In mammals, iodine functions biochemically as a component of thyroid hormones, which are required for many aspects of normal metabolism, growth, and development. In addition to the iodine that is required as a central component of the thyroid hormones, selenium is essential for thyroid hormone synthesis and activation and for their proper metabolism. The role of selenium in biological systems is much more complex than previously supposed. Selenium deficiency may cause abnormal thyroid hormone metabolism1–3 that then results in decreased growth rate, reduced fertility, and impaired immune responses and resistance to infectious diseases.4 Thyroxine, which is the major hormone product of the thyroid gland, is biologically inactive and needs to be converted to its active metabolite T3 via 5a-monodeiodination.5,6 Deiodination of T4 to T3 is catalyzed by selenoenzymes, namely deiodinase types I, II, and III. These enzymes contain selenium as seleno cystein at their active sites.7–9 Another effect of selenium on thyroid hormone metabolism is derived from its action on selenium-containing GSH-Pxs and thioredoxin reductase, which protect the thyroid gland from H2O2 that is released during the synthesis of thyroid hormones.10–12
The discovery of the influence of selenium on thyroid hormone metabolism has concentrated considerable research into the effects of selenium deficiency on thyroid hormone homeostasis in humans and other species. Nutritional deficiency of selenium in rats results in decreases in the activity of deiodinase type I and serum T3 concentration.13 A similar situation has been observed in calves that are fed diets low in selenium.14,15 Effects of a low-selenium diet on serum concentrations of T4 and T3 in calves,1 heifer calves on pasture,15 Angus and crossbred beef cows,16 and housed dairy cows consuming preserved food17 have also been reported. However, there is limited information about thyroid hormone status in association with naturally occurring clinical forms of selenium deficiency in farm animals kept under field conditions. The purpose of the study reported here was to assess changes in serum concentrations of thyroid hormones associated with selenium deficiency myopathy in lambs. The lambs included in the study were affected with a subacute form of selenium deficiency myopathy (nutritional myodegeneration or so-called stiff-lamb disease), which is the most well-described and common clinical form of selenium deficiency in large animals.
Materials and Methods
Animals—The study proposal was reviewed and approved by the Department of Clinical Sciences' review board at the Veterinary Faculty of Urmia University (reference No. 003.D.84). The study was conducted in Urmia (northwest Iran), where selenium deficiency myopathy is a common disease in lambs. Lambs with subacute selenium deficiency myopathy, which had been either referred to the Veterinary Teaching Clinic and Hospital of Urmia University by clients or identified by veterinarians during farm visits, were the target population. Tentative diagnosis was made on the basis of clinical signs including stiffness; trembling of the limbs; weakness; inability to stand; and large, firm muscle masses. These lambs were from flocks in which previous cases of selenium deficiency myopathy had been confirmed through determination of circulating concentration of selenium and activities of GSH-Px and CK, response to treatment, or necropsy findings.
The group of 35 affected lambs was comprised of 11 males and 24 females that were 3 weeks to 4 months old; each lamb had clinical and clinicopathologic characteristics of selenium deficiency myopathy. Affected lambs that had both low erythrocyte GSH-Px activity and high plasma CK activity were classified as selenium deficient and included in the study. Thirty healthy agematched lambs (6 males and 24 females) from herds without any evidence of the disease were also included (with owner consent) in the study. Erythrocyte GSH-Px and plasma CK activities in these healthy lambs were within reference ranges; these lambs were therefore classified as non-selenium deficient. Data collected from each group during the study were compared.
Sample collection—Ten milliliters of blood from the jugular vein of each lamb was collected into tubes that contained no anticoagulant or heparin (1 U/mL of blood). Heparin-treated blood samples were centrifuged at 1,500 × g for 15 minutes to separate plasma from erythrocytes. The plasma was used for CK activity determination; erythrocytes were washed 3 times with saline (0.9% NaCl) solution, and the hemolysate was stored frozen at ×20°C (for < 4 days) until GSHPx activity determination was performed. Serum was harvested from blood samples with no anticoagulant by centrifugation at 1,500 × g for 15 minutes; serum aliquots were frozen at ×20°C for subsequent determination of TSH, tT4, and tT3 concentrations. The duration of storage (interval from sample collection to assessment) was < 8 days. Samples (plasma and serum) with hemolysis were excluded from analysis.
