Molecular and Cellular Endocrinology, 0 1991 Elsevier Scientific Publishers
MOLCEL
Cl93
81 (1991) C193-Cl95 Ireland, Ltd. 0303.7207/91/$03.50
02638
At the Cutting Edge
Cretinism, thyroid hormones and selenium B. Contemprk, J. Vanderpas and J.E. Dumont I.R. I.B.H. N., lJnk:ersite Libre de Bruxelles3 B-1070 Brussels, Belgium (Accepted
Key words: Selenium
deficiency;
Iodine
deficiency;
Cretinism;
Thyroid metabolism and the epidemiology of selenium deficiency would at first sight appear to be clearly distinct fields. Recently, however, they have converged as a result of investigations linking the molecular biology of thyroid hormone metabolizing enzymes and the pathogenesis of endemic cretinism. Among other things, these results have very practical consequences for the prophylaxis and treatment of both iodine and selenium deficiencies *. In terms of epidemiology and pathophysiology the story begins with a study of endemic cretinism in Central Africa. Endemic cretinism is a disease characterized by mental retardation, and occurs in areas of severe iodine deficiency. In Northern Zaire, it was found that the endemic cretins were myxoedematous (hypothyroid) and that their characteristic deficits (both developmental and in terms of mental retardation) were proportional to the severity of their hypothyroidism (Dumont et al., 1963). The clinical aspects of the disease were similar to those of congenital hypothyroidism, and thus could be explained by thyroid deficiency starting around birth (Vanderpas et al., 1986). Endemic cretins, in contrast with the general population, had no goitre and in fact were char-
Address for correspondence: Dr. B. Contempre, I.R.I.B.H.N., Universite Libre de Bruxelles, Campus Erasme, Bldg. C, Route de Lennik 808, B-1070 Brussels, Belgium. * WHO - ICCIDD Consultation on Interrelationship between Iodine Deficiency, Selenium Deficiency and Thyroid Hormones (Aberdeen, March 25-27th 19911.
29 July 19911
Thyroid
hormone
metabolism
acterized by a small thyroid with a small, rapidly turning-over iodine pool (Dumont et al., 1969). The cause of the degeneration of the gland remains unknown. Studies of other areas of endemic goitre revealed another form of cretinism in which thyroid function was normal, but where neurological deficits such as deaf-mutism were prevalent (Delong et al., 1985). Why iodine deficiency should lead to such different syndromes in different areas, and why the thyroid is destroyed in myxoedematous cretinism, have remained mysteries for 20 years. From studies of thyroid biochemistry we know that relatively high levels of H,O, are generated in the gland, particularly in response to thyrotropin (Corvilain et al., 1991). This H,O, serves as substrate for the thyroperoxidase that catalyzes iodide oxidation and binding to thyroglobulin and the oxidative coupling of iodotyrosines in iodothyronines. In leucocytes, H,O, is known to be toxic (Rous et al., 1980); it might therefore be possible that under conditions of high thyroid stimulation, such as normally occurs at birth and to a greater extent in iodine deficient areas, such H,O, might kill the thyroid cells if protective mechanisms were deficient (Goyens et al., 1987). H,O, is detoxified by glutathione peroxidase (an enzyme known to contain selenium) and catalase; other related 0, radicals such as O;- are detoxified by superoxide dismutases (one containing Zn2+, another Cu2+) (Ganther et al., 1976; Carmagnol et al., 1983; Levander et al., 1983). We therefore decided to ascertain the Se, Cu and Zn status of populations in an African en-
Cl94
