CANALICULAR BILE FLOW AND BROMOSULFOPHTHALEIN TRANSPORT MAXIMUM: THE EFFECT OF A BILE SALT-INDEPENDENT CHOLERETIC, SC-2644

Transcription

1 GASTROENTEROLOGY 66: , 1974 Copyright 197~ 1l\' The William, & Wilkim Co. Vol. 66. )lo. ~ Printed in U.S.A. CANALICULAR BILE FLOW AND BROMOSULFOPHTHALEIN TRANSPORT MAXIMUM: THE EFFECT OF A BILE SALT-INDEPENDENT CHOLERETIC, SC-2644 G. E. GIBSON, M,D., M,RA.C,P" AND E. L, FORKER, M,D. Department of Physiology and Biophysics and Gastrointestinal Research Laboratories, Department of Internal Medicine, The University of Iowa, Iowa City, Iowa Both taurocholate and (3-(2, 4-diamethoxy-5-cyclohexylbenzoyl)propionic acid (SC-2644) produce an increase in bile flow and an equivalent increase in the biliary clearance of erythritol Only the former, however, produces an increase in bromosulfophthalein (BSP) transport maximum and an increase in the fraction of unconjugated dye appearing in bile, We conclude that the taurocholate-dependent changes in BSP excretion are not attributable to the associated choleresis, but instead represent a specific effect of the bile acid, From the present experiments, it is not possible to determine whether an effect of taurocholate on the membrane transport of BSP or micellar sequestration of dye in the canalicular lumen is the more important mechanism, but indirect evidence is reviewed which suggests that alterations in the intracellular conjugation of BSP do not account for the bile acid-dependent changes in BSP transport maximum, In 1966, O'Maille et al 1 reported that an infusion of taurocholate could increase the hepatic excretory capacity for bromosulfophthalein (BSP) in dogs, Ritt et al 2 later suggested that the increase in BSP output was largely accounted for by the appearance in bile of additional unconjugated dye, Similar observations have since been Received April ~3, 1973, Accepted October 9, 197:3. This work was presented in part to the American Association for the Study of Liver Disease, November, 1972, Chicago, Illinois. Address requests for reprints to: E. L. Forker, M,D" Department of Medicine, University of Iowa, Iowa City, Iowa This work was supported in part hy United States Public Health Service Grants AM and AM-05:390. Dr. Gibson was a Postdoctoral Fellow in gastroenterology at The University of Iowa, His present address is: Department of Gastroenterology, Royal Adelaide Hospital, Adelaide 5000, Australia, Dr. Forker is a Markle Scholar in academic medicine and National Institutes of Health Research Career Development Awardee AM reported from other laboratories for a variety of other animals species,3-6 One interpretation of this phenomenon which appears to have gained wide acceptance attributes the effect of bile acids to the associated choleresis, which, by diluting the BSP in bile, permits the excretion of additional dye without exceeding a putative concentration maximum, Based on a kinetic analysis of BSP transport in the rat,7, 8 we have previously suggested that the dependency between BSP excretion and taurocholate choleresis may have little to do with the changes in bile flow per se, but instead probably represents a specific effect of taurocholate on the membrane transport of the dye, The experiments to be reported here were designed to reexamine the flow postulate by determining whether or not the apparent transport maximum (Tm) for BSP increases when canalicular choleresis occurs without a change in bile acid output. If net BSP transport is dependent on canalicular bile flow per se, one should expect to find 1046

2 May 1974 SC-2644 AND ESP TRANSPORT MAXIMUM 1047 similar changes in BSP excretion, no matter how the canalicular choleresis is produced. On the other hand, if the change i.n BSP Tm observed during taurocholate choleresis is a specific effect of bile acid transport, it should be possible to increase canalicular flow by other means without affecting the output of BSP. The choleretic we have chosen for this purpose is {1-( 2, 4-diamethoxy -5-cyclohexylbenzoyl)propionic acid (SC-2644) (fig. 1), the most potent of a large group of drugs originally synthesized by the G. D. Searle Corporation (Columbus, Ohio) by linking a "{-oxobutyric acid side chain to various aromatic hydrocarbon nuclei. Original reports of the choleretic activity of this material were published by Cook et a1., who kindly supplied the material for the present study. Little is known about the mechanism of action of SC-2644, but as recently emphasized by Wheeler and King" and confirmed by our own studies, the duration and magnitude of the choleretic response are such that its effect cannot be accounted