This chapter has been superceeded by 4 newer chapters, 6a, 6b, 6c, and 6d. However this Chapter, written originally by Dr Samuel Refetoff and updated by Drs Franklyn and Shephard, remains a treasure trove of information on many now-obscure thyroid tests, and references. For that reason we maintain it as a part of our Archive for use of MDs who may wish to investigate a bit of the history of thyroid testing. L De Groot, MD
The possibility of thyroid disease is considered when signs or symptoms suggest hyper- or hypothyroidism or some physical abnormality of the thyroid gland. Evaluation of the patient should include a thorough history and physical examination. Since most thyroid diseases require prolonged periods of treatment, it is crucial that a firm diagnosis be established before embarking on such a program. Further, a number of medications, in particular those used in the treatment of thyroid disease, may alter the results of thyroid function tests in such a way that reinvestigation after therapy has begun may provide ambiguous results.
During the past three decades, clinical thyroidology has witnessed the introduction of a plethora of diagnostic procedures. These laboratory procedures provide greater choice, sensitivity, and specificity which have enhanced the likelihood of early detection of occult thyroid diseases presenting with only minimal clinical findings or obscured by coincidental nonthyroid diseases. They also assist in the exclusion of thyroid dysfunction when symptoms and signs closely mimic a thyroid ailment. On the other hand, the wide choice of complementary and overlapping tests indicates that each procedure has its limitations and that no single test is always reliable.
Thyroid tests can be classified into broad categories according to the information they provide at the functional, etiologic, or anatomic levels ( Table 6-1 ).
1. Tests that directly assess the level of the gland activity and integrity of hormone biosynthesis. These tests such as thyroidal radioiodide uptake and perchlorate discharge are carried out in vivo.
2. Tests that measure the concentration of thyroid hormones and their transport in blood. They are performed in vitro and provide indirect assessment of the level of the thyroid hormone dependent metabolic activity.
3. Another category of tests attempts to more directly measure the impact of thyroid hormone on peripheral tissues. Unfortunately, tests available to assess this important parameter are nonspecific, since they are often altered by a variety of nonthyroidal processes.
4. The presence of several substances, such as thyroid autoantibodies, usually absent in healthy individuals, are useful in establishing the etiology of some thyroid illnesses.
5. Invasive procedures, such as biopsy, for histological examination or enzymatic studies are occasionally required to establish a definite diagnosis. Gross abnormalities of the thyroid gland, detected by palpation, can be assessed by scintiscanning and by ultrasonography.
6.The integrity of the hypothalamo-pituitary-thyroid axis can be evaluated by (a) the response of the pituitary gland to thyroid hormone excess or deficiency; (b) the ability of the thyroid gland to respond to thyrotropin (TSH); and (c) the pituitary responsiveness to thyrotropin-releasing hormone (TRH). These tests are intended to identify the primary organ affected by the disease process that manifests as thyroid dysfunction; in other words, primary (thyroid), secondary (pituitary), or tertiary (hypothalamic) malfunction.
7.Lastly, a number of special tests will be briefly described. Some are valuable in the elucidation of the rare inborn errors of hormone biosynthesis, and others are mainly research tools.
Each test has inherent limitations, and no single procedure is diagnostically adequate for the entire spectrum of possible thyroid abnormalities. The choice, execution, application and interpretation of each test requires the understanding of thyroid physiology and biochemistry dealt with in the preceding chapters. Thyroid tests serve not only in the diagnosis and management of thyroid illnesses but also to better understand the pathophysiology underlying a specific disease.
|Table 6-1. Tests of Thyroid Function and Aids in the Diagnosis of Thyroid Diseases|
|In Vivo Tests of Thyroid Gland Activity and Integrity of Hormone Synthesis and Secretion Thyroidal Radioiodide Uptake (RAIU) Early Thyroid RAIU and 99mPertechnetate Uptake Measurements Perchlorate Discharge Test Saliva to Plasma Radioiodide Ratio Measurement of Hormone Concentration and Other Iodinated Compounds and Their Transport in Blood Measurement of Total Thyroid Hormone Concentration in Serum Iodometry Radioligand and Immunometric Assays TT4 TT3 Measurement of Total and Unsaturated Thyroid Hormone-Binding Capacity in Serum In vitro Uptake Tests TBG Measurement Estimation of Free Thyroid Hormone Concentration Dialysable T4 and T3 by Isotopic Equilibrium Free T4 and T3 Index Methods Estimation of FT4 and FT3 by TBG Measurement Two-step Immunoassays Analogue (one-step) Immunoassays Measurements of Iodine-Containing Hormone Precursors and Products of Degradation 3.3′,5′-triiodothyronine of Reverse T3 (rT3) 3,5,-diiodothyronine (3,5-T2) 3,3′,-diiodothyronine (3,3′-T2) 3′,5′,-diiodothyronine (3′,5′,-T2) 3′-monoiodothyronine (3′-T1) 3-monoiodothyronine (3-T1) Tetra- and triiodothyroacetic acid (TETRAC and TRIAC) 3,5,3′-T3 sulfate (T3S) di- and monoiodityrosine (MIT and DIT) Thyroglobulin (Tg) Measurement of Thyroid Hormone and Its Metabolites in Other Body Fluids and in Tissues Urine Amniotic Fluid (AF) Cerebrospinal Fluid (CSF) Milk Saliva Effusions Tissues Tests Assessing the Effects of Thyroid Hormone on Body Tissues Basal Metabolic Rate (BMR) Deep Tendon Reflex Relaxation Time (Photomotogram)||Tests Related to Cardiovascular Function Miscellaneous Biochemical and Physiologic Changes Related to the Action of Thyroid Hormone on Peripheral Tissues Measurement of Substances Absent in Normal Serum Thyroid Autoantibodies Thyroid-Stimulating Immunoglobulins (TSI) Thyroid Stimulation Assays Standard in vivo Mouse Bioassay (LATS) In vitro Bioassays (animal or human tissue and recombinant TSH Receptor) Thyrotropin Binding Assays Thyroid Growth-Promoting Assay Other Substances with Thyroid-Stimulating Activity Exophthalmos-Producing Substance (EPS) Tests of Cell-Mediated Immunity (CMI) Anatomic and Tissue Diagnoses Thyroid Scintiscanning Radioiodide and 99mPertechnitate Scans Other Isotope Scans Fluorescent Scans Ultrasonography X-Ray and Related Procedures Computed Tomography (CT Scanning) Angiography Lymphography Thermography Magnetic Resonance Imaging (MRI) Biopsy of the Thyroid Gland Core Biopsy (Open od Closed) Percutaneous Fine-needle Aspiration (FNA) Evaluation of the Hypothalamic-Pituitary-Thyroid Axis Thyrotropin (TSH) Thyrotropin-Releasing Hormone (TRH) Test Other Tests of TSH Reserve Thyroid Stimulation Test Thyroid Suppression Test Specialized Thyroid Tests Iodotyrosine Deiodinase Activity Test for Defective Hormonogenesis Iodine Kinetic Studies Absorption of Thyroid Hormone Turnover Kinetics of T4 and T3 Metabolic Kinetics of Thyroid Hormones and Their Metabolites Measurement of the Production Rate and Metabolic Kinetics of Other Compounds Transfer of Thyroid Hormone from Blood to Tissues Applications of Molecular Biology in the Diagnosis of Thyroid Diseases|
Common to these tests is the administration to the patient of radioisotopes that cannot be distinguished by the body from the naturally occurring stable iodine isotope (127I). In contrast to all other tests, these procedures provide a means to directly evaluate thyroid gland function. Formerly these tests were used in the diagnosis of hypothyroidism and thyrotoxicosis, but this application has been supplanted by measurement of serum TSH and thyroid hormone concentrations in blood. Also, alterations of thyroid gland activity and in handling of iodine are not necessarily coupled to the amount of hormone produced and secreted. The tests are time consuming, relatively expensive and expose the patient to irradiation. Nevertheless, they still have some speccific applications including the diagnosis of inborn errors of thyroid hormonogenesis. Administration of isotopes is required for thyroid gland scanning used to demonstrate ectopic thyroid tissue and to establish the etiology of some forms of thyrotoxicosis. Finally, measurement of the thyroidal radioiodide uptake can be used as a means for estimating the dose of radioiodide to be delivered in the therapy of thyrotoxicosis and thyroid carcinoma.
To understand the physiological basis of this category of tests, one should remember the following facts. Iodine is an integral part of the thyroid hormone molecule. Although several other tissues (salivary glands, mammary glands, lacrimal glands, the choroid plexus, and the parietal cells of the stomach) can extract iodide from blood and generate a positive tissue to serum iodide gradient, only the thyroid gland stores iodine for an appreciable period of time. 1 Since the kidneys continually filter blood iodide, the final fate of most iodine atoms is either to be trapped by the thyroid gland or to be excreted in the urine. When a tracer of iodide is administered to the patient, it rapidly becomes mixed with the stable extrathyroidal iodide pool and is thereafter handled identically as the stable isotope. Thus, the thyroidal content of radioiodine gradually increases and that in the extrathyroidal body pool gradually declines, until virtually no free iodide is left. Normally this end point is reached between 24 and 72 hours.
From data of the radioiodide uptake by the thyroid gland and/or urinary excretion and/or stable iodide concentration in plasma and urine, the following parameters can be derived: (1) the rate of thyroidal iodine uptake (thyroid iodide clearance), (2) the fractional thyroid radioactive iodide uptake (RAIU), (3) the absolute iodide uptake (AIU) by the thyroid gland, and (4) the urinary excretion of radioiodide, or iodide clearance. After the complete removal of the administered radioiodide from the circulation, depletion of the radioisotope from the thyroid gland can be monitored by direct counting over the gland. Reappearance of the radioiodine in the circulation in protein-bound form can be measured and can be used to estimate the intrathyroidal turnover of iodine and the secretory activity of the thyroid gland.
The foregoing tests can be combined with the administration of agents known either to normally stimulate or to inhibit thyroid gland activity thus providing information on the control of thyroid gland activity. Administration of radioiodide followed by scanning allows us to examine the anatomy of functional tissue. The latter two applications of in vivo tests utilizing radioiodide will be discussed under their respective headings.
The potential hazard of irradiation resulting from the administered radioisotopes should always be kept in mind. Children are particularly vulnerable, and doses of X-rays as small as 20 rads to the thyroid gland are associated with increased risk of developing thyroid malignancies. 2 However, it must be noted that there is no proven danger from isotopes used for the diagnosis of thyroid diseases. 3 In vivo administration of radioisotopes is absolutely contraindicated during pregnancy and in breast-feeding mothers because of placental transport of isotope and excretion into breast milk.
A number of radioisotopes are now available. Furthermore, provision of more sophisticated and sensitive detection devices has substantially decreased the dose required for the completion of the studies. Table 6-2 5-7 lists the most commonly used isotopes for in vivo studies of the thyroid. Isotopes with slower physical decay, such as 125I and 131I, are particularly suitable for long-term studies. Isotopes with faster decay, such as 123I and 132I, usually deliver a lower irradiation dose and are advantageous in short-term and repeated studies. The peak photon energy gamma emission differs among isotopes, allowing the execution of simultaneous studies with two isotopes.
|Table 6-2. Commonly Used Isotopes for In Vivo Studies and Radiation Dose Delivered|
|Nuclide||Principal Photon Energy (keV)||Physical Decay||Estimated Radiation Dose (m rads/ÂµCi)||Average Dose Given for Scanning Purposes (ÂµCi)|
|Mode||Half-Life (Days)||Thyroida||Total Body|
|131I-||364||ÃŸ (0.606 Mev)||8.1||1,340||0.08||50|
|132I-||670||ÃŸ (2.12 MeV)||0.10||15||0.1||50b|
|aCalculations take into account the rate maximal uptake, and residence time of the isotope as well as gland size. For the iodine isotopes, average data for adult euthyroid persons used were: t-1/2 of uptake 5 hours, biologic t-1/2 50 days, maximal uptake 20%, and gland size 15 g (see also refs. [Quimby, 1970 #628;MIRD, 1975 #629;MIRD, 1976 #630]). bDose used for early thyroidal uptake studies.|
This is the most commonly used thyroid test requiring the administration of a radioisotope. It is usually given orally in a capsule or in liquid form and the quantity accumulated by the thyroid gland at various intervals of time is measured using a gamma scintillation counter. Correction for the amount of isotope circulating in the blood of the neck region, by subtracting counts obtained over the thigh, is of particular importance during the early periods following its administration. A dose of the same radioisotope, usually 10%, placed in a neck “phantom” is also counted as a “standard”. The percentage of thyroidal radioactive iodide uptake (RAIU) is calculated from the counts cumulated per constant time unit.
The percentage of RAIU 24 hours after the administration of radioiodide is most useful, since in most instances the thyroid gland has reached the plateau of isotope accumulation, and because it has been shown that at this time, the best separation between high, normal, and low uptake is obtained. Normal values for 24-hour RAIU in most parts of North America are 5 to 30 percent. In many other parts of the world, normal values range from 15 to 50 percent. Lower normal values are due to the increase in dietary iodine intake following the enrichment of foods, particularly mass produced bread (150 Âµg of iodine per slice), with this element. 8 The inverse relationship between the daily dietary intake of iodine and the RAIU test is clearly illustrated in Figure 6-1. The intake of large amounts of iodide (>5 mg/day), mainly from the use of iodine-containing radiologic contrast media, antiseptics, vitamins, and drugs such as amiodarone, suppresses the RAIU values to a level hardly detectable using the usual equipment and doses of the isotope. Depending upon the type of iodine preparation and the period of exposure, depression of RAIU can last for weeks, months, or even years. Even external application of iodide may suppress thyroidal radioiodide uptake. The need to inquire about individual dietary habits and sources of excess iodide intake is obvious.
The test does not measure hormone production and release but merely the avidity of the thyroid gland for iodide and its rate of clearance relative to the kidney. Disease states resulting in excessive production and release of thyroid hormone are most often associated with increased thyroidal RAIU and those causing hormone underproduction with decreased thyroidal RAIU (Figure 6-2, below). Important exceptions include high uptake values in some hypothyroid patients and low values in some hyperthyroid patients. Increased thyroidal RAIU with hormonal insufficiency co-occur in the presence of severe iodide deficiency and in the majority of inborn errors of hormonogenesis (see Chapter 20 and 16 ). In the former, lack of substrate, and in the latter, a specific enzymatic block of hormone synthesis cause hypothyroidism poorly compensated by TSH-induced thyroid gland overactivity. Decreased thyroidal RAIU with hormonal excess is typically encountered in the syndrome of transient thyrotoxicosis (both de Quervain’s and painless thyroiditis), 9 ingestion of exogenous hormone (thyrotoxicosis factitia), iodide-induced thyrotoxicosis (Jod-Basedow disease), 10 and in patients with thyrotoxicosis on moderately high intake of iodide (see Table 6-3 ). High or low thyroidal RAIU as a result of low or high dietary iodine intake, respectively, may not be associated with significant changes in thyroid hormone secretion.
Various factors including diseases that affect the value of the 24-hour thyroidal RAIU are listed in Table 6-3 . Several variations of the test have been devised which have particular value under special circumstances. Some of these are briefly described.
|Table 6-3. Diseases and Other Factors That Affect the 24-Hour Thyroidal RAIU|
In some patients with severe thyrotoxicosis and low intrathyroidal iodine concentration, the turnover rate of iodine may be accelerated causing a rapid initial uptake of radioiodide, reaching a plateau before 6 hours, followed by a decline through release of the isotope in hormonal or other forms (Figure 6-2, above). Although this phenomenon is rare, some laboratories choose to routinely measure early RAIU, usually at 2, 4 or 6 hours. Early measurements require the accurate determination of background activity contributed by the circulating isotope. Radioisotopes with a shorter half-life, such as 123I and 132I, are more suitable.
Since thyroidal uptake in the very early period following administration of radioiodide reflects mainly iodide trapping activity, 99mTc as the pertechnetate ion (99mTcO4-) may be used. In euthyroid patients, thyroid trapping is maximal at about 20 minutes and is approximately 1% of the administered dose 11 . This test, when coupled with the administration of T3, can theoretically be used to evaluate thyroid gland suppressibility in thyrotoxic patients treated with antithyroid drugs (see below).
This test is used to detect defects in intrathyroidal iodide organification. It is based on the following physiological principle. Iodide is “trapped” by the thyroid gland through an energy-requiring active transport mechanism. Once in the gland, it is rapidly bound to thyroglobulin and retention no longer requires active transport. Several ions, such as thiocyanate (SCN-) and perchlorate (ClO4-), inhibit active iodide transport and cause the release of the intrathyroidal iodide not bound to thyroid protein. Thus, measurement of intrathyroidal radioiodine loss following the administration of an inhibitor of iodide trapping would indicate the presence of an iodide-binding defect.
In the standard test, epithyroid counts are obtained at frequent intervals (every 10 or 15 minutes) following the administration of radioiodide. Two hours later, 1g of KClO4 is administered orally and repeated epithyroid counts continue to be obtained for an additional 2 hours. In normal individuals, radioiodide accumulation in the thyroid gland ceases after the administration of the iodide transport inhibitor but there is little loss of the thyroidal radioactivity accumulated prior to induction of the “trapping” block. A loss of 5% percent or more indicates an organification defect (see Chapter 16 ). 12 The severity of the defect is proportional to the extent of radioiodide discharged from the gland and is complete when virtually all the activity accumulated by the gland is lost (see Fig. 16-2, below). The test is positive in the inborn defect of iodide organification, which can be associated with deafness (Pendred’s syndrome), during the administration of iodide organification blocking agents, in many patients with thyroiditis, or following treatment with radioactive iodide.
Measurements of T4 and T3 in serum and the estimation of their free concentration have become the most commonly used tests for the evaluation of the thyroid hormone-dependent metabolic status. This approach results from the development of simple, sensitive, and specific methods for measuring these iodothyronines and because of the lack of specific tests for the direct measurement of the metabolic effect of these hormones. Other advantages are the requirement of only a small blood sample and the large number of determinations that can be completed by a laboratory during a regular workday.
The thyroid gland is the principal source of all hormonal iodine-containing compounds or their precursors and peripheral tissue are the source of the products of degradation. Their chemical structures, and normal concentrations in serum are given in Figure 6-3. It is important to note that the concentration of each substance is dependent not only upon the amount synthesized and secreted but also upon its affinity for carrier serum proteins, distribution in tissues, rate of degradation, and finally, clearance.
The main secretory product of the thyroid gland is t4t3 being next in relative abundance. Both compounds are metabolically active when administered vivo. They synthesized and stored as a part larger moleculethyroglobulin.
18 Under normal circumstances, only minute amounts of Tg escape into the circulation. On a molar basis, it is the least abundant iodine-containing compound in blood. With the exception of T4, Tg, and small amounts of DIT and MIT, all other iodine-containing compounds found in the serum of normal man are produced mainly in extrathyroidal tissues by a stepwise process of deiodination of T4. 19 An alternative pathway of T4 metabolism that involves deamination and decarboxylation but retention of the iodine residues gives rise to TETRAC and TRIAC. 20,21 Conjugation to form sulfated iodoproteins also occurs. 22 Circulating iodoalbumin is generated by intrathyroidal iodination of serum albumin. 23 Small amounts of iodoproteins may be formed in peripheral tissues 24 or in serum 25,26 by covalent linkage of T4 and T3 to soluble proteins. Although the physiological function of circulating iodine compounds other than T4 and T3 remains unknown, measurement of changes in their concentration is of research interest.
Measurement of Total Thyroid Hormone Concentration in Serum
Iodometry. Iodine constitutes an integral part of the thyroid hormone molecule. It is thus not surprising that determination of iodine content in serum was the first method suggested almost six decades ago for the identification and quantitation of thyroid hormone. 27 Measurement of the Protein-Bound Iodine (PBI) was the earliest method used routinely for the estimation of thyroid hormone concentration in serum. This test measured the total quantity of iodine precipitable with the serum proteins, 28 90% of which is T4. The normal range was 4 – 8 Âµg I/dl of serum.
Efforts to measure serum thyroid hormone levels with greater specificity and with lesser interference from nonhormonal iodinated compounds, led to the development of the butanol extractable iodine (BEI) and T4I by column techniques. All such chemical methods for the measurement of thyroid hormone in serum have been replaced by the ligand assays which are devoid of interference by even large quantities of nonhormonal iodine-containing substances.
Radioimmunoassays. Concentrations of thyroid hormones in serum can be measured by radioimmunoassays (RIA). The principle of these assays is the competition of a hormone (H), being measured, with the same isotopically labeled compound (H*) for binding to a specific class of IgG molecules present in the antiserum [antibody (Ab)]. H is the ligand and the Ab is either a polyclonal antiserum to H or a monoclonal IgG. The reaction obeys the law of mass action. Thus, at equilibrium, the amount of H* bound to Ab to form the complex Ab-H* is inversely proportional to the concentration of H, forming the complex Ab-H, provided the amounts of Ab and H* are kept constant.
AbH* + [H] AbH + H*
The radioisotope content in Ab-H* or in the unbound (free) H* is determined after their separation by precipitation of the antibody-ligand complex or adsorption of the free ligand. Some RIAs are carried out with the Ab fixed to a solid support, reacting with H and H* in solution. Increments of known amounts of H are added to a series of reactions to construct a standard curve that describes the curvilinear stoichiometric relationship between Ab-H* and H. It can be converted to a straight line by a number of mathematical transformations, such as the logit-log plot. Blank reactions contain H* but not specific Ab or, a large excess of H in a full reaction. 29 The sensitivity of the assay is dependent upon the affinity of the Ab and specific activity of H*. Under optimal conditions, as little as 1 pg of H can be measured.
In assays for thyroid hormones, the hormone needs to be liberated from serum binding proteins, mainly TBG. Methods to achieve this include extraction, competitive displacement of the hormone being measured, or inactivation of thyroxine-binding globulin (TBG). 31-34 Rarely, some patients develop circulating antibodies against thyronines that interfere with the RIA carried out on unextracted serum samples. Depending on the method used for the separation of bound from free ligand, values obtained may be either spuriously low or high in the presence of such antibodies. 38,39
A wide choice of commercial kits is available for most RIA procedures, making these assays accessible to all medical centers. RIAs have been adapted for the measurement of T4 in small samples of dried blood spots on filter paper and are used in screening for neonatal hypothyroidism. 40
Non-radioactive Methods. More recently, assays have been developed that are based on the principle of the radioligand assay but do not use radioactive material. These assays, which use ligand conjugated to an enzyme have largely replaced RIAs. The enzyme-linked ligand competes with the ligand being measured for the same binding sites on the antibody. Quantitation is carried out by spectrophotometry of the color reaction developed after the addition of the enzyme substrate. 42 Both homogeneous [enzyme-multiplied immunoassay technique (EMIT)] and heterogeneous [enzyme-linked immunosorbent assay (ELISA)] assays for T4 have been developed. 43-45 In the homogeneous assays, no separation step is required, thus providing easy automation. 43 In one such assay, T4 is linked to malate dehydrogenase, inhibiting the enzyme activity. The enzyme is activated when the T4-enzyme conjugate is bound to T4-specific antibody. Active T4 conjugates to other enzymes, such as peroxidase 44 and alkaline phosphatase, 45 have also been developed. The assay has been adapted for the measurement of T4 in dried blood samples used in mass screening programs for neonatal hypothyroidism. 45 Other non-radioisotope immunoassays use fluorescence excitation for detection of the labeled ligand, a technique which is finding increasing application. Such assay methods utilize a variety of chemiluminescent molecules such as 1,2-dioxetanes, luminol and derivatives, acridinium esters, oxalate esters and firefly luciferins, as well as many sensitizers and fluorescent enhancers. 45a One such assay which employes T4 conjugated to ÃŸ-galactosidase and fluoresence measurements of the hydrolytic product of 4-methyl-umbelliferyl-ÃŸD-galactopyranoside has been adapted for use in a microanalytical system requiring only 10Âµl of serum. 45b
Serum Total Thyroxine (TT4). The usual concentration of TT4 in adults ranges from 5 to 12 Âµg/dl (64 – 154 nmol/L). When concentrations are below or above this range in the absence of thyroid dysfunction, they are usually the result of an abnormal level of serum TBG. The hyperestrogenic state of pregnancy and administration of estrogen-containing compounds are the most common causes of a significant elevation of serum TT4 levels in euthyroid persons. Less commonly, TBG excess is inherited. 50 Serum TT4 is virtually undetectable in the fetus until midgestation. Thereafter, it rapidly increases, reaching high normal adult levels during the last trimester. A further acute but transient rise occurs within hours after delivery. 51 Values remain above the adult range until 6 years of age, but subsequent age related changes are minimal so that in clinical practice, the same normal range of TT4 applies to both sexes and all ages.
Small seasonal variations and changes related to high altitude, cold, and heat have been described. Rhythmic variations in serum TT4 concentration are of two types: variations related to postural changes in serum protein concentration 56 and true circadian variation. 31 Postural changes in protein concentration do not alter the free T4 (FT4) concentration.
Although levels of serum TT4 below the normal range are usually associated with hypothyroidism, and above this range with thyrotoxicosis, it must be remembered that the TT4 level may not always correspond to the FT4 concentration which represents the metabolically active fraction (see below). The TT4 concentration in serum may be altered by independent mechanisms: (1) an increase or decrease in the supply of T4 , as seen in most cases of thyrotoxicosis and hypothyroidism, respectively; (2) changes due solely to alterations in T4 binding to serum proteins; and (3) compensatory changes in serum TT4 concentration due to high or low serum levels of T3. Conditions associated with changes in serum TT4 and their relationship to the metabolic status of the patient are listed in Table 6-4.
|Table 6-4. Conditions Associated with Changes in Serum TT4 Concentration and Relation to the Metabolic Status|
|Metabolic Status||Serum TT4 Concentration|
|Thyrotoxic||Hyperthyroidism (all causes, including Graves disease, Plummer’s disease, toxic thyroid adenoma, early phase of subacute thyroiditis)Thyroid hormone leak (early stage of subacute thyroiditis, transient thyrotoxicosis)
Excess of exogenous or ectopic T4 (thyrotoxicosis factitia, struma ovarii)
Predominantly Pituitary resistance to thyroid hormone
|Intake of excessive amounts of T3 (thyrotoxicosis factitia)||Low TBG (congenital or acquired)T3 thyrotoxicosis (untreated or recurrent post therapy); morecommon in iodine deficient areas
Drugs competing with T4-binding to serum proteins (see also entry under euthyroid with low TT4)
Hypermetabolism of nonthyroidal origin (Luft’s syndrome)
|Euthyroid||High TBG (congenital or acquired)T4-binding albumin-like variant
Endogenous T4 antibodies
Replacement therapy with T4 only
Treatment with D-T4
Generalized resistance to thyroid hormone
|Low TBG (congenital or acquired)Endogenous T4 antibodies
Mildly elevated or normal T3 T3 replacement therapy Iodine deficiency Treated thyrotoxicosis Chronic thyroiditis Congenital goiter
Drugs competing with T4-binding to serum proteins (see Table 39-4)
|Hypothyroid||Severe generalized resistance to thyroid hormone||Thyroid gland failurePrimary (all causes, including gland destruction, severe iodine deficiency, inborn error of hormonogenesis)
Secondary (pituitary failure)
Tertiary (hypothalamic failure)
|High TBG (congenital or acquired)?Isolated peripheral tissue resistance to thyroid hormone|
Serum TT4 levels are low in conditions associated with decreased TBG concentration, the presence of abnormal TBG’s with reduced binding affinity (see Chapter 16 ) or when the available T4-binding sites on TBG are partially saturated by competing drugs present in blood in high concentrations (see Table 5-2 ). Conversely, TT4 levels are high when the serum TBG concentration is high. The person remains euthyroid provided the feedback regulation of the thyroid gland is intact.
Although changes in transthyretin (TTR) concentration rarely give rise to significant alterations in TT4 concentration, 57 the presence of a variant serum albumin with high affinity for T4 58,59 or antibodies against T4 38,39 produce apparent elevations in the measured TT4 concentration, whereas the metabolic status remain normal. The variant albumin is inherited as an autosomal dominant trait termed familial dysalbuminemic hyperthyroxinemia (FDH) (see Chapter 16 ).