Sample preparation and enzyme and hormone assays—All samples were run in duplicate. Glutathione peroxidase activity of hemolyzed erythrocytes was measured spectrophotometricallya at 37°C by use of a commercial kit,b according to the modified method of Paglia and Valentine.18 Both intraand interassay coefficients of variation were < 2%. Plasma CK activity was assessed by use of a commercial kitc and an autoanalyzer.d
Serum TSH concentration was measured via radioimmunoassaye; for use in sheep, the method was modified with the inclusion of rabbit anti-sheep TSH,f according to the procedure described by Sapin et al.19 Intra-assay and interassay coefficients of variation were 5.1% and 6.5%, respectively.
Serum tT4 and tT3 concentrations were determined by use of commercial ELISA kits according to the manufacturer's instructions.g,h Briefly, serum was added to microtiter plate wells that were coated with anti-T4 or anti-T3 antibodies and then incubated with T4-horseradish peroxidase or T3-horseradish peroxidase conjugates for 60 minutes before the wells were washed. Horseradish peroxidase substrate was added into each well and incubated for 30 minutes. The intensity of the color formed is inversely proportional to the concentrations of T4 and T3 in the samples. The absorbance was measured by use of a microtiter plate reader. A set of standards was used to plot a standard curve from which the amounts of T4 and T3 in samples were calculated. In our laboratory, the intra-assay and interassay coefficients of variation have been determined to be 5.4% and 7.2% for the T4 assay, respectively, and 3.8% and 4.2% for the T3 assay, respectively.
Statistical analysis—Descriptive statistics were calculated and data were checked for outliers and for normality by use of the Kolmogorov-Smirnov test. Except for serum tT3 concentration and plasma CK activity, which were transformed to normal distribution by logarithmic conversion, the distributions of other variables were considered normal (P > 0.05 [significance set at a value of P < 0.05]). For each variable, independent Student t tests were used to compare the mean values between affected and healthy lambs. Pearson correlation was used to investigate the interrelationships between variables. Data are expressed as mean ± SD. For all comparisons, a value of P < 0.05 was considered significant. Analyses were performed with statistical software.i
Results
Compared with findings in healthy lambs, erythrocyte GSH-Px activity, serum tT3 concentration, and the ratio of tT3 to tT4 were significantly (P < 0.001) decreased in lambs with selenium deficiency myopathy. In addition, serum TSH concentration, plasma CK activity, and tT4 concentration were significantly (P < 0.001) greater in affected lambs than the corresponding values in healthy lambs (Table 1).
Values of erythrocyte GSH-Px and plasma CK activities; serum TSH, tT4, and tT3 concentrations; and tT3:tT4 concentration ratio in 30 healthy lambs and 35 lambs with selenium deficiency myopathy. Data are presented as mean ± SD (range).
Variable | Healthy group | Affected group | Reference range |
---|---|---|---|
GSH-Px (U/g of hemoglobin) | 132.3 ± 29.5 (65.9–185.6) | 45.1 ± 8.0 (25.4–59.1)* | 60–18020 |
CK (U/L) | 159.8 ± 33.4 (115.0–262.0) | 1,993.2 ± 856.9 (890.0–3,750.0)* | 100–54721 |
TSH (mU/L) | 1.47 ± 0.30 (0.84–2.50) | 5.41 ± 1.1 (3.57–8.40)* | NR |
tT4 (nmol/L) | 57.5 ± 6.5 (45.1–70.8) | 77.2 ± 6.0 (62.9–86.3)* | 38.0–79.222 |
tT3 (nmol/L) | 1.91 ± 0.18 (1.56–2.35) | 1.40 ± 0.15 (1.01–1.64)* | 0.97–2.3022 |
tT3:tT4 ratio | 0.033 ± 0.005 (0.025–0.047) | 0.018 ± 0.001 (0.015–0.020)* | NR |
For this variable, value is significantly (P < 0.001) different from that of the healthy lambs. NR = Not reported.