demic goitre area, the island of Idjwi, on Kivu Lake. The results were clearcut, and showed that there was severe selenium deficiency and consequent glutathione peroxidase cell depletion in this area, where myxoedematous endemic cretinism is prevalent (Goyens et al., 1987). Subsequent and more extensive studies revealed a belt of severe selenium deficiency in Central Africa (Vanderpas et al., 1990), further suggesting a link between Se deficiency and thyroid disease. Quite independently, biochemists working on the deiodination of the prohormone thyroxine to the active hormone T, (3,5,3’-triiodothyronine) or the inactive derivative rT, (3,3’,5’-triiodothyronine) found that the nutritional status of the experimental animal modulated this metabolism. Nutritionists working on trace element deficiencies then found that T4 to T, deiodination, and the activity of the liver 5’-deiodinase type I that catalyzes the reaction, were much depressed in selenium deficient animals (Beckett et al., 1987, 1989; Arthur and Beckett, 1989). The explanation of these findings came earlier this year, when this 5’-deiodinase was found to be a selenium-containing enzyme (Arthur et al., 1990; Behne et al., 19901, and the cloning of the enzyme demonstrated a selenocysteine in its structure coded by a triplet which normally yields a stop codon (Berry et al., 1991). An identical mechanism leads to the insertion of selenocysteine into glutathione peroxidase (Chambers et al., 1986). It is not yet known whether the other 5’-deiodinase type II, located mainly in the brain and catabolizing T4 to T, exclusively, is also a selenium enzyme; an effect of Se on the activity of this deiodinase group at central level cannot, however, be excluded (Beckett et al., 1989). Thus selenium deficiency clearly can cause impairment of active thyroid hormone generation in peripheral tissues. As for GSH peroxidase, these effects only occur with severe deficiencies; above a critical level selenium is not rate limiting the synthesis of these enzymes, and therefore does not control their levels. This generation of active thyroid hormone CT,) in the brain is crucial for the development of this organ in the fetus. However, T, generated in the thyroid or elsewhere in the periphery does not gain access from serum to brain, so this organ is
therefore entirely dependent on T, generated in situ from T4 (Calve et al., 1990). Therefore in endemic goiter areas, while a clinically euthyroid pregnant woman is often in a compensated hypothyroid state, characterised by low serum T4 but normal serum T,, the fetus lacks the T4 essential for brain development. Failure to generate sufficient T, may therefore be responsible for the neurological abnormalities and retardation of brain development that occur in nervous endemic cretins. This process could well occur early in utero, at a time when the fetus mostly depends on the mother for T4 supply (Pharoah et al., 1971; Escobar de1 Rey et al., 1986; Vulsma et al., 1989). The deaf mutism of children with generalized thyroid hormone resistance and thus, tissue hypothyroidism in utero before the onset of their own thyroid hormone synthesis, may be a model of what happens in nervous cretinism (Refetoff, 1982). It also suggests that it could be very dangerous for the fetus to treat hypothyroid pregnant women with T,, which would compensate maternal hypothyroidism but further decrease T4 production by both maternal and fetal thyroid glands, and thus deprive the fetal brain of T4 (Calve et al., 1990). How do these four lines of research converge on the pathogenesis of endemic cretinism? A first clue is that in Central African endemic goitre areas myxoedematous cretinism is much more frequent, and nervous cretinism much less frequent, ‘than in other endemic goitre areas. In Darfur, Sudan, where iodine deficiency is not combined with Se deficiency, most cretins are of the nervous type (Moreno