Another possible cause of discrepancy between the observed serum TT4 concentration and the metabolic status of the patient is divergent changes in the serum TT3 and TT4 concentrations with alterations in the serum T3/T4 ratio. The most common situation is that of elevated TT3 concentration. The source of T3 may be endogenous, as in T3 thyrotoxicosis, or exogenous, as during ingestion of T3. In the former situation, contrary to the common variety of thyrotoxicosis, elevation in the serum TT3 concentration is not accompanied by an increase in the TT4 level. In fact, the serum TT4 level is normal and occasionally low. 60 This finding indicates that in T3 thyrotoxicosis the hormone is predominantly secreted as such rather than arising from the peripheral conversion of T4 to T3. Ingestion of pharmacologic doses of T3 results in thyrotoxicosis associated with severe depression of the serum TT4 concentration. A moderate hypersecretion of T3 can be associated with euthyroidism and a low serum TT4 concentration. This circumstance, occasionally referred to as T3 euthyroidism, may be more prevalent than T3 thyrotoxicosis. It is believed to constitute a state of compensatory T3 secretion as a physiologic adaptation of the failing thyroid gland, such as after treatment for thyrotoxicosis, in some cases of chronic thyroiditis, or during iodine deprivation. 61,62 Serum TT4 concentration is also low in normal persons receiving replacement doses of T3. Conversely, serum TT4 levels are above the upper limit of normal in 15-50% of patients treated with exogenous T4. 63 Because of the relatively slow rate of metabolism and large extrathyroidal T4 pool, the serum concentration of the hormone varies little with the time of sampling in relation to ingestion of the daily dose. 64
Serum Total Triiodothyronine (TT3). Normal serum TT3 concentrations in the adult are 80-190 ng/dl (1.2 – 2.9 nmol/L). While sex differences are small, those with age are more dramatic. In contrast to serum TT4, TT3 concentration at birth is low, about one-half the normal adult level. It rises within 24 hours to about double the normal adult value followed by a rapid decrease over the subsequent 24 hours to a level in the upper adult range, which persists for the first year of life. 51 A decline in the mean TT3 level has been observed in old age, although not in healthy subjects. 52,53 so that a fall in TT3 may refelct the prevalence of nonthyroidal illness rather than to age alone. 67 Although a positive correlation between serum TT3 level and body weight has been observed, it may be related to overeating. 68 Rapid and profound reductions in serum TT3 level can be produced within 24-48 hours of total calorie or only carbohydrate deprivation. 69-71
Most conditions causing serum TT4 levels to increase are associated with high TT3 concentrations. Thus, serum TT3 levels are usually elevated in thyrotoxicosis and reduced in hypothyroidism. However, in both conditions the TT3/TT4 ratio is elevated relative to normal euthyroid persons. This elevation is due to the disproportionate increase in serum TT3 concentration in thyrotoxicosis and a lesser diminution in hypothyroidism relative to the TT4 concentration. 72 Accordingly, measurement of the serum TT3 level is a more sensitive test for the diagnosis of hyperthyroidism, and that of TT4 more useful in the diagnosis of hypothyroidism.
There are circumstances in which changes in the serum TT3 and TT4 concentrations are either disproportionate or in opposite direction ( Table 6-5 ). These include the syndrome of thyrotoxicosis with normal TT4 and FT4 levels (T3 thyrotoxicosis). In some patients, treatment of thyrotoxicosis with antithyroid drugs may normalize the serum TT4 but not TT3 level, producing a high TT3/TT4 ratio. In areas of limited iodine supply 62 and in patients with limited thyroidal ability to process iodide, 61 euthyroidism can be maintained at low serum TT4 and FT4 levels by increased direct thyroidal secretion of T3. Although these changes have a rational physiologic explanation, the significance of discordant serum TT4 and TT3 levels under other circumstances is less well understood.
|Table 6-5. Conditions That May be Associated with Discrepancies Between the Concentration of Serum TT3 and TT4|
|Serum( + = up, – = down, N=normal)||Metabolic Status|
|+||+||N||T3-thyrotoxicosis (endogenous)||Endemic iodine deficiency (T3 autoantibodies)a||—-|
|+||N||-||Treated thyrotoxicosis (T4 autoantibodies)||Endemic cretins (severe iodine deficiency)|
|+||+||-||Pharmacologic doses of T3 (exogenous T3-toxicosis) Partially treated thyrotoxicosis||T3 replacement (especially 1 to 3 h after ingestion) Endemic iodine deficiency||(T3 autoantibodies)|
|-||-||N||Most conditions associated with reduced conversion of T4 to T3 Chronic or severe acute illness b Trauma (surgical, burns) Fasting and malnutrition Drugs c (T3 autoantibodies)a|
|-||N||+||Severe nonthyroidal illness associated with thyrotoxicosis||Neonates (first three weeks of life) T4 replacement Familial hyperthyroxinemia due to T4-binding albumin-like variant (T4 autoantibodies)a|
|-||-||+||At birth Acute nonthyroidal illness withtransient hyperthyroxinemia||(T4 autoantibodies)a|
|a Artifactual values dependent upon the method of hormone determination in serum. b Hepatic and renal failure, diabetic ketoacidosis, myocardial infarction, infectious and febrile illness, malignancies c Glucocorticoids, iodinated contrast agents, amiodarone, propranolol, propylthiouracil|
The most common cause of discordant serum concentrations of TT3 and TT4 is a selective decrease of serum TT3 due to decreased conversion of T4 to T3 in peripheral tissues. This reduction is an integral part of the pathophysiology of a number of nonthyroidal acute and chronic illnesses and calorie deprivation (see Chapter 5 ). In these conditions, the serum TT3 level is often lower than that commonly found in patients with frank primary hypothyroidism. Yet, these persons do not present clear clinical evidence of hypometabolism. In some individuals, decreased T4 to T3 conversion in the pituitary gland 75 or in peripheral tissues 76 is thought to be an inherited condition.
A variety of drugs may also produce changes in the serum TT3 concentration without apparent metabolic consequences (see Chapter 6 ). Drugs that compete with hormone binding to serum proteins decrease serum TT3 levels, generally without affecting the free T3 concentration ( Table 5-5 ). Some drugs, such as glucocorticoids, 77 depress the serum TT3 concentration by interfering with the peripheral conversion of T4 to T3. Others, such as phenobarbital, 78 depress the serum TT3 concentration by stimulating the rate of intracellular hormone degradation. The majority have multiple effects. These effects are combinations of those described above, as well as inhibition of the hypothalamic-pituitary axis or thyroidal hormonogenesis. 79
Changes in serum TBG concentration have an effect on the serum TT3 concentration similar to that on TT4 (see Chapter 16 ). The presence of endogenous antibodies to T3 may result in apparent elevation of the serum TT3 but as in the case of high TBG, it does not cause hypermetabolism. 38
Administration of commonly used replacement doses of T3, usually in the order of 75 Âµg/day or 1 Âµg/kg body weight per day, 80 results in serum TT3 levels in the thyrotoxic range. Furthermore, because of the rapid gastrointestinal absorption and relatively fast degradation rate, the serum level varies considerably according to the time of sampling in relation to hormone ingestion. 64
Because the concentration of thyroid hormone in serum is dependent on its supply as well as on the abundance of hormone-binding sites on serum proteins, the estimation of the latter has proved useful in the correct interpretation of values obtained from the measurement of the total hormone concentration. These results have been used to provide an estimate of the free hormone concentration, which is important in differentiating changes in serum total hormone concentration due to alterations of binding proteins in euthyroid patients from those due to abnormalities in thyroid gland activity giving rise to hypermetabolism or hypometabolism.
In Vitro Uptake Tests: In vitro uptake tests measure the unoccupied thyroid hormone-binding sites on TBG. They use labeled T3 or T4 and some form of synthetic absorbent to measure the proportion of radiolabeled hormone that is not tightly bound to serum proteins. Because ion exchange resins are often used as absorbents, the test became known as the resin T3 or T4 uptake test (T3U or T4U), describing the technique rather than the entity measured.
The test is usually carried out by incubating a sample of the patient’s serum with a trace amount of labeled T3 or T4. The labeled hormone, not bound to available binding sites on TBG present in the serum sample, is absorbed onto an anion exchange resin and measured as resin-bound radioactivity. Values correlate inversely with the concentration of unsaturated TBG. Various methods use different absorbing materials to remove the hormone not tightly bound to TBG. 83 Labeled T3 is usually used because of its less firm yet preferential binding to TBG. Depending upon the method, typical normal results for T3U are 25-35% or 45-55%. Thus, it is more valuable to express results of the uptake tests as a ratio of the result obtained in a normal control serum run in the same assay as the test samples. Normal values will then range on either side of 1.0, usually 0.85-1.15.
The uptake of the tracer by the absorbent is inversely proportional to the amount of unsaturated binding sites (unoccupied by endogenous thyroid hormone) in serum TBG. Thus, the uptake is increased when the amount of unsaturated TBG is reduced as a result of excess endogenous thyroid hormone or a decrease in the concentration of TBG. In contrast, the uptake is decreased when the amount of unsaturated TBG is increased as a result of a low serum thyroid hormone concentration or an increase in the concentration of TBG. Since the test can be affected by either or both independent variables, serum total thyroid hormone and TBG concentrations, the results cannot be interpreted without knowledge of the hormone concentration. As a rule, parallel increases or decreases in both serum TT4 concentration and the T3U test indicate hyperthyroidism and hypothyroidism, respectively, whereas discrepant changes in serum TT4 and T3U suggest abnormalities in TBG binding. However, abnormalities in hormone and TBG concentrations may coexist in the same patient. For example, a hypothyroid patient with a low TBG level will typically show a low TT4 level and normal T3U result (Figure 6-4). Several nonhormonal compounds, due to structural similarities, compete with thyroid hormone for its binding site on TBG. Some are used as pharmacologic agents and may thus alter the in vitro uptake test as well as the total thyroid hormone concentration in serum. A list is provided in Table 5-2 .
TBG and TTR Measurements.
The concentrations of TBG and TTR in serum can be either estimated by measurement of their total T4-binding capacity at saturation or more usually measured directly by immunologic techniques. 87,88
TBG concentration in serum can be determined by RIA, 88 and both TBG and TTR can be measured by Laurell’s rocket immunoelectrophoresis, 89,90 by radial immunodiffusion, 91 or by enzyme immunoassay; 87 commercial methods are available. The true mean value for TBG is 1.6 mg/dl (260 nmol/L), with a range of 1.1 – 2.2 mg/dl(180 – 350 nmol/L) serum. In adults, the normal range for TTR is 16 – 30 mg/dl (2.7 – 5.0 Âµmol/L). The concentrations of TBG and TTR in serum vary with age, sex, pregnancy, and posture. Determination of the concentration of these proteins in serum is particularly helpful in evaluation of extreme deviations from normal, as in congenital abnormalities of TBG. In most instances, however, the in vitro uptake test, in conjunction with the serum TT4 level, gives an approximate estimation of the TBG concentration.
A minute amount of thyroid hormone circulates in the blood in a free form, not bound to serum proteins. It is in reversible equilibrium with the bound hormone and represents the diffusible fraction of the hormone capable of traversing cellular membranes to exert its effects on body tissues. 94 Although changes in serum hormone-binding proteins affect both the total hormone concentration and the corresponding fraction circulating free, in the euthyroid person the absolute concentration of free hormone remains constant and correlates with the tissue hormone level and its biologic effect. Information concerning this value is probably the most important parameter in the evaluation of thyroid function as it relates to the metabolic status of the patient.
With few exceptions, the free hormone concentration is high in thyrotoxicosis, low in hypothyroidism, and normal in euthyroidism even in the presence of profound changes in TBG concentration, 97 provided the patient is in a steady state (see Fig. 5-4). Notably, free T4 (FT4) concentration may be normal or even low in patients with T3 thyrotoxicosis and in those ingesting pharmacologic doses of T3. On occasion, the concentration of FT4 may be outside the normal range in the absence of an apparent abnormality in the thyroid hormone-dependent metabolic status. This is frequently observed in severe nonthyroidal illness during which both high and low values have been reported. 98-100 As expected, when a euthyroid state is maintained by the administration of T3 or by predominant thyroidal secretion of T3, the FT4 level is also depressed. More consistently, patients with a variety of nonthyroidal illnesses have low FT3 levels. 101 This decrease is characteristic of all conditions associated with depressed serum TT3 concentrations due to a diminished conversion of T4 to T3 in peripheral tissues (see Chapter 5 ). Both FT4 and FT3 values may be out of line in patients receiving a variety of drugs (see below). Marked elevations in both FT4 and FT3 concentrations in the absence of hypermetabolism are typical of patients with resistance to thyroid hormone (see Chapter 16 ). The FT3 concentration is usually normal or even high in hypothyroid persons living in areas of severe endemic iodine deficiency. Their FT4 levels are, however, normal or low. 62
Direct Measurement of Free T4 and Free T3. Direct measurements of the absolute FT4 and FT3 concentrations are technically difficult and have, until recently, been limited to research assays. In order to minimize perturbations of the relationship between the free and bound hormone, these must be separated by ultrafiltration or by dialysis involving minimal dilution and little alteration of the pH or electrolyte composition. The separated free hormone is then measured directly by radioimmunoassay or chromatography. 97,97a These assays are probably the most accurate available, but small, weakly bound, dialyzable substances or drugs may be removed from the binding proteins and the free hormone concentration measured in their presence may not fully reflect the free concentration in vivo.
Isotopic Equilibrium Dialysis. This method has been the “gold standard” for the estimation of the FT4 or FT3 concentration for almost 30 years. It is based on the determination of proportion of T4 or T3 that is unbound, or free, and is thus able to diffuse through a dialysis membrane, i.e., the dialyzable fraction (DF). To carry out the test, a sample of serum is incubated with a tracer amount of labeled T4 or T3. The labeled tracer rapidly equilibrates with the respective bound and free endogenous hormones. The sample is then dialyzed against buffer at a constant temperature until the concentration of free hormone on either side of the dialysis membrane has reached equilibrium. The DF is calculated from the proportion of labeled hormone in the dialysate. The contribution from radioiodide present as contaminant in the labeled tracer hormone should be eliminated by purification 98 and by various techniques of precipitation of the dialyzed hormone.102 FT4 and FT3 levels can be measured simultaneously by addition to the sample of T4 and T3 labeled with two different radioiodine isotopes.103 Ultrafiltration is a modification of the dialysis technique. 98 Results are expressed as the fraction (DFT4 or DFT3) or percent (%FT4 or %FT3) of the respective hormones which dialyzed and the absolute concentrations of FT4 and FT3 are calculated from the product of the total concentration of the hormone in serum and its respective DF. Typical normal values for FT4 in the adult range from 1.0 to 3.0 ng/dl (13 – 39 pmol/L) and for FT3 from 0.25 to 0.65 ng/dl (3.8 – 10 nmol/L).
Results by these techniques are generally comparable to those determined with the direct, one step, methods (see below) but are more likely to differ with extremely low or extremely high TBG concentrations or in the presence of circulating inhibitors of protein binding, especially in situations of non-thyroidal illness. 104, 104a,104b The measured DF may be altered by the temperature at which the assay is run, the degree of dilution, the time allowed for equilibrium to be reached and the composition of the diluting fluid. 105 The calculated value is dependent on an accurate measurement of total T4 or T3 and may be incorrect in patients with T4 or T3 autoantibodies. Some of these problems, particularly those arising from dilution, may be superceded by commercially available dialysis methods or ultrafiltration methods of free from bound hormone which do not necessitate serum dilution.
Index Methods. As the determination of free hormone by equilibrium dialysis is cumbersome and technically demanding, many clinical laboratories have used a method by which a free T4 index (FT4I) or free T3 index (FT3I) is derived from the product of the TT4 or TT3 (determined by immunoassay) and the value of an in vitro uptake test (see below). While not always in agreement with the values obtained by dialysis, these techniques are rapid and simple. They are more likely to fail at extremely low or extremely high TBG concentrations, in the presence of abnormal binding proteins, in the presence of circulating inhibitors of protein binding , and their reliability has been questioned in patients with non-thyroidal illness.
The theoretical contention that the FT4I is an accurate estimate of the absolute FT4 concentration can be confirmed by the linear correlation between these two parameters. This is true provided results of the in vitro uptake test (T3U or T4U) are expressed as the thyroid hormone binding ratio (THBR), determined by dividing the tracer counts bound to the solid matrix by counts bound to serum proteins. 106 Values are corrected for assay variations using appropriate serum standards and are expressed as the ratio of a normal reference pool. 106,107 The normal range is slightly narrower than the corresponding TT4 in healthy euthyroid patients with a normal TBG concentration. It is 6.0 – 10.5 Âµg/dl or 77 – 135 nmol/l when calculated from TT4 values measured by RIA. In thyrotoxicosis, FT4I is high and in hypothyroidism it is low irrespective of the TBG concentration. Euthyroid patients with TT4 values outside the normal range as a result of TBG abnormalities have a normal FT4I. 83 Lack of correlation between the FT4I and the metabolic status of the patient has been observed under the same circumstances as those described for similar discrepancies when the FT4 concentration was measured by dialysis.
Methods for the estimation of the FT3I are also available 103 but are rarely used in routine clinical evaluation of thyroid function. Like the FT4I, it correlates well with the absolute FT3 concentration. The test corrects for changes in TT3 concentration resulting from variations in TBG concentration.
Estimation of FT4 and FT3 Based on TBG Measurements. Since most T4 and T3 in serum are bound to TBG, their free concentration can be calculated from their binding affinity constants to TBG and molar concentrations of hormones and TBG. 109,110 A simpler calculation of the T4/TBG and T3/TBG ratios yields values that are similar to but less accurate than the FT4I and FT3I, respectively. 106
Two-step Immunoassays. In these assays, the free hormone is first immunoextracted by a specific bound antibody (first step), frequently fixed to the tube (coated tube). 111,112 After washing, labeled tracer is added and allowed to equilibrate between the unoccupied sites on the antibody and those of serum thyroid hormone-binding proteins. The free hormone concentration will be inversely related to the antibody bound tracer and values are determined by comparison to a standard curve. Values obtained with this technique are generally comparable to those determined with the direct methods. They are more likely to differ in the presence of circulating inhibitors of protein binding and in sera from patients with non-thyroidal illness.
Analog (One-Step) Immunoassays. In these assays, a labeled analog of T4 or T3 directly competes with the endogenous free hormone for binding to antibodies. 113 In theory, these analogs are not bound by the thyroid hormone binding proteins in serum. However, various studies have found significant protein binding to the variant albumin-like protein, 113a to transthyretin and to iodothyronine autoantibodies. 114 This results in discrepant values to other assays in a number of conditions including non-thyroidal illness, pregnancy and in individuals with familial dysalbuminemic hyperthyroxinemia (FDH). 113a A growing number of commercial kits is available some of which have been modified to minimize these problems, 113b . Nonetheless, their accuracy remains controversial, although such comercial methods are being increasingly adopted in the routine clinical chemistry laboratory. 112
Considerations in Selection of Methods for the Estimation of Free Thyroid Hormone Concentration. None of the available methods for the estimation of the free hormone concentration in serum is infallible in the evaluation of the thyroid hormone-dependent metabolic status. Each test possesses inherent advantages and disadvantages depending upon specific physiologic and pathologic circumstances. For example, methods based on the measurement of the total thyroid hormone and TBG concentrations cannot be used in patients with absent TBG due to inherited TBG deficiency. Under such circumstances, the concentration of free thyroid hormone is dependent upon the interaction of the hormone with serum proteins that normally play a negligible role (TTR and albumin). When alterations of thyroid hormone binding do not equally affect T4 and T3, discrepant results of FT4I are obtained when using labeled T4 or T3 in the in vitro uptake test. For example, euthyroid patients with the inherited albumin variant (FDH) or having endogenous antibodies with greater affinity for T4 will have high TT4 but a normal T3U test which will result in an overestimation of the calculated FT4I. In such instances, calculation of the FT4I from a T4U test may provide more accurate results. Conversely, reduced overall binding affinity for T4 which affects T3 to a lesser extent will underestimate the FT4I derived from a T3U test. Similarly, use of the T4U and T3U for estimation of the free hormone concentration, is satisfactory in the presence of alterations in TBG concentration but not alterations of the affinity of TBG for the hormone. 116,117
Methods based on equilibrium dialysis are most appropriate in the estimation of the free thyroid hormone level in patients with all varieties of abnormal binding to serum proteins provided the true concentration of total hormone has been accurately determined. All methods for the estimation of the FT4 concentration may give either high or low values in patients with severe nonthyroidal illness. 96-100 , 119 , 120 This has been attributed to the presence of inhibitors of thyroid hormone binding to serum proteins as well as to the various adsorbents used in the test procedures. 121,122 Some of these inhibitors have been postulated to leak from the tissues of the diseased patient. 123,124 Such discrepancies are even more pronounced during transient states of hyperthyroxinemia or hypothyroxinemia associated with acute illness, after withdrawal of treatment with thyroid hormone and in acute changes in TBG concentration (see Chapters 5 and 16 ).
The contribution of various drugs that interfere with binding of thyroid hormone to serum proteins or with the in vitro tests should also be taken into account in the choice and interpretation of tests (see Table 5-2 ). Although the free thyroid hormone concentration in serum seems to determine the amount of hormone available to body tissues, factors that govern their uptake, transport to the nucleus and functional interactions with nuclear receptors ultimately determine their biological effects.
Measurements of Iodine-Containing Hormone Precursors and Products of Degradation
The last two decades have witnessed the development of RIAs for the measurement of a number of naturally occurring, iodine-containing substances that possess little if any thyromimetic activity. Some of these substances are products of T4 and T3 degradation in peripheral tissues. Others are predominantly, if not exclusively, of thyroidal origin. Since they are devoid of significant metabolic activity, measurement of their concentration is of value only in the research setting in detecting abnormalities in the metabolism of thyroid hormone in peripheral tissues, as well as defects of hormone synthesis and secretion.
3,3′,5′-Triiodothyronine or Reverse T3 (rT3). rT3 is principally a product of T4 degradation in peripheral tissues (see Chapter 3). It is also secreted by the thyroid gland, but the amounts are practically insignificant. 126 Thus, measurement of rT3 concentration in serum reflects both tissue supply and metabolism of T4 and identifies conditions that favor this particular pathway of T4 degradation.
When total rT3 (TrT3) is measured in unextracted serum, a competitor of rT3 binding to serum proteins must be added. 127 Several chemically related compounds may cross-react with the antibodies. The strongest cross-reactivity is observed with 3,3′-T2 but this does not present a serious methodologic problem because of its relatively low levels in human serum. Though cross-reactivity with T3 and T4 is lesser, these compounds are more often the cause of rT3 overestimation due to their relative abundance, particularly in thyrotoxicosis. 128 Free fatty acids interfere with the measurement of rT3 by RIA. 129
The normal range in adult serum for TrT3 is 14-30 ng/dl (0.22 – 0.46 nmol/L) although varying values have been reported. It is elevated in subjects with high TBG and in some individuals with FDH. 132 Serum TrT3 levels are normal in hypothyroid patients treated with T4, indicating that peripheral T4 metabolism is an important source of circulating rT3. 126 , 133 Values are high in thyrotoxicosis and low in untreated hypothyroidism. High values are normally found in cord blood and in newborns. 133,134
With only a few exceptions, notably uremia, serum TrT3 concentrations are elevated in all circumstances that cause low serum T3 levels in the absence of obvious clinical signs of hypothyroidism. These conditions include, in addition to the newborn period, a variety of acute and chronic nonthyroidal illnesses, calorie deprivation, and the influence of a growing list of clinical agents and drugs (see Table 5-3 ).
Current clinical application of TrT3 measurement in serum is in the differential diagnosis of conditions associated with alterations in serum T3 and T4 concentrations when thyroid gland and metabolic abnormalities are not readily apparent.
The dialyzable fraction of rT3 in normal adult serum is 0.2 – 0.32%, or approximately the same as that of T3. The corresponding serum FrT3 concentration is 50 – 100 pg/dl (0.77 – 1.5 pmol/L). In the absence of gross TBG abnormalities, variations in serum FrT3 concentration closely follow those of TrT3. 101
3,5-Diiodothyronine (3,5-T2). The normal adult range for total 3,5-T2 in serum measured by direct RIAs is 0.20 – 0.75 ng/dl (3.8 – 14 pmol/L). 135 That 3,5-T2 is derived from T3 is supported by the observations that conditions associated with high and low serum T3 levels have elevated and reduced serum concentrations of 3,5-T2, respectively. 136 Thus, high serum 3,5-T2 levels have been reported in hyperthyroidism, and low levels in serum of hypothyroid patients, newborns, during fasting, and in patients with liver cirrhosis.
3,3′-Diiodothyronine (3,3′-T2). Normal concentrations in adults probably range from 1 to 8 ng/dl (19 – 150 pmol/L). 137 Levels are clearly elevated in hyperthyroidism and in the newborn. Values have been found to be either normal or depressed in nonthyroidal illnesses, 137 in agreement with the demonstration of reduced monodeiodination of rT3 to 3,3′-T2. 138 In vivo turnover kinetic studies and measurement of 3,3′-T2 in serum after the administration of T3 and rT3 have clearly shown that 3,3′-T2 is the principal metabolic product of these two triiodothyronines.
3′,5′-Diiodothyronine (3′,5′-T2). Reported concentrations in serum of normal adults have a mean overall range of 1.5 – 9.0 ng/dl (30 – 170 pmol/L). 139,140 The substances that principally cross react in the assay are rT3, 3,3-LT2 and 3-T1. Values are high in hyperthyroidism and in the newborn. 139,140 Being the derivative of rT3 monodeiodination, 139 3′,5′-T2 levels are elevated in serum during fasting 140,141 and in chronic illnesses 133 in which the level of the rT3 precursor is also high. Administration of dexamethasone also produces an increase in the serum 3′,5′-T2 level. 139
3′-Monoiodothyronine (3′-T1). The concentration of 3′-T1 in serum of normal adults, measured by RIA, has been reported to range from 0.6 to 2.3 ng/dl (15 – 58 pmol/L) 133 and from <0.9 to 6.8 ng/dl (<20 – 170 pmol/L). Its two immediate precursors, 3,3,’-T2 and 3′,5′-T2 are the main cross-reactants in the RIA. Serum levels are very high in hyperthyroidism and low in hypothyroidism. The concentration of 3′-T1 in serum is elevated in all conditions associated with high rT3 levels, including newborns, nonthyroidal illness, and fasting. 134 This finding is not surprising since the immediate precursor at 3′-T1 is 3′,5′-T2, 142 a product of rT3 deiodination, which is also present in serum in high concentration under the same circumstances. The elevated serum levels of 3′-T1 in renal failure are attributed to decreased clearance since the concentrations of its precursors are not increased.
3-Monoiodothyronine (3-T1). Experience with the measurement of 3-T1 in serum is limited. Normal values in serum of adult humans using 3H labeled 3-T1 in a specific RIA ranged from <0.5 – 7.5 ng/dl (<13 – 190 pmol/L). 143 The mean concentration of 3-T1 in serum of thyrotoxic patients and in cord blood was significantly higher. 3-T1 appears to be a product of in vivo deiodination of 3,3′-T2.