for by active excretion of the material itself. Of particular importance to the present work are the findings that the increment in bile flow arises in the canaliculi and that the choleresis occurs without a change in bile acid excretion. Experimental Methods and Design Animals for these studies were 15 female, mongrel dogs (11 to 15 kg) anesthetized with sodium pentobarbital. After an overnight fast, cannulae were placed in the common bile duct, a femoral vein, and the contralateral femoral artery. The cystic duct was ligated, and to minimize absorption of endogenous bile acids, the contents of the gallbladder were aspirated. The abdomen was closed, and, after a recovery period of approximately 30 min, each animal was given a loading dose of BSP (Hynson, Westcott, and Dunning, Baltimore, Md.; 18,umoles kg- 1 ), followed by a constant intravenous infusion of the following composition: Na taurocholate (Calbiochem, San Diego, Calif.; 0.18,umole min- 1 kg- 1 ), BSP (0.32,umole min- 1 kg- 1 ), and isotonic NaCI (0.5 ml min-i). This infusion was continued throughout the rest of the experiment, and subsequent procedures and observations are referenced to this starting point, taken as zero time. The background OCH, ~oc", ~-CH2-CH2-COOH o FIG. l. {3-( 2, 4-diamethoxy-5-cyclohexylbenzoyl) propionic acid (SC infusion of Na taurocholate was assumed to approximate normal fasting bile acid secretion in the dog. ' 11 The BSP infusion in every case proved sufficient to ensure a gradually rising plasma level of the dye throughout the remainder of the experiment. The anticholinergic, pipenzolate methylbromide (Lakeside Laboratories, Milwaukee, Wisc.; 0.1 mg kg- 1 every 20 min after a loading dose of 0.5 mg kg- ') was given to minimize spontaneous variations in the bile flow. At t = 30 min, the first of three consecutive 10-min bile samples was collected to determine the basal level of bile flow, BSP Tm, and the fraction of the dye present as the free or unconjugated form. Bile flow and BSP excretion were determined for each 10-min period, and agreement of the individual values to within 57c of the mean was taken as an acceptable criterion of steady state biliary excretion, having previously determined that an interval of 30 min (3 '\ 10) would be sufficient to reveal dead space errors attributable to the volume of the biliary tree and the collecting cannula. The fraction of unconjugated BSP was determined chromatographically (vide infra) from a pooled bile sample representing the entire 30-min collection. The secretory response to subsequent infusions of additional taurocholate (1.5, 2.0, or 3.0,umoles min- 1 kg- 1 ), SC-2644 (0.5,umole min- 1 kg-i), or dehydrocholate (Dohme Research Laboratories, West Point, Pa.; 1.9,umoles min -1 kg- 1 ) was determined in the same manner after a 60-min interval required to achieve a new steady state. The sequence in which these materials were given to each dog is summarized in table 1. Usually each dog was studied during two additional periods following the initial base line or control observation (period 1). In some cases, the responses to two choleretics were evaluated separately in the same dog by first giving either taurocholate or dehydrocholate (period 2), followed by an interval of60 min in which bile flow was allowed to return toward the control level followed, in turn, by a third period in which the response to SC-2644 was evaluated. In this type of experiment, the infusion of SC-2644 always followed the infusion of bile acid, because

3 1048 GIBSON AND FORKER Vol. 66, No.5 TABLE 1. Choleretic infusion a Experiment Period 1 Period 2 Period 3 Infusion I'moles min-'kg- 1 Infusion I'moles min-1kg- 1 Infusion I'moles min-1kg- 1 1 " TC 0.18 DHC 1.9 SC 0.5 2" TC 0.18 DHC 1.9 SC b TC 0.18 DHC 1.9 SC TC 0.18 TC 2.0 TC TC 0.18 TC TC 0.18 TC TC 0.18 SC TC 0.18 TC 3.0 SC TC 0.18 TC 3.0 SC " TC 0.18 TC 1.5 TC + SC " TC 0.18 TC 1.5 TC +SC 1.5 '+).5 12 C TC 0.18 TC 1.5 TC + SC TC 0.18 SC 0.5 SC +TC TC 0.18 SC 0.5 SC +TC TC 0.18 SC 0.5 SC +TC a TC, taurocholate; DHC, dehydrocholate; SC, SC "Received HC-erythritol. C Received 'H-taurocholate. SC-2644 choleresis persists for many hours after even the smallest effective dose. To obviate this bias, a second kind of experiment was conducted, in which either taurocholate or SC-2644 was evaluated alone during period 2 and the second choleretic was superimposed during period 3. In three experiments of this type, 3H_ taurocholate (New England Nuclear Corporapon, Boston, Mass.) was employed to evaluate the possibility that the addition of SC-2644 might alter the previously determined excretion rate of