et al., submitted). There is, however, as yet no proof (and proof will be difficult to obtain) that the thyroid dysgenesis in myxoedematous endemic cretinism is due to enhanced stimulation and H,O, generation combined with decreased GSH peroxidase and H,O, detoxification. By decreasing thyroid hormone catabolism, Se deficiency in a pregnant woman may protect the supply of T4 to the fetal brain, an organ which in rat is very well protected against selenium deficiency (Behne et al., 1988; Golstein et al., 1988); in this way it may therefore prevent the neurological consequences of early fetal hypothyroidism, and thus nervous cretinism (Contempre et al., 1991). Selenium deficiency would
Cl95
thus protect the fetus against some of the effects of iodine deficiency (Golstein et al., 1988; Contempt+ et al., 19911, and selenium supplementation alone in a severe iodine and selenium deficiency could lead to a shift of the picture of cretinism from the myxoedematous to the neurological form. This latter hypothesis has some experimental support. Selenium supplementation in either iodide-depleted rats or endemic cretins, i.e. under conditions of severe iodine depletion and thyroid decompensation, greatly decreases serum thyroid hormones levels (both T4 and T,), and induces catastrophic thyroid failure (Golstein et al., 1988; Contempt+ et al., 1991). This effect is presumably due to increased 5’-deiodinase activity involving increased catabolism of both T4 and rT,, and thus iodide loss by the kidney. Decreased thyroid oxidation of iodide by H,O, reduction, and increased GSH peroxidase levels leading to thyroid inefficiency, presumably also contribute to these inopportune effects of selenium supplementation (Golstein et al., 1988). Whatever its exact mechanism, the adverse effects of selenium supplementation in iodide deficiency show that one deficiency may temper the deleterious effects of another (Golstein et al., 1988). It is tempting to speculate that iodine deficiency, and the consequently lower thyroid hormone levels and metabolic rate, may prevent some of the consequences of selenium deficiency such as the cardiac decompensation called Keshan disease in China (Johnson et al., 1981; Giangqui et al., 1988). In epidemiology as in ecology, changing one factor in an intricate interdependent network may have quite unexpected consequences. In clinical practice, public health authorities should avoid correcting selenium deficiency without prior exclusion of any possibility of concurrent iodine deficiency. References Arthur, J.R. and Beckett, G.J. (1989) in Selenium in Biology and Medicine (Wendel, A., ed.), pp. 90-95, SpringerVerlag, Heidelberg. Arthur, J.R., Nicol, F. and Beckett, G.J. (1990) Biochem. J. 272, 537-540. Beckett, G.J., Beddows, S.E., Morrice, P.C., Nicol, F. and Arthur, J.R. (1987) Biochem. J. 248, 443-447.
Beckett, G.J., MacDougall, D.A., Nicol, F. and Arthur, J.R. (1989) Biochem. J. 259, 887-892. Behne, D., Hilmert, H., Scheid, S., Gessner, H. and Elger, W. (1988) Biochim. Biophys. Acta 966, 12-21. Behne, D., Kyriakopoulos, A., Meinhold, H. and Kohrle, J. (1990) Biochem. Biophys. Res. Commun. 173, 1143-1149. Berry, M., Banu, L. and Larsen, P.R. (1991) Nature 349, 438-440. Calvo, R., Obregon, M.J., Ruiz de Ona, C., Escobar de1 Rey, F. and Morreale de Escobar, G. (1990) J. Clin. Invest. 86, 889-899. Carmagnol, F., Sinet, P.M. and Jerome, H. (1983) Biochim. Biophys. Acta 759, 49-57. Chambers, I., Frampton, J., Goldfarb, P., Affara, N., McBain, W. and Harrison, P.R. (1963) EMBO J. 5(6), 1221-1227. Contempre, B., Dumont, J.E., Ngo, B., Thilly, C.H., Diplock, A.T. and Vanderpas, J. (1991) J. Clin. Endocrinol. Metab. 