In rats and human beings, anticonvulsant drugs have considerable

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1 J Vet Intern Med 2000;14: Effects of Long-Term Phenobarbital Treatment on the Thyroid and Adrenal Axis and Adrenal Function Tests in Dogs Peter B. Müller, Karen J. Wolfsheimer, Joseph Taboada, Giselle Hosgood, Beth P. Partington, and Frédéric P. Gaschen Phenobarbital can interfere with the thyroid axis in human beings and rats by accelerating hepatic thyroxine metabolism because of enzyme induction. In human beings, it also can interfere with the low-dose dexamethasone suppression test (LDDST) used to assess adrenal function by accelerating dexamethasone metabolism. This effect can cause a lack of suppression of pituitary ACTH and subsequent adrenal cortisol release after dexamethasone administration. 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, mg/kg) PO q12h for 29 weeks, resulting in therapeutic serum concentrations (20 40 g/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.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. With the methods employed, we did not find any effects of the drug on the hormonal equilibrium of the adrenal axis. Key words: Adrenal axis, Adrenal function tests, Phenobarbital, Thyroid axis. In rats and human beings, anticonvulsant drugs have considerable effects on the thyroid gland and thyroid hormone metabolism. Phenobarbital increases hepatic metabolism and biliary excretion of thyroxine by inducing hepatic enzymes, such as thyroxine-glucuronosyltransferase and cytochrome P450 enzymes. 1 5 In rats, a primary increase of hepatic thyroxine uptake stimulates hepatic deiodinative and excretory processes. Increased fecal and deiodinative clearance of thyroxine and triiodothyronine activates the thyroid axis, resulting in increased pituitary thyrotropin (TSH) secretion and subsequent establishment of a new steady state with normal thyroxine concentrations and somewhat increased TSH concentrations. 2,4,6 Thyroid gland weight remains increased, indicating chronic stimulation. 2 Several textbooks and review articles in the veterinary literature mention that phenobarbital decreases basal serum thyroxine concentrations in dogs However, more recent data suggest that phenobarbital treatment at mg q12h for 3 weeks has no significant effect on total thyroxine From the Department of Veterinary Physiology, Pharmacology and Toxicology (Müller, Wolfsheimer), and Department of Veterinary Clinical Sciences (Taboada, Hosgood, Partington), School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA; and the Clinic for Companion Animals, University of Berne, Berne, Switzerland (Gaschen). Previously presented at the ACVIM forum 1998 in San Diego, CA, and at the Phi Zeta Research Emphasis Day 1998 at the School of Veterinary Medicine, Louisiana State University. It represents a portion of a thesis submitted to the Veterinary Medical Faculty of Bern (Switzerland) to fulfill the requirements for a Swiss Dr med vet degree. Reprint requests: Joseph Taboada, DVM, Dipl ACVIM, Department of Veterinary Clinical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803; jtaboada@ mail.vetmed.lsu.edu. Submitted June 26, 1998; Revised March 12, 1999, and July 1, 1999; Accepted October 19, Copyright 2000 by the American College of Veterinary Internal Medicine /00/ /$3.00/0 (TT4), free thyroxine (FT4), and TSH in Beagles. Serum phenobarbital concentrations in this study ranged from g/ml 11 and were below the therapeutic range of g/ml. To our knowledge, no data investigating a possible interference of phenobarbital with the thyroid axis in dogs after an extended period of treatment have been published (results of 2 studies concerning this topic have recently been published). 12,13 Two tests employed in our study (TSH concentration and FT4 concentration by equilibrium dialysis) have only recently become available for use in dogs. In human beings, long-term treatment with anticonvulsants can interfere with the low-dose dexamethasone suppression test (LDDST) used for the assessment of adrenal function. Because of induction of hepatic enzymes involved in steroid metabolism, dexamethasone is metabolized more rapidly and subsequently fails to adequately suppress pituitary ACTH release, which is followed by inadequate serum cortisol suppression. 14,15 A lack of cortisol suppression after dexamethasone injection also occurs in dogs with spontaneous hyperadrenocorticism. Phenobarbital also may interfere with the adrenal axis itself by accelerating the metabolism of endogenous steroids. 16,17 Few studies have evaluated possible similar interferences in dogs, 18,19 indicating that the ACTH stimulation test (ACTHST) is not affected by concurrent phenobarbital administration. It is unclear whether or not this is also the case for the LDDST and whether hormonal equilibrium of the adrenal axis is affected by phenobarbital in normal dogs The purpose of the study reported here was to further elucidate the effects of phenobarbital treatment at therapeutic dosages on the thyroid axis, adrenal axis, and adrenal function tests in normal dogs. Materials and Methods Dogs and Diagnostic Tools Twelve adult, neutered, male dogs of various breeds, ranging in weight from 11 to 23 kg and 1 to 6 years of age, were housed in

