J. Physiol. (1976), 261, pp. 337-357 With 7 text-figure8 Printed in Great Britain

337

THE SECRETORY CHARACTERISTICS OF DEHYDROCHOLATE IN THE DOG: COMPARISON WITH THE NATURAL BILE SALTS

BY E. R. L. O'MAILLE AND T. G. RICHARDS From the Department of Physiology, University of Liverpool, Liverpool L69 3BX

(Received 17 February 1976) SUMMARY

1. During dehydrocholate administration in the taurine replete dog, the maximum excretory rate of total bile salt (almost entirely dehydrocholate derivative, mostly conjugated) was 3-84 + 0- 53 (S.D.) gimole/min. kg body wt. (eleven experiments). This was much less than the excretory maximum previously obtained for taurocholate (8.64 + 1 31 (S.D.) mole/ min. kg) or actively conjugated cholate (6.83 + 1-31 (S.D.) Jamole/min. kg total cholate, mostly conjugated). 2. The superimposition of taurocholate infusion did not cause any significant change in the 'dehydrocholate' maximum but taurocholate itself was excreted into bile at no more than about half its normal maximum. When taurocholate maximum excretion was established first, it was reduced by dehydrocholate administration. In both types of experiment the joint bile salt excretory maximum was of the same order as that of taurocholate alone, provided taurocholate made up at least 40-50 % of the total bile salt. 3. When taurocholate administration was stopped, the maximum excretory rate of 'dehydrocholate' rose to values up to 63 % above the initially determined excretory maximum; the enhanced 'dehydrocholate' excretory maximum, when calculated for optimal conditions, approached that of actively conjugated cholate, even though the effective 'dehydrocholate' concentration in bile was ten to twenty times the critical micellar concentration of taurocholate. This suggests that the effective bile salt concentration in bile is not an important determinant of the secretary performance of a bile salt. 4. To explain findings (2) and (3) it is necessary to postulate that taurocholate has both a facilitatory and an inhibitory action on 'dehydrocholate' excretion. The facilitatory action, which persists after taurocholate has left the animal, may consist either of an increase in the

E. R. L. O'MAILLE AND T. G. RICHARDS 338 maximum rate at which modification of dehydrocholate takes place within the liver cell, or an increase in the number of functioning 'carriers 'for 'dehydrocholate' transfer. The data suggest that the inhibitory effect is due to the competitive interaction that also appears to exist between the two bile salts. 5. The increase in bile flow rate per unit increase in 'dehydrocholate' excretion (15 ml./m-mole) was about twice that obtained for taurocholate. There was no significant formation of micellar aggregates during 'dehydrocholate' excretion, as judged from the total electrolyte concentration of bile and its osmolality. 6. During the excretion of 'dehydrocholate'-taurocholate mixtures (approximately 1: 1) at submaximal rates the associated bile flow rate was not less than the sum of the separate components, thus suggesting that 'dehydrocholate' was not being incorporated in taurocholate mixed

micelles. INTRODUCTION

The principal bile salt normally present in the bile of the dog is sodium taurocholate. When dissolved in water, this 'natural' bile salt, above a certain critical concentration and temperature, forms micellar aggregates; if lecithin and cholesterol are also present, as in bile, 'mixed' micelles, containing all three components are formed. Taurocholate is actively transferred into bile, a process upon which the excretion of lecithin and cholesterol is almost entirely dependent. It is not established whether: (i) taurocholate is first transferred into bile with the lipids following secondarily in accordance with a physicochemical equilibrium, as has been suggested by Wheeler & King (1972); or (ii) all three components are transferred together as a complex; or (iii) combinations of these processes occur. By contrast, sodium dehydrocholate is a synthetic bile salt, which although closely similar in molecular structure to the natural bile salts, differs from them in not forming micellar aggregates either on its own or with lipids. The biliary excretion of dehydrocholate derivatives leads to no increase in the excretion of lecithin and cholesterol (Wheeler & King, 1972). The purposes of studying the secretary characteristics of these two classes of bile salt when administered singly or in combination were the following. The first object was to determine whether both bile salts were excreted into bile by the same (active) transport system; the taurine conjugate of dehydrocholate derivative, which presents for excretion, is very similar in structure to taurocholate, so that sharing of a common transport system might reasonably be expected; on the other hand, if taurocholate is transferred as a complex with lecithin and cholesterol it is conceivable that a bile salt without the potential to form such complexes

DEHYDROCHOLATE SECRETION 339 might be prevented from using the same transport system. It was also wondered whether any special kind of interaction occurred between the two classes of bile salt, such as has been observed between bile salts and other large organic anions actively transferred into bile; for example it has been shown that taurocholate administration enormously enhances the maximum excretory rate of bromsulphthalein (O'Maille, Richards & Short, 1966; Forker & Gibson, 1973). The second purpose was to see if some indication might be obtained on the extent to which the secretary performance of a bile salt was limited by its effective concentration in bile. Because of the micellar aggregates which the natural bile salts form in bile, the effective bile salt concentration (i.e. the critical micellar concentration) is very much less than the total concentration. An expected consequence would be that the back transfer of a bile salt from bile to liver cell or blood would be reduced. This may be an important factor in the very impressive ability of the liver to effect net transfer of the natural bile salts from blood to bile (in the dog, taurocholate is almost completely (92+5% (S.D.)) removed from the blood flowing through the liver (O'Maille, Richards & Short, 1967). With dehydrocholate (including dehydrocholate derivative in bile (see below)) however, the total concentration is also approximately the effective concentration, which (in bile) is much higher than the critical micellar concentration of the natural bile salts. The third object was to determine whether there was any interaction between the bile salts in bile, for instance whether dehydrocholate derivative could be incorporated in taurocholate mixed micelles. An abstract describing some of the findings has already been published (O'Maiille & Richards, 1975). METHODS All experiments were performed on adult mongrel dogs which had been premedicated with diethylthiambutene (Themlon, Burroughs Wellcome and Co.) (5 mg/kg body wt. I.M.) and anaesthetized with pentobarbitone (Nembutal. Abbott Laboratories) (13.2 mg/kg i.v.). The abdomen was opened by a median incision, the common bile duct cannulated and the cystic duct ligated. A catheter was inserted into a radicle of the splenic vein to provide for portal administration of taurine. Two 'angiocaths' (Desert Angiocath, 16 ga., 0 044 in. (0.112 cm) i.d., 2-25 in. (5.7 cm), Bard-Davol Ltd), with three-way taps attached, were placed in the left femoral vein to provide for infusions of dehydrocholate and taurocholate. Bile salt and taurine solutions were administered by constant infusion pumps. An 'angiocath' was placed in the right femoral vein for intermittent administration of anaesthetic. Blood samples were also taken from this catheter in some experiments, and in others from a second angiocath inserted into the right femoral artery. The blood was expelled from the sampling syringes into dry tubes which were centrifuged shortly afterwards; serum was removed and stored either in a deep freezer or an ordinary refrigerator. Bile was collected in graduated tubes, in some cases under oil, and stored in a refrigerator before and between analyses.

