Diagnostic Evaluation of The Hypothalamic-Pituitary-Thyroid Axis

• Canine Hypothyroidism

• Feline Hyperthyroidism

Canine Hypothyroidism

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Hypothyroidism is a multisystemic disorder associated with reduced cellular metabolic function caused by deficiency of thyroid hormone. In more than 95% of cases, this is associated with destruction of the thyroid gland itself (primary hypothyroidism) as a consequence of immune-mediated destruction (lymphocytic thyroiditis) or idiopathic atrophy. Clinical signs of hypothyroidism are very variable (see Table 1) and may occur together or a single sign may predominate. As a consequence, hypothyroidism is a differential diagnosis in very many conditions. Problems of overdiagnosis can arise because reduced thyroid hormone output is a very common finding in a wide variety of systemic non-thyroidal illnesses (NTI). In addition, some cases of obesity, exercise intolerance and dermatopathy may respond to some extent to thyroid hormone supplementation, even in the absence of hypothyroidism.

Diagnosis therefore currently requires:

• Presence of compatible clinical signs
• Exclusion of other systemic disease
• Demonstration of low (low normal) total and/or free T4
• TSH measurement
• Evaluation of the clinical response to the introduction and withdrawal of T4 supplementation

Currently, a reliable source of TSH is not available in the UK. However, initial trials using human recombinant TSH in normal dogs are encouraging. TRH is available, but, as discussed below, the finding of a poor response to TRH is not specific for hypothyroidism.

Long-term studies into the significance of thyroglobulin autoantibodies in normal dogs are not complete. However, autoantibody presence in dogs with compatible signs and equivocal laboratory results significantly increases the index of suspicion for hypothyroidism associated with lymphatic thyroiditis.

Table 1: Clinical Manifestations of Hypothyroidism in the adult dog

Profile Changes in Hypothyroidism

Routine laboratory test abnormalities are very common in hypothyroidism and include mild normocytic, normochromic, non-regenerative anaemia (40% of cases), hypercholesterolaemia (60-80%), which can be mild to marked; hypertrigylceridaemia (80%), mild increases in alanine transferase (ALT), alkaline phosphatase (ALP) and creatinine kinase (CK) (35%).

While none of these changes are specific, their presence is supportive of hypothyroidism in a dog with appropriate signs. Profiles are also useful in excluding other differential diagnosis and for identifying other disease conditions which may significantly affect thyroid function (Cushing's, diabetes mellitus, other systemic disease).

Where Cushing's could be a differential diagnosis clinically, it is important to exclude this endocrinopathy before evaluating thyroid function. Cushing's can significantly reduce basal T4, free T4, TSH and TRH responsiveness, resulting in a potentially erroneous diagnosis of hypothyroidism.

Total Thyroxine (T4)

Thyroid gland hormone production comprises T4 predominantly with small amounts of triiodothyroinine (T3). More than 99% of T4 is bound to serum proteins (thyroid binding globulin (TBG) and albumin) leaving 0.1% unbound and available to tissues for intracellular conversion to T3, the metabolically active form. Measurement of total T4 includes both the free and bound T4.

Hypothyroidism, Cushing's, systemic illness and drug therapy can all be associated with alterations in the relative proportions of bound and free T4.

In early hypothyroidism, basal T4 is often within normal limits but declines as the condition progresses. There is however, complete overlap between the T4 concentrations seen in hypothyroidism, NTI and those associated with drug therapy (see Table 2).

Table 2: Factors and conditions associated with reduced total T4

Therefore, middle range or higher basal T4 concentrations help to exclude hypothyroidism whereas low normal or reduced concentrations are not specific for hypothyroidism and require further diagnostic evaluation (see Table 3).

Sample required: 1 ml separated serum or heparin plasma.

Accuracy: As assessed against results of TSH stimulation and therapeutic response, T4 has been reported to correlate with hypothyroidism or euthyroidism in approximately 85% of cases.

Table 3: Interpretation of Basal T4 Concentration (Immulite Canine Thyroxine)

Total Triiodothyronine (T3)

Although T3 is the metabolically active hormone at a cellular level, most of the conversion of T4 to T3 occurs intracellularly. Consequently, serum T3 is not a good guide to its level within cells. Low, normal and occasionally increased T3 concentrations have been reported in canine hypothyroidism; the latter reflecting the relative shift to T3 synthesis from T4 in the failing thyroid.

