A simple method is described for determing thyroxine binding proteins in human serum by electrophoresis at pH 8.6, using cellulose acetate membrane as the supporting medium. The procedure had high reliability in sera of normal subjects, pregnant women and patients with decreased thyroxine binding capacity of thyroxine binding globulin.</p

Arima, Terukatsu

Imai, Masanobu

Kondo, Tadasuke

Kosaka, Kiyowo

Takata, Shinji

Okayama University Digital Information Repository

Rapid determination of thyroxine binding proteins of human serum

Okayama University Scientific Achievement Repository

Acta Medica OkayamaVolume 30, Issue 1 1976 Article 4FEBRUARY 1976Rapid determination of thyroxine bindingproteins of human serumTerukatsu Arima∗ Shinji Takata† Masanobu Imai‡Tadasuke Kondo∗∗ Kiyowo Kosaka††∗Okayama University,†Okayama University,‡Mitoyo General Hospital,∗∗Okayama University,††Okayama University,Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.Rapid determination of thyroxine bindingproteins of human serum∗Terukatsu Arima, Shinji Takata, Masanobu Imai, Tadasuke Kondo, and KiyowoKosakaAbstractA simple method is described for determing thyroxine binding proteins in human serum byelectrophoresis at pH 8.6, using cellulose acetate membrane as the supporting medium. The pro-cedure had high reliability in sera of normal subjects, pregnant women and patients with decreasedthyroxine binding capacity of thyroxine binding globulin.∗PMID: 61711 [PubMed - indexed for MEDLINE] Copyright c©OKAYAMA UNIVERSITYMEDICAL SCHOOLActa Med. Okayama 30, 49-55 (1976)RAPID DETERMINATION OF THYROXINE BINDINGPROTEINS OF HUMAN SERUMTerukatsu ARIMA, Shinji TAKATA, Masanobu IMAI*,Tadasuke KONDO and Kiyowo KOSAKAFirst Department of Internal Medicine, Okayama University Medical School,Okayama 700 and *Mitoyo General Hospital, Toyohama, Kagawa 769, JapanReceived for publication, November 19, 1975Abstract: A simple method is described for determining thyroxinebinding proteins in human serum by electrophoresis at pH 8.6, usingcellulose acetate membrane as the supporting medium. The procedurehad high reliability in sera of normal subjects, pregnant women andpatients with decreased thyroxine binding capacity of thyroxinebinding globulin.The T 3-resin sponge uptake test (T3-RSU) (1) and T 4 -resin spongetechnique (T4 -RST) (2) for determining serum thyroxine concentrations havebeen widely employed. The former depends on the principle that circulatingthyroxine is bound to thyroxine binding globulin whereas added 131I-triiodo-thyronine is less firmly bound to the same protein. Therefore, radiothyronineunbound to protein is taken up by exogeneous materials. The T 4 -RST isbased on in vitro competition between circulating thyroxine extracted withethanol and tracer 125I-thyroxine for binding sites of exogeneous thyroxinebinding globulin. The resin sponge acts as a secondary binding site forunbound thyroxine.A discrepancy is sometimes encountered between the procedures. Serumwith an abnormal T3-RSU value and a normal T 4 -RST value should beexamined for thyroxine binding capacity of thyroxine binding globulin.A decreased or an absence of serum thyroxine binding globulin has beenreported since 1959 in certain cases (3, 4, 5). The genetic aspects of thisdefect have been analysed (6, 7).Methods for determining thyroxine binding capacity of serum proteinhave been developed using reverse-flow paper electrophoresis (8,9, 10), starchgel electrophoresis (9, 10, 11) and Laurell rocket immunoelectrophoresis (12).A simple method of electrophoresis on cellulose acetate membrane at pH 8.9in Tris-acetate buffer was reported by Marshall and