Quantitative Determination of Total and Free Triiodothyronine and Thyroxine

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1 Tohoku J. exp. Med., 1969, 99, Quantitative Determination of Total and Free Triiodothyronine and Thyroxine Toshiro Sakurada, Shintaro Saito, Kiyoshi Inagaki, Sumio Tayama and Tatsuo Torikai Department of Internal Medicine (Prof. T. Torikai), Tohoku University School of Medicine, Sendai A new method for the quantitative determination of free triiodothyronine (ft3) and an accurate method for the quantitative determination of free thyroxine (ft4) in human plasma were developed. The percentages of T3 and T4 in plasma were studied by thin-layer chromato graphy. An elutable fraction (EF) of T3, ratio of free 125I-T3 to protein-bound 125I-T3, was obtained using Sephadex-microcolumn chromatography. EF of T4 was obtained by the same method. Mean normal total T3 (tt3) and total T4 (tt4), calculated from plasma butanol extractable iodine (BEI) and molar percentages of T3 and T4, were 0.63 and 5.59 ƒêg per 100ml plasma, respectively. Mean normal ft3 and ft4, calculated from tt3, tt4 and EF values of T3 and T4, were 2.43 and 2.21mƒÊg per 100ml plasma, respectively. The ft3 values can be obtained by the present method more easily and speedily than by the method of Nauman et al. The present ft4 values, in con sideration of BEI and the percentage of T4 in plasma, represent a more accurate one than the earlier data by Lee et al. Recently, free thyroxine in serum had been known to play an important part in the metabolism of the human body.1-5 Free triiodothyronine, another active form of iodoamino acid, was first detected in human serum by Nauman et al.6 in This paper describes a new method for the quantitative determination of free triiodothyronine and an accurate method for the quantitative determination of free thyroxine in human plasma. MATERIALS AND METHODS 125I-labeled triiodothyronine (125I-T3, specific activity mCi/mg) and 125I -labeled thyroxine (125I-T4, specific activity mCi/mg) were obtained weekly from Abbott Laboratories and stored in a deep freezer. Nonradioactive 3-monoiodotyrosine (MIT), 3,5-diiodotyrosine (DIT), 3,5,3'-triiodothyro nine (T3) and thyroxine (T4) were obtained from Nutritional Biochemical Corporation (U. S. A.) and dissolved in ethanol-2 N ammonia (1 F1) as 1mM solution. Plasma protein-bound iodine (PBI) was determined by the alkali-ashing method of Barker and Humphrey7 and plasma butanol-extractable iodine (BEI) by the method of Kontaxis and Pickering.8 Received for publication, June 17,

2 180 T. Sakurada et at. Extraction of iodinated compounds Twenty ml each of plasma were obtained from patients 24, 48 and 72 hrs after the oral administration of 200ƒÊCi of 131I. Iodinated compounds were extracted by West et al.'s method9 from 20ml of plasma with nine fold volume of acetone containing 0.2% HCI. Acid-acetone extracts were concentrated in vacuo to 15ml and the lipids were removed by three times washing with 5ml of hexane in separatory funnel. Combined aqueous layers were extracted three times with equal volume of ethylacetate. Ethylacetate layers were combined and dried in vacuo and the residue was dissolved in 0.2ml of ethanol-2 N ammonia (9 F1) for thin-layer chromatography. Separation of T3 and T4 by thin-layer chromatography ml of the extracts of iodinated compounds was applied on a thin-layer plate (20 ~ 10 ~0.05cm) of Kieselguhr G (Merck) and developed with n-butanol-chloroform (4 F7) saturated with conc. ammonia water (ammonia atmosphere) solvent system. After 50 min run at 30 KC, the chromatoplate was dried with warm air and the radioactivity of each iodinated compound was scanned by an automatic thin-layer chromatogram scanner (Aloka, Japan). The percentages of iodinated compounds in plasma were estimated by planimetry. At the same time, they were detected by Gmelin's colour xeagent.12 Rf values obtained were 0.87 for inorganic iodide, 0.36 for MIT, 0.18 for DIT, 0.73 for T, and 0.55 for T4, respectively. Molar percentages of T3 and T4 in plasma (molar % of T3 and T4) are calculated as follows: _??_ % T3 of and T4: Percentages of radioactivities of T3 and T4 in plasma thyronines Molar % of T4=100-Molar % of T3 Elutable fractions of T3 and T4 The method of Lee et a1.13 for quantitative determination of free thyroxine was applied to determine the elutable fraction (EF) of T3 and T4. Microcolumns (45 ~6mm) were prepared with 100mg of Sephadex (G-25, coarse) swollen in 5ml of disodium EDTA (3.7 ~10-4 M, ph 7.0). One ml of plasma containing 0.5ƒÊCi (15-24mƒÊg) of 125I-T3 was added to this column after incubation for 1 hr at 25 KC. Seven ml of 0.5M NaCI were used to elute the protein bound125i-t3 and 3ml of 0.1 N NaOH to elute the free 125I-T3. Radioactivity of each eluate was counted in an automatic well-type scintillation counter (Aloka, Japan, back ground cpm). The ratio of net count of free 125I-T3 to net count of protein bound 1251-T3 was defined as EF of T3. EF of T4 was measured by the same method._??_ RESULTS Extraction of iodinated compounds When 0.5ƒÊCi of 125I-T3 and of 125I-T4 was added individually to 15ml of plasma to investigate the recovery of West's methods, large volumes of hexane resulted in the decreased recovery of iodinated compounds. So 5ml of hexane were used in the present study, and 32% of recoveries were obtained for both 125I-T3 and 125I -T4.

