(Roche, Michel & Tata, 1954). Bilirubin is excreted by rats as the glucuronide (Lathe & Walker, 1958). This similarity in the mode of excretion

Transcription

1 16 J. Phy8iol. (1962), 163, pp With 4 text-figure8 Printed in Great Britain THE EFFET OF BLRUBN ON THE EXRETON OF THYROXNE N THE BLE OF RATS BY A. UAR6N*, N. B. MYANT AND. OSORO From the Medical Research ouncil Experimental Radiopathology Research Unit, Hammersmith Hospital, London, W. 12 (Received 12 March 1962) Thyroxine is excreted in the bile of rats partly as the free hormone, but mainly as the glucuronide (Taurog, Briggs & haikoff, 1952) together with small quantities of metabolites of thyroxine other than the glucuronide (Roche, Michel & Tata, 1954). Bilirubin is excreted by rats as the glucuronide (Lathe & Walker, 1958). This similarity in the mode of excretion of thyroxine and bilirubin raises the possibility that the two substances may compete with one another for the same excretory mechanism. n this paper we describe experiments designed to test whether alterations in the amount of bilirubin excreted in the bile of rats have any effect on the formation of the glucuronide of thyroxine. METHODS The rats used were albino males of the Wistar strain weighing about 2 g. They were anaesthetized by an intraperitoneal injection of amylobarbitone (1 mg/1 g) given 2 min before operation; further injections were given to keep the animals anaesthetized while the bile was draining. For the cannulation of the bile duct the abdomen was opened by a midline incision, and a fine flexible polythene tube, 1 cm long, was inserted into the bile duct and tied with two ligatures. The abdomen was closed with sutures and the bile allowed to drain into weighed collecting tubes with the open end of the cannula about 1 cm below the level of the bile duct. The collecting tubes were changed at intervals of about 15 min and weighed. After closure of the abdomen, the femoral artery and vein were exposed on both sides and kept covered with cotton wool moistened with warm saline solution. ntravenous injections were given into the femoral veins; blood samples, each of about -5 ml., were taken from the femoral arteries into heparinized syringes at intervals of 2-3 min. A88ay of radioactivity. -5 ml. of bile from each sample was transferred to a 2 cm2 polythene planchette and dried on a steam-bath. Radioactivity on the dried planchette was measured with a mica-window counter (EHM 2/S, G.E.. Research Laboratories). From each blood sample 5 ml. of whole blood was transferred to a planchette and assayed in the same way as the bile samples; self-absorption of radioactivity was found to be negligible with this quantity of blood. hromatography. For analysis of the radioactive components in the bile, samples of about -1 ml. were transferred to strips of Whatman No. 3iM filter paper and submitted to descending chromatography in freshly prepared n-butanol equilibrated with an equal volume * British ouncil Scholar

2 BLRUBN AND THYROXNE 161 of 2N acetic acid. Radioactivity on the paper strips was measured with a continuously recording scanner. The peaks of radioactivity corresponding to iodide and thyroxine were identified by comparing their RF values with those of the peaks given by known samples of radioactive iodide and thyroxine added to non-radioactive rat bile and run in the same solvent system. The peak corresponding to thyroxine glucuronide was identified as the one immediately behind the thyroxine peak by showing that more than 98 % of the radioactive material, eluted from this region and incubated with,b-glucuronidase for 2 hr at 37, was converted to free radioactive thyroxine. No attempt was made to identify any of the other radioactive substances appearing in the bile. Material. L-thyroxine labelled with 131 (2-3 uc/pg) was obtained from Abbott Laboratories Pacific Ltd (hicago). The dose of radioactive thyroxine was dissolved in 1 ml. physiological saline for injection. The bilirubin (British Drug Houses) was dissolved in 1 ml. of 5M-Na2O3 in physiological saline. The f3-glucuronidase (from molluscs) was obtained from L. Light and o. Ltd (olnbrook, England). RESULTS Observations were first made on the excretion of total radioactivity in the bile over a period of 5 hr after an intravenous injection of radioactive thyroxine (1,uc with less than 1,g of carrier) given immediately after cannulation of the bile duct. Radioactivity was present in the earliest sample of bile at a concentration 2-3 times that in the blood. The concentration of total activity in the bile then fell continuously throughout the period of observation in parallel with the fall in the blood concentration (Fig. 1). When 1 ml. of the Na2O3 solution used for dissolving the bilirubin was injected intravenously between the second and third hours, there was no change in the rate of fall of activity in the bile (Fig. 1 a). Nor was there any change when doses of 3-1 mg bilirubin were injected (Fig. 1 b). Within a few minutes of the injection of bilirubin the bile became dark yellow and remained so for at least 1 hr. hromatographic analysis of the radioactive bile showed seven distinct peaks of activity on the paper strips, in addition to a variable amount of activity at the origin (Fig. 2). From the concentration of total activity in each sample of bile and the proportion of the total in the peaks revealed by chromatography it was possible to estimate separately the activity due to thyroxine and to thyroxine glucuronide. Figure 3 shows the results of a typical experiment in which the radioactivity was measured in the thyroxine and thyroxine glucuronide fractions in serial samples of bile collected after an intravenous injection of radioactive thyroxine. Activity in the thyroxine fraction was highest in the first sample taken and fell continuously throughout the 5-hr period. n the thyroxine glucuronide fraction activity began at a relatively low level, rose to a maximum between the 2nd and 3rd hour and then fell in parallel with the activity in the thyroxine fraction, but at a higher level. The injection of the Na2O3 solution had no effect on the curve of excretion 11 Physiol. 163

