Effects of selenium supplementation on thyroid hormone metabolism in phenylketonuria subjects on a phenylalanine restricted diet

Type I 5′-deiodinase was recently characterized as a selenocysteine-containing enzyme in humans and other mammals. Up to now, the effect of selenium (Se) supplementation on thyroid hormone metabolism in humans has only been reported in the very peculiar nutritional environment of Central Africa, where combined severe iodine and Se deficiency occurs. In this study, a group of phenylketonuria subjects with a low selenium status, but a normal iodine intake were supplemented with selenium to investigate changes in their thyroid hormone metabolism. After 3 wk of selenium supplementation (1 μg/kg/d), both the concentrations of the prohormone thyroxine (T4) and the metabolic inactive reverse triiodothyronine (rT3) decreased significantly. Clinically, the phenylketonuria subjects remained euthyroid before and after selenium supplementation. The individual changes of plasma Se and glutathione peroxidase activity were closely associated with individual changes of plasma T4 and rT3.     

The effect of a selenium supplementation on the outcome of patients with severe systemic inflammation, burn and trauma

Patients with systemic inflammatory response syndrome (SIRS) and sepsis exhibit decreased plasma selenium and glutathione peroxidase activity. This has been shown in several clinical studies. Moreover, the degree of selenium deficiency correlates with the severity of the disease and the incidence of mortality. Patients with SIRS and sepsis are exposed to severe oxidative stress. Selenoenzymes play a major role in protecting cells against peroxidation, especially lipid peroxidation and are involved in the regulation of inflammatory processes. Therefore, selenium substitution in those patients might be effective in the prevention of multiorgan failure. The results of randomised clinical trials investigating selenium substitution in critical ill patients with inflammation are reviewed. In two independently performed randomised, prospective clinical trials, including patients with systemic inflammatory response syndrome or sepsis, the supplementation of selenium revealed a significant reduction in multiorgan failure and, especially, a lower incidence of acute renal failure and respiratory distress syndrome. One of those trials also could demonstrate a significant reduction of mortality in the most severely ill patients. Two other studies, where selenium together with other trace elements or a mixture of antioxidants were used in the treatment of patients with severe burn injuries or trauma showed a significant reduction in the secondary infection rate, including sepsis. Thus, selenium supplementation seems to improve the outcome of patients with SIRS, sepsis and severe injury, however, pivotal prospective clinical trials with sufficient statistical power are now necessary to finally prove the efficacy of a selenium supplementation in these diseases.     

The effects of selenium depletion and repletion on the metabolism of thyroid hormones in the rat

Rats were fed selenium-deficient (less than 0.005 mg selenium/kg) or selenium-supplemented diets (0.1 mg selenium/kg, as Na2SeO2) for up to five wks from weaning to assess the effects of developing selenium deficiency on the metabolism of thyroid hormones. Within two wks 3:5,3'-triiodothyronine (T3) production from thyroxine (T4) in liver homogenates from selenium-deficient rats was significantly lower compared with the activity in liver homogenates from selenium-supplemented rats. This decreased activity was probably responsible, in part, for the higher T4 and lower T3 concentrations in plasma from the selenium-deficient rats after 3, 4, and 5 weeks of experiment. Repletion of selenium-deficient rats with single intra-peritoneal injections of 200 micrograms selenium/kg body wt. (as Na2SeO3) 5 days before sampling reversed the effects of the deficiency on thyroid hormone metabolism and significantly increased liver and plasma glutathione peroxidase activities. However a dose of 10 micrograms selenium/kg body wt given to rats of similar low selenium status had no effect on thyroid hormone metabolism or glutathione peroxidase activity but did reverse the increase in hepatic glutathione S-transferase activity characteristic of severe selenium deficiency. Imbalances in thyroid hormone metabolism are an early consequence of selenium deficiency and are probably not related to changes in hepatic xenobiotic metabolizing enzymes associated with severe deficiency.     

