion. Conversely, if Cb > C,, JV,> JV,so that the difference between these two parameters now represents the rate at which fluid is reabsorbed at the distal epithelial structures. By these criteria, it also follows that any increase or decrease in bile flow will be entirely canalicular in origin if the change in solute biliary clearance is of the same magnitude as that in total bile flow. That is, when AJy = A (Jv . cb/c,). Determination

of Canalicular

Permeability

To obtain information about the permeability characteristics of the bile canaliculus, biliary entry of the inert carbohydrate must again be confined to the canalicular network. Moreover, (+ for the solute in question must now be between 0 and 1, possibly closer to 0 than to 1.That is, solute movement into the canalicular lumen must be severely restricted. Under these conditions, solute entry into the biliary system can be defined by Equation (2), written as (1

ch c,=

,I

_

o.

-

4

el~~--~l~lv~~c~Ac

(5) I ’

in which P, (cm/s) is the permeability coefficient for the canalicular membrane and A, (cm’) is the canalicular surface area. If the marker solute enters the canalicular lumen via the transcellular route, A,, P,, and (+will refer to the canalicular membrane only if the sinusoidal membrane and intracellular milieu provide no resistance to solute movement. Because this is unlikely and cannot be tested, these parameters must be assumed to represent average values for the sinusoidal and canalicular membranes, and for the intracellular medium. For the sake of clarity, however, they will be treated here as though they referred solely to the canalicular membrane. As for Equation (a), Equation (5) holds under the condition that Cb/C, is constant for a given rate of JV. Figure 1 shows the form of Equation (5) when A, is given an arbitrary value, and C&, is plotted as a function of JY.It is readily apparent that when JV+ 0, Cb/C, + 1, consistent with the concept that, indeed, solute will enter the bile canaliculus solely by diffusion in the absence of water flow. Conversely, when JV --f a~, C&, ---f (1 - uj, so that C&r now represents the solute sieving coefficient for the canalicular membrane, and convection is virtually the only mechanism of solute entry into the lumen. Because C&,

Figure

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I. Theoretical relationship between solute bile-to-plasma concentration ratio (C&Z,) and bile flow II,, cm3/s. g liver) as defined by Equation (5). Solid line is obtained (a) by giving an arbitrary value to the canalicular surface area (A,, cm’lg liver] and to the solute permeability coefficient (PC, cm/s) for the canalicular membrane and (b) by assuming that both A, and P, are constant during changes in Jy and that 0 < (T < 1. Dotted lines are obtained when D = 0 (C&, = 1) or when Jy = = @b/c, = 1 - cr). Note that, when o = 0, solute will enter canalicular bile freely so that C& will always be unity, regardless of the value of JY.When JV = 00,solute will enter the canalicular lumen exclusively by convection so that the bile-to-plasma concentration ratio now represents the solute sieving coefficient for the canalicular membrane.

cannot be measured at these extreme values of JV (0 or m), Equation (5) can be solved if A, and either P, or o are known. Theoretically, none of these parameters are known, so that a solution to Equation (5) would seem impossible. In reality, however, there are a number of ways to obtain a satisfactory estimate of (T and P,. First of all, A, need not necessarily be known as it can be lumped together with P,. In such a case, P, will no longer be expressed in its conventional units (cm/s), but in units of flow rate (cm3/s . g liver). This will not allow a comparison with the permeability coefficients determined in other transporting epithelia in which the tissue surface area is ordinarily measured. Yet, it will provide a relative measure of canalicular membrane permeability, which is of value in studies of bile secretory physiology and pathophysiology (see below). Second, although in theory C&, = (1 - ~7) only when JV= ~0, in some animal species solute biliary excretion appears to be determined almost exclusively by convection at rates of bile flow that are within the physiologic range. Under these conditions, therefore, solute C&r provides a rough estimate of the coefficient of molecular sieving for the canalicular membrane so that P, can simply be calculated from Equation (5) following substitution of (1 - cb/&) for u. Finally, u and P, can be obtained by curve-fitting analysis, which is the most appropriate and elegant approach. In such a case, Cb/Cr is measured at different rates of JV, and Equation (5) is fitted to the

220 TAVOLONI

couples of values obtained this way. Using this procedure, a critical condition that must be met is that changes in JV must be produced without affecting the membrane permeability; that is, u and P, must remain constant during changes in JV. If the marker molecule does not cross the hepatocyte plasma membrane, but enters canalicular bile, and diffusion and solvent drag are the only mechanisms of permeation, a paracellular mechanism can be evoked and Equation (5) can now be used to describe solute convection or diffusion, or both, through such a route. In this case, (+ and P, will define the permeability characteristics of the transjunctional shunt pathway.

