Introduction.....Actions..... Hormones..... Regulation ...Non-Thyroidal illness
Function Tests

Introduction:

The thyroid gland consists of a small (several millimeters), bilobulated structural tissue partially surrounding an area of the primary airway in the neck (the trachea). (Sometimes, functional thyroidal tissue is also found in other locations.) This gland synthesizes the hormone thyroxine which, itself, is converted to other, active or inactive hormones. The synthesis and metabolism of thyroxine, its function and the functions of its metabolites in general, the regulation of its synthesis and activity...all this will be discussed below. Following this discussion of the basics of thyroid hormones and the effects of non-thyroidal illnesses on circulating hormone measurements, the reader should focus on the clinically important thyroid syndromes in dogs (hypothyroidism) and cats (hyperthyroidism).

Thyroid Hormone Actions:

The thyroid hormones affect virtually every metabolic activity in the body. These include the concentration and functionality of numerous enzymes, all aspects of the metabolism of fats, carbohydrates, protein, vitamins, the utility of minerals, the secretion and breakdown of all other hormones, as well as the response of tissues to these other hormones. The thyroid hormones have marked influence on the contractile strength and rhythm of the heart, they enhance respiratory drive especially when tissue/blood oxygen levels are suboptimal (i.e. promotes breathing!), they stimulate the marrow to create more red blood cells, if needed (erythropoiesis), and are active in the regulation of bone synthesis and turnover. They are important for processes where cell turnover and resynthesis are ongoing (e.g. the hair follicle). According to Feldman and Nelson* “...no tissue or organ system escapes the adverse effects of thyroid hormone excess or insufficiency.”

The Important Thyroid Hormones:

These consist of thyroxine, (also called T₄ ), 3,5,3’-triiodo L-thyronine (also known as T3) and 3,3’,5’-triiodo L-thyronine (known as “reverse” T₃ ...or rT₃ ). All of these contain iodine bound to one or more carbons of complex rings that comprise the backbone structure of the hormones. The structure of each of these is shown in the diagram.

During metabolism, T₄ is converted to T₃ or to rT₃ via removal of an iodine atom from one of the hormonal rings. The T₃ is the biologically active thyroid hormone, whereas rT₄ has no biological activity. It is presumed that factors that regulate whether T₄ is converted to T₃ or rT₃ are pivotal in determining and thus regulating the functional activity of the thyroid at the level of the gland, itself. As we shall see, control and regulation of thyroid hormone activity is also accomplished at other levels.

It should be noted that the primary hormone created in the thyroid is T₄ ..and that only about 20% of the T₃ produced from T₄ is produced in the thyroid gland. Instead, circulating free T4 is converted to T3 at the level of the target tissues...when hormone diffuses into cells or binds to tissue-specific receptors. T₃ enters the cell, where is asserts its influence on function at the cytological or nuclear level, accordingly. It is important to add that there are other pathways of metabolism of thyroid hormones in target tissues not involving iodine removal or addition...and that these are essential to the functional influence of the thyroid upon those specific tissues.

One more thing that will be important, when we discuss clinical diseases and testing of thyroid function is the form of circulating T₄ . Most...nearly all (99.9%)... of T₄ in the circulation is bound to protein. Barely ~0.1% of T4 is free. Only the free T4 can be converted to T₃ at the target tissue; however as free T₄ is consumed, in principle more is released from protein to replace it. However, this process can be affected by other complicating factors (see non-thyroidal illness) that, also, will be discussed under clinical diseases and testing methods.

Regulation of Thyroid Hormone Function-Synthesis:

Regulation of thyroid hormones occurs from regions external to the gland and from within the thyroid itself.
The extrathyroidal pathways are illustrated here, and described below.