Tompkins (13).We recently had the opportunity of studing two euthyroid patients whodisplayed elevated T s-RSU. The purpose of this paper is to report a newmethod for rapid determination of thyroxine binding capacity of thyroxine491Arima et al.: Rapid determination of thyroxine binding proteins of human serumProduced by The Berkeley Electronic Press, 197650 T.ARIMA, S. TAKATA, M.IMAI, T. KONDO and K. KOSAKAbinding globulin. Decreased capacity was confirmed in the sera of the euthy-roid patients. The method is simple to apply in a clinical laboratory with anelectrophoretic system for routine serum protein fractionation.MATERIALS AND METHODSSubjects. The subjects were 20 euthyroids, S pregnant women and 2 eu-thyroids with increased T a RSU. A history of clinical thyroid disease wasabsent from all subjects.Blood samples. Blood samples were obtained by venipuncture. The serumwas separated and stored at -25°C until use.Laboratory methods. Ta-RSU and T 4-RST were performed by using com-mercial products obtained from Abbott Laboratories, Oak Ridge. Tennessee.125I-labeled thyroxine in SO% propylene glycol (Abbott Laboratories) wasdiluted with water, extracted with n-butanol and repurified by an extractionprocedure (8). Chromatographic analyses of these preparations revealed thatthyroxine_125J comprised from 8S-90% of total radioactivity. Thyroxine-1251thus purified was diluted to 3.87,ug/3l6pCi/ml with cold L.thyroxine. Thepurity of L-thyroxine (Wako-junyaku, Osaka) was verified in a silica gel TLCsystem of n-butanol: acetone: methanol: 2N-NH40H (3: 5: 1 : 1).Assay of tlryroxine binding capaciiJl. Five microliters of 1251-thyroxine solutionwere added to SOpl of serum and incubated for 6::> minutes at 25 u C. After incu-bation lpl of the incubation mixture was applied to a cellulose acetate mem-brane (Separax, joko.Sangyo, Osaka), 1.0 cm x 6.0 cm and electrophoresed inVeronal buffer at pH 8.6 with an ionic strength of 0.06 at a constant current of0.473 rnA for each strip for 30 minutes (14). After electrophoresis, proteins onthe strip were stained by Ponceau 3R solution (Ponceau 3R: trichloroaceticacid: water, 0.8g: 6g: 100ml) (14). Each protein fraction was removed, and theradioactivity was counted in an Aloka well-type scintillation counter.Autoradiographs were prepared by overnight exposure to Fuji X-ray films.Their radioactivity on the strips was measured with an end windo w Geiger-Muller counter, employing a continuous-recording, counting-rate meter.RESULTSTBG was separated from thyroxine binding albumin (TBA) and thyroxinebinding prealbumin (TBPA) by electrophoresis on cellulose acetate membranein barbital buffer at pH 8.6 (Fig. I). Radioactive-thyroxine distributions inserum are shown in Fig. 2. Radioactive bands corresponding to TBG, TBAand TBPA were present. Radioactive recovery after electrophoresis was98~d and the recovery after protein staining was 97%. The radioactivityof the protein fraction was counted after cutting out the bands.Reproducibility of the method was determined by ten retests of the samesample for TBPA, TBA and TBG. The results are shown in Table 1.When increasing quantities of thyroxine were added to the reaction2Acta Medica Okayama, Vol. 30 [1976], Iss. 1, Art. 4http://escholarship.lib.okayama-u.ac.jp/amo/vol30/iss1/4Thyroxine Binding Proteins 5132tBATBG f\(I, r(/ \ (J ~r .'': (V'..,; '. TBPA~"\ \~./'V' TBA '.....-.....,./1\/ \~oJ.,N \, '-JV"'",__'L___ TBG(0.\.! \.jI/ i! \\ J~~\.!"./ \\"-\.....,~32Fig. 1. Autoradiograph of thyroxine-bindingproteins separated by cellulose acetate membraneelectrophoresis. Anode is at the right. 