Thyroid function. John R Arthur* and Geoffrey J Beckett*

Transcription

1 Thyroid function John R Arthur* and Geoffrey J Beckett* *Division of Micronutrient and Lipid Metabolism, Rowett Research Institute, Aberdeen, UK f University Department of Clinical Biochemistry, The Royal Infirmary, Edinburgh, UK Correspondence to: DrJR Arthur, Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB21 9SB. UK Normal thyroid status is dependent on the presence of many trace elements for both the synthesis and metabolism of thyroid hormones. Iodine is most important as a component of the hormones, thyroxine and 3,3',5-tri-iodothyronine (T 3 ) and iodine deficiency may affect approximately one billion people throughout the world. Selenium is essential for normal thyroid hormone metabolism being involved with selenium-containing iodothyronine de-iodinases that control the synthesis and degradation of the biologically active thyroid hormone, T 3. Additionally, selenoperoxidases and thioredoxin reductase protect the thyroid gland from peroxides produced during the synthesis of hormones. The roles of iron, zinc and copper in the thyroid are less well defined but sub- or supraoptimal dietary intakes of all these elements can adversely affect thyroid hormone metabolism. The importance of the thyroid gland in maintaining human health is well recognised. Since iodine is a crucial constituent of thyroid hormones, it is not surprising that thyroid dysfunction is very common in geographical areas of iodine deficiency. However, even when this trace element is present in adequate supply, thyroid disease is present in 3-5% of the population. Furthermore, the regulated supply of thyroid hormone to specific tissues is crucial during fetal development 1. There has been much research into thyroid gland biochemistry and control, both under normal conditions and when thyroid hormone metabolism is abnormal. Thyroid hormone metabolism and function is a complex process and includes synthesis of the hormones and prohormones in the thyroid gland, their inter-conversion by iodothyronine de-iodinases in the organs of the body, and the binding of T 3 to nuclear receptors to control gene expression 1. Thyroid hormone metabolism and action are dependent on a multitude of enzymes and proteins and the expression or function of many of these can be influenced by trace elements. The elements most closely associated with the thyroid are iodine and selenium and their roles in thyroid hormone homeostasis are relatively well defined. In addition, levels of iron, zinc and copper in the diet have British Medical Bulletin 1999; 55 (No. 3): The British Council 1999

2 Trace elements and thyroid function all been shown to influence thyroid metabolism, although the mechanism of these effects is far from clear. This review will consider the role of these trace elements in maintaining normal thyroid hormone metabolism and in preventing dysfunction. Thyroid disorders and iodine The effects of excess iodine intake Iodine is the trace element that is most likely to influence thyroid function since it is an integral constituent of thyroid hormones. The recommended daily requirement is (ig. Iodine deficiency and its associated clinical manifestations such as goitre, hypothyroidism, and impaired mental and physical development (cretinism) present a major world health problem 2. The most common thyroid disorders in areas of adequate iodine intake are the autoimmune thyroid diseases and nodular goitre. In these areas, hypothyroidism is commonly due to Hashimoto's thyroiditis or results from atrophy of the gland (primary atrophic hypothyroidism). Graves' disease, solitary nodular goitre, multinodular goitre and autoimmune thyroiditis comprise the vast majority of patients who present with an over active gland. The increased use of iodine supplements in salt and bread and of iodine containing food preservatives has resulted in a marked increase in iodine intake in some countries including the US, Great Britain and Scandinavia. This may be 4 times the recommended daily intake in certain regions and there is now compelling evidence that such excess can give rise to thyroid dysfunction 3. Following the acute administration of iodine, thyroid hormone production is transiently inhibited (Wolff-Chaikoff effect). This effect has been used clinically to render patients euthyroid prior to thyroid surgery, by giving potassium iodide in combination with propranolol 4. Indeed, before the discovery of antithyroid drugs, patients with Graves' disease were treated with high doses of iodide. In areas that have been previously iodine deficient, iodine supplements frequently give rise to hyperthyroidism which commonly manifests as multinodular goitre. This has led to the suggestion that, in these patients, there were pre-existing autonomous micronodules or macronodules but that the hyperthyroidism was masked because of low iodine supply, only to be uncovered when iodine was given 3. There is evidence, particularly in animals, that high levels of iodine can be a contributing factor in the development of autoimmune thyroid British Medical Bulletin 1999,55 (No. 3) 659

