The Relationship of Thyroid-stimulating Hormone (TSH), Thyroxine (T4), and Triiodothyronine (T3) in Primary Thyroid Failure

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1 The Relationship of Thyroid-stimulating Hormone (TSH), Thyroxine (T4), and Triiodothyronine (T3) in Primary Thyroid Failure MANJULA S. KUMAR, PH.D., ALI M. SAFA, M.D., SHARAD D. DEODHAR, M.D., PH.D., AND O. PETER SCHUMACHER, M.D., PH.D. Kumar, Manjula S., Safa, Ali M., Deodhar, Sharad D., and Schumacher, O. Peter: The relationship of thyroid-stimulating hormone (TSH), thyroxine (T4), and triiodothyronine (T3) in primary thyroid failure. Am J Clin Pathol 68: ,1977. Thyroid-stimulating hormone (TSH), serum thyroxine (T4) and triiodothyronine (T3) in sera of 100 patients with primary thyroid failure were measured by radioimmunoassay. Effective thyroxine ratio (ETR) was also measured in 69 of these sera. TSH was elevated in all, with a mean of 76.7 ± 55 fiv/m\ (range 11 to 2 ju.u/ml), and was used to confirm the diagnosis. T4 and T3 levels in this group were 1.8 ± 1.5 /u.g/100 ml (range < 0.2 to 7.0 fig/100 ml) and 76.7 ± 76 ng/100 ml (range < 10 to 600 ng/100 ml), respectively. ETR was 0.81 ± A moderate inverse relationship was observed between TSH and T4 (r = 0.73), in contrast to TSH and T3 and TSH and ETR, which showed comparatively poor relationships (r = 0.41 and 0.43). This observation, in addition to the finding that 17 patients had subnormal T4 but normal or elevated T3, suggests that normal T3 levels alone may not be sufficient to maintain euthyroidism. In contrast, there were only three clinically hypothyroid patients who had elevated TSH, normal T4, but subnormal T3 levels. This study indicates that T4 and T3 may function together to maintain euthyroidism, and that in addition to serum TSH, T4 determination has more diagnostic value than serum T3 or ETR in these patients. (Key words: Thyroid-stimulating hormone; Triiodothyronine; Radioimmunoassay; Primary thyroid failure; Effective thyroxine ratio.) HUMAN PITUITARY thyroid-stimulating hormone (TSH) and its responses in disorders of the thyroid gland have been thoroughly studied. 8-8 It is generally accepted that the serum TSH concentrations are almost invariably elevated in patients with primary hypothy- Received September 17, 1976; accepted for publication November 18, Supported in part by the Carl T. Reinberger Fund. Presented at the Fall Meeting of the American Society of Clinical Pathologists, September Address reprint requests to Dr. Kumar: Department of Immunopathology, Cleveland Clinic, 9500 Euclid Ave., Cleveland, Ohio Departments of Immunopathology and Endocrinology, Cleveland Clinic Foundation, Cleveland, Ohio roidism. 6-8,10 Elevation of TSH is now considered a sine qua non for this diagnosis. 16 It is also appreciated that the level of serum TSH in hypothyroid individuals is a function of serum thyroxine (T4) level. 710 The role of triiodothyronine (T3) in the regulation of thyroid function has generated considerable interest in recent years, 5,911,12,15 but the diagnostic value of serum T3 determinations in hypothyroidism is not clear. The present study was undertaken to assess the relationship between the serum concentrations of TSH, T4, and T3 and effective thyroxine ratio (ETR) in untreated patients with primary thyroid failure, and also to evaluate the diagnostic value of these tests in these patients. Materials and Methods Patients included in this study were evaluated at the Cleveland Clinic between