Separation of T3 and T4 by TLC Triiodothyronine and Thvroxine in Human Plasma 181 Thin-layer chromatograms and radioscannograms of iodinated compounds in normal human plasma are shown in Fig. 1. Three peaks of radioactivity were found corresponding to inorganic iodide, T3 and T4.

3 Separation of T3 and T4 by TLC Triiodothyronine and Thvroxine in Human Plasma 181 Thin-layer chromatograms and radioscannograms of iodinated compounds in normal human plasma are shown in Fig. 1. Three peaks of radioactivity were found corresponding to inorganic iodide, T3 and T4. The mean molar percentages of T3 in iodothyronines (Fig. 2) were 9.1 for euthyroidism, 11.8 for hyperthyroidism, 13.8 for hypothyroidism, 16.3 for simple goiter, 17.9 for nontoxic nodular goiter and 18.1 for chronic thyroiditis. There was no difference among molar percentages of T3 obtained 24, 48 and 72 hrs after the single oral administration of 131I. Elutable fraction of T3 and T4 Binding of T3 to Sephadex was tested applying 0.5ƒÊCi of free 125I-T3 to a Sephadex G-25 microcolumn. About 90 per cent of free T3 were proved to be retained on Sephadex and eluted with 0.1 N NaOH (Table 1, Exp. 1). When plasma containing 125I-T3 was applied to Sephadex G-25 microcolumn, 0.37 per cent of 125I-T3 was found to be free (Table 1, Exp. 2). As shown in Table 2, EF of T3 was constant until an added amount of T3 reached twice as much as the value of PBI. Beyond this dose, EF of T; increased gradually. Fig. 1. Thin-layer chromatogram and radioscannogram of icdinated compounds extracted from normal human plasma obtained 24 hrs after the oral administratior of 200ƒÊCi of 131I. From origin (O) to the solvent front (F) the compounds separated in the order of diiodotyrosine (DIT), monoiodotyrosine (MIT), thyroxine (T4), triiodothyronine (T3) and inorganic iodide (I-), their pure samples were added to the extract. Stationary phase; Kieselguhr G (Merck). Solvent system: n-butanol-chloroform (4 F7) saturated with cone. ammonia (ammonia atmosphere) at 30 KC.

4 182 T. Sakurada et at. Fig. 2. Molar percentages of T3 and T4 in various thyroid conditions. TABLE 1. Elution pattern for 125I-T3, 125I-T4 and plasma containing 125-T3 and 125I-T4 Exp. 1. Elution pattern for 0.5ƒÊCi of free 125I-T3. Exp. 2. Elution pattern for one ml of plasma (PBI F4.3ƒÊg/100ml) containing 0.5ƒÊCi of 125I-T3. Exp. 3. Elution pattern for 0.5ƒÊCi of free 125I-T4. Exp. 4. Elution pattern for one ml of plasma (PBI F4.3ƒÊg/100ml) containing 0.5ƒÊCi of 125I-T4. * Elution with 0.5M NaCi õ Elution with 0.1 N NaOH About 90 per cent of 0.5ƒÊCi of 125I-T4 were proved to bind Sephadex G-25 microcolumn (Table 1, Exp. 3). When plasma containing 125I-T4 was applied to Sephadex G-25 microcolumn, per cent of 125I-T4 was found to be free (Table 1, Exp. 4). These findings were in agreement with earlier work.13 The mean values of EF of T3 and T4 are summarized in Figs. 3 and 4. They were 3.9 ~10-3 and 4.7 ~10-4 in euthyroidism, 5.8 ~10-3 and 19.6 ~10-4 in hyperthyroidism, 1.9 ~10-3 and 2.9 ~10-4 in hypothyroidism, 2.6 ~10-3 and 3.3 ~ 10-4 in simple goiter, 2.8 ~10-3 and 4.O ~10-4 in nontoxic nodular goiter, 2.9 ~10-3

