Scandinavian Journal of Clinical and Laboratory Investigation
ISSN: 0036-5513 (Print) 1502-7686 (Online) Journal homepage: http://www.tandfonline.com/loi/iclb20
On the selection of in vitro thyroid function tests Kristian Liewendahl To cite this article: Kristian Liewendahl (1977) On the selection of in vitro thyroid function tests, Scandinavian Journal of Clinical and Laboratory Investigation, 37:5, 379-384, DOI: 10.1080/00365517709091495 To link to this article: http://dx.doi.org/10.1080/00365517709091495
Published online: 28 Aug 2009.
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Date: 23 March 2016, At: 17:30
Scund, J . din. Lub. Invest. 37, 379-384, 1977.
EDITORIAL
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On the selection of in vitro thyroid function tests The introduction, over ten years ago, of clinically applicable methods for the measurement of serum total and free thyroxine started a fruitful period in thyroid function assessment. Since then the popularity of in vitro thyroid tests has grown steadily, whereas the use of in vivo procedures has declined. Obvious reasons for this development are that in vivo tests demand attendance of the patient at the laboratory, require the administration of radioactive isotopes potentially hazardous to the patient, and are diagnostically less accurate than in vitro tests because of a large normal variation partly due to differences in dietary iodine. Unlike in vitro tests, in vivo tests cannot be automated and processed in large series. The vast number of thyroid tests introduced in recent years and the confusion caused by the use of different terms for similar tests have had a frustrating effect on the clinician. The problem is essentially a result of rapid progress in thyroid research, which makes nomenclatures for thyroid tests obsolete in a few years. The nomenclature proposed by the American Thyroid Association and amended by McGowan [15] is the most useful recommendation now available. Thyroxine
The determination of serum thyroxine (T4) will probably remain the standard thyroid function test. Laboratories are now switching from the T4 competitive protein-binding method of Murphy to the more specific, sensitive and precise T4 radioimmunoassay. This process has recently been accelerated by the observation of Rootwelt [20] that the T4 level measured with the competitive proteinbinding method, but not with the radioimmunoassay technique, rises markedly when serum is kept at room temperature for a few days. The reason for this is the release from triglycerides of free fatty acids (FFA), which compete with thyroxine for binding sites on T4-binding proteins. The FFA generated in vitro also increase other tests based on protein-binding of thyroid hormones such as the serum triiodothyronine uptake test (T3U) and the single stage method for the estimation of serum free T4 [13, 171. Unsaturated FFA are much more effective than saturated FFA in raising these tests [13, 221. On the other hand, the in vivo variations of FFA occurring in health and disease are usually too small to be of importance for the interpretation of thyroid tests in clinical practice [13]. The enzyme multiplied immunoassay technique has recently been applied to the determination of serum T4 in a fully automated form [8]. This method has several potential advantages over the T4 radioimmunoassay, but its clinical usefulness has yet to be determined.
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Free thyroxine
Serum T4 is known to vary with the concentration of T4-binding proteins, particularly T4-binding globulin (TBG). Common reasons for increased TBG are pregnancy and the use of oestrogens in the form of oral contraceptives. Impaired synthesis or increased catabolism of TBG occurs in many diseases such as severe liver disease, nephrotic syndrome and malignancy, and when corticosteroids or androgens are administered. High amounts of salicylates decrease serum T4 by interfering with the binding of thyroxine to TBG and T4binding prealbumin. Under these circumstances the evaluation of thyroid function benefits from the determination of the serum free T4 concentration, which is influenced much less than the total T4 by variations in the binding proteins. The metabolic significance of the free fraction is generally recognized and clinical experience has proved free T4 to be the single most accurate thyroid test. Diphenylhydantoin decreases serum total and free T4 probably by increasing the turnover of thyroxine in the liver but eumetabolism is maintained [14]. This is a good example that free T4 does not always accurately reflect the thyroidal