March 1972 and November Sixty-five patients were clinically hypothyroid; the diagnosis of probable hypothyroidism was considered in the rest of the cases and confirmed by high TSH levels. TSH values of more than 10 /^u/rnl were considered elevated (normal 2.8 ± 1.0). Patients who were partially treated or taking estrogen or oral contraceptives were not included. The age and sex distribution of these patients is summarized in Table 1. The patients were classified into the following categories: 1. Idiopathic hypothyroidism (n =42). These patients had no goiter and the thyroglobulin antibody (TG-AB) titer was negative. 2. Hashimoto's thyroiditis (n = 22). These patients either had positive TG-AB titers greater than

2 748 KUMAR ETAL. A.J.C.P. December ^ 8 6 O !. r =0.73 >j 4 \ 80 \ 2 - ay.. ill" SUBNORMAL 1 NORMAL M.1S.D. 6.4 ±1.2 SUPRANORMAL FIG. 1. Distribution of serum T4 levels in 100 primary hypothyroid patients. The solid horizontal line indicates the mean normal level, and the dotted lines indicate ±2 SD. 1:2,500 with or without diffuse goiter, or the diagnosis was confirmed histologically by needle biopsy or surgery. A group of patients (n = 25) who had TG-AB titers less than 1:2,500 or negative titers, but had diffuse firm goiters, were classified as probable Hashimoto's thyroiditis. 3. Post m I therapy hypothyroidism (n = 4). These patients received conventional doses of 131 I for treatment of hyperthyroidism due to Graves' disease. 4. Post-thyroidectomy hypothyroidism (n = 7). Radioimmunoassay of TSH, T4 and T3 Serum TSH, T4 and T3 were measured by doubleantibody radioimmunoassay technic. Methods previously described 4-5,7 were used, with slight modifications. Human TSH for labeling and rabbit anti-tsh were obtained from the National Institute of Arthritis, Me- Age (Yr.) TOTAL Table 1. TSH, T4 and T3 Levels in Primary Hypothyroid Patients at Different Ages Total No. (Female + Male) 9 (6 + 3) 23 (18 + 5) 42 ( ) 26 (18 + 8) 100 ( ) * Significantly higher, P = <.01. TSH, MU/ml ± 70* 75 ± ± ± ± 55 T4, /xg/100 ml 1.5 ± ± ± ± ± 1.5 T3, ng/100 ml 144 ± ± ± ± ± TSH 0.2 I T 4 /ig/100 FIG. 2. Relationship between serum TSH and T4. Shaded areas indicate the normal ranges for TSH and T4, respectively. tabolism and Digestive Diseases, National Institutes of Health, Bethesda, Maryland. TSH research standard-b was obtained from the Medical Research Council, National Institute for Medical Research, Mill Hill, London. Interassay variation at the level of 3 //.U/ml was 8%. The mean serum TSH concentration in sera of 45 normal subjects was 2.8 ±1.0 (SD) /uu/ml. The antiserum to T4 was made in rabbits by immunizing with T4 conjugated to bovine serum albumin. It showed 4.8% cross reaction with T3 and less than 0.1% with diiodotyrosine and monoiodotyrosine. T4-125 I was purchased from Industrial Nuclear, St. Louis. 8-Anilino naphthelene sulfonic acid (ANS, 75 jug/tube) was used to inhibit the binding of thyroid-binding globulin (TBG). The interassay variation at the level of 0.6 /u,g/ 100 ml was 8%. In sera of 65 normal subjects the mean T4 level was 6.4 ± 1.2 (SD) jug/100 ml. The antiserum production to T3 was identical to that of T4. The cross reaction with T4 was 0.2%, and with reverse T3, diiodotyrosine and monoiodotyrosine was less than 0.1%. T3-125 I was purchased from Abbott Laboratories, Chicago, Illinois. ANS (25 jitg/tube) was used to inhibit the TGB binding. The interassay variation at the level of 160 ng/100 ml was 9%. The mean T3 level in sera of 55 normal subjects was 173 ± 39 (SD) /xg/100 ml. Thyroid antibodies were measured with a thyroglob-