Anti-T3 antibodies are more common than anti-T4 antibodies and are often associated with lymphocytic thyroiditis. When present, spuriously high T3 concentrations are evident in some RIA and chemiluminescent assay systems. Their presence is not pathognomic for hypothyroidism however.

Sample required: 1 ml separated serum

Free T4 (FT4)

FT4 theoretically has greater diagnostic value than T4 in that it better reflects available hormone at a cellular level and is less likely to be affected by drugs or NTI. As consequence, in human medicine, it is used extensively to the virtual exclusion of T4.

Two general methods are available for determination of FT4; equilibrium dialysis (EQ) and analogue FT4 assay.

A new non-isotopic direct dialysis method for FT4(EQ) has recently been developed, which is suitable for routine use and appears to correlate well with standard isotopic equilibrium dialysis.

Using this method, a low FT4 EQ has better specificity but lower sensitivity than T4.

Analogue (non-dialysis) FT4 methods can be fully automated and have been optimised for use in human medicine. The degree of automation associated with these methods makes them significantly faster and cheaper to perform, but provides no further diagnostic information beyond the T4.

Sample required: 1ml separated serum

Interpretation: Reductions in T4(EQ) are more specific than reduced T4. However, prior glucocorticoid or phenobarbitone therapy or severe NTI can be associated with low concentrations as well as hypothyroidism. FT4(EQ) can be a useful additional test, however, either in combination with cTSH or as part of a Thyroid Function Panel (see below).

Accuracy: As assessed on the basis of the TSH stimulation test and therapeutic response.

Direct dialysis: Sensitivity 80%, specificity 93%

Analogue methods: Identical to T4

Endogenous Canine TSH assay (cTSH)

In human medicine, detection of increased TSH concentrations in combination with low FT4(EQ) reliably diagnoses primary hypothyroidism.

In dogs, only 63-80% of hypothyroid cases have increased cTSH, while 8-13% of dogs with NTI have increased cTSH concentrations. As a result, cTSH results should be interpreted only in the light of simultaneous T4 measurement after clinical evaluation for the presence of NTI.

T4 with TSH, which is sometimes expressed as T4:TSH ratio, has been shown to be as accurate as FT4(EQ) and is now the recommended screening test for canine hypothyroidism.

Sample required: 1 ml separated serum.

Interpretation: Combination of normal cTSH and T4 is very likely to reflect the presence of normal thyroid function. Low T4 with markedly increased cTSH (>2.5ng/ml) is consistent with primary hypothyroidism. Low/low normal T4 with normal/mildly increased cTSH may reflect hypothyroidism, presence of NTI or effects of prior sulphonamide, glucocorticoid or chronic anticonvulsant therapy.

Accuracy: cTSH sensitivity 87%, specificity 82%

T4 and cTSH: Sensitivity 87%, specificity 92%

Thyroid Autoantibodies

Approximately half of all naturally occurring cases of hypothyroidism result from lymphocytic thyroiditis. This condition, analogous to Hashimoto's thyroiditis in human medicine, is considered to be immune-mediated and is associated with serum autoantibodies directed against the thyroid products thyroglobulin, T3 and less commonly T4.

Previous methods of measuring thyroglobulin antibodies (TGAA) were cumbersome and associated with frequent false positive results in dogs with NTI.

However, newer, more specific ELISA methods have been shown to be suitable for diagnostic use however.

Sample required: 1ml serum

Interpretation: Presence of TGAA with clinical and laboratory findings consistent with hypothyroidism confirm lymphocytic thyroiditis.

In dogs with low/low normal T4 and normal cTSH, presence of TGAA assists differentiation of hypothyroidism from NTI.

6% of normal dogs have TGAA. In human medicine, there is a correlation between presence of TGAA and development of hypothyroidism. However, the predictive value of TGAA for the subsequent development of hypothyroidism in dogs is not yet clear.

Thyroid Function Panel

Includes T4, T3, FT4, FT3 (analogue methods or EQ) T4, T3, thyroglobulin autoantibodies and cTSH.

In the USA, difficulty in obtaining TSH has resulted in extensive use of panels both for diagnosis of individuals and breed screening programs. In this country, breeders often express interest in their use. But, while they are helpful for identification of autoantibodies that affect assayed results, their value in predicting the development of disease is not clear.

Advantages of the thyroid panel include relatively low costs per test and the convenience of a bundled package of tests. Disadvantages include slower turnaround of results and the potential difficulties in interpretating a single abnormality in an otherwise normal panel.