In the brain there exists an important system-wide regulatory center known as the hypothalamus. The hypothalamus synthesizes and releases a hormone called thyrotropin-releasing-hormone (or TRH). The release of TRH by the hypothalamus causes release of thyrotropin-stimulating-hormone (or TSH) by the nearby pituitary gland. TSH from the pituitary travels via circulation to the thyroid tissues and is the most significant factor for stimulating the synthesis and release of biologically active thyroid hormone molecules.

The regulation of TRH synthesis and release from the hypothalamus is incompletely understood, but probably involves complex neurological signals and interactions that establish the initial “set-point” for TRH release. However, the hypothalamus is also sensitive to levels of TSH; when TSH levels are increased, there is feedback of this information to the hypothalamus resulting in the cessation of further synthesis and release of more TRH (this process is called feedback inhibition). It is also believed that the presence of adequate circulating levels of free T₄ , and T₃ also affect a feedback inhibition on the hypothalamus to cease TRH release AND on the pituitary to cease TSH release. A diagram illustrates these interactions..the lines in the image from the thyroid hormones reflect back to illustrate that thyroid hormones inhibit further stimulation of their own synthesis by the hypothalamus and the pituitary glands (dashed [----] lines and minus [-] signs).

The thyroid gland itself is able to affect some regulation of its activity ("autoregulation"). This can occur via controlling its uptake of iodine (essential to the synthesis and function of thyroid hormones), controlling its sensitivity to the influence of TSH, failure to synthesize adequate T₄ when iodine availability is reduced, reducing T₄ in the presence of excessive iodine, and possibly synthesizing rT₃ in preference to T₃ when some conditions warrant less biological activity.

The conditions which instigate and promote this thyroid gland autoregulation are poorly understood and probably more complicated than necessary for this discussion. They are only mentioned so that the reader is aware they exist.

The Effects of Non-Thyroidal Illness on Circulating Thyroid Hormones:

Given this background, the reader is encouraged to read the pages that deal with clinically relevant forms of aberrant thyroid gland activity: Hypothyroidism in dogs; Hyperthyroidism in cats.

This portion of the discussion will lead to first principles pertaining to how we test and interpret tests for aberrant thyroid hormone activity in dogs and cats . This will become relevant and important when the reader reviews the pages on Hypothyroidism (dogs) and on Hyperthyroidism (cats). The details of actual tests are found near the end of this page

Evaluation of thyroid gland hormone output (hypothyroid = not enough output; hyperthyroid = excessive output) can be accomplished via measurements of circulating thyroid hormone levels. The simplest and easiest to measure is total T₄ (i.e bound to protein). It is also possible to measure freeT₄ , T₃ , and rT₃ . Sometimes, it is useful to measure stimulating hormone levels (TRH or TSH) or the response of the thyroid gland to administration of one or the other of these stimulating hormones (TRH-Stimulation Test; TSH Stimulation Test) or to the administration of T₃ on the level of T₄ (T₃ -Suppression Test). Details about these tests are discussed below. The specific indication for one or the other complex tests of thyroid function will be elaborated upon in the discussions of hypo- and hyperthyroidism.

Sometimes measured levels of circulating hormones are altered by illnesses NOT related to the thyroid gland. In many non-thyroidal illnesses, total T4 measurements reveal lower than normal values; T₃ may be anything (T₃ alone is never reliable for evaluation of thyroid function), and freeT₄ may be low, normal or high....but the animal is not truly hypothyroid OR hyperthyroid.

Causes for lowered T₄ with non-thyroidal illnesses include:

• impaired binding to plasma protein
• reduced affinity for binding to plasma protein
• decreased production of T4 due to effect of illness on
  • thyroid
  • pituitary
  • hypothalamus

Incidences of lowered T₄ due to non-thyroidal illness (false positives):**

• With mild non-thyroidal disease: 8% false positives
• With moderate non-thyroidal disease: 28% false positives
• With Severe disease: 60% false positives

Causes for Normal or Elevated T₄ when true hypothyroidism is present

• Antibody to thyroid hormone precursor (thyroglobulin)...[not antibody towards T₄ or T₃ )

