J. clin. Pth. (1967), 2, 5 Comprison of three simple methods for the ssessment of 'free' thyroid hormone T. M. D. GIMLETTE1 From the Rdio-Isotope Lbortory, St. Thoms's Hospitl, London SYNOPSIS A dilysis method for estimting 'free' thyroxine is compred with two indirect methods using the uptke of triiodothyronine by the red cells nd by Sephdex column. The three methods give results which correlte well nd the Sephdex method is the simplest. The theoreticl bsis of such tests nd their role in the ssessment of thyroid sttus is briefly discussed. A number of direct nd indirect methods for evluting the free frction of the totl plsm thyroxine hve been described. The most direct method, using rdioiodine-lbelled thyroxine, is equilibrium dilysis (Sterling nd Hegedus, 1962); simpler techniques for estimting 'dilysble thyroxine' s rte of trnsfer cross semi-permeble membrne (Christensen, 1959; Gimlette, 1964) nd by Sephdex chromtogrphy (Lee, Henry, nd Golub, 1964) hve lso been described. More indirect methods using lbelled triiodothyronine hve included the estimtion of the uptke of triiodothyronine by the red cells (Hmolsky, Stein, nd Freedberg, 1957), by ion exchnge resins (Mitchell, 1958) nd resin sponge (Mitchell, Hrden, nd O'Rourke, 196), Sephdex columns (Shpiro nd Rbinowitz, 1962; Gimlette, 1967), nd chrcol (Herbert, Gottlieb, Lu, Gilbert, nd Silver, 1965). This pper reports comprison of three simple methods, nmely, the dilysble thyroxine technique, the red cell uptke of triiodothyronine, nd Sephdex column chromtogrphy. METHODS 1 Sixteen-hour dilysble thyroxine ws estimted by method previously described (Gimlette, 1964). This is the estimtion of the percentge of trcer quntity of 1311 thyroxine dded to the plsm under investigtion which is dilysed, during hours t 37 C., out of smll cellophne bg immersed in smple of the sme plsm in test tube. The norml rnge ws -46--8 %. 2 The red-cell uptke of triiodothyronine ws estimted by the method of Hmolsky et l. (1957) with some 'Present ddress: Deprtment of Nucler Medicine, The Liverpool Clinic, I Myrtle St., Liverpool 7. Received for publiction 18 August 1966. modifictions: 2 ml. smples of blood were used, nd incubtion nd wshing were crried out in glss test tubes. The results were clculted in terms of triiodothyronine-binding coefficient (Adms, Specht, nd Woodwrd, 196). Red cell binding coefficient -H X U H 1-U Where H Hemtocrit, % Red cell = U uptke 1 1 (uncorrected) The norml rnge found in extensive previous studies ws -7 to -12 with the technique used. 3 Sephdex chromtogrphy ws crried out by method previously described (Gimlette, 1967). The results were expressed in terms of triiodothyroninebinding coefficient nd the norml rnge ws -5-.1. MATERIAL One hundred nd twenty ptients were studied by the three methods described. All the ptients hd been referred for the estblishment of their thyroid sttus nd this ws done by routine methods including rdioiodine studies. Fifty-one ptients were euthyroid nd without significnt orgnic disese, 12 were euthyroid but suffering from serious orgnic disese of vrious kinds, 38 were hyperthyroid, nd 19 were hypothyroid. In 39 of these ptients serum thyroxine ws lso estimted by modifiction of the method of Ekins, previously described (Gimlette, 1965). The norml rnge ws 5 5-14 pg./1 ml. In the mjority of these p.tients thyroxine-binding globulin nd prelbumin mximum binding cpcities were estimted (Oppenheimer, Squef, Surks, nd Huer, 1963). The norml rnges were 15- tzg./1 ml. nd 11-3 ug./1 ml. 5 J Clin Pthol: first published s 1.1136/jcp.2.2.5 on 1 Mrch 1967. Downloded from http://jcp.bmj.com/ on 15 September 218 by guest. Protected by
