Clinical Endocrinology (1976) 5, 587-594.
INHIBITION OF CONVERSION O F THYROXINE TO T R I I O D O T H Y R O N I N E I N PATIENTS WITH SEVERE C H R O N I C ILLNESS J. N. C A R T E R , C. J. E A S T M A N , J. M. CORCORAN A N D L. L A Z A R U S Garvan Institute of Medical Research, St Vincent's Hospital, Sydney, Australia (Receiced 17 February 1976; revised 2 June 1976; accepted 4 June 1976)
SUMMARY
Many clinically euthyroid patients with severe, chronic, non-thyroidal illnesses (i.e. sick euthyroid patients) have very low circulating concentrations of total and absolute free triiodothyronine (T3), low-normal concentrations of total thyroxine (T4), elevated concentrations of absolute free T4, and circulating concentrations of thyrotrophin (TSH) that are either normal or subnormal. This study was undertaken to elucidate the mechanism of the low circulating T3 concentrations. The disappearance rate of 1251-T3from the circulation of five representative sick euthyroid patients was studied and found to be slower, but not significantly SO, compared with three control subjects, thus excluding an increased destruction rate as the cause of the low T3 levels. A selective decrease of T3 secretion from the thyroid gland of these patients was also excluded by the results of TSH stimulation tests. Inhibition of extra-thyroidal conversion of T4 to T3 was suggested by studies of the thyroid function in a hypothyroid woman with a Grade IV lymphoma on T4 replacement therapy. When the lymphoma was in remission, her circulating T3 concentration was 2-55 nmol/l but when it relapsed it fell to 0.55 nmol/l. The T4 concentrations were 124.7 nmol/l and 126 nmol/l respectively. Decreased monodeiodination of T4 to T3 in sick euthyroid patients was confirmed by paper chromatography of extracted serum obtained 48 h after an i.v. injection of lfSI-T4 into two severely ill patients from the intensive therapy unit and a control subject. Peaks of radioactivity corresponding to 12%T4 and "'I-T3 were detected in the control subject, but only a single peak corresponding to 12%T4 was detected in the ill patients. INTRODUCTION It has recently been established that many clinically euthyroid patients with severe, chronic, non-thyroidal illnesses (i.e. sick euthyroid patients) have very low circulating concentrations Correspondence: Dr C. J. Eastman, Endocrine Unit, Woden Valley Hospital, Canberra, A.C.T., Australia 2606. B
587
588
J . N. Carter et al.
of total and absolute free triiodothyronine (T3), low-normal concentrations of total thyroxine (T4), elevated concentrations of absolute free T4, and normal or subnormal circulating concentrations of thyrotrophin (TSH) (Carter et al., 1974; Oppenheimer et al., 1963). These alterations of thyroid function are not related to a specific disease process as they &cur in a wide variety of illnesses, such as severe chronic liver disease, disseminated malignancy, severe chronic obstructive airways disease, chronic renal failure and in patients with miscellaneous illnesses requiring intensive care (Carter et al., 1974). The predominant source of circulating T3 in man is peripheral conversion from T4 (Brown et al., 1974). Since the degree of conversion varies directly with the circulating T4 concentration (at least in athyreotic patients on replacement T4 therapy) (Eastman et al., 1975), and since sick euthyroid patients have a relatively greater reduction of total T3 than total T4 concentrations, it has been postulated that a defect in monodeiodination of T4 to T3 is the major cause of the low T3 concentrations in these patients (Carter et al., 1974; Chopra et al., 1974). The present study was undertaken to clarify the mechanism of the depressed circulating T3 concentrations in sick euthyroid patients. PATIENTS A N D METHODS Studies of the peripheral metabolism of '"1 labelled T3 were performed in five sick euthyroid patients and three euthyroid control subjects. Of the former, two had decompensated chronic liver disease, one severe chronic obstructive airways disease, one disseminated malignancy and one chronic renal failure. Informed consent was obtained from all the conscious patients and from the responsible medical officers when the patients were comatose. Each individual was given an i.v. injection of 30 pCi (approximately 55 ng T3) of I2'I-T3 (specific activity 416 pCi/pg; Abbott Laboratories) in 2 ml of a polygeline solution ('Haemaccel', Bekringwerke AG). The solution was sterilized via a Millipore Filter (pore size 0.22 pm) and the 'Haemaccel' solution was used to decrease adherence of the labelled hormone to the syringe and filter (Kraegan et al., 1975). Blood samples were taken l-), 24 and 48 h after the administration of the tracer. The serum from these blood samples was frozen and stored at -20°C until analysed 24 h after the last blood sample was drawn. The amount of labelled T3 present in each aliquot of serum was determined by precipitating the T3 with an excess of specific T3 antiserum in the presence of B-anilino-l-naphthalenesulphonicacid and barbitone buffer to inhibit binding of T3 to endogenous thyroid hormone binding proteins. The antibody bound and free fractions were separated with charcoal. TSH stimulation tests, using 10 u of bovine TSH ('Thytropar') i.m., were performed in three sick euthyroid patients (two with decompensated chronic liver disease and one from the intensive therapy unit) and in three control subjects. Basal samples of sera were obtained for T3 and T4 estimations and further samples were drawn 24 h after the administration of the TSH. The thyroid function of a 53-year-old housewife suffering from Stage IV lymphoma and primary hypothyroidism was studied whilst the lymphoma was in remission and again 3 months later when she was critically ill following relapse. On each occasion she was clinically euthyroid and her only medication was 0.2 mg T4 daily. Assessment of the degree of peripheral conversion of T4 to T3 was made by determining the amount of "'I-T3 in the serum 48 h after an i.v. injection of '"I-T4. 50 pCi (approximately 40 ng T4) of 12'I-T4 (specific activity 1300 pCi/pg; Abbott Laboratories) in 2 ml
