David Bruyette United States
Over the past few years, we have learned a great deal about the pathogenesis and diagnosis of canine hypo-thyroidism. This has been due in large part to improvements in the diagnostic tests available to the clinician. Autoimmune thyroiditis is the most common cause of hypothyroidism. A recent study looked at the value of autoantibody testing in dogs. Serum samples from dogs with various endocrine abnormalities and from 30 obese adult females had Tyroglobulin Autoantibodies (TgAA) concentrations determined by use of the ELISA. Six experiments were done: 1) definition of positive results for TgAA using samples from normal and T3 autoantibody (T3AA) positive dogs; 2) establishment of prevalence of positive results in 91 clinically normal dogs; 3) evaluation of positive results for sera from dogs with nonthyroidal illnesses; 4) testing of samples from dogs with primary hypo-thyroidism but absence of T4AA or T3AA, or both; 5) determination of prevalence of false-negative results in dogs that are T4AA and/or T3AA positive, which were (18 dogs) or were not (22 dogs) receiving L-thyroxine replacement therapy; and 6) examination of thyroid biopsy specimens from 18 dogs (8 TgAA positive and 10 TgAA negative).
Positive results were defined as at least twice (200%) the optical density of the negative-control sample. False-positive results were obtained for only 3.4% of 146 dogs with nonthyroidal illness. Thirty-seven percent of dogs with primary hypothyroidism, but no evidence of T4AA or T3AA, or both, were TgAA positive. False-negative results were found in one of 22 and two of 18 T3AA-positive dogs with and without thyroid replacement therapy, respectively. Thyroid biopsy specimens from eight TgAA-positive dogs had evidence of lymphocytic thyroiditis, whereas those from 10 TgAA-negative dogs did not. The assay was sensitive and specific for identification of lymphocytic autoimmune thyroiditis in dogs, and has potential for aiding early diagnosis of thyroiditis in dogs and identifying dogs likely to perpetuate hypothyroidism in breeding programs.
A number of medications and non-thyroidal illness can interfere with the various thyroid function tests. Several papers examined the effects of anti-convulsant therapy on the pituitary-thyroid axis. A multicentric prospective study was conducted to monitor the effect of phenobarbital on serum total thyroxine (T4) and thyroid-stimulating hormone (TSH) concentrations in epileptic dogs. Serum T4 concentrations were determined for 22 epileptic dogs prior to initiation of phenobarbital therapy (time 0) and at three weeks, six months, and 12 months after the start of phenobarbital. Median T4 concentration was significantly lower at three weeks and six months compared to time 0.
Thirty-two percent of dogs had T4 concentrations below the reference range at six and 12 months. Nineteen of the 22 dogs had serum TSH concentrations determined at all sampling times. A significant upward trend in median TSH concentration was found. No associations were found between T4 concentration, dose of phenobarbital, and serum phenobarbital concentration. No signs of overt hypothyroidism were evident in dogs with low T4, with one exception. TSH stimulation tests were performed on six of seven dogs with low T4 concentrations at 12 months, and all but one had normal responses.
In conclusion, phenobarbital therapy decreased serum T4 concentration but did not appear to cause clinical signs of hypothyroidism. Serum TSH concentrations and TSH stimulation tests suggest that the hypothalamic-pituitary-thyroid axis is functioning appropriately. A second study evaluated the changes in serum total T4 (TT4), free T4 (FT4), thyroid-stimulating hormone (TSH), cholesterol and albumin concentrations, and activities in serum of alanine aminotransferase (ALT), alkaline phosphatase (ALP), and gamma-glutamyl transferase (GGT) after discontinuation of long-term phenobarbital administration in normal dogs. Twelve normal dogs were administered phenobarbital at a dosage of approximately 4.4-6.6 mg/kg q12h PO for 27 weeks. Blood was collected for analysis before and after 27 weeks of phenobarbital administration and then weekly for 10 weeks after discontinuation of the drug. The dogs were clinically normal throughout the study period. Serum ALT and ALP activity and TSH and cholesterol concentrations were significantly higher than baseline at week 27. Serum T4 and FT4 were significantly lower. Serum albumin and GGT were not changed from baseline at week 27. Changes in estimate of thyroid function (TT4, FT4, TSH) persisted for one to four weeks after discontinuation of phenobarbital, whereas changes in hepatic enzyme activity (ALT, ALP) and cholesterol concentration resolved in three to five weeks.