6 RESULTS The results of the study re indicted in Tbles I nd II nd in Figures 1, 2, nd 3. Clerly there is correltion between the three methods of ssessing 'free' thyroxine, but this is not precisely liner. TABLE if THYROID STATUS ESTIMATED BY THREE METHODS Euthyroid Hyper- Hypo- Method Etthyroid nd Ill thyroid thyroid Totl Sephdex 51(2) 12(5) 38 (5) 19(4) 12() Red cell uptke 51(2) 12(7) 38 (3) 19(4) 12() Dilysble T4 51(3) 12(9) 38 (3) 19(7) 12() 'The figures indicte the number of ptients in ech group nd the figures in brckets those incorrectly ssigned. The results with the red cell method nd the Sephdex method both indicted the correct dignosis in 87 %. The dilysble thyroxine method indicted the correct dignosis in 82%. The difference between the results is probbly not significnt Ptients 1 Euthyroid 2 Euthyroid 3 Euthyroid 4 Euthyroid 5 Euthyroid 6 Euthyroid 7 Euthyroid 8 Euthyroid 9 Euthyroid 1 Euthyroid 11 Euthyroid, ill 12 Euthyroid, ill 13 Euthyroid, ill 14 Euthyroid, ill 15 Euthyroid, ill Euthyroid, ill Hyperthyroid 18 Hyperthyroid 19 Hyperthyroid 2 Hyperhtyroid 21 Hyperthyroid Hyperthyroid 23 Hyperthyroid 24 Hyperthyroid Hyperthyroid Hyperthyroid 27 Hyperthyroid 28 Hyperthyroid 29 Hyperthyroid 3 Hypothyroid 31 Hypothyroid 32 Hypothyroid 33 Hypothyroid 34 Hypothyroid 35 Hypothyroid 36 Hypothyroid 37 Hypothyroid 38 Hypothyroid 39 Hypothyroid T. M. D. Gimlette nd my be due to the norml rnge in the ltter, estblished in n erlier study (Gimlette, 1964), being little low. With ll three methods the dignostic ccurcy ws better in hyperthyroid thn in hypothyroid ptients nd lest good in ill ptients; over one-third of the incorrect results were high vlues in this reltively smll group, in whom bnorml levels of binding proteins, prticulrly of thyroxine-binding prelbumin, re frequently present. The group of ptients in whom plsm thyroxine hs been estimted is smll, but promising results from combining this test with n estimtion of 'free' thyroxine by using the product of plsm thyroxine nd Sephdex-binding coefficient my be seen (Tble TI). Misleding results when only one test ws used were not infrequent, prticulrly in the euthyroid, ill group; but when the bove product ws used correct seprtion of ll the ptients ws chieved. In the euthyroid, ill ptients plsm thyroxine nd the mximum binding cpcity of thyroxine- BLE II RESULTS OF ESTIMATIONS OF PLASMA AND FREE THYROXINE Binding Coefficient Thyroxine (g./1 ml.) Dilysble Thyroxine Thyroxine x Sephdex Red Cells Sephdex Binding Coefficient 9 11-7 1-3 127 7-4 11-7-7 1-5 6-8 11-2 7 6-3 4 5.7 5.3 8-7 21-2*5 13-5 15-8 19-9 14-2 245 13-7 15-15-5 18-21-6 2-2 2-2-2 6-6-2 1-7 - 1-1-8 5-7 53 55-69 -67-63 7-71 *5-51 53-89 -81 1 49 99-86 -68 79-69 95-91 1-1 -84-86 93 95 1-1 -84-92 -78 49-48 -41 54 49 O55 35-43 52 54-68 -75-86 -78-89 9-86 7-71 77-137 -138-158 -121 75-8 -128-18 -13-13 -151-134 -136-13 -13-131 -134-133 -113-69 -72-64 -66 75-66 53-61 -63-66 -63 o66-7 -66-66 -65-76 56-62 -62-18 -118-124 -18-64 -7-98 79 99-18 -15-12 1 1-121 -96-15 -138-1 -96 44-48 -39 4-52 -51 34 44-43 -48 57 77 72-84 49-71 59 59-42 -69-76 74 O5-62 34-81 2-6 1-62 1-34 1-71 2-99 1-71 3.9 1-61 1*44 2-33 2-48 2-51 1-94 9-11 -23-1 -32-8 * 4-24 -27 Mximum Binding Cpcity T.B.G. 2 23 21 2 14 14 23 18 18 31 19 32 27 37 28 2 24 T.B.P.A. 14 21 245 11 5 1 9 6 4 11 18 18 7 23 18 18 24 21 f 24 15 J Clin Pthol: first published s 1.1136/jcp.2.2.5 on 1 Mrch 1967. Downloded from http://jcp.bmj.com/ on 15 September 218 by guest. Protected by