2 158 Müller et al outdoor concrete runs. They were cared for according to the Guide for the Care and Use of Laboratory Animals. 22 During the study, effects of phenobarbital treatment on the liver were investigated simultaneously with the same group of dogs, necessitating repeated general anesthesia for collection of liver biopsy specimens. Results of this second part of the study are published in a companion paper. Dogs received water and a commercial dry dog food a ad libitum and were introduced into their environment for acclimation 1 month before study onset. All dogs were examined before phenobarbital treatment began to detect any clinical or biochemical abnormalities and to obtain multiple baseline results for the tests evaluated during the study because every dog was used as its own control. Pretreatment evaluation included physical examination, CBC, and urinalysis (4 weeks before treatment), and 2 serum biochemical panels (4 weeks before and immediately before treatment). A Knott concentration test for microfilaria and an enzyme-linked immunosorbent assay for antigen of adult heartworms b were performed to rule out dirofilariasis. Pretreatment evaluation of thyroid gland function included 3 determinations of TT4, FT4 by equilibrium dialysis, TT3, and TSH at 3, 2, and 1 week before treatment. To evaluate adrenal function before treatment, we performed 3 ACTHST and 3 LDDST with at least 2 days in between every test. Therefore, pretreatment adrenal function testing was completed within 14 days. The adrenal glands were evaluated ultrasonographically with a 7.5-MHz transducer. c Based on these evaluations, all dogs were considered to be normal. After collection of baseline data, phenobarbital treatment d at a dosage of approximately 5 mg/kg (range, mg/kg) PO q12h was initiated. The drug was administered in meatballs of canned dog food. e Serum phenobarbital concentrations were found to be below the therapeutic range in 1 dog and at the low end of the therapeutic range in several other dogs after 5 weeks. The dosage of these animals was increased at the beginning of week 6 (new dosage range, mg/ kg). Phenobarbital treatment was maintained for 29 weeks during the months of May through December. Evaluation during phenobarbital treatment included daily observations for activity level and behavior, weekly weight measurements, and monthly physical examinations. Phenobarbital serum trough concentrations (12 hours after administration) were determined after 5, 9, 17, and 27 weeks. Thyroid function tests (TT4, FT4, TT3, and TSH) were determined after 5, 9, 13, 17, 21, and 27 weeks. Serum cholesterol concentrations were determined after 17 and 27 weeks. LDDST and ACTHST were performed after 12 and 29 weeks, with 2 days between the 2 tests. Adrenal gland thickness was determined ultrasonographically with a 7.5-MHz transducer after 10 and 27 weeks. The order of the dogs for every testing before and during phenobarbital application was randomized. Methods of Sample Collection and Processing Blood Samples. Blood for serum biochemical analysis, thyroid tests, phenobarbital serum concentrations, and basal cortisol concentration before ACTHST and LDDST was collected between 8:00 AM and 10:00 AM in order to avoid possible diurnal variations. The dogs were fasted for at least 15 hours before all blood sampling. Serum for biochemical analysis was processed immediately, and serum for all endocrine assays was separated immediately from the blood clot and frozen at 62 C within 1 hour for later analysis. ACTHST. ACTH gel (2.2 IU/kg, maximum 40 IU per dog f ) was injected in the thigh musculature of the left hind leg. Blood was sampled for determination of serum cortisol concentrations before and 2 hours after ACTH injection. LDDST. Dexamethasone g (0.015 mg/kg) was injected in a cephalic vein. Blood was sampled for determination of serum cortisol concentrations before and 3 and 8 hours after dexamethasone injection. Laboratory Evaluation. Serum biochemical analysis, CBC, urinalyses, Knott, and occult heartworm tests were performed with standard laboratory equipment and assay techniques validated for the respective laboratory. Phenobarbital serum concentrations were determined with a fluorescence polarization immunoassay. h TT4 concentrations were determined with a commercially available radioimmunoassay, previously validated for use in dogs in our laboratory. i Biological specificity was demonstrated by determining that injected TSH resulted in an increase in serum TT4 concentrations ( 48 nmol/l) after 6 hours in 12 healthy dogs. Low-end sensitivity for the assay, defined as the apparent concentration 2 SD below the counts at maximum binding, was 3.2 nmol/l. Intra-assay coefficient of variation (CV) for 30 replicates with a mean concentration of 32.5 nmol/l was 4.9%. Interassay CV for a single sample performed in duplicate in 15 assays, with a mean concentration of 30.8 nmol/l, was 7.1%. A normal reference range of 52 healthy dogs (32 pets belonging to students, faculty, or staff and 20 research dogs of various breeds) of both genders, ranging in age from 1 12 years, was nmol/l (mean 2 SD; range, nmol/l). FT4 was determined with a commercially available radioimmunoassay, previously validated for use in dogs in our laboratory. j Biological specificity was demonstrated by determining that injected TSH resulted in an increase in serum FT4 concentrations ( 34.5 pmol/l) after 6 hours in 12 healthy dogs. Low-end sensitivity for the assay, as previously defined, was 1.9 pmol/l. Intra-assay CV for 20 replicates with a mean concentration of 13.6 pmol/l was 12.4%. Interassay CV for a single sample performed in duplicate in 9 assays, with a mean concentration of 31.8 pmol/l, was 12.8%. A normal reference range of 72 healthy dogs (52 pets belonging to students, faculty, or staff and 20 research dogs of various breeds) of both genders, ranging in age from 1 12 years, was pmol/l (mean 2 SD; range, pmol/l). TSH concentrations were determined with a commercially available immunoradiometric assay. k Biological specificity was demonstrated by observing that there was a 35-fold increase in mean TSH concentration after the administration of I 131 to ablate the thyroid glands in 6 healthy dogs. TSH concentrations returned to normal in these dogs after oral administration of thyroxine. 23 High TSH concentrations ( 0.8 ng/ml) were demonstrated in our laboratory in 10 dogs with clinical signs of hypothyroidism, very low concentrations of TT4 ( 3.2 nmol/l) and FT4 ( 1.9 pmol/l), and clinical response to thyroxine supplementation. Low-end sensitivity for the assay, as previously defined, was 0.03 ng/ml. Intra-assay CV for 24 replicates, with a mean concentration of 0.31 ng/ml, was 6.0%. Interassay CV for a single sample performed in duplicate in 14 assays, with a mean concentration of 0.30 ng/ml, was 7.9%. A normal reference range of 44 healthy dogs (pets of various breeds, belonging to students, faculty, or staff) of both genders and ranging in age from 1 12 years, was ng/ml (mean 2 SD; range, ng/ml). TT3 concentrations were determined with a commercially available radioimmunoassay, previously validated for use in dogs in our laboratory. l Low-end sensitivity, as previously defined, was 0.1 nmol/l. Intra-assay CV for 20 replicates, with a mean concentration of 2.07 nmol/l, was 9.0%. Interassay CV for a single sample performed in duplicate in 12 assays, with a mean concentration of 2.00 nmol/l, was 8.2%. A normal reference range of 52 healthy dogs (32 pets belonging to students, faculty, or staff and 20 research dogs of various breeds) of both genders ranging in age from 1 12 years, was nmol/l (mean 2 SD; range, nmol/l). Cortisol serum concentrations were determined with a commercially available radioimmunoassay, previously validated for use in dogs in our laboratory. m Biological specificity was demonstrated by observing that cortisol concentrations increased ( nmol/l) 2 hours after injection of ACTH in 14 healthy dogs. In addition, cortisol concentrations decreased ( 12.1 nmol/l) 8 hours after injection of dexamethasone in the same 14 dogs. Low-end sensitivity, as previously defined, was 6 nmol/l. Intra-assay CV of 15 replicates, with a mean concentration of nmol/l, was 6.2%. Interassay CV of a single sample performed in duplicate in 15 assays, with a mean concentration of nmol/l, was 7.1%. A normal reference range of 48 healthy dogs

3 Phenobarbital and the Thyroid and Adrenal Axis in Dogs 159 Fig 1. Total serum thyroxine concentrations. Values are graphed as mean SD. * Significantly less than before treatment (week 0). Dotted lines indicate upper and lower limits of reference range. (28 pets belonging to students, faculty, or staff and 20 research dogs of various breeds) of both genders and ranging in age from 1 12 years, was nmol/l (mean 2 SD; range, nmol/l). A normal reference range for cortisol concentrations 2 hours after ACTH injection in 14 healthy dogs was nmol/l (mean 2 SD; range, nmol/l). A normal reference range for cortisol concentrations 3 and 8 hours after dexamethasone injection of 14 healthy dogs was nmol/l (mean 2 SD; range, 6 43 nmol/l). For each endocrine function test, samples were analyzed in several assays because of the large number of samples. Statistical Analysis Body weights, serum phenobarbital concentrations, adrenal thickness, and serum biochemical data were considered continuous and were evaluated for normality with the Shapiro-Wilk statistic. The data for multiple baseline periods were combined and is referred to as week 0. The data were considered to follow a normal distribution if there was failure to reject the null hypothesis of normality at P.05. Data that were not normally distributed were transformed (log transformation) for