bromsulphthalein is excreted in bile partly as the free dye and partly as a intravenously administered bromsulphthalein and found that simultaneous

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1 Quart. J. exp. Physiol. (1966) 51, BLRUBN, BROMSULPHTHALEN AND NDOCYANNE GREEN EXCRETON N BLE. Bv T. HARGREAVES. From the Department of Chemical Pathology, St. George's Hospital Medical School, Hyde Park Corner, London, S.W.1. (Received for publication 29th November 1965) Bromsulphthalein inhibits conjugation in rat liver slices and rabbit liver homogenates. ndocyanine green inhibits conjugation in rabbit liver homogenates only. Bromsulphthalein is excreted in rat bile in preference to bilirubin and indocyanine green. ndocyanine green and bilirubin mutually depress the biliary excretion of each other. t is suggested that indocyanine green may be excreted into the bile by a slightly different path from that taken by bromsulphthalein and bilirubin. PREVOUS studies have suggested that various substances excreted in the bile compete with one another at several stages in their transport from plasma to bile. Dragstedt and Mills [1936] examined the rate of clearance of intravenously administered bromsulphthalein and found that simultaneous administration of bilirubin reduced the rate of clearance by per cent. Coesar et al. [1961] found that the patterns of peak excretion and biliary recovery of indocyanine green were not significantly altered in rats following simultaneous injection with bromsulphthalein. Hunton et al. [1961] showed that in unanaesthetized dogs the clearance of bromsulphthalein is reduced by indocyanine green, rose bengal, bilirubin and sodium dehydrocholate. These authors suggested that, although the structures of these compounds are diverse, similarities in their rates of plasma disappearance and their mutual competition for biliary excretion indicate a common excretory mechanism. Bilirubin is conjugated with glucuronic acid before excretion, whereas bromsulphthalein is excreted in bile partly as the free dye and partly as a glutathione conjugate. ndocyanine green is excreted in bile as the free dye Ṫhe effect of bilirubin, conjugated bilirubin, bromsulphthalein and indocyanine green on the maximum biliary excretion of each other has been investigated in male white rats and the effect of the dyes on conjugation has been studied in liver preparations in vitro. This has been done in order to determine whether these substances are excreted by the same path in the liver and if possible to shed some light on the mechanism of this pathway. METHODS Materials. - Acceptor substances: (1) o-aminophenol (Koch-Light Ltd.) was resublimed before use, and ascorbic acid (analytical grade) was added to the o-aminophenol solution to minimize oxidation. (2) 10 mg. bilirubin (British Drug Houses, Ltd.) was dissolved in 0'15 ml N-NaOH with stirring ml. phosphate-bicarbonate solution was added and mixed, the solution was centrifuged to remove any material not in solution, and this solution was used in the in vitro experiments. 184