taurocholate. In three other experiments, HC-erythritol (New England Nuclear) was given with the priming dose of BSP to provide a measure of erythritol clearance during SC-2644 choleresis. The radioactivity of plasma and bile was measured by liquid scintillation counting after adding 100 ~tl of plasma or 20 ~tl of bile to 15 ml of a 1:5 (v:v) mixture of Scintisol-GP and Scintolute (Isolab, Inc., Akron, Ohio). Corrections for quenching were determined by internal standardization. The concentrations of total BSP in bile and plasma were determined spectrophotometrically according to the method of Seligson et al. 12 BSP conjugates in bile were separated by ascending thin layer chromatography on silica gel plates with acetone-h 2 0-NH3 (81: 15: 4) as the solvent system. 13 Four bands of conjugated dye were separated from the more rapidly moving conjugated material by this method. All four bands were identified by exposure to NH3 vapor, eluted from the silica gel with H 2 0, and measured spectrophotometrically. The values to be reported here are the amounts of unconjugated material expressed as fractions of the total. The purity of labeled taurocholate was evaluated by thin layer chromatography,14 followed by scanning with a thin-window gas flow counter. A single radioactive peak was identified which migrated with authentic taurocholate run in parallel on the same plate. Results A typical experiment in which taurocholate and SC-2644 were given separately is illustrated in figure 2. The data for bile flow, BSP Tm, and the unconjugated fraction of biliary BSP in this and subsequent figures are shown as ratios, with the corresponding base line or control observations arbitrarily taken as unity. The infusion of taurocholate (3 Jlmoles min -1 kg- 1 in this case) increased bile flow approximately 2.5 times, and there were associated increases of 35% and 50% in total BSP output and the fraction of unconjugated dye, respectively. Subsequently, when these values had returned toward their original levels, an infusion of SC-2644 produced nearly a 3-fold change in total bile flow but no

4 May 1974 SC-2644 AND BSP TRANSPORT MAXIMUM 1049 further change in either total BSP excretion or the unconjugated fraction. The data from three similar experiments with dehydrocholate in which erythritol clearances were also measured appear in table 2. In all 3 dogs, dehydrocholate choleresis was associated with an increase in the unconjugated fraction of BSP. One dog exhibited a small increase in BSP Tm, while the other 2 experienced an appreciable decline in dye output. SC-2644 choleresis produced only minor changes in these parameters, although the erythritol clearances clearly suggest that the additional fluid originated in the canaliculi. SSP O.:U.. M ".,.~ K'" I Touroc~:ol~. I f: M "'... 1(9 Plasma BSP mg. % ---~~ o RATIO FIG. 2. Each vertical bar represents the mean value for three consecutive lo-min bile collections expressed as a ratio to the corresponding control value determined at 45 min. Absolute control values are: flow ~ 13.4!Ll min -! kg-i; bromosulfophthalein (BSP) transport maximum (TM) ~ 0.14!Lmole min-! kg-i; unconjugated fraction ~ 26%. Background infusion of taurocholate (solid line) ~ 0.18!Lmole min-! kg-i. SC 2644, ~-(2, 4-diamethoxy-5-cyclohexylbenzoyl)propionic acid. TABLE 2. Dehydrocholate (DHC; 1.9!J.M min-! kg-i) and SC-2644 (SC; O.5!LM min-! kg-i) To be sure that the failure of SC-2644 choleresis to increase either total BSP excretion or the unconjugated fraction was not attributable to a simultaneous decrease in taurocholate excretion, three experiments, of the sort illustrated in figure 3, were done using 3H-taurocholate. The recovery of radioactivity from bile was in every case within 5% of the infusion rate and was unchanged by the addition of SC Taurocholate choleresis was again associated with substantial increases in BSP Tm and the unconjugated fraction, but no further change in either of these values was seen during a subsequent period when SC-2644 was used to triple the rate of bile production. Three additional experiments of this type, in which the choleretics were used in reverse order, yielded a similar result. Figure 4 summarizes the data for taurocholate and SC-2644 from all 15 experiments in which one or both of these choleretics was studied. Inasmuch as each dog served as its own control, the summary data are presented as mean percentage changes from the initial base line values observed in the same animal. The changes in BSP Tm and the unconjugated fraction scatter around zero for the periods in which SC-2644 was responsible for the increment in bile flow, while these changes were always substantially greater than zero during infusions of taurocholate at three different rates. In all dogs, the increase in total BSP output during taurocholate chol- Experiment Total bile flow BSP Tm (I'M min -I kg-i) Unconjugated fraction (1'1 min- I kg-i) Ratio to control Ratio to control Ratio to control Erythritol clearance (jllmin-'kg-') Control Control Control t.ce " DHC SC DHC SC DHC SC Control SC -- t.f Mean a t!.cp/t!.f ~ (change in erythritol c1earance)/(change in total bile flow). %

