Cambridge CB2 3EG. ['25I]L-thyroxine. Experiments were performed after 24 hr had elapsed.

Transcription

1 J. Physiol. (1971), 212, pp With 2 text-ftgurea Printed in Great Britain AN EXAMINATION OF THE EXTENT OF REVERSIBILITY OF THYROXINE BINDING WITHIN THE THYROXINE DISTRIBUTION SPACE IN THE RABBIT By P. W. NATHANIELSZ From the Physiological Laboratory, University of Cambridge, Cambridge CB2 3EG (Received 6 July 197) SUMMARY 1. New Zealand white rabbits were injected intraperitoneally with ['25I]L-thyroxine. Experiments were performed after 24 hr had elapsed. 2. Thyroxine distribution space was calculated as 129 ml. kg-'. Half-life of disappearance of thyroxine from the blood of the urethane anaesthetized rabbit was 653 min. 3. After exchange of approximately 2 ml. kg-' whole blood with heparinized blood from a non-radioactive donor, equilibration of radioactivity in the recipient had produced a peak radioactivity by 2 hr. 4. The thyroxine space in which complete exchange took place within 2 hr was ml. kg-' in six rabbits. This is 79 6 % of the thyroxine distribution space. 5. These results are compared with observations in the rat. INTRODUCTION Recently experimental data have been published by Harland & Orr (197) in the rat using an exchange transfusion technique to investigate the permanence or reversibility of the binding of thyroxine to tissues. These authors conclude that the volume of the exchangeable thyroxine pool is only about 4 ml. kg-' in this species. They state that the "'free T4 ' control concept will require re-examination". These results have a profound bearing on the physiological significance of the protein binding of other hormones as well as thyroxine. For this reason the experiments have been repeated in the rabbit to allow more careful control of certain parameters whose values have to be assumed in work on the rat due in particular to the practical difficulties of sampling the small blood volume. 15-2

2 448 P. W. NATHANIELSZ METHODS New Zealand white rabbits weighing kg were used. All animals were injected intraperitoneally with approx. 2,uc [125I]L-thyroxine, equivalent to -48 /sg thyroxine (Radiochemical Centre, Amersham). The labelled thyroxine was made up in 2 % fresh calf serum (v/v in physiological saline) and equilibrated in a water-bath for 1 min at 45 C before injection. The rabbits were anaesthetized with 5-15 ml. 25 % urethane intravenously 24 hr after the administration of the tracer thyroxine. Throughout the experiment the animals were maintained on heated operating tables. A glass tracheal cannula was inserted into the trachea. Polyethylene catheters were introduced into the right femoral vein and right carotid artery. The experiments were performed on two separate days and on each day two injected animals were prepared as described above and -5 ml. blood samples taken at 3 min intervals for 6 hr to permit calculation of the half-life of disappearance of thyroxine in the urethane anaesthetized rabbit under the conditions of the experiment. Two heparinized venous samples were obtained from the remaining rabbits at 5 min intervals and then blood was withdrawn from the right carotid artery and replaced with freshly drawn heparinized blood from a donor rabbit anaesthetized with urethane. The volume of blood exchanged was measured accurately and usually amounted to about 2 ml. kg-' body weight. The haematocrit of the blood removed and that used to replace it was measured in triplicate in an Adam's Autocrit centrifuge. Further heparinized venous samples were taken for measurement of [125J]- L-thyroxine radioactivity at 3 min intervals for 3 hr after the exchange transfusion. Total [125I]radioactivity in -2 ml. plasma was measured in a Nuclear Chicago Autogamma 4222 well-type scintillation counter to a counting accuracy of 1 %, and the sample was then recounted after precipitation with 5 ml. 2% trichloroacetic acid (TCA) with removal of the supernatant after centrifugation. TCA precipitable radioactivity was taken as [1251]L-thyroxine radioactivity. Calculations. The half-life for disappearance of [251I]L-thyroxine from the blood was calculated by graphically plotting the radioactivity per -2 ml. in the four rabbits in which no exchange transfusion was performed. The regression line was calculated for the disappearance of radioactivity (Snedecor, 195) and the theoretical initial concentration ml. of radioactivity obtained (C). A 1 % solution of the injected solution was counted to obtain the total counts injected (T). The Thyroxine Distribution Space (T.D.S.) was calculated according to Ingbar & Freinkel (1955). T.D.S. = -C. RESULTS Disappearance of [125I]L-Thyroxine radioactivity from the plasma. Fig. 1 illustrates the disappearance of [1251]L-thyroxine radioactivity in one of the four rabbits which did not undergo exchange transfusion. The half-lives for disappearance of radioactivity from the blood in these four animals were 629, 615, 538, 829 min (mean 653 min). Radioactivity in the blood after exchange transfusion. In all six of the exchanged animals the radioactivity was declining by 2 hr after the exchange transfusion and the 2 hr plasma radioactivity was used to calculate the