status of the patient. In severe, acute or chronic non thyroidal illness, and as a response to surgical stress, serum free T4 is quite often moderately increased. The diagnosis of thyroid disease should, when possible, be postponed until the acute illness has been resolved. In chronic disease the establishment of an associated thyroid disease often requires several thyroid parameters carefully considered together with the clinical evaluation. Free thyroxine index The equilibrium dialysis or ultrafiltration methods necessary for the determination of serum free T4 are rather cumbersome for routine use and instead a free T4 index is usually employed. The rationale of this index is that in euthyroid subjects, variations in T4-binding proteins result in opposite variations in T4 and T3U (the triiodothyronine uptake test, which is a test for the estimation of unsaturated thyroxine-binding capacity) and their product, the free T4 index, therefore remains constant. The correlation between free T4 concentration and free T4 index is mostly satisfactory, but there are exceptions to this rule. Premachandra, Gossain & Perlstein [18] reported high free T4 concentration and normal free T4 index in an euthyroid patient with inherited TBG deficiency. It is also known from other studies that because of the limitations of the T3U test, the free T4 index is able to correct for variations in TBG only to a degree. Also in non thyroidal illness with decreased TCbinding prealbumin, the free T4 index sometimes underestimates the true free concentration. The reason is probably that the T3U test does not correctly reflect variations in T4-binding prealbumin because of the relatively low affinity of this protein for triiodothyronine. We have observed that in this situation a free T4 index based on a thyroxine uptake test (T4U) is sometimes in better agreement with the clinical diagnosis, though its use has not proved to be a major solution to this problem [12]. The free T4 index with a close linear relationship to the free T4 concentration reported by Hamada et al. [I 11 is an interesting attempt to optimize this test. More work of this type is needed if the diagnostic value of free T4 index is to improve from what it is today.
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Because of frequent variations in serum T4-binding capacity also in euthyroid patients and the disturbing effect of many drugs T3U and T4U are of poor diagnostic accuracy as independent tests for discrimination between normal and abnormal thyroid states, especially in hospital practice [I 21. Their use should be restricted to the calculation of free T4 indices. Quantitation of TBG with immunoelectrophoresis or radioimmunoassay does not seem to offer advantages over the uptake tests in thyroid function assessment. The inconvenience and potential errors involved in performing two separate tests for obtaining the free T4 index have inspired the design of single-stage tests based on a dual competitive protein-binding principle introduced by Mincey, Thorson & Brown [16], with the effective thyroxine ratio. Several modifications of this test have appeared under various names, but the term ‘free thyroxine index (single stage)’ in the nomenclature of McGowan [ 151 seems to be the most informative one. In a study made at this laboratory the single stage indices gave twice as many incorrect classifications of both hyper- and hypothyroidism as the dual-stage tests [12]. It is the author’s impression that in a study of patients not only with overt thyroid disease but also with mild cases the diagnostic accuracy of single-stage free T4 indices is inferior to that of the dual-stage methods. Triiodothyronine
Triiodothyronine (T3) is secreted from the thyroid but most of the circulating T3 is produced from monodeiodination of T4 in the liver and other peripheral tissues. There is now convincing evidence that T3 is metabolically more active than T4, and the question of whether T4 possesses any intrinsic hormone activity is still unsolved. According to Chopra, Solomon & Chua Teco [7], clinical hypothyroidism when serum free T3 is normal and free T4 is subnormal, is not consistent with T4 functioning only as a prohormone. Nuclear receptors in peripheral tissues for both T3 and T4 also indicate that T4 has metabolic potency. However, this is not a crucial problem for clinical chemistry where the selection of appropriate tests is based more on clinical observations than on theoretical considerations. Usually in toxic diffuse and toxic nodular goitre serum T3 and T4 are both elevated and one test is sufficient for confirmation of hyperthyroidism. Often