5 _??_ Triiodothyronine and Thyroxine in Human Pl asma 183 Fig. 3. Elutable fractions of triiodothyronine in various thyroid conditions. Fig. 4. Elutable fractions of thyroxine in various thyroid conditions. and 4.O ~10-4 in chronic thyroiditis, 2.5 ~10-3 and 2.7 ~10-4 in pregnancy and 7.3 ~1O-3 and 15.4 ~1O-4 in subacute thyroiditis, respectively. Calculation of total T3, total T4, free T3 and free T4 follows : Total T3 (tt3), total T4 (tt4), free T3 (ft3) and free T4 (ft4) were calculated as

6 184 T. Sakurada et al. =ft4 ~(EF of T4) The mean values of tt3, ft4, ft3 and ft4 are shown in Table 3. They are high in hyperthyroidism, low in hypothyroidism and normal in simple goiter, nontoxic nodular goiter and chronic thyroiditis. DISCUSSION It is preferable to adopt BEI instead of PBI to estimate free and total T3 and T4, since BEI represents iodothyronines (T3 and T4), while PBI represents sum of iodoamino acids (iodothyronines and iodotyrosines), thyroglobulin, iodoprotein and contaminated inorganic iodide.14 Influences of iodotyrosines, thyroglobulin, iodoprotein and contaminated inorganic iodide to free and total T3 and T4 could be avoided by the use of BET instead of PBI. Moreover, ft4 obtained derectly from BEI is theoretically inaccurate and higher than those obtained from tt4, because BEI is a sum of ashed T3 and T4. Therefore, tt3 and ft4 should be calculated before the estimation of tt3 and ft4. The kinetics of T3 and T4 are known to be quite different,15-17 so their molar percentages in plasma seem to vary at different time intervals after a single dose of tracer, even if one assumes that they started out with the same specific activity at the moment of secretion into the blood from the thyroid gland. In some reports,131i had been given daily until the achievement of isotopic equilibrium. Pitt-Rivers and Rall15 reported that isotopic equilibrium in rat thyroid gland was obtained 48 hrs after the injection of 131I, and Werner and Block18 reported that equilibrium in human thyroid gland was not necessarily attained by 72 hrs after the administration of 131I. But, Tong and Chaikoff19 reported that the 131I compostion (MIT, DIT, T3, T4 and I-) of the rat thyroid gland was constant over a period of 8 hrs to 9 days after a single injection of carrier free 131Ị All T3 in blood was proved to be derived from T3 in the thyroid20 and T3 is secreted from the thyroid gland in the same ratio to T4 as they occur in the thyroid. So the result obtained by Tong and Chaikoff shows that 131I composition of blood as well as that of thyroid gland are constant after single injection of 131I. And the present results show that there is no difference among molar percentages of T3 obtained 24, 48 and 72 hrs after the single oral administration of 131I, in agreement with the findings of Shinlaoka and Jasani29 who investigated the T3 and T4 ratio in the human plasma taken on the 1st, 2nd, 4th and 6th days after the oral administration of 131Ị For these reason, the plasma obtained 24 hrs after the oral administration of 131I was adopted in the present study to obtain the molar percentages of T 3 and T4 in plasma. West at al.'s method9 was very convenient to determine the molar percentages of T3, and T4 in human plasma, because the recovery rate was the same for both 131I-T 3 and 131I-T4 and the procedure can be carried out easily in a short time. And it is not necessary to calculate back to 100% from this recovery of 32%, as the West's method is employed in the present study only to estimate the percentages of T3 and T4 in plasma.