3 Vol. 68 No. 6 TSH, T4 AND T3 IN PRIMARY THYROID FAILURE ' h 100 ^ 80 r»0.4l 600 * 1 ^ ? * ;? t & SUBNORMAL NORMAL SUPRANORMAL M.±S.D. I73±39 FIG. 3. Distribution of serum T3. The solid horizontal line indicates the mean normal level, and the dotted lines indicate ±2 SD. ulin hemagglutination test kit, purchased from Wellcome Reagents, Ltd., England. Effective thyroxine ratio (ETR) was measured using a kit from Mallinckrodt Diagnostics, Missouri. The normal range was 0.86 to 1.13 (mean 0.98 ± 0.65). Results The age and sex distribution of the patients, and the mean levels for TSH, T4 and T3 in different age groups, are shown in Table 1. Serum TSH levels were elevated in sera from all these patients. The mean was 76.7 ± 55 (SD) ju,u/ml, with a range of 11 to 2 /u,u/ml. In sera of nine hypothyroid patients less than years old the mean TSH level was ± 70 (SD) ju,u/ml, with a range of 36.6 to 2.0 /itu/ml, which was significantly higher than the average for older age groups (P = <.01). A slightly higher mean T3 level was also observed for this age group, but the difference was not statistically significant (P = >.05), while no difference in T4 levels was found. The average T4 level was 1.8 ± 1.5 (SD) /Ag/100 ml, with a range of 0.2 to 7.0 /u,g/100 ml. As illustrated in Figure 1, 86 patients had subnormal T4 levels (< /Ag/100 ml), 14 patients had normal T4 levels ( /j,g/100 ml), and 13 of these 14 had T4 levels in the range of ju.g/100 ml, which was below the mean normal level. Fair inverse correlation was observed between serum TSH and T4 (Fig. 2), with a correlation coefficient (r) of The distribution of serum T3 values is shown in Fig- 60 TSH 0 T T 3 ng/100 ml F,G Relationship between serum TSH and T3. Shaded areas reflect the normal range. ure 3. The mean T3 level in these patients was 76.7 ± 76 ng/100 ml, with a range of to 600 ng/100 ml. Seventy-two patients had subnormal T3 levels, 26 had normal, and two had elevated T3 values more than 2 SD from the mean. The correlation of TSH with T3 was I 100 T 4 >io FIG. 5. Relationship between serum T4 and T3. The shaded areas reflect normal range. The dotted lines indicate the sensitivities of the assay for T4 and T3, respectively.

4 750 KUMAR ETAL. A.J.C.P.. December 1977 Table 2. Distribution of Patients According to T4 and T3 Levels No. of Patients T4 * One patient had slightly elevated T3 level. T3 Supranormal * poor (Fig. 4), with a correlation coefficient (r) of Interestingly, the patient who had a T3 level of 600 ng/ 100 ml was clinically hypothyroid. She had Hashimoto's thyroiditis with goiter; her serum T4 was 2.5 /xg/100 ml and serum TSH was 190 ^U/ml. Figure 5 illustrates the relationship between T4 and T3 levels (r = 0.64). Sixty-nine patients had both T4 and T3 subnormal. Seventeen had subnormal T4 but normal or elevated T3, and three had subnormal T3 but normal T4; Of these 17 patients with normal T3 but subnormal T4, seven had Hashimoto's thyroiditis, eight had idiopathic hypothyroidism, and two had hypothyroidism due to 131 I treatment and thyroidectomy for Graves' disease. The mean TSH level in sera of these 17 patients was 68.3 ± 52 /xu/ml (range to 190), mean T4 level was 2.5 ± 1 /u.g/100 ml (range 1 to 3.8), and mean T3 level was 155 ± 119 ng/100 ml (range 100 to 600). The remaining 11 patients had T4 and T3 levels in low-normal ranges, eight of these patients had the clinical diagnosis of