the relative increase in T3 is higher than that of T4 and in early hyperthyroidism T3 sometimes rises several months in advance of T4. Serum T3 is therefore a more sensitive indicator of thyroidal hyperfunction and it has been proposed that it should be the first test applied when the clinical pattern fits that of hyperthyroidism. However, the advantage of sensitivity is hampered by a certain lack of specificity because sometimes raised serum T3 is also found in euthyroid patients previously treated for hyperthyroidism or receiving preparations containing triiodothyronine. The not uncommon combination especially in the elderly of hyperthyroidism together with a disease reducing the peripheral T3 production could also lead to erroneous conclusions if T3 were to be applied uncritically as a screening test for hyperthyroidism. The decrease in serum T3 with advancing age further complicates the use of this test in the elderly unless special age-correlated references values are applied. Hyperthyroidism when serum T3 is elevated but total and free T4 are normal is called T3 toxicosis. This term must be accepted only if serum TBG is normal,
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because a similar test pattern can occur in hyperthyroidism with TBG deficiency. Another important condition in which the determining of serum T3 is indicated is in patients with low serum T4 after treatment for hyperthyroidism. In these patients, a normal or increased level of T3 is often consistent with euthyroidism. A similar situation may also occur in Hashimoto's disease and in goitrous subjects. In severe, acute or chronic nonthyroidal diseases serum T3 is quite often subnormal partly because of impaired peripheral conversions of T4 to T3 [l, 61. Another factor diminishing serum T3 in these conditions is the decreased binding of T3 to serum proteins reflected in an increased T3U value [l]. Serum T3 is therefore not a reliable test for the diagnosis of hypothyroidism associated with other diseases. Serum T4 is also more often decreased than T3 in hypothyroidism due to a preferential production of T3 whereby the organism is able to save iodine. Consequently, serum T3 appears to be of no definite value as a test for hypothyroidism. Free triiodothyronine
As for serum T4 the determination of T3 is influenced by variations in TBG and a few laboratories therefore also advocate the determination of free T3 for clinical purposes [25].Tedious dialysis techniques and the need for radioiodinated triiodothyronine with a high specific activity are drawbacks that render the routine application of this test difficult. Because urinary excretion of T3 could reflect serum free T3 its quantitation, as an index of thyroid function, has been attempted with some success [4]. Studies by Shakespear & Burke [21] show that about half the urinary T3 consists of conjugates, and that even unconjugated T3 in urine depends not only on the serum free fraction but also on renal function and the excretion of urinary proteins. Nevertheless, urinary T3 gives a good discrimination of hyperthyroidism. Because of apparent inconvenience and no real advantages compared with serum assays one can agree with the authors that this method is not recommended as a clinical test for hyperthyroidism. Reverse triiodothyronine
Monodeiodination of T4 in the peripheral tissues produces not only T3 but also 3, 3', 5'-triiodothyronine (reverse T3; rT3) via removal of iodine from the inner ring instead of the phenolic ring. Very little of the circulating rT3 comes from thyroidal secretion. Systemic illness, starvation, surgical stress, and treatment with dexamethasone stimulate the production of rT3 and decrease that of T3 [3, 5, 141. As rT3 is metabolically inactive these dual pathways of T4 metabolism appear to be an important control mechanism in the thyroid hormone economy in a variety of conditions. Serum rT3 tends to increase in hyperthyroidism and diminish in hypothyroidism and could therefore be of use in the interpretation of the ordinary thyroid function tests [lo, 191. Particularly in systemic illness a low serum rT3 together with a low T3 would indicate associated hypothyroidism, whereas a normal or high rT3 with a low T3 would not. Very high values of rT3 found in the newborn (cord serum) and the amniotic fluid appear to be of potential value for the diagnosis of neonatal and fetal hypothyroidism [2]. A more exact
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evaluation of the usefulness of rT3 can soon be expected because there is currently a great interest in rT3 in various clinical and experimental conditions.