1, normalserum; 2, serum from patient A; 3, serum froma pregnant woman.Fig. 2. Comparison of the distribu-tion of 125J-thyroxine in serum. Anodeis at the right. 1, normal; 2, patient B;3, a pregnant woman.TABLE 1 REPRODUCIBILITY OF THE RAPID METHOD FOR DETERMINING HUMANTHYROXINE BINDING PROTEIN AT pH 8.6Fraction radioactivity (%)Retest no. TBPA TBA TBG1 4.8 49.6 45.62 4.1 51. 9 45.03 3.0 51. 3 45. 74 3.9 51. 2 44.95 5.0 50.0 45.06 4.1 50.7 45.27 3.0 51. 3 45. 78 4.0 50.8 45.29 3.9 49.9 46.210 3.0 51. 7 46.3Mean 3.8 50. 7 45.5S.D. 0.67 0.89 0.673Arima et al.: Rapid determination of thyroxine binding proteins of human serumProduced by The Berkeley Electronic Press, 197652 T. ARIMA, S. TAKATA, M. IMAI, T. KONDO and K. KOSAKATABLE 2 DISTRIBUTION OF 125I-THYROXINE IN NORMAL SERUM BYTHE PRESENT ASSAY METHODFraction radioactivitySubjects 125I-thyroxine (ng)·TBPA (%) TBA (%) TBG (%)1 5.4 45.4 49.22 7.1 42.8 50.1 69.23 6.1 50.0 43.94 8.2 42.6 49.2 67.95 7.4 44. 1 48.5 70.06 5.0 42.1 52.9 77.97 5.9 50.0 44.18 7.0 46.2 46.89 5.4 40.8 53.8 63.410 5.4 58.7 35.911 5.1 50.8 44.1 68.412 4.3 46.3 49.4 70.7. 13 5.6 43.4 51.0 70.314- 4.6 49.5 45.915 5.9 49.3 44.8 64. 116 4.6 44.4 51.017 5.4 45.8 48.8 75.618 3. 7 47.2 49.119 6.5 39.2 54.320 4.2 43.5 52.3Mean 5.6 46.2 48.2 69.8S.D. 1.1 5.1 4.3 4.2"The amount of 125I-thyroxine in the TBG fraction per mg of aI-globulin on availablesubjects.TABLE 3 DISTRIBUTION OF 125J-THYROXINE IN SERUM OF PREGNANT WOMENBY THE PRESENT ASSAY METHODFraction radioactivityCase 125I-thyroxine (ng)*TBPA (%) TBA (%) TBG (%)1 2. 7 27.3 70.02 7.5 31.3 61. 2 78.23 4.0 16.0 80.0 96.24 5.6 20.4 74.0 82.65 4.3 30.4 65.3Mean 4.8 25.1 70. 1 85. 7S.D. 1.7 7.8 2.1*The amount of 125I-thyroxine in the TBG fraction per mg of aI-globulin on availablewomen.4Acta Medica Okayama, Vol. 30 [1976], Iss. 1, Art. 4http://escholarship.lib.okayama-u.ac.jp/amo/vol30/iss1/4Thyroxine Binding Proteins 53mixture for patient A, a TOO plateau was obtained at a concentration ofO.2pg/ml of thyroxine, while the concentration of thyroxine bound to TBGof a normal subject was still increasing (Fig. 3).Radiothyroxine distributions in serum obtained from normal subjects andpregnant women are shown in Tables 2 and 3, respectively. Data from twopatients with discrepancies between T s-RSU and T 4-RST are shown in Table4. I25I-thyroxine found in the TBG fraction per mg of aI-globulin is shownin Table 2, 3 and 4. The amount bound was in the following order: preg-nant women, euthyroids and the patients.0.2 fR2Ii 0.1TBPA<:<Ii0...c-o...,"C TBA<::::>0LJ<Ii 0.2c:x0...~0.10.3 0.4Thyroxine added to assay( ~g/ml serum)Fig. 3. Distribution of 125I-thyroxine in serumprotein as a function of radiothyroxine added to theassay. 1, normal serum; 2, serum from patient A.TABLE 4 ABNORMAL SERUM DISTRIBUTION OF 125I-THYROXINE BY THE PRESENTASSAY METHOD AND THE T3-RSU AND T4-RST VALUES BY ROUTINECLINICAL LABORATORY METHODSFraction radioacti vity 125I-thyroxinePatient (ng)* TJ-RSU (%) T4-RST (pg/100ml)TBPA (%) TBA (%) TBG(%)A 17.5 55.8 26. 7 43.5 48.3 8.4-B 5.1 83.2 11. 7 20.2 56.3 4.0*The amount of 125I-thyroxine in the TBG fraction per mg of lXI-globulin.5Arima et al.: Rapid determination of thyroxine binding proteins of human serumProduced by The Berkeley Electronic Press, 197654 T. ARIMA, S. TAKATA, M. IMAI, T. KONDO and K. KosAKADISCUSSIONA clinical laboratory method is described for the rapid determination ofthyroxine binding capacity of serum protein. This method uses the sameelectrophoretic system as that in routine laboratory procedures for serumprotein fractionation. TBPA, TBA and TBG were separated (Fig. 1 and 2).The recovery of added radiothyroxine was 97% even after protein staining.The