Hashimoto's thyroiditis, and two had hypothyroidism due to thyroidectomy and one, hypothyroidism due to radioactive iodine treatment. The distribution of patients according to T4 and T3 levels is shown in Table 2. All three patients with normal T4 but low T3 values were clinically hypothyroid. Two of them had myxedema as a result of 131 I therapy and thyroidectomy. The mean ETR for 69 of these patients was 0.81 ±.051. Fourteen (%) of these patients had ETR's in the normal range (0.86 to 0.96), and the correlation with TSH was poor (r = 0.43). Discussion Serum TSH determinations have been found to be the most helpful of various laboratory tests for evaluating primary hypothyroidism. That plasma TSH concentrations are quite constant and do not change in response to a variety of stressful stimuli 814 or variation in serum thyroxine-binding globulin 7 also increases the significance of an elevated TSH. In our experience, patients who had TSH values greater than 25 /tu/ml were either definitely hypothyroid or were suspected clinically to have hypothyroidism. Eleven patients who had slight TSH elevations (11 to 25 /i,u/ml) were found to have low-normal T3 and T4 levels. Eight of these patients had Hashimoto's thyroiditis and three had had thyroidectomy and 131 I therapy for Graves' disease. Our observations confirm those of others 7,14 that hypothyroid patients younger than years of age tend to have higher TSH levels, but no significant difference in T4 and T3 was observed in patients of different ages. Our observation that 17 hypothyroid patients had low T4 but normal or elevated T3 is in agreement with the data of Chopra and associates 2 and Wehner and Gorman, 15 and suggests that in primary thyroid deficiency there may be a sequential failure first of T4 production and then of T3 production while hypothyroidism develops clinically. It is also possible that intense TSH stimulation favors T3 production and release. This observation also indicates that a normal T3 level alone is not enough to keep the patient euthyroid. This observation, in addition to the better correlation of serum T4 with TSH than with serum T3, supports the view that T4 has some intrinsic hormonal activity without prior conversion to T3. Observations by Wehner and Gorman 15 offer convincing evidence that patients with myxedema treated with T3 alone require a much higher concentration of T3 to maintain a euthyroid status. They emphasize that a normal serum concentration of T3 is not sufficient to suppress serum TSH to normal levels. Blackburn and Keating 1 have reported that a maintenance dose of 50 to 75 /xg/day of T3 is needed to render a myxedematous patient normal metabolically. These doses are known to raise serum T3 concentration to twice the normal level. 15 These data and the findings of Chopra and associates 2,3 are in agreement with our observations and support the concept that T3 is not the only metabolically active hormone, and that T3 and T4 may function together to maintain euthyroidism. It is also intriguing that 3% of the patients had low serum T3, normal serum T4, elevated serum TSH and were clinically hypothyroid, suggestive of the possibility of "T3 hypothyroidism" analogous to "T3 euthyroidism" 11 and "T3 hyperthyroidism" 9,18 previously reported. Our study confirms those of others 7,13 that elevated TSH values found in primary hypothyroidism are helpful in establishing the diagnosis and differentiating it from hypothyroidism secondary to hypopituitarism. In addition, this study points out that next to serum TSH, serum T4 has greater diagnostic discriminatory value than serum T3 or ETR. References 1. Blackburn CM, Keating FR: Comparative effectiveness of daily doses of L-triiodothyronine or L-thyroxine in the control of myxedema (abstr). J Clin Invest 33:918, 1954