2 Excretion in Bile Uridine diphosphate glucuronic acid (UDP glucuronic acid) was uridine diphosphoglucuronic acid ( per cent quoted purity, ammonium salt) from Sigma Chemical Co., St. Louis, Mo., U.S.A. Bilirubin for infusion was prepared by dissolving 25 mg. bilirubin in 10 ml. of an isotonic solution containing 0-52 g. Na2CO3 per 100 ml. and 0 5 g. NaCl per 100 ml. Bromsulphthalein (Samoore, Ltd.) was infused in a concentration 5 mg./ml., indocyanine green (Batch No Koch-Light & Co.) was infused in a concentration 5 mg./ml. Conjugated bilirubin was added in the in vitro experiments as undiluted human bile from patients in whom a T-tube had been inserted in the common bile duct. For the in vivo experiments conjugated bilirubin was obtained by infusing bilirubin into a rat and collecting the bile; this was then diluted with 0-85 per cent sodium chloride solution. The colncentration of conjugated bilirubin was determined by the method of Malloy and Evelyn [1937]. Phosphate-bicarbonate solution (ph 7.4) was 27 mm-nahco3 123 mm-nacl, 5 mm-kcl, 1 2 mm-kh2po4 and 1-2 mm-mgcl2. The solution was gassed with C02 (95:5) for 10 min. Animals. - Male white rats of a Wistar strain and male rabbits were obtained commercially. All animals were fed immediately before the experiment. The animals were killed by stunning and cervical dislocation. The livers were placed immediately in phosphate bicarbonate solution in ice. Liver slices approximately 10,t thick were cut by hand with a razor blade. Homogenates were prepared by grinding 1 g. liver in a teflon glass homogenizer with 9 ml M potassium chloride solution containing 3-2 x 10-4M potassium bicarbonate. Rate of Conjugation. - n glucuronide synthesis a glucuronide group is transferred from UDP-glucuronate under the influence of the microsomal enzyme UDP-transglucuronylase (UDP-glucuronate glucuronyl transferase (acceptor unspecific), EC ) to an acceptor which may be one of a wide variety of compounds (e.g. o-aminophenol and bilirubin). The rate of conjugation of bilirubin and o-aminophenol was estimated by determining glucuronide synthesis in vitro. Uridine diphosphate glucuronic acid was added in the homogenate experiments. Bilirubin as an Acceptor Substance. - Synthesis of bilirubin glucuronide in sliced rat liver tissue was determined by the method of Lathe and Walker [1958]. Synthesis in rabbit liver homogenates was by a slight modification of the method of Lathe and Walker [1958] with added UDP-glucuronic acid in a final concentration of 0 3 mm. Bilirubin in tissues was estimated by the method described by Hargreaves [1965]. o-aminophenol as Acceptor Substrate. - Synthesis of o-aminophenol glucuronide in rat liver slices was determined by the method of Levvy and Storey [1949]. Synthesis in rabbit liver homogenates was determined by the method of Stevenson and Dutton (1962]. Uptake of Conjugated Bilirubin. - The rate of uptake of conjugated bilirubin by rat liver slices was determined by shaking liver slices of approximately 200 mg. in conical flasks containing 3 ml. phosphate-bicarbonate solution and 0415 mg. conjugated bilirubin for 1 hr. at 370 with air as the gas phase. Normal and liver slices denatured by heating at 800 C. for 5 min. were used. Conjugated bilirubin was determined in the slices as described by Hargreaves [1965]. Bromsulphthalein was added to give final concentrations of 0 01, 041, 1.0 and 10 mm in the in vitro reaction mixtures, and indocyanine green to give final concentrations of 0-001, 0 01, 041 and 190 mm. Animal Experiments. - Male white rats were anmesthetized with 0 07 ml. pentobarbitone sodium (Nembutal, Abbott Ltd.) per 100 g. and the bile duct cannulated [Weinbren and Billing, 1956]. Bilirubin and the dyes were infused intravenously and the biliary excretion of each determined. Excretion of njected Bilirubin (unconjugated). -7 mg. bilirubin per 100 g. body wt. was given in bicarbonate saline solution intravenously over a period of 30 min.

