Research Article| Volume 13, ISSUE 3, P599-630, September 1993

Download started.


Analytic Measurements of Free Thyroxine

  • Roger Ekins
    Address re-print requests to Roger Ekins, MA, PhD, DSc Department of Molecular Endocrinology University College London Medical School Mortimer Street London WIN 8AA England.
    From the Department of Molecular Endocrinology, University College London Medical School, London, England
    Search for articles by this author
      This paper is only available as a PDF. To read, Please Download here.
      Controversy has centered both on the free hormone hypothesis and the mode of operation and clinical reliability of the newer immunologic methods of free hormone measurement for more than a decade. Consideration of hormone transport kinetics suggests that the free hormone concentration measured in vitro may not always constitute the key determinant of hormone transport. Free hormone assay methods are reviewed, with particular emphasis on methods based on labeled or solid-phase analogues. The reliability of most commercial kits is suspect, yielding what is better viewed as a diagnostic index than an analytically valid free hormone measurement.
      To read this article in full you will need to make a payment

      Purchase one-time access:

      Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'

      Subscribers receive full online access to your subscription and archive of back issues up to and including 2002.

      Content published before 2002 is available via pay-per-view purchase only.


      Subscribe to Clinics in Laboratory Medicine
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect


        • Ain K.B.
        • Mori Y.
        • Refetoff S.
        Reduced clearance of thyroxine-binding globulin (TBG) with increased sialylation: A mechanism for estrogen induced elevation of serum TBG concentration.
        J Clin Endocrinol Metab. 1987; 65: 689
        • Ain K.B.
        • Refetoff S.
        Relationship of oligosaccharide modification to the cause of thyroxine-binding globulin excess.
        J Endocrinol Metab. 1988; 66: 1037
        • Alexander N.M.
        Free thyroxin in serum: Labeled thyroxine-analog methods fall short of their mark [editorial].
        Clin Chem. 1986; 32: 417
        • Amino N.
        • Nishi K.
        • Nakatani K.
        • et al.
        Effect of albumin concentration on the assay of serum free thyroxin by equilibrium radioimmunoassay with labeled thyroxin analog (Amerlex free T4).
        Clin Chem. 1983; 29: 321
        • Bartelena L.
        Recent achievements in studies on thryoxine-binding proteins.
        Endocr Rev. 1990; 11: 47
        • Bartelena L.
        • Robbins J.
        Variations in thyroid hormone transport proteins and their clinical implications.
        Thyroid. 1992; 2: 237
        • Bayer M.
        Free thyroxine results are affected by albumin concentration and nonthyroidal illness.
        Clin Chim Acta. 1983; 130: 391
        • Beck-Peccoz P.
        • Romelli P.B.
        • Cattaneo M.G.
        • et al.
        Free T4 and free T3 measurement in patients with antiiodothyronine autoantibodies.
        in: Albertini A. Ekins R.P. Free Hormones in Blood. Elsevier Biomedical Press, Amsterdam1982: 231
        • Chopra I.J.
        • Chua Teco G.N.
        • Mead J.F.
        • et al.
        A competitive ligand binding assay for measurement of thyroid hormone binding inhibitor in serum and tissues.
        J Clin Endocrinol Metab. 1985; 58: 619
        • Christofides N.D.
        • Sheehan C.P.