Selenium, selenoenzymes, oxidative stress and risk of neoplastic progression from Barrett's esophagus: results from biomarkers and genetic variants

Clinical trials have suggested a protective effect of selenium supplementation on the risk of esophageal cancer, which may be mediated through the antioxidant activity of selenoenzymes. We investigated whether serum selenium concentrations, selenoenzyme activity, oxidative stress and genetic variation in selenoenzymes were associated with the risk of neoplastic progression to esophageal adenocarcinoma (EA) and two intermediate endpoints, aneuploidy and tetraploidy. In this prospective cohort study, during an average follow-up of 7.3 years, 47 EA cases, 41 aneuploidy cases and 51 tetraploidy cases accrued among 361 participants from the Seattle Barrett's Esophagus Research Study who were free of EA at the time of blood draw and had at least one follow-up visit. Development to EA was assessed histologically and aneuploidy and tetraploidy by DNA content flow cytometry. Serum selenium concentrations were measured using atomic absorption spectrometry, activity of glutathione peroxidase (GPX) 1 and GPX3 by substrate-specific coupled test procedures, selenoprotein P (SEPP1) concentrations and protein carbonyl content by ELISA method and malondialdehyde concentrations by HPLC. Genetic variants in GPX1-4 and SEPP1 were genotyped. Serum selenium was not associated with the risk of neoplastic progression to EA, aneuploidy or tetraploidy (P for trend?=?0.25 to 0.85). SEPP1 concentrations were positively associated with the risk of EA [hazard ratio (HR)?=?3.95, 95% confidence intervals (CI)?=?1.42-10.97 comparing the third tertile with the first] and with aneuploidy (HR?=?6.53, 95% CI?=?1.31-32.58), but not selenoenzyme activity or oxidative stress markers. No genetic variants, overall, were associated with the risk of neoplastic progression to EA (global p?=?0.12-0.69). Our results do not support a protective effect of selenium on risk of neoplastic progression to EA. Our study is the first to report positive associations of plasma SEPP1 concentrations with the risk of EA and aneuploidy, which warrants further investigation.     

Dietary selenium status and plasma thyroid hormones in chicks

Experiments were conducted to study the effect of marginal levels of selenium and vitamin E on plasma thyroid hormones of meattype chicks. Plasma thyroxine (T₄) was significantly increased when a semipurified diet was supplemented with either selenium or vitamin E. Triiodothyronine (T₃) was also significantly increased by vitamin E and in one experiment with selenium supplementation. No significant increase in these hormones was observed in birds fed a corn-soybean-meal diet supplemented with these nutrients. Plasma corticosterone level was reduced and weight of the bursa of Fabricius increased by selenium or vitamin E supplementation. These nutrients may be necessary for providing the optimum thyroid conditions for activity of thyroid peroxidase.     

Thyroid function and plasma selenium in chronic uremic patients on hemodialysis treatment

It has been shown recently that Selenium (Se), an essential trace element for humans, is involved in the regulation of thyroid function, since the enzyme that catalyzes the liver conversion of the thyroid hormone T4 to the more active form T3 is a selenoenzyme. In chronic uremic patients, low blood Se levels as well as thyroid function abnormalities are often found. The present study was carried out to verify whether any correlation exists between Se levels and thyroid function, and to evaluate possible changes in hormonal pattern during Se supplementation in 10 chronic uremic patients on hemodialysis (HD) treatment. Se was supplemented orally as sodium selenite over six consecutive months. Basic plasma Se levels were significantly lower in patients than in normal controls. Right from the start of Se supplementation, plasma Se concentration promptly normalized and leveled off in the normal range throughout the study. Significant increase of FT3 and reduction of TSH levels were detected during Se supplementation. In Se-supplemented patients, a significant direct correlation was also found between reverse T3 (rT3) and TSH, and a significant inverse correlation was found between Se and TSH. Our results suggest that Se deficiency in chronic uremic patients represents a factor influencing the thyroid function and that the Se status should be determined in the evaluation of thyroid metabolism in these patients.     

Protection against reactive oxygen species by selenoproteins

Reactive oxygen species (ROS) are derived from cellular oxygen metabolism and from exogenous sources. An excess of ROS results in oxidative stress and may eventually cause cell death. ROS levels within cells and in extracellular body fluids are controlled by concerted action of enzymatic and non-enzymatic antioxidants. The essential trace element selenium exerts its antioxidant function mainly in the form of selenocysteine residues as an integral constituent of ROS-detoxifying selenoenzymes such as glutathione peroxidases (GPx), thioredoxin reductases (TrxR) and possibly selenoprotein P (SeP). In particular, the dual role of selenoprotein P as selenium transporter and antioxidant enzyme is highlighted herein. A cytoprotective effect of selenium supplementation has been demonstrated for various cell types including neurons and astrocytes as well as endothelial cells. Maintenance of full GPx and TrxR activity by adequate dietary selenium supply has been proposed to be useful for the prevention of several cardiovascular and neurological disorders. On the other hand, selenium supplementation at supranutritional levels has been utilised for cancer prevention: antioxidant selenoenzymes as well as prooxidant effects of selenocompounds on tumor cells are thought to be involved in the anti-carcinogenic action of selenium.     

