Thyrotropin Isoforms: Implications for Thyrotropin Analysis and Clinical Practice

Thyrotropin Synthesis

Thyrotropin (TSH) is a heterodimeric 28-kDa-glycoprotein hormone released from the anteromedial pituitary and is a regulator of thyroid function. Its synthesis is controlled by the hypothalamic neuropeptide TSH-releasing hormone (TRH). The two peptide subunits of TSH are noncovalently linked and cotranslationally glycosylated with mannose-rich oligosaccharides (1). Posttranslationally, the two subunits are combined and the attached oligosaccharides are further processed. Synthesis of a mature TSH molecule requires the excision of signal peptides from both TSH α- and β-subunits, followed by trimming of mannose and further addition of fucose, galactose, and sialic acids (2). Thus, mature TSH molecules are asparagine-(N)-linked [Asp(N)-linked] complex carbohydrate structures capped with sulfate and/or sialic acid molecules (3,4) (Fig. 1). TSH oligosaccharide structures vary according to the source of TSH: human pituitary–derived TSH comprises fucosylated biantennary glycans with terminal N-acetylgalactosamine sulfate and low sialic acid content (5–7), while recombinant human TSH (rhTSH), synthesized in Chinese hamster ovary cells, specifically terminates in α2,3-linked sialic acid (8–11) and rhTSH formed in yeast lacks sialic acid residues altogether (12).

Glycosylation Patterns and TSH Bioactivity

Appropriate glycosylation is necessary to maintain normal TSH bioactivity (13). Indeed, both α- and β-subunits of TSH have functionally important domains associated with TSH receptor (TSHR) binding and activation. The α-subunit has two Asp(N)-linked oligosaccharide chains with a typical biantennary structure, while TSH β-subunit has only one chain (Fig. 2). The transcriptional and posttranscriptional mechanisms involved in TSH glycosylation result in folding of these subunits, leading to their heterodimerization. These mechanisms also qualitatively regulate TSH secretion, prevent intracellular degradation, and influence TSH clearance rate from the circulation (14,15). A three-dimensional TSH structure has been proposed in the 1990s based on crystallographic studies and comparisons with other glycoprotein hormones (16), but the schemes have not been fully confirmed by more recent studies, including structures of the follicle stimulating hormone extracellular domain and the TSH extracellular domain (17–19).

Human pituitary–derived TSH carbohydrate chains are subject variations in glycan structures, effecting TSH bioactivity (6,7). Such variations in glycosylation can be normal and have been observed in healthy subjects during the nocturnal TSH surge, normal fetuses during the last trimester of pregnancy, primary hypothyroidism, nonthyroidal illnesses, and in TSH-secreting pituitary adenomas (6,7).

TSH can be modified by changes in its terminal mannose, N-acetylgalactosamine sulfate, GlcNAc galactose, and/or core fucose. Specific hepatic receptors rapidly capture these modified TSH forms (20–23), resulting in blood enriched with highly branched and sialylated TSH glycoforms. Thus, patients with primary hypothyroidism exhibit TSH molecules with increased sialylation (6,7,24,25) and decreased inner fucosylation (26). Despite the loss of N-linked oligosaccharides, TSH isoforms maintain effective binding to the cognate receptor. However, loss of glycosylation leads to the loss of TSH biological activity (27). Moreover, deglycosylated isoforms may disrupt the biological activity of normal TSH and human thyroid-stimulating immunoglobulins (28), possibly by preventing the binding of other isoforms (29,30).

On the other hand, a higher glycosylation rate lowers the TSH clearance rate from the circulation; therefore, modulations of the TSH molecule result in changes in hepatic and renal clearance. Because of the nonhepatocyte cellular location of various liver lectin receptors, even in cases of hepatic insufficiency, such as chronic liver disease, fatty liver and cirrhosis serum TSH concentrations, as assessed by immunoassay, remain normal (31–33). In cases of chronic kidney disease patients, impaired glycosylation is associated with a change in TSH clearance rate, resulting in increased TSH biological half-life, decreased pulsatility, and a response to hypothalamic TRH feedback. Yet, in these patients as well, normal TSH levels are maintained (34–36).

