reciprocal of the rate of deiodination being proportional to the reciprocal

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1 J. Phy&iol. (1972), 222, pp With 6 text-figuree Printed in Great Britain DEIODINATION OF THYROID HORMONES BY THE PERFUSED RAT LIVER BY A. P. HILLIER From the Physiological Laboratory, University of Cambridge, Downing Street, Cambridge CB2 3EG (Received 6 December 1971) SUMMARY 1. An investigation has been made into the deiodination of thyroid hormones by the perfused rat liver. The hormones were labelled with 125I in the phenolic ring and the rate of deiodination was estimated by measuring the release of radio-iodide into the perfusate. 2. At tracer concentrations, 0-98 % of the liver thyroxine is deiodinated/ 5 min. The deiodination of tri-iodothyronine is considerably faster, 3-3 %/5 min. 3. Deiodination is very sensitive to changes in temperature. 4. The reaction shows saturation kinetics typical of many enzymes, the reciprocal of the rate of deiodination being proportional to the reciprocal of the hormone concentration in the tissue. The maximum rate of deiodination of each hormone is about 1-5,ug/min for a whole liver preparation weighing 16 g. 5. Tri-iodothyronine inhibits thyroxine deiodination and vice versa, suggesting that a single enzyme is responsible for both reactions. 6. Propyl thiouracil (PTU) at high concentrations inhibits the deiodination of both hormones. 7. An abnormally high rate of deiodination is associated with the actual injection of hormone into the preparation. This suggests that only the free (unbound) hormone in the tissue is directly available to the deiodinating enzyme. 8. About half of the whole body deiodination of thyroxine is relatively insensitive to PTU. It is suggested that most of this type of deiodination is performed in the liver and that the process is one of inactivation. INTRODUCTION Deiodination (the removal of iodine atoms) is a major metabolic pathway for thyroid hormones. The little knowledge we have of this process comes mainly from studies in vivo. These involve measuring the urinary

2 476 A. P. HILLIER excretion of inorganic radio-iodide following injection into the animal of thyroid hormones labelled in the phenolic ring with radioactive iodine. Although a number of attempts have been made to study deiodination in simple in vitro systems, the results with tissue slices and homogenates have proved very difficult to interpret. There is considerable doubt about the basic mechanism of the reaction and the particular tissues involved. The subject has recently been reviewed by Galton (1968). Further understanding of this metabolic pathway could be especially valuable since it has often been suggested that deiodination is in some way linked to the hormonal action of thyroxine. The main evidence for this view is the observation that PTU competitively inhibits both the deiodination and the metabolic actions of thyroxine (Morreale de Escobar & Escobar del Rey, 1967). This paper reports experiments designed to gather basic information about the deiodination of thyroid hormones by the liver. In general, the results suggest the presence of a single enzyme responsible for deiodinating (and probably inactivating) both thyroxine and tri-iodothyronine. METHODS The preparation used in these experiments has already been described in a study of thyroxine secretion into bile and a detailed account of the animals, materials and methods will be found there (Hillier, 1972a). Briefly, it consists of a rat liver perfused via the hepatic portal vein with Tyrode solution at a pressure head of 60 cm water. The bile is collected through a fine polyethylene cannula and discarded. The thyroxine binding sites in the tissue are labelled by injecting, over a 10-sec period, a trace amount of radioactive hormone into the perfusion fluid entering the preparation. The Tyrode solution spends only 2-3 sec within the liver substance, but this is long enough for % of the injected hormone to be captured by the binding sites in the tissue. In the present experiments, deiodination of this tissue-bound hormone is followed by measuring the release into the perfusion fluid of radio-iodide generated by the deiodination reaction. The standard procedure adopted in most experiments. A rat liver preparation was set up with the perfusate initially draining into a sink. A trace amount (0-01,ug) of radioactive hormone in 0-5 ml. Tyrode solution was injected into the fluid entering the tissue and 10 min allowed for the preparation to settle (see Results). The perfusate was then switched from draining into the sink to being pumped up into a reservoir of 100 ml. Tyrode solution. From this reservoir the fluid recycled back to the liver under a pressure head of 60 cm water. Zero time was taken as the instant at which the perfusion circuit was completed. At 5 min intervals thereafter, 5 ml. samples of perfusate were taken from the reservoir for estimation of their radioiodide content. The fluid removed was replaced immediately by ordinary Tyrode solution. The flow rate was normally ml./min and the total volume of fluid in the circuit was 220 ml. so the circulation time was between 3 and 4 min. The radioactivity of the liver was monitored throughout the course of each experiment. At the end, the liver was removed from the apparatus, cut up into small pieces and its total radioactivity determined. This was done with the same counter that