3 Micronutrients in health and disease The effects of low iodine intake Selenium disease. How iodine produces such an effect is unclear but it may involve the production of iodine-rich thyroglobulin, which may be particularly immunogenic. Some, but not all, studies in humans have shown that the presence of serum anti-microsomal antibodies is more prevalent in areas adequate in iodine than in areas of mild iodine deficiency 3. A role for iodine in modifying the effects of growth factors is also possible. For example, epidermal growth factor receptors in thyrocytes are greatly diminished in the presence of iodine. Up to one billion people live in areas where they are at risk from the effects of iodine deficiency. Of these, approximately 211 million have goitre and about 20 million have impaired brain function. There are many iodine deficiency diseases (IDDs) which can affect human subjects of all ages, but they are particularly severe in fetal development and during stages of rapid growth in infants giving rise to cretinism 1. The clinical outcomes of iodine deficiency thus range from mild hypothyroidism to severe endemic cretinism. Cretinism manifests in two quite different forms. In myxoedematous cretinism, there is severe hypothyroidism and stunted growth but the prevalence of goitre is much lower than that in non-cretins 1. There is often thyroid atrophy with no thyroid tissue being palpable. Typically plasma thyroxine (T 4 ) and 3,3',5-tri-iodothyronine (T 3 ) are very low whilst thyrotrophin (TSH) is extremely high. Thyroid destruction in these subjects may start in utero or soon after birth. In neurological cretinism, which is more common than the myxoedematous form of the disease, mental deficiency may be accompanied by neurological problems including hearing and speech defects. Growth and thyroid function are usually normal. Whilst the pathogenesis of cretinism is unclear, there is no doubt that iodine deficiency plays the major role since the diseases may be prevented by iodine supplementation of salt used for cooking by an injection of iodised oil. However, other factors, which may vary with geographical location, also influence the prevalence and type of the disease. These may include goitrogens and selenium status 1-5. Concentrations of selenium are higher in the thyroid than in any other tissues other than liver and kidney, indicating that it has important functions within the gland. In 1987, it became apparent that selenium could exert marked effects on thyroid hormone metabolism in tissues 660 British Medical Bulletin 1999,55 (No 3)

4 Trace elements and thyroid function other than the thyroid 6. It, therefore, differs in its action from iodine whose effects on thyroid status are largely confined to the thyroid. Selenium is an essential component of many selenoproteins that regulate thyroid hormone synthesis, preserve thyroid integrity in conditions of marked oxidative stress, and control hormone metabolism in nonthyroidal tissues where the prohormone T, is converted to biologically active T 3 or its inactive isomer rt 3 5 " 7. Selenium and thyroid hormone de-iodination The discovery of increased T 4 and decreased T 3 concentrations in plasma from selenium deficient rats and cattle provided the first demonstration that selenium could affect thyroid hormone metabolism. These changes were associated with considerable decreases in hepatic and renal type I iodothyronine de-iodinase (IDI) activity which converts T 4 to T 3. These observations led to the suggestion that IDI was a selenoenzyme; this was subsequently proved by partial purification and cloning. The selenium present as selenocysteine in IDI is coded for by a TGA stop codon 5 " 7. The levels of T 4, T 3 and rt 3 are also regulated by two further deiodinases type II (IDII) and type HI (IDHI) that also contain selenium. IDII shows quite a different tissue distribution from EDI and is found principally in brain, central nervous system, brown adipose tissue and the pituitary. The metabolic function of these tissues depends on T 3 which is 'locally produced' from T 4 by de-iodination catalysed by IDII. This process provides a very sensitive mechanism by which thyroid hormone metabolism can be finely regulated in specific tissues 5 ' 8. To further refine the intracellular control of T 3 at a tissue level, IDm catalyses inner ring de-iodination, which converts T 4 to the inactive rt 3 and also catabolises T 3 to produce the inactive T 2 Thus co-ordinated expression of the three de-iodinases can regulate the concentration of T 3 that is presented to specific tissues 5 ' 8. The ontogeny of the de-iodinases suggests that they are switched on and off in utero and that this may be crucial in regulating and coordinating fetal development in animals and humans. However, the ontogeny of IDI in humans is quite different from that in the rat or the sheep and thus data from animal models should be interpreted with caution 9. Selenium deficiency and thyroid hormones In selenium deficiency, there is a stria hierarchy of selenium supply to specific tissues and also to different selenoenzymes within a tissue. British Medial Bulletin 1999,55 (No. 3) 661