Inhibition of conversion of thyroxine to triiodothyronine
589
'Haemaccel' solution was injected i.v. via a Millipore Filter into two severely ill patients in the intensive therapy unit and into one control subject convalescing in the orthopaedic ward. One of the patients was suffering from bronchopneumonia following perforation of a duodenal ulcer 7 days earlier and the other was comatose following the removal of a cerebral haemangioma 10 days earlier. Blood was drawn 48 h later and 1 ml of the resulting serum was extracted with 3 ml of 95% ethanol. The supernatant was subsequently dried under nitrogen and reconstituted with 1 ml of methanol-ammonia solution (99 volumes of methanol to 1 volume of 2 M ammonium hydroxide). Descending paper chromatography with Whatman 3MM chromatography paper was then performed for 20 h according to the method of Sterling (Rall et al., 1972). Samples of serum to which both '251-T3and 1251-T4 had been added were treated identically to the test samples and chromatographed in the same chromatography chamber. After the paper strips were dried, they were cut into 1 cm wide sections and the radioactivity in each counted in an auto gamma counter for 1 h. From previous chromatography studies it had been shown that there was less than 5% '"I-T3 contamination of the lz5I-T4 solution used. To determine how quickly serum total T3 concentrations fall in patients with severe illnesses, six patients from the coronary care unit who were suffering from proven myocardial infarction of less than 24 h duration were studied. Samples of serum were obtained at 24 h intervals over the ensuing 48-72 h and subsequently assayed for T3 and T4. Serum T3 (Eastman et al., 1975) and T4 (Corcoran et al., 1973) concentrations were measured by specificradioimmunoassays.Statistical analyses were performed using Student's t test. RESULTS Disappearance rates of _"'I-T3 The percentages of precipitable IZ5I-T3in the sera of the five sick euthyroid patients and the three control subjects are shown in Table 1. The mean percentages of '251-T3 present in the circulation 24-48 h after the i.v. injection of the labelled hormone were higher in the ill patients than in the controls. Probably due to the small number of patients involved in the study, these differenceswere not statistically significant. TSH stimulation tests The results of the TSH stimulation tests are tabulated in Table 2. The mean +SEM increases in serum T3 and T4 concentrations following TSH in the sick euthyroid patients were 1-32+0.43 nmol/l and 105-4&33.4nmol/l respectively, and in the control subjects were 1.2*0-18 nmol/l and 50.1+_ 11.6 nmol/l respectively. These differences were not statistically significant (P>O-05). Studies in the hypothyroid patient with lymphoma When the lymphoma was in remission, the circulating T3 concentration was 2-55&0-11 (2 SD)nmol/l and the T4 concentration was 124.7 +23-1 nmol/l. Three months later, following relapse of the lymphoma, the T3 concentration had fallen to 0.55f0.11 nmol/l whilst the T4 concentration was essentially unchanged at 126+ 10-3nmol/l. Extra-thyroidal conversion of T4 to T3 Paper chromatography of extracted serum drawn 48 h after an i.v. injection of '251-T4
J . N . Carter et al.
590
TABLE1. Disappearance rates of '2sI-labelled triiodothyronine Serum T3 f 2 SD (nmol/l)
Group Sick euthyroid 1 2 3 4 5
Mean? SEM Control 1
2 3 Mean f SEM
Percentage of *251-T3in serum after 1.5 h
24 h
48 h
028 f 0-14 0.98 f 0 1 1 0-46f0.12 074 f0 0 9 083k018 0.66f 0.11
100 100 100 100 100
31 29 31
19 19
1.63f 0 15 2-14f0I7 1.38f 0 I2 1.72f 0 1 8
100 100 100
52
64 4126' 22 28 29 26f2*
23 44 49 31 f6**
I5 15
17 1 6 t I**
P>0.05;** P>0.05. TABLE 2. Thyrotrophin stimulation tests
Serum T3 (nmol/l)
Serum T4 (nmol/l)
Group Basal
Post TSH
Basal
Post TSH
1-26 2.61
Mean fSEM
0.66 028 1a9 0-68 f 0 18
2.0f0.32
91.3 61.7 I324 95.1 f 16.7
120.8 23 1.4 248 200f 33.4
Control 1 2 3 Mean fSEM
1.92 3-13 2.29 2.45 f0.29
2-92 4-79 3-24 3.65 k 0-48
79.7 122.1 111.8 104.1f 10.3
119-5 200.5 145.2 155.5f 193
Sick euthyroid 1
2 3
210
was performed in two severely ill patients from the intensive therapy unit and in one control subject. It had been determined from chromatography performed both previously and simultaneously with the above study that the radioactivity peak corresponding to T4 occurred 2-3 cm from the origin whilst the peak for T3 occurred 8-9 cm from the origin. Extracts of serum to which labelled T3 and T4,individually and in combination, had been added were used for these studies. A discrete peak corresponding to T4 with no peak in the T3 region was found in each of the ill patients whilst discrete peaks corresponding to both T4 and T3 were found in the control subject. A superimposed graph of the chromatography results in the control subject, and one of the ill patients is shown in Fig. 1.
