Although hypothyroidism is one of the most common endocrinopathies in dogs, diagnosis can be challenging. Determination of total serum T4 concentration is accepted as the primary screening test for the disease, and low serum T4 concentrations are intuitively suggestive of hypothyroidism. However, it is well-known that low T4 concentrations are frequently encountered in euthyroid dogs with various nonthyroidal diseases and in dogs receiving certain pharmacologic agents such as glucocorticoids, among others.1–4 In humans, determination of endogenous TSH concentrations, which are expected to be increased during states of low functional T4, has become standard practice for the initial assessment of patients with suspected thyroid insufficiency.5 However, the low diagnostic sensitivity of the test for cTSH concentration has precluded its use as a reliable diagnostic test in veterinary medicine.6–8 Determination for serum free-T4 concentration by equilibrium dialyses has been suggested as an alternative, noninvasive means and is considered a more sensitive and specific test than determination of total serum T4 concentration.8 However, 2 major considerations have to be taken into account. Firstly, the equilibrium dialyses method for determination of serum free-T4 concentrations is an expensive analysis that is not offered by many laboratories. Secondly, some dogs without hypothyroidism, especially those with euthyroid sick syndrome, may have serum free-T4 concentrations that are also decreased.
Therefore, the TSH-stimulation test is still considered the most accurate test, and by some authors,9 it is still recognized as the gold standard for the diagnosis of hypothyroidism in dogs. Determination of circulating T4 concentration before and after the administration of TSH provides an assessment of the functional reserve capacity of the thyroid gland with minimal change in post-TSH T4 concentration, compared with the basal concentration, expected in dogs with hypothyroidism. The test has been recommended for affected dogs in which a diagnosis cannot be accurately obtained by use of other assays, and it is anticipated as particularly useful in dogs with nonthyroidal illness as well as in those receiving medication known to affect thyroid function.10 Because the traditionally used bTSH is no longer commercially available as a pharmaceutical preparation, rhTSH has been proposed as an alternative. Several protocols for the TSH-stimulation test in dogs with rhTSH have been described11–13; however, studies have been limited to healthy dogs. Although an equivalent biological activity of rhTSH, compared with bTSH, has been demonstrated in healthy Beagles,11 the response of the thyroid gland to rhTSH in clinically ill dogs or those receiving medications cannot be predicted from the previously acquired data. To our knowledge, no published clinical studies exist evaluating the diagnostic efficacy of the rhTSH-stimulation test in a clinical setting. The purpose of the study reported here was to evaluate rhTSH in the TSH-stimulation test in dogs with clinical signs consistent with hypothyroidism.
Materials and Methods
Animals—Sixty-four dogs, 38 males (11 castrated) and 26 females (19 spayed), with a median body weight of 32.3 kg (range, 4.2 to 76.5 kg) and a median age of 6 years (range, 1 to 14 years) were included in the study. They had been brought to the Clinic for Small Animal Internal Medicine of the University of Zurich between October 2002 and June 2005 for investigation of clinical signs consistent with hypothyroidism. The most common complaints, either alone or in combination, were as follows: exercise intolerance (n = 40), dermatologic problems (24), and weight gain (8). Medication history (with special focus on antimicrobials, anticonvulsants, and anti-inflammatory preparations) was recorded for each dog, and if possible, a follow-up study was performed. The study was conducted in compliance with institutional guidelines for research on animals, and informed consent from pet owners was obtained before testing.
TSH-stimulation tests—Thyroid-stimulating hormone-stimulation tests were performed with rhTSHa (75 μg [resuspended in 0.5-mL sterile injection water]/dog, IV). With owner consent, dogs (n = 5) that had unchanged serum T4 concentrations (ie, no normal stimulation) in response to rhTSH administration and a body weight > 20 kg received a second dose of rhTSH (150 μg/dog, IV). None of the dogs had adverse reactions to rhTSH administration. Blood samples (jugular venipuncture) were collected immediately before and 6 hours after the administration of rhTSH. After clot retraction at room temperature (approx 21°C), serum was harvested via low-speed centrifugation and transferred to tubes for storage at −20°C for subsequent hormone assay.