analysis such that they followed a normal distribution. This procedure was necessary for TT4, FT4, TSH, cholesterol, and all cortisol concentrations. Body weights, phenobarbital concentrations, and thyroid data were analyzed by the general linear model y Dog Week Dog Week, cortisol data during adrenal testing were evaluated using the general linear model y Dog Week Time Week Time Dog Week Time, and adrenal thickness was evaluated using the general linear model y Dog Week Side Week Side Dog Week Side. In all 3 models, the effect of Dog was considered random and the random variance of the Dog interaction was used as the error term for the evaluation of Week (model 1), Week, Time and Week Time (model 2), and Week, Side and Week Side (model 3). These models accounted for the repeated measurements on each dog. The term describes the overall mean, and describes the residual error. A 2- sided hypothesis with P.05 was used to determine significance of the main effects (Week, Time, Side). For model 1, where there were significant main effects, multiple comparisons between data at each week and data at week 0 were made by adjusted least-squares means and a Dunnett test, maintaining an experimentwise error of.05. Thus, where a significant difference between time and baseline is noted in results and graphs, the P value was.05 if not otherwise indicated. For model 2, multiple comparisons were made between pre- and postinjection cortisol data within each week and between weeks by adjusted least-squares means, maintaining an experimentwise error of.05. Thus, where a significant difference between pre- and postinjection cortisol data is noted in results and graphs, the P value was.05 if not otherwise indicated. For model 3, where there were significant main effects, multiple comparisons were made between weeks and sides by adjusted least-squares means, maintaining an experimentwise error of.05. The SAS procedure Proc Mixed n was used for the analysis. Presentation of Data. Data are presented graphically as mean SD of the values of all dogs at the respective time. Results Physical Examinations and Serum Phenobarbital Concentrations The dogs had only little sedation for about 3 days after initiation of phenobarbital treatment. Their behavior thereafter was normal, with no obvious change in physical activity or attitude. The only notable changes on repeated physical examinations were a subjectively mildly enlarged liver on palpation in several dogs during the study. Mean body weights increased significantly over time (P.001) from kg (mean SD) before treatment to kg at week 29 of treatment. Serum phenobarbital trough concentrations during treatment were within the therapeutic range of g/ml at all times in all dogs, with the exception of dog 5 at week 5 (19.4 g/ml). Means and SD recorded were (in g/ml) as follows: week 5, ; week 9, ; week 17, ; week 27, There was no significant change in serum phenobarbital concentrations over time. After the dosage adjustment at week 6, the maintenance dosages for individual dogs ranged from 4.8 to 6.6 mg/kg q12h. Serum Thyroid Function Tests Before treatment, 1 of 36 TT4 concentrations was in the low range, but the mean of the 3 pretreatment TT4 concentrations and all FT4 concentrations were within the normal range in all dogs. Four of 36 TSH concentrations before treatment were slightly above the reference range (0.38, 0.38, 0.40, and 0.41 ng/ml; normal, ng/ml), but the mean of the 3 pretreatment TSH concentrations was within the normal range in all dogs. Four of 36 pretreatment TT3 concentrations were above the reference range, but the mean of the 3 pretreatment TT3 concentrations was within the reference range in all dogs. Four of 24 pretreatment cholesterol concentrations were slightly below the reference range. TT4. TT4 concentrations changed significantly over time (P.0001). With the exception of week 21, TT4 concentrations were significantly lower than pretreatment concentrations at every point during the study (Fig 1). There was a wide variation among the concentrations, and at every time, there were concentrations within the normal and low

4 160 Müller et al Fig 2. Free serum thyroxine concentrations. Values are graphed as mean SD. * Significantly less than before treatment (week 0). Dotted lines indicate upper and lower limits of reference range. Fig 4. Serum thyrotropin concentrations. Values are graphed as mean SD. * Significantly greater than before treatment (week 0). Dotted lines indicate upper and lower limits of reference range. range. Nine of 12 dogs had at least 1 concentration in the low range, with a total of 19 of 72 concentrations (26%) being in the low range during treatment. Five dogs had 1, 2 dogs had 2, 1 dog had 4, and 1 dog had all 6 TT4 concentrations below the reference range during treatment. FT4. FT4 concentrations changed significantly over time (P.0048). The concentrations during the whole treatment period were significantly lower than pretreatment concentrations (Fig 2). As with TT4, FT4 