5 Vol. 68 No. 6 TSH, T4 AND T3 IN PRIMARY THYROID FAILURE 751 Chopra IJ, Solomon DH, Chua Teco GN: Throxine: Just a prohormone or a hormone too? J Clin Endocrinol Metab 36: , 1973 Chopra IJ, Hershman JM, Hornabrook RW: Serum thyroid hormone and thyrotropin levels in subjects from endemic goiter regions of New Guinea. J Clin Endocrinol Metab : , 1975 Chopra IJ: A radioimmunoassay for measurement of thyroxine in unextracted serum. J Clin Endocrinol Metab 34: , Gharib, H, Hyan RJ, Mayberry WE: Radioimmunoassay for triiodothyronine (T3): Affinity and specificity of the antibody for T3. J Clin Endocrinol Metab 33: , 1971 Hershman JM, Pittman JA: Utility of the radioimmunoassay of serum thyrotropin in man. Ann Intern Med 74: , 1971 Mayberry WE, Gharib H, Bilstad JM: Radioimmunoassay for human thyrotrophin. Clinical value in patients with normal and abnormal thyroid function. Ann Intern Med 74: , 1971 Odell WD, Wilber JF, Utiger RD: Studies of thyrotropin physiology by means of radioimmunoassay. Recent Prog Horm Res 23:47-85, 1967 Rappaport B, Ingbar SH: Production of triiodothyronine in normal human subjects and in patients with hyperthyroidism. Contribution of intrathyroidal iodine analysis. Am J Med 56: , ReichlinS, Utiger RD: Regulation of the pituitary-thyroid axis in man: Relationship of TSH concentration to concentration of free and total thyroxine in plasma. J Clin Endocrinol Metab 27: , Sterling K, Bellabarbara D, Newman ES: Determination of triiodothyronine concentration in human serum. J Clin Invest 48: , Surks MI, Schadlow AR, Oppenheimer JH: A new radioimmunoassay for plasma L-triiodothyronine: Measurement in thyroid disease and in patients maintained on hormonal replacement. J Clin Endocrinol Metab 51: , Taunton OD, McDaniel HC, Pittman JA Jr: Standardization of TSH testing. J Clin Endocrinol Metab 25: , Utiger RD: Thyrotrophin radioimmunoassay: Another test of thyroid function. Ann Intern Med 74: , Wehner HW, Gorman CA: Interpretation of serum tri-iodothyronine levels measured by the Sterling technic. N Engl J Med 284: , Wehner HW: T3 hyperthyroidism. Mayo Clin Proc 47: , 1972 ACKNOWLEDGEMENT During the past year articles submitted to the JOURNAL have on occasion been reviewed by individuals other than members of the Board of Editors. These include the following: ADELE AMSDEN, M.D. ROY N. BARNETT, M.D. MYRTON F. BEELER, M.D. JOHN R. CARTER, M.D. RONALD CECHNER, M.D. A. N. D'AGOSTINO, M.D. G. RICHARD DICKERSIN, M.D. L. W. DIGGS, M.D. RONALD J. ELIN, M.D. EVAN R. FARMER, M.D. EDWIN R. FISHER, M.D. ROBERT FREEMAN, M.D. EUGENE P. FRENKEL, M.D. YAO SHI FU, M.D. FRANK GARRITY, M.D. ELSON B. HELWIG, M.D. ELAINE JAFFE, M.D. PETER I. JATLOW, M.D. MICHAEL T. KELLEY, PH.D., M.D. F. M. KING, M.D. MICHAEL KYRIAKOS, M.D. RAFFAELE LATTES, M.D. PETER LEHMANN, M.D. BENJAMIN LEWIS, M.D. LUTHER E., LINDNER, M.D. GEORGE D. LUNDBERG, M.D. P. MANALO-SEARS, M.D. DAVID L. PAGE, M.D. HERBERT F. POLESKY, M.D. HAROLD E. RESINGER, M.D. STANLEY J. ROBBOY, M.D. UROS ROESSMANN, M.D. JON E. ROSENBLATT, M.D. ROBERT E. SCULLY, M.D. RICHARD SIBLEY, M.D. P. BYRD SMITH, PH.D. THOMAS F. SMITH, M.D. EDWARD H. SOULE, M.D. MILES STANDISH, M.D. IRVING SUNSHINE, M.D. ROBERT WEAVER, M.D.