Analytic procedures—Serum T4 concentration was measured by use of a commercially available radioimmunoassay validated for use in dogs (reference range, 1.5 to 3.5 μg/dL).b Serum cTSH concentrations were determined in prestimulation samples by use of a homologous solid-part, 2-site chemiluminescent enzyme immunometric assay validated for use in dogs (upper limit of the reference range, 0.6 ng/mL).c For dogs from which serum was available, assays for autoantibodies against T4, thyroglobulin (n = 49 samples), and triiodothyronine (47) were performed at the Endocrine Section of the Animal Health Diagnostic Laboratory, Michigan State University, as published previously.14,15
Allocation of dogs—Results of TSH-stimulation tests were interpreted with criteria established in 38 healthy dogs.11 On the basis of these criteria, dogs were placed into 1 of 3 groups as follows: those with normal (ie, poststimulation values indicative of euthyroidism), unchanged (ie, poststimulation values indicative of hypothyroidism; no thyroid gland stimulation), or intermediate (ie, poststimulation values between unchanged and normal values) post-TSH T4 concentrations. Normal TSH-stimulation test results were defined as post-TSH T4 concentrations of > 2.5 μg/dL and at least 1.5 times the basal T4 concentration. Dogs with post-TSH T4 concentrations of < 1.6 μg/dL and < 1.5 times the basal T4 concentration were considered to have unchanged post-TSH T4 concentrations; dogs with a post-TSH T4 concentration between 1.6 and 2.5 or post-TSH T4 concentrations of > 2.5 μg/dL but an increase of < 1.5 times basal T4 were considered to have intermediate post-TSH T4 concentrations. Whether the test results confirmed the diagnosis of hypo- or euthyroidism was based on follow-up examinations.
Statistical analysis—Data were analyzed by use of non-parametric methods.d,e The Kruskal-Wallis test for unpaired samples, Friedman repeated-measures test for paired samples, and Dunn multiple comparisons test were used. The Mann-Whitney U test was used to determine differences between 2 groups. To test for an association between categoric variables, the Δ2 statistic was used; for continuous variables, linear correlation and the nonparametric Spearman rank test were calculated. Values of P < 0.05 were considered significant.
Results
Dogs with unchanged post-TSH T4concentrations—Fourteen of the 64 (21.9%) dogs had unchanged post-TSH T4 concentrations with median (range) basal T4 and post-TSH T4 concentrations of 0.5 μg/dL (0.1 to 1.2 μg/dL) and 0.6 μg/dL (0.1 to 1.2 μg/dL), respectively. Median (range) cTSH concentrations determined in 13 dogs were 1.1 ng/mL (0.02 to 6.0 ng/mL), with 9 dogs having a high cTSH concentration of > 1 ng/mL.
Thirteen of 14 dogs with unchanged post-TSH T4 concentrations had improvement of their clinical signs of hypothyroidism after starting T4 supplementation (20 μg/kg, q 12 h), confirming the diagnosis of hypothyroidism. However, in 1 dog, hypothyroidism could not be confirmed during the follow-up period. Laboratory test results of this dog revealed a T4 contration of 0.5 μg/dL, a post-TSH T4 concentration of 0.8 μg/dL, and a cTSH concentration of 0.02 ng/mL. Although T4 supplementation was initiated, the dog had progressive worsening of its clinical signs and the owners elected euthanasia; they declined necropsy examination and histologic examination of the thyroid gland. This dog had been treated with carprofen for > 4 weeks, and it had been considered moderately to severely ill when the test had been performed.
Dogs with normal post-TSH T4concentrations—Thirty-five of the 64 (54.7%) dogs had normal post-TSH T4 concentrations. Median (range) basal T4 and post-TSH T4 concentrations were 1.9 μg/dL (0.6 to 3.0 μg/dL) and 3.7 μg/dL (2.5 to 5.7 μg/dL), respectively. Median (range) cTSH concentrations determined in 33 dogs were 0.17 ng/mL (0.04 to 1.1 ng/mL), with 2 dogs having high cTSH concentrations of > 1 ng/mL. One of these 2 dogs had an unchanged basal T4 concentration.