59 1
Inhibition of conversion of thyroxine to triiodothyronine
Distance from the origin (an)
FIG.1. Paper chromatography of serum obtained 48 h after intravenous injection of
I2'I-
thyroxine into a normal euthyroid adult control patient (unbroken line) and a sick euthyroid patient (broken line). The initial peak represents T4 12'1 and the second peak T3 '"I. 3-Or
2.5
-
2.0
-
1.5
-
1.0
-
-
T
5
c
" 1
0
1
2
3
Oays posl myocardial infaction
FIG.2. Decline in serum total T3 levels in six patients followed for 3 days after admission to hospital with myocardial infarction.
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J. N . Carter et al.
Serum T3 concentrations in patients with acute myocardial infarction
Six patients with acute myocardial infarction were studied serially during the 48-72 h following admission to the coronary care unit. The serum T3 concentrations during this period are graphed in Fig. 2. It can be seen that there is a marked fall in the T3 levels within the first 24-48 h of admission. The mean lowest value for the six patients was 1.57 nmol/l and this was significantly lower than the mean baseline value of 2.44 nmol/l (P>0-005). The mean serum T4 levels did not change over the 72 h period. DISCUSSION There are only three possible explanations for the markedly depressed serum T3 concentrations associated with the mildly decreased serum T4 concentrations present in sick euthyroid patients: (i) increased T3 destruction rate whilst the production rate is not increased; (ii) a selective decrease of T3 secretion from the thyroid gland; (iii) decreased extra-thyroidal conversion of T4 to T3. The differences between the mean values of each group were not statistically significant, however, and the results were not compatible with the hypothesis that an increased disappearance rate of T3 accounts for, or contributes to, the markedly reduced serum T3 concentrations in these patients. In these studies we used single compartment kinetics, rather than subject our sick patients to more complex and demanding kinetic studies, which do not allow us to draw firm conclusions from the data concerning metabolic clearance rates of T3. The latter is dependent upon distribution volume which may be altered when there is a disturbance in thyroid function (Cavalieri et al., 1970, 1971). The second possibility of a selective decrease of T3 secretion from the thyroid gland was tested by performing TSH stimulation tests in three severely ill patients and three control subjects. It was found that the mean maximal increases of both T3 and T4 24 h after TSH were not significantly different, suggesting that there was not a selective decrease of T3 secretion from the thyroid glands of sick euthyroid patients. Consequently, the only possible explanation for the greatly reduced serum T3 concentrations in sick euthyroid patients is a decrease in the peripheral conversion of T4 to T3. This conclusion was supported by the studies performed in the patient with lymphoma and primary hypothyroidism in whom the circulating T3 concentration fell from 2.55 nmol/l when the lymphoma was in remission to 0-55 nmol/l when it relapsed. This marked fall could not be attributed to malabsorption of the ingested T4 since her serum total T4 concentration remained essentially unchanged over this period. As the only source of T3 in this patient was monodeiodination of T4, and as there is not an increased destruction rate of T3 in severely ill patients, the only explanation compatible with the data is a decrease in the conversion of T4 to T3. This defect in monodeiodination was unequivocally confirmed by the paper chromatography studies. As shown in Fig. 1, there was a peak of radioactivity corresponding to T3 in the control subject but there was no such peak in the sick euthyroid patients. A radioactivity peak corresponding to the T4, however, was present in each chromatograph. It is apparent from the study of the patients with acute myocardial infarction that the serum T3 concentrations are significantly reduced within 48 h of the onset of the severe illness (see Fig. 2). Because the half life of circulating T3 in healthy euthyroid subjects is about 24 h (Nicoloff et al., 1972) (as intimated above, it may be longer in sick euthyroid
Inhibition of conversion of thyroxine to triiodothyronine
593
patients), the onset of the reduction in T4 to T3 conversion must occur close to the onset of the illness. The circulating concentrations of reverse T3 (rT3 ; 3,3’,5’-triiodothyronine)have recently been found to be elevated in the fetus (Chopra, 1974). Thyroid function in the fetal and sick euthyroid states are virtually identical (Eastman et al., 1973; Abuid et al., 1973; Erenberg et al., 1974), and sick euthyroid patients also have increased circulating concentrations of rT3 compared with control subjects (Chopra et al., 1975). This raises the possibility that in the sick euthyroid state, T4 may preferentially be monodeiodinated to rT3 whereas under normal conditions it is preferentially converted to T3. As rT3 has little known biological activity (Pittman & Barker, 1959) the possible significance of elevated circulating concentrations of rT3 in sick euthyroid patients is not clear. However, evidence from animal experimentation suggests that the role of rT3 may be to decrease TSH production by interfering with feedback control via occupation of pituitary binding sites (Money et al., 1960; Stasilli et al., 1959) and to inhibit the metabolic action of T3 and/or T4 by occupation of peripheral binding sites (Pittman et at., 1964), leading to an overall decrease in calorigenesis and metabolic rate. This hypotheses requires experimental confirmation in the human. ACKNOWLEDGMENTS
This work was supported by a grant from the National Health and Medical Research Council of Australia. We thank Ms C. Batley and Ms T. Enright and Associate Professor O’Rourke for permission to study patients in the Coronary Care Unit. REFERENCES P.R. (1973) Serum triiodothyronine and thyroxine in the neonate and ABUID,J., STINSON,D.A. & LARSEN. the acute increases in these hormones following delivery. Journal of Clinical Investigation, 52,1195-1 199. BROWN,J., CHOPRA, I.J., CORNELL, J.S., HERSHMAN, J.M., SOLOMON, D.H., ULLER, R.P. & VANHERLE, A.J. (1974) Comparisons and alterations in circulating thyroglobulin, triiodothyronine and thyroxine in response to exogenous (bovine) and endogenous (human) thyrotropin. A n d s of Internal Mcdicine. 81,68-81.