73(l), 213-215. Corvilain, B., Van Sande, J., Laurent, E. and Dumont, J.E. (1991) Endocrinology 128, 779-785. Delong, R., Stanbury, J.B. and Fierro Benitez, R. (1985) Dev. Med. Child. Neurol. 27, 317-322. Dumont, J.E., Ermans, A.M. and Bastenie, P.A. (1963) J. Clin. Endocrinol. Metab. 23, 325-335. Dumont, J.E., Delange, F. and Ermans, A.M. (1969) in Endemic Goiter (Stanbury, J.B., ed.), pp. 91-98, Pan American Health Organisation, Washington, DC. Escobar de1 Rey, F., Pastor, R., Mallol, J. and Morreale de Escobar, G. (1986) Endocrinology 11(4), 13-16. Ganther, H.E., Hafeman, D.G., Lawrence, R.A., Serfa, R.E. and Hoekstra, W.G. (1976) in Vol. 2 (Prasad, A.S., ed.), pp. 165-234, Academic Press, New York. Giangqui, Y., Keyou, G., Junshi, C. and Xiaoshu, C. (1988) World Rev. Nutr. Diet 55, 98-152. Golstein, J., Corvilain, B., Lamy, F., Paquer, D. and Dumont, J.E. (1988) Acta Endocrinol. 118, 495-502. Goyens, P., Golstein, J., Nsombola. B., Vis, H. and Dumont, J.E. (1987) Acta Endocrinol. 114, 497-502. Johnson, R.A., Baker, S.S., Fallon, J.T., Maynard, E.P., Ruskin, J.N., Wen, Z., Ge, K. and Cohen, H.J. (1981) New Engl. J. Med. 304, 1210-1212. Levander, O.A. (1983) Curr. Top. Nutr. Dis. 6, 345-368, Moreno, R., Boelaert, M., El Badawa, S., El Tom, M. and Vanderpas (submitted) J. Am. J. Clin. Nutr. Pharoah, P.O.D., Buttfield, I.H. and Hetzel, B.S. (1971) Lancet i, 308-310. Refetoff, S. (1982) Am. J. Physiol. 243, E88-E98. Rous, D., Weening, R.C., Wyss, S.R. and Aebi, H.E. (1980) J. Clin. Invest. 65, 1515-1522. Vanderpas, J.B., Rivera-Vanderpas, M.T., Bourdoux, P., Luvivilla, K., Lagasse, R., Perlmutter-Cremer, N, Delange, F., Lanoie, L., Ermans, A.M. and Thilly, C.H. (1986) New Engl. J. Med. 315, 791-795. Vanderpas, J.B., Contempre, B., Duale, N.L., Goossens, W., Ngo, B., Thorpe, R., Dumont, J.E., Thilly, C.H. and Diplock, A.T. (1990) Am. J. Clin. Nutr. 52, 1087-1093. Vulsma, T., Gons, M.H. and de Vijlder (1989) New Engl. J. Med. 321, 13-16.
MOLCEL
Cl93
81 (1991) C193-Cl95 Ireland, Ltd. 0303.7207/91/$03.50
02638
At the Cutting Edge
Cretinism, thyroid hormones and selenium B. Contemprk, J. Vanderpas and J.E. Dumont I.R. I.B.H. N., lJnk:ersite Libre de Bruxelles3 B-1070 Brussels, Belgium (Accepted
Key words: Selenium
deficiency;
Iodine
deficiency;
Cretinism;
Thyroid metabolism and the epidemiology of selenium deficiency would at first sight appear to be clearly distinct fields. Recently, however, they have converged as a result of investigations linking the molecular biology of thyroid hormone metabolizing enzymes and the pathogenesis of endemic cretinism. Among other things, these results have very practical consequences for the prophylaxis and treatment of both iodine and selenium deficiencies *. In terms of epidemiology and pathophysiology the story begins with a study of endemic cretinism in Central Africa. Endemic cretinism is a disease characterized by mental retardation, and occurs in areas of severe iodine deficiency. In Northern Zaire, it was found that the endemic cretins were myxoedematous (hypothyroid) and that their characteristic deficits (both developmental and in terms of mental retardation) were proportional to the severity of their hypothyroidism (Dumont et al., 1963). The clinical aspects of the disease were similar to those of congenital hypothyroidism, and thus could be explained by thyroid deficiency starting around birth (Vanderpas et al., 1986). Endemic cretins, in contrast with the general population, had no goitre and in fact were char-
Address for correspondence: Dr. B. Contempre, I.R.I.B.H.N., Universite Libre de Bruxelles, Campus Erasme, Bldg. C, Route de Lennik 808, B-1070 Brussels, Belgium. * WHO - ICCIDD Consultation on Interrelationship between Iodine Deficiency, Selenium Deficiency and Thyroid Hormones (Aberdeen, March 25-27th 19911.