concentrations varied widely during the treatment period, and there were concentrations in the normal and low range at all points, except for week 21 (no low concentrations). Eight of 12 dogs had at least 1 concentration in the low range, with a total of 16 of 72 concentrations (22%) being in the low range during treatment. Five dogs had 1, 1 dog had 3, and 2 dogs had 4 out of 6 FT4 concentrations below the reference range during treatment. TT3. TT3 concentrations changed significantly over time (P.0008). Concentrations at week 21 were significantly higher than pretreatment concentrations (Fig 3). Only 4 dogs had increased TT3 concentrations at any point during treatment; there were 2 dogs with 1 and 2 dogs with 2 increased concentrations during the treatment period. TSH. TSH concentrations changed significantly over time (P.0033). At week 27, TSH concentrations were significantly higher than before treatment. This increase in TSH concentration started at week 13 and became significant by the end of the study (Fig 4). Only 5 dogs had TSH concentrations above the reference range sometime during treatment. Two dogs had 1, 2 dogs had 2, and 1 dog had 3 increased TSH concentrations during the treatment period. Eight of these 9 concentrations were only mildly increased ( 0.67 ng/ml; normal, ng/ml). Serum Cholesterol. Serum cholesterol concentrations changed significantly over time (P.0001). Concentrations at week 17 and 27 were significantly higher than pretreatment concentrations (Fig 5). One dog had cholesterol concentrations above the reference range by week 17, and 3 dogs by week 27. Adrenal Function Tests and Adrenal Ultrasonographic Evaluation ACTHST. The cortisol concentrations before and 2 hours after injection of ACTH did not differ between testing periods. Cortisol concentrations increased significantly after Fig 3. Total serum triiodothyronine concentrations. Values are graphed as mean SD. * Significantly greater than before treatment (week 0). Dotted lines indicate upper and lower limits of reference range. Fig 5. Serum cholesterol concentrations. Values are graphed as mean SD. * Significantly greater than before treatment (week 0). Dotted lines indicate upper and lower limits of reference range.

5 Phenobarbital and the Thyroid and Adrenal Axis in Dogs 161 Fig 6. Results of ACTH stimulation tests. Values are graphed as mean SD. # Significantly greater than the respective pre-acth value. Dotted lines indicate upper and lower limits of post-acth reference range. ACTH administration at every testing period before and during the study (P.0001), but the magnitude of increase did not differ between testing periods (Fig 6). With the exception of 5 preinjection cortisol concentrations below the reference range (3 concentrations at week 0 and 2 concentrations at week 12), all of the cortisol concentrations before and after stimulation were within the respective reference ranges at all times. Therefore, the ACTHST results suggest normal adrenal function for every dog at every point before and during phenobarbital treatment. LDDST. The cortisol concentrations before and 3 and 8 hours after dexamethasone injection did not differ between testing periods. At every testing period before and during phenobarbital treatment, cortisol concentrations were significantly suppressed both 3 and 8 hours after injection, compared with preinjection concentrations (P.0001). The magnitude of suppression both after 3 and 8 hours did not differ between testing periods (Fig 7). One dog at week 0 and 2 dogs at week 12 had preinjection cortisol concentrations above the reference range. However, except for 1 dog at 3 hours after injection (week 0), all dogs had postinjection cortisol concentrations that were suppressed well below the upper limit of the reference range, indicating normal adrenal function at every point before and during treatment. Adrenal Ultrasonographic Evaluation. Ultrasonographic evaluation disclosed no abnormalities or changes in structure, echogenicity, or shape of the adrenal glands at any testing period. The thickness of the adrenal glands did not change significantly during the treatment period, and there was no significant difference in sizes of the left and right adrenal glands. All of the measurements were within normal limits. Means and SD of left and right adrenal glands thicknesses recorded were (in mm) as follows: week 0, left and right; week 10, left and right; week 27, left and right. Discussion Thyroid Axis Several veterinary textbooks mention that anticonvulsant treatment can lower serum thyroxine concentrations in Fig 7. Results of low-dose dexamethasone suppression tests. Values are graphed as mean SD. # Significantly less than the respective predexamethasone value. Dotted lines indicate 138 nmol/l, upper limit of predexamethasone reference range, and 43 nmol/l, upper limit of postdexamethasone reference range. dogs. 7 9 Phenobarbital treatment at therapeutic dosages significantly decreased TT4 and FT4 concentrations in our dogs, whereas