Supporting Experimental Evidence The first attempt to measure the biliary clearance of inert carbohydrates as a means to obtain information about bile secretion was made in 1961 by Schanker and Hogben (4) who obtained indirect but strong evidence that in the rat, mannitol, sucrose, and inulin enter the biliary tree by passive mechanisms, and that the liver is highly permeable to these inert, large, hydrophilic molecules. These experiments set the scenario for the classical studies by Forker (11) and Wheeler et al. (6) who demonstrated that in guinea pigs and dogs, respectively, (a) convection was the primary mechanism of erythritol and mannitol biliary entry and (b) the biliary clearance of these two carbohydrates increased in parallel with the increase in bile flow induced by bile acids, but remained unchanged during secretin choleresis. Thus, inasmuch as secretin was thought to stimulate secretion at the bile ductules and ducts (18), these findings were construed to suggest that erythritol and mannitol enter the biliary system solely at the level of the bile canaliculus, so that these inert carbohydrates could be used to separate canalicular from distal activity. Later results in monkeys (19), baboons (20),and humans (21-23)were consistent with these pioneer observations and interpreted to suggest that, in these species as well, the biliary clearance of erythritol or mannitol, or both, provides an estimate of canalicular bile flow. In the rabbit (24,25) and rat (5,26-28), secretin does not stimulate bile secretion, hence no indirect evidence could be obtained to infer that erythritol and mannitol enter bile solely at the level of the bile canaliculus. At least in the rat, however, the hormone was reported to produce a somewhat more conspicuous increase in bile flow if infused through the portal vein (29) or hepatic artery (29,30), and the choleresis was indeed associated with no significant change in erythritol biliary clearance (29). This finding was thus construed to support the validity of these clearance approaches in this species as well.

94,No.l GASTROENTEROLOGYVol.

Measurements of erythritol and mannitol biliary clearances were not limited to studies in which bile flow was stimulated by a physiologic bile acid or the hormone secretin, but rather extended to a variety of conditions associated with choleresis or cholestasis. And, the anatomic location at which osmotic choleretics (31-34) and other stimulants (35~al), hormones (42-521, and hepatotoxic agents (53-62) affected bile formation was inferred on the basis of their effects on the biliary clearance of erythritol or mannitol, or both. For instance, the conviction, which still holds today, that glucagon choleresis is canalicular in origin has evolved almost exclusively from these clearance approaches (43,44,47,48) and has not been weakened by the notion that glucagon is a hormone of the secretin family, which is thought to stimulate ductular secretion (18)and with which it shares a striking chemical resemblance (63). Most of these studies were carried out in the rat which, because of its poor responsiveness to the choleretic effect of secretin, has never proved to be a suitable animal model for distinguishing canalicular from ductular activity. Yet, perhaps because of the analogy with renal clearance measurements, which had earlier demonstrated their usefulness in studies of urine formation, there was a widespread acceptance of these biliary clearance approaches as though they were assured a leading role in the overall strategy for unraveling the mechanisms of bile formation and cholestasis. As to the significance of the biliary clearance of sucrose and inulin, most of the impetus for carrying out these measurements has come from the finding reported by Forker (64)that, in rats, estrogen cholestasis was associated with a large increase in sucrose biliary permeability. This observation gave functional meaning to these clearance measurements, and lent support to the postulate that cholestasis may involve an increased diffusion of bile constituents from the canaliculus back to plasma. Successive studies have not only extended this intriguing finding to various models of cholestasis (55,59,6567),they have also demonstrated that immaturity of the biliary system (68,69) and even choleresis produced by certain bile acids (70-72)are associated with an increase in sucrose or inulin biliary permeability. The concepts thus evolved that biliary permeation of sucrose and inulin is an important feature of hepatocytic bile formation, and the degree of entry of these large carbohydrates into bile is indicative of the integrity of the bile canaliculus. Moreover, because transhepatic movement of sucrose and inulin was thought to be confined virtually to the transjunctional route between hepatocytes (65,66,70-73), it seemed warranted to propose that the biliary clearance of these large hydrophilic molecules provides a