CARTER, J.N.. EASTMAN, C.J., CORCORAN, J.M. & LAZARUS, L. (1974) Effect of severe chronic illness on thyroid function. Loncet, ii, 971-974. CAVALIERI, R.R., STEINBERG, M. & SWRLE,G.L. (1970) The distribution kinetics of triiodothyronine: studies of euthyroid subjects with decreased plasma thyroxine-binding globulin and patients with Grave’s disease. Journal of Clinical Imestigafian, 49, 1041-1050. CAVAUERI, R.R., STEINBERG, M. & S m u , G.L. (1971) Metabolic clearance rate of L-triiodothyronine in man: a comparison of results by single injection and constant infusion methods. Joiirnbl of Clinical Endocrinology and Metabolism, 33,624-629. CHOPRA, I.J. (1974) A radioimmunoassay for measurement of 3,3‘,5-triiodothyronine (reverse T3). Journal of Clinical Investigation, 54, 583-592. CHOPRA,I.J., SOLOMON, D.H., CHOPRA,U., YOUNG,R.T. & CHUATECO, G.N. (1974) Alterations in circulating thyroid hormones and thyrotropin in hepatic cirrhosis: evidence for euthyroidism despite subnormal serum triiodothyronine. Journal of Clinical Endocrinology and Metabolism, 39, 501-51 1. CHOPRA.I.J., CHOPRA, U..S m , S.R., REZA,M. & SOLOMON, D.H. (1975) Reciprocal changes in serum concentrations of 3,3’,5’-triodothyronine (reverse T3) and 3,3’5-triiodothyronine (T3)-systemic illness. Journal of Clinical Endocrinology. 41, 1043-1049. CORCORAN, J.M.,EASTMAN, C.J., EKINS,R.P. & PAUL, W. (1973) The production of antixra for the radioimmunoassay of thyroxine. Journal of Endocrinology, 58, xxii.
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EASTMAN. C.J., CORCORAN, J.M.. JEQUIER, A., EKINS,R.P. & WILLIAMS,E.S. (1973) Triiodothyronine concentration in cord and maternal sera a t term. Clinical Science and Molecular Medicine, 45, 251-255. EASTMAN, C.J., CORCORAN, J.M., EKINS,R.P., WILLIAMS, E.S. & NABARRO, J.D.N. (1975)The radioimmunoassay of triiodothyronine and its clinical application. Journal of Clinical Pathology, 28,225-230. ERENBERG, A., PHmm, D.L..LAM,R. & FISHER, D.A. (1974)Total and free thyroid hormone concentrations in the neonatal period. Pediatrics, 53,211-216. KRAEGAN. E.W., LAZARUS, L., MELLER, H..CAMPBELL, L. & CHIA,Y.O. (1975)Carrier solutions for lowlevel intravenous insulin infusion. British Medical Journal, iii, 464466. MONEY, W.L., KUMAOKA, S., RAWSON,R.W. & KROC,R.L. (1960) Part IV. Metabolic effects of thyroid hormones and their analogues. Comparative effects of thyroid hormones and their analogues in experimental animals. Annals of New York Academy of Sciences, 86,512-544. NICOLOFF. J.T., Low, J.C., DUSSAULT, J.H. &FISHER, D.A. (1972)Simultaneous measurements of thyroxine and triiodothyronine turnover kinetics in man. Journal of Clinical Inuestigation, 51,473-483. OPPENHEIMER, J.H., ~ U E F R.,, SURKS, M.J. & HAUER,H. (1963)Binding of thyroxine by serum proteins evaluated by equilibrium dialysis and electrophoretic techniques. Alterations in non-thyroidal illness. Journal of Clinical Inuestigation, 42, 1769-1782. PITTMAN, C.S. & BARKER, S.B. (1959) Inhibition of thyroxine action by 3,3',5'-triiodothyronine. Endocrinology, 64,466468. PITTMAN, C.S.. SHINOHARA. M., THRASHER, H.& MCCRAW.E.F. (1964)Effect of thyroxine analogues on the peripheral metabolism of thyroxine: the half life and pattern of elimination. Endocrinology. 74.61 1. RALL,J.R., STERLING, K., GHARIB, H.& MAYBERRY, W.E. (1972)Thyroid hormone analysis. Methods in Investigative and Diagnostic Endocrinology (General Editor S . Berson), Part I: The Thyroid and Biogenic Amines, pp. 204-240. STASILW, N.R., KROC,R.L. & MELTZER, R.I. (1959)Antigoitrogenic and calorigenic activities of thyroxine analogues in rats. Endocrinology, 64, 62-82.