5 Micronutrients in health and disease Concentrations of selenium and selenoenzymes are greatly decreased in liver, kidney and muscle, whereas those in the brain and endocrine organs such as the thyroid gland are less affected. Indeed, the expression of thyroidal IDI can even increase in selenium-deficient rats 5. Within different organs, specific selenoproteins are retained at the expense of others, presumably to preserve the most important aspects of metabolism in selenium deficiency 10. For example, in the selenium-deficient rat, EDI is better retained than cytoplasmic glutathione peroxidase in thyroid, liver and kidney, presumably in order to preserve thyroid function and iodothyronine de-iodination and to limit changes in plasma T 4, T 3 and TSH 5. If selenium deficiency is continued over more than one generation in rats, plasma T 4 concentrations may not be increased. The reasons for this compensation are not fully understood but it may be that homeostasis is achieved by increased metabolism of T 4 by IDIIF. Selenium, thyroid hormone synthesis and oxdidative damage in the thyroid The functional unit for thyroid hormone synthesis is the follicle, a structure made up of clusters of thyrocytes. These synthesise a high molecular weight protein thyroglobulin, which is then exported and stored as a colloid in the follicular lumen. Synthesis of thyroid hormones requires iodination of tyrosyl residues on thyroglobulin and subsequent coupling of these iodinated derivatives. These reactions take place within the follicular lumen at the surface of the apical membrane and not within the thyrocyte 1-5. Iodination of tyrosyl residues on thyroglobulin requires the generation of hydrogen peroxide in high concentrations and also the action of thyroid peroxidase, an enzyme located on the luminal side of the apical membrane. The generation of H 2 O 2 is probably crucial for the control of thyroid hormone synthesis and is regulated by a complex network of interacting second messenger systems. The thyrocyte is thus exposed to high concentrations of H 2 O 2 and consequently of toxic lipid hydroperoxides. However, the availability of H 2 O 2 and the subsequent peroxidative damage are decreased by selenoenzyme systems in the thyrocytes which may be involved in regulating hormone synthesis. Extracellular glutathione peroxidase is secreted by thyrocytes into the follicular lumen and may regulate the availability of H 2 O 2 for thyroid peroxidase at the apical membrane 11. The intracellular glutathione peroxidases may detoxify H 2 O 2 and lipid hydroperoxides within the thyrocyte and protect the gland from oxidative damage. It is significant that glutathione peroxidase activity is decreased by over 99% in the liver but by only 50% in the thyroid of selenium-deficient rats British Medical Bulletin 1999,55 (No. 3)

6 Trace elements and thyroid function Thioredoxin reductase may also protect against the damaging effects of H 2 O 2 in the thyrocyte. This selenoenzyme can also act a growth factor and may have several important and diverse biological effects on the cell, acting either directly or through thioredoxin. Signalling pathways that are associated with increased H^C^ production, stimulate synthesis of thioredoxin reductase in the thyrocyte. As well as having a potential antioxidant role, thioredoxin reductase and thioredoxin can provide reducing equivalents for the activity of IDI 12. It is less easy to demonstrate effects of selenium status on plasma TSH and T 3 concentrations in the heterogeneous human population. Nevertheless, low selenium status has been associated with changes in serum thyroid hormone concentrations with an inverse correlation between plasma selenium and T 4 concentrations consistent with low IDI activity in low selenium status in elderly subjects. Moreover, selenium supplementation decreased plasma T 4, but plasma T 3 and TSH concentrations were unaffected by selenium status and treatment 13. Plasma T 4 concentrations are also elevated in subjects with phenylketonuria and this has been attributed to low selenium status arising from consumption of low protein diets. Selenium supplementation of these patients decreased plasma T 4 concentrations, consistent with an increase in hepatic IDI activity 14. There is some evidence that de-iodination of thyroid hormones is impaired in humans receiving large amounts of selenium, but this has not been studied in detail 15. Selenium and the sick euthyroid syndrome In euthyroid patients with severe illness not attributable to a thyroidal cause (non-thyroidal illness: NTI), plasma concentrations of T 3 are often markedly decreased. This condition is very common in hospitalised patients and is often referred to as the 'sick euthyroid' or 'low T 3 ' syndrome 16. Such patients often also have decreased concentrations of plasma selenium and this has led to the suggestion that the low T 3 may result from impaired expression of hepatic IDI 17. Whilst selenium supplementation can attenuate the fall in T 3 in NTI, the syndrome has complex effects which span the whole of the hypothalamic-pituitary thyroid axis The hormone pattern in NTI is different from that in simple selenium deficiency, where the most marked changes are in plasma T 4 not plasma T 3. In contrast, in many patients with NTI, plasma T 3 concentrations may be very low but with little change in plasma T The fall in plasma selenium, which occurs in acute illness, may be a negative acute phase response 19. British Medical Bulletin 1999;55 (No. 3) 663