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Thyrotropin
Because of the negative feedback system between the thyroid and the pituitary, serum thyrotropin (TSH) also reflects variations in the production of thyroid hormones. Increased serum TSH is an almost invariable finding in primary hypothyroidism already at the subclinical stage of the disease. Elevated values do not, therefore, indicate overt hypothyroidism unless there is a consistency with the clinical pattern. Raised serum TSH concentrations occur quite frequently in euthyroid patients with autoimmune thyroiditis and in subjects euthyroid after treatment for hyperthyroidism, especially when radioiodine has been used, and is thought to indicate subclinical thyroid failure and potential hypothyroidism in the future [9, 231. Serum TSH is usually low or undetectable in pituitary and hypothalanic hypothyroidism but normal or slightly elevated concentrations have occasionally been observed. The basal TSH level cannot therefore be used to differentiate between these two forms of secondary hypothyroidism, though differences in the TSH response to thyrotropin-releasing hormone (TRH) are worth looking for. No diagnosis of hyperthyroidism based on the decrease in serum TSH is possible because the present method is not sensitive enough to distinguish reliably between normal and subnormal TSH concentrations. In suspected hyperthyroidism with equivocally increased thyroid hormone level, the lack of TSH response to TRH is a typical finding, whereas a normal response is inconsistent with this diagnosis. Those using this test must be aware of the possibility that lack of TSH response to TRH may occur also in nonthyrotoxic states, especially in ophthalmic Graves’ disease, and that interference from many drugs can modify the result. Conclusions
Due to differences in the disease panorama and in available diagnostic facilities local differences exist in the diagnosis and management of thyroid diseases. One defined strategy for the assessment of thyroid function is therefore futile. Instead the most useful tests will be recalled. With serum T4, free T4 index (preferably dual-stage), T3 and the TSH response to stimulation with TRH a correct diagnosis can be obtained in the overwhelming majority of patients with hyperthyroidism, although it is seldom necessary to resort to using all of these tests. Primary hypothyroidism can mostly be confirmed with serum T4, free T4 index and TSH. Knowledge of the various factors affecting thyroid hormone metabolism is still a prerequisite for the selection of appropriate in vitro thyroid function tests and for the judicious interpretation of test results that are not in immediate conformity with the clinical impresssion. Department of Clinical Chemistry, Helsinki University Central Hospital, and Minerva Foundation Institute for Medical Research, Helsinki, Finland
Kristian Liewendahl
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REFERENCES I Bermudez, F., Surks, M.I. & Oppenheimer, J.H. High incidence of decreased serum triiodothyronine concentration in patients with nonthyroidal disease. J. elin. Endocr. 41, 27, 1975. 2 Burman, K.D., Read, J., Dimond, R.C., Strum, D., Wright, F.D., Patow, W., Earll, J.M. & Wartofsky, L. Measurements of 3, 3’, 5’- triiodothyronine (reverse T3), 3, 3’-~-diiodothyronine, T3, and T4 in human amniotic fluid and in cord and maternal serum. J. clin. Endocr. 43, 1351, 1976. 3 Burr, W.A., Griffiths, R.S., Ramsden, D.B., Hoffenberg, R.,Meinhold, H. & Wenze1,K.W. Effect of a single dose of dexamethasone on serum concentrations of thyroid hormones. Lancet, 2, 58, 1976. 4 Chan, V., Landon, J., Besser, G.M. & Ekins, R.P. Urinary tri-iodothyronine excretion as index of thyroid function. Lancet, 2, 253, 1972. 5 Chopra, I.J., Chopra, U., Smith, S.R., Reza, M. & Solomon, D.H. Reciprocal changes in serum concentrations of 3,3’, 5’triiodothyronine (reverse T3) and 3, 3’, 5-triiodothyronine (T3) in systemic illness. J. clin. Endocr. 41, 1043, 1975. 6 Chopra, I.J., Solomon, D.H., Chopra, U., Young, R.T. & Chua Teco, G.N. Alterations in circulating thyroid hormones and thyrotropin in hepatic cirrhosis: evidence for euthyroidism despite subnormal serum triiodothyronine. J. din. Endow. 39, 501, 1974. 7 Chopra, I.J., Solomon, D.H. & Chua Teco, G.N. Thyroxine: just a prohormone or a hormone too?J. clin Endocr. 36, 1050, 1973. 8 Galen, R.S. & Forman, D. Enzyme immunoassay of serum thyroxine with “AutoChemist” multichannel analyser. Clin Chem. 23, 119, 1977. 9 Gordin A. & Lamberg, B.-A. Natural course of autoimmune thyroiditis. Lancet, 2, 1234, 1975. 10 Griffiths, R.S., Black, E.G. & Hoffenberg, R. Measurement of serum 3,3’, 5’-(reverse) T3, with comments on its derivation. Clin. Endocr. 5, 679, 1976. I 1 Hamada, S., Nakagawa, T., Mori, T. & Torizuka, K. Re-evaluation of thyroxine binding and free thyroxine in serum by paper electrophoresis and equilibrium dialysis, and a new free thyroxine index. J. clin. Endocr. 31, 166, 1970.. 12 Liewendahl, K. & Helenius, T. Comparison of serum free thyroxine indices and ‘corrected’ thyroxine tests. Clin. chim. Acta, 64, 263, 1975.