3 -, Hargreaves Bile was collected for three 15-min. periods and the bilirubin (total and conjugated) estimated by the method of Malloy and Evelyn [1937]. The maximum rate of bilirubin excretion was determined over the min. period and expressed as tg. bilirubin excreted/100 g. body wt./min. After 45 min. the rat was killed and the total and conjugated bilirubin content of the liver determined. Excretion of njected Bilirubin (conjugated). -Bile containing 20 mg. conjugated bilirubin was diluted with 0-85 per cent sodium chloride solution and given intravenously to rats over a period of 30 min. Bile was collected for three 15-min. periods and the conjugated bilirubin was estimated by the method of Malloy and Evelyn [1937]. The maximum rate of conjugated bilirubin excretion for the min. period was determined and expressed as,ug. conjugated bilirubin excreted/100 g. body wt./min. Excretion of Bromsulphthalein. Bromsulphthalein (5 mg./100 g. body wt.) was infused over a period of 30 min., 15 min. collections of bile were made, and the bromsulphthalein estimated [Varley, 1962]. The results were expressed as pg. bromsulphthalein exereted/100 g. body wt./min. for the period min. Excretion of ndocyanine Green. - ndocyanine green was dissolved in aqueous solvent (5 mg./ml.) and infused intravenously for 30 min. in anaesthetized rats. Fifteen min. collections of bile were made and the indocyanine green estimated by dilution and measurement of the extinction at 805 m,u. The results were expressed as,ig. indocyanine green excreted/100 g. body wt./min. for the period min. RESULTS n vitro Experiments. -Conjugation in Slices. -The addition of bromsulphthalein lowered the rates of conjugation of o-aminophenol in rat liver slices. nhibition of bilirubin conjugation and reduction of the amount of conjugated bilirubin formed in the slices only occurred at a concentration of 10-0 mim bromsulphthalein (Table ). 1.0 mm bromsulphthalein increased the TABLE. EFFECT OF BROMSULPHTHALEN ON CONJUGATON N RAT LVER SLCES Bilirubin o-aminoplienol Rate of Bilirubin Percentage Rate of Bromsulphthalein conjugation in slice conjugated conjugation conen. (mm) myt moles/mg./hr. jtg./g. in slice mit moles/mg./hr. 0 0^ * * Mean of three experiments with each substrate. Using bilirubin as substrate 25 ml. conical flasks contained 0-25 mm bilirubin together with serum 14-6 per cent v/v and phosphate-bicarbonate solution to 3 ml. Using o- aminophenol as substrate 25 ml. conical flasks contained 0-23 mm o-aminophenol and 1 0 mm ascorbic acid. Approximately 200 mg. liver slices were shaken in the medium for 1 hr. with air as the gas phase. unconjugated bilirubin in the slice, suggesting that at this concentration bromsulphthalein increased the permeability of the slice to bilirubin. Addition of indocyanine green to rat liver slices conjugating bilirubin and o-aminophenol did not reduce the amount of conjugates formed by rat liver slices (Table ).

4 Excretion in Bile Conjugation in Homogenates. - The addition of bromsulphthalein and indocyanine green to rabbit liver homogenates lowered the rates of conjugation of bilirubin and o-aminophenol (Table ). TABLE. EFFECT OF NDOCYANNE GREEN ON CONJUGATON N RAT LVER SLCES Bilirubin o-aminophenol ndocyanine Rate of Bilirubin Percentage Rate of green concn. conjugation in slice conjugated conjugation (mm) mjy moles/mg./hr.,ug./g. in slice myi moles/mg./hr Mean of three experiments with each substrate. Using bilirubin as substrate, 25 ml. conical flasks contained 0-25 mm bilirubin together with serum 14-6 per cent (v/v) and phosphate-bicarbonate solution to 3 ml. Using o-aminophenol as substrate, 25 ml. conical flasks contained 0-23 mm o-aminophenol and 1-0 mm ascorbic acid. Approximately 200 mg. liver slices were shaken in the medium for 1 hr. with air as the gas phase. Uptake of Conjugated Bilirubin. - The addition of bromsulphthalein to phosphate-bicarbonate solution containing conjugated bilirubin reduced the uptake of conjugated bilirubin by normal and by heat denatured rat liver slices. The addition of indocyanine green did not reduce the uptake in normal or heat denatured slices (Table V). TABLE. EFFECT OF BROMSULPHTHALEN AND NDOCYANNE GREEN ON CONJUGATON N HOMOGENATES Rate of conjugation my moles/mg./hr. Bromsulphthalein ndocyanine green Conen.,- r,a (mm) Bilirubin o-aminophenol Bilirubin o-aminophenol Mean of three experiments with each substrate. 