Thyroid-stimulating hormone responses after single administration of thyrotropin-releasing hormone and combined administration of four hypothalamic releasing hormones in beagle dogs

DOMESTIC ANIMAL E N D O C R I N O L O G Y Vol. 13(5):465-468, 1996

ELSEVIER

THYROID-STIMULATING HORMONE RESPONSES AFTER SINGLE ADMINISTRATION OF THYROTROPIN-RELEASING HORMONE AND COMBINED ADMINISTRATION OF FOUR H Y P O T H A L A M I C R E L E A S I N G H O R M O N E S IN B E A G L E D O G S B.P. Meij, J.A. Mol, and A. Rijnberk Department of Clinical Sciences of Companion Animals Faculty of Veterinary Medicine, Utrecht University PO Box 80. 154, NL-3508 TD Utrecht The Netherlands Received Janua~ 23,1996

Thyrotropin (TSH) responses were determined in eight healthy male beagle dogs after a single administration of thyrotropin-releasing hormone (TRH) and the combined administration of four hypothalamic releasing hormones, i.e., corticotropin-releasing hormone, growth hormone-releasing hormone, gonadotropin-releasing hormone, and TRH. In both tests, TRH was administered in a dose of l0 p~g/kg. Basal TSH concentrations ranged form 0.07 to 0.27 p,g/1 (mean -+ SE, 0.14 -4-0.02 Ixg/1). The administration of TRH, alone or in the combined test, resulted in a prompt and significant increase in TSH with mean (_+SE) plasma TSH peaks of 1.26 ± 0.22 Izg/1 at 10 min and 0.85 _+0.17 p~g/1 at 30 rain, respectively. The area under the curve (0-120 min) was significantly lower in the combined test than in the single TRH test, whereas the increments were not significantly different. It is concluded that measurements of TSH responses to TRH alone and in combination with other releasing hormones can be used for the assessment of pituitary thyrotropic cell function. In the combined test, the TSH response is slightly lower than that in the single test.

INTRODUCTION The measurement o f basal concentrations of serum thyroid hormones has certain limitations in the diagnosis of canine hypothyroidism, because of the overlap in the ranges found in hypothyroid and euthyroid dogs and the effects of various drugs and nonthyroidal illness (1-3). Serum thyroid hormone responses in dynamic tests, i.e., stimulation with thyrotropin (TSH) or thyrotropin-releasing hormone (TRH), are commonly used to assess thyroid function because an assay for canine TSH has not been available until very recently (1-3). Only in 1995 were preliminary reports published on the validation of assays for canine TSH (4-6). A n assessment of TSH responses to T R H alone and TRH combined with corticotropinreleasing hormone (CRH), growth hormone-releasing hormone (GHRH), and gonadotropin-releasing hormone (GnRH) has been described in the rat (7) and in humans (8-15). A combined anterior pituitary function test was also introduced for use in the dog. It comprises the rapid sequential intravenous administration of CRH, GHRH, GnRH, and TRH and measurements in plasma of adrenocorticotropin, GH, luteinizing hormone (LH), and prolactin (PRL), respectively (16). In the latter study, TSH measurements were not included because at that time, there was no reliable TSH assay for use in dogs. T R H was used because of its PRL-releasing properties. The objectives o f this study were to determine the TSH responses to T R H alone and to TRH in combination with other releasing hormones in normal beagle dogs as a reference for future studies of pituitary thyrotropic and thyroid function and dysfunction. © Elsevier Science Inc. 1996 655 Avenue of the Americas, New York, NY 10010