serum TSH concentrations increased significantly only after 27 weeks of treatment. Serum cholesterol concentrations increased during the treatment period, whereas TT3 fluctuated minimally. Little is known about the mechanisms by which enzyme inducers alter thyroid hormone metabolism and the observed changes in the thyroid axis in dogs. We therefore cannot explain the exact mechanism of the observed changes. Several potential mechanisms could have contributed to the observed results. Phenobarbital may accelerate thyroxine elimination by hepatic enzyme induction as proposed in studies on rats and human beings, 1,4,24,27 which results in a primary decrease of FT4. As a compensatory mechanism, thyroxine moves from the protein-bound state into the free pool to normalize FT4 concentrations. However, because of the constant removal of thyroxine from the free fraction into the liver, FT4 concentrations remain decreased and thyroxine storage in the serum (ie, protein-bound T4) decreases as manifested by a decrease in TT4. By week 5, a steady state with increased T4 turnover and decreased TT4 and FT4 concentrations was established in our dogs. By week 27, a compensatory increase in TSH secretion seemed to occur. It is unclear why the compensatory mechanism of increased TSH secretion started so late, was observed only to such a minor degree, and was ineffective in increasing TT4 and FT4 at week 27. Increased TSH concentrations have been observed as early as 14 days after induction of hypothyroidism in dogs, and TSH was increased 35-fold above baseline concentrations after 4 weeks of induced hypothyroidism. 23 Possibilities for the lack of an instant TSH increase would be concomitant central inhibition of TSH release by phenobarbital, accelerated TSH clearance, or failure of the pituitary gland to recognize hypothyroxinemia because of the normal T3 concentrations, which in turn could remain at pretreatment concentrations because of increased thyroxine deiodination in the pituitary gland (see following text). The rise of TT4 and TT3 at week 21 could be attributed to variability of hepatic thyroxine metabolism, or it might be a random occurrence. Phenobarbital could act as a thyroxine analog in vivo.

6 162 Müller et al This would simulate a hyperthyroxinemic state at the pituitary gland, resulting in a decrease of TSH secretion and subsequent decrease of TT4 and FT4. Serum TSH concentrations were not significantly less at week 5 when compared with pretreatment concentrations in our dogs, which makes this mechanism unlikely. Phenobarbital could displace thyroxine from plasma protein-binding sites, resulting in a decrease of the proteinbound fraction. FT4 concentrations may rise temporarily, causing an inhibition of TSH release and resulting in decreased thyroxine secretion by the thyroid gland. Subsequently, a new hormonal steady state would be established with decreased TT4 concentrations and normal TSH concentrations. This mechanism would have to take place before week 5, and it would contradict the in vitro finding that 10 g/ml of phenobarbital does not alter T4 serum protein binding in dogs. 28 Furthermore, FT4 would return to pretreatment concentrations during the new hormonal steady state, which was not the case in our dogs. Phenobarbital could act as an inhibitor of TSH release as proposed in a study in rats, 5 causing a decrease in thyroxine secretion by the thyroid gland and subsequently a decrease in TT4 and FT4. However, a significant decrease in TSH concentrations was not observed in our dogs. The lack of a contemporaneous control group in our study could make it difficult to expand our findings on a random population of dogs (gender, fasting status, and time of blood sampling). However, the observed statistically significant changes were independent from these parameters, because every dog served as its own control. Lowered thyroxine concentrations are not likely caused by the 15 hours of fasting before blood collection because there is no effect of up to 36 hours of fasting on basal serum TT4 concentrations and the response to TSH injection in dogs. 29 Further, the time of blood collection is unlikely to be a cause for decreased TT4 concentrations because blood was drawn at the same time before and during the study. Although TT4 was shown to fluctuate during the day, there is no circadian rhythmicity. 29,30 Seasonality is unlikely to be a cause for decreased TT4 concentrations in our dogs. Although it was shown that TT4 serum concentrations in dogs are highest in autumn and lowest in summer, 31 TT4 concentrations in our study were decreased in autumn. Triiodothyronine (T3) is the biologically active thyroid hormone in peripheral tissues. It is predominantly produced at those sites by thyroxine deiodination; very little T3 is secreted by the thyroid gland itself. Pituitary TSH output is partly regulated by T3, which originates from intrapituitary T4 deiodination and from the serum T3 pool. 32 Therefore, measurement of serum T3 concentrations seems only to be a crude indicator of thyroid function and deiodinative enzyme activity. In rats, phenobarbital increases thyroxine deiodination to T3. 