5 1050 GIBSON AND FORKER Vol. 66, No.5 14 Plasma SSP 12 t 10 a mg. % SSP 0 32 ), ,... o(i l I H-3-Tourocholole 1, $ u'-l "',... 1(."'!BSC-2644D'! O$ )oim INII~ KO~ RATIO 4 FIG. 3. Each uertical bar represents the mean value for three consecutive lo-min bile collections, expressed as a ratio to the corresponding control value determined at 45 min. Absolute control values are: flow ~ 7.90 /11 min - I kg I; bromosulfophthalein (BSP) transport maximum (Tm) /1mole min- I kg-i; unconjugated fraction ~ 31%. Background infusion of unlabeled taurocholate (solid line) ~ 0.18 /1mole min -I kg-i. SC-2644, /1-(2,4-diamethoxy-.5- cyclohexylbenzoyl)propionic acid. 100 o asp Tm WJ aile FLOW ~~~GATEO Te TC Te 5C-2644 ls um m... 1<9 2 um min 1<9 3uM min Kg 0.5 ~ mm K9 n<~ n04 n;5 n~12 FIG. 4. Summary of data from 1.5 dogs. Each group of vertical bars show the means and SE'S for n observations, expressed as percentage increases from control values in the same dog. Mean control values for 1.5 dogs are: bile flow ~ 11.2 ± 2.2/11 min -I kg-i; bromosulfophthalein transport maximum (BSP Tm) ~ 0.18 ± 0.04 /1mole min- I kg-i; unconjugated fraction ~ 24 ± 7'1'. TC. taurocholate; SC /1-(2, 4-diamethoxy-5-cyclophexylbenzoyl )propionic acid. eresis was nearly accounted for by an increase in the output of unconjugated dye. The mean percentage increase in the output of conjugated material for all three doses of taurocholate was 0.08 ± 0.08 (SE, n = 12). Discussion At least four possible explanations have been suggested to account for the effect of taurocholate choleresis on BSP excretion. O'Maille et al. 1 originally suggested that the excretory capacity for BSP was set by the reversible dissociation of a BSP-carrier complex at the biliary face of the canalicular membrane. According to this view, the net excretion of BSP would be limited by a maximum biliary concentration of the dye, and the effect of increasing canalicular flow would be to allow for the excretion of more dye within this concentration limit. The recognition by Ritt et al. 2 that taurocholate increased not only the apparent Tm for BSP but the fraction of the dye appearing in bile as unconjugated material led to the suggestion that BSP might be subject to back diffusion across the canalicular membrane. Since unconjugated material was postulated to diffuse more readily than conjugated dye, an increase in bile flow would be expected to produce a preferential increase in the excretion of the unconjugated form. In both of these reports, it was noted that the bile aciddependent increase in BSP Tm was roughly proportional to the increase in bile flow-an observation which appears to have led to widespread acceptance of the idea that the increase in bile flow is responsible for the increase in BSP excretion. 1-5 In both of the hypotheses outlined above, the putative cause and effect relation between dye excretion and bile flow depends fundamentally on the view that BSP normally moves in both directions across the canalicular membrane, and specifically on the idea that movement in the backward direction, i.e., away from the lumen, is an important determinant of net excretion. According to this postulate, the effect of increasing bile flow is to promote a favorable shift in the flux ratio by more rapid removal of dye from the site of secretion. The two remaining explanations represent alternatives to the flow hypothesis. They suggest that the effect of taurocholate is unrelated to the change in bile flow, but instead represents a specific effect of the bile acid-either a direct effect of' taurocholate on the active transport of BSp s or a physicochemical association between taurocholate and BSP in the canalicular lumen. s, 15 The possibility that bile acid-