In all lambs with selenium deficiency myopathy, erythrocyte GSH-Px activity was < 60 U/g of hemoglobin (reference range, 60 to 180 U/g of hemoglobin), which is the lower reference limit suggested by other researchers.20 Mean ± SD plasma CK activity of affected lambs was 1,993.2 ± 856.9 U/L, a value that was greatly increased from the accepted normal plasma activity of 52 ± 10 U/L20 in sheep (reference range, 100 to 547 U/L).21 In addition, in lambs with selenium deficiency myopathy, mean serum tT4 concentration was 77.2 ± 6.0 nmol/L; this value was increased, compared with the published mean value of 56.8 ± 14.5 nmol/L (reference range, 38.0 to 79.2 nmol/L).22 In 14 of 35 (40%) affected lambs, serum tT4 concentration was greater than the upper reference limit (79.2 nmol/L)22 reported for sheep. Mean serum concentration of tT3 in the affected lambs was significantly less than that in the healthy lambs; however, the value in any individual affected lamb was not less than the lower reference limit for this hormone concentration in sheep (reference range, 0.97 to 2.30 nmol/L22). Mean serum tT3 concentration in lambs with selenium deficiency myopathy also did not differ markedly from the mean value reported for sheep (1.40 ± 0.15 nmol/L and 1.53 ± 0.43 nmol/L, respectively).22
Evaluation of the interrelationships between assayed variables in lambs affected with selenium deficiency myopathy revealed significant negative correlations between erythrocyte GSH-Px activity and each of the following: plasma CK activity (r = −0.443; P < 0.01), serum TSH concentration (r = −0.599; P < 0.001), serum tT4 concentration (r = −0.577; P < 0.001), and serum tT3 concentration (r = −0.621; P < 0.001; Table 2). In healthy lambs, there was no significant correlation between erythrocyte GSH-Px activity and any variable. However, a significant negative correlation (r = −0.779; P < 0.001) between serum tT4 concentration and T3:T4 concentration ratio was detected in healthy lambs but not in affected lambs.
Correlations between erythrocyte GSH-Px and plasma CK activities; serum TSH, tT4, and tT3 concentrations; and tT3:tT4 concentration ratio in 30 healthy lambs and 35 lambs with selenium def-ciency myopathy.
Variable | Group | GSH-Px | CK | TSH | tT4 | tT3 | tT3:tT4 ratio |
---|---|---|---|---|---|---|---|
GSH-Px | Healthy | ||||||
PC | 1 | 0.113 | −0.025 | −0.116 | −0.036 | 0.067 | |
P value | NA | 0.552 | 0.897 | 0.542 | 0.849 | 0.724 | |
Affected | |||||||
PC | 1 | −0.443 | −0.599 | −0.577 | −0.621 | −0.291 | |
P value | NA | 0.008 | <0.001 | <0.001 | <0.001 | 0.090 | |
CK | Healthy | ||||||
PC | 0.113 | 1 | −0.178 | −0.481 | 0.155 | 0.474 | |
P value | 0.552 | NA | 0.364 | 0.007 | 0.412 | 0.008 | |
Affected | |||||||
PC | −0.443 | 1 | 0.276 | 0.066 | 0.278 | 0.341 | |
P value | 0.008 | NA | 0.108 | 0.706 | 0.105 | 0.045 | |
TSH | Healthy | ||||||
PC | −0.025 | −0.178 | 1 | 0.217 | −0.195 | −0.278 | |
P value | 0.897 | 0.346 | NA | 0.248 | 0.301 | 0.137 | |
Affected | |||||||
PC | −0.599 | 0.276 | 1 | 0.322 | 0.372 | 0.184 | |
P value | < 0.001 | 0.108 | NA | 0.059 | 0.028 | 0.290 | |
tT4 | Healthy | ||||||
PC | −0.116 | −0.481 | 0.217 | 1 | −0.032 | −0.779 | |
P value | 0.542 | 0.007 | 0.248 | NA | 0.867 | < 0.001 | |
Affected | |||||||
PC | −0.557 | 0.066 | 0.322 | 1 | 0.767 | 0.032 | |
P value | < 0.001 | 0.706 | 0.059 | NA | < 0.001 | 0.856 | |
tT3 | Healthy | ||||||
PC | −0.036 | 0.155 | −0.195 | −0.032 | 1 | 0.642 | |
P value | 0.849 | 0.412 | 0.301 | 0.867 | NA | < 0.001 | |
Affected | |||||||
PC | −0.621 | 0.278 | 0.372 | 0.767 | 1 | 0.664 | |
P value | < 0.001 | 0.105 | 0.028 | < 0.001 | NA | < 0.001 | |
tT3:tT4 ratio | Healthy | ||||||
PC | 0.067 | 0.474 | −0.278 | −0.779 | 0.642 | 1 | |
P value | 0.724 | 0.008 | 0.137 | < 0.001 | < 0.001 | NA | |
Affected | |||||||
PC | −0.291 | 0.341 | 0.184 | 0.032 | 0.664 | 1 | |
P value | 0.090 | 0.045 | 0.290 | 0.856 | < 0.001 | NA |
PC = Pearson correlation. NA = Not applicable.