Tetraiodothyroacetic Acid (TETRAC or T4A) and Triiodothyroacetic Acid (TRIAC or T3A). The iodoamino acids T4A and T3A, products of deamination and oxidative decarboxylation of T4 and T3, respectively, have been detected in serum by direct RIA measurements. 21 , 76 , 144 Reported mean concentrations in the serum of healthy adults have been 8.7 ng/dl 144 and 2.6 ng/dl (range, 1.6 – 3.0 ng/dl or 26 – 48 pmol/L)) 21 for T3A and 28 ng/dl (range <8 – 60 mg/dl or <105 – 800 pmol/L) 76 for T4A. Serum T4A levels are reduced during fasting and in patients with severe illness, 145 although the percentage of conversion of T4 to T4A is increased. 20 , 146 The concentration of serum T3A remains unchanged during the administration of replacement doses of T4 and T3. 21 It has been suggested that intracellular rerouting of T3 to T3A during fasting is responsible for the maintenance of normal serum TSH levels in the presence of low T3 concentrations. 147
3,5,3′-T3 Sulfate (T3S). A RIA procedure to measure T3S in ethanol extracted serum samples is available. 22 Concentrations in normal adults range from 4-10 ng/dl (50-125 pmol/L). Although the principal source of T3S is T3, and the former binds to TBG, values are high in newborns and low in pregnancy. This suggests different rates of T3S generation or metabolism in mother and fetus. T3S values are high in thyrotoxicosis and in nonthyroidal illness.
Diiodotyrosine (DIT) and Monoiodotyrosine (MIT). Although RIA methods for the measurement of DIT and MIT have been developed, due to limited experience, their value in clinical practice remains unknown. Early reports gave a normal mean value for DIT in serum of normal adults of 156 ng/dl (3.6 nmol/L), 148 with progressive decline due to refinement of techniques to values as low as 7 ng/dl with a range of 1 – 23 ng/dl (0.02 – 0.5 nmol/L). 149 Thus, the normal range for MIT of 90 – 390 ng/dl (2.9 – 12.7 nmol/L) 150 is undoubtedly an overestimation. Iodotyrosine that has escaped enzymatic deiodination in the thyroid gland appears to be the principal source of DIT in serum. Iodothyronine degradation in peripheral tissues is probably a minor source of iodotyrosines since administration of large doses of T4 to normal subjects produces a decline rather than an increase in the serum DIT level. 149 DIT is metabolized to MIT in peripheral tissues. Serum levels of DIT are low during pregnancy and high in cord blood.
Thyroglobulin (Tg). RIA methods were those first used routinely for measurement of Tg in serum, 151 , although other assays methods employing IRMA, ICMA, and ELISA technology have been reported 151a-d and are gaining increasing popularity. They are specific and, depending upon the sensitivity of the assay, capable of detecting Tg in the serum of approximately 90% of the euthyroid healthy adults. When antisera are used in high dilutions, there is virtually no cross-reactivity with iodothyronines or iodotyrosines. Results obtained from the analysis of sera containing Tg autoantibodies may be inaccurate, depending upon the antiserum employed. 152 The presence of thyroid peroxidase antibodies does not interfere with the Tg RIA. Despite the relaibility of measurements of serum Tg, it is clear that different assay methods may result in values discrepant by up to 30%, even though refernce preparations are available. 152a Typically, IMA methods underestimate the serum Tg value, while RIA methods overestimate it, so it is essential that clinical decisions are based upon serial measurements using the same assay.
Tg concentrations in serum of normal adults range from <1 to 25 ng/ml (<1.5 – 38 pmol/L), with mean levels of 5 – 10 ng/ml. 151 , 153-155 On a molar basis, these concentrations of Tg are minute relative to the circulating iodothyronines; 5,000-fold lower than the corresponding concentration of T4 in serum. Values tend to be slightly higher in women than in men. 151 In the neonatal period and during the third trimester of pregnancy, mean values are approximately 4- and 2-fold higher. 154,156 They gradually decline throughout infancy, childhood and adolescence. 157 The positive correlation between the levels of serum Tg and TSH indicates that pituitary TSH regulates the secretion of Tg.
Elevated serum Tg levels reflect increased secretory activity by stimulation of the thyroid gland or damage to thyroid tissue, whereas values below or at the level of detectability indicate a paucity of thyroid tissue or suppressed activity. Tg levels in a variety of conditions affecting the thyroid gland have been reviewed 158 and are listed in Table 6-6.
|Table 6-6 Conditions Associated with Changes in Serum Tg Concentration Listed According to the Presumed Mechanism|
|IncreasedTSH mediated Acute and transient (TSH and TRH administration, neonatal period) Chronic stimulation Iodine deficiency, endemic goiter, goitrogens Reduce thyroidal reserve (lingual thyroid) TSH-producing pituitary adenoma Generalized resistance to thyroid hormone TBG deficiency
Non-TSH mediated Thyroid stimulators IgG (Graves’ disease) hCG (trophoblastic disease) Trauma to the thyroid (needle aspiration and surgery of the thyroid gland, 131I therapy) Destructive thyroid pathology Subacute thyroiditis “Painless thyroiditis” Postpartum thyroiditis Abnormal release Thyroid nodules (toxic, nontoxic, multinodular goiter) Differentiated nonmedullary thyroid carcinoma Ab normal clearance (renal failure)
|DecreasedTSH suppression Administration of thyroid hormone
Decreased synthesis Athyreosis (postoperative, congenital) Tg synthesis defect
Interpretation of a serum Tg value should take into account the fact that Tg concentrations may be high under normal physiologic conditions or altered by drugs. Administration of iodine and antithyroid drugs increase the serum Tg level, as do states associated with hyperstimulation of the thyroid gland by TSH or other substances with thyroid-stimulating activity. This is due to increased thyroidal release of Tg rather than changes in its clearance. 159 Administration of TRH and TSH also transiently increases the serum level of Tg. 160 Trauma to the thyroid gland, such as that occurring during diagnostic and therapeutic procedures including percutaneous needle biopsy, surgery, or 131I therapy, can produce a striking, although short-lived, elevation in the Tg level in serum. 154 , 161,162 Pathological processes with destructive effect on the thyroid gland also produce transient, though more prolonged increases. 163 Tg is undetectable in serum after total ablation of the thyroid gland as well as in normal persons receiving suppressive doses of thyroid hormone. 158 It is thus a useful test in the differential diagnosis of thyrotoxicosis factitia, 164 especially when transient thyrotoxicosis with a low RAIU or suppression of thyroidal RAIU by iodine are alternative possibilities.
The most striking elevations in serum Tg concentrations have been observed in patients with metastatic differentiated nonmedullary thyroid carcinoma even after total surgical and radioiodide ablation of all normal thyroid tissue. 154 , 165 It usually persists despite full thyroid hormone suppressive therapy, suggesting excessive autonomous release of Tg by the neoplastic cells. The determination is thus of particular value in the follow-up and management of metastatic thyroid carcinomas, particularly when they fail to concentrate radioiodide. 153 , 165 Follow-up of such patients with sequential serum Tg determinations helps the early detection of tumor recurrence or growth and the assessment of the efficacy of treatment. Measurement of serum Tg is also useful in patients with metastases, particularly to bone, in whom there is no evidence of a primary site and thyroid malignancy is being considered in the differential diagnosis. 154 , 165 On the other hand, serum Tg levels are of no value in the differential diagnosis of primary thyroid cancer because levels may be within the normal range in the presence of differentiated thyroid cancer and high in a variety of benign thyroid diseases. 153,155 , 165 Whether early detection of recurrent thyroid cancer after initial ablative therapy could be achieved by serum Tg measurement without cessation of hormone replacement therapy is debated because Tg secretion by the tumor is modulated by TSH and is suppressed by the administration of thyroid hormone. 166-168 Detectable serum thyroglobulin during thyroid hormone suppression reliably indicated the presence residual or recurrent disease.
Tg levels are high in the early phase of subacute thyroiditis. 163 Declining serum Tg levels during the course of antithyroid drug treatment of patients with Graves’ disease may indicate the onset of a remission. 162 , 169 Tg may be undetectable in the serum of neonates with dyshormonogenetic goiters due to defects in Tg synthesis 170 but are very high in some hypothyroid infants with thyromegaly or ectopy. 171 Measurement of serum Tg in hypothyroid neonates is useful in the differentiation of infants with complete thyroid agenesis from those with hypothyroidism due to other causes, and thus in most cases obviates the need for the diagnostic administration of radioiodide. 171 , 172
Measurement of Thyroid Hormone and Its Metabolites in Other Body Fluids and in Tissues
Clinical experience with measurement of thyroid hormone and its metabolites in body fluids other than serum and in tissues is limited for several reasons. Analyses carried out in urine and saliva do not appear to give additional information, not obtained from measurements carried out in serum. Amniotic fluid, cerebrospinal fluid, and tissues are less readily accessible for sampling. Their likely application in the future will depend on information they could provide beyond that obtained from similar analyses in serum.
Because thyroid hormone is filtered in the urine predominantly in free form, measurement of the total amount excreted over 24 hours offers an indirect method for the estimation of the free hormone concentration in serum. The 24-hour excretion of T4 in normal adults ranges from 4 to 13 Âµg and from 1.8 to 3.7 Âµg, depending upon whether total or only conjugated T4 is measured. Corresponding normal ranges for T3 are 2.0 – 4.0 Âµg and 0.4 – 1.9 Âµg. 173-175 Striking seasonal variations have been shown for the urinary excretion of both hormones, with a nadir during the hot summer months, in the absence of significant changes in serum TT4 and TT3. As expected, values are normal in pregnancy and in nonthyroidal illnesses, and are high in thyrotoxicosis and low in hypothyroidism. 174 , 175 The test may not be valid in the presence of gross proteinuria and impairment of renal function. 176
Amniotic Fluid (AF)
All iodothyronines measured in blood have also been detected in AF. With the exception of T3, 3,3′-T2 and 3′-T2, the concentration at term is lower than that in cord serum. 139,140 , 142 , 177-179 This fact cannot be fully explained by the low TBG concentration in AF. Although the source of iodothyronines in AF is unknown, the general pattern more closely resembles that found in the fetal than in the maternal circulation.
The TT4 concentration in AF average 0.5 Âµg/dl (65 nmol/L) with a range of 0.15 – 1.0 Âµg/dl and is thus very low when compared to values in maternal and cord serum. 177-179 The FT4 concentration is, however, twice as high in AF relative to serum. The TT3 concentration is also low relative to maternal serum being on the average 30 ng/dl (0.46 nmol/L) in both AF and cord serum. 179 rT3, on the other hand, is very high in AF, on average 330 ng/dl (5.1 nmol/L) during the first half of gestation, declining precipitously at about the 30th week of gestation to an average of 85 ng/dl (1.3 nmol/L) which is also found at term. 178,179
Cerebrospinal Fluid (CSF)
T4, T3, and rT3 concentrations have been measured in human CSF. 180-182 The concentrations of both TT4 and TT3 are approximately 50-fold lower than those found in serum. However, the concentrations of these iodothyronines in free form are similar to those in serum. In contrast, the level of TrT3 in CSF is only 2.5-fold lower than that of serum, whereas that of FrT3 is 25-fold higher. This is probably due to the presence in CSF of a larger proportion of TTR which has high affinity for rT3. 181 All the thyroid hormone-binding proteins present in serum are also found in CSF, although in lower concentrations. 181 The concentrations of TT4 and FT4 are increased in thyrotoxicosis and depressed in hypothyroidism. Severe nonthyroidal illness gives rise to increased TrT3 and FrT3 levels. 182
TT4 concentration in human milk is of the order of 0.03 – 0.5 Âµg/dl. 183 Analytical artifacts were responsible for the much higher values formerly reported. 183,184 TT3 concentrations range from 10 to 200 ng/dl (015 – 3.1 nmol/L). 184,185 The concentration of TrT3 ranges from 1 – 30 ng/dl (15 – 460 pmol/L). 184 Thus, it is unlikely that milk would provide a sufficient quantity of thyroid hormone to alleviate hypothyroidism in the infant.
It has been suggested that only the free fraction of small nonpeptide hormones which circulate predominantly bound to serum proteins would be transferred to saliva and that their measurement, in this easily accessible body fluid, would provide a simple and direct means to determine their free concentration in blood. This hypothesis was confirmed for steroid hormones, 186 not tightly bound to serum proteins. Levels of T4 in saliva range from 4.2 – 35 ng/dl (54 – 450 pmol/L) and do not correlate with the concentration of free T4 in serum. 187 This finding is, in part, due to the transfer of T4 bound to the small but variable amounts of serum proteins that reach the saliva.
TT4 measured in fluid obtained from serous cavities bears a direct relationship to the protein content and the serum concentration of T4. Limited experience with Tg measurement in pleural effusions from patients with thyroid cancer metastatic to lungs suggests that it may be of diagnostic value. 165
Since the response to thyroid hormone is expressed at the cell level, it is logical to assume that hormone concentration in tissues should correlate best with its action. Methods for extraction, recovery, and measurement of iodothyronines from tissues have been developed but, for obvious reasons, data from thyroid hormone measurements in human tissues are limited. Preliminary work has shown that under several circumstances, hormonal levels in tissues such as liver, kidney, and muscle usually correlate with those found in serum. 188
Measurements of T3 in cells most accessible for sampling in humans, namely, red blood cells gave values of 20 – 45 ng/dl (0.31 – 0.69 nmol/L) or one-fourth those found in serum. 189 They are higher in thyrotoxicosis and lower in hypothyroidism.
The concentrations of all iodothyronines have been measured in thyroid gland hydrolysates. 18 , 133 , 139 In normal glands, the molar ratios relative to the concentration of T4 are on average as follows: T4/T3 = 10; T4/rT3 = 80; T4/3,5′-T2 = 1,400; T4/3,3′-T2 = 350; T4/3′,5′-T2 = 1,100; and T4/3′-T1 = 4,400. Information concerning the content of iodothyronines in hydrolysates of abnormal thyroid tissue is limited, and the diagnostic value of such measurements has not been established.
Measurement of Tg in metastatic tissue obtained by needle biopsy may be of value in the differential diagnosis, especially when the primary site is unknown and the histological diagnosis is not conclusive.
Tests Assessing the Effects of Thyroid Hormone on Body Tissues
Thyroid hormone regulates a variety of biochemical reactions in virtually all tissues. Thus, ideally, the adequacy of hormonal supply should be assessed by the tissue responses rather than by parameters of thyroid gland activity or serum hormone concentration which are several steps removed from the site of thyroid hormone action. Unfortunately, the tissue responses (metabolic indices) are nonspecific because they are altered by a variety of physiologic and pathologic mechanisms unrelated to thyroid hormone deprivation or excess. The following review of biochemical and physiologic changes mediated by thyroid hormone has a dual purpose: (1) to outline some of the changes that may be used as clinical tests in the evaluation of the metabolic status, and (2) to point out the changes in various determinations commonly used in the diagnosis of a variety of nonthyroidal illnesses, which may be affected by the concomitant presence of thyroid hormone deficiency or excess.
The BMR has a long history in the evaluation of thyroid function. It measures the oxygen consumption under basal conditions of overnight fast and rest from mental and physical exertion. Since standard equipment for the measurement of BMR may not be readily available, it can be estimated from the oxygen consumed over a timed interval by analysis of samples of expired air. 190 The test indirectly measures metabolic energy expenditure or heat production.
Results are expressed as the percentage of deviation from normal after appropriate corrections have been made for age, sex, and body surface area. Low values are suggestive of hypothyroidism, and high values reflect thyrotoxicosis. The various nonthyroidal illnesses and other factors affecting the BMR, including sources of errors, have been reviewed. 191 Although this test is no longer a part of the routine diagnostic armamentarium, it is still useful in research.
Delay in the relaxation time of the deep tendon reflexes, visible to the experienced eye, occurs in hypothyroidism. Several instruments have been devised to quantitate various phases of the Achilles tendon reflex. Although normal values vary according to the phase of the tendon reflex measured, the apparatus used and individual laboratory standards, the approximate adult normal range for the half-relaxation time is 230-390 msec. Diurnal variation, differences with sex, and changes with age, cold exposure, fever, exercise, obesity, and pregnancy have been reported. However, the main reason for the failure of this test as a diagnostic measure of thyroid dysfunction is the large overlap with values obtained in euthyroid patients and alterations caused by nonthyroidal illnesses. 192
Thyroid hormone induced changes in the cardiovascular system can be measured by noninvasive techniques. One such test measures the time interval between the onset of the electrocardiographic QRS complex (Q) and the arrival of the pulse wave at the brachial artery, detected by the Korotkoff sound (K) at the antecubital fossa. 193 Related tests which determine the systolic time interval (STI) measure the preejection period (PEP), obtained by subtraction of the left ventricular ejection time (LVET) from the total electromechanical systole (Q-A2). 194 The left ventricular ejection time (LVET) which is also affected by the thyroid status can be measured by the M mode echocardiogram 195 (Figure 6-5). The PEP/LVET ratio is also useful in the assessment of thyroid hormone action in the cardiovascular system. 196 As with other tests of thyroid hormone action, the principal deficiency of these measurements is their alteration in a variety of nonthyroidal illnesses.
Miscellaneous Biochemical and Physiologic Changes Related to the Action of Thyroid Hormone on Peripheral Tissues
Thyroid hormone affects the function of a variety of peripheral tissues. Thus, hormone deficiency or excess may alter a number of determinations used in the diagnosis of illnesses unrelated to thyroid hormone dysfunction. Knowledge of the determinations which may be affected by thyroid hormone is important in the interpretation of laboratory data (Table 6-7).
|Table 6-7. Biochemical and Physiologic Changes Related to Thyroid Hormone Deficiency and Excess ( + = up, – = down, N = normal)|
|Entity Measured||During Hypothyroidism||During Thyrotoxicosis|
|Metabolism of various substances and drugs Fractional turnover rate (antipyrine,197 dipyrone,198 PTU, and methimazole,197 albumin,199 low-density lipoproteins,200 cortisol,201,202 and Fe203,204 )||-||+|
|Sex hormone- binding globulin14,208,209||-||++|
|Factor VIII-related antigen212||+|
|Response to glucose214||-||-|
|Response to glucagon215||+||-|
|Estradiol-17ÃŸ216 , testosterone14,208,216 and gastrin217||- or N||+|
|Parathyroid hormone concentration218,219||+||-|
|Response to PTH administration219||-||+|
|Calcitonin response to Ca++ infusion221||-|
|Renin activity and aldosterone222,223||-||+|
|Catecholamines224 and noradrenaline225||+||+|
|Atrial naturetic peptide226,227||-||+|
|Erythropoietin204||N or -||+|
|LH216||N or +|
|Response to GnRH228||+||N|
|Prolactin and response to stimulation with TRH, arginine, and chlorpromazine229,230||+ or N||-|
|Response to insulin231,232||-||N or -|
|Response to TRH233||No change|
|Epidermal growth factor234|
|Creatine-phosphokinase,235,236 lactic dehydrogenase,236 and glutamic oxaloacetic transminase236||+||-|
|Alkaline phosphatase219,239 a||a||+|
|Angiotensin-converting enzyme,212,241 alanine aminotransferase,242 and glutathione S-transferase242,243||N||+|
|Carotene, vitamin A246|
|cAMP,247 cGMP,248 and Fe203,249||+ N or -||- N or +|
|Fractional turnover during iv tolerance test214||-|
|Creatinine256||N or +||-|
|Creatine256||N or +||+|
|Cholesterol,246,257 carotene,246,257 phospholipids and lethicin,246,257 and triglycerides257,258||+||-|
|Type IV collagen260||+||+|
|Type III Pro-collagen 260||-||+|
|Free fatty acids261||+|
|after epinephrine infusion264||No change||+|
|cGMP248||N or -||+|
|Tyrosine206||N or -||+|
|MIT (after) administration of 131IMIT265||+|
|Tyramine, tryptamine, and histamine268||+|
|17-hydroxycorticoids and ketogenic steroids269||-||+|
|Pyridinoline (PYD), deoxypyridinoline (DPD)270||+|
|Hydroxyproline,271 and hydroxylysyl glycoside272||+|
|Red blood cells|
|Glucose-6-phosphate dehydrogenase activity275||N or -||+|
|Reduced glutathione276 and carbonic anhydrase277||+||-|
|White blood cells||-||-|
|ATP production in mitochondria280||?+||-|
|Sebium excretion rate282||-||N|
|Intestinal system and absorption|
|Basic electrical rhythm of the duodenum283||-||+|
|Intestinal transit and fecal fat286,287||-|
|Pulmonary function and gas exchange|
|Dead space,288 hypoxic ventilatory drive,289 and arterial pO2288||-|
|Neurologic system and CSF|
|Relaxation time of deep tendon reflexes (phomotogram)290||+||-|
|Cardiovascular and circulatory system|
|Timing of the arterial sounds (QKd)193||+||-|
|Left ventricular ejection time (LVET), preejection period (PEP) ratio194||-||-|
|Heart rate and QRS voltage||+|
|T wave||Flat or inverted||Transient abnormalities|
|Common arrhythmias||Atrioventricular block||Atrial fibrillation|
|Osseous maturation (bone age by X-ray film)294,295||Delayed (epiphysial dysgenesis)||Advanced|
|N = normal; + = increased; – = decreased. aIn children bIn neonates.|
Measurement of Substances Absent in Normal Serum
Tests that measure substances present in the circulation only under pathologic circumstances do not provide information on the level of thyroid gland function. They are of value in establishing the cause of the hormonal dysfunction or thyroid gland pathology.
The humoral antibodies most commonly measured in clinical practice are directed against thyroglobulin (Tg) or thyroid cell microsomal (MC) proteins. The latter is principally represented by the thyroid peroxidase (TPO). 296-298 More recently, immunoassays have been developed using purified and recombinant TPO. 299, 299a, 299b Other circulating immunoglobulins, which are less frequently used as diagnostic markers, are those directed against a colloid antigen, T4 and T3. Antibodies against nuclear components are not tissue specific. Immunoglobulins possessing the property of stimulating the thyroid gland will be discussed in the next section.
A variety of techniques have been developed for the measurement of Tg and MC antibodies. These procedures include a competitive binding radioassay, complement fixation reaction, 300 tanned red cell agglutination assay, 301 the Coon’s immunofluorescent technique, 302 enzyme-linked immunosorbent assay. 299 , 303 Although the competitive binding radioassay 304,305 is a sensitive test, agglutination methods combine sensitivity and simplicity and have now largely superceded other methods. Current commercial kits utilize synthetic gelatin beads rather than red cells. 305a
In the assay of Tg and MC antibodies by hemagglutination (TgHA and MCHA), particulate material is coated with either human Tg or solubilized thyroid MC proteins (TPO) and exposed to serial dilutions of the patient’s serum. Agglutination of the coated particulates occurs in the presence of antibodies specific to the antigen attached to their surface. To detect false-positive reactions, it is important to include a blank for each sample using uncoated particles. Because of the common occurrence of prozone or blocking phenomenon, it is necessary to screen all serum samples through at least six consecutive two-fold dilutions. 306 Results are expressed in terms of the highest serum dilution, or titer, showing persistent agglutination. The presence of immune complexes, particularly in patients with high serum Tg levels, may mask the presence of Tg antibodies. Assays for the measurement of such Tg-anti-Tg immune complexes have been developed. 307
Normally, the test response is negative but results may be positive in up to 10% of the adult population. The frequency of positive test results is higher in women and with advancing age. The presence of thyroid autoantibodies in the apparently healthy population is thought to represent subclinical autoimmune thyroid disease rather than false-positive reactions. Nonetheless, it is difficult to compare results from such studies since some laboratories using agglutination methods report low titres (1/100-1/400) as positive. It is important when reporting values that a method-specific normal range is utilized and assays calibrated against internationally available refernce preparations. The availability of such preparations allows the reporting of results in International Units. 305a TPO antibodies are detectable in approximately 95% of patients with Hashimoto’s thyroiditis and 85% of those with Graves’ disease, irrespective of the functional state of the thyroid gland. Similarly, Tg antibodies are positive in about 60 and 30% of adult patients with Hashimoto’s thyroiditis and Graves’ disease, respectively. 305,306 , 308,309 Tg antibodies are less frequently detected in children with autoimmune thyroid disease. 310 Although higher titers are more common with Hashimoto’s thyroiditis, quantitation of the antibody titer carries little diagnostic implication. The tests are of particular value in the evaluation of patients with atypical or selected manifestations of autoimmune thyroid disease (ophthalmopathy and dermopathy). Positive antibody titers are predicative of post partum thyroiditis. 311 Low antibody titers occur transiently in some patients after an episode of subacute thyroiditis. 312 There is no increased incidence of thyroid autoantibodies in patients with multinodular goiter, thyroid adenomas, or secondary hypothyroidism. In some patients with Hashimoto’s thyroiditis and undetectable thyroid autoantibodies in their serum, intrathyroidal lymphocytes have been demonstrated to produce TPO antibodies.
Other antibodies directed against thyroid components or other tissues have been described in the serum of some patients with autoimmune thyroid disease. They are less frequently measured, and their diagnostic value in thyroid disease has not been fully evaluated. Circulating antibodies capable of binding T4 and T3 have also been demonstrated in patients with autoimmune thyroid diseases which may interfere with the measurement of T4 and T3 by RIA techniques. 38,39 , 314
Antibodies reacting with nuclear components, which are not tissue specific, and with cellular components of parietal cells and adrenal, ovarian, and testicular tissues are more commonly encountered in patients with autoimmune thyroid disease. 315 Their presence reflects the frequency of coexistence of several autoimmune disease processes in the same patient (see Chapter 7 ).
A large number of names have been given to tests which measure abnormal ?-globulins present in the serum of some patients with autoimmune thyroid disease, in particular Graves’ disease. 317 The interaction of these unfractionated immunoglobulins with thyroid follicular cells usually results in a global stimulation of thyroid gland activity and only rarely causes inhibition. It has been recommended that these assays all be called TSH receptor antibodies (TRAb) with a phrase “measured by ……………… assay” to identify the type of method used for their determination. 106 The tests will be described under three general categories: (1) those measuring the thyroid stimulating activity using in vivo or in vitro bioassays; (2) tests based on the competition of the abnormal immunoglobulin with binding of TSH to its receptor; and (3) measurement of thyroid growth promoting activity of immunoglobulins. Tests employ both human and animal tissue material or cell lines.
The earliest assays employed various modifications of the McKenzie mouse bioassay. 318,319 The abnormal ?-globulin with TSH-like biological properties has relatively longer in vivo activity, hence its name, long-acting thyroid stimulator (LATS). The assay measures the LATS induced release of thyroid hormone from the mouse thyroid gland prelabeled with radioiodide. The presence of LATS in serum is pathognomonic of Graves’ disease. However, depending upon the assay sensitivity, a variable percent of untreated patients will show a positive LATS response. LATS may be found in the serum of patients with Graves’ disease even in the absence of thyrotoxicosis. Although it is more commonly present in patients with ophthalmopathy, especially when accompanied by pretibial myxedema, 320 LATS does not appear to correlate with the presence of Graves’ disease, its severity, or course of complications. LATS crosses the placenta and may be found transiently in newborns from mothers possessing the abnormal ? globulin. 321
Attempts to improve the ability to detect thyroid stimulating antibodies (TSAb) in autoimmune thyroid disease lead to the development of several in vitro assays using animal as well as human thyroid tissue. The ability of the patient’s serum to stimulate endocytosis in fresh human thyroid tissue is measured by direct count of intracellular colloid droplets formed. Using such a technique, human thyroid stimulator (HTS) activity has been demonstrated in serum samples from patients with Graves’ disease that were devoid of LATS activity measured by the standard mouse bioassay. 322 TSAb can be detected by measuring the accumulation of cyclic adenosine monophosphate (cAMP) or stimulation of adenylate cyclase activity in human thyroid cell cultures and thyroid plasma membranes, respectively. 323 Accumulation of cAMP in the cultured rat thyroid cell line FRTL5 has also been used as an assay for TSAb. 324 Stimulation of release of T3 from human 325 and porcine 326 thyroid slices is another form of in vitro assay for TSAb. An in vitro bioassay using a cytochemical technique depends upon the ability of thyroid-stimulating material to increase lysosomal membrane permeability to a chromogenic substrate, leucyl-ÃŸ-naphthylamide, which then reacts with the enzyme naphthylamidase. Quantitation is by scanning and integrated microdensitometry. 327
The cloning of the TSH receptor 328,329 lead to the development of an in vitro assay of TSab using cell lines that express the recombinant TSH receptor. 330,331 This assay, based on the generation if cAMP, is specific for the measurement of human TSH receptor antibodies that have thyroid stimulating activity and thus contrasts with assays based on binding to the TSH receptor (see below) that cannot distinguish between antibodies with thyroid-stimulating and TSH-blocking activity. Accordingly, the recombinant human TSH receptor assay measures antibodies relevant to the pathogenesis of autoimmune thyrotoxicosis and is more sensitive than formerly used TSab assays. 331a For example, 94% of serum samples were positive for TSab compared to 74% when the same samples were assayed using FRTL5 cells. 332
Thyrotropin-Binding Inhibition Assays. The principal of binding-inhibition assays dates to the discovery of another class of abnormal immunoglobulins in patients with Graves’ disease; those which neutralize the bioactivity of LATS tested in the mouse. 333 This material, known as LATS protector (LATS-P), is species specific having no biologic effect on the mouse thyroid gland but capable of stimulating the human thyroid. 334 The original assay was cumbersome, limiting its clinical application.