I 3 Liewendahl, K. & Helenius, T. Effect of fatty acids on thyroid function tests in uitro and in uiuo. Clin. ehim. Acta, 72, 301, 1976. 14 Liewendahl, K. & Majuri, H.Thyroxine, triiodothyronine, and thyrotropin in serum during long-term diphenylhydantoin therapy. Scand. J. clin. Lab. Invest. 36, 141, 1916. 15 McGowan, G.K. Nomenclature in the laboratory assessment of thyroid function. The proceedings of a workshop symposium held at St Bartholomew’s Hospital, (Ed. G.K. McGowan), London, November 1972. J. elin. Path. 28, 207, 1975. 16 Mincey, E.K., Thorson, S.C. & Brown, J.L. A new in uitro blood test for determining thyroid status-the effective thyroxine ratio. Clin.Biochem. 4, 286, 1971. 17 Nosslin, B. & Thorell, J.I. Effects of fat meal, heparin, and fatty acids on the triiodothyronine uptake test. Scand. J. clin. Lab. Inuest. 27, 67, 1971. 18 Premachandra, B.N., Gossain, V.V. & Perlstein, I.B. Increased thyroxine in a euthyroid patient with thyroxine-binding globulin I elin. . Endocr. 42, 309, 1976. deficiency. . 19 Ratcliffe, W.A., Marshall, J. & Ratcliffe, J.G. The radioimmunoassay of 3, 3’, 5’- triiodothyronine (reverse T3) in unextracted human serum. CIin Endocr. 5,631, 1976. 20 Rootwelt, K. The influence of fatty acids on serum thyroxine determination by competitive protein-binding radioassay. Scand. J. clin. Lab. Inuest. 35, 649, 1975. 21 Shakespear, R.A. & Burke, C.W. Triiodothyronine and thyroxine in urine. I. Measurement and application. J. clin. Endocr. 42, 494, 1976. 22 Shaw, W., Hubert, I.L. & Spierto, F.W. Interference of fatty acids in the competitive protein-binding assay for serum thyroxine. Clin. Chem. 22, 673, 1976. 23 Toft, A.D., Seth, J., Irvine, W.J. &Cameron, E.H.D. Thyroid function in the long-term follow-up of patients treated with iodine-I31 for thyrotoxicosis. Lancet, 2, 576, 1975. 24 Vagenakis, A.G., Burger, A., Portnay, G.I., Rudolph, M.,O’Brian, J.T., Azizi, F., Arky, R.A., Nicod, P., Ingbar, S.H. & Braverman, L.E. Diversion of peripheral thyroxine metabolism from activating to inactivating pathways during complete fasting. J. clin. Endocr. 41, 191, 1975. 25 Weeke, J. & Orskov, H. Ultrasensitive radioimmunoassay for direct determination of free triiodothyronine concentration in serum Scand. J. elin. Lab. Inuest. 35, 237, 1975.
ISSN: 0036-5513 (Print) 1502-7686 (Online) Journal homepage: http://www.tandfonline.com/loi/iclb20
On the selection of in vitro thyroid function tests Kristian Liewendahl To cite this article: Kristian Liewendahl (1977) On the selection of in vitro thyroid function tests, Scandinavian Journal of Clinical and Laboratory Investigation, 37:5, 379-384, DOI: 10.1080/00365517709091495 To link to this article: http://dx.doi.org/10.1080/00365517709091495
Published online: 28 Aug 2009.
Submit your article to this journal
Article views: 4
View related articles
Citing articles: 1 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=iclb20 Download by: [McMaster University]
Date: 23 March 2016, At: 17:30
Scund, J . din. Lub. Invest. 37, 379-384, 1977.