A value of P < 0.05 was considered significant.
Discussion
Results of the present study provided some information regarding alterations in thyroid hormone metabolism in lambs with selenium deficiency myopathy. Affected lambs had higher serum concentrations of tT4 and lower serum concentrations of tT3, compared with findings in healthy lambs. Similar findings in rats23,24 and calves fed selenium-deficient diets14,15 and in cattle grazing on selenium-deficient pasture25 have been reported. In agreement with results of another study,20 a significant negative correlation between erythrocyte GSH-Px and plasma CK activities was evident in lambs with selenium deficiency myopathy in the present study, which suggests that muscular damage increases with deterioration of selenium status.
Selenium-containing deiodinases regulate the synthesis and degradation of the biologically active thyroid hormone T3, and peripheral deiodination of T4 plays a primary role in regulating thyroid hormones homeostasis.26,27 The tT3:tT4 concentration ratio has been considered as a functional marker of selenium status in humans.12 Conversion of T4 to T3 can be assessed by monitoring the T3:T4 concentration ratio in blood. In humans, progressive reduction of this ratio can be reversed with selenium supplementation.28 In agreement with the findings from animal experiments29 and human studies,28,30 lambs with selenium deficiency myopathy in the present study had significantly increased serum concentration of tT4 and significantly decreased serum concentration of tT3, compared with values in healthy lambs, and there was a significant negative correlation between erythrocyte GSH-Px activity and serum tT4 concentration. These alterations could be attributed to reduced conversion of tT4 to tT3 in peripheral tissues as a result of decreased activity of selenium-containing deiodinases, which in turn decreased the tT3:tT4 concentration ratio.
The mean serum concentration of T4 in lambs with selenium deficiency myopathy was significantly higher than that of healthy lambs; however, in 14 of 35 affected lambs, the serum concentrations of tT4 were higher than the upper reference limit reported for sheep. We do not have an explanation for this finding; nevertheless, the intensity and duration of selenium deficiency and individual variations could be among the probable possibilities.
Although high T4 concentration in the blood of selenium-deficient animals is a well-recognized finding,23,31 there are conflicting reports about alterations in T3 concentration in those affected animals. Golstein et al32 reported that the plasma half-life of T3 in control and selenium-deficient rats was similar and concluded that there is a higher efficiency of thyroid hormone synthesis in the thyroid gland of selenium-deficient rats. In another study,33 serum T3 concentrations in selenium-deficient rats remained within reference range, despite markedly high circulating T4 concentrations; furthermore, the serum half-life of T3 in the selenium-deficient rats was increased by 20%, compared with values in selenium-supplemented rats. From these data, the investigators concluded that increased T3 sulfate generation in selenium-deficient rats may lead to greater T3 availability through enterohepatic recycling of the iodothyronine and that this may explain why there were only minor changes in serum T3 concentration.33 Although selenium-deficient lambs in the present study had significantly lower circulating tT3 concentration, compared with that in healthy lambs, the mean concentration of this hormone in affected lambs was within the reference range defined for sheep. In addition, the serum tT3 concentration was not lower than the minimum reference limit for sheep in any affected lamb. Furthermore, although both erythrocyte GSH-Px activity and serum tT3 concentration in the lambs with selenium deficiency myopathy were significantly lower than the values in healthy lambs, there was a significant negative relationship between these variables in the affected group. The cause of this interrelationship is not clear, but it is likely that the increase in plasma half-life of tT3 that has been identified in selenium-deficient rats32,33 may also occur in selenium-deficient lambs. Our observations suggested that, as in selenium-deficient rats, the alterations in the blood concentration of tT3 are not as remarkable as alterations in blood concentration of tT4 in selenium-deficient lambs. However, because of the significant decrease in serum tT3 concentration in lambs with selenium deficiency myopathy in the present study, compared with the value in healthy lambs, and because of the important roles of this hormone in the regulation of metabolism (eg, stimulation of oxygen utilization, provision of glucose to cells, enhancement of the basal metabolic rate and heat production, stimulation of protein synthesis, and increasing lipid metabolism), growth, and development, it is necessary to evaluate the effects and consequences of alteration of serum T3 concentration in the well-being of selenium-deficient lambs.