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MATERIALS AND METHODS Animals. Eight healthy adult male beagle dogs (median age, 2 yr; range, 1-6 yr) weighing 13-16 kg were used. The dogs were housed individually in indoor kennels under a normal daylight regimen, with access to separate outdoor runs for about 2 hr/d. They were fed a commercial diet, and they had free access to water. The animals were adapted to these conditions for 2 wk before the start of the experiment. Between the single and the combined tests, there was an interval of 1 wk, and the tests were performed after an overnight fast. The dogs and the experimental protocol were the same as in a previous study (16). Materials. Ovine CRH (oCRF; Peninsula Laboratories Inc., Belmont, CA) and human GHRH (hGHRF; Peninsula Laboratories Inc.) were both kept frozen at -25 ° C and thawed at room temperature immediately before use. A GnRH analogue, gonadorelin (Fertagyl; Intervet, Boxmeer, The Netherlands), and TRH (TRH; Hoffman-La Roche, Basel, Switzerland) were kept at 4 ° C. Experimental Protocol. An intravenous catheter was placed in the cephalic vein of each dog to facilitate rapid sequential injections. In the combined test, all four releasing hormones were injected intravenously within 30 sec, immediately after the collection of the zero blood sample from the jugular vein, in the following order and doses: 1 ~g of CRH/kg, 1 p~g of GHRH/kg, 10 ~g of GnRH/kg, and 10 p~g of TRH/kg (16). The clock for blood sampling was started immediately after the administration of the last releasing hormone. Blood samples were collected at -30, -15, 0, 5, 10, 20, 30 45, 60, 90, and 120 min from the jugular vein and transferred to ice-chilled EDTA-coated tubes. Samples were centrifuged at 4 ° C for 10 min. Plasma was stored at -25 ° C until assayed for TSH. In the single test, TRH was injected alone in the same dose as in the combined function test. Blood sampling and processing of samples were the same as in the combined test. The protocol was approved by the Ethical Committee of the Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands. TSH Determination. Plasma TSH was measured by immunoradiometricassay with a commercially available kit (Diagnostic Products Co., Los Angeles, CA) (4,5). The sensitivity of the assay amounted to 0.03 p~g of TSH/I. The intra-assay and interassay variation coefficients were 2.4 and 3.9%, respectively, at a mean TSH concentration of 4.6 and 3.6 ~g/1. There was negligible cross-reactivity of canine LH or follicle stimulating hormone in the assay for canine TSH. Statistical Analysis. Results are expressed as means +- SE. The area under the curve (AUC) values of TSH concentrations after the combined test and the TRH test were calculated by the trapezoidal method after subtraction of the basal level. Increments were calculated between peak levels and basal levels at 0 rain. Differences in AUC and increments between the TRH and the combined tests were assessed for statistical significance (at the 5% level) by the nonparametric Wilcoxon matched-pairs signed-ranks test. RESULTS No serious side effects were observed after TRH administration or the combined administration of releasing hormones. One dog vomited, and in two dogs, hypersalivation and restlessness occurred immediately after the combined administration of releasing hormones. These reactions had disappeared within 5 min after the injection. Basal TSH concentrations ranged from 0.07 to 0.27 i~g/1 (mean _+SE, 0.14 _+0.02 p~g/1). In both the single and the combined tests, the injection of TRH resulted in prompt and significant increases in the plasma TSH levels. In the single TRH test, the maximum plasma TSH concentration (mean _+ SE) was 1.26 + 0.22 I~g/ml at 10 rain, and in the

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combined test, it was 0.85 _+ 0.17 p,g/1 at 30 min (Figure 1). The mean plasma TSH level returned to near-baseline levels by 120 min (Figure 1). In the single test, the mean A U C (0--120 rain) for TSH was 70.1 _+ 14.4 and the mean plasma TSH increment was 1.29 -+ 0.22 Ixg/1. In the combined test, the mean A U C (0-120 min) for TSH was 45.7 -+ 8.7 and the mean plasma TSH increment was 0.78 _+ 0.15 ixg/l. The A U C (0-120 min) values for TSH in the combined test were significantly lower than the A U C values in the T R H test (Wilcoxon, P < 0.05), whereas the plasma TSH increments were not significantly different (P = 0.0687). DISCUSSION The results o f this study demonstrate that in normal dogs, both the single administration o f TRH and the combined rapid sequential administration o f the four hypothalamic releasing hormones, CRH, GHRH, GnRH, and TRH, result in increases in plasma TSH concentrations. The TSH response after TRH alone was higher than in the combined test. The canine TSH assay appears to have been reliable, because prompt TSH responses were observed after the injection o f TRH alone or combined with other releasing hormones. There have been reports of interactions between T R H and other releasing hormones in humans, but some o f the reports were in disagreement with others. One study reported on a G H R H - i n d u c e d central inhibitory mechanism on TSH secretion after G H R H plus TRH (14). In other studies, it was found that G H R H potentiates the TSH-releasing activity of TRH after combinations o f G H R H plus T R H (8,9,15) or G H R H and GnRH plus TRH (15) and after the combination of all four releasing hormones (8,9,11,12). These interactions were not observed in other studies in humans that used the combined administration of four releasing hormones (10,13). A study performed in male rats revealed no significant

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Figure 1. Plasma TSH response (mean ___SE) in eight healthy male beagle dogs after the rapid (30-sec) intravenous injection (arrow) of a combination of four hypothalamic releasing hormones (0) compared with that after the injection of 10 p~g of TRH/kg alone (O). In the combined test, the releasing hormones were injected in the following order and doses: 1 p.g of CRH/kg, 1 I~gof GHRH/kg, 10 ~zgof GnRH/kg, and 10 Ixg of TRH/kg. The AUC was significantly lower in the combined than in the single stimulation test (Wilcoxon, P < 0.05).

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in vitro or in vivo interactions between any of four synthetic hypothalamic releasing factors (CRH, GHRH, GnRH, and TRH) on TSH secretion (7). Unlike in humans, the combined administration of GHRH and TRH in our dogs did not result in higher TSH responses than in the single test. In conclusion, our findings indicate that measurements of TSH responses after TRH administration may offer a rapid, safe, and effective method of investigating anterior pituitary thyrotropic cell function. The test's primary use in the dog may be the assessment of secondary dysfunction of the thyroid, e.g., hypothyroidism of pituitary origin. In the rapid sequential intravenous administration of four hypothalamic releasing hormones, including TRH, a slightly lower TSH response should be expected, as compared with that in the single TRH test. ACKNOWLEDGMENTS/FOOTNOTES Address correspondence and reprint requests to: Dr. B.P. Meij, Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 8, PO Box 80. 154, NL-3508 TD Utrecht, The Netherlands.

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