Effect of selenium depletion and supplementation on the kinetics of type 1 5′-iodothyronine deiodinase and T3/T4 in rats

Presently, the effect of selenium (Se) deficiency and excess of Se (1 ppm) on the activity of selenoenzymes type 1 5′-iodothyronine deiodinase (5′-DI), glutathione peroxidase (GSH-Px), and level of thyroid hormones (T₃ and T₄) was studied in rats. Se levels in the serum and liver, T₃ and T₄ in the serum, GSH-Px levels in the liver, and 5′-DI activity in the liver/aorta/thyroid were estimated after 1, 2, and 3 mo of Se-deficient (0.02 ppm), Se-adequate (0.2 ppm), and Se-excess (1 ppm) diet feeding. All of these parameters decreased significantly in the Se-deficient group as compared to the adequate group. Within the deficient group, as the Se deficiency progressed, all of the parameters except 5′-DI decreased after 2 and 3 mo in comparison to 1-mo data. Thyroidal 5′-DI activity in Se deficiency showed the maximum increase. A significant increase was observed in all of the above parameters in the 1 ppm Se-supplemented diet group when compared with the adequate Se group; also, as the Se deposition increased within the Se-excess diet group, a significant increase was observed in all of the above parameters. However, as observed by others, the intake of excess of Se (i.e., 2 ppm in the diet) did not elevate the activities of selenoenzymes and thyroid hormones; rather, it had adverse effects. The present study concludes that Se supplementation at least up to 1 ppm enhances the selenoenzyme activities, and above this level, it may not be considered as an indicator of selenoenzyme activities.     

Effect of selenium on thyroid hormone metabolism in filial cerebrum of mice with excessive iodine exposure

The effects of supplementing selenium on thyroid hormone metabolism were studied on mice with excessive iodine exposure. The serum concentrations of thyroxine (T₄) and triiodothyronine (T₃) and the activities of iodothyronine 5′ and 5-deiodinase (D2, D3) were measured in the brain of filial mice to study the influence of selenium on thyroid hormone metabolism. Measurements were carried out on postnatal day 0, 14, and 28. It was found that selenium supplementation alleviated the adverse effects of excessive iodine on progeny. The serum TT₄ level as well as TT₄ and TT₃ concentrations and D3 activity in cerebrum of progeny decreased, whereas D2 activity increased in the cerebrum of progeny on postnatal day 0 and 14. Selenium supplementation exerted some favorable effects on thyroid hormone metabolism in cerebrum of progeny of dam with excessive iodine intake.     

Selenoproteins in human thyroid tissues

The aim of the present work was to clarify whether the activities of selenoenzymes can serve as markers for different tumors or goiters, as classified by histological criteria. The following parameters were determined: 1) selenium content of plasma (Se), 2) activities of the selenoenzymes: plasma glutathione peroxidase (plGSHPx), cytosolic glutathione peroxidase (cGSHPx), type I and type II iodothyronine deiodinases (ID-I, ID-II), thioredoxin reductase (THRR) in human thyroid tissues. The material came from follicular neoplasm, papillary carcinoma, struma nodosa, struma lymphomatosis Hashimoto, other thyroid surgery specimens, and normal tissues. There was no difference in Se nor in plGSHPx between patients and healthy volunteers. No significant differences were found for any parameter in thyroid carcinoma versus normal or goitrous thyroid tissue. In the whole group of thyroid surgery specimens the statistically significant correlations were found between ID-I and ID-II and between THRR and selenoperoxidases. Principal components analysis confirmed the above correlation and moreover revealed correlation between Se and plGSHPx, but did not detect any clear distinction between patients with the different diagnoses.     