Techniques used currently, which may be collectively termed radioreceptor assays, are based on the competition of the abnormal immunoglobulins and TSH for a common receptor-binding site on thyroid cells. The test is akin in principle to the radioligand assays, in which a natural membrane receptor takes the place of the binding proteins or antibodies. Various sources of TSH-receptors are employed including, human thyroid cells, 335 their particulate or solubilized membrane, 336,337 and cell membranes from porcine thyroids 338 or from guinea pig fat cells 339 or recombinant human TSH receptor expressed in mammalian cells. 340 Since the assays do not directly measure thyroid-stimulating activity, the abnormal immunoglobulins determined have been given variety of names, such as thyroid binding inhibitory immunoglobulins (TBII) or antibodies (TBIAb) and thyrotropin-displacing immunoglobulins (TDI). This type of assay has indicated that not all the antibodies detected do stimulate the thyroid, and some are inhibitory. Even using modern techniques, 305a the presence of inhibitory antibody is less sensitive and specific for Graves’ disease than the presence of styimulatory antibody activity. 331 The stimulatory and inhibitory effects can be differentiated only by functional assays, typically measuring the production of cyclic AMP.
Thyroid Growth-Promoting Assays.
Assays have been also developed that measure the growth promoting activity of abnormal immunoglobulins. One such assay is based on the staining by the Feulgen reaction of nuclei from guinea pig thyroid cells in S-phase. 341 Another assay measures the incorporation of 3H-thymidine into DNA in FRTL cells. 342 Whether the thyroid growth stimulating immunoglobulins (TGI) measured by these assays represent a population of immunoglobulins distinct from that with stimulatory functional activity remains a subject of active debate.
Clinical Applications. Measurement of abnormal immunoglobulins that interact with thyroid tissue by any of the methods described above is not indicated as a routine diagnostic test for Graves’ disease. It is useful, however, in a few selected clinical conditions: (1) in the differential diagnosis of exophthalmos, particularly unilateral exophthalmos, when the origin of this condition is otherwise not apparent; the presence of TSI would obviate the necessity to undertake more complex diagnostic procedures described elsewhere; 343 (2) in the differential diagnosis of pretibial myxedema, or other forms of dermopathy, when the etiology is unclear and it is imperative that the cause of the skin lesion be ascertained; (3) in the differentiation of Graves’ disease from toxic nodular goiter, when both are being considered as the possible cause of thyrotoxicosis, when other tests such as thyroid scanning and thyroid autoantibody tests have been inconclusive, and particularly when such a distinction would play a role in determining the course of therapy; (4) when non-autoimmune thyrotoxicosis is suspected in a patient with hyperthyroidism and diffuse or nodular goiter 344,345 ; (5) in Graves’ disease during pregnancy, when high maternal levels of TSAb are a warning for the possible occurrence of neonatal thyrotoxicosis; (6) in neonatal thyrotoxicosis, where serial TSAb determinations showing gradual decrease may be helpful to distinguish between intrinsic Graves’ disease in the infant and transient thyrotoxicosis resulting from passive transfer of maternal TSAb. 321 , 346 Some investigators have found the persistence of TSAb’s to be predicative of the relapse of Graves’ thyrotoxicosis following a course of antithyroid drug therapy. 347
Some patients with trophoblastic disease develop hyperthyroidism as a result of the production and release of a thyroid stimulator which has been termed molar or trophoblastic thyrotropin or big placental TSH. 348 It is likely that the thyroid-stimulating activity in patients with trophoblastic disease is entirely due to the presence of high levels of human chorionic gonadotropin (hCG). 350 Thus, the RIA of hCG can be useful in the differential diagnosis of thyroid dysfunction.
Exophthalmos-Producing Substance (EPS)
A variety of tests have been developed for measuring exophthalmogenic activity in serum. 351-354 Although a great uncertainty still exists regarding the pathogenesis of thyroid associated eye disease, the role of the immune system appears to be central. Exophthalmogenic activity has also been detected in IgG fractions of some patients with Graves’ ophthalmopathy. The role of assays to detect specific antibodies is discussed further in Chapter 7 .
Delayed hypersensitivity reactions to thyroid antigens are present in autoimmune thyroid diseases (see Chapters 7 ). CMI was measured in several ways: (1) the migration inhibition test, which measured the inhibition of migration of sensitized leukocytes when exposed to the sensitizing antigen; (2) the lymphotoxic assay, which measured the ability of sensitized lymphocytes to kill target cells when exposed to the antigen; (3) the blastogenesis assay, which scored the formation of blast cells after exposure of lymphocytes to a thyroid antigen; and (4) thymus-dependent (T) lymphocyte subset quantitation utilizing monoclonal antibodies. More recently, measures of T-cell proliferation, determined by uptake of 3Hthymidine, has become the standard test of CMI employed in the research setting. 354a, 354b The tests require fresh leukocytes from the patient, are variable in their response, and are difficult to perform.
Anatomic and Tissue Diagnoses
The purpose of the procedures described in this section is to evaluate the anatomic features of the thyroid gland, localize and determine the nature of abnormal areas and eventually provide a pathologic or tissue diagnosis. All of these tests are performed in vivo.
Normal and abnormal thyroid tissue can be externally imaged by three scintiscanning methods: (1) with radionuclides that are concentrated by normal thyroid tissues such as iodide isotopes, and 99mTc given as the pertechnetate ion; (2) by administration of radiopharmaceutical agents which are preferentially concentrated by abnormal thyroid tissues; and (3) fluorescent scanning, which uses an external source of 241Am and does not require administration of radioactive material. Each has specific indications, advantages, and disadvantages.
The physical properties, dosages, and radiation delivered by the most commonly used radioisotopes are listed in Table 6-2 . The choice of scanning agents depends on the purpose of the scan, the age of the patient, and the equipment available. Radioiodide scans cannot be performed in patients who have recently ingested iodine-containing compounds. 123I and 99mTcO4- are the radionuclides of choice because of the low radiation exposure. 355-357 Iodine-131 is still used for the detection of functioning metastatic thyroid carcinoma by total body scanning.
Radioiodide and 99mPertechnetate Scans. 99mTcO4- is concentrated, and all iodide isotopes are concentrated and bound, by thyroid tissue. Depending upon the isotope used, scans are carried out at different times after administration: 20 minutes for 99mTcO4-, 4 or 24 hours for 123I-; 24 hours for 125I- and 131I-; and 48, 72, and 96 hours when 131I- is used in the search for metastatic thyroid carcinoma. The appearance of the normal thyroid gland on scan may be best described as a narrow-winged butterfly. Each “wing” represents a thyroid lobe, which in the adult measures 5 ? 1 cm in length and 2.3 ? 0.5 cm in width. 358 Common variants include the absence of a connecting isthmus, a large isthmus, asymmetry between the two lobes, and trailing activity extending to the cricoid cartilage (pyramidal lobe). The latter is more commonly found in conditions associated with diffuse thyroid hyperplasia. Occasionally, collection of saliva in the esophagus during 99mTcO4- scanning may simulate a pyramidal lobe, but this artifact can be eliminated by drinking water.
The indications for scanning are listed in Table 6-8 . In clinical practice, scans are most often requested for evaluation of the functional activity of solitary nodules. Normally, the isotope is homogeneously distributed throughout both lobes of the thyroid gland. This distribution occurs in the enlarged gland of Graves’ disease and may be seen in Hashimoto’s thyroiditis. A mottled appearance may be noted in Hashimoto’s thyroiditis and can occasionally be seen in Graves’ disease especially after therapy with radioactive iodide. Irregular areas of relatively diminished and occasionally increased uptake are characteristic of large multinodular goiters. The traditional nuclear medicine jargon classifies nodules as “hot”, “warm,” and “cold,” according to their isotope-concentrating ability relative to the surrounding normal parenchyma (Figure 6-6). Hot, or hyperfunctioning, nodules are typically benign, although the presence of malignancy has been reported. 359,360 Cold, or hypofunctioning, nodules may be solid or cystic. Some may prove to be malignant, but the great majority are benign. This differentiation cannot be made by scanning. 77 , 361 Occasionally, a nodule which is functional on a 99mTcO4- scan will be found to be cold on an iodine scan; this pattern is found with both benign and malignant nodules. The scan is of particular value in identifying autonomous thyroid nodules since the remainder of the gland is suppressed. Search for functioning thyroid metastases is best accomplished using 2-10 mCi of 131I after ablation of the normal thyroid tissue and cessation of hormone therapy to allow TSH to increase above the upper limit of normal. Recent studies have addressed the question of whether recombinant human TSH allows scanning without requiring cessation of hormone therapy. 362 Uptake is also found outside the thyroid gland in patients with lingual thyroids and in the rare ovarian dermoid tumor containing functioning thyroid tissue.
|Table 6-8. Indications for Radionuclide Scanning|
|Detection of anatomic variants and search for ectopic thyroid tissue (thyroid hemiagenesis, lingual thyroid, struma ovarii) Diagnosis of congenital athyreosis Determination of the nature of abnormal neck or chest (mediastinal) masses Evaluation of solitary thyroid nodules (functioning or non-functioning) Evaluation of thyroid remnants after surgery Detection of functioning thyroid metastases Evaluation of focal functional thyroid abnormalities (suppressed or nonsuppressible tissue)|
The scan can be used as an adjunct during TSH stimulation and T3 suppression tests to localize suppressed normal thyroid tissue or autonomously functioning areas, respectively (see below). Applications other than those listed in Table 6-8 are of doubtful benefit and are rarely justified considering the radiation exposure, expense, and inconvenience. 123I single photon emission computed tomography (SPECT) may also be useful in the evaluation of thyroid abnormalities. 363
Other Isotope Scans. Because most test procedures, short of direct microscopic examination of thyroid tissue, fail to detect thyroid malignancy with any degree of certainty, efforts have been made to find other radioactive materials that would hopefully be of diagnostic use. Several such agents that are concentrated by metabolically active tissues, irrespective of whether they have iodide-concentrating ability, have been tried. However, despite claims to the contrary, they have either had only limited value or their diagnostic usefulness has not been fully evaluated. These agents include 75Se methionine, 125Ce, 67Ga, citrate, 32P, pyrophosphate 99mTc, and 201Thallium. 364
Scanning with 131I-labeled anti-TG for the detection of occult metastatic thyroid malignancy that fails to concentrate 131I showed early promising results. 365 However, the procedure has not proved clinically useful.
Ultrasonography, or echography, is used to outline the thyroid gland and to characterize lesions differing in density from the surrounding tissue. The technique differentiates interphases of different acoustic densities, using sound frequencies in the megahertz range that are above the audible range. A transducer fitted with a piezoelectric crystal produces and transmits the signal and receives echo reflections. Interfaces of different acoustic densities reflect dense echoes, liquid transmits sound without reflections, and air-filled spaces do not transmit the ultrasound. 368
One of the most useful applications of the ultrasonogram is the differentiation of solid from cystic lesions. 368,369 Purely cystic lesions are entirely sonolucent, whereas solid lesions produce multiple echoes due to multiple sonic interphases. Many lesions, however, are mixed (solid and cystic) called complex lesions. Some tumors may have the same acoustic characteristics as the surrounding normal tissue thus, escaping echographic detection. While high-resolution ultrasonography can detect thyroid nodules of the order of few millimeters, 370 lesions need to be larger than 0.5 cm to allow differentiation between solid and cystic structures. A sonolucent pattern is frequently noted in glands with Hashimoto’s thyroiditis, but this has also been described in multinodular glands and in patients with Graves’ disease. 368 , 371 , 372
Because sonography localizes the position as well as the depth of lesions, the procedure has been used to guide the needle during aspiration biopsy. 373 In complex lesions, the sonographic guiding insures sampling from the solid portion of the nodule. With experience and proper calibration, sonography can be used for the estimation of thyroid gland size. 374,375 Several recent reports have described treatment of toxic nodules by the injection of alcohol under sonographic guidance. 376 Although ultrasonography has found virtually the same applications as scintiscanning, claims that the former may differentiate benign from malignant lesions are unfounded. Also, ultrasonography cannot be used for the assessment of substernal goiters because of interference from overlying bone.
The procedure is simple and painless, and at the frequencies of sound used, do not produce tissue damage. Since it does not require the administration of isotopes, it can be safely used in children and during pregnancy. Also, because the procedure is independent of iodine-concentrating mechanisms, it is valuable in the study of suppressed glands.
A simple X-ray film of the neck and upper mediastinum may provide valuable information regarding the location, size, and effect of goiter on surrounding structures. X-rays may show an asymmetric goiter, an intrathoracic extension of the gland, and displacement or narrowing of the trachea. If there is any suggestion of posterior extension of the mass, it is useful to take films during the swallow of X-ray contrast material. The soft tissue X-ray technique may disclose calcium deposits. Large deposits in flakes or rings are typical of an old multinodular goiter, whereas foci of finely stippled flecks of calcium are suggestive of papillary adenocarcinoma.
Information, not related to anatomic abnormalities of the thyroid gland may be obtained from X-ray studies. In children with a history of hypothyroidism, an X-ray film of the hand to determine the bone age could aid in estimating the onset and duration of thyroid dysfunction. 294,295 Hypothyroidism leads to retardation in bone age and in infants produces a dense calcification of epiphyseal plates most easily seen at the distal end of the radius. Long-standing myxedema produces pituitary hypertrophy which, especially in children but also in adults, causes enlargement of the sella turcica demonstrable on imaging of the pituitary region.
Computed Tomography (CT) and Magnetic Resonance Imaging (MRI). These techniques provide useful information on the location and architecture of the thyroid gland as well as its relationship to surrounding tissues. 378 They are, however, too costly relative to other procedures which provide similar information. An important application of CT is the assessment and delineation of obscure mediastinal masses and large substernal goiters. 379 The necessity to infuse iodine containing contrast agents limits the application of CT in patients being considered for radioiodide therapy. CT and MRI have found firm application in another area of thyroid diseases, namely, in the evaluation of ophthalmopathy 343 and mediastinal masses. 379
A barium swallow may be useful in evaluating impingement of a goiter on the esophagus, while a flow volume loop 380 may be useful in documenting functional impingement on the upper airway.
Biopsy of the Thyroid Gland
Histologic examination of thyroid tissue for diagnostic purposes requires some form of an invasive procedure. The biopsy procedure depends on the intended type of microscopic examination. Core biopsy for histologic examination of tissue with preservation of architecture is obtained by closed needle or open surgical procedure; aspiration biopsy is performed to obtain material for cytologic examination.
Core Biopsy. Closed core biopsy is an office procedure carried out under local anesthesia. A large (about 15-gauge) cutting needle of the Vim-Silverman type is most commonly used. 384 The needle is introduced under local anesthesia through a small skin nick and firm pressure is applied over the puncture site for 5-10 minutes after withdrawal of the needle. In experienced hands, complications are rare, but may include transient damage to the laryngeal nerve, puncture of the trachea, laryngospasm, jugular vein phlebitis, and bleeding.385 Because of the fear of disseminating malignant cells, biopsy was restricted for many years to the differential diagnosis of diffuse benign diseases. With the improvement of cytology and biopsy techniques, open biopsy carried out under local or general anesthesia has been virtually abandoned. 385
Percutaneous Fine Needle Aspiration (FNA). The development of more sophisticated staining techniques for cytologic examination, the realization that fear of tumor dissemination along the needle tract was not well founded, and especially the high diagnostic accuracy of the technique are responsible for the increasing popularity of percutaneous fine needle aspiration. 385 , 388,388a,388b
The procedure is exceedingly simple and safe. The patient lays supine, with the neck hyperextended by placing a small pillow under the shoulders. Local anesthesia is usually not required. The skin is prepared with an antiseptic solution. The lesion, fixed between two gloved fingers, is penetrated with a fine (22- to 27-gauge) needle attached to a syringe. Suction is then applied while the needle is moved within the nodule. A non-suction technique using capillary action has also been developed. The small amount of aspirated material, usually contained within the needle or its hub, is applied to glass slides and spread. Some slides are air dried and others are fixed before staining. As biopsy of small nodules may be technically more difficult, the use of ultrasound to guide the needle is preferred. 373 , 376 It is important that the slides be properly prepared, stained and read by a cytologist experienced in the interpretation of material from thyroid gland aspirates.
The yield of false-positive and false-negative results is variable from one center to another, but both are acceptably low. Various centers have reported that the accuracy of this technique in distinguishing benign from malignant lesions may be as high as 95%. 385 , 388 In one clinic in which the procedure is used routinely, the number of patients operated upon decreased by one-third, whereas the percentage of thyroid carcinomas among the patients who underwent surgery doubled. 389 When results are suggestive of a follicular neoplasia, surgery is required as follicular adenoma cannot be differentiated from follicular cancer by cytology alone. As the sample obtained may not always be representative of the lesion, surgical treatment is indicated for lesions highly suspicious of being malignant on clinical grounds. Other uses of aspiration biopsy include presumed lymphoma or invasive anaplastic carcinoma when biopsy may spare the patient an unnecessary neck exploration. Another application of needle aspiration is in the confirmation and treatment of thyroid cysts and autonomous thryoid nodules. 389a
Evaluation of the Hypothalamic-Pituitary-Thyroid Axis
The development of an RIA for the routine measurement of TSH in serum and the availability of synthetic TRH 390,391 have placed increased reliance on tests assessing the hypothalamic-pituitary control of thyroid function. These tests allow the diagnosis of mild and subclinical forms of thyroid dysfunction, and provide a means to differentiate between primary, pituitary (secondary) or hypothalamic (tertiary), thyroid gland failure.
The routine measurement of TSH in clinical practice used initially RIA techniques. These first generation assays had a sensitivity level of 1 mU/L which did not allow the separation of normal from reduced values. A major problem with early TSH RIAs was cross-reactivity with gonadotropins (LH, FSH, and hCG) sharing with TSH a common a-subunit. 399 Nevertheless, even older RIA methods for measurement of pituitary TSH correlated well with values obtained using bioassay techniques. 401 Another uncommon source of error is the presence in the serum sample of heterophilic antibodies induced by vaccination with materials contaminated with animal serum, 402 or endogenous TSH antibodies. 403 RIA techniques for measurement of TSH in dry blood spots on filter paper are used for the screening of neonatal hypothyroidism. 33
Newer techniques have been developed using multiple antibodies to produce a “sandwich” type assay in which one antibody (usually directed against the a subunit) serves to anchor the TSH molecule and an other (usually monoclonal antibodies directed against the ÃŸ subunit) is either radioiodinated (Immunoradiometric assay, IRMA) or is conjugated with an enzyme (Immunoenzymometric, IEMA) or a chemiluminescent compound (Chemiluminescent assay, ICMA). 112 , 404 In these assays, the signal should be directly related to the amount of the ligand present rather than being inversely related as in RIAs measuring the bound tracer. 405 This results in decreased background “noise” and a greater sensitivity, decreased interference from related compounds as well as an expanded useful range. 112 , 404 , 406 Initial improvements of the TSH assay resulted in assays with sensitivity limit of 0.1 mU/L, a normal range of approximately 0.5 – 4.5 mU/L and the ability to distinguish between low and normal TSH values. Recently, commercial assays have been developed with even higher sensitivity limit of 0.005 – 0.01 mU/L and a similar normal range but an expanded range between the lower limit of normal and the lower limit of sensitivity. 407,408
The nomenclature for differentiating these various assays has not been standardized with manufacturers applying various combinations of “high(ly)”, “ultra” and “sensitive”. It has been recommended that the sensitivity limit be used in defining the assays with the early radioimmunoassays detecting values ?1 mU/L designated “first generation assays”, those with a lower sensitivity limit of 0.1 mU/L designated as “second generation assays” and those with a lower sensitivity limit of ? 0.01 mU/L designated as “third generation assays”. 112 The determination of the appropriate sensitivity level has also been controversial. Some define it based on the level with a coefficient of variation less than 20% and others as the lowest level which can be reliably differentiated from the zero TSH standard.112,406 At a minimum, for a TSH assay to be considered “sensitive”, the overlap of TSH values in sera from clinically hyperthyroid and euthyroid individuals should be less than 5% and preferably less than 1%. 112
In a number of these “third generation” assays, TSH detected in clinically toxic patients and elevated values found in euthyroid subjects were not confirmed when the samples were measured in other assays. In some cases, this has been attributed to the presence of antibodies directed against the animal immunoglobulins used in the assay. 409-411 These act to bind the anchoring and detecting antibodies and lead to an over-estimation of TSH. In some cases, this effect may be blocked by the addition of an excess of non-specific immunoglobulin of the same species. 411
TSH appears abruptly in the pituitary and serum of the fetus at midgestation, and can also be detected in amniotic fluid. 51 , 412,413 The mean TSH level is higher in cord than in maternal blood. A substantial increase, to levels several fold above the upper range in adults, is observed during the first half-hour of life. 413 Levels decline to near the normal adult range by the third day of life. Minimal changes reported to occur during adult life and in early adolescence 414 have no significant effect on the overall range of normal. In the absence of pregnancy, no significant sex differences have been observed. Although early studies failed to show diurnal TSH variation, 415 significantly higher values have been recorded during the late evening and early night which are partially inhibited by sleep. 416 This diurnal rhythm of TSH is superimposed upon continuous high-frequency, low-amplitude variations. The nocturnal TSH surge persists in patients with mild primary hypothyroidism, 417,418 and is abolished in hypothalamic hypothyroidism 417 , 419 and in some patients during fasting 420 and with non-thyroidal illness. 421,422 It is enhanced by oral contraceptives, 423 and is abolished by high levels of glucocorticoids. 424 The presence of seasonal variation has not been a uniform finding, but it is unlikely to affect the clinical interpretation of serum values. 425 Various types of stressful stimuli have no significant effect on the basal serum TSH level, except for a rise during surgical hypothermia in infants but not in adults. 426 Various stimuli, such as administration of insulin, vasopressin, glucagon, bacterial pyrogens, arginine, prostaglandins, and chlorpromazine, which elicit in normal humans a secretory response of some pituitary hormones, have no effect on serum TSH. However, administration of any of a growing list of drugs has been found to alter the basal concentration of serum TSH and/or its response to exogenous TRH (see Table 5-4 ).
In the presence of a normally functioning hypothalamic-pituitary system, there is an inverse correlation between the serum concentration of FT4 and TSH. Changes in the serum concentration of TT4 as a result of TBG abnormalities, or drugs competing with T4 binding to TBG, have no effect on the level of serum TSH. The pituitary is exquisitely sensitive to both minimal decreases and increases in thyroid hormone concentration, with a logarithmic change in TSH levels in response to changes in T4 404 , 408 , 427 , 428 (Figure 6-7) Thus, serum TSH levels should be elevated in patients with primary hypothyroidism and low or undetectable in thyrotoxicosis. Indeed, in the absence of hypothalamic pituitary disease, illness or drugs, TSH is an accurate indicator of thyroid hormone status and the adequacy of thyroid hormone replacement. 404 , 429
In patients with primary hypothyroidism of whatever cause, levels may reach 1,000 ÂµU/ml or higher. The magnitude of serum TSH elevation grossly correlates with the severity and in part with the duration of thyroid hormone deficiency. 430,431 TSH concentrations above the upper limit of normal have been observed in the absence of clinical symptoms and signs of hypothyroidism and in the presence of serum T4 and T3 levels well within the normal range. 430 , 432 This condition is most commonly encountered in patients developing hypothyroidism due to Hashimoto’s thyroiditis or with limited ability to synthesize thyroid hormone because of prior thyroid surgery, radioiodide treatment, or severe iodine deficiency. 430 , 433 There is disagreement on whether such patients have subclinical hypothyroidism or a “compensated state” in which euthyroidism is maintained by chronic stimulation of a reduced amount of functioning thyroid tissue through hypersecretion of TSH. Transient hypothyroidism, may occur in some infants during the early neonatal period. 434 There are two circumstances in which the usual reverse relationship between the serum level of TSH and T4 is not maintained in patients with proven primary hypothyroidism. Treatment with replacement doses of T4 may normalize or even produce serum levels of thyroid hormone above the normal range before the high TSH levels have reached the normal range. 404 , 431 , 435 This is particularly true in patients with severe or long-standing primary hypothyroidism who may require three to six months of hormone replacement before TSH levels are fully suppressed. Conversely, serum TSH concentration may remain low or normal for up to five weeks after withdrawal of thyroid hormone replacement when serum levels of T4 and T3 have already declined to values well below the lower range of normal. 404 , 436 Causes for discrepancies between TSH and free T4 and T3 levels are listed in Table 6-9 .
|Table 6-9. Discrepancies Between TSH and Free Thyroid Hormone Levels|
|Elevated Serum TSH Value Without Low FT4 or FT3 Values|
|Subclinical hypothyroidism (inadequate replacement therapy, mild thyroid gland failure)|
|Recent increase in thyroid hormone dosage|
|Inappropriate TSH secretion syndromes|
|Subnormal Serum TSH Value Without Elevated FT4 or FT3 Values|
|Subclinical hyperthyroidism (excessive replacement therapy, mild thyroid gland hyperfunction, autonomous nodule)|
|Recent decrease in suppressive thyroid hormone dosage|
|Recent treatment of thyrotoxicosis (Graves’ disease, toxic multinodular goiter, toxic nodule)|
|Resolution thyrotoxic phase of thyroiditis|
At this time, it is uncertain as to what TSH level is appropriate for suppressive thyroid hormone therapy. The frequency with which patients have subnormal, but detectable, TSH values depends on both the population studied and the sensitivity of the assay (Figure 6-8, below). Using an assay with a sensitivity limit of 0.1 mU/L, 3 to 4% of hospitalized patients have been noted to have a subnormal TSH. 432 , 437 When patients with an undetectable TSH in such an assay were re-evaluated in an assay with a sensitivity limit of 0.005 mU/L, 3 of 77 (4%) with thyrotoxicosis and 32 of 37 (86%) with non-thyroidal illness or on drugs were found to have a subnormal but detectable TSH level. 407 Thus, the more sensitive the assay, the more likely that patients with clinical thyrotoxicosis will have undetectable serum TSH while those with illness will have a subnormal but detectable level. However, with progressively more sensitive assays, the likelihood of a clinically toxic patient to have a detectable TSH will increase, and if patients on suppressive therapy are treated until the TSH is undetectable, the more likely they will have symptoms of thyrotoxicosis.
A persistent absence of a reverse correlation between serum thyroid hormone and TSH concentration has a very different connotation. A low serum level of thyroid hormone without clear elevation of the serum TSH concentration is suggestive of trophoprivic hypothyroidism, especially when associated with obvious clinical stigmata of hypothyroidism. 433 An inherited defect of the TSH receptor has been shown to produce marked persistent hyperthyrotropinemia in the presence of normal thyroid hormone levels. 438 In some cases, a mild elevation of the serum TSH level measured by RIA is probably due to the presence of immunoreactive TSH with reduced biologic activity. 397 Distinction between pituitary and hypothalamic hypothyroidism can be made on the basis of the TSH response to the administration of TRH (see below).