January 1988

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was first obtained by substituting (1 - C&,) for (T, measure of paracellular pathway permeability. Choand by measuring C&, and Jy under spontaneous lestasis could therefore be explained in terms of secretory conditions. Subsequently, a k value for the increased junctional permeability by postulating cholestatic liver was calculated by substituting the that “leaky” junctions between hepatocytes would same value of (1 - C&,) for 0; and measuring C&, allow regurgitation of solutes from the bile canalicand JV during the induction of cholestasis. The ulus back to plasma, and ultimately lead to dissiparesults from this analysis have unanimously shown a tion of the osmotic gradient underlying vectorial drastic increase in sucrose and inulin biliary permetransport of bile water (64-75). ability coefficients during various models of choleThe conviction that sucrose and inulin enter the stasis (64,65,72,91,92) but, as discussed below, one bile canaliculus primarily via the transjunctional would do well to reserve judgment on whether the shunt pathway has three main experimental origins. assumptions included in this approach are correct. First, studies in rats have localized lanthanum More appropriately, Strasberg et al. (94)attempted to within the canalicular lumen and junctional comdetermine u and k for sucrose and inulin in the plexes, but not within hepatocytes (70,76). Second, biliary system of the monkey by curve-fitting analyGraf et al. (77) reported that the electrical resistance sis. They changed the bile flow rate by infusing of isolated hepatocyte couplets is low, and lowvarious amounts of choleretic bile acids, and fitted resistance epithelia, conventionally classified as Equation (5)to the values of C&$ and Jy measured “leaky,” have fewer sealing strands in their “zonula experimentally. Their results were fully consistent occludens” (78,79), are highly permeable to ions and with the theoretical model and, for the first time, nonelectrolytes, and do not have unusually permeprovided direct estimates of both (T and k for these Third, studies in able plasma membranes (80-85). two carbohydrates. However, these investigators did several animal species have demonstrated that sunot extend their observations to studies of biliary crose and inulin enter bile in significant amounts permeability during cholestasis, so that it was not when infused intravenously (1,4,6,7), and equilibrate more readily with bile than with liver cell established whether k or U, or both, change during bile secretory failure. This was done a few years later water (12). Although indirect, this evidence was in accord with results obtained in other studies of by Jaeschke et al. (95) using ethinylestradiol and a-naphthylisothiocyanate as cholestatic agents. transporting epithelia (86-88)and with the common belief that these large inert carbohydrates do not Their results made the intriguing point that cycross the plasma membrane of biological cells, It is naphthylisothiocyanate, but not ethinylestradiol, increases sucrose and inulin biliary permeability coefnot surprising, therefore, that the concept of paracellular solute movement was championed by ficients. More importantly, they demonstrated that biliary physiologists, and the transjunctional shunt the increase in k produced by c-u-naphthylisothiocyapathway was given an important role in bile secrenate was associated with a significant decline in (+ tory physiology and pathophysiology (65-76,89,90). for both solutes, a finding fully consistent with the An increase in sucrose or inulin biliary permeabilpredictions from the theoretical model (see below). ity associated with cholestasis can be claimed when Recently, Reichen et al. (92) have suggested that, solute biliary clearance increases above control levwhen using Equation (5) for assessing the permeabilels. Most often, however, biliary excretion of either ity characteristics of the biliary tree, determination carbohydrate declines during the acute phase of of biliary recovery of labeled sucrose relative to that cholestasis, even though solute C& increases sigof tritiated water is somewhat more advantageous nificantly. Thus, whether the increased C&, is than the conventional measurement of sucrose expressive of an augmented permeation or simply of C&,. Furthermore, by perfusing the isolated rat an increased contribution of solute diffusion due to liver via the portal vein or the hepatic artery, the reduction in bile flow rate (see Figure 1) can be Reichen and Sagesser (96) have claimed that changes established only by solving Equation (5). As previin permeability at the portal venous and hepatic ously discussed, there are two ways of solving Equaarterial beds can be assessed separately with such a tion (5) and, indeed, both of them have been atmultiple-indicator dilution technique. As already tempted. In most instances (64,65,72,91-931, two mentioned, however, water enters bile throughout assumptions were made: (a) solute C&, observed at the biliary system (e.g., bile canaliculi, intrahepatic physiologic rates of bile flow roughly estimates the and extrahepatic bile ductules and ducts) and by canalicular membrane sieving coefficient so that D = multiple mechanisms (e.g., diffusion, convection, (1 - C&,) and (b) the sieving coefficient is not exocytosis), and tritiated water does not selectively affected by the cholestatic insult. Thus, a physiologic label a single route or mechanism. Thus, the validity estimate of the permeability coefficient k, which is the equivalent of the expression (PC . A,) used here, of these novel approaches remains to be proved.