To avoid false positive results, it is recommended that thyroid testing be performed at least four weeks after discontinuation of phenobarbital administration. Elevated serum activity of hepatic enzymes six to eight weeks after discontinuation of phenobarbital may indicate hepatic disease. The effects of phenobarbital were again assessed on thyroid as well as on adrenal function. The effects of phenobarbital on the thyroid axis, the adrenal axis, and adrenal function tests were prospectively investigated in 12 normal, adult dogs. Phenobarbital was administered at 5 mg per kilogram of body weight (range, 4.8-6.6 mg/kg q12h PO for 29 weeks, resulting in therapeutic serum concentrations (20-40 microg/mL). Serum total thyroxine (TT4), free thyroxine (FT4) by equilibrium dialysis, total triiodothyronine (TT3), thyrotropin (TSH), and cholesterol were determined before and during phenobarbital treatment. LDDST, ACTH stimulation tests, and ultrasonographic evaluation of the adrenal glands were performed before and during treatment. TT4 and FT4 decreased significantly (P < or = .05), TT3 had minimal fluctuation, TSH had only a delayed compensatory increase, and cholesterol increased during phenobarbital treatment.
The delayed increase in TSH, despite persistent hypothyroxinemia, suggests that accelerated hepatic thyroxine elimination may not be the only effect of phenobarbital on the thyroid axis. There was no significant effect of phenobarbital on either of the adrenal function tests. A larger study examined 78 epileptic dogs receiving phenobarbital (group 1) and 48 untreated epileptic dogs (group 2). Serum biochemical analyses, including T4 and TSH concentrations, were performed for all dogs. Additional in vitro analyses were performed on serum from healthy dogs to determine whether phenobarbital in serum interferes with T4 assays or alters free T4 (fT4) concentrations. Mean serum T4 concentration was significantly lower, and mean serum TSH concentration significantly higher, in dogs in group 1, compared with those in group 2. Thirty-one (40%) dogs in group 1 had serum T4 concentrations less than the reference range, compared with 4 (8%) dogs in group 2. All dogs in group 2 with low serum T4 concentrations had recently had seizure activity. Five (7%) dogs in group 1, but none of the dogs in group 2, had serum TSH concentrations greater than the reference range. Associations were not detected between serum T4 concentration and TSH concentration, age, phenobarbital dosage, duration of treatment, serum phenobarbital concentration, or degree of seizure control. Signs of overt hypothyroidism were not evident in dogs with low T4 concentrations. Addition of phenobarbital in vitro to serum did not affect determination of T4 concentration and only minimally affected fT4 concentration. The authors concluded that clinicians should be aware of the potential for phenobarbita treatment to decrease serum T4 and increase TSH concentrations and should use caution when interpreting results of thyroid tests in dogs receiving phenobarbital.
Lastly, another study determined whether administration of phenobarbital, potassium bromide, or both drugs concurrently was associated with abnormalities in baseline serum total thyroxine (T4), triiodothyronine (T3), free T4, or thyrotropin (thyroid-stimulating hormone; TSH) concentrations in epileptic dogs.