340

E. R. L. O'MAILLE AND T. G. RICHARDS

Dehydrocholic acid (Calbiochem Ltd, Los Angeles; Schwarz/Mann, New York; Ward Blenkinsop and Co., London) solutions, which ranged in concentration from 25 to 75 mm, were prepared as follows. The material was first dissolved in 0 15 m-NaOH, the volume of which was a little above the calculated equivalent required to form the sodium salt of dehydrocholic acid; 32-5 ml. 0-154 M-KCl was added per litre (final volume), the pH adjusted to 7-6-7-7 with 0 15 M-HCl and the solution brought to the correct volume with 0-154 M-NaCl. The osmolality of the solutions was 256-278 m-osmole/kg H20. Solutions of pure synthetic sodium taurocholate (L. Light and Co. or its successor Koch-Light and Co., Colnbrook, Bucks; Maybridge Chemical Co. Tintagel, Cornwall) were prepared essentially as described previously (O'Maille et al. 1966). Concentrations of taurocholate ranged from 15 to 66 mM; the osmolality of the solutions infused was 283 ± 18 (S.D.) m-osmole/kg H20 (ten observations). Taurine (B.D.H. or Hopkins and Williams) was dissolved in distilled water to give, most commonly, 0-275 M solutions. Before bringing to final volume with distilled water the solutions were adjusted to pH 7-2-7-5 with 0-15 m-NaOH; The osmolality of these solutions was 279+8 (S.D.) m-osmole/kg H20 (nine observations). Analy8es The concentration of taurocholate in bile was determined by the furfural-H2S04 reaction as described previously (O'Maille, Richards & Short, 1965). The concentration of total bile salt in bile was estimated by the electrolyte difference method (i.e concentration of total bile salt anion in bile = ([Na+] + [K+]) - ([Cl-] + [HCO3-]) as described by Wheeler & Ramos (1960). These authors found that during bile salt ('taurocholate') infusion the bile salt concentration in bile, as calculated by the above equation was in such close agreement with that obtained by direct analysis that thereafter they used the indirect method for bile salt estimation. More recently Rutishauser & Stone (1975) have also found close agreement between the indirect method for bile salt estimation in bile (during taurocholate or taurodeoxycholate infwion) and several direct methods. We have also found very good agreement between the concentrations of taurocholate in bile (during taurocholate administration) obtained by direct analysis and that obtained by the indirect method (during the infusion of any bile salt at the rates employed in these experiments the infused bile salt (or its derivatives) is the only significant bile salt present in bile). It is clear that during dehydrocholate infusion the total bile salt concentration in bile, as estimated by the electrolyte difference method, is made up almost entirely of dehydrocholate derivative (see Results section). The concentrations of sodium and potassium in bile were determined by flame photometry (either an EEL Model A or a ' 170 ' digital flame photometer was used). The concentration of chloride in bile was obtained by coulometric titration, using an EEL chloride meter. Total CO2 in bile was determined with a Van Slyke manometric apparatus: the concentration of bicarbonate in the bile samples collected under oil was obtained from this by subtracting the concentration of CO2 in physical solution (1.206 mm at an assumed Pco, of 40 mmHg and a solubility coefficient of 0.51 ml. C02/ml. bile per atmosphere). No allowance for CO2 in physical solution was made for bile samples exposed to the atmosphere during collection; the total CO2 in such samples, collected on either side of bile samples under oil, was in satisfactory agreement with the corrected values of the latter. The osmolality of bile and serum was obtained with osmometers which operate by measuring the temperature at which freezing occurs; either an 'osmette F' (Precision Instruments Inc., Massachusetts, U.S.A.) or an 'Advanced Osmometer' Model 3L (Advanced Instruments Inc., Massachusetts, U.S.A.) was used.