3 THYROXINE DEIODINATION 477 was used to estimate the concentration of radio-iodide in the perfusion fluid. The amount of radio-iodide released from the liver during each 5 min period was then expressed as a percentage of the amount of radioactive hormone in the liver during the same period. This quantity was used as a measure of the deiodination rate. In some experiments, stable (non-radioactive) hormone was injected into the preparation; the method has already been described (Hillier, 1972b). In other experiments, PTU was introduced into the system by injecting into the reservoir known volumes of a stock solution of PTU containing 1 mg/ml. An alternative procedure was adopted in the experiments reported in Fig. 2. Here, the concentration of radio-iodide in the perfusate was measured in the period immediately following the injection of hormone. It was necessary to collect the perfusate in clearly separated, precisely timed fractions and therefore to collect it as soon as it left the tissue. This was done by supporting the liver on a plastic grid and collecting the perfusate with a funnel placed beneath the grid. The perfusate was not recycled at any stage. The hormone injection took only 2 sec and, by injecting solutions of radio-iodide and suspensions of red blood cells, it was shown that, within 15 sec, all of this injected solution had perfused through the liver and into the sink. Samples of perfusate were collected in 10-sec fractions and the first sample was taken between 25 and 35 sec after the hormone injection. This ensured that no traces of the injected material were present even in the first sample. Any radio-iodide detected in this sample must therefore have arisen by deiodination. The rate of deiodination at different hormone concentrations. The absolute rate of deiodination was estimated at each dose level as follows. First, an initial level of deiodination at tracer concentrations of hormone was established using the standard procedure. After 20 min a second injection of a known amount of stable hormone was given. If the dose was large, it caused the deiodination rate (expressed as %/5 min) to fall. The new rate was established within 10 min of the injection and was measured for a further 20 min; it was then expressed as a percentage of the initial value. The initial value at tracer concentration was taken as 098 %/5 min for thyroxine and 3-3 %/5 min for tri-iodothyronine (see Results). Using these values, the absolute rate of deiodination at the known dose level was calculated in,ig/min. For example, in one experiment, injection of 100 pg tri-iodothyronine reduced the rate of deiodination to 52 % of its initial value (i.e. to 1 72 %/5 min). Similar measurements were made over a wide range of dose levels for both hormones. Estimation of radio-iodide in the perfusate. Paper chromatography with butanol: acetic acid:water (78:10:12) showed only two radioactive materials in the perfusate, iodide and hormone. Iodide always constituted more than 80 % of the total radioactivity. The hormona was removed from the sample by adding plasma followed by tri-chloroacetic acid. The plasma protein binds the hormone and is then precipitated out by the acid. Control experiments showed that more than 97 % of the hormone and less than 1% of the iodide was removed by the procedure. The details were as follows. Five ml. of perfusate was squirted into 2-5 ml. citrated human plasma, followed a minute later by 2-5 ml. of an 80 / solution of tri-chloroacetic acid in water. The mixture was centrifuged for 3 min and 4 ml. supernatant taken for estimation of its radioactivity. The radioactive material remaining in solution after this procedure was called radio-iodide and this supposition was confirmed by comparing its properties to those of a known sample of radio-iodide. The two materials had the follovwing properties in common. (1) They were not precipitated out by tri-chloroacetic acid in the presence of plasma protein. (2) They both diffused rapidly and at the same rate through a Visking dialysis membrane. (3) This diffusion was not impeded by the presence of