7 Micronutrients in health and disease Combined selenium and iodine deficiencies Several animal studies have been conducted to assess the effects of combined selenium and iodine deficiencies. Selenium deficiency can exacerbate the hypothyroidism and goitre due to iodine deficiency. In contrast, selenium deficiency can increase or decrease brain IDII activity, which is a crucial enzyme for the supply of T 3 required for normal brain development. Such animal studies illustrate the complex interactions between selenium and iodine deficiency and provide some basis for interpretation of the effects of the deficiencies in humans 5. The most comprehensive studies of the roles of selenium and iodine deficiency in human thyroid metabolism have been carried out in Zaire in areas where there has been a high incidence of myxoedematous cretinism. Cretinism was endemic in particular geological areas that coincided with areas of low selenium soil content. This led to the proposal that the thyroid atrophy was due to loss of protection from toxic levels of hydrogen peroxide. In low selenium conditions the thyrocyte was hypothesised to express less glutathione peroxidase and hence to lose protection from the peroxidative stress generated by the gland in iodine deficiency 5 ' Further support for this hypothesis has come from selenium supplementation trials in the endemic goitre belt of Northern Zaire. Serum selenium levels in children and cretins in this area were much lower than levels in adults living in villages on the border of the endemia area and, indeed, were similar to levels in severely selenium-deficient children in China. When schoolchildren and cretins were given 50 ug Se/day (as selenomethionine) for 2 months, serum selenium and red cell glutathione peroxidase returned to normal. In the cretins, there was also a marked fall in serum total T 4 levels from an initial mean of 12.8 nmol/1 to 2.8 nmol/1, accompanied by a rise in TSH. These T 4 concentrations can be compared with a lower limit of normal of approximately 60 nmol/1 in many European countries. In the normal schoolchildren, selenium supplementation also reduced serum T 4 but without a concomitant rise in TSH concentrations 5-20 ' 21. The fall in serum T 4, which occurred, on giving selenium may have resulted from an increase in the expression of hepatic IDI, which would increase the metabolism of plasma T 4 to T 3. In the normal children with adequate amounts of functional thyroid tissue, the gland would be able to meet the subsequent increased requirement for T 4 synthesis. However, in cretins there was probably insufficient functional thyroid tissue to meet the increased requirement for thyroid hormone 5 ' 20. These studies led to the hypothesis that selenium deficiency may actually protect the brain from some of the detrimental effects of iodine deficiency. Since the brain depends on plasma T, for production of T 3, which is 664 British Medial Bulletin 1999,55 (No. 3)

8 Trace elements and thyroid function essential for normal brain development, the increased plasma T 4 concentrations in selenium deficiency may help to protect the fetal brain during development. Thus it is now considered unethical to supplement a population deficient in both selenium and iodine with selenium alone because of the theoretical risk of exacerbating abnormal brain development 20. Selenium deficiency can not always totally explain the onset of endemic cretinism in iodine-deficient areas. In certain provinces of Africa where the degree of selenium deficiency is similar to that in Zaire but the iodine deficiency is more severe, myxoedematous cretinism does not occur. Similarly in areas of Tibet where iodine and selenium deficiencies are both severe the incidence of myxoedematous cretinism is very low and neurological cretinism predominates 22. Thus, combined iodine and selenium deficiencies are not sufficient alone to explain the high frequency of myxoedematous cretinism in Central Africa. The possible role of other factors such as thiocyanates must again be considered. The effects of thyroid disease on selenium status Zinc and thyroid function Thyroid status appears to have an influence on selenium metabolism. Patients with hyperthyroidism have lower levels of plasma selenium and red cell glutathione peroxidase than do euthyroid subjects, regardless of the cause of the hyperthyroidism. When treatment restores a euthyroid state, these markers of selenium status also return to normal 23. Low selenium status is not the cause of the hyperthyroidism but rather the hyperthyroidism diminishes the size of the plasma pool of selenium, possibly by modifying the half-life of the selenoproteins. There are indications that zinc is also important for normal thyroid homeostasis. Its roles are complex and may include effects on both the synthesis and mode of action of the hormones. Thyroid hormone binding transcription factors, which are essential for modulation of gene expression, contain zinc bound to cysteine residues 24. However, it is not known whether dietary zinc deficiency has a direct effect on this aspect of thyroid hormone metabolism. In cultured cells, very strong chelators of zinc are required to influence binding of transcription factors to DNA. In the thyroid gland itself, transcription factor 2, which interacts with the promoters for the thyroglobulin and thyroperoxidase genes, is a zinc-containing protein. The binding of transcription factor 2 is affected by redox state, but again it is not known whether this can be changed by dietary zinc intake 24. British Medical Bulletin 1999;55 (No. 3) 665