DEHYDROCHOLATE SECRETION

341

Chromatograpy of bile It has been established in several species including the human (Soloway, Hofmann, Thomas, Schoenfield & Klein, 1973) and dog (Desjeux, Erlinger & Dumont, 1973) that dehydrocholate is modified in its passage through the liver. The modification in the human consists of the sequential conversion of the keto groups at 3, 7 and 12, in that order, to hydroxyl groups, although only a small proportion (10%) is converted all the way to trihydroxycholanic acid (cholic acid). Conversion of the 3 keto group (at least) to a hydroxyl group also occurs in the dog. In addition these derivatives are conjugated with glycine (human) or taurine (human and dog). In these experiment the main object was to separate conjugated from free dehydrocholate derivatives. This was carried out by descending chromatography using the Ta phase system (amylacetate 85:heptane 15, equilibrated with an equal volume of 70% formic acid) of Sjovall (1959), after which the developed papers (Whatman 3Mm) were airdried with a fan heater. The bile salt derivatives were located by dipping the papers in a dinitrophenylhydrazine reagent (see Sjovall, 1964) or in an ethanolic solution of iodine. Ketonic bile acids (dehydrocholic acid and derivatives), but not trihydroxy bile acids such as cholic or taurocholic acids, show up as orange or yellow spots following treatment with dinitrophenylhydrazine. Both ketonic and trihydroxy bile acids appear as brown spots after dipping in the iodine solution. The Ta phase system described above, which had previously (O'MAille et al. 1965) been found to give wide separation of taurocholic from free cholic acid, also gave wide separation of taurodehydrocholic fromfree dehydrocholic acid (RFs of about 0 *3 and 0 *8, respectively). The RF of taurodehydrocholic acid was somewhat less than that of taurocholic acid but the RF of free dehydrocholic acid was about the same as that of free cholic acid. Following dehydrocholate administration, two (sometimes three) ketonic bile acid spots were readily seen in the taurodehydrocholic-taurocholic acid zone of the paper and another ketonic bile acid spot was readily visible in the free bile acid zone; the latter spot included all unconjugated forms of 'dehydrocholate'. The upper spot in the ' conjugated' zone of the paper had an RF approximately the same as taurodehydrocholate, but unlike the latter, it took several hours to develop its full colour. The second ketonic bile salt readily seen in the conjugated zone had an RF approximately the same as that of taurocholate but reacted immediately with the dinitrophenylhydrazine reagent. We have not determined the exact identity of these dehydrocholate derivatives, as reference substances have not been available to us, but it has been possible to deduce some features of their chemical nature both from the kind of experiments we have performed and from data already published (see Results section). RESULTS

Determination of the maximum excretory rate of total dehydrocholate derivative The maximum rate at which dehydrocholate derivative could be excreted by the liver was determined either by infusing dehydrocholate at a series of ascending rates until no further increase in the rate of bile salt excretion took place, or by infusing dehydrocholate from the beginning at a rate well above the expected excretory maximum. Such systemic infusion of dehydrocholate was accompanied by intraportal infusion of taurine at an equimolar (or greater) rate, to prevent depletion of the animal's store

E. R. L. o'MAILLE AND T. G. RICHARDS 342 of taurine as a result of its loss in the bile as taurine conjugates of dehydrocholate derivatives (see O'Ma'ille et at. 1965). The relation between dehydrocholate infusion rate and the excretory rate of total bile salt (almost entirely dehydrocholate derivative, mostly conjugated - see below) is shown in Fig. 1. At low rates of dehydrocholate administration (less than about 2 ,tmolelmin. kg) the excretory rate of total bile salt was only slightly less than the infusion rate. As the administration rate of dehydrocholate was increased above this, the excretory rate of total bile V

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Fig. 1. Note that at low infusion rates of dehydrocholate the excretory rate of total bile salt (almost entirely dehydrocholate derivative, mostly conjugated) was almost equal to the infusion rate. Increasing infusion rates eventually led to the bile salt excretory rate reaching a maximum of 3-84 ± 0 53 (S.D.) /smole/min.kg. The vertical line above and below the mean value represents ± 1 (S.D.). The point on the ordinate encloses ± 1 S.D. of the control excretory rate of total bile salt (data from fifteen experiments in the taurine replete dog).

salt fell progressively below the infusion rate; administration rates greater than about 6-7 gtmole/min. kg led to no further increases in the excretory rate of total bile salt. The maximum excretory rate of total bile salt (which closely approximates total dehydrocholate derivative) obtained in this way was 3 84 + 0 53 (S.D.) /smole/min .kg body wt. (eleven experiments). This is much lower than the excretory maximum previously found for taurocholate (8.64 + 1.31 (S.D.) ,umole/min.kg) or actively conjugated cholate (6-83 + 1-31 (S.D.) ,smole/min.kg total cholate, mostly conjugated) (Richards & O'Maille, 1973). The above 'dehydrocholate' excretory maximum is also much lower than that of the other common

DEHYDROCHOLATE SECRETION

343 natural bile salt, glycocholate. The latter bile salt, though not normally present in dog bile, has an excretory maximum of the same order as that of taurocholate (O'Maiille et at. 1967; O'Maille, Richards & Short, 1969). During 'dehydrocholate' maximum excretion, the total bile salt concentration in bile was 57-5 + 5-2 (S.D.) mM (eleven experiments) while during taurocholate maximum excretion the biliary concentration of DHCin

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Fig. 2. Dehydrocholate (together with an approximately equimolar mntraportal taurine infusion) was infused systemically to the end of the experiment . -at a supramaximal rate of 9-92 cflmole/min.kg body wt. ( DHC in). Taurocholate was infused systemically at 1.98 ,umole/min .kg from 1F54 to 2*75 hr and at 4 30 ,smole/min .kg from 2-75 to 4 hr (-*-*-*, TO in). Bile flow rate divided by 10 (A*-As BFR). Total bile salt excretory rate into bile (0OO, total BS out). Taurocholate excretory rate (A-A, TO out). Total bile salt excretory rate minus taurocholate excretory rate (e--, 'DRO' out); this value is comprised almost entirely of the excretory rate of dehydrocholate derivative into bile; it includes a negligible amount of endogenous bile salt other then taurocholate. Note that taurocholate administration caused little or no reduction in the maximum excretory rate of 'dehydrocholate' ('DHC' out).

taurocholate was 131@10 ±21@0 (S.D.) mM (eleven experiments). The determined bile salt concentration in bile during 'dehydrocholate ' excretion is likely also to be approximately the effective concentration (see below). By contrast, during taurocholate excretion, due to the formation of micellar aggregates, the effective biliary concentration is the critical micellar concentration, which is only a very small fraction of the

E. R. L. o'MAILLE AND T. G. RICHARDS experimentally measured taurocholate concentration in bile. The precise value of the critical micellar concentration during taurocholate excretion in these experiments is not known but about 3-5 mm (see Hofmann, 1965; Hofmann & Small, 1967) would represent its upper limit. 344

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Fig. 3. Symbols and notation as in Fig. 2. The 'dehydrocholate' maximum excretory rate of about 3-2 psmole/min . kg was not reduced (apart from a brief, early phase) by supramaximal taurocholate infusion, despite the fact that taurocholate itself was being excreted into bile at about half its own excretory maximum. When taurocholate infusion was stopped the 'dehydrocholate' maximum excretory rate increased progressively to reach a value about 63 % greater than the initial (control) maximum.