In another group of pathologic conditions, serum TSH levels may not be suppressed despite a clear elevation of serum free thyroid hormone levels. Because such a finding is incompatible with a normal thyroregulatory control mechanism of the pituitary, which is preserved in the more common forms of thyrotoxicosis, it has been termed inappropriate secretion of TSH. 439 It implicitly suggests a defective feedback regulation of TSH. When associated with the classical clinical and metabolic changes of thyrotoxicosis, it is usually due to TSH-secreting pituitary adenoma or isolated pituitary resistance to the feedback suppression by thyroid hormone. 439 The existence of hypothalamic hyperthyroidism can be questioned. 440 Precise diagnosis requires further studies, including radiologic examination of the pituitary gland and a TRH test. In addition, the presence of high circulating levels of the a-subunit of pituitary glycoprotein hormones (a-SU), giving rise to a disproportionately high a-SU/TSH molar ratio in serum, is characteristic, if not pathognomonic, of TSH-secreting pituitary tumors. 439 , 441 Normal, and occasionally high serum TSH levels, associated with a clear elevation in serum FT4 and FT3 but no clear clinical evidence of hypothyroidism or symptoms and signs suggestive of both thyroid hormone deficiency and excess are typical of resistance to thyroid hormone (RTH) 442 (see Chapter 16 ).
Although TSH has been implicated in the pathogenesis of simple, nontoxic goiter, unless hypothyroidism supervenes or iodide deficiency is very severe, TSH levels are characteristically normal. Elevated TSH levels may occur in the presence of normal thyroid hormone levels and apparent euthyroidism in nonthyroidal diseases 437 , 443 (see also Chapter 5 ) and with primary adrenal failure. 444 A more common occurrence in severe acute and chronic illnesses is a normal or low serum TSH concentration despite low levels of T3 and even low T4 levels. 407 , 429 , 445 TSH values may be transiently elevated during the recovery phase.446 Various hypotheses to explain these anomalous findings have been proposed, but a satisfactory explanation is not at hand.
A specific RIA for the ÃŸ subunits of human TSH is also available but has not found clinical application. 447
TRH. The hypothalamic tripeptide TRH (protirelin) plays a central role in the regulation of pituitary TSH secretion. 391 , 419 It is thus not surprising that attempts have been made to measure its concentration in a variety of body fluids, with the purpose of deriving information relevant to the function of the thyroid gland in health and in disease. Several methods have been used for quantitation of TRH, 448-451 but for many reasons, measurement in humans has failed to provide information of diagnostic value. These include, high dilution of TRH by the time it reaches the systemic circulation, rapid enzymatic degradation and ubiquitous tissue distribution. 448 , 450,451 Mean serum TSH levels of 5 and 6 pg/ml have been reported. It is uncertain whether measurements carried out in urine truly represent TRH. 449
The TRH test measures the increase of pituitary TSH in serum in response to the administration of synthetic TRH. The magnitude of the TSH response to TRH is modulated by the thyrotroph response to active thyroid hormone and is thus almost always proportional to the concentration of free thyroid hormone in serum. The response is exquisitely sensitive to minor changes in the level of circulating thyroid hormones, which may not be detected by direct measurement. 427,428 A direct correlation between basal serum TSH values and the maximal response to TRH has been observed even in the absence of thyroid hormone abnormalities, suggesting that there may be a fine modulation of pituitary sensitivity to TRH in the euthyroid state. 452
TRH normally stimulates pituitary prolactin secretion and, under certain pathologic conditions, the release of GH and ACTH. 391 Accordingly, the test has been used for the assessment of a variety of endocrine functions, some unrelated to the thyroid. In clinical practice, the TRH test is used mainly (1) to assess the functional integrity of the pituitary thyrotrophs and thus to aid in differentiating hypothyroidism due to intrinsic pituitary disease from hypothalamic dysfunction and (2) in the diagnosis of mild thyrotoxicosis when results of other tests are equivocal, and (3) in the differential diagnosis of inappropriate TSH secretion, in particular when a TSH-secreting adenoma is suspected.
TRH is effective when given intravenously as a bolus or by infusion, 414 , 453 intramuscularly, 454 or orally 455 in single or repeated doses. Doses as small as 6 Âµg can elicit a significant TSH response, and there is a linear correlation between the incremental changes in serum TSH concentrations and the logarithm of the administered TRH dose. 414 The standard test uses a single TRH dose of 400 Âµg/1.73 m2 body surface area, given by rapid intravenous injection. Serum is collected before and at 15 minutes and then at 30 minute intervals over 120-180 minutes although many clinicians chose to obtain a single post-injection sample at 15, 20 or 30 minutes. In normal persons there is a prompt increase in serum TSH, with a peak level at 15-40 minutes, which is, on the average, 16 ÂµU/ml, or fivefold the basal level. The decline is more gradual, with a return of serum TSH to the preinjection level by three to four hours. 414 , 453 Results can be expressed in terms of the peak level of TSH achieved, the maximal increment above the basal level (?TSH), the peak TSH value expressed as a percentage of the basal value, or the integrated area of the TSH response curve. Determination of TSH before and 30 minutes after the injection of TRH provides information concerning the presence or absence of TSH responsiveness but cannot detect delayed or prolonged responses.
The stimulatory effect of TRH is specific for pituitary TSH, its free a- and ÃŸ- subunits, 447 and prolactin. Under normal circumstances, no significant changes are observed in the serum levels of other pituitary hormones 456 or potential thyroid stimulators. 457 Responsiveness is present at birth, 458 is greater in women than in men, particularly in the follicular phase of the menstrual cycle, 459 and may be blunted in older men, 414 , 454,455 but this is not a consistent finding. 460 On the average, the magnitude of the response is greater at 11 P.M. than at 11 A.M., 452 in accordance with the diurnal pattern of the basal TSH level which correlates to its response to TRH. Repetitive administration of TRH to the same subject at daily intervals causes a gradual obtundation of the TSH response, 453 presumably due to the increase in thyroid hormone concentration 461 and also in part due to TSH “exhaustion”. 462 However, more than one hour must elapse between the increase in thyroid hormone concentration and TRH administration for inhibition of the TSH response to occur. A number of drugs (see Table 5-4 ) and nonendocrine diseases (see Chapter 5 ) may affect to various extents the magnitude of the response.
TRH-induced secretion of TSH is followed by a release of thyroid hormone that can be detected by direct measurement of serum TT4 and TT3 concentrations. 160 Peak levels are normally reached approximately four hours after the administration of TRH and are accompanied by an increase in serum Tg concentration. The incremental rise in serum TT3 is relatively greater, and the peak is, on the average, 50% above the basal level. Measurement of changes in serum thyroid hormone concentration after the administration of TRH has been proposed as an adjunctive test and is useful in the evaluation of the integrity of the thyroid gland or bioactivity of endogenous TSH. 463 Increase in RAIU is minimal and occurs only with high doses of TRH given orally. 455
Side effects from the intravenous administration of TRH, in decreasing order of frequency, include nausea, flushing or a sensation of warmth, desire to micturate, peculiar taste, light-headedness or headache, dry mouth, urge to defecate, and chest tightness. They are usually mild, begin within a minute after the injection of TRH, and last for a few seconds to several minutes. A transient rise in blood pressure has been observed on occasion, but there are no other changes in vital signs, urine analysis, blood count, or routine blood chemistry tests. 456 , 464 The occurrence of circulatory collapse is exceedingly rare. 465
The test provides a means to distinguish between secondary (pituitary) and tertiary (hypothalamic) hypothyroidism ( Fig. 6-9 ). Although the diagnosis of primary hypothyroidism can be easily confirmed by the presence of elevated basal serum TSH levels, secondary and tertiary hypothyroidism are typically associated with TSH levels that are low or normal. On occasion the serum TSH concentration may be slightly elevated due to the secretion of biologically less potent molecules, 397 but it remains inappropriately low for the degree of thyroid hormone deficiency. Differentiation between secondary and tertiary hypothyroidism cannot be made with certainty without the TRH test. A TSH response is suggestive of a hypothalamic disorder, and a failure to respond is compatible with intrinsic pituitary dysfunction. 466 Furthermore, the typical TSH response curve in hypothalamic hypothyroidism shows a delayed peak with a prolonged elevation of serum TSH before return to the basal value (Figure 6-9). The lack of a TSH response in association with normal prolactin stimulation may be due to isolated pituitary TSH deficiency. 467 Caution should be exercised in the interpretation of test results after withdrawal of thyroid hormone replacement or after treatment of thyrotoxicosis when, despite a low serum thyroid hormone concentration, TSH may remain low and not respond to TRH for several weeks. 404 , 433 , 436 , 468
In the most common forms of thyrotoxicosis, the mechanism of feedback regulation of TSH secretion is intact but is appropriately suppressed by the excessive amounts of thyroid hormone. Thus, both the basal TSH level and its response to TRH are suppressed unless thyrotoxicosis is TSH induced. 404 , 407 , 417 With the development of more sensitive TSH assays, the TRH test is generally not needed in the evaluation of a thyrotoxic patient with an undetectable TSH.407 Differential diagnosis of conditions leading to inappropriate secretion of TSH may be aided by the TRH test result. Elevated basal TSH values that do not respond by a further increase to TRH are typical of TSH-secreting pituitary adenomas. 439 , 441 Patients with inappropriate secretion of TSH due to resistance to thyroid hormone have a normal or exaggerated TSH response to TRH that, in most instances, is suppressed with supraphysiologic doses of thyroid hormone. 442
Because of the high sensitivity of the pituitary gland to the feedback regulation by thyroid hormone, small changes in the latter profoundly affects the response of TSH to TRH. Thus, patients with non-TSH-induced thyrotoxicosis of the mildest degree have a reduced TSH response to TRH whereas those with primary hypothyroidism exhibit an accentuated response that is prolonged (Figure 6-9, see above). These changes may occur in the absence of clinical or other laboratory evidence of thyroid dysfunction.
The TSH response to TRH, is subnormal or absent in one-third of apparently euthyroid patients with autoimmune thyroid disease, and even members of their family, may not respond to TRH. 469,470 Most, but not all patients with reduced TSH response to TRH, will also show thyroid activity that is nonsuppressible by thyroid hormone. A common dissociation between these two tests is typified by a normal TRH response in a nonsuppressible patient. This finding is not surprising since patients with nonsuppressible thyroid glands often have limited capacity to synthesize and secrete thyroid hormone, due to prior therapy or partial destruction of their glands by the disease process. Clinically, euthyroid patients, who do not respond to TRH, admittedly have a slight excess of thyroid hormone. It is less easy to reconcile the rare occurrence of TRH unresponsiveness in a patient who is suppressible by exogenous thyroid hormone. It should be remembered, however, that a suppressed pituitary may take a variable amount of time to recover, a phenomenon that may be the basis of such discrepancies. 404 , 436 , 468 Despite discrepancies between the results of the TRH and T3 suppression tests, 469,470 the use of the former is much preferred particularly in elderly patients in whom administration of T3 can produce untoward effects.
The maintenance of thyroid gland activity that is independent of TSH can be demonstrated by the thyroid suppression test. Under normal conditions, administration of thyroid hormone in quantities sufficient to satisfy the body requirement suppresses endogenous TSH resulting in reduction of thyroid hormone synthesis and secretion. Since thyrotoxicosis due to excessive secretion of hormone by the thyroid gland implies that the feedback control mechanism is not operative or has been perturbed, it is easy to understand why under such circumstances the supply of exogenous hormone would also be ineffective in suppressing thyroid gland activity. The test is of particular value in patients who are euthyroid or only mildly thyrotoxic but suspected of having abnormal thyroid gland stimulation or autonomy.
Usually the test is carried out with 100 Âµg of L-T3 (liothyronine) given daily in two divided doses over a period of 7-10 days. 24 hour RAIU is obtained before and during the last two days of T3 administration.476 Normal persons show a suppression of the RAIU by at least 50% compared to the pre-L-T3 treatment value. No change or lesser reduction is not only typical of Graves’ disease but also other form of endogenous thyrotoxicosis, including toxic adenoma, functioning carcinoma, and thyrotoxicosis due to trophoblastic diseases. The presence of nonsuppressibility indicates thyroid gland activity independent of TSH but not necessarily thyrotoxicosis. Euthyroid patients with autonomous thyroid function have a normal TSH response to TRH before the administration of L-T3. However, inhibition of TSH secretion by the exogenous T3 does not suppress the autonomous activity of the thyroid gland. This is the most commonly encountered discrepancy between the results of the two related tests. When the T3 suppression test is used in conjunction with the scintiscan, localized areas of autonomous function can be identified. The test can be carried out without the administration of radioisotopes by measuring serum T4 before and two weeks following the ingestion of L-T3. Although total suppression of T4 secretion never occurs, even after prolonged treatment with L-T3, a reduction by at least 50% is normal. 477
Variants of the test have been proposed to reduce the potential risks of L-T3 administration in elderly patients and in those with angina pectoris or congestive heart failure. With the availability of sensitive TSH determinations and the TRH test, which are less dangerous, thyroid suppression tests are no longer indicated.
Specialized Thyroid Tests
A number of specialized tests are available for the evaluation of specific aspects of thyroid hormone biosynthesis, secretion, turnover, distribution, and absorption. Their primary application is of investigative nature. They are only briefly mentioned here for the sake of completeness.
The test involves the intravenous administration of tracer MIT or DIT labeled with radioiodide. Urine, collected over a period of four hours, is analyzed by chromatography or resin column separation. Normally, only 4-8% of the radioactivity is excreted as such; the remainder appears in the urine in the form of iodide. 480 Excretion of larger amounts of the parent compound indicates inability to deiodinate iodotyrosine. The test is useful in the diagnosis of a dehalogenase defect (see Chapter 16 ).
After administration of RAI, the isotopically labeled compounds synthesized in the thyroid gland and those secreted into the circulation can be analyzed by immunologic, chromatographic, electrophoretic, and density gradient centrifugation techniques. 481 Such tests serve to evaluate the synthesis and release of thyroid hormone, as well as to delineate the formation of abnormal iodoproteins.
The iodine kinetic procedure is used to evaluate overall iodide metabolism and to elucidate the pathophysiology of thyroid diseases. The analysis involves follow-up of the fate of administered radioiodide tracer by measurement of thyroidal accumulation, secretion into blood, and excretion in the urine and feces. 482 Double tracer techniques and programs for computer-assisted analysis of data are available.
Failure to achieve normal serum thyroid hormone concentration after administration of replacement doses of thyroid hormone is usually due to poor compliance, occasionally to the use of inactive preparations, and rarely, if ever, to malabsorption. The last can be evaluated by the simultaneous oral and intravenous administration of the hormone labeled with two different iodine isotope tracers. The ratio of the two isotopes in blood is proportional to the net absorbed fraction of the orally administered hormone. 483,484 Under normal circumstances, approximately 80% of T4 and 95% of T3 administered orally are absorbed. Hypothyroidism and a variety of other unrelated conditions have little effect on the intestinal absorption of thyroid hormones. Absorption may be diminished in patients with steatorrhea, in some cases of hepatic failure, during treatment with cholestyramine, and with diets rich in soybeans. The absorption of thyroid hormone can also be evaluated by the administration of a single oral dose of 100 Âµg T3 or 1 mg T4, followed by their measurement in blood sampled at various intervals. 485,486
Turnover kinetic studies require the intravenous administration of isotope-labeled tracer T4 or T3. 487-491 The half-time (t1/2) of disappearance of the hormone is calculated from the rate of decrease in serum trichloroacetic acid precipitable, ethanol extractable, or antibody precipitable isotope counts. Compartmental analysis can be used for the calculation of the turnover parameters. 488,489 The calculated daily degradation (D) or production rate (PR) is the product of the fractional turnover rate (K), the extrathyroidal distribution space (DS), and the average concentration of the hormone in serum. Noncompartmental analysis may be used for the calculation of kinetic parameters. 488 The metabolic clearance rate (MCR) is defined as the dose of the injected labeled tracer divided by the area under its curve of disappearance. The PR is then calculated from the product of the MCR and the average concentration of the respective nonradioactive iodothyronine measured in serum over the period of the study. Simultaneous studies of the T4 and T3 turnover kinetics can be carried out by injection of both hormones, labeled with different iodine isotopes. 488 , 490,491
Average normal values in adults for T4 and T3, respectively, are: t1/2 = 7.0 and 0.8 days; K = 10% and 90% per day; DS = 11 and 30 liters of serum equivalent; MCR = 1.1 and 25 liters/day; and PR = 90 and 25 Âµg/day.
The hormonal PR is accelerated in thyrotoxicosis and diminished in hypothyroidism. In euthyroid patients with TBG abnormalities, the PR remains normal, since changes in the serum hormone concentration are accompanied by compensatory changes in the fractional turnover rate and the extrathyroidal hormonal pool. 492 A variety of nonthyroidal illnesses may alter hormone kinetics 491 , 493 (see Chapter 5 ).
The kinetics of production of various metabolites of T4 and T3 in peripheral tissues and their further metabolism can be studied. Most methods use radiolabeled iodothyronine tracers injected intravenously. 489-491 Their disappearance is followed in serum samples obtained at various intervals of time after injection of the tracers by means of chromatographic and immunologic techniques of separation. Kinetic parameters can be calculated by noncompartmental analysis or by two or multiple compartment analysis. Estimates have been made by the differential measurement in urine of the isotopes derived from the precursor and its metabolite. They are in agreement with measurements carried out in serum. 494 Conversion rates (CR) of iodothyronines, principally generated in peripheral tissues, can be calculated from the ratio of their PR, and that of their respective precursors. Some iodothyronines, such as T3, are secreted by the thyroid gland as well as generated in peripheral tissues. Studies to calculate the CR require administration of thyroid hormone to block thyroidal secretion. 493
On the average 35% and 45% of T4 are converted to T3 and rT3, respectively, in peripheral tissues. The conversion of T4 to T3 is greatly diminished in a variety of illnesses (see Chapter 5 ) of nonthyroidal origin and in response to many drugs ( Table 5-3 ). Degradation and monodeiodination of iodothyronines can be estimated without the administration of isotopes. They are, however, less accurate. The conversion of T4 to T3 can be estimated semiquantitatively by the measurement of serum TT3 concentration after treatment with replacement doses of T4. 493
The metabolism and PRs of a variety of compounds related to thyroid physiology can be studied using their radiolabeled congeners and application of the general principles of turnover kinetics. Studies of TSH have demonstrated changes related not only to thyroid dysfunction but also associated with age, kidney, and liver disease. 495,496 Studies of the turnover kinetics of TBG have shown that the slight increases and decreases of serum TBG concentration associated with hypothyroidism and thyrotoxicosis, respectively, are due to changes in the degradation rate of TBG rather than synthesis. 492
Transfer of hormone from blood to tissues can be estimated in vivo by two techniques. A direct method follows the accumulation of the administered labeled hormone tracer by surface counting over the organ of interest. 497 An indirect method follows the early disappearance from plasma of the simultaneously administered hormone and albumin, labeled with different radioisotope tracers. 498 The difference between the rates of disappearance of the hormone and albumin represents the fraction of hormone that has left the vascular (albumin) space and presumably has entered the tissues.
1. Brown- Grant K: Extrathyroidal iodide concentrating mechanisms. Physiol Rev 4l:189-211, 1961.
2. Modan B , Mart H, Baidatz D: Radiation-induced head and neck tumors. Lancet 1:277-299, 1974.
3. Hall P, Boice JD, Berg G, Bjelkkengren G, Ericsson U-B, Hallquist A et al. Leukaemia incidence after iodine-131 exposure. Lancet 340:1-4, 1992.
5. Quimby EH , Feitelberg S, Gross W: Radioactive nuclides in medicine and biology (ed 3). Lea & Febiger, 1970.
6. MIRD: D ose estimate report no. 5: Summary of current radiation dose estimates to humans from 123I, 124 I, 126 I, 130 I, 131 I, and 132 I as sodium iodide. J Nucl Med 16:857-860, 1975.
7. MIRD: Dose estimate report no. 8: Summary of current radiation dose estimates to normal humans from 99m Tc as sodium pertechnetate. J Nucl Med 17:74-77, 1976.
8. Pittman JA Jr., Dailey GE III, Beschi RJ: Changing normal values for thyroidal radioiodine uptake. N Engl J Med 280:1431-1434, 1969.
9. Gluck FB, Nusynowitz ML, Plymate S: Chronic lymphocytic thyroiditis, thyrotoxicosis, and low radioactive iodine uptake: Report of four cases. N Engl J Med 293:624-628, 1975.
10. Savoie JC , Massin JP, Thomopoulos P, Leger F: Iodine-induced thyrotoxicosis in apparently normal thyroid glands. J Clin Endocrinol Metab 41:685-691, 1975.
11. Higgins HP , Ball D, Estham S: 20-min 99m Tc thyroid uptake: A simplified method using the gamma camera. J Nucl Med 14:907-911, 1973.
12. Baschieri L, Benedetti G, deLuca F, Negri M: Evaluation and limitations of the perchlorate test in the study of thyroid function. J Clin Endocrinol Metab 23:786-791, 1963.
18. Chopra IJ , Fisher DA, Solomon DH, Beall GN: Thyroxine and triiodothyronine in the human thyroid. J Clin Endocrinol Metab 36:311-316, 1973.
19. Engler D , Burger AG: The deiodination of iodothyronines and of their derivatives in man. Endocr Rev 5:151-184, 1984.
20. Pittman CS , Shimizu T, Burger A, Chambers JB Jr.: The nondeiodinative pathways of thyroxine metabolism: 3,5,3′,5′-tetraiodothyroacetic acid turnover in normal and fasting human subjects. J Clin Endocrinol Metab 50:712-716, 1980.
21. Gavin LA , Livermore BM, Cavalieri RR, al e: Serum concentration, metabolic clearance, and production rates of 3,5,3′-triiodothyroacetic acid in normal and athyreotic man. J Clin Endocrinol Metab 51:529-534, 1980.
22. Chopra IJ , Wu S-Y, Teco GNC, Santini F: A radioimmunoassay for measurement of 3,5,3′-triiodothyronine sulfate: Studies in thyroidal and nonthyroidal diseases, pregnancy, and neonatal life. 75:189-194, 1992.
23. deVijlder JJM , Veenboer GJM: Thyroid albumin originates from blood. Endocrinology 131:578-584, 1992.
24. Surks MI , Oppenheimer JH: Formation of iodoprotein during the peripheral metabolism of 3,5,3′-triiodo-L-thyroxine- 125 I in the euthyroid man and rat. J Clin Invest 48:685-695, 1969.
25. Refetoff S , Matalon R, Bigazzi M: Metabolism of L-thyroxine (T4) and L-triiodothyronine (T3) by human fibroblasts in tissue culture: Evidence for cellular binding proteins and conversion of T4 to T3. Endocrinology 91:934-947, 1972.
26. Koerner D , Surks MI, Oppenheimer JH: In vitro formation of apparent covalent complexes between L-triiodothyronine and plasma protein. J Clin Endocrinol Metab 36:239-245, 1973.
27. Trevorrow V : Studies on the nature of the iodine in blood. J Biol Chem 127:737-750, 1939.
28. Barker SB: Determination of protein-bound iodine. J Biol Chem 173:715-724, 1948.
29. Refetoff S : Principles of competitive binding assay and radioimmunoassay. A. Gottschalk and E. J. Potchen (eds), Diagnostic Nuclear Medicine (Golden’s Diagnostic Radiology), Williams & Wilkins, Baltimore, pp. 215-236,1976.
31. O’Connor JF , Wu GY, Gallagher TF, Hellman L: The 24-hour plasma thyroxin profile in normal man. J Clin Endocrinol Metab 39:765-771, 1974.
32. Fang VS , Refetoff S: Radioimmunoassay for serum triiodothyronine: Evaluation of simple techniques to control interference from binding proteins. Clin Chem 20:1150-1154, 1974.
33. Larsen PR , Dockalova J, Sipula D, Wu FM: Immunoassay of thyroxine in unextracted human serum. J Clin Endocrinol Metab 37:117-182, 1973.
34. Sterling K , Milch PO: Thermal inactivation of thyroxine-binding globulin for direct radioimmunoassay of triiodothyronine in serum. J Clin Endocrinol Metab 38:866-875, 1974.
35. Mitsuma T , Nihei N, Gershengorn MC, Hollander CS: Serum triiodothyronine: Measurements in human serum by radioimmunoassay with corroboration by gas-liquid chromatography. J Clin Invest 50:2679-2688, 1971.
38. Ikekubo K , Konishi J, Endo K, et al: Anti-thyroxine and anti-triiodothyronine antibodies in three cases of Hashimoto’s thyroiditis. Acta Endocrinol 89:557-566, 1978.
39. Sakata S , Nakamura S, Miura K: Autoantibodies against thyroid hormones or iodothyronine. Implications in diagnosis, thyroid function, treatment, and pathogenesis. Ann Intern Med 103:579-589, 1985.
40. Canadian Task Force on the periodic health examination. Periodic health examination, 1990 Update: 1. Early detection of hyperthyroidism and hypothyroidism in adults and screening of newborns for congenital hypothyroidism. J Can Med Assoc 142:955-961, 1990.
42. Schuurs AWM , Van Weemen BK: Enzyme-immunoassay. Clin Chim Acta 81:1-40, 1977.
43. Galen RS , Forman D: Enzyme immunoassay of serum thyroxine with AutoChemist” multichannel analyzer. Clin Chem 23:119-121, 1977.
44. Schall RF , Fraser AS, Hausen HW, al e: A sensitive manual enzyme immunoassay for thyroxine. Clin Chem 24:1801-1804, 1978.
45. Miyai K , Ishibashi K, Kawashima M: Enzyme immunoassay of thyroxine in serum and dried blood samples on filter paper. Endocrinol Jpn 27:375-380, 1980.
45a. Rongen HA , Hoetelmans RM, Bult A, van Bennekom: Chemiluminescence and immunoassays. J Pharmaceut Biomed Anal 12:433-62, 1994
45b Gonzalez RR , Robaina R, Rodriguez ME, BlancaS : An enzyme immunoassay for determining total thyroxine in human serum using an ultramicroanalytical system. Clin Chim Acta 197:159-170, 1991
50. Refetoff S: Inherited thyroxine-binding globulin (TBG) abnormalities in man. Endocr Rev 10:275-293, 1989.
51. Abuid J , Klein AH, Foley TP Jr., Larsen PR: Total and free triiodothyronine and thyroxine in early infancy. J Clin Endocrinol Metab 39:263-268, 1974.
52. Franklyn JA, Ramsden DB & Sheppard MC: The influence of age and sex on tests of thyroid function. Annal Clin Biochem 22:502-505, 1985.
53. Westgren U , Burger A, Ingemanssons S, Melander A, Tibblin S, Wahlin E: Blood levels of 3,5,3′-triiodothyronine and thyroxine: Differences between children, adults, and elderly subjects. Acta Med Scand 200:493-495, 1976.
56. DeCostre P , Buhler U, DeGroot LJ, Refetoff S: Diurnal rhythm in total serum thyroxine levels. Metabolism 20:782-791, 1971.
57. Bartalena L : Recent achievements in studies on thyroid hormone-binding proteins. Endocr Rev 11:47-64, 1990.
58. Stockigt JR, Topliss DJ, Barlow JW, White EL, Hurley DM, Taft P: Familial euthyroid thyroxine excess: An appropriate response to abnormal thyroxine binding associated with albumin. J Clin Endocrinol Metab 53:353-359, 1981.
59. Sunthornthepvarakul T , Angkeow P, Weiss RE, Hayashi Y, Refetoff S: A missense mutation in the albumin gene produces familial disalbuminemic hyperthyroxinemia in 8 unrelated families. Biochem Biophys Res Commun 202:781-787, 1994.
60. Sterling K, Refetoff S, Selenkow HA: T3 toxicosis: Thyrotoxicosis due to elevated serum triiodothyronine levels. JAMA 213:571-575, 1970.
61. Sterling K , Brenner MA, Newman ES, al e: The significance of triiodothyronine (T3) in maintenance of euthyroid status after treatment of hyperthyroidism. J Clin Endocrinol Metab 33:729-731, 1971.
62. Delange F , Camus M, Ermans AM: Circulating thyroid hormones in endemic goiter. J Clin Endocrinol Metab 34:891-895, 1972.