4,33 In our study, increased thyroxine deiodination could maintain T3 concentrations at the level of the pituitary gland despite low serum thyroxine concentrations, which in turn would prevent an appropriate rise of TSH. We cannot rule out that increased peripheral thyroxine deiodination contributed to the decrease of TT4 and FT4 in our study. The rise of TSH at the end of the study then could have been caused by a decrease of thyroxine deiodination and subsequent increase in TSH because of lowered intrapituitary T3 concentrations. To further evaluate the possibility of actual hypothyroidism, we determined serum cholesterol concentrations after 17 and 27 weeks of treatment. Increased cholesterol concentrations are commonly seen in hypothyroid dogs. 21 Although cholesterol significantly increased during the study, only 4 of 24 concentrations during treatment were above the reference range. The significance of this finding remains unclear because there could be a direct effect of phenobarbital on serum cholesterol concentrations. Studies in human beings found an increase in cholesterol concentrations during phenobarbital treatment, 34,35 whereas a study in dogs found a decrease in cholesterol concentrations during phenobarbital treatment of 1 year, which was attributed to hepatic dysfunction. 18 Although there were no abnormalities on physical examinations, it remains unclear whether some of our dogs actually became hypothyroid during the study as indicated by the observed hypothyroxinemia and increase of serum cholesterol concentrations. Hypothyroxinemia could be compensated at a cellular level by increased thyroxinemonodeiodination to T3, making determinations of TT4, FT4, and TSH meaningless in the assessment of thyroid status in dogs treated with phenobarbital. In this case, a false diagnosis of hypothyroidism would be made in an euthyroid dog based on abnormal serum thyroid function tests. It also remains unclear whether or not dogs that become hypothyroxinemic during phenobarbital treatment need thyroxine supplementation. The observed increased serum cholesterol concentrations could reflect hypothyroidism or could be caused by a direct effect of phenobarbital on serum lipids. We are unable to assess which of the observed tests would be better for evaluation of thyroid function in dogs during phenobarbital treatment because it is unclear whether or not our dogs became hypothyroid. Assuming euthyroidism in our dogs, TSH would seem to be a more reliable test to determine normal thyroid function as indicated by fewer abnormal values than for TT4 and FT4. The TSH assay however has been shown to have a high specificity but low sensitivity for the diagnosis of hypothyroidism in dogs. 36,37 Total T3 remained stable during treatment if compared with TT4, FT4, and TSH, but T3 is of minimal value in the evaluation of thyroid function of dogs 21 and can therefore not be recommended as a diagnostic tool in dogs during phenobarbital treatment. A minority of the dogs in this study had consistently low thyroxine concentrations. Repeated measurements of TT4, FT4, and TSH, and repeated physical examinations could therefore be of additional value to assess thyroid status in dogs treated with phenobarbital. Phenobarbital was administered for a limited time in our study, whereas epileptic dogs receive the drug throughout the remainder of their lives. Changes in the hormonal situation of these dogs at a later point are possible, especially because TSH concentrations started to increase by week 27. More studies in dogs receiving phenobarbital are needed to verify the aforementioned possibilities, the hormonal situation after several years of treatment, and the occurrence of actual hypothyroidism versus isolated hypothyroxinemia. Investigation of thyroxine kinetics and the hormonal situ-

7 Phenobarbital and the Thyroid and Adrenal Axis in Dogs 163 ation in the pituitary gland during phenobarbital treatment, as well as thyrotropin-releasing hormone or TSH stimulation tests, may provide further information. Adrenal Glands All dogs appeared to have normal adrenal function before phenobarbital treatment. Although 4 of 36 baseline cortisol concentrations were above (n 1) or below (n 3) the reference range, and 1 dog showed inappropriate cortisol suppression at 3 hours postdexamethasone before treatment, all of the other data collected before phenobarbital treatment (serum biochemical analyses, CBC, urinalyses, and ultrasonographic and physical examinations) were within the respective reference ranges. The 4 baseline cortisol concentrations outside of the reference range were likely caused by episodic cortisol secretion, 21 individual variation, or stress. The 1 inappropriately high cortisol concentration 3 hours postdexamethasone administration (47.3 nmol/l; normal, 6 43 nmol/l) was interpreted as individual variation because the dog had appropriate suppression at 8 hours and all of the other cortisol concentrations in its pretreatment LDDST were within normal limits. The results of ACTHST and LDDST were within normal limits in all the dogs at all testing periods during phenobarbital treatment. In human beings, it was found that anticonvulsant treatment with enzyme-inducing drugs (phenytoin, primidone, phenobarbital) can cause a lack of cortisol suppression after dexamethasone application because of accelerated elimination of the exogenous steroid. 