All of the 35 dogs with normal post-TSH T4 concentrations were considered only mildly ill. Four of them had received medication prior to the test as follows: pimobendan for 2 weeks (n = 1 dog), enalapril for 3 months (1), amoxicillin for 1 week (1), and 1 dose of prednisolone 2 days before the test (1). During the follow-up period, a diagnosis other than hypothyroidism was made for 18 of the 35 dogs; 4 dogs had improvement in the clinical signs of hypothyroidism without treatment, and 13 dogs were lost to follow-up. Therefore, in at least 22 of the 35 dogs with follow-up information, hypothyroidism could be clearly excluded.
Dogs with intermediate post-TSH T4concentrations—Fifteen of the 64 (23.4%) dogs had intermediate post-TSH T4 concentrations with median (range) basal T4 and post-TSH T4 concentrations of 1.4 μg/dL (0.7 to 3.2 μg/dL) and 2.2 μg/dL (1.7 to 3.2 μg/dL), respectively. Median (range) cTSH concentrations determined in 15 dogs were 0.22 ng/mL (0.02 to 0.55 ng/mL); none of the 15 dogs had a cTSH concentration above the upper limit of the reference range.
Nine of the 15 dogs had received medication (carprofen [n = 1 dog], prednisolone [6], and cyclosporine [2]) for > 4 weeks prior to the test. Two of the dogs treated with prednisolone had additionally received amoxicillin and cephalosporin, respectively. Two dogs had improvement of their clinical signs without further treatment, and no final diagnosis was made; in 3 of the 9 dogs, further workup led to another diagnosis and improvement of the clinical signs with appropriate treatment. Two of the 9 dogs not only had received medication during endocrine testing but also had moderate nonthyroidal disease. Because of deteriorating clinical signs despite treatment, the owners of these 2 dogs elected euthanasia without further diagnostic workup. Necropsy and histologic examinations of the thyroid glands were not permitted. Two dogs were lost to follow-up.
Six of the dogs with intermediate post-TSH T4 concentrations had not received any medication prior to the test. In 3 of the dogs, clinical signs of hypothyroidism resolved without further treatment and no final diagnosis was made. In 2 dogs, another diagnosis was made and clinical signs improved with appropriate treatment. One dog was treated with T4, resulting in no improvement of clinical signs. Follow-up of this dog revealed CNS neoplasia 8 months later.
Taken together, in 10 of the 15 dogs with intermediate post-TSH T4 concentrations, hypothyroidism could be clearly excluded with follow-up information (another diagnosis or resolved clinical signs without T4 supplementation), whereas in 5 of the dogs, neither hypothyroidism could be clearly excluded nor could euthyroidism be confirmed.
Comparison of the 3 groups—Dogs with intermediate post-TSH T4 concentrations had received medication known to influence thyroid function significantly more often and had been considered moderately ill significantly more often than dogs with normal post-TSH T4 concentrations. Although basal T4 and post-TSH T4 concentrations were significantly different among the 3 groups, sex, age, and body weight were not (data not shown; Figure 1). A weak (r = 0.4) but significant (P < 0.001) inverse correlation was found between post-TSH T4 concentrations and age of the 64 dogs. Significant differences in cTSH concentration were only detected between dogs with normal post-TSH T4 concentrations and unchanged post-TSH T4 concentrations and between dogs with intermediate post-TSH T4 concentrations and unchanged post-TSH T4 concentrations (Figure 2). Interestingly, only 2 of the 49 tested dogs (1 with an intermediate post-TSH T4 concentration and 1 with an unchanged post-TSH T4 concentration) had autoantibodies against thyroglobulin; none of the tested dogs had detectable serum autoantibodies against T4, and only 1 of the 47 dogs tested for autoantibodies against T3 had a positive result (the dog with an unchanged post-TSH T4 concentration and a positive result for autoantibodies against thyroglobulin).