Sample required: 2 ml serum

Provocative Thyroid Function Tests

Protocol:

• Obtain a baseline separated serum sample (1ml)
• Inject TRH 0.1mg/kg iv
• Obtain a second separated serum sample 4 hours post injection

Table 4: Guidelines for TRH dosage on 200µg/2ml vials (Protirelin, Cambridge Labs)

Interpretation: Post-TRH T4 should be 1.2 times the pre-TRH T4 and exceed 30 nmol/L in normal dogs. Pre-TRH T4 less than 25nmol/l with post-TRH T4 less than 20nmol/l is consistent with hypothyroidism. Post-TRH T4 in the range 20-30nmol/l is equivocal.

Reduced stimulation is seen with hypothyroidism but also in some euthyroid dogs and dogs with NTI. As a result, normal TRH stimulation reliably excludes hypothyroidism, but reduced stimulation test results can be seen with conditions other than hypothyroidism.

Monitoring T4 Replacement Therapy

Pre- and post-pill sampling for T4 or FT4 can be very useful in the assessment of therapeutic adequacy in dogs on replacement therapy with T4. Very considerable variation has been reported in intestinal absorption and rate of metabolism of T4, and in the bioavailability of T4 in differing preparations. As a consequence, each dog on replacement therapy should be sampled after two weeks on therapy and as necessary thereafter to optimise the dose and formulation for the individual (see table 6).

Peak concentrations should be in the upper half or slightly above the top of the normal range. The exact time of peak concentrations post-pill also shows considerable individual variation. However, single sampling 4-6 hours post-pill has been recommended. Pre and post-pill testing can be useful in dogs showing a poor response to therapy to check for poor absorption and rapid metabolism.

Table 5: Guidelines for adjusting synthetic thyroxine supplementation based on results of evaluation of serum T4 concentrations

Feline Hyperthyroidism

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Hyperthyroidism is a common, multisystemic disorder resulting from excessive circulating concentrations of T4 and T3. In 98-99% of cases, it is caused by benign thyroid neoplasia (adenomas {adenomatous hyperplasia}). However, 1-2% of cases are associated with functional thyroid carcinoma.

While the condition is recognised in cats as young as 6 years, more than 90% of cases are aged 10 or older. Clinical findings are very variable (see Table 6). Palpable goitre, weight loss and involvement of more than one body system are common features. Common laboratory profile changes in hyperthyroidism are listed in Table 7. Increases in ALT and ALP are very common findings. However, normal concentrations of these enzymes do not rule out hyperthyroidism. A variety of other conditions (gastrointestinal disease, diabetes mellitus, cardiac disease, progressive lymphocytic cholangitis) may show similar profile changes, however, and are important differentials clinically.

Table 6: Clinical findings in feline hyperthyroidism

Table 7: Routine profile changes in feline hyperthyroidism

HAEMATOLOGY	BIOCHEMISTRY
Normal or increased PCV
Mild microcytosis
Mature neutrophilia	ALT AST
Lymphopenia or lymphocytosis	ALP
Monocytosis	Urea
Eosinophilia	Fructosamine

Diagnosis is normally straightforward and is based on the history, clinical findings and presence of increased basal T4. More challenging cases include those with apathetic hyperthyroidism in which unusual clinical sings predominate (reduced activity, anorexia, ventral neck flexion), those in which hyperthyroidism is an incidental finding unrelated to the presenting illness, and those cases with classic clinical signs but an apparently normal basal T4.

Further diagnostic evaluation of the latter group may include a repeat T4 after 7-14 days, checking for concurrent illness or drug therapy, which may reduce T4 concentration into the normal range in a hyperthyroid individual or occasionally the use of suppression or stimulation tests.

Where cases are managed medically, periodic monitoring of T4 is useful in assessing the adequacy of therapy. T4 concentrations should be in the lower half of the reference range in cats on neomercazole therapy.

Total T4

Sample required: 1ml separated serum

Interpretation: Increased T4 in cats aged 6 years or greater is diagnostic of hyperthyroidism where there are consistent clinical signs. However, 10-15% of hyperthyroid cats have T4 concentrations in the normal range. This reflects the fluctuating T4 levels into and above the normal range seen in early disease. Other causes include effects of concurrent non-thyroidal illness or drug therapy, which may depress basal T4 into the normal range.