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Conflicting Evidence The first experimental observation in conflict with the dogma that biliary entry of inert carbohydrates the size of erythritol or larger is restricted to the canaliculus was made by Peterson and Fujimoto (97) who demonstrated that recoveries of labeled erythritol, mannitol, and inulin from rat bile were not complete after these solutes were injected retrogradely via the common bile duct cannula. However, these results did not alarm biliary physiologists and, in fact, only when secretin was found to partially increase the biliary clearance of erythritol or mannitol, or both (98-101), was the likelihood considered that these solutes also permeate the biliary epithelial structures. And even then, another possibility was left open. Secretin may in part stimulate canalicular secretion so that both of these inert carbohydrates would still be valid markers of However realistic hepatocytic water flow (98-100). this latter alternative seemed, it did not generate controversy. Indeed, the finding that erythritol, mannitol, sucrose, and inulin were all capable of permeating the extrahepatic bile duct of the rat (102) and guinea pig (103) has virtually swept away any realistic hope that biliary entry of these carbohydrates is confined to the bile canaliculus. These observations have not only raised serious questions as to the validity of erythritol and mannitol biliary clearances for estimating canalicular bile flow, they have also obscured the significance of sucrose and inulin biliary entry. In fact, an increase in sucrose and inulin permeation may not be solely expressive of an augmented leakiness of junctions between hepatocytes, but may also involve structural or functional alterations at sites distal to the hepatic parenchyma. Even though these new results had already jeopardized the usefulness of these clearance approaches, the challenge was far from over. First, Javitt (104) reported that entry of polyethylene glycol 900 in rat bile was much greater than that of erythritol or mannitol, as values of polyethylene glycol 900 C&Z, as high as 4 were observed under spontaneous secretory conditions. The significance of this result has never been clarified, but he construed it to suggest that the biliary clearance of erythritol or mannitol, or both, greatly underestimates canalicular secretion as these carbohydrates, unlike polyethylene glycol 900, equilibrate too rapidly between plasma and canalicular bile and do not provide a measure of net water reabsorption. Second, recent studies in guinea pigs have demonstrated that several hydroxy bile acids diminish erythritol and mannitol biliary permeation during choleresis, whereas they increase or fail to modify that of sucrose and inulin (7,105). As summarized in

GASTROENTEROLOGY Vol. 94, No. 1

I

,

,

,

,

,

0 +10 +20+30+40+50+60 A BILE

FLOW

,

+70+00

(jd/mm/kgl

Figure 2. Relationship between changes (A) in erythritol biliary clearance and those in bile flow during choleresis produced by various bile acids in the guinea pig. TDC, taurodehydrocholate; TDX, taurodeoxycholate: TUDX, tauroursodeoxycholate; TCDX, taurochenodeoxycholate; CH, cholate; and TC, taurocholate. Note that if no change in permeability occurs and erythritol enters bile primarily by convection, the slope of such a relationship should equal solute C&Z,. In the guinea pig, this is the case only when bile flow is stimulated by TDC (and its unconjugated form; see Reference 195). During TDX choleresis, erythritol biliary clearance increases at a rate smaller than that of bile flow (slope < C&r), whereas during TUDX infusion solute biliary clearance is essentially unaffected (slope -0). When bile flow is stimulated by TCDX, CH, and TC, erythritol biliary clearance actually declines below control values (negative slope). (Reproduced with permission from Tavoloni, Am J Physiol 1984;247:G527-36.)