Role of the type 2 iodothyronine deiodinase (D2) in the control of thyroid hormone signaling

Background

Thyroid hormone signaling is critical for development, growth and metabolic control in vertebrates. Although serum concentration of thyroid hormone is remarkable stable, deiodinases modulate thyroid hormone signaling on a time- and cell-specific fashion by controlling the activation and inactivation of thyroid hormone.

Scope of the review

This review covers the recent advances in D2 biology, a member of the iodothyronine deiodinase family, thioredoxin fold‐containing selenoenzymes that modify thyroid hormone signaling in a time- and cell-specific manner.

Major conclusions

D2-catalyzed T3 production increases thyroid hormone signaling whereas blocking D2 activity or disruption of the Dio2 gene leads to a state of localized hypothyroidism. D2 expression is regulated by different developmental, metabolic or environmental cues such as the hedgehog pathway, the adrenergic- and the TGR5-activated cAMP pathway, by xenobiotic molecules such as flavonols and by stress in the endoplasmic reticulum, which specifically reduces de novo synthesis of D2 via an eIF2a-mediated mechanism. Thus, D2 plays a central role in important physiological processes such as determining T3 content in developing tissues and in the adult brain, and promoting adaptive thermogenesis in brown adipose tissue. Notably, D2 is critical in the T4-mediated negative feed-back at the pituitary and hypothalamic levels, whereby T4 inhibits TSH and TRH expression, respectively. Notably, ubiquitination is a major step in the control of D2 activity, whereby T4 binding to and/or T4 catalysis triggers D2 inactivation by ubiquitination that is mediated by the E3 ubiquitin ligases WSB-1 and/or TEB4. Ubiquitinated D2 can be either targeted to proteasomal degradation or reactivated by deubiquitination, a process that is mediated by the deubiquitinases USP20/33 and is important in adaptive thermogenesis.

General significance

Here we review the recent advances in the understanding of D2 biology focusing on the mechanisms that regulate its expression and their biological significance in metabolically relevant tissues. This article is part of a Special Issue entitled Thyroid hormone signalling.     

Selenoenzymes,Laboratory Parameters,and Trace Elements in Different Types of Thyroid Tumor

This study was performed to investigate selenoenzyme activities and trace element concentrations in thyroid tissues, with reference to other parameters routinely used to characterize thyroid function. This was to reveal relevant parameters as possible additional markers of tumor grade, clinical course, and prognosis of thyroid disorders. The tissue samples were obtained during surgical treatment (total or near total thyroidectomy) of 122 patients with different types of thyroid tumor. For most of the investigated parameters in different groups of patients, we did not find statistically significant differences. In the majority of cases, thyroid benign or malignant tumors were not accompanied by significant derangement of the gland selenoenzymes and of either intrathyroidal or plasma concentration of selenium. Nevertheless, types I and II iodothyronine deiodinases were the most promising (among selenoenzymes) targets for diagnoses and possibly therapy of thyroid tumors. Higher activities of both enzymes in cases with Graves’ disease, as compared with other thyroid lesions, suggest their involvement in the pathogenesis of this condition. Patients with struna nodosa had higher levels of thyroid Zn, Cu, and Pb as compared with papillary carcinoma subjects and also a higher level of Cu than follicular carcinoma cases. The above diagnostics may play a similar role to some of the general thyroid function indices, TSH, anti-TG, anti-TPO, and calcitonin, which can partially distinguish between various thyroid tumors. In conclusion, some of selenium status markers, when accompanied with general parameters, and trace elements can serve as factors with pathophysiologic relevance and be helpful in the identification of malignant disease. Multivariate statistical methods should be employed to tackle a broad array of thyroid tumor diagnostic data in a short time. Partial least squares model and other pattern recognition methods seem to be the most appropriate methods for that task. The miniaturization of all the steps of complex analytical procedure should be developed in a way to allow its completion as sensitive, robust, and efficient for use of the small quantity of material provided by fine-needle biopsy.     

Selenium intake and cardiovascular risk: what is new?