3 THYROXINE BINDING IN THE RABBIT 449 x. 6 X E 6 E (C4 o 4 C ~~~~~ o N N C o X~~~~~~~ Time (min) Fig. 1. Disappearance of [1251]L-thyroxine radioactivity from femoral vein blood in the urethane-anaesthetized rabbit. xi - r 6, E o N M~~ N E 5 N IN B - 4_ N~ ~ ~ ~ ~ Z3 N N C -C Time (min) Fig. 2. Effect of exchange transfusion in rabbit 3. A, disappearance of radioactivity with no exchange transfusion, calculated on the basis of a half-life of 653 min. B, radioactivity after transfusion.

4 45 P. W. NATHANIELSZ effect of the exchange transfusion. At 2 hr after the resting samples only 88 % of the initial radioactivity would be present in the blood had no exchange transfusion been made (see Fig. 2). This point is of fundamental significance and follows from the half-life of 653 min in the non-exchanged animals. Failure to allow for this and any differences in the haematocrits of the blood exchanged will introduce marked errors. Table 1 shows the results obtained and the difference in the calculated volume of the exchangeable thyroxine pool if the natural disappearance of [1251]L-thyroxine is not allowed for. If the exchangeable thyroxine pool = x ml. Plasma volume removed = a ml. Plasma volume replaced = b ml. Initial counts [125I]thyroxine = c x2 ml. plasma-' 2 min-'. 88 % initial counts [125I]thyroxine = d -2 ml. plasma-' 2 min-'. 12 min counts [1251]thyroxine = f *2 ml. plasma-' 2 min-. Then column I shows the results calculated from the formula Cx = (x-a+b)f+ac, i.e. not allowing for the continuing use of thyroxine throughout the experimental period, and column II shows the results calculated from the formula dx = (x-a+b)f+ad, making the necessary correction to column I. Mean values + S.E. of mean are 55* ml. plasma kg-' from formula I and from formula II. Total thyroxine distribution space calculated in four rabbits without exchange transfusion. This was found to be 137 ml. kg-', 11 ml. kg-', 129 ml. kg-', 141 ml. kg-' in the four animals (mean 129 ml. kg-'). DISCUSSION Much experimental evidence exists to suggest that the level of free thyroxine is an important parameter in regulating the level of activity in the tissues. As a corollary it is held that the level of free thyroxine is determined by equilibration with the various binding proteins in the blood. Thus pharmacological agents which affect the binding of thyroxine affect thyroid function (Hetzel, Good, Welby & Charnock, 196); similarly, physiological alteration of the level of binding proteins can affect the disappearance of thyroxine from the blood (Tata & Shellabarger, 1959). In certain pathological and surgical states alterations in thyroxine binding also occur (Oppenheimer & Bernstein, 1965). In vitro experiments with