Figure 2, only dehydrocholate and its taurine conjugate do increase the biliary clearance of erythritol and mannitol in parallel with the increase in bile flow. All the others invariably decrease either solute C&Z, during choleresis and, with some of them [trihydroxy and 3a,7a-dihydroxy bile acids (105)], the biliary clearance actually declines below control values. This unconventional finding has provided strong evidence for multiple pathways or mechanisms of solute biliary entry for, if erythritol, mannitol, sucrose, and inulin all shared the same route and mechanism of permeation, their biliary clearances should similarly be affected by any given bile acid. At the same time, however, these results have invalidated the fundamental tenet that changes in erythritol and mannitol biliary clearances relative to those in water flow are indicative solely of the anatomic site at which bile is being secreted or reabsorbed. Now, we must add another factor to the equation; changes in biliary clearance of these small carbohydrates may also reflect alterations in canalicular membrane permeability. Third, we have recently shown that in the guinea pig, spontaneously reversible cholestasis produced

January 1988

by low doses of taurolithocholate is associated with an irreversible increase in sucrose and inulin biliary permeation (103). That is, biliary clearance of sucrose and inulin averaged, respectively, 13% and 4% of water flow before taurolithocholate was injected, but 28% and 16% 1 h after 10 pmol/kg of the cholestatic bile acid was given, when bile flow had fully returned to its precholestatic rate. This finding raises serious questions regarding the suggestion that changes in canalicular membrane (junctional) permeability are expressive of altered secretory function (64-66,71,73,74). Fourth, criticism must be raised with respect to the use of Equation (5) for estimating changes in membrane permeability during cholestasis. As previously discussed, most investigators have calculated sucrose and inulin biliary permeability coefficients (k’s) for the normal and cholestatic liver by assuming that (a) (1 - C&,) measured under spontaneous secretory conditions roughly estimates the solute reflection coefficient u for the canalicular membrane and (b) c does not change during the induction of cholestasis (64,65,72,91-93). First of all, both of these assumptions have been proved to be incorrect when (+and k (95) or g and P, (103) were

Figure

3. Fluorescent photomicrograph of rat hepatocytes in culture incubated with fluoresceinated dextran (mol wt -70,000) for 12-16 h. Note the presence of fluorescence within vesiclelike structures, suggesting that such a large molecule is internalized through an endocytotic mechanism. (Reproduced from Lake et al., J Clin Invest 1985;76:676-84, by copyright permission of the American Society for Clinical Investigation.]

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0

-0

-0

Froct~on Number

Figure

4. Distribution of [3H]methoxyinulin and various marker enzymes in fractions of rat liver microsomes separated by isopycnic centrifugation through a linear sucrose gradient. Note that [3H]methoxyinulin is recovered primarily in a fraction enriched in galactosyltransferase activity, an enzyme associated with Golgi membranes. (Reproduced with permission from Lorenzini et al., Gastroenterology 1986;91:1278-88.)

calculated by curve-fitting analysis. The second assumption, then, includes a logical fallacy. Equation (5), in fact, defines solute distribution between plasma and bile only if permeation through biliary membranes is assumed to be via aqueous pores. If the permeability of the solute in question increases during cholestasis, two explanations can be considered. Either a new pathway has been formed or an increase in pore radius or a change in pore architecture, or both, as to allow increased permeation, has been produced by the cholestatic injury. The first possibility is highly unlikely if the new pathway involved a lipid phase, inasmuch as sucrose and inulin are highly hydrophilic molecules. In such a case, then, Equation (5) would no longer be applicable to describe solute biliary entry. If a new polar route is formed, as some preliminary studies would suggest (106), solute diffusion and convection would still be the mechanisms of solute biliary permeation. Similarly, if the size or the architecture of the existing membrane pores has changed, both diffusion and convection would be responsible for solute permeation. In either case, therefore, both of these processes would be affected when solute biliary permeability is increased. This means that (Tmust decrease when P, (or k) increases. As already mentioned, data from our own work (103) and from that of Jaeschke et al. (95) are fully consistent with this line of reasoning. Finally, and perhaps most importantly, recent studies have provided evidence that inert hydrophilic molecules are internalized by hepatocytes and eventually excreted into bile via an endocytoticexocytotic route (107-109). Thus, exposure of cultured rat hepatocytes to fluoresceinated dextran (mol wt -70,000)results in fluorescence accumulation within intracellular vesicles (Figure 3), and kinetic studies of sucrose and inulin biliary excretion in the

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define entry of polar nonelectrolytes bile.

into canalicular

Perspective and Future Directions

120 Time. mm

Figure 5. Effect of 10 PM colchicine (W] on inulin uptake (37°C) by rat hepatocytes in culture. Control cell batches were incubated with 10 PM lumicolchicine (0). In either case, curves are the best fits of the equation describing solute entry into a two-compartment system, a rapidly turning-over compartment (presumably endosomal) and a slowly turning-over compartment (storage]. Note that colchicine, an inhibitor of microtubular function, significantly inhibits total inulin accumulation, but has no effect on initial rate of solute uptake. This indicates that only that fraction of endocytosed inulin destined for intracellular storage or biliary excretion, or both, requires intact microtubular function. (Reproduced with permission from Scharschmidt et al., Proc Nat1 Acad Sci USA 1986;83:9488-92.)