72 x 12 mm. tubes contained 36 mm phosphate-buffer ph 7-4; 12mM Mg.C12; mm bilirubin; 0-3 mm UDPGA and serum 14 per cent (v/v) and 15 mg. liver homogenate. ncubation 20 min. at 37. n vivo Experiments. - Effect of Bromsulphthalein. - Bilirubin Excretion. - The mean value for the maximum hepatic clearance of bilirubin in fourteen experiments was 73-6 ptg. bilirubin/100 g. body wt./min. Simultaneous administration of an equimolecular quantity of bromsulphthalein (0-043 m-mole) with bilirubin reduced the maximum hepatic clearance of bilirubin to 37-7,tg. bilirubin/100 g. body wt./min. Doubling this dose of bromsulphthalein reduced the maximum hepatic clearance of bilirubin to 15-6,ug. bilirubin/100 g. body wt./min. (Table V). 187

5 188 Hargreaves The mean value for the maximum hepatic clearance of bromsulphthalein in eight rats was 73,ug. bromsulphthalein/100 g. body wt./min. When bromsulphthalein was administered with an equimolecular amount of TABLE V. EFFECT OF BROMSUPHTHALEN AND NDOCYANNE GREEN ON CONJUGATED BLRUBN UPTAKE BY RAT LVER Conjugated bilirubin pg./200 mg. wet wt. liver Bromsulphthafein ndocyanine green Concn., -_A,_A r (mm) Normal Heat-denatured Normal Heat-denatured Mean of three experiments with each substance. The flasks contained approximately 200 mg. wet wt. rat liver slices in 3 ml. phosphatebicarbonate solution containing 150,ug. conjugated bilirubin. ncubation at 370 for 1 hr. bilirubin, the maximum hepatic clearance was 100 pg. bromsulphthalein/100 g. body wt./min. When twice the equimolecular amount of bromsulphthalein was administered together with bilirubin, the maximum hepatic clearance of bromsulphthalein was 66 Mg./100 g. body wt./min. TABLE V. EFFECT OF BROMSULPHTHALEN ON BLRUBN EXCRETON Maximum hepatic clearance pug./100 g. body wt./min. B.S.P. No. of, -A m-mole expts. Bilirubin Conjugated bilirubin Bromsulphthalein Bilirubin (0-043 m-mole/min.) Conjugated bilirubin (0.019 m-mole/30 min.) i nfusions into male Wistar rats, anaesthetized with Veterinary Nembutal; the common bile duct was cannulated. 15 min. collections of bile were made, and bilirubin was estimated by the method of Malloy and Evelyn [1937]. Bromsulphthalein by the method of Varley [1962]. The experiments suggest that bromsulphthalein is excreted in the bile in preference to bilirubin. This was investigated further by infusing bilirubin into rats continuously to produce maximum hepatic clearance of bilirubin for a period of 90 min. During this time an equimolecular amount of bromsulphthalein was infused and the biliary excretion of both substances determined. Bromsulphthalein reduced the maximum hepatic clearance of bilirubin when given during a constant infusion of bilirubin (fig. 1).

6 Excretion in Bile 189 Conjugated Bilirubin Excretion. - The mean value for the maximum hepatic clearance of conjugated bilirubin in eight rats was 43,ug. conjugated bilirubin/100 g. body wt./min. The mean hepatic clearance of conjugated bilirubin when an equimolecular amount of bromsulphthalein (0.019 m-mole) was given simultaneously was r BLRUBN BROMSULPHTHALEN 7H. MNUTES FG. 1. Effect of bromsulphthalein on bilirubin excretion. Bilirubin was infused into a rat to produce a maximum concentration in bile (-) an equimolecular amount of bromsulphthalein was infused and the biliary concentration measured (--- ). Mean of three experiments with Wistar rats anaesthetized with pentobarbitone sodium. 39 jug. conjugated bilirubin/100 g. body wt./min. When twice the equimiolecular amount of bromsulphthalein (0.038 m-mole) was given with conjugated bilirubin, the maximum hepatic clearance of bilirubin was 28 jpg. conjugated bilirubin/100 g. body wt./min. Administration of three times the equimolecular amount of bromsulphthalein (0.057 m-mole) together with conjugated bilirubin reduced the mean maximum hepatic clearance of conjugated bilirubin to 19,pg. conjugated bilirubin/100 g. body wt./min. The mean clearance of bromsulphthalein, when given in equimolecular, twice equimolecular, and thrice equimolecular quantities with conjugated bilirubin, was 78,ag. bromsulphthalein, 111 jug. bromsulphthalein and 105,g. bromsulphthalein/100 g. body wt./min. respectively (Table V).