        • Midgley J.E.M.
        One-step, labeled-antibody assay for measuring free thyroxin. 1. Assay development and validation.
        Clin Chem. 1992; 38: 11
        • Dussault J.H.
        • Hobel C.J.
        • Fisher D.A.
        Maternal and fetal thyroxine secretion during pregnancy in the sheep.
        Endocrinology. 1971; 88: 47
        • Ekins R.P.
        Methods for the measurement of free thyroid hormones.
        in: Ekins R. Faglia G. Pennisi F. Free Thyroid Hormones. Excerpta Medica, Amsterdam1978: 72
        • Ekins R.P.
        The direct measurement of free (non-proteiri bound) hormones in body fluids.
        in: Hunter W.M. Corrie J.E.T. Immunoassays for Clinical Chemistry. Churchill Livingstone, Edinburgh1983: 319
        • Ekins R.P.
        Hypothesis: The roles of serum thyroxine binding proteins and maternal thyroid hormones in foetal development.
        Lancet. 1985; 1: 1129
        • Ekins R.
        Validity of analog free thyroxin immunoassays.
        Clin Chem. 1987; 32: 2137
        • Ekins R.
        Measurement of free hormones in blood.
        Endocr Rev. 1990; 11: 5
        • Ekins R.P.
        One-step labeled-antibody assay for measuring free thyroxine. 1. Assay development and validation [letter].
        Clin Chem. 1992; 38: 2355
        • Ekins R.P.
        • Edwards P.R.
        Plasma protein-mediated transport of steroid and thyroid hormones: A critique.
        Ann NY Acad Sci. 1988; 538: 193
        • Ekins R.P.
        • Edwards P.R.
        Plasma protein-mediated transport of steroid and thyroid hormones: A critique [letter].
        Am J Physiol. 1988; 255 (response to reply) ibid E406-408: E403
        • Ekins R.P.
        • Edwards P.R.
        • Jackson T.M.
        • et al.
        Interpretation of labeled-analog free hormone assay.
        Clin Chem. 1984; 30: 491
        • Ekins R.P.
        • Edwards P.R.
        • Newman B.
        The role of binding proteins in hormone delivery.
        in: Albertini A. Ekins R. Free Hormones in Blood. Elsevier Biomedical Press, Amsterdam1982: 3
        • Ekins R.P.
        • Ellis S.M.
        The radioimmunoassay of free thyroid hormones in serum.
        in: Robbins J. Braverman L.E. Thyroid research, Proceedings of the Seventh International Thyroid Conference, Boston. Excerpta Medica, Amsterdam1975: 597
        • Ekins R.P.
        • Filetti S.
        • Kurtz A.B.
        • et al.
        A simple general method for the assay of free hormones (and drugs): Its application to the measurement of serum-free thyroxine levels and the bearing of assay results on the Tree thyroxine’ concept.
        J Endocrin. 1980; 85: 29
        • Ekins R.P.
        • Jackson T.M.
        • Edwards P.R.
        • et al.
        Euthyroid sick syndrome and free thyroxine assay.
        Lancet. 1983; 2: 402
        • Escobar del Rey F.
        • Pastor R.
        • Mallol J.
        • et al.
        Effects of maternal iodine deficiency on the L-thyroxine and 3,5,3′-triiodo-L-thyronine contacts of rat embryonic tissues before and after onset of fetal thyroid function.
        Endocrinology. 1986; 118: 1259
        • Farer L.S.
        • Robbins J.
        • Blumberg B.S.
        • et al.
        Thyroxine-serum protein complexes in various animals.
        Endocrinology. 1962; 70: 686
        • Franklyn J.A.
        • Davis J.R.
        • Ramsden D.B.
        • et al.
        Oestrogen administration increases nuclear T3 receptor number in the rat anterior pituitary [abstr].
        J Endocrinol. 1985; 104: 97
        • Glinoer D.