PURPOSE OF REVIEW: Selenium is an essential element with a narrow safety margin. Adequate selenium intake is needed to maximize the activity of glutathione peroxidases and other selenoproteins. This review discusses recent experimental and epidemiologic contributions on the role of selenium for the prevention of atherosclerotic cardiovascular disease. RECENT FINDINGS: Few randomized trials have evaluated the efficacy of selenium supplementation on cardiovascular endpoints. Most trials, conducted in selenium-replete populations, found no evidence of cardiovascular protection. A meta-analysis of 13 prospective cohort studies found a moderate inverse relationship between plasma/serum selenium and coronary heart disease. The interpretation of these data is complicated, however, by potential residual confounding and publication bias. In contrast, recent data from trials of selenium-containing supplements and from epidemiologic studies suggest that chronically increased selenium intake in selenium-replete populations can induce diabetes and maybe also hypercholesterolemia. SUMMARY: Current evidence is insufficient to support a protective role for selenium in cardiovascular prevention. Large high-quality randomized controlled trials and observational studies are needed across populations with different levels of selenium intake. Furthermore, subjects living in regions with high selenium intake should be aware that selenium supplements may increase their risk of diabetes and hypercholesterolemia.     

Selenium and thyroid function in infants, children and adolescents

Selenium is an integral component of the enzymes glutathione peroxidase (GPx) and iodothyronine deiodinases. Although selenium nutrition could conceivably affect thyroid function in infants, children and adolescents, available data suggest that the effect of selenium deficiency on thyroid function is relatively modest. In patients with isolated selenium deficiency (such as patients with phenylketonuria receiving a low-protein diet), peripheral thyroid hormone metabolism is impaired but there are no changes in thyrotropin (TSH) or clinical signs of hypothyroidism, suggesting that these patients are euthyroid. Selenium supplementation may be advisable to optimize tissue GPx activity and prevent potential oxidative stress damage. In areas where combined selenium and iodine deficiencies are present (such as endemic goiter areas in Central Africa), selenium deficiency may be responsible for the destruction of the thyroid gland in myxoedematous cretins but may also play a protective role by mitigating fetal hypothyroidism. In these areas, selenium supplementation should only be advocated at the same time or after iodine supplementation. In patients with absent or decreased production of thyroid hormones and who rely solely on deiodination of exogenous L-thyroxine for generation of the active triiodothyronine (such as patients with congenital hypothyroidism), selenium supplementation may optimize thyroid hormone feedback at the pituitary level and decrease stimulation of the residual thyroid tissue.     

Selenium Supplementation in Autoimmune Thyroiditis Female Patient—Effects on Thyroid and Ovarian Functions (Case Study)

Recently published biochemical data suggest the significant role of selenium compounds as the adjuvants combined with L: -thyroxine therapy, which can reduce antithyroid peroxidase antibodies' (TPOAb) levels in patients with Hashimoto disease. The study was undertaken to document in a more detailed way the changes in parameters expressing the thyroid and ovarian function brought about by selenium supplementation (50-100 microg/day) in a woman undergoing autoimmune thyroiditis (AIT) therapy. This prospective observational case study lasted for 14 months plus additional 5 months as a follow-up period. Parameters reflecting selenium status, thyroid metabolism, and sex hormones secretion were determined at the onset and end of the study period, as well as in some of its middle points. During the supplementation trial, serum selenium (Se) increased by 45% and plasma glutathione peroxidase (GPX3) by 21%. TPOAb decreased by 76%. All other parameters also fluctuated during the supplementation period, but all results were always within normal physiological ranges. After withdrawal of the supplementation, the sharp fall of Se and GPX3 promptly occurred, and this phenomenon was accompanied with a marked increase in TPOAb. This report stresses the importance of selenium supplementation in AIT treatment. However, the efficiency and durability of the effect of Se supplementation on the TPOAb titer remain an open question. The clarification of mechanism(s) underlying Se interaction with autoimmune processes should throw new light on this issue.     

The importance of selenium and the effects of its deficiency in animal health

Selenium (Se) is an essential trace element in animal nutrition, and exerts multiple actions related to animal production, fertility and disease prevention. Glutathione peroxidase (GSH-PX) enzyme was the first proven selenoenzyme that can prevent oxidative damage of the cellular membrane. Actually more than 30 selenoenzymes have been described and a hierarchy process for expression in the animal has been established. White muscle disease (WMD) was the first recognized condition associated with Se deficiency. WMD causes new born mortality, especially in ruminants, and impaired production condition in growing and adult animals. Selenium is critical to thyroid hormone synthesis and it is also very important for converting T4 (thyroxin inactive form) to T3 (active form). A good immune response requires Se too. Selenium status in soil, plants and animal blood and tissue can be used in the diagnosis of Se deficiency. Diverse forms of Se supplements are available, but many factors affect their activity and efficacy, such as its chemical form and animal's health and production condition. The relationships between foetus Se metabolism and pregnant dam Se status are critical for productivity and need further research.     