7 190 Hargreaves Effect of ndocyanine Green. - Bilirubin Excretion. - Simultaneous administration of one-tenth the equimolecular amount of indocyanine green together with bilirubin did not reduce the mean maximum hepatic clearance of bilirubin. n five rats the mean maximum hepatic clearance of bilirubin given simultaneously with indocyanine green was 72,tg. bilirubin/100 g. body wt./min. Simultaneous administration of half equimolecular amounts (0.017 m-mole) of indocyanine green together with bilirubin (0.033 m-mole) slightly reduced the mean maximum hepatic clearance of bilirubin. n four rats the mean maximum hepatic clearance of bilirubin was 53,ug. bilirubin/100 g. body wt./min. (Table V). TABLE V. EFFECT OF NDOCYANNE GREEN ON BLARY EXCRETON Maximum hepatic clearance pg./100 g. body wt./min..c.g. No. of, s m-mole expts. Bilirubin Conjugated bilirubin B.S.P..C.G. Control values (73.6) (43) (73) (4.7) Bilirubin (0.033 m-mole/30 min.) Conjugated bilirubin (0.019 m-mole/30 min.) 0* *7 Bromsulphthalein (0.015 m-mole/30 min.) nfusions into male Wistar rats, anmesthetized with Veterinary Nembutal; the common bile duct was cannulated. 15 min. collections of bile were made, and bilirubin was estimated by the method of Malloy and Evelyn [1937], indocyanine green by dilution and determination of the extinctions. The mean value for the maximum hepatic clearance of indocyanine green in sixteen rats was 4.7 jig. indocyanine green/100 g. body wt./min. When one-tenth of the equimolecular amount was administered with bilirubin, the maximum hepatic clearance of indocyanine green was 1.05 pg. indocyanine green/100 g. body wt./min. When half the equimolecular amount of indocyanine green (0-017 m-mole) was administered together with bilirubin (0.033 m-mole) the maximum hepatic clearance was 3.2,ug. indocyanine green/100 g. body wt./min. Conjugated Bilirubin Excretion. - The maximum hepatic clearance of conjugated bilirubin when administered simultaneously with one-fifth of the equimolecular amount of indocyanine green ( m-mole) was 39,ug. conjugated bilirubin/100 g. body wt./min. When 0.5 equimolecular amounts of indocyanine green (0.009 m-mole) were administered with conjugated bilirubin (0-018 m-mole) the maximum hepatic clearance was 19 jug. conjugated bilirubin/100 g. body wt./min. The maximum hepatic clearance of indocyanine green when administered simultaneously in 0.2 equimolecular and 0.5 equimolecular amounts together with conjugated bilirubin was

8 Excretion in Bile /g. indocyanine green and 1 7 /tg. indocyanine green/100 g. body wt./min. respectively. These results suggest that when bilirubin and indocyanine green are infused together in the rat the biliary excretion of each substance is decreased. The Effect of ndocyanine Green on Biliary Excretion of Bromsulphthalein. - The mean maximum hepatic clearances of indocyanine green and bromsulphthalein were 4.7,ug. indocyanine green and 73 jug. bromsulphthalein/100 g. c== C.G. B.S.P UJ uj z 4tY 4 uj cl U 4-0. x MNUTES FG. 2. Effect of bromeulphthalein on indocyanine green excretion. ndocyanine green was infused into a rat to produce a maximum concentration in bile (-). Bromsulphthalein was infused and the biliary concentration measured (- -). Mean of four experiments in Wistar rats anaesthetized with pentobarbital sodium. body wt./min. respectively. The simultaneous administration of indocyanine green in a dose (0.003 m-mole) 0.2 times the equimolecular amount of bromsulphthalein (0.015 m-mole) reduced the mean maximum hepatic clearance of indocyanine green to 0.32,ug. indocyanine green/100 g. body wt./min. in five experiments. The mean maximum hepatic clearance of bromsulphthalein in these experiments was 73,ug. bromsulphthalein/100 g. body wt./min. When equimolecular amounts (0-015 m-mole) of indocyanine green and bromsulphthalein were given to rats and their biliary excretion determined,