63. Parle JV , Franklyn JA, Cross KW, Jones SR, Sheppard MC: Thyroxine prescription in the community: serum TSH level assays as an indicator of undertreatment or overtreatment. Br J Gen Pract 43:107-109, 1993.
64. Saberi M , Utiger RD: Serum thyroid hormone and thyrotropin concentrations during thyroxine and triiodothyronine therapy. J Clin Endocrinol Metab 39:923-927, 1974.
67. Olsen T , Laurberg P, Weeke J: Low serum triiodothyronine and high serum reverse triiodothyronine in old age: An effect of disease not age. J Clin Endocrinol Metab 47:1111-1115, 1978.
68. Welle S , O’Conell M, Danforth D Jr., Campbell R: Decreased free fraction of serum thyroid hormones during carbohydrate over-feeding. Metabolism 33:837-839, 1984.
69. Portnay GI , O’Brian JT, Bush J, al e: The effect of starvation on the concentration and binding of thyroxine and triiodothyronine in serum and on the response to TRH. J Clin Endocrinol Metab 39:191-194, 1974.
70. Azizi F : Effect of dietary composition on fasting-induced changes in serum thyroidhormones and thyrotropin. Metabolism 27:935-942, 1978.
71. Scriba PC, Bauer M, Emmert D, al e: Effects of obesity, total fasting and re-alimentation of L-thyroxine (T4), 3,5,3′-L-triiodothyronine (T3), 3,3′,5′-L-triiodothyronine (rT3), thyroxine binding globulin (TBG), cortisol, thyrotrophin, cortisol binding gloublin (CBG), transferrin, a 2 -haptoglobin and complement C’3 in serum. Acta Endocrinol 91:629-643, 1979.
72. Larsen PR : Triiodothyronine. Review of recent studies of its physiology and pathophysiology in man. Metabolism 21:1073-1092, 1972.
75. RÃ¶sler A , Litvin Y, Hage C, Gross J, Cerasi E: Familial hyperthyroidism due to inappropriate thyrotropin secretion successfully treated with triiodothyronine. J Clin Endocrinol Metab 54:76-82, 1982.
76. Maxon HR , Burman KD, Premachandra BN, et al: Familial elevation of total and free thyroxine in healthy, euthyroid subjects without detectable binding protein abnormalities. Acta Endocrinol 100:224-230, 1982.
77. Chopra IJ , Williams DE, Orgiazzi J, Solomon DH: Opposite effects of dexamethasone on serum concentrations of 3,3′,5′-triiodothyronine (reverse T3) and 3,3′,5-triiodothyronine (T3). J Clin Endocrinol Metab 41:911-920, 1975.
78. Cavalieri RR , Sung LC, Becker CE: Effects of phenobarbital on thyroxine and triiodothyronine kinetics in Graves’ disease. J Clin Endocrinol Metab37 308-316:1973.
79. Davies PH, Franklyn JA: Effects of drugs on tests of thyroid function. Eur J Clin Pharmacol 40:439-451, 1991.
80. Busnardo B , Vangelista R, Girelli ME, al. e: TSH levels and TSH response to TRH as a guide to the replacement treatment of patients with thyroid carcinoma. J Clin Endocrinol Metab 42:901-906, 1976.
83. Refetoff S , Hagen S, Selenkow HA: Estimation of the T4 binding capacity of serum TBG and TBPA by a single T4 load ion exchange resin method. J Nucl Med 13:2-12, 1972.
87. Miyai K , Ito M, Hata N: Enzyme immunoassay of thyroxine-binding globulin. Clin Chem 28:2408-2411, 1982.
88. Refetoff S , Murata Y, Vassart G, Chandramouli V, Marshall JS: Radioimmunoassays specific for the tertiary and primary structures of thyroxine-binding globulin (TBG): Measurement of denatured TBG in serum. J Clin Endocrinol Metab 59:269-277, 1984.
89. Freeman T , Pearson JD: The use of quantitative immunoelectrophoresis to investigate thyroxine-binding human serum proteins. Clin Cheim Acta 26:365-368, 1969.
90. Nielsen HG , Buus O, Weeke B: A rapid determination of thyroxine-binding globulin in human serum by means of the Laurell Rocket immunoelectrophoresis. Clin Chim Acta 36:133-138, 1972.
91. Mancini G , Carbonara AO, Heremans JF: Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry 2:235-254, 1965.
92. Chopra IJ , Solomon DH, Ho RS: Competitive ligand-binding assay for measurement of thyroxine-binding globulin (TBG). J Clin Endocrinol Metab 35:565-573, 1972.
93. Marshall JS , Levy RP, Steinberg AG: Human thyroxine-binding globulin deficiency: A genetic study. N Engl J Med 274:1469-1473, 1966.
94. Ekins R : Measurement of free hormones in blood. Endocrine Rev 11:5-6, 1990.
97. Nelson JC , Tomel RT: Direct determination of free thyroxin in undiluted serum by equilibrium dialysis/radioimmunoassay. Clin Chem 34:1737-1744, 1988.
98. Surks MI, Hupart KH, Pan C, Shapiro LE: Normal free thyroxine in critical nonthyroidal illnesses measured by ultrafiltration of undiluted serum and equilibrium dialysis. J Clin Endocrinol Metab 67:1031-1039, 1988.
99. Melmed S , Geola FL, Reed AW, al e: A comparison of methods for assessing thyroid function in non-thyroidal illness. J Clin Endocrinol Metab 54:300-306, 1982.
100. Wong TK , Pekary E, Hoo GS, Bradley ME, Hershman JM: Comparison of methods for measuring free thyroxin in nonthyroidal illness. Clin Chem 38:720-724, 1992.
101. Chopra IJ , Chopra U, Smith SR, al e: Reciprocal changes in serum concentration of 3,3′,5′-triiodothyronine (reverse T3) and 3,3′,5-triiodothyronine (T3) in systemic illnesses. J Clin Endocrinol Metab 41:1043-1049, 1975.
102. Oppenheimer JH , Squef R, Surks MI, Hauer H: Binding of thyroxine by serum proteins evaluated by equilibrium dialysis and electrophoretic techniques. Alterations in non-thyroidal illness. J Clin Invest 42:1769-1782, 1963.
103. Snyder SM , Cavalieri RR, Ingbar SH: Simultaneous measurement of percentage free thyroxine and triiodothyronine: Comparison of equilibrium dialysis and Sephadex chromatography. J Nucl Med 17:660-664, 1976.
104. Nelson JC , Bruce WR, Pandian MR: Dependence of free thyroxine estimates obtained with equilibrium tracer dialysis on the concentration of thyroxine-binding gloublin. Clin Chem 38:1294-1300, 1992.
104a Nelson JC , Weiss R, Wilcox RB: Underestimates of serum free T4 concentrations by free T4 immunoassays. J Clin Endocrinol Metab 79:76-79, 1994
104b Nelson JC , Nayak SS, Wilcox RB: Variable underestimates of serum free T4 immunoassays of free t4 concentrations in simple solutions. J Clin Endocrinol Metab 79:1373-1375, 1994
105c Faber J , Waetjen I, Siersbaek-Nielsen K: Free T4 measured in undiluted serum by dialysis and ultrafiltration: effects of non-thyroidal illness and an acute load of salicylate or heparin. Clin Chim Acta 223:159-167, 1993
105. Van der Sluijs Veer G, Vermes I, Bonte HA, Hoorn RKJ: Temperature effects on free-thyroxine measurements: Analytical and clinical consequences. Clin Chem 38:1327-1331, 1992.
106. Larsen PR , Alexander NM, Chopra IJ, et al: Revised nomenclature for test of thyroid hormones and thyroid-related proteins in serum. Clin Chem 33:2114-2116, 1987.
107. Felicetta JV , Green WL, Mass LB, al e: Thyroid function and lipids in patients with chronic liver disease treated by hemodialysis with comments on the free thyroxine index. Metabolism 28:756-763, 1979.
109. Glinoer D , Fernandez-Deville M, Ermans AM: Use of direct thyroxine-binding gloublin measurement in the evaluation of thyroid function. J Endocrinol Invest 1:329-335, 1978.
110. Attwood EC : The T3/TBG ratio and the biochemical investigation of thyrotoxicosis. Clin Biochem 12:88-92, 1979.
111. Nuutila P , Koskinen P, Irjala K, et al: Two new two-step immunoassays for free thyroxin evaluated: Solid-phase radioimmunoassay and time-resolved fluoroimmunoassay. Clin Chem 36:1355-1360, 1990.
112. Hay ID , Bayer MF, Kaplan MM, Klee GG, Larsen PR, Spencer CA: American Thyroid Association assessment of current free thyroid hormone and thyrotropin measurements and guidelines for future clinical assays. Clin Chem 37:2002-2008, 1991.
113. Wilkins TA , Midgley JEM, Barron N: Comprehensive study of a thyroxin-analog-based assay for free thyroxin (“Amerlex FT4″). Clin Chem 31:1644-1653, 1985.
113a Stockigt JR , Stevens V, White E, Barlow JW: Unbound analog radioimmunoassays for free thyroxin measure the albumen-bound hormone fraction. Clin Chem 29:1408-10, 1983.
113b Christofides ND , Sheehan CP: Enhanced chemiluminescence labeled-antibody immunoassay (Amerlite-MAB) for free thyroxine: design, development and technical validation. Clin Chem 41: 17-23, 1995
113c Christofides ND , Sheehan CP: Multicenter evaluation of enhanced chemiluminescence labeled-antibody immunoassay (Amerlite-MAB) for free thyroxine. Clin Chem 41: 24-31, 1995
114. John R : Autoantibodies to thyroxin and interference with free-thyroxin assay. Clin Chem 29:581-582, 1983.
116. Sarne DH, Refetoff S, Murata Y, Dick M, Watson F: Variant thyroxine-binding globulin in serum of Australian Aborigines. A comparison with familial TBG deficiency in Caucasians and American Blacks. J Endocrinol Invest 8:217-224, 1985.
116a Samuels MH, Pillote K, Asher D et al. Variable effects of nonsteroidal antiinflammatory agents on thyroid test results . J Clin Endocrinol Metab 2003; 88: 5710-6.
117. Murata Y , Refetoff S, Sarne DH, Dick M, Watson F: Variant thyroxine-binding globulin in serum of Australian Aborigines: Its physical, chemical and biological properties. J Endocrinol Invest 8:225-232, 1985.
119. Kaptein EM , Macintyre SS, Weiner JM, al e: Free thyroxine estimates in nonthyroidal illness: Comparison of eight methods. J Clin Endocrinol Metab 52:1073-1077, 1981.
120. Lehotay DC , Weight CW, Seltman JH, al e: Free thyroxin: A comparison of direct and indirect methods and their diagnostic usefulness in nonthyroidal illness. Clin Chem 28:1826-1829, 1982.
121. Oppenheimer JH , Schwartz HL, Mariash CN, Kaiser FE: Evidence for a factor in the sera of patients with nonthyroidal illness which inhibits iodothyronine binding by solid matrices, serum proteins, and rat hepatocytes. J Clin Endocrinol Metab 54:757-766, 1982.
122. Woeber KA , Maddux BA: Thyroid hormone binding in nonthyroidal illness. Metabolism 30:412-416, 1981.
123. Chopra IJ , Solomon DH, Teco GNC, Eisenberg JB: An inhibitor of the binding of thyroid hormones to serum proteins is present in extrathyroidal tissues. Science 215:407-409, 1982.
124. Chopra IJ , Chua Teco GN, Mead JF, al. e: Relationship between serum free fatty acids and thyroid hormone binding inhibitor in nonthyroidal illnesses. J Clin Endocrinol Metab 60:980-984, 1985.
126. Chopra IJ : An assessment of daily production and significance of thyroidal secretion of 3,3′,5′-triiodothyronine (reverse T3) in man. J Clin Invest 58:32-40, 1976.
127. Nicod P , Burger A, Staeheli V, Vallotton MB: A radioimmunoassay for 3,3′,5′-triiodo-L-thyronine in unextracted serum: Method and clinical results. J Clin Endocrinol Metab 42:823-829, 1976.
128. Chopra IJ : A radioimmunoassay for measurement of 3,3′,5′-triiodothyronine (reverse T3). J Clin Invest 54:583-592, 1974.
129. O’Connell M , Robbins DC, Bogardus C, al. e: The interaction of free fatty acids in radioimmunoassays for reverse triiodothyronine. J Clin Endocrinol Metab 55:577-582, 1982.
132. Weiss RE , Angkeow P, Sunthornthepvarakul T, et al: Linkage of familial dysalbuminemic hyperthyroxinemia to the albumin gene in a large Amish family. J Clin Endocrinol Metab 80:1995.
133. Chopra IJ : A radioimmunoassay for measurement of 3′-monoiodothyronine. J Clin Endocrinol Metab 51:117-123, 1980.
134. Chopra IJ , Sack J, Fisher DA: Circulating 3,3′,5′-triiodothyronine (reverse T3) in the human newborn. J Clin Invest 55:1137-1141, 1975.
135. Engler D , Markelbach U, Steiger G, Burger AG: The monodeiodination of triiodothyronine and reverse triiodothyronine in man: A quantitative evaluation of the pathway by the useof turnover rate techniques. J Clin Endocrinol Metab 58:49-61, 1984.
136. Pangaro L , Burman KD, Wartofsky L, al. e: Radioimmunoassay for 3,5-diiodothyronine and evidence for dependence on conversion from 3,5,3′-triiodothyronine. J Clin Endocrinol Metab 50:1075-1081, 1980.
137. Faber J , Kirkegaard C, Lumholtz IB, al. e: Measurements of serum 3′,5′-diiodothyronine and 3,3′-diiodothyronine concentrations in normal subjects and in patients with thyroid and nonthyroid disease: Studies of 3′,5′-diiodothyronine metabolism. J Clin Endocrinol Metab 48:611-617, 1979.
138. Geola F, Chopra IJ, Geffner DL: Patterns of 3,3′,5′-triiodothyronine monodeiodination in hypothyroidism and nonthyroidal illnesses. J Clin Endocrinol Metab 50:336-340, 1980.
139. Chopra IJ, Geola F, Solomon DH, Maciel RMB: 3′,5′-diiodothyroxine in health and disease: Studies by a radioimmunoassay. J Clin Endocrinol Metab 47:1198-1207, 1978.
140. Burman KD , Wright FD, Smallridge RC, al. e: A radioimmunoassay for 3′,5′-diiodothyronine. J Clin Endocrinol Metab 47:1059-1064, 1978.
141. Jaedig S , Faber J: The effect of starvation and refeeding with oral versus intravenous glucose on serum 3,5-,3,3′- and 3′,5′-diiodothyronine and 3′-monoiodothyronine. Acta Endocrinol 100:388-392, 1982.
142. Smallridge RC , Wartofsky L, Green BJ, al. e: 3′-L-monoiodothyronine: Development of a radioimmunoassay and demonstration of in vivo conversion from 3′,5′-diiodothyronine. J Clin Endocrinol Metab 48:32-36, 1979.
143. Corcoran JM , Eastman CJ: Radioimmunoassay of 3-L-monoiodothyronine: Application in normal human physiology and thyroid disease. J Clin Endocrinol Metab 57:66-70, 1983.
144. Nakamura Y , Chopra IJ, Solomon DH: An assessment of the concentration of acetic acid and proprionic acid derivatives of 3,5,3′-triiodothyronine in human serum. J Clin Endocrinol Metab 46:91-97, 1978.
145. Burger A, Suter P, Nicod P, al. e: Reduced active thyroid hormone levels in acute illness. Lancet 1:163-655, 1976.
146. Pittman CS, Suda AK, Chambers JB Jr., al. e: Abnormalities of thyroid hormone turnover in patients with diabetes mellitus before and after insulin therapy. J Clin Endocrinol Metab 48:854-860, 1979.
147. Dlott RS , LoPresti JS, Nicoloff JT: Evidence that triiodoacetate (TRIAC) is the autocrine thyroid hormone in man. Thyroid 2(Suppl):S-94, 1992.
148. Nelson JC , Weiss RM, Lewis JE, al. e: A multiple ligand-binding radioimmunoassay of diiodothyrosine. J Clin Invest 53:416-422, 1974.
149. Nelson JC, Lewis JE: Radioimmunoassay of iodotyrosines. G. E. Abraham (eds), Handbook of Radioimmunoassay, Marcel Dekker, New York, pp. p 705,1979.
150. Meinhold H , Beckert A, Wenzel W: Circulating diiodotyrosine: Studies of its serum concentration, source, and turnover using radioimmunoassay after immunoextraction. J Clin Endocrinol Metab 53:1171-1178, 1981.
151. Van Herle AJ , Uller RP, Matthews NL, Brown J: Radioimmunoassay for measurement of thyroglobulin in human serum. J Clin Invest 52:1320-1327, 1973.
151a Spencer CA , Takeuchi M, Kazarosyan M: Current status and performance goals for serum thyroglobulin assays. Clin Chem 42:164-173, 1996
151b Marquet PY , Daver A, Sapin R et al. Highly sensitive immunoradiometric assay for serum thyroglobulin with minimal interference from autoantibodies. Clin Chem 42: 258-262, 1996
151c Erali M , Bigelow RB, Meikle AW. ELISA for thyroglobulin in serum: recovery studies to evaluate autoantibody interference and relaibility of thyroglobulin values. Clin Chem 42: 766-770, 1996
151d Dai J , Dent W, Atkinson JW, Cox JG, Dembinski TC. Comparison of three immunoassay kits for serum thryoglobulin in patients with thyroid cancer. Clin Biochem 29:461-465, 1996
152. Spencer CA , Takeuchi M, Kazarosyan M, al. e: Serum thryoglobulin autoantibodies:prevalence, influence on serum thryoglobulin measurement, and prognostic significance in patients with differentiated thryoid carcinoma. J Clin Endocrinol Metab 83: 1121-27, 1998.
152a Spencer CA, Wang CC. Thyroglobulin measurement. Techniques, clinical benefits and pitfalls. Endocrinol Metab Clin North Am 24:841-63, 1995
153. Ozata M, Suzuki S, Miyamoto T, al e.: Serum thyroglobulin in the follow-up of patients with treated differentiated thyroid cancer. J Clin Endocrinol Metab 79:98-105, 1994.
154. Pacini F, Pinchera A, Giani C, Grasso L, Dover F, Baschieri L: Serum thyroglobulin in thyroid carcinoma and other thyroid disorders. J Endocrinol Invest 3:283-292, 1980.
155. Black EG , Cassoni A, Gimlette TMD, al. e: Serum thyroglobulin in thyroid cancer. Br Med J 3:443-445, 1981.
156. Pezzino V , Filetti S, Belfiore A, al. e: Serum thyroglobulin levels in the newborn. J Clin Endocrinol Metab 52:364-366, 1981.
157. Penny R , Spencer CA, Frasier D, Nicoloff JT: Thyroid-stimulating hormone and thyroglobulin levels decrease with chronological age in children and adolescents. J Clin Endocrinol Metab 56:177-180, 1983.
158. Refetoff S , Lever EG: The value of serum thyroglobulin measurement in clinical practice. JAMA 250:2352-2357, 1983.
159. Izumi M , Kubo I, Taura M, al. e: Kinetic study of immunoreactive human thyroglobulin. J Clin Endocrinol Metab 62:400-412, 1986.
160. Uller RP , Van Herle AJ, Chopra IJ: Comparison of alterations in circulating thyroglobulin, triiodothyronine and thyroxine in response to exogenous (bovine) and endogenous (human) thyrotropin. J Clin Endocrinol Metab 37:741-745, 1973.
161. Lever EG , Refetoff S, Scherberg NH, Carr K: The influence of percutaneous fine needle aspiration on serum thyroglobulin. J Clin Endocrinol Metab 56:26-29, 1983.
162. Uller RP , Van Herle AJ: Effect of therapy on serum thyroglobulin levels in patients with Graves’ disease. J Clin Endocrinol Metab 46:747-755, 1978.
163. Smallridge RC , DeKeyser FM, Van Herle AJ, al. e: Thyroid iodine content and serum thyroglobulin: Clues to the national history of destruction-induced thyroiditis. J Clin Endocrinol Metab 62:1213-1219, 1986.
164. Mariotti S , Martino E, Cupini C, al. e: Low serum thyroglobulin as a clue to the diagnosis of thyrotoxicosis factitia. N Engl J Med 307:410-412, 1982.
165. Van Herle AJ , Uller RP: Elevated serum thyroglobulin: A marker of metastases in differentiated thyroid carcinoma. J Clin Invest 56:272-277, 1975.
166. Schneider AB , Line BR, Goldman JM, Robbins J: Sequential serum thyroglobulin determinations, 131 I scans, and 131 I uptakes after triiodothyronine withdrawal in patients with thyroid cancer. J Clin Endocrinol Metab 53:1199-1206, 1981.
167. Colacchio TA, LoGerfo P, Colacchio DA, Feind C: Radioiodine total body scan versus serum thyroglobulin levels in follow-up of patients with thyroid cancer. Surgery 91:42-45, 1982.
168. Black EG, Sheppard MC: Serum thyroglobulin measurements in thyroid cancer: evaluation of “false” positive results. Clin Endocrinol (Oxf) 35:519-20, 1991.
169. Kawamura S , Kishino B, Tajima K, al. e: Serum thyroglobulin changes in patients with Graves’ disease treated with long term antithyroid drug therapy. J Clin Endocrinol Metab 56:507-512, 1983.
170. Black EG , Bodden SJ, Hulse JA, Hoffenberg R: Serum thyroglobulin in normal and hypothyroid neonates. Clin Endocrinol 16:267-274, 1982.
171. Heinze HJ , Shulman DI, Diamond FB Jr., Bercu BB: Spectrum of serum thyroglobulin elevation in congenital thyroid disorders. Thyroid 3:37-40, 1993.
172. Czernichow P , Schlumberger M, Pomarede R, Fragu P: Plasma thyroglobulin measurements help determine the type of thyroid defect in congenital hypothyroidism. J Clin Endocrinol Metab 56:242, 1983.
173. Burke CW, Shakespear RA, Fraser TR: Measurement of thyroxine and triiodothyronine in human urine. Lancet 2:1177-1179, 1972.
174. Chan V , Landon J: Urinary thyroxine excretion as index of thyroid function. Lancet 1:4-6, 1972.
175. Chan V , Besser GM, Landon J, Ekins RP: Urinary tri-iodothyronine excretion as index of thyroid function. Lancet 2:253-256, 1972.
176. Burke CW , Shakespear RA: Triiodothyronine and thyroxine in urine. II. Renal handling, and effect of urinary protein. J Clin Endocrinol Metab 42:504-513, 1976.
177. Sack J , Fisher DA, Hobel CJ, Lam R: Thyroxine in human amniotic fluid. J Pediatr 87:364-368, 1975.
178. Chopra IJ , Crandall BF: Thyroid hormones and thyrotropin in amniotic fluid. N Engl J Med 293:740-743, 1975.
179. Burman KD , Read J, Dimond RC, al. e: Measurement of 3,3′,5′-triiodothyronine (reverse T3), 3,3′-L-diiodothyronine, T3, and T4 in human amniotic fluid and in cord and maternal serum. J Clin Endocrinol Metab 43:1351-1359, 1976.
180. Siersbaek-Nielsen K, Hansen JM: Tyrosine and free thyroxine in cerebrospinal fluid in thyroid disease. Acta Endocrinol 64:126-132, 1970.
181. Hagen GA , Elliott WJ: Transport of thyroid hormones in serum and cerebrospinal fluid. J Clin Endocrinol Metab 37:415-422, 1973.
182. Nishikawa M , Inada M, Naito K, al. e: 3,3′,5′-triiodothyronine (reverse T3) in human cerebrospinal fluid. J Clin Endocrinol Metab 53:1030-1035, 1981.
183. Mallol J , ObregÃ³n MJ, Morreale de Escobar G: Analytical artifacts in radioimmunoassay of L-thyroxin in human milk. Clin Chem 28:1277-1282, 1982.
184. Varma SK , Collins M, Row A, al. e: Thyroxine, tri-iodothyronine, and reverse tri-iodothyronine concentrations in human milk. J Pediatr 93:803-806, 1978.
185. Jansson L , Ivarsson S, Larsson I, Ekman R: Tri-iodothyronine and thyroxine in human milk. Acta Paediatr Scand 72:703-705, 1983.
186. Riad-Fahmy D , Read GF, Walker RF, Griffiths K: Steroids in saliva for assessing endocrine function. Endocr Rev 3:367-395, 1982.
187. Elson MK , Morley JE, Shafer RB: Salivary thyroxine as an estimate of free thyroxine: Concise communication. J Nucl Med 24:700-702, 1983.
188. Reichlin S , Bollinger J, Nejad I, Sullivan P: Tissue thyroid hormone concentration of rat and man determined by radioimmunoassay: Biologic significance. Mt Sinai J Med 40:502-510, 1973.
189. Ochi Y , Hachiya T, Yoshimura M, al. e: Determination of triiodothyronine in red blood cells by radioimmunoassay. Endocrinol Jpn 23:207-213, 1976.
190. Lim VS , Zavata DC, Flanigan MJ, Freeman RM: Basal oxygen uptake: A new technique for an old test. J Clin Endocrinol Metab 62:863-868, 1986.
191. Becker DV : Metabolic indices. S. C. Werner and S. H. Ingbar (eds), The Thyroid: A Fundamental and Clinical Text., Harper & Row, New York, pp. 524-533,1971.
192. Waal-Manning HJ : Effect of propranolol on the duration of the Achiles tendon reflex. Clin Pharmacol Ther 10:199-206, 1969.
193. Rodbard D, Fujita T, Rodbard S: Estimation of thyroid function by timing the arterial sounds. JAMA 2010:884-887, 1967.
194. Nuutila P , Irjala K, Saraste M, SeppÃ¤lÃ¤ P, Viikari J: Cardiac systolic time intervals and thyroid hormone levels during treatment of hypothyroidism. Scand J Clin Lab Invest 52:467-477, 1992.
195. Lewis BS , Ehrenfeld EN, Lewis N, Gotsman MS: Echocardiographic LV function in thyrotoxicosis. Am Heart J 97:460-468, 1979.
196. Tseug KH, Walfish PG, Persand JA, Gilbert BW: Concurrent aortic and mitral valve echocardiography permits mesurements of systolic time intervals as an index of peripheral tissue thyroid functional status. 69:633-638, 1989.
197. Vesell ES, Shapiro JR, Passananti GT, al. e: Altered plasma half-lives of antipyrine, propylthiouracil, and methimazole in thyroid dysfunction. Clin Pharmacol Ther 17:48-56, 1975.
198. Brunk SF , Combs SP, Miller JD, al. e: Effects of hypothyroidism and hyperthyroidism on dipyrone metabolism in man. J Clin Pharmacol 14:271-279, 1974.
199. Kekki M : Serum protein turnover in experimental hypo- and hyperthyroidism. Acta Endocrinol suppl. 91:1-139, 1964.
200. Walton KW , Scott PJ, Dykes PW, Davies JWL: The significance of alterations in serum lipids in thyroid dysfunction. II. Alterations of the metabolism and turnover of 131 I-low-density lipoproteins in hypothyroidism and thyrotoxicosis. Clin Sci 29:217-238, 1965.
201. Hellman L , Bradlow HL, Zumoff B, Gallagher TF: The influence of thyroid hormone on hydrocortisone production and metabolism. J Clin Endocrinol Metab 21:1231-1247, 1961.
202. Gallagher TF , Hellman L, Finkelstein J, al. e: Hyperthyroidism and cortisol secretion in man. J Clin Endocrinol Metab 34:919-927, 1972.
203. Kiely JM , Purnell DC, Owen CA Jr.: Erythrokinetics in myxedema. Ann Intern Med 67:533-538, 1967.
204. Das KC , Mukherjee M, Sarkar TK, al. e: Erythropoiesis and erythropoietin in hypo- and hyperthyroidism. J Clin Endocrinol Metab 40:211-220, 1975.
205. Rivlin RS , Melmon KL, Sjoerdsma A: An oral tyrosine tolerance test in thyrotoxicosis and myxedema. N Engl J Med 272:1143-1148, 1965.