Excretory rate of taurocholate before and during dehydrocholate infu8ion The excretory rate of taurocholate in the control state, i.e. before dehydrocholate infusion, was 0-0639 + 0020 (S.D.) psmole/min. kg (eleven experiments). During supramaxinal dehydrocholate infusion the taurocholate excretory rate fell to 00280 + 0-0183 (s.D.) Iumole/min. kg, which formed only 0-7 % of the total bile salt excretory rate. At submaximal rates of dehydrocholate administration the excretory rate of taurocholate, which showed no consistent trend upwards or downwards compared with the control rate, also formed an insignificant fraction (average 3-3 %) of the total bile salt excretory rate. In these experiments, therefore, there was no significant conversion of infused dehydrocholate into cholate.

DEIHYDROCHOLATE SECRETION

345

Effect of taurocholate administration on maximum 'dehydrocholate' excretion Sodium dehydrocholate was administered continuously at a supramaximal rate in these experiments; when 'dehydrocholate 'excretionhad reached a maximum, a taurocholate infusion at either submaximal (Fig. 2) or supramaximal rate (Fig. 3) was superimposed. It can be seen (Figs. 2 and 3) 18 1-6 °

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Fig. 4. Collected data from seven experiments (bottom panel) and six experiments (middle and top panels) to show the effect of taurocholate administration on the 'dehydrocholate' maximum excretory rate. Bottom panel: 'dehydrocholate' maximum excretory rate obtained during taurocholate infusion divided by that obtained before taurocholate infusion (ordinates) plotted against taurocholate excretory rate obtained during its infusion (abscissae). The 'dehydrocholate' excretory maximum was approximately the same during taurocholate administration as before it. The point with a question mark beside it was obtained during the infusion of a taurocholate solution of abnormally high pH (8 2). Middle panel: the 'dehydrocholate' maximum in the period after taurocholate infusion was stopped was almost invariably greater than that during taurocholate infusion. The relative 'dehydrocholate' maximum is plotted against the declining taurocholate excretory rates after taurocholate infusion had been stopped. Top panel: the 'dehydrocholate' excretory maximum in the period after taurocholate infusion was stopped was almost invariably greater than the initial (control) maximum before taurocholate was given. The relative 'dehydrocholate' excretory maximum is plotted against the declining taurocholate excretory rates after taurocholate infusion had been stopped.

E. R. L. O'MJILLE AND T. G. RICHARDS that taurocholate administration caused little or no reduction in the 'dehydrocholate' maximum even when taurocholate was being transferred into bile at about half its own excretory maximum. Similar results from all seven experiments in which the effects of taurocholate administration on 'dehydrocholate' maximum excretion were tested are summarized in Fig. 4A. When taurocholate was administered at half-maximal rates (or less) (as in Fig. 2) it was excreted in bile at a rate only slightly less than the infusion rate. However the biliary excretion of taurocholate during its administration at supramaximal rates (Fig. 3) was only about half that which would be expected if no dehydrocholate were being administered at the same time. It may also be noted here that in four experiments in which taurocholate was given at a sufficiently high rate (as in Figs. 2 and 3, for example) the joint bile salt maximum (average 7-6,umole/min.kg, approximately half taurocholate, half 'dehydrocholate') approached that of taurocholate alone (8.6 ,umole/min. kg), indicating that in these circumstances at least, dehydrocholate derivative could substitute for and be excreted almost as well as taurocholate, if in fact both bile salts have a common transport system (see Discussion). (Conversely, in the one experiment performed, when taurocholate maximum excretion was established first, the subsequent administration of dehydrocholate at a supramaximal rate led to progressive decreases in taurocholate excretory rate together with progressive increases in 'dehydrocholate' excretory rate; there was no diminution in the joint bile salt excretory maximum until the taurocholate excretory rate fell to less than 43 % of the total). When taurocholate infusions were stopped in the above experiments, the 'dehydrocholate' maximum almost invariably increased (e.g. Fig. 3), sometimes to well above initial control values. This increase in the 'dehydrocholate' maximum took place as the taurocholate remaining in the animal (after its infusion) was 'offloaded' into bile at progressively diminishing rates. The enhanced dehydrocholate excretion persisted after the excretion of taurocholate had declined to initial control rates. The results from six experiments in which the dehydrocholate maxima obtained after stopping taurocholate infusion are compared with those which prevailed during or before its administration are summarized in Fig. 4 (middle and top panels). The possibility that the elevated 'dehydrocholate' maximum obtained after stopping taurocholate administration could have been merely what the initial 'dehydrocholate' maximum might have climbed to in that time, had there been no intervention, can be excluded: in the control case, i.e. where dehydrocholate (with taurine) alone was given throughout the experiment for the same length of time, its maximum excretory rate declined slightly after about 2-5 hr for the rest of the 346

DEHYDROCHOLATE SECRETION 347 experiment. The enhanced 'dehydrocholate' maximum excretory rate, after taurocholate administration at rates of 2-10,umole/min.kg had been stopped, was 5-19+0-43 (S.D.) gzmole/min.kg (six experiments), which was associated with a simultaneous taurocholate excretion of 1-07 + 0-88 (S.D.) Fmole/min.kg. This represents an average increase of about 36% (range 15-63%) over the initial control 'dehydrocholate' maximum. The greatest enhancement was seen after taurocholate had earlier been given at a supramaximal rate.