9 Micronutrients in health and disease The role of zinc in thyroid metabolism has been investigated in animals but with conflicting results. Zinc deficiency can decrease plasma T 3 levels but in other studies no effect was observed. Similarly, zinc deficiency has been reported to both increase and lower hepatic IDI activity 25 * 26. These conflicting reports may reflect differences in the severity of the zinc deficiency and subsequent effects on food intake, since this by itself would decrease plasma T 3 levels and hepatic IDI activity. Marginal zinc deficiency appears to have no influence on the effects of iodine deficiency in rats 27. However, in a more complex study with combined selenium, iodine and zinc deficiencies, there was an interaction between selenium and zinc deficiencies on thyroid follicle cell architecture, which was compatible with apoptosis 28. Zinc has also been connected with thyroid metabolism in humans. Many patients with Down's syndrome have low serum zinc levels which have been associated with diarrhoea and resultant malabsorption of zinc. Those patients also had raised plasma TSH concentrations and autoantibodies to thyroglobulin. When patients with Down's syndrome were supplemented with zinc, plasma TSH levels were decreased to normal and plasma reverse T 3 was increased to normal 29. In other studies, low zinc status was associated with decreased thyroid hormone levels. The biochemical basis of such changes has yet to be established 30. Other trace elements and the thyroid Acknowledgement Iron and copper status have also been linked to decreased plasma T 3 concentrations in animals and man 31 ' 32. As with zinc, these changes have not yet been associated with specific changes in enzymes involved in thyroid hormone metabolism. It remains to be determined whether the changes in thyroid metabolism are a direct result of the iron and copper deficiencies or a non-specific response to poor health. JRA is grateful to the Scottish Office Agriculture Environment and Fisheries Department for financial support. References 1 Hetzel BS, Wellby ML. Iodine. In: O'Dell BL, Sunde RA (Eds) Handbook of Nutritionally Essential Mineral Elements, New York: Marcel Delcker, 1997; Delange FM, Ermans AM. Iodine deficiency. In: Braverman LE, Utiger RD (Eds) Werner and Ingbar's The Thyroid, 7th edn. Philadelphia: lippincot-raven, 1996; British Medial Bulletin 1999,55 (No. 3)