In cats with suspicious signs in which an initial basal T4 is normal (but within the upper half of the normal range), further diagnostic evaluation may still be indicated. Repeat basal T4 after a minimum of 7 days may yield a diagnosis. In other cases in which an early diagnosis is required, the T3 suppression test or TRH stimulation test may be useful.

Total T3

Sample required: 1 ml separated serum

Interpretation: Increased T3 levels are evident in approximately 75% of cases. In 25% however T3 is within normal limits. As a consequence, measurement of T3 has less diagnostic value than T4.

Free T4

Sample required: 1ml separated serum

Interpretation: Increased FT4 concentrations, as determined by equilibrium dialysis, are found in a similar proportion of hyperthyroid cats to those with elevated T4, i.e., sensitivity of FT4(EQ) and T4 for hyperthyroidism is similar. However, small numbers of cats with non-thyroidal illness have increased FT4 concentrations despite having normal thyroid function. As a consequence, the finding of increased FT4(EQ) is less specific for hyperthyroidism than increased T4.

T4 is therefore preferred over FT4(EQ) as a diagnostic screening test for hyperthyroidism.

Provocative Thyroid Function Tests

T3 Suppression Test

Principle: Release of TSH from the pituitary is inhibited by increased concentrations of T3, which may be induced by administration of oral T3 tablets. In euthyroid cats, T4 concentration falls following the reduction in TSH secretion induced by increased T3. In hyperthyroid cats however, in which T4 secretion is relatively independent of TSH, little change in T4 is noted following T3 administration.

Protocol:

• Take a baseline separated serum sample and store it frozen
• T3 (liothyronine 25µg) is given tid per os for 2 days
• On the morning of the third day, give a seventh dose
• Take a second sample 2-4 hours post-pill.

Samples are assayed for T4 and T3.

Interpretation: T3 concentrations rise following liothyronine administration and are used to monitor owner compliance with drug administration. Normal cats show a 50% or greater reduction in T4 concentration following liothyronine and have concentrations of 20 nmol/l or less. Hyperthyroid cats show minimal change in T4 concentration and have post liothyronine T4 levels in excess of 25 nmol/l.

Values between 20-25 nmol/L are non diagnostic.

Accuracy: T3 suppression test is useful in differentiating hyperthyroid from euthyroid cats and has similar sensitivity and specificity to the TRH stimulation test. However, it is a prolonged test and very dependent on good owner compliance. Failure to ingest the liothyronine may result in apparent failure of T4 to suppress and false positive diagnosis of hyperthyroidism.

TRH Stimulation Test

Principle: TRH stimulates the release of TSH from the pituitary and results in increased T4 concentrations in normal cats. In hyperthyroid cats, release of TSH is inhibited by high T4 concentrations. As a result, hyperthyroid cats stimulate very poorly in response to TRH.

Protocol:

• Obtain a baseline separated serum sample
• Administer TRH (0.1 mg/kg iv)
• Take a second sample 4 hours later

T4 only is routinely assayed in this test.

Interpretation: Normal cats and those with non-thyroidal illness have been demonstrated to show an approximately two-fold increase in T4 in response to TRH. Hyperthyroid cats show little increase (<50%) in response to TRH. Increases in T4 >60% are seen in cats with normal thyroid function. Responses between 50-60% are regarded as equivocal.

Accuracy: TRH stimulation and T3 suppression tests evaluate the pituitary thyroid axis in different ways but have been shown to have similar diagnostic value. However, both tests potentially may produce equivocal results in a small percentage of cases.

Warning: Administration of TRH iv is commonly associated with central nervous system signs including salivation, vomiting, tachypnoea and defaecation. These signs are transient and resolve by the time of the second sample.

Suggested Reading

Feldman, E.C., Nelson R W 1996. Second Edition Canine and Feline Endocrinology and Reproduction, W.B. Saunders, Philadelphia

Nelson, R.W., Couto G G, 1998, Small Animal Internal Medicine Second Edition, Mosby Year Book, Missouri

Peterson, M.E., Endocrine and Metabolic Disorders in Current Veterinary Therapy XII, Bonagura (Ed) 1995, W.B. Saunders, Philadelphia

Bruyette, D.S., Endocrine and Metabolic Diseases in Consultations in Feline Internal Medicine Volume 2, 1994 August, J.R. (Ed)., W.B. Saunders, Philadelphia

Torrence, A.O.; Mooney, C.T., Manual of Small Animal Endocrinology 1998 Second Edition, BSAVA Publications, Cheltenham