9 192 Hargreaves the maximum hepatic clearance of indocyanine green was reduced to 2.5,g./ 100 mg./min.; the maximum hepatic clearance of bromsulphthalein was 70,ug./100 g. body wt./min. The results show that bromsulphthalein is excreted preferentially to indocyanine green by the liver into the bile. This was further investigated by infusing indocyanine green into rats to maintain maximal hepatic secretion of indocyanine green. The indocyanine green was infused for a 'C.G. w z w U *B.S.P * g; S0 50 w MNUTES FG. 3. Effect of simultaneous infusion of bromsulphthalein and indocyanine green on biliary excretion in aroesthetized Wistar rats. Bromsulphthalein (--- - ). ndocyanine green (-). 90 min. period; during this time bromsulphthalein was infused for 30 mm. and the biliary concentration of each substance was determined. Figure 2 is a mean of the results of four experiments. Bromsulphthalein reduced the maximal hepatic clearance of indocyanine green during infusion of indocyanine green, confirming that of the two dyes bromsulphthalein is excreted preferentially. n one experiment, in which equimolecular amounts of indocyanine green and bromsulphthalein were injected simultaneously, the biliary excretion of each substance was followed for 135 min. (fig. 3). Bromsulphthalein initially depressed the excretion of indocyanine green, but as the excretion of bromsulphthalein decreased the excretion of indocyanine green increased.

10 Excretion in Bile 193 DscUSSON Bilirubin is excreted by the liver into bile. n mammals exciretion of bilirubin depends on prior conjugation with glucuronic acid [Schmid et. al., 1958]. Conjugation occurs in liver microsomes under the influence of the enzyme UDP-transglucuronylase; bilirubin diglucuronide is then excreted from the liver cells to the biliary canaliculi, and this appears to be the rate limiting step. Bromsulphthalein has been extensively used for testing hepatic function. Little has been known of its hepatic transport and excretory mechanism until recent years. Disappearance of bromsalphthalein from the blood after intravenous administration involves a complex process; it is affected by the interrelated functions of hepatic uptake, storage, conjugation and biliary excretion, each of which can be influenced by dynamic factors such as blood circulation and protein availability [Combes and Stakelum, 1961]. The dye is excreted by the liver into the bile at a maximum rate which is not increased when the dose of bromsulphthalein is increased. n rats conjugation with glutathione represents the major metabolic pathway of bromsulphthalein in the liver; the enzyme is in the soluble fraction of rat liver [Combes and Stakelum, 1960] and some bromsulphthalein is excreted unchanged in the bile İndocyanine green, a tricarbocyanine dye, is excreted in an unconjugated form in the bile [Caesar et al., 1961]. Hunton et al. [1960] found that bromsulphthalein clearance was reduced by indocyanine green and bilirubin in dogs; in rats they found that bromsulphthalein and indocyanine green each affected the clearance of the other. Caesar et at. [1961] found that, in rats, the patterns of peak excretion of bromsulphthalein and indocyanine green were not altered following their simultaneous injection. Wheeler et al. [19601 found that indocyanine green did not reduce bromsulphthalein excretion in dogs. Fauvert [1959] suggested that there was competition for hepatic uptake between bromsulphthalein and bilirubin. Each of the three substances under investigation, bilirubin, bromsulphthalein, and indocyanine green, is excreted in the bile by potentially different. mechanisms. Bilirubin must be conjugated before excretion, conjugation occuring in the microsomes. Bromsulphthalein is not necessarily conjugated before