Scatterplot of serum T4 concentrations before (time 0) and 6 hours after administration of rhTSH IV to 64 dogs as follows: dogs with unchanged (ie, no stimulation) post-TSH T4 concentrations (n = 14; squares), dogs with normal post-TSH T4 concentrations (35; circles), and dogs with intermediate post-TSH T4 concentrations (15; triangles). *Significant (P < 0.05) difference between sample collection times. Median values are indicated by horizontal lines.
Citation: American Journal of Veterinary Research 67, 12; 10.2460/ajvr.67.12.2012
Scatterplot of serum cTSH concentrations in dogs with unchanged (ie, no stimulation) post-TSH T4 concentrations (n = 13; squares), normal post-TSH T4 concentrations (33; circles), and intermediate post-TSH T4 concentrations (15; triangles). Upper limit of the reference range (gray area) for cTSH is 0.6 ng/mL.*Significant (P< 0.05) difference between groups.
Citation: American Journal of Veterinary Research 67, 12; 10.2460/ajvr.67.12.2012
Effect of doubling the rhTSH dose—To exclude a dose-related phenomenon as a cause for a decrease in thyroid gland stimulation, 5 of the 64 dogs (2 with unchanged post-TSH T4 concentrations and 3 with intermediate post-TSH T4 concentrations) with a median body weight of 33.5 kg (range, 23.5 to 76.5 kg) received a higher dose of rhTSH (150 μg/dog, IV) that was twice the original dose. However, in none of the dogs did this higher dose lead to a significant improvement in thyroid gland stimulation (Figure 3).
Scatterplot representing the influence of dose of rhTSH on serum T4 concentrations in 5 dogs given 75 and 150 μg of rhTSH, IV. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 67, 12; 10.2460/ajvr.67.12.2012
Discussion
Because the TSH-stimulation test remains the gold standard for the diagnosis of hypothyroidism in dogs and bTSH is no longer available as pharmaceutical preparation, we wanted to determine whether rhTSH with the proposed protocol could be used for the test in a broad population of dogs suspected of having hypothyroidism.
None of our dogs had adverse or anaphylactic reactions to the rhTSH, even after repeated administration. Also, in previous studies11,13 on healthy dogs, no adverse reactions had been observed despite repeated administration of rhTSH. Although the number of studied dogs is too small to draw any general conclusions, we assume that rhTSH might be safer than the traditionally used bTSH. The recombinant product is not only purer, compared with the bTSH preparation, but recombinant proteins are also smaller than purified proteins and therefore tend to be less antigenic.16
In our study and in accordance with previous reports,7,10,17 determination of basal T4 and cTSH concentrations was useful to discriminate most of the hypothyroid from euthyroid dogs. However, at least 1 dog with hypothyroidism would have been missed on the basis of basal T4 and cTSH concentrations as sole criteria. In addition, 1 euthyroid dog would have been misjudged as having hypothyroidism had the TSH-stimulation test not been included in the diagnostic measures.
Fifteen of our dogs had intermediate post-TSH T4 concentrations; during follow-up studies in most of these dogs, hypothyroidism could be excluded. There are several reasons for intermediate post-TSH T4 concentrations that have to be considered. Many of these dogs had received medication prior to the test, although several drugs are known to alter thyroid function,1,2,9,18 the most important of these are glucocorticoids, which have been shown not only to inhibit the hypothalamic-pituitary axis but also to influence peripheral metabolism of thyroid hormones. Six of our dogs with intermediate post-TSH T4 concentrations had received prednisolone for > 4 weeks before the TSH-stimulation test was performed. Moreover, 2 of these dogs were also considered ill. Of course, usefulness of performing diagnostics tests on dogs receiving medication or with concurrent disease can be considered questionable; however, situations exist in which diagnosis should be made without delay. Because the biologically active free-T4 is less influenced by nonthyroidal disorders, determination of this parameter by use of equilibrium dialyses can be recommended as a noninvasive first-line test. However, in some of the euthyroid-sick dogs, free-T4 concentrations can be decreased even in the range of true hypothyroid dogs. Although more invasive, the TSH-stimulation test is supposed to be useful in those instances in which one has to differentiate true hypothyroidism from euthyroid-sick syndrome; dogs with primary hypothyroidism are nonresponsive and those with decreased baseline thyroid hormone concentration caused by other factors such as illness or drug administration usually respond to TSH, although often in a suppressed manner. Therefore, in addition to the determination of the free-T4, the rhTSH-stimulation test might be recommended in dogs with concurrent illness or in those receiving medication in which diagnosis has to be established immediately. Nevertheless, one should consider that severe systemic illness can lead to not only low free-T4 concentrations, but also to unchanged post-TSH T4 concentrations in the range considered diagnostic for primary hypothyroidism, which was observed in one of our dogs with an unchanged post-TSH T4 concentration. It has not been addressed in our study which of the diagnostic approaches (determination of free-T4 concentration or performing a TSH-stimulation test) finally offers the higher diagnostic sensitivity and specificity. However, exploration of this issue might be relevant given concerns about the expense of injectable rhTSH and the invasiveness of the test.