Seventy-eight dogs with seizure disorders that did not have any evidence of a thyroid disorder (55 treated with phenobarbital alone, 15 treated with phenobarbital and bromide, and eight treated with bromide alone) and 150 clinically normal dogs that were not receiving any medication were evaluated. Serum total T4, total T3, free T4, and TSH concentrations, as well as serum concentrations of anticonvulsant drugs, were measured in the 78 dogs with seizure disorders. Reference ranges for hormone concentrations were established based on results from the 150 clinically normal dogs. Total and free T4 concentrations were significantly lower in dogs receiving phenobarbital (alone or with bromide), compared with concentrations in clinically normal dogs. Administration of bromide alone was not associated with low total or free T4 concentration. Total T3 and TSH concentrations did not differ among groups of dogs. These results indicate that serum total and free T4 concentrations may be low (i.e., in the range typical for dogs with hypothyroidism) in dogs treated with phenobarbital. Serum total T3 and TSH concentrations were not changed significantly in association with phenobarbital administration. Bromide treatment was not associated with any significant change in these serum thyroid hormone concentrations.
The advent of validated assays for cTSH and fT4ED has greatly improved our ability to distinguish between hypothyroidism and sick euthyroidism. In the first paper on cTSH testing, hypothyroidism was induced in dogs by IV administration of sodium iodide I131 solution. Subsequently, L-thyroxine was administered orally to normalize serum thyroxine concentrations. The cTSH assay appeared to be specific and was sufficiently sensitive to detect cTSH in the serum of these dogs prior to induction of hypothyroidism. There was a 35-fold increase in mean serum cTSH concentration following induction of hypothyroidism, and 35 days after initiation of thyroid replacement therapy, mean serum cTSH concentration was not significantly greater than mean baseline value. The authors conclude that assay of serum cTSH is likely to prove helpful in the differential diagnosis of primary, secondary, and tertiary hypothyroidism in dogs, and in monitoring response to thyroid hormone replacement treatment.
The same group of workers then looked at 62 healthy dogs and 49 dogs with clinical signs consistent with hypothyroidism (16 were hypothyroid and 33 were euthyroid with concurrent disease). Samples from healthy dogs were used to establish a reference range for serum cTSH concentration. The 49 dogs were categorized as hypothyroid or euthyroid with concurrent disease on the basis of clinical signs, results of additional diagnostic and thyroid-stimulating hormone (TSH) response tests, and response to administration of levothyroxine sodium. Function of the thyroid gland was considered normal when serum total thyroxine (T4) concentration six hours after TSH administration was > 2.5 micrograms/dl. Hypothyroidism was diagnosed when serum T4 concentration after TSH administration was < or = 1.5 microgram/dl. RESULTS: Serum cTSH concentration differed significantly among all three groups. Four of 33 (12%) euthyroid dogs had cTSH concentrations that were greater than the reference range, whereas six of 16 (38%) hypothyroid dogs had cTSH concentrations within the reference range. Specificity for serum cTSH concentration was 0.88 and sensitivity was 0.63. When interpreted in combination with serum T4 concentration, specificity increased to 1.0. The authors suggested that the cTSH assay had good specificity for use in the diagnosis of hypothyroidism in dogs. Because this assay had low sensitivity, a diagnosis of hypothyroidism could not be excluded based on a serum cTSH concentration that was within the reference range.