3 162 A. UARBN, N. B. MYANT AND. OSORO 5 E 4 o 3 -E 2 u-, -o 1 -o i 5. m - F ) J o o o o a 1* 5s -- -E co 5 4 E r 6 3 E 2 :3 n 1 a -o - m 5 -. D. i u m - t b 1. 5? 7E -3 ~ co Fig. 1. Total radioactivity (counts/min/o1 ml.) in bile () and blood () after injection of radioactive thyroxine given within 5 min of insertion of cannula into bile duct; rate of flow of bile ( [l ). Values for radioactivity in bile and for bile flow plotted at mid point of sampling interval. Arrows show intravenous injection (a) of 1 ml. Na2O3 solution or (b) of 6 mg bilirubin dissolved in 1 ml. Na2O3 solution. Vertical scales logarithmic.

4 BLRUBN AND THYROXNE 163 of either of the two radioactive fractions (Fig. 3a). When bilirubin (3-1 mg) was injected intravenously between the 2nd and 3rd hour there was no detectable change in the excretion curve of thyroxine glucuronide (Fig. 3 b). However, there was a small, though constant, rise in the excretion of radioactive thyroxine in the first sample taken after the injection of bilirubin. Thyroxine Thyroxine glucuronide odide i -4 Fig. 2. Distribution of radioactivity on paper chromatogram made from a sample of bile taken 2 hr after injection of radioactive thyroxine. Arrow to right shows origin; arrow to left shows solvent front. Positions of thyroxine, thyroxine glucuronide and iodide shown above their respective peaks. Solvent mixture: n-butanol equilibrated with 2N acetic acid. n order to test for a possible effect of bilirubin on glucuronide excretion when more complete equilibration had been achieved, the radioactive thyroxine was given 4 hr before cannulation of the bile duct. After cannulation, bile samples were collected for a period of 4-5 hr. Under these conditions radioactivity in the thyroxine and thyroxine glucuronide fractions of the bile fell in parallel at a constant proportional rate throughout the sampling period, activity in the glucuronide fraction exceeding that in the thyroxine fraction. njections of Na2O3 solution or of bilirubin (3-1 mg) given 2-3 hr after cannulation had no detectable effect on the excretion curve of thyroxine glucuronide (Fig. 4a, b). Bilirubin, however, caused a slight but temporary rise in the excretion of thyroxine (Fig. 4b) DSUSSON t is well established that the glucuronides of substituted phenols are formed in the liver by a reaction between uridine diphosphate glucuronic acid (UDPGA) and the phenol, the reaction being catalysed by UDPGA transferase (Dutton & Storey, 1954). For reasons discussed elsewhere (Myant, 1958) the glucuronide of thyroxine, itself a substituted phenol, is 11-2

5 v 164 A. UABON, N. B. MYANT AND. OSOBO almost certainly formed by this reaction, though complete proof of this is lacking. The formation of the glucuronide is an obligatory step in the excretion of bilirubin in the bile of rats, since rats of the Gunn strain, which cannot form bilirubin glucuronide from UDPGA (Lathe & Walker, 1958), are unable to excrete bilirubin, although they can excrete exogenous bilirubin c 33-1 O,x E, a >1 _~ ve. o >' = t 1 2 3, U.b_.ox E b - - ru 5 )_ xv c _-,3 *' d 2 t Fig. 3. Radioactivity (countsfmin/o1 ml.) in thyroxine () and thyroxine glucuronide (A) fractions of bile after injection of radioactive thyroxine given within 5 min of insertion of cannula. Arrows show intravenous injection (a) of 1 ml. Na2O3 solution or (b) of 3 mg bilirubin solution. Vertical scale logarithmic.

6 BLRUBN AND THYROXNE 165 glucuronide at the normal rate (Schmid, Axelrod, Hammaker & Swarm, 1958). f, therefore, the glucuronides of thyroxine and bilirubin are formed by the same enzyme system, an increase in the excretion of bilirubin might be expected to diminish the excretion ofthyroxine glucuronide. The results reported here show, however, that the formation of thyroxine 3 o. 'E2 boo- A A A a xe 1 o o 4 o 5 O Time afi ter injection of radioactive thyroxine (hr) *2 3 S 21 _) - x o E 1 -o u _ o 5. E AA o A A A b A,, A '&i oaa o o - 'U *-. 4) 4 J Fig. 4. Radioactivity (counts/min/*l ml.) in blood (-), and in thyroxine () and thyroxine glucuronide (A) fractions of bile excreted through cannula inserted 4 hr after an injection of radioactive thyroxine. Arrows to left show insertion of cannula; arrows to right show injection of (a) 1 ml. Na2O3 solution or (b) 1 mg bilirubin solution. Vertical scale logarithmic.