TSH Measurements

As a single hormone determination, serum TSH immunoassays provide the most sensitive index to reliably detect thyroid function abnormalities (37–39). The current standards of care call for the use of “third-generation” TSH assays with a functional sensitivity of <0.02 mIU/L (37,40–42) in order to detect differing degrees of TSH suppression. Of note, not all immunoassays perform consistently in measuring very low levels of TSH, and the sensitivity of TSH assays used in clinical laboratories (functional sensitivity) may differ significantly from that stated by the manufacturer (analytical sensitivity) (38).

The usefulness of population-based TSH reference intervals to detect thyroid dysfunction in individuals may be somewhat limited (43,44). This is because it has been demonstrated that for TSH the between-person variability is more variable than within-person variability (45–47). Several epidemiological studies indicate that within-person (intraindividual) TSH variability is relatively narrow, and varies by only about 0.5 mIU/L when tested every month over a span of 1 year. Theoretically, it may be important to evaluate individuals with marginally (yet confirmed) low (e.g., 0.3–0.4 mIU/L) or high (3.0–4.5 mIU/L) TSH levels relative to patient-specific risk factors for cardiovascular disease, rather than relative to the normal TSH reference interval (48). However, it has not been demonstrated that serum TSH levels outside a person's individual range (but still within the population reference interval) can result in increased morbidity and mortality.

TSH is used in some U.S. states and regions of the world to screen newborns for underactive thyroid. It is also used for monitoring thyroid replacement therapy, in the workup of female infertility, and to screen adults for thyroid disorders. An abnormal serum TSH concentration indicates an excess or deficiency in thyroid hormone levels but does not provide information about the underlying etiology.

Baseline serum TSH concentrations are important when thyroid abnormalities are first suspected, and then again when equilibrium has been reached or the clinical status has changed (Table 1). Close monitoring of TSH and free thyroxine (FT4) is recommended for pregnant women with thyroid disease, for severely ill patients with nonthyroidal illness (49), and for severely ill hypothyroid patients with malabsorption of oral levothyroxine (LT4).

Nonthyroidal illness can frequently alter thyroid hormone peripheral metabolism and hypothalamic–pituitary–thyroidal function, resulting in thyroid test abnormalities, including decreased or increased serum TSH levels (50). It is important to distinguish the generally mild, transient TSH alterations typical of nonthyroidal illness from the more profound and persistent TSH changes associated with hyper- or hypothyroidism (51).

Primary hypothyroidism is the most common cause of elevated serum TSH. In such patients, serum FT4 is low-normal or reduced, and serum free triiodothyronine (FT3) concentrations usually remain normal until thyroid function level has markedly declined (52). In an iodine-sufficient patient, a transient elevation in TSH occurs during the recovery phase after a severe illness (15), when the hypothalamic–pituitary–thyroid axis is temporarily overactive. In general, such patients do not have an underlying thyroid dysfunction.

Hypothalamic–pituitary dysfunction may be associated with normal or modest increases in TSH, resulting from abnormal TSH glycosylation in the TRH-deficient patient. TSH resistance is associated with molecular defects that hinder adequate transmission of TSH stimulatory signals into thyrocytes. The defect may affect steps along the cascade after TSH binding to its receptor. Resistance to TSH because of either TSHR or G_s alpha mutations (53) and the resulting TSH elevations does not necessarily indicate hypothyroidism (54). Compound heterozygotes or homozygotes for partially inactivating mutations of the TSHR may have an elevated TSH, but with normal peripheral hormones, and be euthyroid, while monoallelic mutations in the G_s alpha subunit are associated with pseudohypoparathyroidism. If the mutation is on the maternal allele, then this can be associated with mild TSH elevations. Because of silencing of the paternal allele caused by imprinting, there is no thyroid phenotype if the mutation is on the paternal allele (55).

Isolated TSH elevations (with normal serum FT4) may be observed in a variety of conditions, including subclinical (mild) hypothyroidism, recovery from hypothyroxinemia of nonthyroidal illnesses, partial TSH resistance, hypothalamic hypothyroidism, and the presence of medications (such as amiodarone), which can inhibit thyroid hormone synthesis and metabolism and may cause transient reversible elevation of serum TSH (56).