isolated perfused rat liver (107) and in the intact bile fistula rat (108)are consistent with the concept that at least a fraction of solute entry into bile involves a vesicular mechanism. Evidence has also been obtained to indicate that endosomelike vesicles, presumably derived from the Golgi apparatus, are involved in the transcellular movement of such a solute (108). Thus, as illustrated in Figure 4, isopycnic centrifugation of rat liver microsomes through a linear sucrose gradient has revealed that [3H]-methoxyinulin distributes primarily within a subcellular fraction enriched in galactosyl transferase, an enzyme associated with Golgi membranes (108). Furthermore, as colchicine (Figure 51, but not chloroquine, inhibits inulin uptake by cultured rat hepatocytes, it would appear that intact microtubular function, but not vesicle acidification, is required for this vesicular transport pathway (109). At present, the contribution of this transcytotic route to the total biliary entry of sucrose and inulin seems to be small, for Scharschmidt et al. (109) have obtained evidence that as much as 80% of the endocytosed inulin is regurgitated back into plasma and only 2% is transported into bile. Yet, the existence of such a mechanism implies that movement of these inert carbohydrates into the bile canaliculus can no longer be confined to the transjunctional shunt pathway. Perhaps more importantly, it cannot be described solely in terms of diffusion and convection so that, in a strict sense, Equation (5) is no longer applicable to

In this report, we have attempted to summarize most of the relevant work on biliary entry of inert carbohydrates as it pertains to the study of bile secretory physiology and pathophysiology. It has also been our intention to provide a somewhat critical discussion of the interpretation of these biliary clearance data based on both theoretical grounds and existing knowledge of the structure and function of bile secretory apparatus. The salient point that we have made is that, whereas early evidence supported the validity of the biliary clearance of erythritol or mannitol for estimating canalicular bile flow and that of sucrose or inulin for characterizing the permeability of the transjunctional shunt pathway between hepatocytes, new data are in conflict with these postulates and have delineated the usefulness of these clearance measurements much more narrowly. In effect, these approaches have rapidly descended from “Olympian Heights” and settled into a limited, even uncertain, role in studies of bile formation and cholestasis. As a result of these new findings, the following two questions are now in order: (a) Where do we presently stand? (b) Where do we go from here? The answer to the first question is not a difficult one. The fact that all of these inert carbohydrates permeate the biliary epithelial structures invalidates, in a strict sense, the use of erythritol and mannitol biliary clearances for separating canalicular from ductular and ductal activity. It must be pointed out, though, that the bile ductules and ducts do not form a tridimensional network as the canaliculi do, so that distal permeation of these molecules can be quite small when compared with the amount of solute entering the canalicular lumen. Also, the extent of solute entry at the biliary epithelium can roughly be estimated using a model in which the bile ductules and ducts are included as permeable structures (103). Thus, if the clearance data are interpreted on the basis of existing limitations, some qualitative information about the site at which bile water is being secreted or reabsorbed can still be obtained for these clearance techniques. As to the significance of sucrose and inulin biliary clearances, the present picture is somewhat more complicated. These large carbohydrates not only permeate the distal biliary epithelium, they also enter canalicular bile via a vesicular route, and the degree of solute biliary permeation does not necessarily correlate with bile secretory activity. However, the contributions of transcytotic (109) and distal entries may be small, or even negligible in species