14,15 Although we cannot rule out a similarly accelerated dexamethasone metabolism in our treated dogs, this effect would not be pronounced enough to alter the results of the LDDST with the protocol that we employed. We cannot rule out an interference of phenobarbital with the LDDST if dexamethasone is injected at the dose of 0.01 mg/kg instead of the higher dose of mg/kg that was used in our study. In human beings, appropriate cortisol suppression, despite enzyme induction, was observed when the dexamethasone dose was increased 4-fold. 14 However, all cortisol concentrations still were suppressed profoundly at 8 hours in the dogs of this study. This makes interference with the LDDST protocol with 0.01 mg/kg dexamethasone unlikely. Our results confirm previous studies in dogs that found no effect of phenobarbital on the ACTHST 18,19 and LDDST. 17 The protocol for the LDDST used in our study is different from the protocols used in studies of human beings, 14,15 which might explain the difference in the respective results. It seems unlikely that phenobarbital treatment for a period exceeding 29 weeks would affect the 2 tests, because all of the cortisol concentrations at week 29 were not different from the respective pretreatment values in these dogs. Our results suggest that a lack of appropriate cortisol suppression during a LDDST or an exaggerated increase in cortisol during an ACTHST in dogs treated with phenobarbital may be caused by adrenal hyperfunction, nonadrenal illness, or individual variation 18 rather than drug interaction. Both tests therefore would seem useful as tools to evaluate adrenal function in dogs treated with phenobarbital. In human beings and rats, phenobarbital can cause an increase in the metabolism of various endogenous steroids, including cortisol, by inducing hepatic steroid hydroxylation. 16,38 A study in rats concluded that endogenous corticosteroid concentrations in the blood are strongly dependent on hepatic steroid clearance in addition to the wellknown hormonal negative feedback system. 16 Veterinary literature mentions that hepatic enzyme-inducing agents can alter cortisol metabolism, leading to an increased ACTH release and adrenal hypertrophy. 20 Mean endogenous ACTH concentrations were found to increase significantly during a 1-year phenobarbital treatment in dogs. 18 In our study, no changes in the baseline cortisol concentration before ACTH or dexamethasone injections throughout the study could be observed. No exaggerated response to ACTH or impaired cortisol suppression after dexamethasone was found, and there was no ultrasonographic evidence of adrenal enlargement based on measurements of adrenal gland thickness. Measurement of adrenal gland thickness has been shown to represent gross adrenal size more accurately than measurements of length or width. 39 With the techniques used in our study, we found no evidence of a stimulated adrenal axis caused by increased cortisol metabolism in normal dogs. However, additional studies determining cortisol kinetics are needed to investigate the possibility of an induced cortisol turnover with subsequent increased ACTH release during phenobarbital treatment of longer duration. Footnotes a Canine P41, PMI Feeds Inc, St Louis, MO b Occult test; DiroCHEK, Synbiotics Corp, San Diego, CA c Ultramark 8, Advanced Technology Laboratories, Bothell, WA d Phenobarbital, Vintage Pharmaceuticals Inc, Charlotte, NC e Hill s Science Diet (Canine Maintenance), Hill s Pet Nutrition Inc, Topeka, KS f HP ACTHar gel, 80 IU/mL, Rhone-Poulenc Rorer Pharmaceuticals Inc, Collegeville, PA g Azium, 2 mg/ml, Schering-Plough Animal Health Corp, Kenilworth, NJ h TDx/TDxFLx Phenobarbital II, Abbott Laboratories, Abbott Park, IL i Coat-A-Count Canine T4, Diagnostic Products Corp, Los Angeles, CA j FT4 by Equilibrium Dialysis, Nichols Institute, San Juan Capistrano, CA k Coat-A-Count Canine TSH IRMA, Diagnostic Products Corp, Los Angeles, CA l Coat-A-Count Canine T3, Diagnostic Products Corp, Los Angeles, CA m Coat-A-Count Cortisol, Diagnostic Products Corp, Los Angeles, CA n SAS version 6.12, SAS Institute, Cary, NC Acknowledgments This study was supported by the Endocrine Laboratory, Louisiana Veterinary Medical Diagnostic Laboratory; School of Veterinary Medicine, Louisiana State University. The authors thank Dr Dawn Boothe for performing the phenobarbital assays and Gaye Gomila and Susan Masson for their technical support.

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