In older adults, serum TSH may be higher than the normal reference interval for healthy adults (57–60). Epidemiological studies indicate elevated TSH levels with aging (61,62). Theoretically, this may be because of the presence of TSH isoforms with low bioactivity, especially when there are no accompanying clinical symptoms of hypothyroidism. The treatment of subclinical hypothyroidism in the elderly (48) may be resolved once the issue of declining TSH bioactivity in aging is resolved, by creating appropriate reference intervals for TSH for older adults, or by new technologies for TSH testing that can differentiate and quantify only TSH isoforms with normal bioactivity (63).

Fine-tuning the degree of TSH suppression plays a critical role in the management of thyroid cancer (64,65). It is well recognized that in patients with advanced-stage differentiated thyroid cancer, maintaining serum TSH below the normal range ensures greater progression-free disease and longevity (64). Suppression may be persistent for a prolonged period. In such cases, it is important to note that TSH concentrations ranging between 0.01 and 0.1 mIU/L can indicate a significant risk for atrial fibrillation and bone loss in older patients (66,67).

In the case of hyperthyroidism, including cases of Graves' disease, toxic adenoma and nodular goiter, subacute and lymphocytic (silent, postpartum) thyroiditis, iodine-induced hyperthyroidism, and exogenous thyroid hormone excess, serum FT4 and FT3 measurements are indicated when TSH levels are less than 0.1 mIU/L and also when TSH is in the range of 0.1–0.4 mIU/L. Further, in cases of Graves' disease, TSH should not be used to monitor therapy, as it may not respond to alterations in circulating thyroid hormones in an appropriate and rapid fashion. Patients with overt hyperthyroidism are expected to have serum TSH concentrations of less than 0.01 mIU/L except in TSH-induced thyrotoxicosis and T4/T3 resistance. In the case of resistance to thyroid hormones, the patients may have a mixed phenotype, with signs of hypothyroidism in some tissues and thyrotoxicosis in others. Because of the impaired feedback at the level of the thyrotrophs, the TSH levels are normal or mildly elevated serum. Isolated TSH suppression may be seen in hyperthyroidism resulting from causes such as subclinical hyperthyroidism, recovery from overt hyperthyroidism, nonthyroidal illnesses, during the first trimester of pregnancy, and in patients using medications such as dopamine and high-dose glucocorticoids.

Methods for Serum TSH Analysis

Laboratory-Related Variability in TSH Measurements

Although assay antibodies recognize peptide determinants of TSH, studies indicate that the presence or absence of specific glycans modify the immunoreactivity of the hormone, particularly when these changes occur in the β-subunit—the subunit specific to TSH (77–79). Furthermore, although pituitary and recombinant TSH usually share high cross-reactivity, clearly, they do not behave in an identical manner (10). Differences between the glycosylation patterns of a pituitary TSH calibrator and serum samples have been shown to be responsible for differences in epitope conformation and for introducing discrepancies in TSH measurements (10).

TSH is the primary test for thyroid function and one of the most common tests performed in the clinical chemistry laboratory (80). Inter- and intralaboratory variations in TSH measurements have been observed depending on the different methods used for analysis (41,81–85). Assays typically measure a nondetermined, highly heterogeneous mixture of glycoforms in addition to degradation products of TSH against a distinct internal standard or reference material (86). Although both preparations may share common glycoforms, they should not be assumed to behave identically (87). There are indications that circulating forms of TSH differ between euthyroid and hypothyroid persons (6,7,24–26). Under pathophysiologic conditions, TSH, similar to many other circulating glycoproteins, exhibits large structural variations (88). For instance, TSH glycosylations related to hypothyroidism may alter epitope expression between serum samples and the calibrator resulting in discordances in TSH measurements (79,89–91).

Monoclonal anti-TSH antibodies can be divided into two groups depending on their capacity to bind different forms of TSH (92). Some antibodies are unable to bind sialylated forms of TSH, whereas others fail to detect nonfucosylated forms of TSH (92). Both forms are seen in patients developing common thyroid disorders, such as hypothyroidism.