The influence of body weight and dose on the magnitude of thyroid gland stimulation has been discussed for bTSH19–21 and rhTSH.11–13 In a previous study,11 we found that 75 μg of rhTSH led to adequate thyroid gland stimulation in dogs, independent of body weight. In our study, no significant difference was found in body weight between the groups and no correlation was found between body weight and post-TSH T4 concentration. Moreover, selected dogs with unchanged or intermediate post-TSH T4 concentrations and body weights > 20 kg received a higher dose of rhTSH, which was twice the original dose, but this did not lead to a significant improvement in thyroid gland stimulation. Therefore, we assume that the dose is an unlikely cause for intermediate post-TSH T4 concentrations. Because a major limiting factor for the use of rhTSH resides in the cost of the product, it would of course be preferable to use the lower dose. However, because the number of dogs in our study was small, the question on adequate dose probably deserves further empirical studies before recommendations for general use can be made.
Influence of age, not only on basal T4 concentration but also on post-TSH T4 concentration, on lower values in older dogs has been demonstrated.17 As no significant difference in age was found among our groups, this seems an unlikely explanation for the high number of dogs with intermediate post-TSH T4 concentrations.
Hypothyroidism is generally not an all-or-nothing condition but is characterized by a gradual loss of thyroid gland function and thyroid gland responsiveness to TSH. Prior to a total loss of T4 secretion, results of a TSH-stimulation test may resemble findings for a dog with euthyroid-sick syndrome. The term subclinical hypothyroidism has been introduced in human medicine to describe a condition with mild to no clinical signs of thyroid dysfunction and serum T4 concentrations within reference range, but with consistently high serum concentrations of endogenous TSH.22 A markedly reduced functional reserve of the thyroid gland is suspected in these people. At least some of our dogs with intermediate post-TSH T4 concentrations might have been in a subclinical hypothyroid state, in which the thyroid gland was stimulated with a high exogenous dose of TSH, although the response was decreased, compared with that of a euthyroid dog.
At this time, it only can be speculated that an intermediate response to TSH was most likely the result of concurrent disease and medication. To prove this assumption, follow-up studies of those dogs would be necessary with repeated testing after cessation of the medication and recovery from other diseases.
In conclusion, rhTSH can be recommended as a valuable substitute for the bTSH preparation. The test can be used in selected dogs in which a diagnosis of hypothyroidism cannot be based solely on serum T4 and cTSH concentrations. Concurrent disease or administration of medication might lead to a suppressed simulation with rhTSH.
ABBREVIATIONS
| T4 | Thyroxine |
| TSH | Thyroid-stimulating hormone |
| cTSH | Canine TSH |
| bTSH | Bovine TSH |
| rhTSH | Recombinant human TSH |
Thyrogen, Genzyme Corp, Haverhill, Suffolk, UK.
Canine DPC radioimmunoassay, Coat-A-Count, Diagnostic Products Corp, Los Angeles, Calif.
Immulite canine TSH, Diagnostic Products Corp, Los Angeles, Calif.
SPSS, Statistical Package for the Social Sciences, Software Packets for Windows, version 11, SPSS Inc, Chicago, Ill.
GraphPad PRISM for Windows, version 3.0, GraphPad Software Inc, San Diego, Calif.
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