excretion, but it is mostly excreted as the glutathione conjugate; conjugation is catalyzed by an enzyme in the soluble liver fraction. ndocyanine green is excreted unchanged in the bile. 10 mm bromsulphthalein inhibited conjugation in vitro in the rat liver slice experiments, but 1 mm bromsulphthalein consistently increased the free bilirubin in the slice. n infusion experiments bromsulphthalein reduced the biliary excretion of bilirubin and conjugated bilirubin, without the biliary excretion of bromsulphthalein being altered. When a constant infusion of bilirubin was given, infusion of bromsulphthalein reduced the maximum hepatic clearance of bilirubin. The results suggested that bromsulphthalein was excreted in the bile in preference to conjugated bilirubin, but the lowering of conjugated VOL. L, NO

11 194 Hargreaves bilirubin excretion suggested that there was an effect on the secretory mechanism. Bromsulphthalein retarded the uptake of conjugated bilirubin in normal and heat denatured rat liver slices, suggesting that this was a physical effect. ndocyanine green did not reduce the rates of conjugation of o-aminophenol and bilirubin in rat liver slices, but inhibited conjugation in rabbit liver homogenates at the concentrations tested. t is unusual for a substance to inhibit conjugation in liver homogenate preparations alone; indocyanine green may only get to the conjugation site or its entry or exit, in the homogenate preparations. There was mutual depression of excretion when indocyanine green and bilirubin were injected together into rats and the biliary excretion measured. Bromsulphthalein inhibited conjugation in vitro and decreased the biliary excretion of pigments. Either bromsulphthalein has a dual action on conjugation and excretion, or else both processes are inextricably linked. ndocyanine green may be excreted by the same path, but does not involve UDP-transglucuronylase. t is difficult to understand why conjugation in homogenates is inhibited unless conjugation and excretion are inextricably linked. n the case of indocyanine green the effect may be only on the excretory path which is only in a readily accessible state in broken cell in vitro preparations; the end results appear as inhibition of conjugation. Javitt [1965] has recently investigated the mode of excretion of phenoltetrabromsulphthalein monosulphate and the tetrasulphonate. Bromsulphthalein is phenoltetrabromsulphthalein disulphonate. Javitt found that the monosulphonate is excreted in rats as a glutathione-glucuronide conjugate, but the tetrasulphonate is excreted unchanged. t is conceivable then that the bromsulphthalein excretion may be linked with UDP-transglucuronylase. The results show that bromsulphthalein is excreted in rat bile in preference to bilirubin and indocyanine green. The lack of inhibitory effect on conjugation in slice preparations bv indocyanine green suggests that indocyanine green may be excreted by a slightly different path from that taken by bilirubin and bromsulphthalein. This is supported by the observation that male fern extract prevents the excretion of bromsulphthalein and conjugated bilirubin but not of indocyanine green [Hargreaves, 1965]. ndocyanine green may not require UDP-transglucuronylase for its excretion, but bilirubin requires the enzyme, and bromsulphthalein may require the enzyme for excretion. The problem could be resolved by preparing a soluble enzyme preparation and determining whether conjugation is inhibited by bromsulphthalein or indocyanine green. ACKNOWLEDGMENTS wish to thank Professor N. H. Martin for his advice and encouragement, Miss V. Price for her skilled technical assistance, and the British Empire Cancer Campaign and the Board of Governors, St. George's Hospital, for financial assistance.

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