w z R lx I w m U) 4 1 5 -r - HYPE R - o EUo EU., ILL x HYPO- I 4-5 'I 1 o5-2 SEPHADEX BINDING COEFFICIENT 1-5 - Comprison of three simple methods for the ssessment of 'free' thyroid hormone 7 - o.. * I * O.- : - SXsp - -L I - x. * ~ 9'. *. * FIG. 1. Comprison of Sephdex binding coefficient nd dilysble thyroxine. (The norml rnges re shown by the verticl nd horizontl lines.) FIG. 2. Comprison of Sephdex binding coefficient nd red cell binding coefficient. J Clin Pthol: first published s 1.1136/jcp.2.2.5 on 1 Mrch 1967. Downloded from http://jcp.bmj.com/ SEPHADEX BINDING COEFFICIENT on 15 September 218 by guest. Protected by
w z o I- w -i 4 4C IC 8 2-O RED CELL BINDING COEFFICIENT FIG. 3. Comprison of red cell binding coefficient nd dilysble thyroxine. binding prelbumin ws usully decidedly low, but the cpcity of thyroxine-binding globulin ws only slightly reduced compred with the norml. This suggests tht reduced thyroxine-binding pre-lbumin my ply n importnt prt in producing the rised vlues for 'free' thyroxine found by ll three tests in this smll group of ill ptients. DISCUSSION T. M. D. Gimlette The object of the tests described bove, nd of others bsed on the sme principle, is to obtin vlue which is proportionl to the frction of the totl plsm thyroxine which is 'free', or not bound to protein. The vlue obtined cn be multiplied by the totl serum thyroxine or chemicl protein-bound iodine (P.B.I.) to give product which is proportionl to the bsolute level of free thyroxine, quntity too smll to be mesured by direct mens. It is generlly believed tht only 'free' hormone, or hormone unbound to lrge protein molecules, is ble to enter cells nd therefter hve its effect on metbolism (Robbins nd Rll, 196; Sterling nd Brenner, 1966), nd therefore the level of free thyroxine in the plsm probbly constitutes the best vilble index of thyroid sttus, prticulrly in the bsence of dequte knowledge of the behviour nd mode of ction of the hormone within the cell under norml nd bnorml conditions. The direct methods using equilibrium dilysis give vlue for the free frction of the serum thyroxine under the prticulr experimentl conditions of the technique, lthough this is not necessrily identicl with the vlue under physiologicl conditions. The simpler techniques of estimting the rte of dilysis cross semipermeble membrne (Christensen, 1959; Gimlette, 1964) cn be used in the sme wy nd give results which re probbly proportionl to those obtined by equilibrium dilysis. The indirect methods on the other hnd compre the binding power of serum or plsm for triiodothyronine with tht of the red cells, resin, Sephdex, or whtever indictor substnce is used. The binding power of plsm is proportionl to the concentrtion of free binding sites vilble (Mynt nd Osorio, 1959) nd this is inversely proportionl to the frction of dded rdioctive triiodothyronine