7 Triiodothyronine and Thyroxine in Human Plasma 185 TABLE 2. Effect of added T3 on EF of T3 In our previous papers,10,11 it was shown that iodinated compounds could be separated very well by one dimensional TLC. Iodinated compounds in human plasma, separated in this study, were inorganic iodide, T3 and T4. MIT and DIT were not detectable, though their existence in some pathologic states was reported 21,22 Higher radioactivity of inorganic iodide was observed in hypothy roidism than in euthyroidism indicating a disturbance of the iodide-organification mechanism. As T3 has three iodine atones and T4 has four iodine atoms, their percentages obtained from radioactivity on TLC do not represent accurate values. So molar percentages of T3 and T4 in plasma should be calculated from their radioactive iodine ratio by a factor of 4 F3. Molar percentages of T3 are slightly higher than the radioactive iodine percentage of T3. The molar percentages of T3 were 9.1 in euthyroidism and 11.8 in hyperthyroidism. These results coincided with the report of Klein.23 It was found (Table 2) that EF of T3 was constant until 5.82ƒÊg of T3 were added to 100ml plasma. Accordingly, it was clear that the addition of 125I-T3, used to determine the EF of T3, did not affect the binding pattern of T3 to the triiodothyronine-binding protein. EF of T3 reflected various thyroid states as well as EF of T424,25; it was high in hyperthyroidism and subacute thyroiditis, low in hypothyroidism and pregnancy, and normal in simple goiter, nontoxic nodular goiter and chronic thyroiditis. In pregnancy and various thyroid diseases, significant changes in both binding capacity and concentration of thyroxine-binding protein have been observed,24,25 so it might be thought that EF of T3 would be influenced by both the concentra tion of T3 in plasma and the binding capacity of T3-binding protein. In these respects, EF of T3 as well as EF of T424,25 might be used to evaluate easily and and precisely the various thyroid states. As shown in Figs. 3 and 4, EF of T3 was even more reliable than EF of T4 for the diagnosis of hypothyroidism. Figs. 3 and 4 show that EF of T3 was about ten times as much as EF of T4.

8 186 T. Sakurada et al. TABLE 3. Mean tt3 tt4, ft3, and ft4, values in various thyroid conditions SD; standard deviation This might be explained by the fact that the affinity of binding protein for T3 was weaker than that for T4.26t T3 values were about one tenth of tt4 values (Table 3). Significant differences were observed in tt4 values among euthyroidism, hyper- and hypothyroidism, but not so clear difference was observed in (T3 between euthyroidism and hypo thyroidism. It was found (Table 3) that the ft4 values obtained by the present method were lower than those obtained by the method of Lee et al.13 But the present data, calculated from BEI and per cent of T4 in plasma, may represent theoretically more accurate ft4 values than the earlier data13 calculated from PBI. Ultimately, ft3 values were about the same as ft4 values; mean normal ft3 and ft4 were 2.43 and 2.21mƒÊg per 100ml plasma, respectively. These values were fairly similar to those of Nauman et al.3; their mean normal value of ft3 was 1.51mƒÊg per 100ml. The procedure of the present method was more simple and was carried out more speedily than the elution method of Nauman et al.,6 so our method might be adopted to investigate the free T3 and T4. The biological activity of T3 is known to be three to five times higher than T4.21,28 So it is evident that ft3, as well as ft4, plays an important role in human metabolism. From this point of view, consideration should be given to ft3 as well as to ft4 when a problem of thyroid hormones is discussed. Acknowledgment We are indebted to Prof. M. Uchiyama and Assoc. Prof. Y. Suzuki, Institute of Pharmacy, Tohoku University School of Medicine, for their advice on TLC. References 1) Robbins, J. & Rall, J.E. The interaction of thyroid hormones and protein in biological fluids. Recent Progr. Hormone Res., 1957, 13,

Triiodothyronine and Thyroxine in Human Plasma 187 2) Sterling, K. & Hegedus, A. Measurement of free thyroxine concentration in human serum. J. clin. Invest., 1962, 41, 1031-1040. 3) Oppenheimer, J.H., Squef, R.