Serum selenium,glutathione peroxidase,lipids, and human liver microsomal enzyme activity

This study evaluated selenium status in relation to lipid peroxidation, liver microsomal function, and serum lipids in humans. Serum selenium concentration, glutathione peroxidase (GSH-Px) activity, liver microsomal enzyme activity, assessed by plasma antipyrine clearance (AP-CL) rate, and serum lipids were determined in 23 healthy subjects in a double-blind placebo-controlled trial of selenium supplementation. The low selenium concentration (74.0±14.2 μg/L, mean±SD) is attributable to the low selenium content of the diet. Subjects with the lowest selenium levels (n=11) had reduced serum GSH-Px activity, AP-CL rate, high-density lipoprotein cholesterol (HDL-C), and total cholesterol (T-C) as compared with subjects with higher selenium concentrations (n=12). Low AP-CL rates were associated with low HDL-C: T-C ratios. Selenium supplementation, 96 μg/d for 2 wk, increased serum selenium, GSH-Px activity, and the HDL-C: T-C ratio. The results suggest that a low serum selenium level is associated with a decrease in liver microsomal enzyme activity and serum HDL-C and T-C concentrations. Selenium supplementation in subjects with low serum selenium may favorably influence relations between serum lipoproteins connected with the development of atherosclerotic vascular disease.     

Selenium supplement alleviated the toxic effects of excessive iodine in mice

The relationship between the iodine intake level of a population and the occurrence of thyroid diseases is U-shaped. When excessive iodine is ingested, hypothyroidism or hyperthyroidism associated with goiter might develop. The aim of the study was to evaluate the effect of Se supplementation on the depression of type 1 deiodinase (D1) and glutathione peroxidase (GSHPx) activities caused by excessive iodine. D1 activity was assayed by the method with ¹²⁵I-rT₃ as a substrate. Compared to the effect of iodine alone, iodine in combination with selenium increased the activities of D1 and GSHPx. The addition of selenium alleviated the toxic effects of iodine excess on the activities of D1 and GSHPx.     

Selenium, the element of the moon, in life on earth

The present status of selenium biochemistry is reviewed with particular emphasis on biomedical problems related to the selenium status of humans and experimental animals. Historical milestones of selenium biochemistry starting from the identification of the first selenoenzymes up to the elucidation of prokaryotic and eukaryotic selenoprotein biosynthesis are compiled. Topical hypotheses on the biological role of selenium in general and of individual selenoproteins in respect to antioxidant defense, redox regulation of metabolic processes, thyroid function, spermatogenesis, oncogenesis, and atherogenesis are critically evaluated.     

The Influence of Supplements of Selenite,Selenate and Selenium Yeast on the Selenium Status of Dairy Heifers

The aim of the study was to define possible differences between selenite, selenate and selenium yeast on various aspects of selenium status in growing cattle. Twenty-four Swedish Red and White dairy heifers were fed no supplementary selenium for 6 months. The basic diet contained 0.026 mg selenium/kg feed dry matter (DM). After the depletion period the animals were divided into 4 groups; group I–III received 2 mg additional selenium daily as sodium selenite, sodium selenate, and a selenium yeast product, respectively. Group IV, the control group, received no additional selenium. The total dietary selenium content for groups I–III during the supplementation period was 0.25 mg/kg DM. After the depletion period the mean concentration of selenium in blood (640 nmol/l) and plasma (299 nmol/l) and the activity of GSH-Px in erythrocytes (610 µkat/l) were marginal, but after 3 months of supplementation they were adequate in all 3 groups. The concentration of selenium in blood and plasma was significantly higher in group III than in groups I and II, but there was no significant difference between groups I and II. The activity of GSH-Px in erythrocytes did not differ between any of the supplemented groups. The animals in the control group had significantly lower concentrations of selenium in blood and plasma and lower activities of GSH-Px in erythrocytes than those in the supplemented groups. The activity of GSH-Px in platelets was also increased by the increased selenium intake. There was no difference in the concentration of triiodothyronine (T3) between any of the groups, but the concentration of thyroxine (T4) was significantly higher in the unsupplemented control group.