The effect of thyroid hormone deficiency on erythropoiesis in dogs

Abstract

This research aimed to evaluate the effect of thyroid hormone deficiency on the erythrocytic system in dogs. Dogs with clinical symptoms of hypothyreosis such as obesity, hyperpigmentation, and lethargy were selected. The dogs demonstrating breed predisposition to hypothyreosis were incorporated in the analysis: Dachshunds, Retrievers, and mixed-breed dogs. A detailed history was taken and clinical, hormonal, biochemical and haematological blood tests were performed. Peripheral blood samples were taken from 53 dogs. Finally, the dogs with the initial T4 (thyroxine) concentration < 1.3 μg/dl and animals demonstrating clinical improvement after a 2-month therapy with levothyroxine at a dose of 10 μg/kg administered per os two times a day were qualified. The animals between 10 months to 13 years of age were divided into two groups: clinically healthy (control group, n = 35) and dogs presenting clinical symptoms of hypothyreosis (experimental group, n = 18). In this research, the broadly described normocytic normochromic non-regenerative anaemia was not diagnosed in dogs with hypothyreosis. However, a positive correlation between T4 and red blood cell indices such as the average mass of haemoglobin per red blood cell, concentration of haemoglobin in a given volume of packed red blood cells as well as a negative correlation with haematocrit value was discovered in the experimental group after the 2-month therapy with levothyroxine. These results point to the influence of thyroid hormones on erythropoiesis. This observation is partially consistent with other studies, which noted the casual link between the changes in red blood cell system and the function of thyroid in dogs and humans. Anaemia, thyroxine, haematopoiesis Anaemia is a common condition accompanying thyroid disorders in humans and animals (Szczepanek-Parulska et al. 2017). Due to their frequent occurrence both the anaemia and thyroid dysfunctions are considered as serious disorders (Rijnberg and Kooista 2010; Vitale et al. 2010; den Elzen et al. 2015; Refaat 2015; Ziglioli et al. 2017). The causal link still remains unclear. It is known that the thyroid hormones have a stimulating effect on erythropoiesis and that they stimulate the production of erythropoietin in iron deficiency anaemia. Therefore, various forms of anaemia may manifest themselves in the course of thyroid dysfunctions. Normocytic normochromic anaemia in the course of hypothyreosis in dogs is the most frequently described type of anaemia in the literature. It may result from bone marrow suppression and deficiencies in erythropoietin, iron, vitamin B12 and folic acid. Additionally, it has been noted that hypoxia resulting from the anaemia disturbs the peripheral conversion of T4 to T3 triiodothyronine) (Omar et al. 2010; Taylor and Rampton 2015). Iron deficiency anaemia and hypothyreosis are considered the causes of mental and behavioural deficiency in humans and animals (Wilson and Curry 2005; Gianoukakis et al. 2009). The thyroid hormones play a key role in haematopoiesis, especially in erythropoiesis. They have a direct stimulating effect on the proliferation of erythrocyte precursors, but they also stimulate the erythropoiesis by increasing the erythropoietin gene expression and erythropoietin production in kidneys (Malgor et al. 1975; Golde et al. 1977; Fandrey et al. 1994; Dorgalaleh et al. 2013). ACTA VET. BRNO 2019, 88: 257–264; https://doi.org/10.2754/avb201988030257 Address for correspondence: Olga Aniołek Lazarski University 43 Swieradowska Str. 02-662 Warsaw, Poland E-mail: [email protected] Phone: +48508084779 http://actavet.vfu.cz/ Experimental research has shown an increased erythroid colony growth induced by fT3 (free triiodothyronine) (Malgor et al. 1995). In reality, in patients with hypothyreosis, the number and proliferation of erythroid cells in bone marrow decreases (Das et al. 1975). In turn, iron deficiency anaemia may have a negative effect on the influence of the state of the thyroid hormone (Khatiwada et al. 2016). Iron is essential for the functioning of thyroid peroxidase (TPO), which plays an important role in thyroid hormone synthesis. The evaluation of the influence of thyroid hormone deficiency on the haematopoietic system in dogs was the subject of this study. Materials and Methods Animals The research was carried out on dogs of Polish origin. The study was conducted in accordance with EU-Convention of the protection of animals