4 478 A. P. HLLER plasma protein. (4) Both were quantitatively precipitated out by silver nitrate solution. (5) Both had an Rf of about 01 during paper chromatography with butanol: acetic acid: water. The radioactive hormones, obtained from Radiochemical Centre, Amersham, were labelled in the outer (phenolic) ring with 125I. Tri-iodothyronine has only the 3'- iodine in this ring. Thyroxine has a 3'- and a 5'-iodine and both are equally labelled. The two iodine atoms in the inner ring of both hormones are not labelled. Between 3 and 6% of the radioactivity of the hormone solutions was due to contaminating radio-iodide. No attempt was made to remove this iodide since it had no effect on the results. Control experiments showed that inorganic radio-iodide was not concentrated by the liver and that all of an injected dose of iodide passed through the liver and into the sink within 15 sec of its injection. In no experiment was a sample of perfusate for radio-iodide estimation taken within 25 sec of the hormone injection. Estimations of radioactivity were made with a well-type scintillation counter (Panax). RESULTS The deiodination of thyroid hormones at tracer concentration. In a first series of twenty experiments (ten with each hormone) the standard procedure described in the Methods was adopted and the deiodination reaction followed for periods of up to 90 min; the shortest experiment lasted 40 min. A typical result for thyroxine is illustrated in Fig. 1. Zero time in this figure refers to the time at which the perfusion fluid began to be recycled; this was 10 min after the actual injection of hormone. Throughout the course of all experiments the concentration of radioactive hormone in the liver gradually declined (Fig. la) and radio-iodide gradually accumulated in the perfusion fluid (Fig. lb). This release of radio-iodide by the liver was used as a measure of the rate of deiodination. The amount of radio-iodide released during each 5 min period was expressed as a percentage of the amount of hormone in the tissue during the same period (Fig. 1c). In each experiment and with both hormones this rate remained remarkably constant for as long as it was measured. In ten preparations the rate of thyroxine deiodination was (s.e.) %/ 5 min. The deiodination of tri-iodothyronine was considerably faster, %/5 min. The mean weight of the liver preparations in these experiments was 15-7 g. The release of radio-iodide into the perfusion fluid immediately after the hormone injection. In these experiments the alternative (non-recycling) procedure was adopted as described in the Methods. This allowed perfusion fluid to be collected as soon as it left the tissue. In all cases the flow rate remained absolutely constant so that changes in the concentration of radio-iodide in the perfusate directly reflected changes in its rate of release from the tissue. Four experiments were performed (two with each hormone) and two results are illustrated in Fig. 2. In all cases there was an extremely high rate of radio-iodide release just

5 THYROXINE DEIODINATION 479 after the injection, with a peak at about 60 sec and the rate falling back to basal levels within 10 min. Using the various tests described in the Methods, it was confirmed that these large amounts of radioactive material released during the first 2 min were indeed radio-iodide. The significance of this observation is discussed later. 40 a u C -0 0 m 20 O b C' 6 V 4 r E CU C.o I I o a t o T (mi n00 C Time (min.) Fig. 1. The metabolism of a trace amount of radioactive thyroxine by the perfused rat liver, a, The total radioactivity of the liver. b, The concentration of radio-iodide in the perfusion fluid. c, The rate of deiodination estimated at 5 min intervals. The effect of temperature on deiodination. Four experiments (two with each hormone) were performed. The standard procedure was adopted except that during the course of perfusion the temperature was reduced to 18 C for a period of 20 min. In each case the fall in temperature caused

6 480 a massive fall A. P. HLLEER in the rate of deiodination; there was an equally rapid recovery when the temperature was returned to 38 C (Fig. 3). Kinetics of the deiodination reaction. In these experiments the absolute rate of deiodination was estimated over a wide range of hormone concentrations for both thyroxine and tri-iodothyronine. The details of the procedure are described in the Methods. The results are illustrated in Fig. 4; each point represents a separate preparation and the conventional double reciprocal plot is used _o C. c 0 5 0!~ Time after injection (min) Fig. 2 The concentration of radio-iodide in the perfusion fluid following the injection of radioactive thyroxine (0) and tri-iodothyronine ( ) into rat liver preparations. For both hormones the deiodination reaction showed saturation kinetics typical of many enzymes. The reciprocal of the rate was proportional to the reciprocal of the substrate concentration (in this case the total hormone content of the tissue). By extrapolation to infinite substrate concentration, the maximum rate of deiodination of both hormones

7 THYROXINE DEIODINATION 481 was estimated at about 1.5 /,g/min for a whole liver preparation. The mean weight of the preparations used in these experiments was 16-3 g. Competitive inhibition between the two hormones. A series of experiments were performed to determine whether tri-iodothyronine inhibited the deiodination of thyroxine and vice versa. The standard procedure was adopted and a steady rate of deiodination established at tracer concentrations of hormone. After 20 min a second injection of the other (stable) 4-380C 18 C 38 C 3 0 -oto Fig Time (min) The effect of a reduction in temperature on the rate of deiodination of thyroxine (0) and tri-iodothyronine (0). hormone was given. The effect of tri-iodothyronine on thyroxine deiodination was investigated in five experiments and three of these are illustrated in Fig. 5. A further three experiments were performed with the reverse combination and the results were very similar. Each hormone clearly inhibited the deiodination of the other, suggesting that both were competing for the same enzyme. The effect of PTU on deiodination. The standard procedure was again adopted and a steady rate of deiodination established at tracer concentrations of hormone. After 20 min a known amount of PTU was added to the perfusion fluid in the reservoir and its effect determined. It invariably