6 May 1974 SC-2644 AND ESP TRANSPORT MAXIMUM 1051 dependent changes in BSP excretion might be explained by changes in the activity of the conjugating enzyme appears unlikely to us for the following reason. While Boyer et al. 3 have shown that high concentrations of taurocholate may depress the conjugation of BSP in rat liver, several other studies l6-19 suggest that this effect by itself would be expected to depress rather than to enhance the maximum capacity for total dye excretion. The major conclusion to be drawn from the present experiments is that it now appears difficult, if not impossible, to attribute the bile salt-dependent changes in BSP Tm to the associated changes in bile flow. SC-2644 produces a striking increase in canalicular fluid production, as measured by the plasma clearance of erythritol, but is without effect on either total BSP excretion or the distribution of conjugates, both of which characterize the response to taurocholate. It is clear from the experiments in which taurocholate excretion rate was measured during SC-2644 choleresis that failure of this drug to alter BSP excretion cannot be attributed to a simultaneous decrease in bile acid output. One other possible source of confusion should be considered, however. It is conceivable that a fortuitous combination of events occurs in such a way that an accelerating effect of SC-2644 choleresis is masked by an underlying inhibitory effect of the drug on BSP excretion. The theoretical possibilities include a depressant effect of SC-2644 on intracellular conjugation of the dye, inhibition of active BSP transport, and an increase in the passive permeability of the canalicular membrane. Judging from previous work in rats, the expected effect of an isolated defect in BSP conjugation is a depression of BSP Tm and an increase in the unconjugated fraction appearing in bile. The important question with regard to the present experiments is whether a defect in conjugation could mask a separate effect of choleresis which, if present alone, would have resembled the response to taurocholate. Assuming that dogs are similar to rats in this respect, the answer is no, inasmuch as the superimposition of a defect in conjugation and a flow-dependent increase in the unconjugated fraction should have led, if anything, to an even more pronounced increase in the unconjugated fraction than was seen with taurocholate. The opposite effect was actually observed. No data are available to exclude explicitly the possibility that SC-2644 competes with, or in some other way inhibits, the active transport of dye, although Cook et al. 10 have reported previously that the drug has no affect on the plasma clearance of BSP. It is clear, in any event, that active excretion of SC-2644 is not responsible for its striking choleretic effect, and to this extent, at least, competition between SC-2644 and BSP for a common membrane carrier appears unlikely. This can be readily appreciated from a comparison of the molar activity of SC-2644 with that for taurocholate (fig. 4). Taurocholate is virtually completely cleared from sinusoidal blood, and owing to micelle formation, can be assumed to exert an osmotic activity roughly equivalent to the concentration of the associated cations. Thus, even a supremely potent monovalent osmotic choleretic, i.e., one which is completely extracted from hepatic blood and exerts its full osmotic potential in the canaliculi, would be expected to produce, at most, an increment in bile flow roughly twice as great as an equivalent molar dose of taurocholate. SC-2644 clearly exceeds these theoretical expectations,' even ignoring the fact that its effect lasts much longer than does a comparable dose of bile acid. The canalicular reflection factor for sucrose, as estimated by Wheeler and King, 11 was unchanged by SC-2644 in their dogs; to that extent, at least, it appears unlikely that an increase in passive permeability could be an important determinant of our results. Final resolution of these questions should be sought in detailed steady state experiments from which one could determine the actual kinetics of BSP transport in the presence of SC It would be an extraordinary coincidence, however, if any of the inhibitory effects mentioned above should happen to balance exactly an accel-

7 1052 GIBSON AND FORKER Vol. 66, No.5 erative effect of choleresis in such a way as to yield no net effect on dye excretion. We are confident, therefore, that bile flow per se is not a determinant of BSP Tm. From the present experiments, it is impossible to draw any firm conclusions about the relative importance of the other two mechanisms which might account for the effect of taurocholate on BSP output. In particular, at the bile acid infusion rates we have used, neither dehydrocholate or SC-2644 produced a large enough change in bile flow to reduce canalicular bile acid concentration below the presumed critical micellar concentration. Thus, it is not possible to attribute the failure of