Taurocholate and 'dehydrocholate' concentrations in bile during the superimposition of taurocholate administration on the 'dehydrocholate' maximum excretory rate The taurocholate concentration in bile in these experiments ranged from 4-4 mM (simultaneous 'dehydrocholate' concentration, about 53 mM) at the lowest taurocholate infusion rate to about 57 mm (simultaneous 'dehydrocholate' concentration, about 41 mM) during supramaximal infusion. During the latter state, therefore, the 'dehydrocholate' concentration, at 41 mm, made up only about 42 % of the total bile salt concentration; this state comes closest to that described by Soloway et al. (1973) for the participation of dehydrocholate derivative in mixed micelles. When supramaximal taurocholate infusion was superimposed on 'dehydrocholate' maximum excretion it was noticed that the increase in bile flow rate divided by the increase in bile salt (taurocholate) excretory rate was only 2-6-4-8 ml./,umole, which was much less than that obtained when taurocholate was being excreted on its own (see below). In these circumstances 'dehydrocholate' may have been incorporated in taurocholate mixed micelles, thereby apparently diminishing the extra bile flow attributable to taurocholate excretion.

'Dehydrocholate' excretion and bile flow The relation between total bile salt excretory rate, obtained during dehydrocholate infusion, and bile flow rate is shown in Fig. 5, which is based on thirty observations drawn from fifteen experiments. The choleresis obtained is contrasted with that obtained during taurocholate infusion. The data for the taurocholate regression line (Fig. 5) consist of sixty-four observations drawn from twenty-seven experiments, the results of many of which have already been published (O'Maille et al. 1965, 1967). The increase in bile flow rate per unit increase in bile salt excretion rate for 'dehydrocholate' excretion (15 ml./m-mole) was about twice that obtained for taurocholate excretion (7.4 ml./m-mole). The leading theory for the formation of canalicular bile is that of local osmosis, i.e. that solute is actively transferred into the bile canaliculus,

348 E. R. L. O'MAILLE AND T. G. RICHARDS with fluid following secondarily along an osmotic gradient. The finding that the increase in bile flow rate per unit increase in bile salt excretion was greater for 'dehydrocholate' than for taurocholate is therefore to be expected, as there was no significant reduction in osmotic activity by micellar aggregation of 'dehydrocholate' in bile compared with 110

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300 2010 6 7 5 4 2 3 Bile salt excretion rate (1umole/min. kg) Fig. 5. The greater bile flow rate evoked by 'dehydrocholate'excretion compared with taurocholate (TC). The abscissae for the line labelled 'DHC' consist of the excretory rates of total bile salt (which consisted almost entirely of dehydrocholate derivative) obtained during dehydrocholate infusion in the taurine replete dog. Details of data in text (regression lines of best fit: 'DHC', y = 15x+7-7; TC, y = 74x+7 8).

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taurocholate (see below). The pronounced hydrocholeresis following administration of dehydrocholate is well established (see Bizard & Vanlerenberghe, 1956) but we are unaware of any previous publication in which the relation between bile flow rate and the excretory rate of total 'dehydrocholate' in bile (as in Fig. 5) is displayed, although Sperber (1965) does supply figures for 'dehydrocholate' concentration in bile.

DEH YDROCHOLATE SECRETION

349

Osmolality and total electrolyte concentration of bile during 'dehydrocholate' excretion The osmolality of bile during 'dehydrocholate' excretion was 279 + 7 (S.D.) m-osmole/kg H20 (thirty-four observations, nine experiments). The 'total' electrolyte concentration (i.e.[Na+] + [K+] + [Cl-] + [HCO3-] + [bile salt anion-]), which represents the sum of the osmotically important solutes in bile, in these samples was 304 ± 8 (S.D.) m-molell., thus giving an osmotic coefficient of 279/304 or 0-92. The osmotic behaviour of the total solute in these bile samples was not significantly different from that of an ordinary inorganic salt solution (such as NaCl or KCl) of similar concentration, in which it can be assumed there is no micellar aggregation; thus the osmolality of 0-154 M-NaCl or 04154 M-KCl (308 m-mole/l.) which was analysed with the above bile samples was 285 + 3 (S.D.) m-osmole/kg H20 (sixteen observations), giving an osmotic coefficient of 0-925. Essentially similar findings were obtained by Reinhold & Wilson (1934) following single injections of dehydrocholate. These results, which indicate that micellar aggregation of 'dehydrocholate' was not taking place on an important scale, may not necessarily be inconsistent with those of Desjeux et al. (1973) whose ultracentrifugation studies suggested that dehydrocholate derivatives formed macromolecular complexes in bile. Dehydrocholate derivatives may be polydisperse with respect to aggregate number, with the greater fraction of the total being in the monomeric form. The huge disparity that may exist between total electrolyte concentration and osmolality of bile during taurocholate infusion is already well established. During taurocholate infusion at submaximal rates of 1-5 #tmole/min. kg, for example, the osmolality of bile (280+5 (S.D.) m-osmole/kg H20, nine observations, two experiments) was similar to that of 'dehydrocholate' bile samples (above) but the total electrolyte concentration in them was 376 + 11 (S.D.) m-mole/l. (osmotic coefficient, 0.74); much lower coefficients have been obtained during supramaximal taurocholate infusions. These osmotic coefficients are very much less than those obtained for ordinary electrolyte solutions of similar concentration. The osmotic ineffectiveness of much of the solute in bile samples such as the above is due largely to the formation of micellar aggregates.

Mixed 'dehydrocholate '-taurocholate excretion and bile flow The purpose of administering dehydrocholate-taurocholate mixtures at submaximal rates as described below was to see if the bile flow rate evoked was merely the same as that predicted from the sum of the separate components or whether it reflected some interaction in bile e.g., the 12