5 ,C qc X THYROXINE BINDING IN THE RABBIT 451 C1 N1- tn C tn X cq1 C1-O 5= C= C) C) eo C; X x C.- C) Q ci Ns CO_.i cc _ :O _ 1 CO CO 1 C. rci.u> W._ X X Lt X Nt $~ C; -~ Nrn Om NcC O "-i1 m =

6 452 P. W. NATHANIELSZ organ systems also suggest that by changing the level of binding proteinor its affinity-equilibration of thyroxine with tissues can be altered (Hillier, 1968). The classical concept has been challenged in the rat using the technique employed in the experiments reported here on the rabbit. It is possible that the difference is due to species variation. The results reported here show the volume of the rapidly exchangeable thyroxine pool to be 43 % of the total thyroxine distribution space calculated on the basis of the injected [1251]L-thyroxine from formula I. Harland & Orr (197) obtain 15-9 % using the same formula in which no allowance is made for the disappearance of [125I]L-thyroxine during the experiment. The discrepancy between these two figures requires investigation. In calculating the T.D.S. from the total injected thyroxine the disappearance rate over the 24 hr of equilibration is taken by Harland & Orr as 1-2 days. This is the value they observe for the whole body distribution as measured in the conscious rat (Harland & Orr, 1969). Calculation of the plasma halflife from the same correspondence (Harland & Orr, 1969) gives a value of about 65 days. At this plasma disappearance rate the plasma radioactivity will be 36*8 % of the theoretical initial radioactivity at the time of injection. If calculated on the basis of whole body disappearance, the value is 56 %. The concept of thyroxine distribution space is based on the plasma concentration. The T.D.S. is defined as the volume required to hold the total body thyroxine at the concentration present in the plasma. Calculating T.D.S. from the formula 56 % of total injected [125I]L-thyroxine 24 hr concentration, rather than 36 8 % of total injected [125I]L-thyroxine 24 hr concentration, will produce a value for T.D.S. which is 152 % of that calculated by the usual method of extrapolating the plasma radioactivity. Harland & Orr (197) obtain a value of 73-1 ml. (261 ml. kg-') for the T.D.S. in rats weighing 26-3 g. If the T.D.S. is calculated from their observed blood disappearance rather than whole body disappearance it is 171*7 ml. kg-'. This value is nearer the usual figure for man and other mammals. Ingbar & Freinkel (1955) give the value for man as approx. 15 % body weight-15 ml. kg-'. This also suggests that the calculated figure of 261 ml. kg-' in the rat is high for the reasons given above. Irvine (1967) observed a T.D.S. of ml. kg-' in the horse. In the opinion of this author the difference in whole body and blood disappearance curves depends on the different time courses of loss of radioactivity from the blood and from the faeces. Differences in the two curves also reflect the fact that measurement of blood radioactivity can differentiate between thyroxine and iodide whereas whole body counting cannot.