10 Trace elements and thyroid function 3 Roti E, Vagenakis AG,. Effects of excess iodine. Braverman LE, Uriger RD (Eds) Werner and Ingbar's The Thyroid, 7th edn. Philadelphia: Lippincot-Raven, 1996; Feek CM, Sawers SA, Irvine J, Ratcliffe WA, Toft AD. Combination of potassium iodide and propranolol in preparation of patients with Graves' disease. N Engl J Med 1980; 302: Arthur JR, Beckett GJ, Mitchell JH. The interactions between selenium and iodine deficiencies in man and animals. Nutr Res Rev 1999; 12: Beckett GJ, Arthur JR.. The lodothyronine deiodinases and 5'-deiodination. Baillieres Clin Endocrinol Metab 1994; 8: Larsen PR, Berry MJ. Nutritional and hormonal regulation of thyroid hormone deiodinases. Annu Rev Nutr 1995; 15: St Germain DL, Galton VA. The deiodinase family of selenoproteins. Thyroid 1997; 7: Richard K, Hume R, Kaptein E et al. Ontogeny of iodothyronine deiodinases in human liver. / Clin Endocrinol Metab 1998; 83: Bermano G, Nicol F, Dyer JA et al. Tissue-specific regulation of selenoenzyme gene expression during selenium deficiency in rats. Biochem ] 1995; 311: Howie AF, Walker SW, Akesson B, Arthur JR, Beckett GJ. Thyroidal extracellular glutathione peroxidase: a potential regulator of thyroid-hormone synthesis. Biochem } 1995; 308: Howie AF, Arthur JR, Nicol F, Walker SW, Beech SG, Beckett GJ. Identification of a 57- kilodalton selenoprotein in human thyrocytes as thioredoxin reductase and evidence that its expression is regulated through the calcium-phosphoinositol signalling pathway. / Clin Endocrinol Metab 1998; 83: Olivieri O, Girelli D, Azzini M et al. Low selenium status in the elderly influences thyroid hormones. Clin Sci 1995; 89: Calomme MR, Vanderpas JB, Francois B et al. Thyroid function parameters during a selenium repletion depletion study in phenylketonuric subjects. Experientia 1995; 51: Bratter P, Negretti de Bratter VE. Influence of high dietary selenium intake on thyroid hormone level in humans. / Trace Elem Med Biol 1997; 10: Beckett GJ, Wilkinson E. Thyroid hormone metabolism and thyroid function tests in nonthyroidal illness. CPD Bull 1998; 1: Berger MM, Lemarchand-Beraud T, Cavadini C, Chiolero R. Relationship between selenium status and the low T 3 syndrome after major trauma. Intensive Care Med 1996; 22: Wardle CA, Berger MM, Raymond MJ et al. Early intravenous selenium supplements modulate serum thyroid hormone concentrations after major trauma. In: Association of Clinical Biochemists (Pub). Proc XVI lnt Congress Clin Chem 1996; Abstract 352 p Nicol C, Herdman J, Sattar N et al. Changes in the concentration of plasma selenium after minor elective surgery: further evidence for a negative acute phase response. Clin Chem 1998; 44: Vanderpas JB, Contempre B, Duale M et al. Selenium deficiency mitigates hypothyroxinemia in iodine-deficient subjects. Am J Clin Nutr 1993; 57: S Vanderpas JB, Dumont JE, Contempre B, Diplock AT. Iodine and selenium deficiency in Northern Zaire. Am J Clin Nutr 1992; 56: Morenoreyes R, Suetens C, Mathieu F et al. Iodine and selenium deficiency in Kashin Beck disease (Tibet). N Engl] Med 1998; 339: Beckett GJ, Peterson FE, Choudhury K et al. Inter-relationships between selenium and thyroid hormone metabolism in the rat and man. / Trace Elem Dis 1991; 5: Civitareale D, Saiardi A, Falasca P. Purification and characterization of thyroid transcription factor 2. Biochem ] 1994; 304: Kralik A, Eder K, Kirchgessner M. Influence of zinc and selenium deficiency on parameters relating to thyroid hormone metabolism. Horm Metab Res 1996; 28: Lukaski HC, Hall CB, Marchello MJ. Impaired thyroid hormone status and thermoregulation during cold exposure of zinc-deficient rats. Horm Metab Res 1992; 24: Smit JGG, Van der Heide D, Van Tintelen G, Beynen AC, Thyroid function in rats with iodine deficiency is not further unpaired by concurrent, marginal zinc deficiency. Br ] Nutr 1993; 70: Ruz M, Codoceo J, Galgani J et al. Single and multiple selenium-zinc-iodine deficiencies affect rat thyroid metabolism and ultrastructure. J Nutr 1999; 129: British Medial Bulletin 1999;S5 (No. 3) 667

11 Micronutrients in health and disease 29 Kanavin O, Scott H, Fausa O, Ek J, Gaarder PL, Brandtzaeg P Immunological studies of patients with Down's syndrome. Measurements of autoantibodies and serum antibodies to dietary antigens in relation to zinc levels. Acta Med Scand 1998; 224: Licastro F, Mocchegiani E, Masi M, Fabris N. Modulation of the neuroendocrine system and immune functions by zinc supplementation in children with Down's syndrome. / Trace Elem Electro Health Dis 1993; 7: Beard JL, Brigham DE, Kelley SK, Green MH, Plasma thyroid hormone kinetics are altered in iron-deficient rats J Nutr 1998; 128: Ohn KL, Walter RM, Keen CL. Copper deficiency affects selenoglutathione peroxidase and selenodeiodinase activities and antioxidant defense in weanling rats. Am J Clin Nutr 1994; 59: British Medical Bulletin 1999;55 (No. 3)