        • Naeije R.
        • Golstein J.
        • et al.
        Alterations in circulating thyroid hormones and thryoxine-binding globulin levels during diabetic ketoacidosis.
        J Endocrinol Invest. 1980; 3: 67
        • Hay I.D.
        • Bayer M.F.
        • Kaplan M.M.
        • et al.
        American Thyroid Association assessment of current free thyroid hormone and thyrotropin measurements and guidelines for future clinical assays.
        Clin Chem. 1991; 37: 2002
        • Helenius T.
        • Liewendahl K.
        Improved dialysis method for free thyroxin in serum compared with five commercial radioimmunoassays in non-thyroidal illness and subjects with abnormal concentrations of thyroxin-binding globulin.
        Clin Chem. 1983; 29: 816
        • Hennemann G.
        Endocrinol Invest. 1986; 9: 1
        • Hoffenberg R.
        • Ramsden D.B.
        The transport of thyroid hormones.
        Clin Sci. 1983; 65: 337
        • Kaptein E.
        Thyroid hormone metabolism in illness.
        in: Hennemann G. Thyroid Hormone Metabolism. Marcel Dekker, New York1986: 296
        • Kurtz A.
        • Dwyer K.
        • Ekins R.P.
        Serum free thyroxine in pregnancy [letter].
        B Med J. 1979; 2: 550
        • Lambert M.
        • Zech F.
        • De Nayer P.
        • et al.
        Elevation of serum thyroxine-binding globulin (but not of cortisol-binding globulin and sex hormone-binding globulin) associated with the progression of human immunodeficiency virus infection.
        Am J Med. 1990; 89: 748
        • Memed S.
        • Geola F.L.
        • Reed A.W.
        • et al.
        A comparison of methods assessing thyroid function in nonthyroidal illness.
        J Clin Endocrinol Metab. 1982; 54: 300
        • Mendel C.M.
        The free hormone hypothesis: A physiologically based mathematical model.
        Endocr Rev. 1989; 10: 232
        • Mendel C.M.
        • Cavalieri R.R.
        • Weisiger R.A.
        Uptake of thyroxine by the perfused rat liver: Implications for the free hormone hypothesis.
        Am J Physiol. 1988; 255: E110
        • Mendel C.M.
        • Cavalieri R.R.
        • Weisiger R.A.
        On plasma protein-mediated transport of steroid and thyroid hormones, [letter].
        Am J Physiol. 1988; 255: E221
        • Mendel G.M.
        • Laughton C.W.
        • McMahon F.A.
        • et al.
        Inability to detect an inhibitor to thyroxine-serum protein binding in sera from patients with non-thyroidal illness.
        Metabolism. 1991; 40: 491
        • Mendel C.M.
        • Weisiger R.A.
        • Cavalieri R.R.
        Uptake of 3,5,3′-triiodothyronine by the perfused rat liver: Return to the free hormone hypothesis.
        Endocrinology. 1988; 123: 1817
        • Mendel C.M.
        • Weisiger R.A.
        • Jones A.L.
        • et al.
        Thyroid hormone-binding proteins in plasma facilitate uniform distribution of thyroxine within tissues: A perfused rat liver study.
        Endocrinology. 1987; 120: 1742
        • Midgley J.E.M.
        • Moon C.R.
        • Wilkins T.A.
        Validity of analog free thyroxin immunoassays. Part II.
        Clin Chem. 1987; 33: 2145
        • Midgley J.E.M.
        • Wilkins T.A.
        Effect of albumin concentration on equilibrium radioimmunoassay of serum free thyroxin with labeled thyroxin analog (Amerlex free T4).
        Clin Chem. 1983; 29: 1861
      1. Midgley JEM, Wilkins TA: A method for determining the free portions of substances in biological fluids. European Pat. No 0026103, 1985