trnsferred to the indictor substnce, the binding power of which is constnt under the experimentl conditions (Robbins nd Rll, 196). These techniques give vlue which my be expressed s binding coefficient, nd which is relted to the free hormone without being direct estimte (Sterling nd Brenner, 1966). Triiodothyronine is used since it hs greter ffinity for the indictor substnces thn thyroxine hs, nd when quntities of the order of -1,tg./1 ml. re dded to serum, n esily mesurble proportion of this rdioctive triiodothyronine is bound by the indictor substnces, while the distribution of thyroxine on the plsm-binding sites is not significntly ltered. The binding properties of triiodothyronine with plsm proteins re not exctly the sme s those of thyroxine. A decrese in binding power of prelbumin, which does not itself bind triiodothyronine, will tend to increse binding of triiodothyronine by the indictor substnce, since such decrese mkes fewer plsm binding sites vilble for thyroxine nd so increses the competition for the limited cpcity protein sites which thyroxine nd triiodothyronine shre (Robbins nd Rll, 196). The increse in the 'free' frction of thyroxine, estimted by equilibrium dilysis, noted in mny ill ptients nd ttributed to decresed prelbumin binding (Oppenheimer et., 1963), is lso noted when triiodothyronine binding by red cells or Sephdex is used s the method of ssessment. In the selection of suitble test for ssessment of 'free' thyroid hormone, the ese nd convenience of those vilble must be considered, since there ppers to be no gross disprity in the reltive vlues obtined by the vrious methods in different conditions. A number of direct nd indirect methods, some of them rther complex, for the ssessment of 'free' hormone hve been described. For simple routine purposes, the estimtion of triiodothyronine- J Clin Pthol: first published s 1.1136/jcp.2.2.5 on 1 Mrch 1967. Downloded from http://jcp.bmj.com/ on 15 September 218 by guest. Protected by
Comprison of three simple methods for the ssessment of 'free' thyroid hormone binding coefficient by the Sephdex method described (Gimlette, 1967) is suggested s convenient method which hs much to recommend it for the purpose. REFERENCES Adms, R., Specht, N., nd Woodwrd, I. (196). J. clin. Endocr., 2, 1366. Christensen, L. K. (1959). Scnd, J. clin. Lb. Invest., 11, 3. Gimlette, T. M. D. (1964). J. clin. Pth.,, 58. (1965). Ibid., 18, 293. (1967). Ibid. In the press. 9 Hmolsky, M. W., Stein, M., nd Freedberg, A. S. (1957). J. clin. Endocr.,, 33. Herbert, V., Gottlieb, C. W., Lu, K.-S., Gilbert, P., nd Silver, S. (1965). J. Lb. clin. Med., 66, 814. Lee, N. D., Henry, R. J., nd Golub,. J. (1964). J. clin. Endocr., 24, 486. Mitchell, M. L. (1958). Ibid., 18, 1437., Hrden, A. B., nd O'Rourke, M. E. (196). Ibid., 2, 1474. Mynt, N. B., nd Osorio, C. (1959). J. Physiol. (Lond.), 146, 344. Oppenheimer, J. H., Squef, R., Surks, M. I., nd Huer, H., (1963). J. clin. Invest., 42, 69. Robbins, J., nd Ril, J. E. (196). Physiol. Rev., 4, 415. Shpiro, B., nd Rbinowitz, J. L. (1962). J. nucl. Med., 3, 4. Sterling, K., nd Hegedus, A. (1962). J. clin. Invest., 41, 131., nd Brenner, M. A. (1966). Ibid., 45, 153. J Clin Pthol: first published s 1.1136/jcp.2.2.5 on 1 Mrch 1967. Downloded from http://jcp.bmj.com/ on 15 September 218 by guest. Protected by