used for scientific purpose (Revised Directive 86/609/the EEC – Directive of the European Parliament and of the Council on the protection of animals used for scientific purpose. We analysed the patients of the Small Animal Clinic of the Faculty of Veterinary Medicine at the Warsaw University of Life Sciences in Poland. Animals qualified for the research had not been treated or vaccinated over the period of two months prior to the tests. Breeds prone to canine hypothyroidism were selected for the test: Dachshunds, Retrievers, and cross-breeds. The animals were not castrated or sterilized. Animal histories were taken and the subjects had to undergo clinical, hormonal, biochemical tests and morphology in all dogs. Blood was collected from a vein. In the end, animals with an initial level of T4 < 1.3 μg/dl and those which had shown clinical improvement after a 2-month treatment with levothyroxine 10 μg/kg (Forthyron, Pfizer, Warsaw, Poland) given daily in two doses per os were qualified for the research. All dogs responded to therapy. Healthy dogs were used as the control group. Their condition was estimated on the basis of medical history and clinical examination. Results of morphological examination of blood and the biochemical profile were used in order to rule out diseases affecting the respective systems. Pregnant females were removed from the tests. Dogs with hypothyroidism have one or more of these symptoms: weight gain without an increase in appetite, lethargy and lack of desire to exercise, cold intolerance, slow heart rate, increased dark pigmentation in the skin, very thin to nearly bald hair coat (Campora et al. 2011). Dogs with the classification to the experimental group showed clinical symptoms of hypothyroidism, did not respond in the TRH stimulation test, in thyroid gland biopsy they had lymphocytic thyroiditis. Two groups of animals aged between 10 months to 13 years of age were created: clinically healthy (control group, n = 18) and a group showing clinical symptoms of hypothyroidism (experimental group, n = 35). The group of clinically healthy dogs had the same blood tests. The experimental group consisted of: Dachshunds (n = 4), Golden Retrievers (n = 6), and cross-breeds (n = 8). The control group consisted of Dachshunds (n = 3), Golden Retrievers (n = 6), and cross-breeds (n = 26). The iron and vitamin B12 concentrations and folic acid levels were not analysed. Haematological examinations Haematological examinations were carried out within 20 min from the moment the blood sample was taken. The tests determined the values of: haemoglobin (Hg), haematocrit (Ht), total red cell count (RBC), total white cell count (WBC), mean cell volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelet count (PLT) and included qualitative evaluation of the differential leukocyte count and differential blood count. Determination of Ht, RBC, WBC, MCV, MCH and MCHC was performed using impedance methods (Abacus, Diatron, France). Haemoglobin concentration was measured using the colorimetric method at a wavelength of 540 nm. The mentioned parameters were determined using a haematology analyser (Abacus). The differential leukocyte count was evaluated manually at a ×1000 magnification using the Olympus CX 40 microscope (France) according to Schilling on the basis of blood smears stained using the Pappenheim method with May-Grünwald-Giemsa (MGG) reagents (Campora et al. 2011). Marking of T4, TSH levels Levels of the thyrotropic hormone (TSH) were marked using chemiluminescent, two-point solid phase immunometric test (Immulite canine TSH, Diagnostic Products Corporation, DPC, Los Angeles, CA). Coefficients within one variability amounted to 5, 4 and 3.8% for TSH levels of 0.2, 0.5 and 2.6 μg/l, respectively. Mid-test deviation reached 6.3 and 8.2% for TSH of 0.16 and 2.8 μg/l, respectively. The TSH levels measured in Immulite canine TSH test after a serial dilution of samples taken from three dogs showed correct linearity in the whole spectrum of calibration. Cross-reactivity with the follicle stimulating hormone (FSH) and luteinizing hormone (LH) was infinitesimal. The test unit contained a sphere coated with mice monoclonal antibody anti-c-TSH. The reagent strip contained 6.5 ml alkaline phosphatase isolated from calf intestine coupled with rabbit anti-c-TSH polyclonal antibody with buffer in preservative. Total concentration of T4 using species-specific anti-T4 monoclonal antibodies (mouse monoclonal antibody anti canine T4) was marked using chemiluminescent, 258