INHIBITION OF CONVERSION O F THYROXINE TO T R I I O D O T H Y R O N I N E I N PATIENTS WITH SEVERE C H R O N I C ILLNESS J. N. C A R T E R , C. J. E A S T M A N , J. M. CORCORAN A N D L. L A Z A R U S Garvan Institute of Medical Research, St Vincent's Hospital, Sydney, Australia (Receiced 17 February 1976; revised 2 June 1976; accepted 4 June 1976)
SUMMARY
Many clinically euthyroid patients with severe, chronic, non-thyroidal illnesses (i.e. sick euthyroid patients) have very low circulating concentrations of total and absolute free triiodothyronine (T3), low-normal concentrations of total thyroxine (T4), elevated concentrations of absolute free T4, and circulating concentrations of thyrotrophin (TSH) that are either normal or subnormal. This study was undertaken to elucidate the mechanism of the low circulating T3 concentrations. The disappearance rate of 1251-T3from the circulation of five representative sick euthyroid patients was studied and found to be slower, but not significantly SO, compared with three control subjects, thus excluding an increased destruction rate as the cause of the low T3 levels. A selective decrease of T3 secretion from the thyroid gland of these patients was also excluded by the results of TSH stimulation tests. Inhibition of extra-thyroidal conversion of T4 to T3 was suggested by studies of the thyroid function in a hypothyroid woman with a Grade IV lymphoma on T4 replacement therapy. When the lymphoma was in remission, her circulating T3 concentration was 2-55 nmol/l but when it relapsed it fell to 0.55 nmol/l. The T4 concentrations were 124.7 nmol/l and 126 nmol/l respectively. Decreased monodeiodination of T4 to T3 in sick euthyroid patients was confirmed by paper chromatography of extracted serum obtained 48 h after an i.v. injection of lfSI-T4 into two severely ill patients from the intensive therapy unit and a control subject. Peaks of radioactivity corresponding to 12%T4 and "'I-T3 were detected in the control subject, but only a single peak corresponding to 12%T4 was detected in the ill patients. INTRODUCTION It has recently been established that many clinically euthyroid patients with severe, chronic, non-thyroidal illnesses (i.e. sick euthyroid patients) have very low circulating concentrations Correspondence: Dr C. J. Eastman, Endocrine Unit, Woden Valley Hospital, Canberra, A.C.T., Australia 2606. B
587
588
J . N. Carter et al.
of total and absolute free triiodothyronine (T3), low-normal concentrations of total thyroxine (T4), elevated concentrations of absolute free T4, and normal or subnormal circulating concentrations of thyrotrophin (TSH) (Carter et al., 1974; Oppenheimer et al., 1963). These alterations of thyroid function are not related to a specific disease process as they &cur in a wide variety of illnesses, such as severe chronic liver disease, disseminated malignancy, severe chronic obstructive airways disease, chronic renal failure and in patients with miscellaneous illnesses requiring intensive care (Carter et al., 1974). The predominant source of circulating T3 in man is peripheral conversion from T4 (Brown et al., 1974). Since the degree of conversion varies directly with the circulating T4 concentration (at least in athyreotic patients on replacement T4 therapy) (Eastman et al., 1975), and since sick euthyroid patients have a relatively greater reduction of total T3 than total T4 concentrations, it has been postulated that a defect in monodeiodination of T4 to T3 is the major cause of the low T3 concentrations in these patients (Carter et al., 1974; Chopra et al., 1974). The present study was undertaken to clarify the mechanism of the depressed circulating T3 concentrations in sick euthyroid patients. PATIENTS A N D METHODS Studies of the peripheral metabolism of '"1 labelled T3 were performed in five sick euthyroid patients and three euthyroid control subjects. Of the former, two had decompensated chronic liver disease, one severe chronic obstructive airways disease, one disseminated malignancy and one chronic renal failure. Informed consent was obtained from all the conscious patients and from the responsible medical officers when the patients were comatose. Each individual was given an i.v. injection of 30 pCi (approximately 55 ng T3) of I2'I-T3 (specific activity 416 pCi/pg; Abbott Laboratories) in 2 ml of a polygeline solution ('Haemaccel', Bekringwerke AG). The solution was sterilized via a Millipore Filter (pore size 0.22 pm) and the 'Haemaccel' solution was used to decrease adherence of the labelled hormone to the syringe and filter (Kraegan et al., 1975). Blood samples were taken l-), 24 and 48 h after the administration of the tracer. The serum from these blood samples was frozen and stored at -20°C until analysed 24 h after the last blood sample was drawn. The amount of labelled T3 present in each aliquot of serum was determined by precipitating the T3 with an excess of specific T3 antiserum in the presence of B-anilino-l-naphthalenesulphonicacid and barbitone buffer to inhibit binding of T3 to endogenous thyroid hormone binding proteins. The antibody bound and free fractions were separated with charcoal. TSH stimulation tests, using 10 u of bovine TSH ('Thytropar') i.m., were performed in three sick euthyroid patients (two with decompensated chronic liver disease and one from the intensive therapy unit) and in three control subjects. Basal samples of sera were obtained for T3 and T4 estimations and further samples were drawn 24 h after the administration of the TSH. The thyroid function of a 53-year-old housewife suffering from Stage IV lymphoma and primary hypothyroidism was studied whilst the lymphoma was in remission and again 3 months later when she was critically ill following relapse. On each occasion she was clinically euthyroid and her only medication was 0.2 mg T4 daily. Assessment of the degree of peripheral conversion of T4 to T3 was made by determining the amount of "'I-T3 in the serum 48 h after an i.v. injection of '"I-T4. 50 pCi (approximately 40 ng T4) of 12'I-T4 (specific activity 1300 pCi/pg; Abbott Laboratories) in 2 ml