Another finding of the present study was that, compared with the findings in healthy lambs, the serum concentration of TSH in lambs with selenium deficiency myopathy was increased, despite an increased circulating concentration of tT4. Similar findings in seleniumdeficient euthyroid rats have been reported.26,31 Under normal circumstances, plasma TSH concentration decreases as plasma T4 concentration increases, but in rats with hyperthyroxinemia of selenium deficiency, plasma TSH concentration is unchanged or increased, indicating an inability of the pituitary gland to respond to the increased plasma T4 concentration.26 The absence of significant correlation between serum TSH and T4 concentrations in selenium-deficient lambs in our study is in agreement with results of studies26,31 in seleniumdeficient rats. Chanoine et al31 reported that serum T4 concentrations increased by 38%, serum T3 concentrations decreased by 22%, and serum TSH concentration increased by 52% in selenium-deficient euthyroid rats, compared with values in rats receiving a selenium supplement. Because T3 was the only thyroid hormone for which circulating concentration was decreased in selenium-deficient rats, those authors suggested that basal TSH secretion was modulated by circulating T3. Although serum TSH concentration was only increased by 52% in selenium-deficient rats in that study,31 serum TSH concentration in lambs with selenium deficiency myopathy was increased by 370%, compared with values in healthy lambs, in the investigation reported here. In addition, the significant positive correlation between serum TSH and serum tT3 concentrations in seleniumdeficient lambs in our study was unexpected. The cause and consequences of such an extraordinary increase in serum TSH concentration and its interaction with T3 need to be determined through further investigation.
Overall, the results of the present study suggest that in sheep with selenium deficiency, as in seleniumdeficient laboratory animals, thyroid hormone metabolism is altered. Those alterations may contribute to economically important disorders, such as reproductive abnormalities (abortion, early embryonic loss, and stillbirth), neonatal death, and septic processes, which develop frequently in selenium-deficient flocks in the geographic region of this investigation.
ABBREVIATIONS
T3 | Triiodothyronine |
T4 | Thyroxine |
tT4 | Total thyroxine |
tT3 | Total triiodothyronine |
GSH-Px | Glutathione peroxidase |
CK | Creatine kinase |
TSH | Thyroid-stimulating hormone |
Ultraspec Plus, UV visible 4054, LKB Pharmacia, Uppsala, Sweden.
Ransel, Randox Laboratories Ltd, Crumlin, Ireland.
CK kit, Pars Azmoun, Tehran, Iran.
Technicon RA 1000 system, Technicon Instruments Corp, New York, NY.
TSH enzyme immunoassay kit, Amersham Pharmacia Biotechnology, Piscataway, NJ.
Accurate Chemical & Scientific Corp, Westbury, NY.
Total T4 ELISA kit, Human Gesellschaft für Biochemica und Diagnostica GmbH, Wiesbaden, Germany.
Total T3 ELISA kit, Human Gesellschaft für Biochemica und Diagnostica GmbH, Wiesbaden, Germany.
SPSS software, version 9.0, SPSS Inc, Chicago, Ill.
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