January 1988

like the guinea pig (103), and it is not readily conceivable that the large increase in soluble biliary permeation often associated with cholestasis is entirely due to increased hepatocellular vesicular transport or distal translocation, or both. Furthermore, recent work from our laboratory has provided direct evidence that these two large carbohydrates are not capable of diffusing through the plasma membrane of the isolated rat hepatocyte (110). Thus, it is reasonable to assume that the increase in solute biliary permeation associated with changes in secretory activity is expressive, at least in part, of increased junctional permeability, so the early hypothesis that leaky junctions are an important factor in the pathogenesis of bile secretory failure need not necessarily be rejected. Within this context, then, another point must be stressed. Recent studies of sucrose and inulin biliary translocation have made a pivotal contribution to our understanding of the vesicle-mediated transcytosis, which may prove to be an important mechanism for transhepatic solute movement and water flow. It must therefore be recognized that, although invalidating some of the classical tenets about the mechanisms and pathways of biliary entry of polar nonelectrolytes, these late studies have paved the way for new concepts of hepatic transport and bile formation. The answer to the second question-where do we go from here-is clearly more complicated. First, we must establish whether we really need a means for accurately separating canalicular secretion from ductular and ductal secretion or reabsorption. The concept that the biliary epithelium is involved in bile formation has evolved from indirect evidence, so one could argue that hepatic bile is formed solely at the bile canaliculus; the collecting bile ductules and ducts may be inert structures with respect to bile formation. However, ongoing research in our laboratory indicates that, at least in the rat, the biliary ductules and ducts secrete a bicarbonate-enriched bile and are the site of secretin choleresis (111).We do not know as yet the physiologic contribution of this distal activity, but it does not appear to be trivial. Thus, if we want to obtain information on the mechanisms of bile formation and cholestasis by sampling bile at the common bile duct, we do need to have a means for accurately separating that fraction of fluid originated at the canaliculi from that secreted or reabsorbed at the biliary epithelium. As we have seen, erythritol and mannitol biliary clearances do not provide a rigorous separation of these activities, thus we must seek for either new suitable markers or alternative strategies to solve such a problem. Similarly, we must decide whether it is important to have an accurate marker of transjunctional permeability between hepatocytes. Again, there is no direct evidence for a paracellular mech-

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anism of bile formation, but a number of observations are consistent with this view. If we want to establish the role of this route in bile secretory physiology and pathophysiology, we have three options to consider. First, measuring sucrose or inulin biliary clearance, or both, may still be a valid approach so that, before permanently dismissing these techniques, we may want to find out how much of either solute biliary entry is due to vesicular transport and distal permeation both during spontaneous secretion and under conditions associated with choleresis or cholestasis. These experiments will have served their purpose should they prove that a major, measurable fraction of sucrose and inulin biliary entry is via the transjunctional shunt pathway between hepatocytes. Second, we can look for other suitable markers of pamcellular fluid movement. Finally, we can confront this problem using other approaches. If we elect to venture into the search for new, valid markers of canalicular bile flow and paracellular water movement, we must keep in mind that the future may not look very promising. There are no good reasons to believe that a certain molecule(s) will freely permeate the bile canaliculus but will not cross the biliary epithelial structures at all, nor that it will enter the biliary tree exclusively via the junctions between hepatocytes. Thus, other strategies should be considered for separating canalicular from distal activity, and for unraveling the role of the transjunctional shunt pathway in bile formation and cholestasis. In particular, recent reports have demonstrated that isolated hepatocyte couplets are an invaluable model for studying hepatocytic secretory processes (77,112-114),and current research in our laboratory (115,116)and published work from other groups (117-121)suggest that biliary epithelial cells can be isolated in satisfactory yield and purity as to allow in vitro studies of bile ductular cell function. Furthermore, studies of hepatocyte junction morphology have contributed significantly to our present understanding of paracellular fluid movement (65,70,89,90,122-125), so that future research in this area and, hopefully, on tight junction biochemistry may shed new light on the function of the transjunctional shunt pathway. These and other approaches should help banish our ignorance of bile secretory physiology and pathophysiology, and ultimately contribute new understanding to the treatment of cholestasis. References 1. Forker EL. Bile formation

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Received November 26, 1986. Accepted July 27, 1987. Address requests for reprints to: N. Tavoloni, Ph.D., Division of Hematology, Atran Building, 3rd Floor, Box 1079, Mount Sinai School of Medicine, Madison Avenue at 100th Street, New York, New York 10029. The studies carried out in the author’s laboratory and the writing of this review were supported by National Institute of Child Health and Human Development grant HD-17556. The author thanks Dr. P.D. Berk for the encouragement, support, and guidance he has provided during the many fruitful years of their association. He also thanks his colleagues who have contributed to the work carried out in his laboratory, in particular Mary Jane T. Jones who, with her patience and skillful technical help, has been a major force in obtaining most of the experimental data dealing with this topic. Finally, he acknowledges Dr. J.L. Boyer and Dr. B.F. Scharschmidt for kindly providing prints of Figure 4 and Figures 3 and 5, respectively, and Mary Barrett for excellent assistance in editing this manuscript.