reliability of the method was confirmed for clinical usage (Table 1).Marshall and Tompkins (13) described a method using an electrophoreticsystem on cellulose acetate membrane at pH 8.9, in which thyroxine bindingproteins were separated, but electrophoresis at this pH is not usually per-formed in the clinical laboratory.The method described in this paper was developed primarily to deter-mine thyroxine binding capacity of TBG when a discrepancy between T 3-RSUand T4-RST was found in serum. The capacity of TBG could be calculatedafter reaching a plateau from the following equation: capacity = (a+ b) xc,where a is the 125I-thyroxine added to the assay, b is the endogeneous thyro-xine determined by T4-RST and c is the percentage of 125I-thyroxine foundin TBG fraction.Acknowledgment: W~ wish to express grateful appreciation to Dr. Yumoto for his usefuldiscussion, and to Mr. Y. Ishii, Mr. S. Yokoyama and Mrs. M. Ishii for their technicalassistance.REFERENCES1. Goolden, A. W. G., Gartside, J. M. and Osorio, C.: An evaluation of the 131I-triiodo-thyronine resin sponge test. ]. Clin. Endocrinol. 25, 127-133, 1965.2. Van Oers, R.J.M. and Gijzen, A.H.J.: A systematic investigation on the competitiveprotein-binding assay of thyroxine in serum with ion exchange strips. Clin. Chim. Acta38, 363-370, 1972.3. Tanaka, S. and Starr, P.: Euthyroid man without thyroxine-binding globulin. ]. Clin.Endocrinol. Metab. 19, 485-487, 1959.4. Ingar, S. H.: Clinical and physiological observations in patient with idiopathic decreasein thyroxine binding globulin of plasma. ]. Clin. Invest. 40, 2053-2063, 1961.5. Beisel, W. R., Zainal, H., Hane, S., DiRaimondo, V. G. and Forsham, P. H.: Low thyroi-dal iodine upta~e with euthyroidism associated with deficient thyroid-binding globulinbut normal cortisol binding. ]. Clin. Endocrinol. Metab. 22, 1165-1170, 1962.6. Nicoloff, J. T., Dowling, J. T. and Patton, D. D.: Inheritance of decreased thyroxine-binding globulin. ]. Clin. Endocrinol. Metab. 24, 294-298, 1964.7. Marshall, J. S., Richard, P. L. and Steinberg, A. G.: Human thyroxine-binding globulindeficiency: a genetic study. New Engl.]. Med. 274, 1469-1473, 1966.8. Robbins, J.: Reverse-flow zone electrophoresis: method for determining thyroxine-binding capacity of serum protein. Arch. Biochem. 63, 461-469, 1956.9. Blumberg, B. C. and Robbins, J.: Thyroxine-serum protein complexes: single dimen-sion gel and paper electrophoresis studies. Endocrinology 67, 368-378, 1960.10. Blumberg, B. C., Farer, L., RaIl, J. E. and Robbins, J.: Thyroxine-serum protein com-6Acta Medica Okayama, Vol. 30 [1976], Iss. 1, Art. 4http://escholarship.lib.okayama-u.ac.jp/amo/vol30/iss1/4Thyroxine Binding Proteins 55plexes: two dimension gel and paper elctrophoresis studies. Endocrinology 68, 25-35, 1961.11. Rich, C. and Beam, A. G.: Localization of the thyroxine-binding protein of serum bystarch gel electrophoresis. Endocrinology 62, 687-689, 1958.12. Nielsen, H. G., Buus, O. and Weeke, B.: A rapid determination of thyroxine-bindingglobulin in human serum by means of the Laurell rocket immunoelectrophoresis. Glin.Ghim. Acta 36, 133-138, 1972.13. Marshall, J. S. and Tompkins, L. S.: Effect of 0, p-DDD and similar compounds onthyroxine binding globulin. ]. Glin. Endocrinol. Metab. 28, 386-392, 1968.14. Kohn, J.: A cellulose acetate supporting medium for zone electrophoresis. Glin. Ghim.Acta 2, 297-303, 1957.7Arima et al.: Rapid determination of thyroxine binding proteins of human serumProduced by The Berkeley Electronic Press, 1976

Rapid determination of thyroxine binding proteins of human serum

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