EDITORIAL
Downloaded by [McMaster University] at 17:30 23 March 2016
On the selection of in vitro thyroid function tests The introduction, over ten years ago, of clinically applicable methods for the measurement of serum total and free thyroxine started a fruitful period in thyroid function assessment. Since then the popularity of in vitro thyroid tests has grown steadily, whereas the use of in vivo procedures has declined. Obvious reasons for this development are that in vivo tests demand attendance of the patient at the laboratory, require the administration of radioactive isotopes potentially hazardous to the patient, and are diagnostically less accurate than in vitro tests because of a large normal variation partly due to differences in dietary iodine. Unlike in vitro tests, in vivo tests cannot be automated and processed in large series. The vast number of thyroid tests introduced in recent years and the confusion caused by the use of different terms for similar tests have had a frustrating effect on the clinician. The problem is essentially a result of rapid progress in thyroid research, which makes nomenclatures for thyroid tests obsolete in a few years. The nomenclature proposed by the American Thyroid Association and amended by McGowan [15] is the most useful recommendation now available. Thyroxine
The determination of serum thyroxine (T4) will probably remain the standard thyroid function test. Laboratories are now switching from the T4 competitive protein-binding method of Murphy to the more specific, sensitive and precise T4 radioimmunoassay. This process has recently been accelerated by the observation of Rootwelt [20] that the T4 level measured with the competitive proteinbinding method, but not with the radioimmunoassay technique, rises markedly when serum is kept at room temperature for a few days. The reason for this is the release from triglycerides of free fatty acids (FFA), which compete with thyroxine for binding sites on T4-binding proteins. The FFA generated in vitro also increase other tests based on protein-binding of thyroid hormones such as the serum triiodothyronine uptake test (T3U) and the single stage method for the estimation of serum free T4 [13, 171. Unsaturated FFA are much more effective than saturated FFA in raising these tests [13, 221. On the other hand, the in vivo variations of FFA occurring in health and disease are usually too small to be of importance for the interpretation of thyroid tests in clinical practice [13]. The enzyme multiplied immunoassay technique has recently been applied to the determination of serum T4 in a fully automated form [8]. This method has several potential advantages over the T4 radioimmunoassay, but its clinical usefulness has yet to be determined.
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Free thyroxine
Serum T4 is known to vary with the concentration of T4-binding proteins, particularly T4-binding globulin (TBG). Common reasons for increased TBG are pregnancy and the use of oestrogens in the form of oral contraceptives. Impaired synthesis or increased catabolism of TBG occurs in many diseases such as severe liver disease, nephrotic syndrome and malignancy, and when corticosteroids or androgens are administered. High amounts of salicylates decrease serum T4 by interfering with the binding of thyroxine to TBG and T4binding prealbumin. Under these circumstances the evaluation of thyroid function benefits from the determination of the serum free T4 concentration, which is influenced much less than the total T4 by variations in the binding proteins. The metabolic significance of the free fraction is generally recognized and clinical experience has proved free T4 to be the single most accurate thyroid test. Diphenylhydantoin decreases serum total and free T4 probably by increasing the turnover of thyroxine in the liver but eumetabolism is maintained [14]. This is a good example that free T4 does not always accurately reflect the thyroidal status of the patient. In severe, acute or chronic non thyroidal illness, and as a response to surgical stress, serum free T4 is quite often moderately increased. The diagnosis of thyroid disease should, when possible, be postponed until the acute illness has been resolved. In chronic disease the establishment of an associated thyroid disease often requires several thyroid parameters carefully considered together with the clinical evaluation. Free thyroxine index The equilibrium dialysis or ultrafiltration methods necessary for the determination of serum free T4 are rather cumbersome for routine use and instead a free T4 index is usually employed. The rationale of this index is that in euthyroid subjects, variations in T4-binding proteins result in opposite variations in T4 and T3U (the triiodothyronine uptake test, which is a test for the estimation of unsaturated thyroxine-binding capacity) and their product, the free T4 index, therefore remains constant. The correlation between free T4 concentration and free T4 index is mostly satisfactory, but there are exceptions to this rule. Premachandra, Gossain & Perlstein [18] reported high free T4 concentration and normal free T4 index in an euthyroid patient with inherited TBG deficiency. It is also known from other studies that because of the limitations of the T3U test, the free T4 index is able to correct for variations in TBG only to a degree. Also in non thyroidal illness with decreased TCbinding prealbumin, the free T4 index sometimes underestimates the true free concentration. The reason is probably that the T3U test does not correctly reflect variations in T4-binding prealbumin because of the relatively low affinity of this protein for triiodothyronine. We have observed that in this situation a free T4 index based on a thyroxine uptake test (T4U) is sometimes in better agreement with the clinical diagnosis, though its use has not proved to be a major solution to this problem [12]. The free T4 index with a close linear relationship to the free T4 concentration reported by Hamada et al. [I 11 is an interesting attempt to optimize this test. More work of this type is needed if the diagnostic value of free T4 index is to improve from what it is today.