7 A. cuar6n, N. B. MYANT AND. OSORO 166 glucuronide is unaffected by doses of bilirubin close to the maximal excretory capacity of a normal rat (Weinbren & Billing, 1956). This suggests that thyroxine and bilirubin do not compete for the same UDPGA transferase. t would not be surprising if this were so, since glucuronic acid is joined to bilirubin by ester bonds (Billing, ole & Lathe, 1957) but to phenols by an ether bond. Absence of competition between thyroxine and bilirubin is also in keeping with the finding oflathe & Walker (1958) that slices of liver from rats of the Gunn strain cannot form bilirubin glucuronide but can form the glucuronide of o-aminophenol. However, the existence of two transferases in rat liver, one catalysing the formation of phenolic glucuronides and the other catalysing that of bilirubin glucuronide, is difficult to reconcile with the observations of Flock & Bollman (1959). These workers found that Gunn rats, in addition to their known inability to form bilirubin glucuronide, are also unable to form thyroxine glucuronide. f there are, in fact, two glucuronideforming enzymes, this finding would imply that Gunn rats inherit a deficiency of two different enzymes. No doubt, the question will be settled when purified preparations of UDPGA transferase (sselbacher, 1961) become available. n the mean time it is worth pointing out that in normal rats the excretion of thyroxine as the glucuronide does not seem to be influenced by large alterations in the load of bilirubin presented to the liver, whether this is because the two substances do not compete for the same enzyme system or because the final step in their conversion to glucuronides is not the rate-limiting step in vivo or, perhaps, because the affinity of thyroxine for UDPGA transferase is much greater than that of bilirubin. n any case it is probable that the excretion of thyroxine in the bile, known to be so important for the elimination of this hormone in rats (Gross & Leblond, 195; Albert & Keating, 1952), does not depend on the formation of the glucuronide (Myant, 1957). f thyroxine glucuronide were an essential intermediate in the transport of thyroxine from the plasma to the bile, the specific activity of the glucuronide should be higher than that of unconjugated thyroxine in the earliest samples of bile obtained after an injection of radioactive thyroxine. The results illustrated in Fig. 3 show that this is not the case. SUMMARY ntravenous injections of bilirubin into rats, in amounts close to the maximal excretory capacity of the liver, have no effect on the excretion of the glucuronide of thyroxine in the bile.

8 BLRUBN AND THYROXNE 167 REFRENES AimRT, A. & KEATrG, F.R., Jr. (1952). The role of the gastrointestinal tract, including the liver, in the metabolism of radiothyroxine. Endocrinology, 51, BunMG, B. H., ole, P. G. & LATrH, G. H. (1957). The excretion of bilirubin as a diglucuronide giving the direct van den Bergh reaction. Biochem. J. 65, DurroN, G. J. & STOREY,. D. E. (1954). Uridine compounds in glucuronic acidmetabolism. 1. The formation of glucuronides in liver suspensions. Biochem. J. 57, Foc, E. V. & Bot., J. L. (1959). Altered thyroxine conjugation in Gunn rats. Fed. Proc. 18, 227. GROSs, J. & LxBLOiD,. P. (195). Metabolism of the thyroid hormone in the rat as shown by physiological doses of labeled thyroxine. J. biol. hem. 184, SSELBAHER, K. J. (1961). Solubilization and purification of glucuronyl transferase from rabbit liver microsomes. Biochem. Biophys. Res. omm. 5, LATHE, G. H. & WALmx, M. (1958). The synthesis of bilirubin glucuronide in animal and human liver. Biochem. J. 7, MYANT, N. B. (1957). Relation between the biliary clearance rate of thyroxine and the binding of thyroxine by the serum proteins. J. Physiol. 135, MYANT, N. B. (1958). The biliary excretion of thyroid hormone. hapter in Lectures on the Scientific Basi8 of Medicine, Vol. vi: , pp London: Athlone Press. RocEa, J., MHEL, R. & TATA, J. (1954). Sur la nature des combinaisons iod6es excr6t6es par le foie et le rein apr6s administration de L-thyroxine et de L-3:5:3'-triiodothyronine. Biochim. biophys. acta, 15, ScHmD, R., AxOD, J.,,AMMAWwR L. & SwARM, R. L. (1958). ongenital jaundice in rats, due to a defect in glucuronide formation. J. lin. nvest. 37, TAUROG, A., BRGGS, F. N. & HAJXOPr,. L. (1952) labelled L-thyroxine.. Nature of the excretion product in bile. J. biol. hem. 194, WENBREN, K. & BLLNG, B. H. (1956). Hepatic clearance of bilimbin as an index of cellular function in the regenerating rat liver. Brit. J. eap. Path. 37,