PHY

26i

E. R. L. O'MAILLE AND T. G. RICHARDS 350 incorporation of 'dehydrocholate' in taurocholate mixed micelles; the latter interaction, on the osmotic theory of bile formation, should lead to

mixtures of 'dehydrocholate' and taurocholate evoking less bile flow per mole of bile salt excreted. In these experiments, in any individual dog, the relation between bile salt excretory rate and bile flow rate was established for (i): taurocholate alone; (ii) 'dehydrocholate' alone; and (iii) a bile salt mixture, approximately half taurocholate, half 'dehydrocholate'. Each bile salt, or bile salt mixture, was administered at a series of constant, submaximal rates, with the order of administration of the single bile salts being varied from experiment to experiment. The bile flow rate elicited from infusing a dehydrocholate-taurocholate mixture was in all cases greater than that predicted from the sum of the separate components - the excess ranged from 4 to 25 % (mean 13 %) above the predicted values (eight observations, four experiments). Two of these experiments provided sufficient observations for the reliable derivation of regression lines for the relation between the excretory rate of the 'dehydrocholate '-taurocholate mixtures and bile flow rate. In one of them (Fig. 6) the slope of the line for the bile salt mixture was the same as that predicted, in the other experiment the slope was only a little (15%) greater than that predicted. Because the intercepts on the bile flow rate axis of the individual 'dehydrocholate' and taurocholate regression lines were not identical the following procedure was adopted to obtain the mean predicted bile flow rate for the bile salt mixtures. The procedure consists of making two predicted estimates of the bile flow rate and using the mean value. First the bile flow rate due to each component was read off from its own individual regression line. To each of these values was added the bile flow rate attributable to the other bile salt obtained by multiplying its excretory rate by the slope, of its regression line. The mean of these two estimates, in the case of every observation for the bile salt mixture, was used for the construction of the regression line for the predicted relation between the excretory rate of the 'dehydrocholate-taurocholate' mixture and bile flow rate (see Fig. 6). In most cases even the higher predicted estimate of bile flow rate was still less than that obtained experimentally. In the above predictions of bile flow rate during the excretion of bile salt mixtures, the intercept on the bile flow rate axis was taken as being independent of bile salt excretion, i.e. would occur in the absence of bile salt excretion and should therefore be the same regardless of which bile salt salt was being excreted. It has been noted, however, that if the total bile flow rate read off directly from the excretory rate of one bile salt on its regression line is added to the total bile flow rate similarly formed from the

351 DEH YDROCHOLATE SECRETION excretory rate of the other bile salt, the value obtained is in approximate agreement with that experimentally observed during the excretion of 'dehydrocholate '-taurocholate mixtures. In the eight observations made, the experimental values ranged from 5 % above to 9 % below (average, 0.6 % below) the sum of the individual total bile flow rates. This procedure, 60

50

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30

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0 IV

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10

0 Bile salt excretion rate

(pumole/min. kg)

Fig. 6. The bile flow rate evoked by the excretion of a 'dehydrocholate' taurocholate (1:1 approx.) mixture (I-f, ('DHC' + TO)) was greater than that predicted (-------, P) from the sum of the separate components (0-0 'DHC ', A -A, TC). See text for method used to obtain 'predicted' line. Note that the slope8 of the experimentally obtained and predicted lines for the 'dehydrocholate '-taurocholate mixture are similar (regression lines of best fit: ('DHC'+TC), experimental: y = 13 7x+9 1; ('DHC'+TC) predicted: y = 13A4x+4 6; 'DHC', y = 17 2x+3 8; TC, y = 9-8x+4-8).

whereby totals are summed, implies that each bile salt stimulates an additional bile flow (each intercept) that is not dependent on the osmotic effect of its own excretion into bile, e.g. a flow dependent on the active T2-2

E. R. L. O'MwAILLE AND T. G. RICHARDS 352 excretion into bile of inorganic electrolyte such as sodium chloride. This bile flow rate 'intercept' component for each bile salt might be elicited by quite low excretory rates of each bile salt and is similar to the suggestion put forward by Soloway et al. (1973) to explain the bile flow rate that was too great in their experiments to be accounted for by the osmotic effect of 'dehydrocholate' excreted at very low rates. However analysed, the results of these experiments with submaximal infusions of a bile salt mixture strongly suggest that 'dehydrocholate' was not being incorporated in taurocholate mixed micelles.

Taurocholate and 'dehydrocholate' concentrations in bile during combined infusion at submaximal rates The concentration of taurocholate in bile in these experiments, which was always very much above its critical micellar concentration, varied from 22 to 45 mm (mean, 33 mM). 'Dehydrocholate' concentration was about the same as that of taurocholate or 49 + 5 (S.D.) % of total concentration (eight observations, four experiments). The lack of incorporation of 'dehydrocholate' in taurocholate mixed micelles, as suggested by the bile flow responses described in the preceding section, may have been due to the fact that it formed too high a proportion (about half) of the total bile salt concentration (see Soloway et al. (1973)). Dehydrocholate derivatives appearing in bile During dehydrocholate infusion in the taurine-replete dog (all of the experiments described so far fall into this category) most of the dehydrocholate derivative in bile appears on paper chromatograms in the section occupied by conjugated bile salts (i.e. the taurodehydrocholatetaurocholate zone, see 'Methods'); a little of the dehydrocholate derivative appears in the free bile acid section of the papers. The ketonic bile salt having an RF approximately the same as taurodehydrocholate appears to be a taurine conjugate on the basis of the following experimental results. When dehydrocholate alone was infused continuously for several hours the spot referred to above eventually diminished in intensity and became very faint just as has been previously observed to happen to the taurocholate spot during prolonged cholate infusion (O'Maille et al. 1965). Furthermore as this spot diminished the ketonic bile acid spot with an RF in the free bile acid zone intensified (as with free cholate during cholate infusion). When taurine was administered intraportally, the spot which previously had been very faint promptly intensified. The second ketonic bile salt in the 'conjugated zone' of paper chromatograms, with an RF approximately the same as that of taurocholate, appears not to be a taurine conjugate: it intensified during

DEHYDROCHOLATE SECRETION

353

prolonged infusion of free dehydrocholate and diminished following taurine administration. It may be a dehydrocholate derivative conjugated with some other substance. In previous experiments (O'Maille et al. 1965) we established that the maximum excretory rate for synthetic taurocholate or actively conjugated

10 o

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E Z._

41(4

0

x

40 1

CZ ._

41

0 2 3 1 Time (hr) after start of supramaximal dehydrocholate infusion

Fig. 7. The maximum excretory rate of total bile salt (largely free 'dehydrocholate', after taurine depletion) was much less than that obtained after extensive conjugation had been restored by intraportal administration of taurine (dose in text) at arrow. Acute taurine depletion was first produced by infusing dehydrocholate alone at 3-35 /imole/min.kg for 3-88 hr. The dehydrocholate infusion was then raised to 1t0- I mole/min . kg, a rate well above the expected maximum excretion of either free or conjugated 'dehydrocholate', for the remainder of the experiment.