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Because of frequent variations in serum T4-binding capacity also in euthyroid patients and the disturbing effect of many drugs T3U and T4U are of poor diagnostic accuracy as independent tests for discrimination between normal and abnormal thyroid states, especially in hospital practice [I 21. Their use should be restricted to the calculation of free T4 indices. Quantitation of TBG with immunoelectrophoresis or radioimmunoassay does not seem to offer advantages over the uptake tests in thyroid function assessment. The inconvenience and potential errors involved in performing two separate tests for obtaining the free T4 index have inspired the design of single-stage tests based on a dual competitive protein-binding principle introduced by Mincey, Thorson & Brown [16], with the effective thyroxine ratio. Several modifications of this test have appeared under various names, but the term ‘free thyroxine index (single stage)’ in the nomenclature of McGowan [ 151 seems to be the most informative one. In a study made at this laboratory the single stage indices gave twice as many incorrect classifications of both hyper- and hypothyroidism as the dual-stage tests [12]. It is the author’s impression that in a study of patients not only with overt thyroid disease but also with mild cases the diagnostic accuracy of single-stage free T4 indices is inferior to that of the dual-stage methods. Triiodothyronine
Triiodothyronine (T3) is secreted from the thyroid but most of the circulating T3 is produced from monodeiodination of T4 in the liver and other peripheral tissues. There is now convincing evidence that T3 is metabolically more active than T4, and the question of whether T4 possesses any intrinsic hormone activity is still unsolved. According to Chopra, Solomon & Chua Teco [7], clinical hypothyroidism when serum free T3 is normal and free T4 is subnormal, is not consistent with T4 functioning only as a prohormone. Nuclear receptors in peripheral tissues for both T3 and T4 also indicate that T4 has metabolic potency. However, this is not a crucial problem for clinical chemistry where the selection of appropriate tests is based more on clinical observations than on theoretical considerations. Usually in toxic diffuse and toxic nodular goitre serum T3 and T4 are both elevated and one test is sufficient for confirmation of hyperthyroidism. Often the relative increase in T3 is higher than that of T4 and in early hyperthyroidism T3 sometimes rises several months in advance of T4. Serum T3 is therefore a more sensitive indicator of thyroidal hyperfunction and it has been proposed that it should be the first test applied when the clinical pattern fits that of hyperthyroidism. However, the advantage of sensitivity is hampered by a certain lack of specificity because sometimes raised serum T3 is also found in euthyroid patients previously treated for hyperthyroidism or receiving preparations containing triiodothyronine. The not uncommon combination especially in the elderly of hyperthyroidism together with a disease reducing the peripheral T3 production could also lead to erroneous conclusions if T3 were to be applied uncritically as a screening test for hyperthyroidism. The decrease in serum T3 with advancing age further complicates the use of this test in the elderly unless special age-correlated references values are applied. Hyperthyroidism when serum T3 is elevated but total and free T4 are normal is called T3 toxicosis. This term must be accepted only if serum TBG is normal,
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because a similar test pattern can occur in hyperthyroidism with TBG deficiency. Another important condition in which the determining of serum T3 is indicated is in patients with low serum T4 after treatment for hyperthyroidism. In these patients, a normal or increased level of T3 is often consistent with euthyroidism. A similar situation may also occur in Hashimoto's disease and in goitrous subjects. In severe, acute or chronic nonthyroidal diseases serum T3 is quite often subnormal partly because of impaired peripheral conversions of T4 to T3 [l, 61. Another factor diminishing serum T3 in these conditions is the decreased binding of T3 to serum proteins reflected in an increased T3U value [l]. Serum T3 is therefore not a reliable test for the diagnosis of hypothyroidism associated with other diseases. Serum T4 is also more often decreased than T3 in hypothyroidism due to a preferential production of T3 whereby the organism is able to save iodine. Consequently, serum T3 appears to be of no definite value as a test for hypothyroidism. Free triiodothyronine