8 482 A.. P. HILLER caused an immediate inhibition of deiodination (the results of individual experiments looked very like those shown in Fig. 5). This new rate was measured over a 20 min period and an estimate made of the percentage inhibition caused by the PTU. Both hormones were studied over a wide range of PTU concentrations and all of the results are illustrated in Fig. 6; each point represents a separate preparation. It was found that triiodothyronine deiodination was slightly more sensitive to PTU than 50- E 40 c 30 o0 I / / o 20 O - / / * l Reciprocal of total amount of hormone in liver (,mg)-' Fig. 4. The reciprocal of the rate of deiodination plotted against the reciprocal of the total hormone content of the tissue for thyroxine (0) and tri-iodothyronine (@). Each point represents a separate preparation. thyroxine but high dose levels of the blocking agent were required for both hormones. There was no evidence for two components in the inhibition (see Discussion). DISCUSSION The deiodinating enzyme in the liver probably acts only on the minute proportion of hormone in the tissue that is present in the free (unbound) state. This is indicated by the following observation. During the injection period the proportion of free hormone in the tissue will be abnormally high; and it was observed that there is an abnormally high rate of deiodi-

9 THYROXINE DEIODINATION 483 nation associated with the injection of both hormones into the preparation (Fig. 2). An exactly analogous effect was observed in a study of thyroxine secretion into bile (Hillier, 1972a), where a more detailed experimental analysis and argument are presented. How much of the total deiodination in the rat can be attributed to the liver? It is possible to make a rough estimate by assuming that the deiodination rate measured in vitro is similar to the rate in vivo. The liver of a 1.2 c E Ln c C0 o S >-.c Time (min) Fig. 5. The effect of injections (at the arrow) of stable tri-iodothyronine on thyroxine deiodination in three preparations. The values against each curve indicate the amount of tri-iodothyronine injected. 100 g rat contains some /g thyroxine (Albright, Heninger & Larson, 1965). Assuming a rate of deiodination of 12 %/hr, the total amount of thyroxine deiodinated by the liver in a day is approximately 0-25,g. The rate of thyroxine deiodination in the whole animal is 0-6 /ug/day (Morreale de Escobar & Escobar del Rey, 1967). The liver could therefore be responsible for roughly 40 % of the total deiodination. Not all of the deiodination measured in the whole animal is equally sensitive to PTU. One fraction, about 50 %, is very sensitive, being strongly inhibited by only mg/100 g rat.day. The other fraction is only weakly inhibited by 10 mg/100 g rat. day (Morreale de Escobar & Escobar del Rey, 1967). Hepatic deiodination of both hormones is certainly

10 484 A. P. HILLIER blocked by PTU but only at high concentrations; and there is no evidence for two components in the inhibition. It appears therefore to belong exclusively to the PTU insensitive fraction. In fact, from the foregoing discussion, the liver may be responsible for most, if not all, of this sort of deiodination. The PTU sensitive component, which may be linked to hormonal action, appears to be confined to extra-hepatic tissues. 100 r 90 H H t- 4._ rt v 0 o o 0 c _o 0 r- _c h 30 F 20-10o Dose of propyl thiouracil (mg) Fig. 6. The effect of PTU on the rate of deiodination of thyroxine (0) and tri-iodothyronine ()). Each point represents a separate preparation. This liver enzyme removes the 3'-iodine atom from tri-iodothyronine, probably to generate 3,5-di-iodothyronine, which is inactive (Barker, 1964). The enzyme may also remove the 3- and 5-iodine atoms from the inner ring-a reaction which cannot be detected since these atoms are not labelled. Removal of the 3'-iodine from thyroxine generates 3,5,5'-triiodothyronine which is a thyroxine antagonist (Barker, 1964). The function of this hepatic enzyme therefore seems to be one of inactivation.

11 THYROXINE DEIODINATION 485 REFERENCES ALBRIGHT, E. C., HENIaGER, R. W. & LARSON, F. C. (1965). Effect of cold induced hyperthyroidism on iodine containing compounds in extra-thyroidal tissue. In Thyroid Research, ed. CASSANO, C. & ANDREOL, M. New York: Academic Press. BARKER, S. B. (1964). Physiological activity of thyroid hormones and analogues. In The Thyroid Gland, vol. I. ed. PITr-RIVERS, R. & TRoTTER, W. R. London: Butterworths. GALTON, V. A. (1968). Thyroid hormone metabolism. In Recent Advance in Endocrinology, ed. JAMES, V. H. T. London: Churchill. HTrrIeR, A. P. (1972a). Active secretion of thyroxine into bile: the role of tissue thyroxine-binding sites. J. Physiol. 221, HILLER, A. P. (1972b). Autoregulation of thyroxine secretion into bile. J. Physiol. 221, MORREALE DE ESCOBAR, G. & ESCOBAR DEL REY, F. (1967). Extrathyroidal effects of some anti-thyroid drugs and their metabolic consequences. Recent Prog. Horm. Re8. 23,