cholate was greater than that of free cholate. We carried out one experiment in the present series to see if this was also true for conjugated and free 'dehydrocholate'. In this experiment acute taurine depletion was first produced by infusing dehydrocholate alone at 3-35,umole/min.kg for 3-88 hr; the infusion rate was then raised to 10-1 #smole/min.kg, a value well above the expected maximum excretory rate of either free or

E. R. L. O'MwILLE AND T. G. RICHARDS conjugated 'dehydrocholate'. When maximum 'dehydrocholate' excretion had been obtained (after taurine depletion) (Fig. 7), taurine was administered portally (priming dose of 0-5m-mole/kg, followed by a constant infusion at 292,umole/min.kg) to the end of the experiment. It can be seen (Fig. 7) that the maximum 'dehydrocholate' excretory rate after taurine depletion (about 2 0 Fzmolelmin. kg) was much less than that obtained after extensive conjugation had been restored by taurine administration (3.8,umole/min.kg). The possible explanations for this finding may be similar to those already discussed in relation to the difference between the free and conjugated cholate excretory maxima (O'Matille et al. 1965, 1967; Richards & O'Maille, 1973). 354

DISCUSSION

The maximum excretory rate of a bile salt is a net rate and therefore represents the difference between the unidirectional transfer rates between blood or liver cell and bile. The lower excretory maximum found for 'dehydrocholate' compared with the natural bile salts may therefore result from a lower transfer rate from blood or liver cell to bile (mainly active) or a greater bile to liver cell or blood transfer rate (back-diffusion) or both. Since dehydrocholate may be modified by partial 'hydroxylation' (reduction) and conjugation before excretion and since the secretary characteristics of these derivatives (and also presumably of unchanged dehydrocholate) probably differ from each other (see Fig. 7), it is clear that the observed total 'dehydrocholate' maximum may be seriously influenced by the maximum rate at which either or both of the modifying processes can take place. Even with the modifying processes fully engaged, it is reasonable to assume that, during supramaximal dehydrocholate infusion, the excretory membrane which actively transfers bile salt into bile is saturated with the several species of bile salt; that is, if the modifying processes themselves are saturated, the unchanged dehydrocholate will 'overflow' to the excretory membrane. But if unchanged dehydrocholate were excreted very poorly compared with the modified derivatives, the observed excretory maximum might largely reflect the maximum rate at which modification could take place. It is clear from the results that dehydrocholate administration had an inhibitory action on taurocholate excretion. Taurocholate also had an inhibitory action on 'dehydrocholate' excretion, since the 'dehydrocholate' excretory maximum rose progressively after taurocholate infusion was stopped. These results, which showed an increase in the excretory rate of one bile salt in association with a decrease in the excretory rate of the other, provide qualitative evidence in support of transport competition.

DEHYDROCIIOLATE SECRETION 355 However, when this mutual inhibitory action was quantitatively analysed, it was not found to be uniformly competitive; that is, the extent to which the excretion of one bile salt replaced that of the other (i.e. the change in 'dehydrocholate' excretion per unit change in taurocholate excretion) was not constant. One explanation for this lack of constancy might be that the individual dehydrocholate derivatives have different secretary properties as discussed in the preceding paragraph. Thus the conjugated, 'hydroxylated' derivatives might readily replace taurocholate but very little of the unmodified or relatively unmodified dehydrocholate might be excreted instead of taurocholate. The converse of this process might also partly explain why superimposed taurocholate infusions had little effect on the 'dehydrocholate' excretory maximum; in this case taurocholate would first replace the poorly excreted fraction of 'dehydrocholate'. However, a possibly more important explanation for the co-existence of an unchanged 'dehydrocholate' maximum together with the excretion of taurocholate at a considerable rate might be the simultaneous enhancing action of taurocholate on 'dehydrocholate' transfer (see below). Finally, non-uniform competition might be explained by some of the membrane carriers being available to taurocholate only, with the remainder being shared between the two bile salts. It is uncertain whether the pronounced reduction of the control excretion of endogenous taurocholate, caused by &upramaximal dehydrocholate infusion (see Results), was due to its displacement from the excretory membrane or to inhibition of its hepatic synthesis. It should be pointed out that the papers (Reinhold & Wilson, 1934; Bradley & Ivy, 1940) referred to by Sperber (1959) in his review article do not provide satisfactory evidence of transport competition; for example, it is clear from Tables 1 and 2 of the paper by Reinhold & Wilson (1934) that the excretion rate of endogenous (tauro)cholate, following single injections of dehydrocholate, was at all times greater (not less as stated by Sperber (1959)) than control values. In addition to the inhibitory action discussed above, taurocholate also had a facilitatory action on 'dehydrocholate' excretion. This was inferred from the finding that the 'dehydrocholate' maximum after earlier taurocholate administration was almost invariably greater than before. The enhancing action of taurocholate on the 'dehydrocholate' excretory maximum differed from that on the bromsulphthalein maximum; enhancement persisted after taurocholate had left the animal in the former case but not in the latter (O'Maille et al. 1966). Taurocholate may increase the 'dehydrocholate' maximum either by increasing the maximum rate of the 'hydroxylating'/conjugating reactions, or if these are not limiting, by increasing, in effect, the number of functional carrying