Antibody Recognition of TSH

Most existing antibodies target three antigenic regions in the TSH molecule. Each cluster comprises at least two close epitopes (92), with a main immunogenic region that has been characterized as dependent on changes in glycosylation (92). Some antibodies display a preference for specific glycoforms and thereby induce discordances among assays, particularly when these assays have been calibrated against the normal pituitary TSH standard.

Both the presence and the nature of specific linkages of the sialic acid residues are of high importance for optimizing antibody recognition. Moreover, fucosylation adds a further level of complexity as evidenced by the preference of some antibodies for nonfucosylated forms of TSH. This indicates that discordance in measurements can arise when an internal calibrator lacks fucose, such as in rhTSH. Thus, the identity of the TSH calibrator is of eminent importance to ensure that serum TSH can be measured with higher accuracy and possibly on a molar basis. Most of the formats tested were quite divergent when the calibrator was missing sialic acid, as in pituitary TSH, or a core fucose, as in nonfucosylated rhTSH fractions (98). The use of a pituitary international reference preparation is likely to mask part, if not all, of the influence of sialic acid and may explain the variable measurements encountered with rhTSH in 1999 and noted in most third-generation assays (41,99).

The use of monoclonal antibodies in TSH immunoassays has virtually eliminated cross-reactivity with other glycoprotein hormones (such as luteinizing hormone or human chorionic gonadotropin [hCG]). However, because monoclonal antibodies differ in their specificity for recognizing various circulating TSH isoforms, these antibody differences can result in the reporting of TSH values that may differ by as much as 1.0 mIU/L between different laboratories (who use different TSH assays) for the same serum specimen (100).

TSH Bioactivity Assays

Serum TSH concentration measurement alone may fail to detect the presence of pituitary and/or hypothalamic disease such as central hypothyroidism or TSH-secreting pituitary tumors, because TSH levels are often within the normal reference interval in these circumstances (105,106). Moreover, current TSH immunoassays cannot distinguish between normal and biologically altered TSH isoforms. TSH isoforms with impaired biologic activity are typically secreted in central hypothyroidism, whereas TSH isoforms with enhanced biologic activity are often secreted by TSH-secreting pituitary tumors (105,106). These abnormal TSH isoforms can result in paradoxically low, normal, or high reported TSH in the face of clinical and biochemical hypo- or hyperthyroidism (106). Therefore, in cases of suspected pituitary or hypothalamic disease, it is important to obtain serum FT4 and TSH concentrations concomitantly.

TSH bioactivity assays indicate the total amount of the biologic potencies of the various circulating TSH isoforms (107). Two methodologies are used to detect TSH antibodies, namely, bioassays and TSH-binding or receptor assays. Bioassays assess the functionality of the antibodies, and as such are able to distinguish between thyroid-stimulating immunoglobulins and thyroid-blocking antibodies (TBAb) (108). On the other hand, TSH-binding inhibiting immunoglobulin (TBII) assays are able to detect antibodies at the TSHR on membranes of the thyroid follicular cells, but they do not measure their functionality, and are thus unable to distinguish between stimulating and blocking antibodies (109).

TSH bioactivity measured in vitro provides a different parameter than the bioactivity of TSH measured in vivo. For example, in vitro, sialic acid (a negatively charged residue) tends to lower the affinity of TSH for the negatively charged TSHR. In comparison, in vivo, sialic acid binds and covers a galactose residue in the oligosaccharides, which then “hides” that galactose from hepatic galactose receptors, prolonging the half-life of circulating TSH. Thus, adding sialic acid to TSH tends to decrease intrinsic in vitro bioactivity slightly, but tends to increase in vivo bioactivity dramatically. This may be the reason why organisms evolved a mechanism to increase the sialylation of TSH when hypothyroid, a biochemical effect demonstrated in hypothyroid patients (25).