206. BÃ©langer R, Chandramohan N, Misbin R, Rivlin RS: Tyrosine and glutamic acid in plasma and urine of patients with altered thyroid function. Metabolism 21:855-865, 1972.
207. Lamberg BA , GrÃ¤sbeck R: The serum protein pattern in disorders of thyroid function. Acta Endocrinol 19:91-100, 1955.
208. Anderson DC: Sex-hormone-binding globulin. Clin Endocrinol 3:69-96, 1974.
209. DeNayer P , Lambot MP, Desmons MC, Rennotte B, Malvaux P, Beckers C: Sex hormone-binding protein in hypothyroxinemic patients: a discriminator for thyroid status in thyroid hormone resistance and familial dysalbuminemic hyperthyroxinemia. J Clin Endocrinol Metab 62:1309-1312, 1986.
210. Macaron C I, Macaron ZG: Increased serum ferritin levels in hyperthyroidism. Ann Intern Med 96:617-618, 1982.
211. Takamatsu J , Majima M, Miki K, Kuma K, Mozai T: Serum ferritin as a marker of thyroid hormone action on peripheral tissues. J Clin Endocrinol Metab 61:672-676, 1985.
212. Graninger W , Pirich KR, Speiser W, al. e: Effect of thyroid hormones on plasma protein concentration in man. J Clin Endocrinol Metab 63:407-411, 1986.
213. Oppenheimer JH : Role of plasma proteins in the binding, distribution, and metabolism of the thyroid hormones. N Engl J Med 278:1153-1162, 1968.
214. Shah JH , Cechio GM: Hypoinsulinemia of hypothyroidism. Arch Intern Med 132:657-661, 1973.
215. Levy LJ, Adesman JJ, Spergel G: Studies on the carbohydrate and lipid metabolism in thyroid disease: Effects of glucagon. J Clin Endocrinol Metab 30:372-379, 1970.
216. Chopra IJ , Tulchinsky D: Status of estrogen-androgen balance in hyperthyroid men with Graves’ disease. J Clin Endocrinol Metab 38:269-277, 1974.
217. Seino Y , Matsukura S, Miyamoto Y, al. e: Hypergastrinemia in hyperthyroidism. J Clin Endocrinol Metab 43:852-855, 1976.
218. Bouillon R , DeMoor P: Parathyroid function in patients with hyper- or hypothyroidism. J Clin Endocrinol Metab 38:999-1004, 1974.
219. Castro JH, Genuth SM, Klein L: Comparative response to parathyroid hormone in hyperthyroidism and hypothyroidism. Metabolism 24:839-848, 1975.
220. Kojima N , Sakata S, Nakamura S, et al: Serum concentrations of osteocalcin in patients with hyperthyroidism, hypothyroidism and subacute thyroiditis. J Endocrinol Invest 15:491-496, 1992.
221. Body JJ , Demeester-Mirkine N, Borkowski A, al. e: Calcitonin deficiency in primary hypothyroidism. J Clin Endocrinol Metab 62:700-703, 1986.
222. Hauger-Klevene JH , Brown H, Zavaleta J: Plasma renin activity in hyper- and hypothyroidism: Effect of adrenergic blocking agents. J Clin Endocrinol Metab 34:625-629, 1972.
223. Ogihara T , Yamamoto T, Miyai K, Kumahara Y: Plasma renin activity and aldosterone concentration of patients with hyperthyroidism and hypothyroidism. Endocrinol Jpn 20:433-438, 1973.
224. Stoffer SS , Jiang NS, Gorman CA, Pikler GM: Plasma catecholamines in hypothyroidism and hyperthyroidism. J Clin Endocrinol Metab 36:587-589, 1973.
225. Christensen NJ: Plasma noradrenaline and adrenaline in patients with thyrotoxicosis and myxoedema. Clin Sci Mol Med 45:163-171, 1973.
226. Zimmerman RS, Gharib H, Zimmerman D, al. e: Atrial naturetic peptide in hypothyroidism. J Clin Endocrinol Metab 64:353-355, 1987.
227. Rolandi E , Santaniello B, Bagnasco M, et al: Thyroid hormones and atrial natriuretic hormone secretion: Study in hyper- and hypothyroid patients. Acta Endocrinol 127:23-26, 1992.
228. Distiller LA , Sagel J, Morley JE: Assessment of pituitary gonadotropin reserve using luteinizing hormone-releasing hormone (LRH) in states of altered thyroid function. J Clin Endocrinol Metab 40:512-515, 1975.
229. Refetoff S , Fang VS, Rapoport B, Friesen HG: Interrelationships in the regulation of TSH and prolactin secretion in man: Effects of L-DOPA, TRH and thyroid hormone in various combinations. J Clin Endocrinol Metab 38:450-457, 1974.
230. Honbo KS, Van Herle AJ, Kellett KA: Serum prolactin levels in untreated primary hypothyroidism. Am J Med 64:782-787, 1978.
231. Brauman H , Corvilain J: Growth hormone response to hypoglycemia in myxedema. J Clin Endocrinol Metab 28:301-304, 1968.
232. Rosenfield PS , Wool MS, Danforth E Jr.: Growth hormone response to insulin-induced hypoglycemia in thyrotoxicosis. J Clin Endocrinol Metab 29:777-780, 1969.
233. Hamada N , Uoi K, Nishizawa Y, al. e: Increase of serum GH concentration following TRH injection in patients with primary hypothyroidism. Endocrinol Jpn 23:5-10, 1976.
234. Kung AEC , Hui WM, Ng ESK: Serum and plasma epidermal growth factor in thyroid disorders. Acta Endocrinol 127:52-57, 1992.
235. Graig FA , Smith JC: Serum creatine phosphokinase activity in altered thyroid states. J Clin Endocrinol Metab 25:723-731, 1965.
236. Fleisher GA , McConahey WM, Pankow M: Serum creatine kinase, lactic dehydrogenase, and glutamic-oxalacetic transminase in thyroid diseases and pregnancy. Mayo Clin Proc 40:300-311, 1965.
237. Doran GR , Wilkinson JH: Serum creatine kinase and adenylate kinase in thyroid disease. Clin Chim Acta 35:115-119, 1971.
238. Stolk JM , Hurst JH, Nisula BC: The inverse relationship between serum dopamine-ÃŸ-hydroxylase activity and thyroid function. J Clin Endocrinol Metab 51:259-264, 1980.
239. Talbot NB , Hoeffel G, Shwachman H, Tuohy EL: Serum phosphatase as an aid in the diagnosis of cretinism and juvenile hypothyroidism. Am J Dis Child 62:273-278, 1941.
240. Lieberthal AS , Benson SG’, Klitgaard HM: Serum malic dehydrogenase in thyroid disease. J Clin Endocrinol Metab 23:211-214, 1963.
241. Yotsumuto H , Imai Y, Kuzuya N, al. e: Increased levels of serum angiotensin-converting enzyme activity in hyperthyroidism. Ann Intern Med 96:326-328, 1982.
242. Gow SMG , Caldwell G, Toft AD, al. e: Relationship between pituitary and other target organ responsiveness in thyroid patients receiving thyroxine replacement. J Clin Endocrinol Metab 64:364-370, 1987.
243. Beckett G J, Kellett HA, Gow SM, Hussey AJ, Hayes JD, Toft AD: Elevated plasma glutathione S-transferase concentrations in hyperthyroidism and in hypothyroid patients receiving thyroxine replacement: Evidence for hepatic damage. Br Med J 2:427-429, 1985.
244. Ogura F , Morii H, Ohmo M, al. e: Serum coenzyme Q 10 levels in thyroid disorders. Horm Metab Res 12:537-540, 1980.
245. Bouillon R , Muls E, DeMoor P: Influence of thyroid function on the serum concentration of 1,25-dihydroxy vitamin D3. J Clin Endocrinol Metab 51:793-796, 1980.
246. Walton KW , Campbell DA, Tonks EL: The significance of alterations in serum lipids in thyroid function. I. The relation between serum lipoproteins, carotenoids, and vitamin A in hypothyroidism and thyrotoxicosis. Clin Sci 29:199-215, 1965.
247. Karlberg BE, Henriksson KG, Andersson RGG: Cyclic adenosine 3′,5′-monophosphate concentration in plasma, adipose tissue and skeletal muscle in normal subjects and in patients with hyper- and hypothyroidism. J Clin Endocrinol Metab 39:96-101, 1974.
248. Peracchi M , Bamonti-Catena F, Lombardi L, al. e: Plasma and urine cyclic nucleotide levels in patients with hyperthyroidism and hypothyroidism. J Endocrinol Invest 6:173-177, 1983.
249. Rivlin RS , Wagner HN Jr.: Anemia in hyperthyroidism. Ann Intern Med 70:507-516, 1969.
250. Feldman DL , Goldberg WM: Hyperthyroidism with periodic paralysis. Can Med Assoc J 101:667-671, 1969.
251. Pettinger WA, Talner L, Ferris TF: Inappropriate secretion of antidiuretic hormone due to myxedema. N Engl J Med 272:362-364, 1965.
252. Jones JE , Deser PC, Shane SR, Flink EB: Magnesium metabolism in hyperthyroidism and hypothyroidism. J Clin Invest 45:891-900, 1966.
253. Baxter JD , Bondy PK: Hypercalcemia of thyrotoxicosis. Ann Intern Med 65:429-442, 1966.
254. Weldon AP, Danks DM: Congenital hypothyroidism and neonatal jaundice. Arch Dis Child 47:469-471, 1972.
255. Greenberger NJ , Milligan FD, DeGroot LJ, Isselbacher KJ: Jaundice and thyrotoxicosis in the absence of congestive heart failure: A study of four cases. Am J Med 36:840-846, 1964.
256. KuhlbÃ¤ch B : Creatine and creatinine metabolism in thyrotoxicosis and hypothyroidism. Acta Med Scand suppl. 331:1-70, 1957.
257. Adlkofer F, Armbrecht U, Schleusener H: Plasma lecithin: Cholesterol acyltransferase activity in hypo- and hyperthyroidism. Horm Metab Res 6:142-146, 1974.
258. PykÃ¤listÃ¶ O , Goldberg AP, Brunzell JD: Reversal of decreased human adipose tissue lipoprotein lipase and hypertriglyceridemia after treatment of hypothyroidism. J Clin Endocrinol Metab 43:591-600, 1976.
259. De Bruin TWA, Van Barlingen H, Van Linde-Sibenius Trip M, Van Vuurst De Vries A-RR, Akveld MJ, Erkelens DW: Lipoprotein (a) and Apolipoprotein B Plasma Concentrations in Hypothyroid, Euthyroid, and Hyperthyroid Subjects. J Clin Endocrinol Metab 76:121-126, 1993.
260. Inui T , Ochi Y, Chen W, Nakajima Y, Kajita Y: Increased serum concentration of type IV collagen peptide and type III collagen peptide in hyperthyroidism. Clin Chem Acta 205:181-186, 1992.
261. Rich C, Bierman EL, Schwartz IL: Plasma nonesterified fatty acids in hyperthyroid states. J Clin Invest 38:275-278, 1959.
262. Amino N , Kuro R, Yabu Y, al. e: Elevated levels of circulating carcinoembryonic antigen in hypothyroidism. J Clin Endocrinol Metab 52:457-462, 1981.
263. Tucci JR, Kopp L: Urinary cyclic nucleotide levels in patients with hyper- and hypothyroidism. J Clin Endocrinol Metab 43:1323-1329, 1976.
264. Guttler RB , Shaw JW, Otis CL, Nicoloff JT: Epinephrine-induced alterations in urinary cyclic AMP in hyper- and hypothyroidism. J Clin Endocrinol Metab 41:707-711, 1975.
265. MacFarlane S , Papadopoulos S, Harden RM, Alexander WD: 131 I and MIT- 131 I in human urine, saliva and gastric juice: A comparison between euthyroid and thyrotoxic patients. J Nucl Med 9:181-186, 1968.
266. HellstrÃ¶m K , Schuberth J: The effect of thyroid hormones on the urinary excretion of taurine in man. Acta Med Scand 187:61-65, 1970.
267. Maebashi M , Kawamura N, Sato M, al. e: Urinary excretion of carnitine in patients with hyperthyroidism and hypothyroidism: Augmentation by thyroid hormone. Metabolism 26:351-356, 1977.
268. Levine RJ , Oates JA, Vendsalu A, Sjoerdsma A: Studies on the metabolism of aromatic amines in relation to altered thyroid function in man. J Clin Endocrinol Metab 22:1242-1250, 1962.
269. Copinschi G , Leclercq R, Bruno OD, Cornil A: Effects of altered thyroid function upon cortisol secretion in man. Horm Metab Res 3:437-442, 1971.
270. Harvey RD , McHardy KC, Reid IW, et al: Measurement of bone collagen degradation in hyperthyroidism and during thyroxine replacement therapy using pyridinium cross-links as specific urinary markers. J Clin Endocrinol Metab 72:1189-1194, 1991.
271. Kivirikko K I, Laitinen O, Lamberg BA: Value of urine and serum hydroxyproline in the diagnosis of thyroid disease. J Clin Endocrinol Metab 25:1347-1352, 1965.
272. Askenasi R , Demeester-Mirkine N: Urinary excretion of hydroxylysyl glycosides and thyroid function. J Clin Endocrinol Metab 40:342-344, 1975.
273. Golden AWG , Bateman D, Torr S: Red cell sodium in hyperthyroidism. Br Med J 2:552-554, 1971.
274. Weinstein M , Sartorio G, Stalldecker GB, al. e: Red cell zinc in thyroid dysfunction. Acta Endocrinol 20:147-152, 1972.
275. Pearson HA , Druyan R: Erythrocyte glucose-6-phosphate dehydrogenase activity related to thyroid activity. J Lab Clin Med 57:343-349, 1961.
276. Vuopio P, Viherkoski M, NikkilÃ¤ E, Lamberg BA: The content of reduced glutathione (GSH) in the red blood cells in hypo- and hyperthyroidism. Ann Clin Res 2:184-186, 1970.
277. Kiso Y , Yoshida K, Kaise K, et al: Erythrocyte carbonic anhydrase-I concentrations in patients with Graves’ disease and subacute thyroiditis reflect integrated thyroid hormone levels over the previous few months. J Clin Endocrinol Metab 72:515-518, 1991.
278. Dube MP , Davis FB, Davis PJ, al. e: Effects of hyperthyroidism and hypothyroidism on human red blood cells Ca 2+ -ATPase activity. J Clin Endocrinol Metab 62:253-257, 1986.
279. Gwinup G , Ogundip O: Decreased leukocyte alkaline phosphatase in hyperthyroidism. Metabolism 62:253-257, 1974.
280. Jemelin M , Frei J, Scazziga B: Production of ATP in leukocyte mitochondria from hyperthyroid patients before and after treatment with a ÃŸ-adrenergic blocker and antithyroid drugs. Acta Endocrinol 66:606-610, 1971.
281. Strickland AL: Sweat electrolytes in thyroid disorders. J Pediatr 82:284-286, 1973.
282. Goolamali SK , Evered D, Shuster S: Thyroid disease and sebaceous function. Br Med J 1:432-433, 1976.
283. Christensen J, Schedl HP, Clifton JA: The basic electrical rhythm of the dudodenum in normal human subjects and in patients with thyroid disease. J Clin Invest 43:1659-1667, 1964.
284. Levy G, MacGillivray MH, Procknal JA: Riboflavin absorption in children with thyroid disorders. Pediatrics 50:896-900, 1972.
285. Singhelakis P , Alevizaki CC, Ikkos DG: Intestinal calcium absorption in hyperthyroidism. Metabolism 23:311-321, 1974.
286. Thomas FB , Caldwell JH, Greenberger NJ: Steatorrhea in thyrotoxicosis: Relation to hypermotility and excessive dietary fat. Ann Intern Med 78:669-675, 1973.
287. Wegener M, Wedmann B, Langhoff T, Schaffstein J, Adamek R: Effect of hyperthyroidism on the transport of a solid-liquid meal through the stomach, intestine, and the colon in man. J Clin Endocrinol Metab 75:745-749, 1992.
288. Scherrer M , KÃ¶nig MP: Pulmonary gas exchange in hypothyroidism. Pneumonologie 151:105-113, 1974.
289. Zwillich CW , Pierson DJ, Hofeldt FD, et al: Ventilatory control in myxedema and hypothyroidism. N Engl J Med 292:662-665, 1975.
290. Lawson JD : The free Achilles reflex in hypothyroidism and hyperthyroidism. N Engl J Med 259:761-764, 1958.
291. Hall R , Owen SG: Thyroid antibodies in cerebrospinal fluid. Br Med J 2:710-711, 1960.
292. Hoffman I, Lowrey RD: The electrocardiogram in thyrotoxicosis. Am J Cardiol 6:893-904, 1960.
293. Lee JK, Lewis JA: Myxoedema with complete A-V block and Adams-Stokes disease abolished with thyroid medication. Br Heart J 24:253-265, 1962.
294. Wilkins L : Epiphysial dysgenesis associated with hypothyroidism. Am J Dis Child 61:13-34, 1941.
295. Bonakdarpour A , Kirkpatrick JA, Renzi A, Kendall N: Skeletal changes in neonatal thyrotoxicosis. Radiology 102:149-150, 1972.
296. Mariotti S , Anelli S, Ruf J, Bechi R, Czarnocka B, Lombardi A: Comparison of serum thyroid microsomal and thyroid peroxidase autoantibodies in thyroid diseases*. J Clin Endocrinol Metab 65:987-993, 1987.
297. Portmann L , Hamada N, Neinrich G, DeGroot LJ: Antithyroid peroxidase antibody in patients with autoimmune thyroid disease: Possible identity with anti-microsomal antibody. J Clin Endocrinol Metab 61:1001-1003, 1985.
298. Rinke R , Seto P, Rapoport B: Evidence for the highly conformational nature of the epitope(s) on human thyroid peroxidase that are recognized by sera from patients with Hashimoto’s thyroiditis. J Clin Endocrinol Metab 71:53, 1990.
299. Kaufman KD , Filetti S, Seto P, Rapoport B: Recombinant human thyroid peroxidase generated in eukaryotic cells: A source of specific antigen for the immunological assay of antimicrosomal antibodies in the sera of patients with autoimmune thyroid disease. J Clin Endocrinol Metab 70:724-728, 1990.
299a Chang CC , Huang CN, Chuang LM. Autoantibodies to thyroid peroxidase in patients with type 1 diabetes in Taiwan. Eur J Endocrinol 139:44-48, 1998
299b Smyth PP , Shering SG, Kilbane MT et al. Serum thryoid peroxidase antibodies, thyroid volume, and outcome in breast carcinoma. J Clin Endocrinol Metab 83:2711-2716, 1998
300. Trotter WR , Belyavin G, Waddams A: Precipitating and complement fixing antibodies in Hashimoto’s disease. Proc R Soc Med 50:961-962, 1957.
301. Boyden SV : The adsorption of proteins on erythrocytes treated with tannic acid and subsequent hemagglutination by antiprotein sera. J Exp Med 93:107-120, 1951.
302. Holborrow EJ , Brown PC, Roitt IM, Doniach D: Cytoplasmic localization of complement-fixing auto-antigen in human thyroid epithelium. Br J Exp Pathol 40:583-588, 1959.
303. Hamada N , Jaeduck N, Portmann L, al. e: Antibodies against denatured and reduced thyroid microsomal antigen in autoimmune thyroid disease. J Clin Endocrinol Metab 64:230-238, 1987.
304. Mori T, Kriss JP: Measurements by competitive binding radioassay of serum anti-microsomal and anti-thyroglobulin antibodies in Graves’ disease and other thyroid disorders. J Clin Endocrinol Metab 33:688-698, 1971.
305. Mariotti S , Pinchera A, Vitti P, al. e: Comparison of radioassay and haemagglutionation methods for anti-thyroid microsomal antibodies. Clin Exp Immunol 34:118-125, 1978.
305a Miles J , Charles P, Riches P. A review of methods available for the identification of both organ-specific and non-organ-specific autoantibodies. Ann Clin Biochem: 35:19-47, 1998
306. Amino N , Hagen SR, Yamada N, Refetoff S: Measurement of circulating thyroid microsomal antibodies by the tanned red cell haemagglutination technique: Its usefulness in the diagnosis of autoimmune thyroid disease. Clin Endocrinol 5:115, 1976.
307. Ohtaki S , Endo Y, Horinouchi K, al. e: Circulating thyroglobulin-antithyroglobulin immune complex in thyroid diseases using enzyme-linked immunoassays. J Clin Endocrinol Metab 52:239-246, 1981.
308. Miles J, Charles P, Riches P: A review of methods available for the identification of both organ-specific and non-organ-specific autoantibodies. Ann Clin Biochem 35:19-47, 1998.
309. Feldt-Rasmussen U : Analytical and clinical performance goals for testing autoantobodies to thyroperoxidase, thyroglobulin, and thryotropin receptor. Clin Chem 42:160-63, 1996.
310. Loeb PB , Drash AL, Kenny FM: Prevalence of low-titer and “negative” antithyroglobulin antibodies in biopsy-proved juvenile Hashimoto’s thyroiditis. J Pediatr 82:17-21, 1973.
311. Tamaki H , Katsumaru H, Amino N, Nakamoto H, Ishikawa E, Miyai K: Usefulness of thyroglobulin antibody detected by ultrasensitive enzyme immunoassay: A good parameter for immune surveillance in healthy subjects and for prediction of post-partum thyroid dysfunction. Clin Endocrinol (Oxf) 37:266-273, 1992.
312. VolpÃ© R , Row VV, Ezrin C: Circulating viral and thyroid antibodies in subacute thyroiditis. J Clin Endocrinol Metab 27:1275-1284, 1967.
313. Balfour BM , Doniach D, Roitt IM, Couchman KG: Fluorescent antibody studies in human thyroiditis: Auto-antibodies to an antigen of the thyroid distinct from thyroglobulin. Br J Exp Pathol 42:307-316, 1961.
314. Staeheli V , Vallotton MB, Burger A: Detection of human anti-thyroxine and anti-triiodothyronine antibodies in different thyroid conditions. J Clin Endocrinol Metab 41:669-675, 1975.
315. Bastenie PA , Bonnyns M, Vanhaelst L, NÃ¨ve P: Diseases associated with autoimmune thyroiditis. P. A. Bastenie and A. Ermans (eds), Thyroiditis and Thyroid Function., Pergamon Press, Oxford, pp. 1972.
317. Gupta MK : Thyrotropin receptor antibodies: Advances and importance of detection techniques in thyroid disease. Clin Biochem 25:193-199, 1992.
318. McKenzie JM: The bioassay of thyrotropin in serum. Endocrinology 63:372-381, 1958.
319. Furth ED , Rathbun M, Posillico J: A modified bioassay for the long-acting thyroid stimulator (LATS). Endocrinology 85:592-593, 1969.
320. Kriss JP , Pleshakov V, Rosenblum AL, al. e: Studies on the pathogenesis of the ophthalmopathy of Graves’ disease. J Clin Endocrinol Metab 27:582-593, 1967.
321. Sunshine P , Kusumoto H, Kriss JP: Survival time of circulating long-acting thyroid stimulator in neonatal thyrotoxicosis: Implications for diagnosis and therapy of the disorder. Pediatrics 36:869-876, 1965.
322. Onaya T , Kotani M, Yamada T, Ochi Y: New in vitro tests to detect the thyroid stimulator in sera from hyperthyroid patients by measuring colloid droplet formation and cyclic AMP in human thyroid slices. J Clin Endocrinol Metab 36:859-866, 1973.
323. Hinds WE , Takai N, Rapoport B, al. e: Thyroid-stimulating activity and clinical state in antithyroid treatment of juvenile Graves’ disease. Acta Endocrinol 94:46-52, 1981.
324. Leedman PJ , Frauman AG, Colman PG, Michelangeli VP: Measurement of thyroid-stimulating immunoglobulins by incorporation of tritiated-adenine into intact FRTL-5 cells: A viable alternative to radioimmunoassay for the measurement of cAMP. Clin Endocrinol (Oxf) 37:493-499, 1992.
325. Takata I , Suzuki Y, Saida K, Sato T: Human thyroid-stimulating activity and clinical state in antithyroid treatment of juvenile Graves’ disease. Acta Endocrinol 94:46-52, 1980.
326. Kendall-Taylor P , Atkinson S: A biological method for the assay of TSAb in serum. J. R. Stockigt and S. Nagataki (eds), Thyroid Research VIII, Australian Academy of Science, Canberra, pp. 763,1980.
327. Petersen V, Rees Smith B, Hall R: A study of thyroid-stimulating activity in human serum with the highly sensitive cytochemical bioassay. J Clin Endocrinol Metab 41:199-202, 1975.
328. Libert F , Lefort A, Gerard C, et al: Cloning, sequencing and expression of the human thyrotropin (TSH) receptors: Evidence for binding of autoantibodies. Biochem Biophys Res Commun 165:1250-1255, 1989.
329. Nagayama Y, Kaufman KD, Seto P, Rapoport B: Molecular cloning, sequence and functional expression of the cDNA for the human thyrotropin receptor. Biochem Biophys Res Commun 165:1184-1190, 1989.
330. Ludgate M, Perret J, Parmentier M, et al: Use of the recombinant human thyrotropin receptor (TRHr) expressed in mammalian cell lines to assay TSHr autoantibodies. Mol Cell Endocrinol 73:R13-R18, 1990.
331. Filetti S , Foti D, Costante G, Rapoport. B: Recombinant human thyrotropin (TSH) receptor in a radioreceptor assay for the measurement of TSH receptor antibodies. J Clin Endocrinol Metab 72:1096-1101, 1991.
331a Botero D , Brown RS. Bioassay of TSH receptor antibodies with Chinese hamster ovary cells transfected with recombinant human TSH receptor: clinical utility in children and adolescents with Graves’ disease. J Pediatr 132:612-618, 1998
332. Vitti P , Elisei R, Tonacchera M, et al: Detection of thyroid-stimulating antibody using Chinese hamster ovary cells transfected with cloned human thyrotropin receptor. J Clin Endocrinol Metab 76:499-503, 1993.
333. Adams DD , Kennedy TH: Occurrence in thyrotoxicosis of a gamma globulin which protects LATS from neutralization by an extract of thyroid gland. J Clin Endocrinol Metab 27:173-177, 1967.
334. Shishiba Y , Shimizu T, Yoshimura S, Shizume K: Direct evidence for human thyroidal stimulation by LATS-protector. J Clin Endocrinol Metab 36:517-521, 1973.
335. Rapoport B , Greenspan FS, Filetti S, Pepitone M: Clinical experience with a human thyroid cell bioassay for thyroid-stimulating immunoglobulins. J Clin Endocrinol Metab 58:332-338, 1984.
336. Smith BR , Hall R: Thyroid-stimulating immunoglobulins in Graves’ disease. Lancet 2:427-431, 1974.
337. Zakarija M , McKenzie JM, Munro DS: Evidence of an IgG inhibitor of thyroid-stimulating antibody (TSAb) as a cause of delay in the onset of neonatal Graves’ disease. J Clin Invest 72:1352-1356, 1983.
338. Shewring G , Smith BR: An improved radioreceptor assay for TSH receptor antibodies. Clin Endocrinol 17:409-417, 1982.
339. Endo K , Amir SM, Ingbar SH: Development and evaluation of a method for the partial purification of immunoglobulin specific for Graves’ disease. J Clin Endocrinol Metab 52:1113-1123, 981.
340. Kosugi S , Ban T, Akamizu T, Konh LD: Identification of separate determinants on the thyrotropin receptor reactive with Graves’ thyroid stimulation antibodies and with thyroid stimulating blocking antibodies in idiopathic myxedema: these determinants have no homologous sequence on gonadotropin receptor. 6:166-180, 1992.
341. Drexhage HA , Bottazzo GF, Doniach D: Thyroid growth stimulating and blocking immunoglobulins. J. Chayen and L. Bitensky (eds), Cytochemical Bioassays, Marcel Dekker, New York, pp. 153,1983.