As for serum T4 the determination of T3 is influenced by variations in TBG and a few laboratories therefore also advocate the determination of free T3 for clinical purposes [25].Tedious dialysis techniques and the need for radioiodinated triiodothyronine with a high specific activity are drawbacks that render the routine application of this test difficult. Because urinary excretion of T3 could reflect serum free T3 its quantitation, as an index of thyroid function, has been attempted with some success [4]. Studies by Shakespear & Burke [21] show that about half the urinary T3 consists of conjugates, and that even unconjugated T3 in urine depends not only on the serum free fraction but also on renal function and the excretion of urinary proteins. Nevertheless, urinary T3 gives a good discrimination of hyperthyroidism. Because of apparent inconvenience and no real advantages compared with serum assays one can agree with the authors that this method is not recommended as a clinical test for hyperthyroidism. Reverse triiodothyronine
Monodeiodination of T4 in the peripheral tissues produces not only T3 but also 3, 3', 5'-triiodothyronine (reverse T3; rT3) via removal of iodine from the inner ring instead of the phenolic ring. Very little of the circulating rT3 comes from thyroidal secretion. Systemic illness, starvation, surgical stress, and treatment with dexamethasone stimulate the production of rT3 and decrease that of T3 [3, 5, 141. As rT3 is metabolically inactive these dual pathways of T4 metabolism appear to be an important control mechanism in the thyroid hormone economy in a variety of conditions. Serum rT3 tends to increase in hyperthyroidism and diminish in hypothyroidism and could therefore be of use in the interpretation of the ordinary thyroid function tests [lo, 191. Particularly in systemic illness a low serum rT3 together with a low T3 would indicate associated hypothyroidism, whereas a normal or high rT3 with a low T3 would not. Very high values of rT3 found in the newborn (cord serum) and the amniotic fluid appear to be of potential value for the diagnosis of neonatal and fetal hypothyroidism [2]. A more exact
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evaluation of the usefulness of rT3 can soon be expected because there is currently a great interest in rT3 in various clinical and experimental conditions.
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Thyrotropin
Because of the negative feedback system between the thyroid and the pituitary, serum thyrotropin (TSH) also reflects variations in the production of thyroid hormones. Increased serum TSH is an almost invariable finding in primary hypothyroidism already at the subclinical stage of the disease. Elevated values do not, therefore, indicate overt hypothyroidism unless there is a consistency with the clinical pattern. Raised serum TSH concentrations occur quite frequently in euthyroid patients with autoimmune thyroiditis and in subjects euthyroid after treatment for hyperthyroidism, especially when radioiodine has been used, and is thought to indicate subclinical thyroid failure and potential hypothyroidism in the future [9, 231. Serum TSH is usually low or undetectable in pituitary and hypothalanic hypothyroidism but normal or slightly elevated concentrations have occasionally been observed. The basal TSH level cannot therefore be used to differentiate between these two forms of secondary hypothyroidism, though differences in the TSH response to thyrotropin-releasing hormone (TRH) are worth looking for. No diagnosis of hyperthyroidism based on the decrease in serum TSH is possible because the present method is not sensitive enough to distinguish reliably between normal and subnormal TSH concentrations. In suspected hyperthyroidism with equivocally increased thyroid hormone level, the lack of TSH response to TRH is a typical finding, whereas a normal response is inconsistent with this diagnosis. Those using this test must be aware of the possibility that lack of TSH response to TRH may occur also in nonthyrotoxic states, especially in ophthalmic Graves’ disease, and that interference from many drugs can modify the result. Conclusions
Due to differences in the disease panorama and in available diagnostic facilities local differences exist in the diagnosis and management of thyroid diseases. One defined strategy for the assessment of thyroid function is therefore futile. Instead the most useful tests will be recalled. With serum T4, free T4 index (preferably dual-stage), T3 and the TSH response to stimulation with TRH a correct diagnosis can be obtained in the overwhelming majority of patients with hyperthyroidism, although it is seldom necessary to resort to using all of these tests. Primary hypothyroidism can mostly be confirmed with serum T4, free T4 index and TSH. Knowledge of the various factors affecting thyroid hormone metabolism is still a prerequisite for the selection of appropriate in vitro thyroid function tests and for the judicious interpretation of test results that are not in immediate conformity with the clinical impresssion. Department of Clinical Chemistry, Helsinki University Central Hospital, and Minerva Foundation Institute for Medical Research, Helsinki, Finland
Kristian Liewendahl
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