Another study also looked at cTSH and fT4ED. Fifty-four dogs with hypothyroidism, 54 euthyroid dogs with nonthyroidal disease initially suspected to have hypothyroidism, and 150 clinically normal dogs were studied. In the 54 dogs with hypothyroidism, diagnosis was established based on clinical signs, results of routine laboratory and TSH stimulation tests, exclusion of concurrent nonthyroidal disease, and a good clinical response to treatment with L-thyroxine. Blood samples were collected from all dogs and were tested for thyroid hormone and TSH concentrations. Reference ranges for hormone concentrations were established based on results for the 150 clinically normal dogs. Of the 54 hypothyroid dogs, 48 (89%) had low total T4 concentrations, three had low-normal concentrations, and three had high concentrations because of T4 autoantibodies. In contrast, only 10 (18%) euthyroid dogs had low total T4 concentrations. Only three of 31 (10%) hypothyroid dogs had low T3 concentrations; 23 had concentrations within the reference range, and five had high concentrations because of T3 autoantibodies. Only three of 38 euthyroid dogs had low T3 concentrations. Of the hypothyroid dogs, 53 (98%) had low free T4 concentrations and one had a low-normal concentration. Only four (7%) euthyroid dogs had low free T4 concentrations. Of the hypothyroid dogs, 41 (76%) had high TSH concentrations, and 13 had TSH concentrations within the reference range. Of the euthyroid dogs, only four (8%) had high TSH concentrations. Of all single hormone measurements evaluated, measurement of free T4 concentration had the highest sensitivity (0.98), specificity (0.93), and accuracy (0.95) as a test for hypothyroidism; measurement of total T4 concentration had a lower sensitivity (0.89), specificity (0.82), and accuracy (0.85). Compared with measurement of total or free T4 concentration, measurement of TSH concentration had a lower sensitivity (0.76) and accuracy (0.84) but specificity (0.93) equal to that for measurement of free T4 concentration. When T4 (total or free) and TSH concentrations were evaluated together, specificity was higher than when T4 or TSH concentration was evaluated alone. Only one euthyroid dog had low T4 (total and free) and high TSH concentrations. These results indicate that measurement of serum free T4 and TSH concentrations is useful for diagnosis of hypothyroidism in dogs. Interestingly, a quarter of the dogs with confirmed hypothyroidism had serum TSH concentrations within reference limits. This was investigated in a separate study.
To determine whether this is due to fluctuations in the release of TSH, the plasma profiles of TSH were analyzed in seven Beagle bitches by collecting blood samples every 10 min for six hours, both before and after induction of primary hypothyroidism. After induction of primary hypothyroidism, a 37-fold increase in mean basal plasma TSH concentration and a 34-fold increase in mean area under the curve for TSH were found. Analysis by the Pulsar program demonstrated pulsatile secretion of TSH in the hypothyroid state, characterized by relatively low amplitude pulses (mean [+/-SEM]) amplitude 41 +/- 3% of basal plasma TSH level) and a mean pulse frequency of 2.0 +/- 0.5 pulses/6 hr. In the euthyroid state, significant TSH pulses were identified in only two dogs. The mean basal plasma TSH level correlated positively (r = 0.84) with the mean amplitude of the TSH pulses, and correlated negatively (r = -0.88) with the TSH pulse frequency. The results of this study demonstrate pulsatile secretion of TSH in dogs during hypothyroidism and only small fluctuations in plasma TSH concentrations during euthyroidism. The findings also suggest that the low TSH values occasionally found in dogs with spontaneous primary hypothyroidism, may in some cases, be in part the result of ultradian fluctuations.
Provocative testing is still considered by some to be the gold standard especially when basal thyroid function tests (TT4, fT4ED, cTSH) are equivocal or discordant. A recent paper assessed the effect of human recombinant TSH on thyroid function in normal dogs. Six healthy beagle dogs were used in each of the three phases of this study. Phase I: thyroid-stimulating hormone response tests were performed by using a total dose of 25 micrograms, 50 micrograms, and 100 micrograms of rhTSH, administered intravenously. Phases II and III: thyroid-stimulating hormone response tests were performed by using 50 micrograms of rhTSH administered by intramuscular and subcutaneous routes, respectively. In each phase and following all the administered doses of rhTSH, an increase in the serum TT4 concentration was noted, although it was not always significant. For phase I, there was a significant increase in serum TT4 concentrations. Based on this study, 50 micrograms was judged the optimal intravenous dose of rhTSH. For phases II and III, there was no significant increase in serum TT4 after the administration of rhTSH. Results of this study suggest that rhTSH could be a good substitute for bovine TSH, when used by the intravenous route, for the TSH stimulation test in dogs. Further studies are required to confirm its clinical usefulness.
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