Bioassays Versus Immunometric Assays of TSH and B/I Ratio

TSH bioassays reflect the sum of the biopotencies of the various circulating TSH isoforms, whereas immunometric assays quantify serum TSH concentrations. Analyzed simultaneously by immunometric and biological assays, the ratio between the bioactivity and immunoactivity serves as an index of the overall potency of circulating TSH molecules (120). Thus, variations in the B/I ratio of immunopurified TSH samples result from changes in the amount of biological activity per unit of immunological activity. This serves as an estimate of the circulating TSH molecules' overall biological potency, or intrinsic bioactivity. Multiplying the B/I ratio by the concentration of TSH in serum produces the bioactive TSH concentration or intrinsic TSH bioactivity (107).

Alterations to the TSH structure affect its binding capacity and clearance. Since TSH undergoes modifications before its release from the anterior pituitary, it is subject to a variety of glycosylations that would affect its binding action. In a case of subclinical hypothyroidism in a young adult with Down's syndrome, antibody recognition of TSH significantly increased with sialylation and fucosylation (121). The modified TSH demonstrated a much stronger and more efficient binding to the assay antibodies compared with the normal pituitary TSH. This competitive binding preference for the modified TSH suggests that the normal active TSH was not as accurately detected in serum, at least in this one patient. This sialylation modification is known to prevent liver uptake and metabolic clearance, and therefore prolongs the half-life of serum TSH (121).

TSH isoform bioactivity is similarly subject to modifications that affect bioassay measurements. TSH glycosylation affects antibody binding activity and its ability to induce cAMP release. Altered glycan groups on TSH affect its bioactivity; deglycosylation may increase receptor binding and decrease signal transduction. Sialylation increases receptor binding action on TSH proteins (88). Thus, to reach accurate diagnoses, both bioactivity and biochemical analysis should be taken into account, especially in cases such as TSH-secreting pituitary tumors and hypothalamic hypothyroidism. This potential discordance should also be considered when routine TSH immunoassay values are not congruent to FT4 and T3 and/or the clinical circumstance (14,122).

Of note, in some clinical conditions (Table 1), the measurements of serum TSH alone as a first-line test may yield misleading information; it is difficult for the laboratory to proactively detect interference from a single measurement such as an isolated TSH test. A more common occurrence is for the physician to suspect assay interference when a reported value is inconsistent with the clinical status of a patient. The most practical way to investigate possible interference is to test the specimen by a different manufacturer's method and check for discordance between the test results. This approach is effective because methods vary in their susceptibility to interfering substances. Occasionally, a biological check can be made using TRH stimulation (which is not presently clinically available in the United States) or thyroid hormone suppression to validate a suspected inappropriate serum TSH level. Interferences producing a falsely elevated TSH value will usually be associated with a blunted (<2-fold increase) response to stimulation.

TSH and Aging

TSH Assessment in Pregnancy

Thyroid disorders are relatively frequent in women of childbearing age (140,141); consequently, TSH measurements are useful in detecting subtle thyroid dysfunction associated with reproductive issues and poor pregnancy outcome. During pregnancy, the placenta secretes high levels of hCG, a glycoprotein hormone sharing a common α-subunit with TSH (142). As discussed above, currently used immunometric TSH assays have overcome the problems posed by hCG cross reactivity (141). Moreover, enhanced sensitivity of third-generation assays has established the lower TSH limit for nonpregnant women as ∼0.3 mIU/L and affords the accurate detection of the low serum TSH concentrations often detected in early pregnancy (143). During the first trimester, elevated hCG secretion results in higher T4 and T3 and suppressed serum TSH concentrations. Since hCG concentrations are higher in multiple pregnancies than in singleton pregnancies, a greater downward shift is observed in twin pregnancies (144). However, in the case of maternal hypothyroxinemia (low FT4 accompanied by normal TSH), which is increasingly recognized for its association with neurodevelopmental deficits, TSH is within the normal range and is therefore not a useful assay for detecting this problem (145). FT4 immunoassays are notoriously unreliable during pregnancy (146), but relying on TSH is not sufficient in this case.

Acknowledgments

Dr. O.P. Soldin was supported in part by NIH R01AG033867 and by the Flight Attendants Medical Research Institute. The project was also supported in part by award number P30CA051008 from the National Cancer Institute. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute or the National Institutes of Health. We thank Chen Fei for help with constructing the figures.

Author Disclosure Statement

The authors declare that no competing financial interests exist.