Inhibition of conversion of thyroxine to triiodothyronine
589
'Haemaccel' solution was injected i.v. via a Millipore Filter into two severely ill patients in the intensive therapy unit and into one control subject convalescing in the orthopaedic ward. One of the patients was suffering from bronchopneumonia following perforation of a duodenal ulcer 7 days earlier and the other was comatose following the removal of a cerebral haemangioma 10 days earlier. Blood was drawn 48 h later and 1 ml of the resulting serum was extracted with 3 ml of 95% ethanol. The supernatant was subsequently dried under nitrogen and reconstituted with 1 ml of methanol-ammonia solution (99 volumes of methanol to 1 volume of 2 M ammonium hydroxide). Descending paper chromatography with Whatman 3MM chromatography paper was then performed for 20 h according to the method of Sterling (Rall et al., 1972). Samples of serum to which both '251-T3and 1251-T4 had been added were treated identically to the test samples and chromatographed in the same chromatography chamber. After the paper strips were dried, they were cut into 1 cm wide sections and the radioactivity in each counted in an auto gamma counter for 1 h. From previous chromatography studies it had been shown that there was less than 5% '"I-T3 contamination of the lz5I-T4 solution used. To determine how quickly serum total T3 concentrations fall in patients with severe illnesses, six patients from the coronary care unit who were suffering from proven myocardial infarction of less than 24 h duration were studied. Samples of serum were obtained at 24 h intervals over the ensuing 48-72 h and subsequently assayed for T3 and T4. Serum T3 (Eastman et al., 1975) and T4 (Corcoran et al., 1973) concentrations were measured by specificradioimmunoassays.Statistical analyses were performed using Student's t test. RESULTS Disappearance rates of _"'I-T3 The percentages of precipitable IZ5I-T3in the sera of the five sick euthyroid patients and the three control subjects are shown in Table 1. The mean percentages of '251-T3 present in the circulation 24-48 h after the i.v. injection of the labelled hormone were higher in the ill patients than in the controls. Probably due to the small number of patients involved in the study, these differenceswere not statistically significant. TSH stimulation tests The results of the TSH stimulation tests are tabulated in Table 2. The mean +SEM increases in serum T3 and T4 concentrations following TSH in the sick euthyroid patients were 1-32+0.43 nmol/l and 105-4&33.4nmol/l respectively, and in the control subjects were 1.2*0-18 nmol/l and 50.1+_ 11.6 nmol/l respectively. These differences were not statistically significant (P>O-05). Studies in the hypothyroid patient with lymphoma When the lymphoma was in remission, the circulating T3 concentration was 2-55&0-11 (2 SD)nmol/l and the T4 concentration was 124.7 +23-1 nmol/l. Three months later, following relapse of the lymphoma, the T3 concentration had fallen to 0.55f0.11 nmol/l whilst the T4 concentration was essentially unchanged at 126+ 10-3nmol/l. Extra-thyroidal conversion of T4 to T3 Paper chromatography of extracted serum drawn 48 h after an i.v. injection of '251-T4
J . N . Carter et al.
590
TABLE1. Disappearance rates of '2sI-labelled triiodothyronine Serum T3 f 2 SD (nmol/l)
Group Sick euthyroid 1 2 3 4 5
Mean? SEM Control 1
2 3 Mean f SEM
Percentage of *251-T3in serum after 1.5 h
24 h
48 h
028 f 0-14 0.98 f 0 1 1 0-46f0.12 074 f0 0 9 083k018 0.66f 0.11
100 100 100 100 100
31 29 31
19 19
1.63f 0 15 2-14f0I7 1.38f 0 I2 1.72f 0 1 8
100 100 100
52
64 4126' 22 28 29 26f2*
23 44 49 31 f6**
I5 15
17 1 6 t I**
P>0.05;** P>0.05. TABLE 2. Thyrotrophin stimulation tests
Serum T3 (nmol/l)
Serum T4 (nmol/l)
Group Basal
Post TSH
Basal
Post TSH
1-26 2.61
Mean fSEM
0.66 028 1a9 0-68 f 0 18
2.0f0.32
91.3 61.7 I324 95.1 f 16.7
120.8 23 1.4 248 200f 33.4
Control 1 2 3 Mean fSEM
1.92 3-13 2.29 2.45 f0.29
2-92 4-79 3-24 3.65 k 0-48
79.7 122.1 111.8 104.1f 10.3
119-5 200.5 145.2 155.5f 193
Sick euthyroid 1
2 3
210
was performed in two severely ill patients from the intensive therapy unit and in one control subject. It had been determined from chromatography performed both previously and simultaneously with the above study that the radioactivity peak corresponding to T4 occurred 2-3 cm from the origin whilst the peak for T3 occurred 8-9 cm from the origin. Extracts of serum to which labelled T3 and T4,individually and in combination, had been added were used for these studies. A discrete peak corresponding to T4 with no peak in the T3 region was found in each of the ill patients whilst discrete peaks corresponding to both T4 and T3 were found in the control subject. A superimposed graph of the chromatography results in the control subject, and one of the ill patients is shown in Fig. 1.