Free T₄ may also be artefactually altered with non-thyroidal illnesses

• it may be increased (due to decreased binding to plasma proteins)
• it may be increased due to reduced cell uptake
• it may be decreased...especially in dogs with concurrent Cushings Disease (Hyperadrenocorticism)...due to decreased TSH secretion
• it may be increased or decreased in cats that not hyperthyroid

Incidences of lowered free T₄ by equilibrium dialysis due to non-thyroidal illness (false positives):**

• With mild non-thyroidal disease: 8% false positives
• With moderate non-thyroidal disease: 17% false positives
• With Severe disease: 44% false positives

Reverse T₃ (rT₃ )...may increase with non-thyroidal illnesses....probably because there is decreased enzyme-mediated conversion of T₄ to T₃ , and the alternative pathway (to rT₃ ) predominates...this is an avenue of research that could be helpful in distinguishing true thyroid pathology from artefacts due to non-thyroidal illnesses.

Principles and Practice of Thyroid Function Testing

Testing for thyroid function is not an exact science and is, as discussed earlier, confounded when there is concurrent non-thyroidal illness. In both dogs and cats, several tests have been examined for efficacy, i.e. sensitivity and specificity. These tests include the following: Total T₄ , freeT₄ (by equilibrium dialysis), T₃ Suppression Test, TSH Stimulation Test, TRH Stimulation Test, Serum TSH Concentration, Serum TRH Concentration, Serum T₃ , Serum Reverse T₃ (rT₃ ).

• Serum Total T₄ :
  • Is most common test for evaluation of thyroid function...directly measures circulating hormone levels
  • Requires a single blood sample
  • For animals already receiving medication to control thyroid levels, blood is best drawn 4-6 hours after administration of medication
  • Is frequently decreased with non-thyroidal illnesses.
  • It may be INCREASED or NORMAL when there is true hypothyroidism (due to presence of antibody to thyroid hormone precursor--thyroglobulin)

• Free T₄ (equilibrium dialysis)
  • Measures circulating T₄ that is not bound to protein...this is the active form of T₄ (gets converted to T₃ )
  • Requires a single blood sample
  • For animals already receiving medication to control thyroid levels, blood is best drawn 4-6 hours after administration of medication
  • The "equilibrium dialysis" method is the only reliable assay
  • Is more specific for diagnosing hypothyroidism than Total T₄
  • May be elevated, decreased or normal with non-thyroidal illnesses. Should never be used alone to evaluate thyroid function...especially in cats where it is interpreted with respect to the concurrent Total T₄ value
  • Unfortunately, in late 2005, this test was not available to most laboratories because the manufacturer of the test kit has temporarily halted production pending some quality control issues. The test is back now (2006) but the availability for the months ahead is uncertain again

• T3 Suppression Test:
  • Test is most useful for evaluating hyperthyroidism in cats
  • Principle of test: 
    • Blood sample is obtained to determined basal levels of circulating T₄ and T₃
    • Then oral administration of T₃ several times daily for three days suppresses the synthesis of T₄ in normal animals (i.e. feedback inhibition...again!)
    • Two to 6hr after last dose of oral T₃, a blood sample is taken to measure T₄
    • In a normal cat, T₄ level is decreased from the basal level; in hyperthyroid cat, T₄ is normal or increased
    • Problem with test is that if owner fails to administer even a single dose of T₃ , the measured T₄ will be not be reduced and an erroneous diagnosis of hyperthyroidism may occur. Therefore, it is essential to measure T₃ in the same blood sample (it should be elevated if there has been full compliance)
  • Considered "gold standard" for testing feline hyperthyroidism by some
  • Effect of non-thyroidal illness on sensitivity of this test is not clear at this time