these agents to increase BSP Tm to a reduced canalicular bile acid concentration, even though dilution undoubtedly occurred. It is of interest that, although dehydrocholate and SC-2644 were similarly ineffective in increasing BSP Tm, dehydrocholate, but not SC-2644, appeared to increase the unconjugated fraction in bile to about the same degree as did the lowest dose of taurocholate. The correct interpretation of this finding remains to be clarified, however, since it would appear to be equally compatible with a minor micellar effect of dehydrocholate or an effect on the membrane transport of BSP, albeit quantitatively less pronounced than that observed with taurocholate. While this work was in progress, similar conclusions with respect to the importance of bile flow as a determinant of BSP excretion were reported for two other bile acid-independent choleretics, theophylline 20 and methyl-4-umbelliferone. 21 It is not known whether these agents and hydrocortisone, reported earlier,22 all produce a choleresis in the same way as does SC-2644, but it is now clear that none of them produces an appreciable change in BSPTm. REFERENCES 1. O'Mailie ERL, Richards TG, Short AH: Factors determining the maximal rate of organic anions secretion by the liver and further evidence on the hepatic site of action of the hormone secretion. J Physiol 186: , Ritt DJ, Schenker S, Combes B: Enhancement of apparent excretory maximum of sulfobromophthalein sodium by taurocholate and dehydrocholate (abstr). Gastroenterology 52:322, Boyer JL, Scheig RL, Klatskin G: The effect of sodium taurocholate on the hepatic metabolism of sulfobromophthalein sodium. The role of bile flow. J Clin Invest 49: , Dhumaeux DP, Berthelot P, Preaux A, et al: A critical study of the concept of maximal biliary transport of sulfobromophthalein in the Wistar rat. Rev Eur Etud Clin BioI 15: , Gronwall R, Cornelius CE: Maximal biliary excretion of sulfobromophthalein sodium in sheep. Am J Dig Dis 15:37-46, Kneapler D, Boyer JL: Flow and its effect on hepatic excretion of sulfobromophthalein (abstr). Gastroenterology 46:398, Gibson GE, Forker EL: EfIect of taurocholate on net excretion of S,,-BSP from liver cells to bile (abstr). Fed Proc 21:309, Forker EL, Gibson GE: Interaction between sulfobromophthalein (BSP) and taurocholate: the kinetics of transport from liver cells to bile in rats, The Liver, Quantitative Aspects of Structure and Function. First International Gstaad Symposium. Edited by G Paumgartner, R Preisig. Basel, S. Karger, Cook DL, Bianchi RG, Hambourger WE, et al: Studies on five long-acting choleretics. J Pharmacol Exp Ther 100: , Cooke DL, Lawler CA, Green DM: Studies on the effect of hydrocholeretic agents on hepatic excretory mechanisms. J Pharmacol Exp Ther 110: , Wheeler HO, King K: Biliary excretion of lecithin and cholesterol in the dog. J Clin Invest 51: , Seligson D, Marino J, Dodson E: Determination of sulfobromophthalein in serum. Clin Chern 3: , Whelan JF, Plaa GL: The application of thin layer chromatography to sulfobromophthalein metabolism studies. Toxicol Appl Pharmacol 5: , Eneroth P, Sjovall J: Extraction, purification and chromatographic analysis of bile acids in biological materials, The Bile Acids, vol. I. Edited by TP Nair, D Kritchevsky. New York, Plenum Press, Ware A, Hardt M, Barnhart T, et al: The intersection of bromsulphthalein (BSP) molecules with themselves and with sodium taurocholate (TCA). Clin Res 21:260, Combes B: The importance of conjugation with glutathione for sulfobromophthalein sodium (BSP) transfer from blood to bile. J Clin Invest 44: , Whelan G, Hock J, Combes B: Biliary excretion of

8 May 1974 SC-2644 AND BSP TRANSPORT MAXIMUM 1053 conjugated sulfobromophthalein sodium (ESP) in rats fed a protein-free diet. Proc Soc Exp Bioi Med 132: , Whelan G, Combes B: Competition by unconjugated and conjugated sulfobromophthalein sodium (BSP) for a transport into bile: evidence for a single excretory system. J Lab Clin Med 78: , Ware A, Combes B: The influence of diet on the metabolism and excretion of bromosulphthalein sodium (BSP) in the rat (abstr). Clin Res 21:52, Barnhart.J, Combes B: Theophylline. hepatic disposition and effect on biliary secretory function (abstr). Gastroenterology 64:162, 197:3 2l. Erlinger S, Dumont M: Influence of canalicular bile flow on sulfobromophthalein transport maximum in bile in the dog. The Liver, Quantitative Aspects of Structure and Function. First International Gstaad Symposium. Edited by G Paumgartner, R Preisig. Basel, S. Karger, Macarol V, Morris TQ. Baker K.J, et al: Hydrocortisone choleresis in the dog. J Clin Invest 49: , 1970