59 1
Inhibition of conversion of thyroxine to triiodothyronine
Distance from the origin (an)
FIG.1. Paper chromatography of serum obtained 48 h after intravenous injection of
I2'I-
thyroxine into a normal euthyroid adult control patient (unbroken line) and a sick euthyroid patient (broken line). The initial peak represents T4 12'1 and the second peak T3 '"I. 3-Or
2.5
-
2.0
-
1.5
-
1.0
-
-
T
5
c
" 1
0
1
2
3
Oays posl myocardial infaction
FIG.2. Decline in serum total T3 levels in six patients followed for 3 days after admission to hospital with myocardial infarction.
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J. N . Carter et al.
Serum T3 concentrations in patients with acute myocardial infarction
Six patients with acute myocardial infarction were studied serially during the 48-72 h following admission to the coronary care unit. The serum T3 concentrations during this period are graphed in Fig. 2. It can be seen that there is a marked fall in the T3 levels within the first 24-48 h of admission. The mean lowest value for the six patients was 1.57 nmol/l and this was significantly lower than the mean baseline value of 2.44 nmol/l (P>0-005). The mean serum T4 levels did not change over the 72 h period. DISCUSSION There are only three possible explanations for the markedly depressed serum T3 concentrations associated with the mildly decreased serum T4 concentrations present in sick euthyroid patients: (i) increased T3 destruction rate whilst the production rate is not increased; (ii) a selective decrease of T3 secretion from the thyroid gland; (iii) decreased extra-thyroidal conversion of T4 to T3. The differences between the mean values of each group were not statistically significant, however, and the results were not compatible with the hypothesis that an increased disappearance rate of T3 accounts for, or contributes to, the markedly reduced serum T3 concentrations in these patients. In these studies we used single compartment kinetics, rather than subject our sick patients to more complex and demanding kinetic studies, which do not allow us to draw firm conclusions from the data concerning metabolic clearance rates of T3. The latter is dependent upon distribution volume which may be altered when there is a disturbance in thyroid function (Cavalieri et al., 1970, 1971). The second possibility of a selective decrease of T3 secretion from the thyroid gland was tested by performing TSH stimulation tests in three severely ill patients and three control subjects. It was found that the mean maximal increases of both T3 and T4 24 h after TSH were not significantly different, suggesting that there was not a selective decrease of T3 secretion from the thyroid glands of sick euthyroid patients. Consequently, the only possible explanation for the greatly reduced serum T3 concentrations in sick euthyroid patients is a decrease in the peripheral conversion of T4 to T3. This conclusion was supported by the studies performed in the patient with lymphoma and primary hypothyroidism in whom the circulating T3 concentration fell from 2.55 nmol/l when the lymphoma was in remission to 0-55 nmol/l when it relapsed. This marked fall could not be attributed to malabsorption of the ingested T4 since her serum total T4 concentration remained essentially unchanged over this period. As the only source of T3 in this patient was monodeiodination of T4, and as there is not an increased destruction rate of T3 in severely ill patients, the only explanation compatible with the data is a decrease in the conversion of T4 to T3. This defect in monodeiodination was unequivocally confirmed by the paper chromatography studies. As shown in Fig. 1, there was a peak of radioactivity corresponding to T3 in the control subject but there was no such peak in the sick euthyroid patients. A radioactivity peak corresponding to the T4, however, was present in each chromatograph. It is apparent from the study of the patients with acute myocardial infarction that the serum T3 concentrations are significantly reduced within 48 h of the onset of the severe illness (see Fig. 2). Because the half life of circulating T3 in healthy euthyroid subjects is about 24 h (Nicoloff et al., 1972) (as intimated above, it may be longer in sick euthyroid
Inhibition of conversion of thyroxine to triiodothyronine
593
patients), the onset of the reduction in T4 to T3 conversion must occur close to the onset of the illness. The circulating concentrations of reverse T3 (rT3 ; 3,3’,5’-triiodothyronine)have recently been found to be elevated in the fetus (Chopra, 1974). Thyroid function in the fetal and sick euthyroid states are virtually identical (Eastman et al., 1973; Abuid et al., 1973; Erenberg et al., 1974), and sick euthyroid patients also have increased circulating concentrations of rT3 compared with control subjects (Chopra et al., 1975). This raises the possibility that in the sick euthyroid state, T4 may preferentially be monodeiodinated to rT3 whereas under normal conditions it is preferentially converted to T3. As rT3 has little known biological activity (Pittman & Barker, 1959) the possible significance of elevated circulating concentrations of rT3 in sick euthyroid patients is not clear. However, evidence from animal experimentation suggests that the role of rT3 may be to decrease TSH production by interfering with feedback control via occupation of pituitary binding sites (Money et al., 1960; Stasilli et al., 1959) and to inhibit the metabolic action of T3 and/or T4 by occupation of peripheral binding sites (Pittman et at., 1964), leading to an overall decrease in calorigenesis and metabolic rate. This hypotheses requires experimental confirmation in the human. ACKNOWLEDGMENTS
This work was supported by a grant from the National Health and Medical Research Council of Australia. We thank Ms C. Batley and Ms T. Enright and Associate Professor O’Rourke for permission to study patients in the Coronary Care Unit. REFERENCES P.R. (1973) Serum triiodothyronine and thyroxine in the neonate and ABUID,J., STINSON,D.A. & LARSEN. the acute increases in these hormones following delivery. Journal of Clinical Investigation, 52,1195-1 199. BROWN,J., CHOPRA, I.J., CORNELL, J.S., HERSHMAN, J.M., SOLOMON, D.H., ULLER, R.P. & VANHERLE, A.J. (1974) Comparisons and alterations in circulating thyroglobulin, triiodothyronine and thyroxine in response to exogenous (bovine) and endogenous (human) thyrotropin. A n d s of Internal Mcdicine. 81,68-81.