9 Triiodothyronine and Thyroxine in Human Plasma 187 2) Sterling, K. & Hegedus, A. Measurement of free thyroxine concentration in human serum. J. clin. Invest., 1962, 41, ) Oppenheimer, J.H., Squef, R., Surks, M.I. & Hauer, H.J. Binding of thyroxine by serum proteins evaluated by equilibrium dialysis and electrophoretic techniques. J. clin. Invest., 1963, 42, ) Liewendahl, K. & Lamberg, B.-A. Free thyroxine in serum determined by dialysis and sephadex G-25 filtration. J. clip. Endocr., 1965, 25, ) Sterling, K. & Brenner, M.A. Free thyroxine in human serum: simplified measure ment with the aid of magnesium precipitation. J. clin. Invest., 1966, 45, ) Nauman, J.A., Nauman, A. & Werner, S.C. Total and free triiodothyronine in human serum. J. clin. Invest., 1967, 46, ) Barker, S.B. & Humphrey, M.J. Clinical determination of protein-bound iodine in plasma. J. clin. Endocr., 1950, 10, ) Kontaxis, N.E. & Pickering, D.E. A micro-method for the determination of butyl alcohol extractable hormonal iodine in serum. J. clin. Endocr., 1958, 18, ) West, C.D., Chavre, V.J. & Wolfe, M. A simple method for estimating serum thyroxin concentration in thyroid disease and iodine-treated patients. J. clin. Endocr., 1966, 26, ) Sakurada, T. Separation of human thyroid hormones by thin layer chromatography. Tohoku J. exp. Med., 1965, 85, ) Sakurada, T. Separation and quantitative determination of human thyroid hormones by thin layer chromatography. Tohoku J. exp. Med., 1966, 88, ) Gmelin, R. & Vertanen, A.I. Asensitive reaction for the paper chromatographic detection of iodide, iodinated tyrosines and thyronines. Acta chem. scanul., 1959, 13, ) Lee, N.D., Henry, R.J. & Golub, O.J. Determination of the free thyroxine content of serum. J. clin. Endocr., 1964, 24, ) Ingbar, S.H. & Woeber, K.A. The thyroid gland. In: Textbook of Endocrinology, edited by R.H. Williams, W.B. Saunders Company, Philadelphia, 1968, p ) Pitt-Rivers, R. & Rall, J.E. Radioiodine equilibrium studies of thyroid and blood. Endocrinology, 1961, 68, ) Sterling, K., Lashof, J.C. & Man, E.B. Disappearance from serum of I131-labeled L-thyroxine and L-triiodothyronine in euthyroid subjects. J. clin. Invest., 1954, 33, ) Kuhl, W.J., Halper, I.S. & Dowben, R.M. Thyroxine and triiodothyronine turnover studies in dystrophia myotonica. J. clin. Endocr., 1961, 21, ) Werner, S.C. & Block, R.J. Discrepancy between the distributions of iodine in human serum when estimated by iodine-131 and iodine-127. Nature (Load.), 1959, 183, ) Tong, W. & Chaikoff, I.L. Hydrolysis of I131-thyroprotein by pancreatic enzymes. J. biol. Chem., 1958, 232, ) Lassiter, W.E. & Stanbury, J.B. The in vivo conversion of thyroxine to 3 F5 F3' triiodothyronine. J. clin. Endocr., 1958, 18, ) Shalom, E.S. Iodinated constituents of plasma of normal subjects and of patients suffering from thyrotoxicosis. J. Endocr., 1966, 36, ) Wellby, M.L., Hetzel, B.S. & Good, B.F. The circulating thyroid hormones in thyro toxieosis. Brit. med. J., 1963, 1, ) Klein, E. Uber die Beziehungen zwischen dem thyroidalen und peripherischen Jo dstoffwechsel bei Schilddriisengesunden und Hyperthyreosen. Acta endocr. (Kbh.), 1960, 34, ) Sakurada, T., Saito, S., Inagaki, K., Tayama, S. & Torikai, T. Polyacrylamide gel electrophoretic study on the effect of estrogen on human thyroxine binding prealbu min. Tohoku J. exp. Med., 1967, 93, ) Sakurada, T., Saito, S., Inagaki, K., Tayama, S. & Torikai, T. Concentration and binding capacity of thyroxin-binding prealbumin in pregnancy, hyper and hypothy roidism. Tohoku J. exp. Med., 1968, 96,

10 188 T. Sakurada et al. 26) Deiss, W.P., Albright, E.C. & Larson, F.C. Comparison of in vitro serum protein binding of thyroxine and triiodothyronine. Proc. Soc. exp. Biol. Med., 1953, 84, ) Gross, J. & Pitt-Rivers, R. 3 F5 F3'-Triiodothyronine. 2. Physiological activity. Biochem. J., 1953, 53, ) Frawley, T.F., McClintock, J.C. & Beebe, R.T. Metabolic and therapeutic effects of triiodothyronine. J. Amer. med. Ass., 1956, 160, ) Shimaoka, K. & Jasani, B.M. The application of two-dimensional paper chromato graphy and low-level counting to the study of triiodothyronine in plasma. J. Endocr., 1965, 32,

THYROID FUNCTION EVALUATION IN PATIENTS WITH INCREASED OR DECREASED THYROXINE-BINDING PROTEIN

THE AMEBICAN JOURNAL OF CLINICAL PATHOLOGY Vol. 50, No. 3 Copyright 1968 by The Williams & Wilkins Co. Printed in U.S.A. THYROID FUNCTION EVALUATION IN PATIENTS WITH INCREASED OR DECREASED THYROXINE-BINDING