29 July 19911
Thyroid
hormone
metabolism
acterized by a small thyroid with a small, rapidly turning-over iodine pool (Dumont et al., 1969). The cause of the degeneration of the gland remains unknown. Studies of other areas of endemic goitre revealed another form of cretinism in which thyroid function was normal, but where neurological deficits such as deaf-mutism were prevalent (Delong et al., 1985). Why iodine deficiency should lead to such different syndromes in different areas, and why the thyroid is destroyed in myxoedematous cretinism, have remained mysteries for 20 years. From studies of thyroid biochemistry we know that relatively high levels of H,O, are generated in the gland, particularly in response to thyrotropin (Corvilain et al., 1991). This H,O, serves as substrate for the thyroperoxidase that catalyzes iodide oxidation and binding to thyroglobulin and the oxidative coupling of iodotyrosines in iodothyronines. In leucocytes, H,O, is known to be toxic (Rous et al., 1980); it might therefore be possible that under conditions of high thyroid stimulation, such as normally occurs at birth and to a greater extent in iodine deficient areas, such H,O, might kill the thyroid cells if protective mechanisms were deficient (Goyens et al., 1987). H,O, is detoxified by glutathione peroxidase (an enzyme known to contain selenium) and catalase; other related 0, radicals such as O;- are detoxified by superoxide dismutases (one containing Zn2+, another Cu2+) (Ganther et al., 1976; Carmagnol et al., 1983; Levander et al., 1983). We therefore decided to ascertain the Se, Cu and Zn status of populations in an African en-
Cl94
demic goitre area, the island of Idjwi, on Kivu Lake. The results were clearcut, and showed that there was severe selenium deficiency and consequent glutathione peroxidase cell depletion in this area, where myxoedematous endemic cretinism is prevalent (Goyens et al., 1987). Subsequent and more extensive studies revealed a belt of severe selenium deficiency in Central Africa (Vanderpas et al., 1990), further suggesting a link between Se deficiency and thyroid disease. Quite independently, biochemists working on the deiodination of the prohormone thyroxine to the active hormone T, (3,5,3’-triiodothyronine) or the inactive derivative rT, (3,3’,5’-triiodothyronine) found that the nutritional status of the experimental animal modulated this metabolism. Nutritionists working on trace element deficiencies then found that T4 to T, deiodination, and the activity of the liver 5’-deiodinase type I that catalyzes the reaction, were much depressed in selenium deficient animals (Beckett et al., 1987, 1989; Arthur and Beckett, 1989). The explanation of these findings came earlier this year, when this 5’-deiodinase was found to be a selenium-containing enzyme (Arthur et al., 1990; Behne et al., 19901, and the cloning of the enzyme demonstrated a selenocysteine in its structure coded by a triplet which normally yields a stop codon (Berry et al., 1991). An identical mechanism leads to the insertion of selenocysteine into glutathione peroxidase (Chambers et al., 1986). It is not yet known whether the other 5’-deiodinase type II, located mainly in the brain and catabolizing T4 to T, exclusively, is also a selenium enzyme; an effect of Se on the activity of this deiodinase group at central level cannot, however, be excluded (Beckett et al., 1989). Thus selenium deficiency clearly can cause impairment of active thyroid hormone generation in peripheral tissues. As for GSH peroxidase, these effects only occur with severe deficiencies; above a critical level selenium is not rate limiting the synthesis of these enzymes, and therefore does not control their levels. This generation of active thyroid hormone CT,) in the brain is crucial for the development of this organ in the fetus. However, T, generated in the thyroid or elsewhere in the periphery does not gain access from serum to brain, so this organ is