• TSH Stimulation Test:
  • Is considered the most sensitive test for determining the presence of canine hypothyroidism. Unfortunately, the bovine source of TSH for this test is no longer available; alterantively, a suitable human gene recombinant TSH can be used in dogs though it is extremely expensive. A suitable TSH source for similarly testing thyroid function in cats is not yet available and current options for TSH stimulation testing in cats lack sufficient sensitivity.
  • Principle of Test:
    • Blood is drawn to determine basal concentration of circulating T₄
    • TSH hormone (bovine or human recombinant DNA derived) is administered intravenously
    • Two to 4 hours later, a second blood sample is obtained.
    • In normal animals, there is stimulation of the thyroid by the TSH administered, and T4 levels increase
    • In hypothyroid animals...those with primary hypothyroidism (>95%), T₄ levels do not increase significantly or at all after administration of the TSH.
    • For animals with secondary (pituitary) hypothyroidism, if it is early in the course of the disease...and the thyroid has not yet atrophied (due to lack of natural TSH stimulation) it is possible that stimulation will also be observed (false negative test). However the majority of secondary hypothyroid animals develop thyroid gland atrophy within weeks after cessation of natural TSH
  • Is considered sensitive, even in the presence of non-thyroidal illness

• TRH Stimulation Test
  • Has been used in cats to detect hyperthyroidism; it is not at all helpful in diagnosing aberrant thyroid function in dogs, however.
  • Principles of Test:
    • In normal cats, administration of TRH (presumably via stimulating the pituitary to release TSH) results in increased (. 60% above baseline) T₄ ; In hyperthyroid cats, a subnormal response (less than 50% above baseline) is expected because the the autonomously functioning thyroid gland in synthezising T₄ which, via feedback inhibition, suppresses the full release of TSH
    • In one half of cats with non-thyroidal illness, there was failure to stimulate T4 above the 50% level, however. It is speculated that some non-thyroidal illnesses suppress TSH secretion , so a suboptimal release of TSH occurs, and a suboptimal increase in T4 is measured (false-positive for hyperthyroidism).
    • The procedural details are as described for the TSH stimulation test, above.

• Serum TSH (or Serum TRH Concentrations)

  There is a specific assay for TSH in dogs (not in cats) allowing evaluation of TSH, along with thyroid hormone levels in animals with suspected altered thyroid function

  Principle of Test:

  With low thyroid levels, as in hypothroidim, there is little feedback inhibition of pituitary secretion of TSH; therefore elevated levels of TSH are expected

  With high thyroid levels, as in hyperthyroidism, the presence of excess circulation thyroid hormones inhibits TSH release, and circulating levels of TSH are low

  The sensitivity for predicting hypothyroidism is only 67% to 73% (can be falsely elevated or falsely decreased)

• Serum T₃ and rT₃ Concentrations
  • Serum T3 levels are unreliable when used to evaluate thyroid function
    • It is normal in 25% of hyperthryoid cats
    • It is normal in up to 90% of hypothyroid dogs
    • It is sometimes decreased in animals with non-thyroidal illness.
  • Reverse T3 (rT₃ ) may be useful for assessing non-thyroidal illness.
    • It is higher than normal during non-thyroidal illness
    • However, there have not yet been good studies of rT₃ levels in hypothroid and hyperthryoid animals.
  • These tests may be useful adjuncts when evaluated along with other tests, such as T₄ , free T₄ , TSH levels, TSH stimulation and T₃  Supression
• Technitium⁹⁹ Radionuclide Scan: is used to evaluate location and magnitude of thyroid hormone secreting tissues when hyperthyroidism has already been diagnosied. Remember..the thyroid tissues are normally located in the neck..but sometimes tissues are found in other places...such as the chest. This is important when considering the treatment options for feline hyperthyroidism. Example of technitium scans are shown elsewhere.

*Feldman & Nelson, Canine and Feline Endocrinology & Reproduction, 2nd ed, Saunders 1996

**VIN and Intervet^® Sponsored Symposium on Canine Hypothyroidism 6/26/06