CARTER, J.N.. EASTMAN, C.J., CORCORAN, J.M. & LAZARUS, L. (1974) Effect of severe chronic illness on thyroid function. Loncet, ii, 971-974. CAVALIERI, R.R., STEINBERG, M. & SWRLE,G.L. (1970) The distribution kinetics of triiodothyronine: studies of euthyroid subjects with decreased plasma thyroxine-binding globulin and patients with Grave’s disease. Journal of Clinical Imestigafian, 49, 1041-1050. CAVAUERI, R.R., STEINBERG, M. & S m u , G.L. (1971) Metabolic clearance rate of L-triiodothyronine in man: a comparison of results by single injection and constant infusion methods. Joiirnbl of Clinical Endocrinology and Metabolism, 33,624-629. CHOPRA, I.J. (1974) A radioimmunoassay for measurement of 3,3‘,5-triiodothyronine (reverse T3). Journal of Clinical Investigation, 54, 583-592. CHOPRA,I.J., SOLOMON, D.H., CHOPRA,U., YOUNG,R.T. & CHUATECO, G.N. (1974) Alterations in circulating thyroid hormones and thyrotropin in hepatic cirrhosis: evidence for euthyroidism despite subnormal serum triiodothyronine. Journal of Clinical Endocrinology and Metabolism, 39, 501-51 1. CHOPRA.I.J., CHOPRA, U..S m , S.R., REZA,M. & SOLOMON, D.H. (1975) Reciprocal changes in serum concentrations of 3,3’,5’-triodothyronine (reverse T3) and 3,3’5-triiodothyronine (T3)-systemic illness. Journal of Clinical Endocrinology. 41, 1043-1049. CORCORAN, J.M.,EASTMAN, C.J., EKINS,R.P. & PAUL, W. (1973) The production of antixra for the radioimmunoassay of thyroxine. Journal of Endocrinology, 58, xxii.
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EASTMAN. C.J., CORCORAN, J.M.. JEQUIER, A., EKINS,R.P. & WILLIAMS,E.S. (1973) Triiodothyronine concentration in cord and maternal sera a t term. Clinical Science and Molecular Medicine, 45, 251-255. EASTMAN, C.J., CORCORAN, J.M., EKINS,R.P., WILLIAMS, E.S. & NABARRO, J.D.N. (1975)The radioimmunoassay of triiodothyronine and its clinical application. Journal of Clinical Pathology, 28,225-230. ERENBERG, A., PHmm, D.L..LAM,R. & FISHER, D.A. (1974)Total and free thyroid hormone concentrations in the neonatal period. Pediatrics, 53,211-216. KRAEGAN. E.W., LAZARUS, L., MELLER, H..CAMPBELL, L. & CHIA,Y.O. (1975)Carrier solutions for lowlevel intravenous insulin infusion. British Medical Journal, iii, 464466. MONEY, W.L., KUMAOKA, S., RAWSON,R.W. & KROC,R.L. (1960) Part IV. Metabolic effects of thyroid hormones and their analogues. Comparative effects of thyroid hormones and their analogues in experimental animals. Annals of New York Academy of Sciences, 86,512-544. NICOLOFF. J.T., Low, J.C., DUSSAULT, J.H. &FISHER, D.A. (1972)Simultaneous measurements of thyroxine and triiodothyronine turnover kinetics in man. Journal of Clinical Inuestigation, 51,473-483. OPPENHEIMER, J.H., ~ U E F R.,, SURKS, M.J. & HAUER,H. (1963)Binding of thyroxine by serum proteins evaluated by equilibrium dialysis and electrophoretic techniques. Alterations in non-thyroidal illness. Journal of Clinical Inuestigation, 42, 1769-1782. PITTMAN, C.S. & BARKER, S.B. (1959) Inhibition of thyroxine action by 3,3',5'-triiodothyronine. Endocrinology, 64,466468. PITTMAN, C.S.. SHINOHARA. M., THRASHER, H.& MCCRAW.E.F. (1964)Effect of thyroxine analogues on the peripheral metabolism of thyroxine: the half life and pattern of elimination. Endocrinology. 74.61 1. RALL,J.R., STERLING, K., GHARIB, H.& MAYBERRY, W.E. (1972)Thyroid hormone analysis. Methods in Investigative and Diagnostic Endocrinology (General Editor S . Berson), Part I: The Thyroid and Biogenic Amines, pp. 204-240. STASILW, N.R., KROC,R.L. & MELTZER, R.I. (1959)Antigoitrogenic and calorigenic activities of thyroxine analogues in rats. Endocrinology, 64, 62-82.