E. R. L. O'MAILLE AND T. G. RICHARDS 356 points for 'dehydrocholate' in the excretory membrane. The latter effect may be achieved either by 'opening up' previously non-secreting parts of the liver or by converting carriers in secreting areas into ones, which, after the passage of taurocholate, can be used by 'dehydrocholate'. The enhanced 'dehydrocholate' maximum (5-19 ,smole/min.kg) was 76% of the actively conjugated cholate maximum; if allowance is made for the associated taurocholate excretion (IP07 lsmole/min. kg) the enhanced 'dehydrocholate' maximum approaches that of actively conjugated cholate more closely. Also, if the peak enhancement of the 'dehydrocholate' maximum of 63 %, obtained after supramaximal taurocholate infusion was stopped, is applied to the average 'dehydrocholate maximum (3-84 gmole/min.kg), a figure of 6 26 gtmole/min.kg is obtained, which is over 90 % of the actively conjugated cholate maximum. This suggests that the effective bile salt concentration in bile (ten to twenty-times greater in the case of 'dehydrocholate ') is not an important factor in limiting the secretary performance of a bile salt. A comparative study of the secretary characteristics of micelle-forming and non-micelle-forming bile salts would be simpler to interpret if both classes of bile salt were excreted into bile unchanged. Unfortunately, synthetic taurodehydrocholate (the use of which in the dog would be almost prohibitively expensive), though more suitable than dehydrocholate, also undergoes structural change in at least some species (Sperber, 1965). More recently, however, it has been found that glycodehydrocholate is excreted unchanged by the rat liver (Young & Hanson, 1972). A comparison of the secretary behaviour of glycodehydrocholate with glycocholate in the rat may help to clarify some of the uncertain issues raised by this investigation. We are grateful to Miss Marguerite Bellmon and Mrs Catherine Brady for invaluable technical assistance. REFERENCES

BIzARD, G. & VANLERENBEBOHIE, J. (1956). Chol6rese et chol6r6tiques. J. Physiol., Pari8 48, 207-364. BiAinLEy, W. B. & Ivy, A. C. (1940). Excretion and determination of cinchophen in bile. Proc. Soc. exp. Biot. Med. 45, 143-148. DEsJEux, J. F., ERLINGER, S. & DUmoNT, M. (1973). M6tabolisme et influence sur la secretion biliaire de d6hydrocholate chez le chien. Bsol. & Gastro-enterol. 6, 9-18. FORKEB, E. L. & GIBSoN, G. (1973). Interaction between sulfobromophthalein (BSP) and taurocholate. In The Liver: Quantitative Aspects of Structure and Function, ed. PAUMGARNER, G. & PRESIG, R., pp. 326-336. Basel: Karger. Ho!MANw, A. F. (1965). Clinical implications of physico-chemical studies on bile salts. Gaetroenterology 48, 484-494. HorIANw, A. F. & SMATL, D. M. (1967). Detergent properties of bile salts: correlation with physiological function. A. Rev. Med. 18, 333-376.

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O'MkiLLE, E. R. L. & RicHARis, T. G. (1975). Comparison of micelle-forming and non-micelle-forming bile salt excretion by the liver of the dog. J. Phy8iol. 250, 41-43P. O'MAILLE, E. R. L., RIcHARDs, T. G. & SHORT, A. H. (1965). Acute taurine depletion and maximal rates of hepatic conjugation and secretion of cholic acid in the dog. J. Phy8iol. 180, 67-79. O'MkrTT, E. R. L., RiCARtDs, T. G. & SHORT, A. H. (1966). Factors determining the maximal rate of organic anion secretion by the liver and further evidence on the hepatic site of action of the hormone secretin. J. Phy8iol. 186, 424-438. O'MkT.T, E. R. L., RIcHARDs, T. G. & SHORT, A. H. (1967). The influence of conjugation of cholic acid on its uptake and secretion: hepatic extraction of taurocholate and cholate in the dog. J. Physiol. 189, 337-350. O'MkILLE, E. R. L., RICHARDS, T. G. & SHORT, A. H. (1969). Observations on the elimination rates of single injections of taurocholate and cholate in the dog. Q. JZ exp. Physiol. 54, 296-310. REINHOLD, J. G. & WILSON, D. W. (1934). The acid-base composition of hepatic bile. 3. The effects of the administration of sodium taurocholate, sodium cholate and sodium dehydrocholate (Decholin). Am. J. Physiol. 107, 400-405. RIcHARDs, T. G. & O'MATTLE, E. R. L. (1973). The effect of biliary pH on the maximum excretory rate of free cholate in the dog. In The Liver: Quantitative Aspect of Structure and Function, ed. PAUMGARTNER, G. & PpEIsIG, R., pp. 345354. Basel: Karger. RuTisHAusER, S. C. B. & STONE, S. L. (1975). Comparative effects of sodium taurodeoxycholate and sodium taurocholate on bile secretion in the rat, dog and rabbit. J. Physiol 245, 584-598. SjovAtL, J. (1959). The determination of bile acids in bile and duodenal contents by quantitative paper chromatography. Bile acids and steroids, 71. Clinica chim. Acta 4, 652-664. SJovATL, J. (1 964). Separation and determination of bile acids. In Methods of Biochemical Analysis, vol. 12, ed. GLIcK, D., pp. 97-41. New York: Interscience (Wiley). SOLOWAY, R. D., HoFMANN, A. F., THOMAS, P. J., SCHOENFIELD, L. J. & KLEIN, P. D. (1973). Triketocholanoic (dehydrocholic) acid: hepatic metabolism and effect on bile flow and biliary lipid secretion in man. J. dlin. Invest. 52, 715-724. SPERBER, I. (1959). Secretion of organic anions in the formation of urine and bile. Pharmac. Rev. 11, 109-134. SPERBER, I. (1965). Biliary secretion of organic anions and its influence on bile flow. In The Biliary System, ed. TYALOR, W., pp. 457-467. Oxford: Blackwell. WHEELER, H. 0. & KING, K. K. (1972). Biliary excretion of lecithin and cholesterol in the dog. J. dlin. Invest. 51, 1337-1350. WHEELER, H. 0. & RAMOS, 0. L. (1960). Determinants of the flow and composition of bile in the unanaesthetized dog during constant infusions of sodium taurocholate. J. dlin. Invest. 39, 161-170. YOUNG, D. L. & HANSON, K. C. (1972). Effect of bile salts on hepatic phosphatidylcholine synthesis and transport, into rat bile. J. Lipid Res. 13, 244-252.