therefore entirely dependent on T, generated in situ from T4 (Calve et al., 1990). Therefore in endemic goiter areas, while a clinically euthyroid pregnant woman is often in a compensated hypothyroid state, characterised by low serum T4 but normal serum T,, the fetus lacks the T4 essential for brain development. Failure to generate sufficient T, may therefore be responsible for the neurological abnormalities and retardation of brain development that occur in nervous endemic cretins. This process could well occur early in utero, at a time when the fetus mostly depends on the mother for T4 supply (Pharoah et al., 1971; Escobar de1 Rey et al., 1986; Vulsma et al., 1989). The deaf mutism of children with generalized thyroid hormone resistance and thus, tissue hypothyroidism in utero before the onset of their own thyroid hormone synthesis, may be a model of what happens in nervous cretinism (Refetoff, 1982). It also suggests that it could be very dangerous for the fetus to treat hypothyroid pregnant women with T,, which would compensate maternal hypothyroidism but further decrease T4 production by both maternal and fetal thyroid glands, and thus deprive the fetal brain of T4 (Calve et al., 1990). How do these four lines of research converge on the pathogenesis of endemic cretinism? A first clue is that in Central African endemic goitre areas myxoedematous cretinism is much more frequent, and nervous cretinism much less frequent, ‘than in other endemic goitre areas. In Darfur, Sudan, where iodine deficiency is not combined with Se deficiency, most cretins are of the nervous type (Moreno et al., submitted). There is, however, as yet no proof (and proof will be difficult to obtain) that the thyroid dysgenesis in myxoedematous endemic cretinism is due to enhanced stimulation and H,O, generation combined with decreased GSH peroxidase and H,O, detoxification. By decreasing thyroid hormone catabolism, Se deficiency in a pregnant woman may protect the supply of T4 to the fetal brain, an organ which in rat is very well protected against selenium deficiency (Behne et al., 1988; Golstein et al., 1988); in this way it may therefore prevent the neurological consequences of early fetal hypothyroidism, and thus nervous cretinism (Contempre et al., 1991). Selenium deficiency would
Cl95
thus protect the fetus against some of the effects of iodine deficiency (Golstein et al., 1988; Contempt+ et al., 19911, and selenium supplementation alone in a severe iodine and selenium deficiency could lead to a shift of the picture of cretinism from the myxoedematous to the neurological form. This latter hypothesis has some experimental support. Selenium supplementation in either iodide-depleted rats or endemic cretins, i.e. under conditions of severe iodine depletion and thyroid decompensation, greatly decreases serum thyroid hormones levels (both T4 and T,), and induces catastrophic thyroid failure (Golstein et al., 1988; Contempt+ et al., 1991). This effect is presumably due to increased 5’-deiodinase activity involving increased catabolism of both T4 and rT,, and thus iodide loss by the kidney. Decreased thyroid oxidation of iodide by H,O, reduction, and increased GSH peroxidase levels leading to thyroid inefficiency, presumably also contribute to these inopportune effects of selenium supplementation (Golstein et al., 1988). Whatever its exact mechanism, the adverse effects of selenium supplementation in iodide deficiency show that one deficiency may temper the deleterious effects of another (Golstein et al., 1988). It is tempting to speculate that iodine deficiency, and the consequently lower thyroid hormone levels and metabolic rate, may prevent some of the consequences of selenium deficiency such as the cardiac decompensation called Keshan disease in China (Johnson et al., 1981; Giangqui et al., 1988). In epidemiology as in ecology, changing one factor in an intricate interdependent network may have quite unexpected consequences. In clinical practice, public health authorities should avoid correcting selenium deficiency without prior exclusion of any possibility of concurrent iodine deficiency. References Arthur, J.R. and Beckett, G.J. (1989) in Selenium in Biology and Medicine (Wendel, A., ed.), pp. 90-95, SpringerVerlag, Heidelberg. Arthur, J.R., Nicol, F. and Beckett, G.J. (1990) Biochem. J. 272, 537-540. Beckett, G.J., Beddows, S.E., Morrice, P.C., Nicol, F. and Arthur, J.R. (1987) Biochem. J. 248, 443-447.