7 THYROXINE BINDING IN THE RABBIT 453 The use of exchange transfusion to observe the return of thyroxine to the blood is theoretically sound. However, the time course of the reequilibration must be carefully followed. By 2 hr in the rabbit re-equilibration has occurred and hence is likely to have done so in the rat, a species with a faster metabolic rate. It is important to ascertain this point since if re-equilibration has not occurred then a low value will be obtained for the exchangeable pool. Minor differences will also exist due to differing time courses of re-equilibration in all compartments of the T.D.S. In calculating the percentage of radioactive thyroxine present after reequilibration has occurred following the exchange transfusion, allowance must be made for the utilization of thyroxine which would have occurred during that interval even in the absence of the exchange transfusion (see Fig. 2). Several factors in the experimental situation may in fact increase this utilization during the exchange transfusion when compared with the conscious animal. Conflicting evidence exists as to the effect of different types of stress on the pituitary-thyroid axis (Brown-Grant, Harris & Reichlin, 1954; Irvine, 1967) but steroids do increase the rate of deiodination of thyroxine in the rat (Nathanielsz, 1968). Thus the marked haemorrhage, although rapidly rectified, may shorten the half-life for thyroxine in the blood even further. In one conscious rabbit with an indwelling venous catheter the half-life for thyroxine in the blood was 675 min and 891 min on two separate occasions. This is considerably less than observed by Brown-Grant & Tata (1961) who found a value of 27 hr between 25 and 96 hr after the injection. If the half-life for thyroxine in the blood in the rat is taken as -65 days (Harland & Orr, 1969) then a correction for the % initial value 2 hr after exchange transfusion in the rat can be made since only 9 % of the blood radioactivity would remain if no exchange transfusion had taken place. Harland & Orr (197) observe a mean value of -81 for the fraction of original radioactivity remaining after 2 hr. Using this mean value against an expected 9 % of the original radioactivity remaining the exchangeable volume would become 21- ml. or 75 ml. kg-' for rats 26-3 g. If, as is likely under anaesthetic, the half-life for thyroxine is even less, the exchangeable volume would be greater still. To conclude, how exchangeable is thyroxine in the extravascular compartment? In the rabbit the exchangeable volume is 12-7 ml. kg-' and on the basis of the recalculation of the data from Harland & Orr (197) in the rat approximately 75 ml. kg-'. These figures therefore are more nearly comparable although no accurate measurements of plasma volumes actually exchanged in the rat were made and changes may occur in both donor and recipient when about 5 % of the blood volume is removed. The proportion of the thyroxine pool which is exchangeable

8 454 P. W. NATHANIELSZ within 2 hr in the rabbit is only 79-6 % of the T.D.S. calculated by the classical method. This does suggest that some of the radioactive thyroxine (about 2-4 %) may exist at sites where equilibration does not take place rapidly. This may occur in poorly perfused organs, in the lymphatic system or fluid spaces such as the cerebrospinal fluid space. The absence of rapid equilibration at these sites does not lessen the physiological importance of the existence of rapid equilibration in the other 79-6 % of the T.D.S. REFERENCES BROWN-GRANT, K., HARRIS, G. W. & REICHLIN, S. (1954). The effect of emotional and physical stress on thyroid activity in the rabbit. J. Physiol. 126, BROWN-GRANT, K. & TATA, J. R. (1961). The distribution and metabolism of thyroxine and 3:5:3'-triiodothyronine in the rabbit. J. Physiol. 157, HARLAND, W. A. & ORR, J. S. (1969). Thyroxine in blood and tissues. Lancet i, 121. HARLAND, W. A. & ORR, J. S. (197). The permanence of tissue binding of thyroxine in the rat. J. Phy8iol. 27, HETZEL, B. S., GOOD, B. F., WELBY, M. L. & CHARNOcK, J. S. (196). Salicylateinduced fall in plasma protein-bound iodine in hyperthyroidism. Lancet i, 957. HILLIER, A. P. (1968). The effect of serum on the uptake of thyroid hormones by the perfused rat heart. J. Physiol. 199, INGBAR, S. H. & FREINKEL, N. (1955). Simultaneous estimation of rates of thyroxine degradation and thyroid hormone synthesis. J. clin. Invest. 34, IRVINE, C. H. G. (1967). Thyroxine secretion rate in the horse in various physiological states. J. Endocr. 39, NATHANIELSZ, P. W. (1968). The effect of acute starvation for 48 hr on deiodination of a tracer dose of [131I]thyroxine in the rat compared with the effect of hydrocortisone. J. Physiol. 194, 8-S81 P. OPPENHEIMER, J. H. & BERNSTEIN, G. (1965). The metabolism and significance of thyroxine-binding prealbumin in man. In Current Topics in Thyroid Research, pp , ed. C. CASSANO & M. ANDREOLI. Oxford: Academic Press. SNEDECOR, G. W. (195). Statistical Methods. Ames, Iowa: Iowa State College Press. TATA, J. R. & SHELLABARGER, C. J. (1959). An explanation for the difference between the responses of mammals and birds to thyroxine and tri-iodothyronine. Biochem. J. 72,