342. Valente WA , Vitti P, Rotella CM, al. e: Autoantibodies that promote thyroid growth: A distinct population of thyroid stimulating antibodies. N Engl J Med 309:1028-1034, 1983.
343. Grove AS Jr. : Evaluation of exophthalmos. N Engl J Med 292:1005-1013, 1975.
344. Parma J, Duprez L, van Sande J, et al: Somatic mutations in the thyrotropin receptor gene cause hyperfunctioning thyroid adenomas. Nature 365:649-651, 1993.
345. Duprez L , Parma J, Van Sande J, et al: Germline mutations in the thyrotropin receptor gene cause non-autoimmune autosomal dominant hyperthyroidism. Nature Genet 7:396-401, 1994.
346. McKenzie JM, Zakarija M: Fetal and neonatal hyper- and hypothyroidism due to maternal TSH receptor antibodies. Thyroid 2:155-159, 1992.
347. Cho Y , Shong MH, Yi KH, Lee HK, Koh S, Min HK: Evaluation of serum basal thyrotrophin levels and thyrotrophin receptor antibody activities as prognostic markers for discontinuation of antithyroid drug treatment in patients with Graves’ disease. Clin Endocrinol (Oxf) 36:585-590, 1992.
348. Hershman JM : Hyperthyroidism induced by trophoblastic thyrotropin. Mayo Clin Proc 47:913-918, 1972.
350. Nisula BC , Ketelslegers JM: Thyroid-stimulating activity and chorionic gonadotropin. J Clin Invest 54:494-499, 1974.
351. Bahn R, Heufelder AE: Pathogenesis of Graves’ ophthalmopathy. New Engl J Med 329:1468-75, 1993.
352. Miller A, Arthurs B, Boucher A, al.e:Significance of antibodies reactive with a 64kDa eye muscle membrane antigen in patients with thryoid autoimmunity. Thyroid 2:197-202, 1992.
353. Winand RJ , Kohn LD: Stimulation of adenylate cyclase activity in retro-orbital tissue membranes by thyrotropin and an exophthalmogenic factor derived from thyrotropin. J Biol Chem 250:6522-6526, 1975.
354. Kodama K , Sikorka H, Bandy-Dafoe P, al. e: Demonstration of a circulating antibody against a soluble eye-muscle antigen in Graves’ ophthalmopathy. Lancet 2:1353-1356, 1982.
354a Matsuoka N , Eguchi K, Kawakami A et al. Lack of B7-1/BB1 and B7-2/B70 expression on thyrocytes of patients with Graves’ disease. J Clin Endocrinol Metab 81:4137-4143, 1996
354b Otto EA , Ochs K, Hansen C, Wall JR, Kahaly GJ. Orbital tissue-derived T lymphocytes from patients with Graves’ ophthalmopathy recognize autologous orbital antigens. J Clin Endocrinol Metab 81:3045-50, 1996
355. Ryo UY , Arnold J, Colman M, al. e: Thyroid scintigram: Sensitivity with sodium pertechnetate Tc 99m and gamma camera with pinhole collimator. JAMA 235:1235-1238, 1976.
356. Atkins HL , Klopper JF, Lambrecht RM, Wolf AP: A comparison of technetium 99m and iodine 123 for thyroid imaging. Am J Roentgenol Radium Ther Nucl Med 117:195-201, 1973.
357. Nishiyama H , Sodd VJ, Berke RA, Saenger EL: Evaluation of clinical value of 123 I and 131 I in thyroid disease. J Nucl Med 15:261-265, 1974.
358. Tong ECK , Rubenfeld S: Scan measurements of normal and enlarged thyroid glands. Am J Roentgenol Radium Ther Nucl Med 115:706-708, 1972.
359. Mazzaferri EL: Management of a solitary thyroid nodule. New Engl J Med 328:553-9, 1993.
360. Becker FO, Economou PG, Schwartz TB: The occurrence of carcinoma in “hot” thyroid nodules: Report of two cases. Ann Intern Med 58:877-882, 1963.
361. Ashcraft MW , Van Herle AJ: Management of thyroid nodules I. History and physical examination, blood tests, x-ray tests and ultrasonography. Head Neck Surg 3:2l6-30, 1981.
362. Ladenson PW , Braverman LE, Mazzaferri EL, al. e: Comparison of administration of recombinant human thyrotropin with withdrawal of thryoid hormone for radioactive iodine scanning in patients with thyroid carcinoma. New Engl J Med 337:888-96, 1997.
363. Chen JJS , LaFrance ND, Allo MD, Cooper DS, Ladenson PWJ: Single photon emission computed tomography of the thyroid. J Clin Endocrinol Metab 66:1240-, 1988.
364. Corstens F , Huysmans D, Kloppenborg P: Thallium-210 scintigraphy of the suppressed thyroid: An alternative for iodine-123 scanning after TSH stimulation. J Nucl Med 29:1360-1363, 1988.
365. Fairweather DS, Bradwell AR, Watson-James SF, Dykes PW, Chandler S, Hoffenberg R: Deletion of thyroid tumours using radiolabeled thyroglobulin. Clin Endocrinol 18:563-570, 1983.
368. Barki Y: Ultrasonographic evaluation of neck masses-sonographic pattern in differential diagnosis. Isr J Med Sci 28:212-216, 1992.
369. Watters DAK, Ahuja AT, Evans RM, et al: Role of ultrasound in the management of thyroid nodules. Am J Surg 164:654-657, 1992.
370. Scheible W , Leopold GR, Woo VL, Gosink BB: High resolution real-time ultrasonography of thyroid nodules. Radiology 133:413-417, 1979.
371. Sostre S , Reyes MM: Sonographic diagnosis and grading of Hashimoto’s thyroiditis. J Endocrinol Invest 14:115-121, 1991.
372. Brander A , Viikinkoski P, Nickels J, Kivisaari L: Thyroid Gland: US screening in a random adult population. Radiol 181:683-687, 1991.
373. Danese D , Sciacchitano S, Farsetti A al. e: Diagnostic accuracy of conventional versus sonography-guided fine-needle aspiration biopsy of thryoid nodules. Thyroid 8:15-21, 1998.
374. Szebeni A , Beleznay EJ: New simple method for thyroid volume determination by ultrasonography. Clin Ultrasound 20:329-337, 1992.
375. Jarlov AE , Hegedus L, Gjorup T, Hansen JEM: Accuracy of the clinical assessment of thyroid size. Dan Med Bull 38:87-89, 1991.
376. Paracchi A , Ferrari C, Livraghi T, et al: Percutaneous intranodular ethanol injection: A new treatment for autonomous thyroid adenoma. J Endocrinol Invest 15:353-362, 1992.
378. Blum M , Reede DL, Seltzer TF, Burroughs VJ: Computerized axial tomography in the diagosis of thyroid and parathyroid disorders. Am J Med Sci 287:34-39, 1984.
379. Brown LR , Aughenbaugh GL: Masses of the anterior mediastinum: CT and MR imaging. Amer J Radiol 157:1171-1180, 1991.
380. Gittoes NJL, Miller MR, Daykin J, Sheppard MC, Franklyn JA: Upper airways obstruction in patients presenting with thyroid enlargement. Br Med J 312:484, 1996.
384. Wang C , Vickery AL Jr., Maloof F: Needle biopsy of the thyroid. Surg Gynecol Obstet 143:365-368, 1976.
385. Ashcraft MW, Van Herle AJ: Management of thyroid suppressive therapy, and fine needle aspiration. Head and Neck Surgery 3:297-322, 1981.
388. Matos-Godilho L , Kocjan G, Kurtz A: Contribution of fine needle aspiration cytology to diagnosis and management of thyroid disease. J Clin Path 45:391-395, 1992.
388a Franklyn JA , Daykin J, Young J et al. Fine needle aspiration cytology in diffuse or multinodular goitre compared with solitary thyroid nodules. Br Med J 307:240-1, 1993
388b Mazzaferri E l. Management of a solitary thyroid nodule. New Engl J Med 328:553-9, 1993
389. Hamberger B , Gharib H, Melton LJ 3rd, al. e: Fine-needle aspiration biopsy of thyroid nodules: Impact on thyroid practice and cost of care. Am J Med 73:381-384, 1982.
389a Bennedbaek FN , Karstrup S, Hegedus L: Percutaneous ethanol injection therapy in the treatment of thyroid and parathyroid diseases. Eur J Endocrinol 136:240-50, 1997.
390. Odell WD, Wilber FJ, Utiger RD: Studies on thyrotropin physiology by means of radioimmunoassay. Recent Prog Horm Res 23:47-85, 1967.
391. Jackson IMD : Thyrotropin-releasing hormone. N Engl J Med 306:145-155, 1982.
397. Beck-Peccoz P, Amr S, Menezes-Ferreira M, al. e: Decreased receptor binding of biologically inactive thyrotropin in central hypothyroidism. Effect of treatment with thyrotropin-releasing hormone. J Clin Endocrinol Metab 312:1085-1090, 1985.
399. Pierce JG : The subunits of pituitary thyrotropin: Their relation to other glycoprotein hormones. Endocrinology 89:1331-1344, 1971.
401. Miyai K, Fukuchi M, Kumahara Y: Correlation between biological and immunological potencies of human serum and pituitary thyrotropin. J Clin Endocrinol Metab 29:1438-1442, 1969.
402. Gendrel D , Feinstein MC, Grenier J, al. e: Falsely elevated serum thyrotropin (TSH) in newborn infants: Transfer from mothers to infants of a factor interfering in the TSH radioimmunoassay. J Clin Endocrinol Metab 52:62-65, 1981.
403. Chaussain JL , Binet E, Job JC: Antibodies to human thyreotrophin in the serum of certain hypopituitary dwarfs. Rev Eur Etud Clin Biol 17:95-99, 1972.
404. Nicoloff JT, Spencer CA: The use and misuse of the sensitive thyrotropin assays. J Clin Endocrinol Metab 71:553-558, 1990.
405. Kricka LJ: Chemiluminescent and bioluminescent techniques. Clin Chem 37:1472-1481, 1991.
406. Spencer CA , Schwarzbein D, Guttler RB, LoPresti JS, Nicoloff JT: Thyrotropin-releasing hormone stimulation test responses employing third and fourth generation TSH assays. J Clin Endocrinol Metab 76: 494-98, 1993.
407. Spencer CA , Takeuchi M, Kazarosyan M al. e: Interlaboratory differences in functional sensitivity of immunometric assays of thryotropin and impact on relaibility of measurement of subnormal concnetrations of TSH. Clin Chem 41: 367-74, 1995.
408. Spencer CA, Takeuchi M, Kazarosyan M: Current status and performance goals for serum TSH assays. Clin Chem 42:140-45, 1996.
409. Brennan MD , Klee GG, Preissner CM, Hay ID: Heterophilic serum antibodies: A cause for falsely elevated serum thyrotropin levels. Mayo Clin Proc 62:894-898, 1987.
410. Wood JM , Gordon DL, Rudinger AN, Brooks MM: Artifactual elevation of thyroid-stimulating hormone. Amer J Med 90:261-262, 1991.
411. Zweig MH , Csako G, Reynolds JC, Carrasquillo JA: Interference by iatrogenically induced anti-mouse IgG antibodies in a two-site immunometric assay for thyrotropin. Arch Path Rad Metab 1165:164-168, 1991.
412. Kourides I A, Heath CV, Ginsberg-Fellner F: Measurement of thyroid-stimulating hormone in human amniotic fluid. J Clin Endocrinol Metab 54:635-637, 1982.
413. Fisher DA , Kleinm AH: Thyroid development and disorders of thyroid function in the newborn. N Engl J Med 304:702-712, 1981.
414. Snyder PJ , Utiger RD: Response to thyrotropin releasing hormone (TRH) in normal man. J Clin Endocrinol Metab 34:380-385, 1972.
415. Hershman JM , Pittman JA Jr.: Utility of the radioimmunoassay of serum thyrotrophin in man. Ann Intern Med 74:481-490, 1971.
416. Brabant G , Prank K, Ranft U, et al: Physiological regulation of circadian and pulsatile thyrotropin secretion in normal man and woman. J Clin Endocrinol Metab 70:403-409, 1990.
417. Bartalena L , Martino E, Falcone M, et al: Evaluation of the nocturnal serum thyrotropin (TSH) surge, as assessed by TSH ultrasensitive assay, in patients receiving long term L-thyroxine suppression therapy and in patients with various thyroid disorders. J Clin Endocrinol Metab 65:1265-1271, 1987.
418. Ria AG , Brabant K, Prank E, Endert E, Wiersinga WM: Circadian changes in pulsatile TSH release in primary hypothyroidism. Clin Endocrinol 37:504-510, 1992.
419. Brabant G, Prank C, Hoang-Vu C, Hesch RD, von zur Muhlen A: Hypothalamic regulation of pulsatile thyrotropin secretion. J Clin Endocrinol Metab 72:145-150, 1991.
420. Romijn JA , Adriaanse G, Brabant K, Prank E, Endert E, Wiersinga WM: Pulsatile secretion of thyrotropin during fasting: A decrease of thyrotropin pulse amplitude. J Clin Endocrinol Metab 70:1631-1636, 1990.
421. Bartalena L , Pacchiarotti A, Palla R, et al: Lack of nocturnal serum thyrotropin (TSH) surge in patients with chronic renal failure undergoing regular maintenance hemofiltration: A case of central hypothyroidism. Clin Nephrol 34:30-34, 1990.
422. Romijn JA , Wiersinga WM: Decreased nocturnal surge of thyrotropin in nonthyroidal illness. J Clin Endocrinol Metab 70:35-42, 1990.
423. Van Cauter E , Golstein J, Vanhaelst L, Leclercq R: Effects of oral contraceptive therapy on the circadian patterns of cortisol and thyrotropin (TSH). Eur J Clin Invest 5:115-121, 1975.
424. Brabant G, Brabant A, Ranft U, et al: Circadian and pulsatile thyrotropin secretion in euthyroid man under the influence of thyroid hormone and glucocorticoid administration. J Clin Endocrinol Metab 65:83-88, 1987.
425. Simoni M , Velardo A, Montanini V, Faustini Fustini M, Seghedoni S, Marrama P: Circannual rhythm of plasma thyrotropin in middle-aged and old euthyroid subjects. Horm Res 33:184-189, 1990.
426. Wilber JF , Baum D: Elevation of plasma TSH during surgical hypothermia. J Clin Endocrinol Metab 31:372-375, 1970.
427. Vagenakis AG , Rapoport B, Azizi F, al. e: Hyper-response to thyrotropin-releasing hormone accompanying small decreases in serum thyroid hormone concentration. J Clin Invest 54:913-918, 1974.
428. Snyder PJ , Utiger RD: Inhibition of thyrotropin response to thyrotropin releasing hormone by small quantities of thyroid hormones. J Clin Invest 51:2077-2084, 1972.
429. Ehrmann DA , Weinberg M, Sarne DH: Limitations to the use of a sensitive assay for serum thyrotropin in the assessment of thyroid status. Arch Intern Med 149:369-372, 1989.
430. Ridgway EC , Cooper DS, Walker H, al. e: Peripheral responses of thyroid hormone before and after L-thyroxine therapy in patients with subclinical hypothyroidism. J Clin Endocrinol Metab 53:1238-1242, 1981.
431. Aizawa T , Koizumi Y, Yamada T, al. e: Difference in pituitary-thyroid feedback regulation in hypothyroid patients, depending on the severity of hypothyroidism. J Clin Endocrinol Metab 47:560-565, 1978.
432. Spencer CA : Clinical utility and cost-effectiveness of sensitive thyrotropin assays in ambulatory and hospitalized patients. Mayo Clin Proc 63:1214-1222, 1988.
433. Surks MI, Chopra IJ, Mariash CN, Nicoloff JT, Solomon DH: American Thyroid Association guidelines for use of laboratory tests in thyroid disorders. JAMA 263:1529-1532, 1990.
434. Delange F , Dodion J, Wolter R, al. e: Transient hypothyroidism in the newborn infant. J Pediatr 92:974-976, 1978.
435. Brown ME , Refetoff S: Transient elevation of serum thyroid hormone concentration after initiation of replacement therapy in myxedema. Ann Intern Med 92:491-495, 1980.
436. Sanchez-Franco F , Cacicedo GL, Martin-Zurro A, al. e: Transient lack of thyrotropin (TSH) response to thyrotropin-releasing hormone (TRH) in treated hyperthyroid patients with normal or low serum thyroxine (T4) and triiodothyronine (T3). J Clin Endocrinol Metab 38:1098-1102, 1974.
437. Spencer CA , Elgen A, Shen D, et al: Specificity of sensitive assays of thyrotropin (TSH) used to screen for thyroid disease in hospitalized patients. Clin Chem 33:1301-1396, 1987.
438. Sunthornthepvarakul T , Gottschalk ME, Hayashi Y, Refetoff S: Resistance to thyrotropin caused by mutations in the thyrotropin receptor gene. N Engl J Med 332:(in press), 1995.
439. Weintraub BD , Gershengorn MC, Kourides IA, Fein H: Inappropriate secretion of thyroid stimulating hormone. Ann Intern Med 95:339-351, 1981.
440. Mihailovic V, Feller MS, Kourides IA, Utiger RD: Hyperthyroidism due to excess thyrotropin secretion: Follow-up studies. J Clin Endocrinol Metab 50:1135-1138, 1980.
441. Kourides IA, Ridgway EC, Weintraub BD, al. e: Thyrotropin-induced hyperthyroidism: Use of alpha and beta subunit levels to identify patients with pituitary tumors. J Clin Endocrinol Metab 45:534-543, 1977.
442. Sarne DH , Sobieszczyk S, Ain KB, Refetoff S: Serum thyrotropin and prolactin in the syndrome of generalized resistance to thyroid hormone: Responses to thyrotropin-releasing hormone stimulation and short term triiodothyronine suppression. J Clin Endocrinol Metab 70:1305-1311, 1990.
443. Brent GA , Hershman JM, Braunstein GD: Patients with severe nonthyroidal illness and serum thyrotropin concentrations in the hypothyroid range. Am J Med 81:463-466, 1986.
444. Topliss DJ, White EL, Stockigt JR: Significance of thyrotropin excess in untreated primary adrenal insufficiency. J Clin Endocrinol Metab 50:52-56, 1980.
445. Wehmann RE, Gregerman RI, Burns WH, al. e: Suppression of thyrotropin in the low-thyroxine state of severe nonthyroidal illness. N Engl J Med 312:546-552, 1985.
446. Bacci V, Schussler GC, Kaplan TB: The relationship between serum triiodothyronine and thyrotropin during systemic illness. J Clin Endocrinol Metab 54:1229-1235, 1982.
447. Kourides IA, Weintraub BD, Ridgway EC, Maloof F: Pituitary secretion of free alpha and beta subunit of human thyrotropin in patients with thyroid disorders. J Clin Endocrinol Metab 40:872-885, 1975.
448. Oliver C , Charvet JP, Codaccioni J-L, Vague J: Radioimmunoassay of thyrotropin-releasing hormone (TRH) in human plasma and urine. 39:406-410, 1974.
449. Emerson CH , Frohman LA, Szabo M, Thakker I: TRH immunoreactivity in human urine: Evidence for dissociation from TRH. 45:392-399, 1977.
450. Mitsuma T, Hiraoka Y, Nihei N: Radioimmunoassay of thyrotropin-releasing hormone in human serum and its application. 83:225-, 1976.
451. Mallik TK , Wilber JF, Pegues J: Measurements of thyrotropin-releasing hormone-like material in human peripheral blood by affinity chromatography and radioimmunoassay. 54:1194-1198, 1982.
452. Weeke J: The influence of the circadian thyrotropin rhythm on the thyrotropin response to thyrotropin-releasing hormone in normal subjects. Scand J Clin Lab Invest 33:17-20, 1974.
453. Haigler ED Jr. , Hershman JM, Pittman JA Jr., Blaugh CM: Direct evaluation of pituitary thyrotropin reserve utilizing thyrotropin releasing hormone. J Clin Endocrinol Metab 33:573-581, 1971.
454. Azizi F , Vagenakis AG, Portnay GE, al. e: Pituitary-thyroid responsiveness to intramuscular thyrotropin-releasing hormone based on analyses of serum thyroxine, tri-iodothyronine and thyrotropin concentration. N Engl J Med 292:273-277, 1975.
455. Haigler ED Jr. , Hershman JM, Pittman JA Jr.: Response to orally administerd synthetic thyrotropin-releasing hormone in man. J Clin Endocrinol Metab 35:631-635, 1972.
456. Ormston BJ , Kilborn JR, Garry R, al. e: Further observations on the effect of synthetic thyrotrophin-releasing hormone in man. Br Med J 2:199-202, 1971.
457. Hershman JM , Kojima A, Friesen HG: Effect of thyrotropin-releasing hormone on human pituitary thyrotropin, prolactin, placental lactogen, and chorionic thyrotropin. J Clin Endocrinol Metab 36:497-501, 1973.
458. Jacobsen BB , Andersen H, Dige-Petersen H, Hummer L: Thyrotropin response to thyrotropin-releasing hormone in fullterm, euthyroid and hypothyroid newborns. Acta Paediatr Scand 65:433-438, 1976.
459. Sanchez-Franco F , Garcia MD, Cacicedo L, al. e: Influence of sex phase of the menstrual cycle on thyrotropin (TSH) response to thyrotropin-releasing hormone (TRH). J Clin Endocrinol Metab 37:736-740, 1973.
460. Harman SM , Wehmann RE, Blackman MR: Pituitary-thyroid hormone economy in healthy aging men:Basal indices of thyroid function and thyrotropin responses to constant infusions of thyrotropin releasing hormone. J Clin Endocrinol Metab 58:320-326, 1984.
461. Wilber J , Jaffer A, Jacobs L, al. e: Inhibition of thyrotropin releasing hormone (TRH) stimulated thyrotropin (TSH) secretion in man by a single oral dose of thyroid hormone. Horm Metab Res 4:508, 1972.
462. Wartofsky L , Dimond RC, Noel GL, al. e: Effect of acute increases in serum triiodothyronine on TSH and prolactin responses to TRH, and estimates of pituitary stores of TSH and prolactin in normal subjects and in patients with primary hypothyroidism. J Clin Endocrinol Metab 42:443-458, 1976.
463. Shenkman L , Mitsuma T, Suphavai A, Hollander CS: Triiodothyronine and thyroid-stimulating hormone response to thyrotrophin-releasing hormone: A new test of thyroidal and pituitary reserve. Lancet 1:111-113, 1972.
464. Anderson MS, Bowers CY, Kastin AJ, al. e: Synthetic thyrotropin-releasing hormone: A potent stimulator of thyrotropin secretion in man. N Engl J Med 285:1279-1283, 1971.
465. McFarland KF , Strickland AL, Metzger WT, Smith JS: Thyrotropin-releasing hormone test: An adverse reaction. Arch Intern Med 142:132-133, 1982.
466. Fleischer N , Lorente M, Kirkland J, al. e: Synthetic thyrotropin releasing factor as a test of pituitary thyrotropin reserve. J Clin Endocrinol Metab 34:617-624, 1972.
467. Sachson R , Rosen SW, Cuatrecasas P, al. e: Prolactin stimulation by thyrotropin-releasing hormone in a patient with isolated thyrotropin deficiency. N Engl J Med 287:972-973, 1972.
468. Vagenakis AG, Braverman LE, Azizi F, al. e: Recovery of pituitary thyrotropic function after withdrawal of prolonged thyroid-suppression therapy. N Engl J Med 293:681-684, 1975.
469. Tamai H, Nakagawa T, Ohsako N, al. e: Changes in thyroid function in patients with euthyroid Graves’ disease. J Clin Endocrinol Metab 50:108-112, 1980.
470. Tamai H , Suematsu H, Ikemi Y, al. e: Responses to TRH and T3 suppression tests in euthyroid subjects with a family history of Graves’ disease. J Clin Endocrinol Metab 47:475-479, 1978.
476. Werner SC, Spooner M: A new and simple test for hyperthyroidism employing L-triiodothyronine and the twenty-four hour I-131 uptake method. Bull NY Acad Med 31:137-145, 1955.
477. Duick DS , Stein RB, Warren DW, Nicoloff JT: The significance of partial suppressibility of serum thyroxine by triiodothyronine administration in euthyroid man. J Clin Endocrinol Metab 41:229-234, 1975.
480. Stanbury JB , Kassenaar AAH, Meijer JWA: The metabolism of iodotyrosines. I. The fate of mono- and di-iodotyrosine in normal subjects and in patients with various diseases. J Clin Endocrinol Metab 16:735-746, 1956.
481. Lissitzky S , Codaccioni JL, Bismuth J, Depieds R: Congenital goiter with hypothyroidism and iodo-serum albumin replacing thyroglobulin. J Clin Endocrinol Metab 27:185-196, 1967.
482. DeGroot LJ: Kinetic analysis of iodine metabolism. J Clin Endocrinol Metab 26:149-173, 1966.
483. Hays MT: Absorption of oral thyroxine in man. J Clin Endocrinol Metab 28:749-756, 1968.
484. Hays MT : Absorption of triiodothyronine in man. J Clin Endocrinol Metab 30:675-677, 1970.
485. Valente WA , Goldiner WH, Hamilton BP, al. e: Thyroid hormone levels after acute L-thyroxine loading in hypothyroidism. J Clin Endocrinol Metab 53:527-529, 1981.
486. Ain KB, Refetoff S, Fein HG, Weintraub BD: Pseudomalabsorption of levothyroxine. JAMA 266:2118-2120, 1991.
488. Oppenheimer JH , Schwartz HL, Surks MI: Determination of common parameters of iodothyronine metabolism and distribution in man by noncompartmental analysis. J Clin Endocrinol Metab 41:319-324, 1172-1173, 1975.
489. Curti GI, Fresco GF: A theoretical five-pool model to evaluate triiodothyronine distribution and metabolism in healthy subjects. Metabolism 41:3-10, 1992.
490. Bianchi R , Mariani G, Molea N, et al: Peripheral metabolism of thyroid hormones in man. I. Direct measurement of the conversion rate of thyroxine to 3, 5,3′-triiodothyronine (T3) and determination of the peripheral and thyroidal production of T3. J Clin Endocrinol Metab 56:1152-1163, 1983.
491. Faber J , Heaf J, Kirkegaard C, et al: Simultaneous turnover studies of thyroxine, 3,5,3′-and 3,3′,5-triiodothyronine, and 3′-monoiodothyronine in chronic renal failure. J Clin Endocrinol Metab 56:211-217, 1983.
492. Refetoff S , Fang VS, Marshall JS, Robin NI: Metabolism of thyroxine-binding globulin (TBG) in man: Abnormal rate of synthesis in inherited TBG deficiency and excess. J Clin Invest 57:485-495, 1976.
493. Lim VS, Fang VS, Katz AI, Refetoff S: Thyroid dysfunction in chronic renal failure: A study of the pituitary-thyroid axis and peripheral turnover kinetics of thyroxine and triiodothyronine. J Clin Invest 60:522-534, 1977.
494. LoPresti JS , Warren DW, Kaptein EM, al. e: Urinary immunoprecipitation method for estimation of thyroxine and triiodothyronine conversion in altered thyroid states. J Clin Endocrinol Metab 55:666-670, 1982.
495. Ridgway EC , Weintraub BD, Maloof F: Metabolic clearance and production rates of human thyrotropin. J Clin Invest 895-903:1974.
496. Cuttelod S , Lemarchand-Beraud T, Magnenat P, al. e: Effect of age and role of kidneys and liver on thyrotropin turnover in man. Metabolism 23:101-113, 1974.
497. Cavalieri RR , Searle GL: The kinetics of distribution between plasma and liver of 131 I-labeled L-thyroxine in man: Observations of subjects with normal and decreased serum thyroxine-binding globulin. J Clin Invest 45:939-949, 1966.
498. Oppenheimer JH , Bernstein G, Hasen J: Estimation of rapidly exchangeable cellular thyroxine from the plasma disappearance curves of simultaneously administered thyroxine- 131 I and albumin- 125 I. J Clin Invest 46:762-777, 1967.