Beckett, G.J., MacDougall, D.A., Nicol, F. and Arthur, J.R. (1989) Biochem. J. 259, 887-892. Behne, D., Hilmert, H., Scheid, S., Gessner, H. and Elger, W. (1988) Biochim. Biophys. Acta 966, 12-21. Behne, D., Kyriakopoulos, A., Meinhold, H. and Kohrle, J. (1990) Biochem. Biophys. Res. Commun. 173, 1143-1149. Berry, M., Banu, L. and Larsen, P.R. (1991) Nature 349, 438-440. Calvo, R., Obregon, M.J., Ruiz de Ona, C., Escobar de1 Rey, F. and Morreale de Escobar, G. (1990) J. Clin. Invest. 86, 889-899. Carmagnol, F., Sinet, P.M. and Jerome, H. (1983) Biochim. Biophys. Acta 759, 49-57. Chambers, I., Frampton, J., Goldfarb, P., Affara, N., McBain, W. and Harrison, P.R. (1963) EMBO J. 5(6), 1221-1227. Contempre, B., Dumont, J.E., Ngo, B., Thilly, C.H., Diplock, A.T. and Vanderpas, J. (1991) J. Clin. Endocrinol. Metab. 73(l), 213-215. Corvilain, B., Van Sande, J., Laurent, E. and Dumont, J.E. (1991) Endocrinology 128, 779-785. Delong, R., Stanbury, J.B. and Fierro Benitez, R. (1985) Dev. Med. Child. Neurol. 27, 317-322. Dumont, J.E., Ermans, A.M. and Bastenie, P.A. (1963) J. Clin. Endocrinol. Metab. 23, 325-335. Dumont, J.E., Delange, F. and Ermans, A.M. (1969) in Endemic Goiter (Stanbury, J.B., ed.), pp. 91-98, Pan American Health Organisation, Washington, DC. Escobar de1 Rey, F., Pastor, R., Mallol, J. and Morreale de Escobar, G. (1986) Endocrinology 11(4), 13-16. Ganther, H.E., Hafeman, D.G., Lawrence, R.A., Serfa, R.E. and Hoekstra, W.G. (1976) in Vol. 2 (Prasad, A.S., ed.), pp. 165-234, Academic Press, New York. Giangqui, Y., Keyou, G., Junshi, C. and Xiaoshu, C. (1988) World Rev. Nutr. Diet 55, 98-152. Golstein, J., Corvilain, B., Lamy, F., Paquer, D. and Dumont, J.E. (1988) Acta Endocrinol. 118, 495-502. Goyens, P., Golstein, J., Nsombola. B., Vis, H. and Dumont, J.E. (1987) Acta Endocrinol. 114, 497-502. Johnson, R.A., Baker, S.S., Fallon, J.T., Maynard, E.P., Ruskin, J.N., Wen, Z., Ge, K. and Cohen, H.J. (1981) New Engl. J. Med. 304, 1210-1212. Levander, O.A. (1983) Curr. Top. Nutr. Dis. 6, 345-368, Moreno, R., Boelaert, M., El Badawa, S., El Tom, M. and Vanderpas (submitted) J. Am. J. Clin. Nutr. Pharoah, P.O.D., Buttfield, I.H. and Hetzel, B.S. (1971) Lancet i, 308-310. Refetoff, S. (1982) Am. J. Physiol. 243, E88-E98. Rous, D., Weening, R.C., Wyss, S.R. and Aebi, H.E. (1980) J. Clin. Invest. 65, 1515-1522. Vanderpas, J.B., Rivera-Vanderpas, M.T., Bourdoux, P., Luvivilla, K., Lagasse, R., Perlmutter-Cremer, N, Delange, F., Lanoie, L., Ermans, A.M. and Thilly, C.H. (1986) New Engl. J. Med. 315, 791-795. Vanderpas, J.B., Contempre, B., Duale, N.L., Goossens, W., Ngo, B., Thorpe, R., Dumont, J.E., Thilly, C.H. and Diplock, A.T. (1990) Am. J. Clin. Nutr. 52, 1087-1093. Vulsma, T., Gons, M.H. and de Vijlder (1989) New Engl. J. Med. 321, 13-16.