        • Midgley J.E.M.
        • Wilkins T.A.
        One-step labeled-antibody assay for measuring free thyroxine. 1. Assay development and validation [reply].
        Clin Chem. 1992; 38: 2358
        • Moses A.C.
        • Lawlor J.
        • Haddow J.
        • et al.
        A point mutation in transthyretin increases affinity for thyroxine and produces euthyroid hyperthyroxinemia.
        J Clin Invest. 1990; 86: 2025
        • Nelson J.C.
        • Weiss R.M.
        The effect of serum dilution on free thyroxine (T4) concentration in the low T4 syndrome of nonthyroidal illness.
        J Clin Endocrinol Metab. 1985; 61: 239
        • Nunez J.
        Effects of thyroid hormones during brain differentiation.
        Mol Cell Endocrinol. 1984; 37: 125
        • Obregon M.J.
        • Mallol J.
        • Pastor R.
        • et al.
        L-thyroxine and 3,5,3′-triiodo-L-thyronine in rat embryos before onset of foetal thyroid function.
        Endocrinology. 1984; 114: 305
        • Odstrechel G.
        • Herd W.
        • Ward F.B.
        • et al.
        New concepts for the assay of unbound thyroxine (FT4) and thyroxine binding globulin (TBG).
        In Radioimmunoassay and Related Procedures in Medicine. 1977; 2: 369
        • Oppenheimer J.H.
        The nuclear receptor-triiodothyronine complex: Relationship to thyroid hormone distribution, metabolism, and biological action.
        in: Oppenheimer J.H. Samuels H.H. Molecular Basis of Thyroid Hormone Action. Academic Press, San Diego1983: 1
        • Oppenheimer J.H.
        • Schwartz H.L.
        • Mariash C.N.
        • et al.
        Evidence for a factor in the sera of patients with nonthyroidal disease which inhibits iodothyronine binding by solid matrices, serum proteins, and rat hepatocytes.
        J Clin Endocrinol Metab. 1982; 54: 757
        • Oppenheimer J.H.
        • Surks M.I.
        Determination of free thyroxine in serum: A theoretical and experimental analysis.
        J Clin Endocrinol Metab. 1964; 24: 785
        • Osorio C.
        • Myant N.B.
        The passage of thyroid hormones from mother to foetus and its relation to foetal development.
        Br Med Bull. 1960; 16: 159
        • Pardridge W.M.
        Carrier-mediated transport of thyroid hormones through the rat blood-brain barrier: primary role of albumin-bound hormone.
        Endocrinology. 1979; 105: 605
        • Pardridge W.M.
        Transport of protein-bound hormones into tissues in vivo.
        Endocr Rev. 1981; 2: 102
        • Pardridge W.M.
        Transport of protein-bound thyroid and steroid hormones into tissues in vivo: A new hypothesis on the role of hormone binding plasma proteins.
        in: Albertini A. Ekins R.P. Free Hormones in Blood. Elsevier Biomedical Press, Amsterdam1982: 45
        • Pardridge W.M.
        Plasma protein-mediated transport of steroid and thyroid hormones [editorial].
        Am J Physiol. 1987; 252: E157
        • Pardridge W.M.
        Selective delivery of sex steroid hormones to tissues in vivo by albumin and sex-hormone-binding globulin.
        Ann NY Acad Sci. 1988; 538: 173
        • Pardridge W.M.
        On plasma protein-mediated transport of steroid and thyroid hormones [reply].
        Am J Physiol. 1988; 255: E224
      2. Pardridge WM: Plasma protein-mediated transport of steroid and thyroid hormones: a critique [reply]. Am J Physiol 255:E405, 1988 (rebuttal) ibid E406-408

        • Pardridge W.M.
        • Landaw E.M.
        Tracer kinetic model of blood-brain barrier transport of plasma protein-bound ligands. Empiric testing of the free hormone hypothesis.
        J Clin Invest. 1984; 74: 745
        • Pardridge W.M.
        • Mietus L.J.
        Influx of thyroid hormones into rat liver in vivo. Differential availability of thyroxine and triiodothyronine bound by plasma proteins.
        J Clin Invest. 1980; 66: 367
        • Pharoah P.O.D.
        • Connolly K.
        • Hetzel B.
        • et al.
        Maternal thyroid function and motor competence in the child.
        Dev Med Child Neurol. 1981; 23: 76
        • Pickard M.R.
        • Sinha A.K.
        • Gullo D.
        • et al.
        The effect of 3,5,3′ triiodothyronine on leucine uptake and incorporation into protein in cultured neurons and subcellular fractions of rat central nervous system.
        Endocrinology. 1987; 121: 2018
        • Rajan M.J.R.
        • Samuel A.M.
        A two-step radioimmunoassay for free triiodothyronine in serum.
        Clin Chem. 1987; 33: 372
        • Refetoff S.
        Inherited thyroxine-binding globulin abnormalities in man.
        Endocr Rev. 1989; 10: 275
        • Refetoff S.
        • Dwulet F.E.
        • Benson M.D.
        Reduced affinity fo:: thyroxine in two of three structural thyroxine-binding prealbumin variants associated with Familial Amyloidotic Polyneuropathy.
        J Clin Endocrinol Metab. 1986; 63: 1432
        • Robbins J.
        • Bartalena L.
        Plasma transport of thyroid hormones.
        in: Hennemann G. Thyroid Hormone Metabolism. Marcel Dekker, New York1986: 3
        • Robbins J.
        • Rail J.E.
        The iodine-containing hormones: Thyroid hormone transport in blood and extravascular fluids.
        in: Gray C.H. James V.H.T. Hormones in Blood. Academic Press, London1979: 575
        • Romelli P.B.
        • Pennisi F.
        • Vancheri L.
        Measurement of free thyroid hormones in serum by column adsorption chromatography and radioimmunoassay.
        J Endocrinol Invest. 1979; 2: 25
        • Ross H.A.
        Analog vs non-analog free T4 assay: Effect of sample dilution.
        Clin Chem. 1987; 33: 200
        • Ross H.A.
        • Benraad Th J.
        An indirect method for the estimation of free thyroxine in serum by means of monoclonal T4 antibody-coated tubes.
        Nuc-Compact. 1984; 15: 204
        • Ross H.A.
        • Benraad T.J.
        Is free thyroxine accurately measurable at room temperature?.
        Clin Chem. 1992; 38: 880
        • Seal V.S.
        • Doe R.P.
        Corticosteroid-binding globulin: Biochemistry, physiology and phylogeny.
        in: Pincus G. Nadao T. Tait J.F. Steroid Dynamics. Academic Press, New York1966: 63
        • Siiteri P.K.
        • Murai J.T.
        • Hammond G.L.
        • et al.
        The serum transport of steroid hormones.
        Recent Prog Horm Res. 1982; 38: 457
        • van der Sluijs Veer G.
        • Vermes I.
        • Bonte H.A.
        • et al.
        Temperature effects on free thyroxine measurements: analytical and clinical consequences.
        Clin Chem. 1992; 38: 1327
        • Stoekigt J.R.
        • De Garis M.
        • Csicsmann J.
        • et al.
        Limitations of a new free thyroxine assay (Amerlex free T4) [rapid communication].
        Clin Endocrinol. 1981; 15: 313
        • Stoekigt J.J.
        • Stevens V.
        • White E.L.
        • et al.
        Unbound analog radioimmunoassays for free thyroxin measure the albumin-bound hormone fraction.
        Clin Chem. 1983; 29: 1408
        • Surks M.I.
        • Hupart K.H.
        • Pan C.
        • et al.
        Normal free thyroxine in critical nonthyroidal illnesses measured by ultrafiltration of undiluted serum and equilibrium dialysis.
        J Clin Endocrinol Metab. 1988; 67: 1031
        • Tabachnick M.
        • Korcek L.
        Effect of long-chain fatty acids on the binding of thyroxine and triiodothyronine to human thyroxine-binding globulin.
        Biochimica et Biophysica Acta. 1986; 881: 292
        • Tait J.F.
        • Burstein S.
        In vivo studies of steroid dynamics in man.
        in: Pincus V. Thimann K.V. Astwood E.B. The Hormones, vol 5. Academic Press, New York1964: 441
        • Tait J.F.
        • Tait S.A.S.
        The effect of plasma protein binding on the metabolism of steroid hormones.
        J Endocrin. 1991; 131: 339
        • Wosilait W.D.
        A theoretical analysis of the distribution of thyroxine among sites on thyroid binding globulin, thyroid binding prealbumin and serum albumin.
        Res Comm Chem Pathol Pharmacol. 1977; 16: 541