Cell Biol Toxicol (2009) 25:153–184 DOI 10.1007/s10565-008-9078-6

Abstract Received: 19 March 2008 / Accepted: 19 March 2008 # Springer Science + Business Media B.V. 2008

An invited paper presented in the symposium “Is copper involved in carcinogenesis/carcinostatics?” Possible association of Wilson’s disease with hepatocellular carcinoma Yan-Hong Gu1, Hiroko Kodama2, Tadaaki Kato1, and Chie Fujisawa2, Fumiaya Kaga2 1

Department of Health Policy, National Research Institute for Child Health and Development, Tokyo, 157–8535 Japan 2 Department of Pediatrics, Teikyo University School of Medicine, Tokyo, 173–8605 Japan Corresponding Author: E-mail: [email protected] Background Wilson’s disease (WD, OMIM 277900) is a genetic disorder resulting from a defect in the copper transporting ATPase (ATP7B). In patients with WD, copper accumulates primarily in the liver and brain. During early-stage disease, copper that has accumulated in the liver binds to cytoplasmic metallothioneins and is not toxic. As the disease progresses, however, copper toxicity develops through the generation of oxidant radicals and the inhibition of key proteins of the oxidant defense system. The resulting effects on microtubules or mitochondria lead to multiple types of injury and damage (Kodama et al. 1998). The chelating agents or zinc currently used to treat this disease have enabled long-term survival in many WD patients. Recently, however, reported cases of hepatocellular carcinoma (HCC) in WD patients have been on the rise. In addition, the Long–Evans

Cinnamon rat, an animal model of WD, suffers from HCC with a high frequency (Masuda et al. 1988). To clarify the relation between WD and HCC, we compared the characteristics of previously reported WD patients with HCC with those of HCC patients without WD. Methods We measured the concentration of copper in liver samples from 21 patients with WD according to the method previously reported (Kodama et al. 2005). For 12 of these patients, multiple samples were measured simultaneously. An electronic search of the English (http://www.ncbi.nlm.nih.gov/sites/entrez), Chinese (http://www.toho-shoten.co.jp/index.jsp), and Japanese (http://www.jamas.or.jp/) Medline databases was conducted to ascertain WD cases with HCC. We used the one-sample t test to compare the average age at the time of diagnosis of HCC in the WD patients with that of patients from the liver cancer study group of Japan, 1994–2003 (LCS-J). Results and discussion We set out to determine whether copper can contribute to hepatic cell carcinogenesis by studying WD patients with HCC. However, the prevalence of WD is only about 1:40,000; WD patients with HCC are therefore exceedingly rare. To resolve this dilemma, we collected and analyzed all reported cases and compared these data with the nationwide data from LCS-J. The analysis revealed features that are characteristic of HCC in WD patients. We found 25 WD patients with HCC reported from 1959 to 2007 in Japan, China, USA, Turkey, France, UK, Singapore, Norway, German, Sweden, and Poland (Lygren 1959; Girard et al. 1968; Kamakura et

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al. 1975; Terao et al. 1982; Wilkinson et al. 1983; Buffet et al. 1984; Guan et al. 1985; Imhof et al. 1985; Madden et al. 1985; Polio et al. 1989; Cheng et al. 1992; Qian et al. 2000; Wang et al. 2000; Cao et al. 2002; Walshe et al. 2003; Iwadate et al. 2004; Kumagi et al. 2004; Czlonkowska et al. 2005; Kumagi et al. 2005; Aydinli et al. 2006; Savas et al. 2006; Ozcay et al. 2006; Xu et al. 2007). There were 20 men and five women. Of the 25 patients, 12 presented with neurological onset (48%) and 10 presented with hepatic onset (40%). The average age at onset of WD, average age at diagnosis of WD, average age at diagnosis of HCC, and average period of Dpenicillamine treatment were 25.2 years (n=22; range, 5–48 years), 31.5 years (n=21; range, 6–66 years), 41.2 years (n=20; range, 12–66 years), and 8.3 years (n=21; range, 0–38 years), respectively. Only one patient, a Japanese man, was homozygous for a mutation (Ala874Val) in the ATP7B gene (Kumagi et al. 2005). This mutation has a frequency of only 1.2% in Japanese WD patients (Yamaguchi et al. 1998). We compared the characteristics of WD patients with HCC with those of HCC patients without WD who were obtained from LCS-J. The male-to-female ratio of WD patients with HCC was 4.0:1, whereas it was 2.9:1 in the LCS-J subjects. For males, the age at diagnosis of HCC in WD patients (mean±SD: 39.4± 14.6 years) was significantly earlier than that of LCSJ subjects (63.9±1.4 years; P=0.000); likewise, for women, the age at diagnosis of HCC in WD patients (48.3±19.3 years) was earlier than that of LCS-J subjects (67.3±1.4 years). At the time of this report, 12 of the patients had died, and 10 were still living. The average age at the time of death in WD patients with HCC was 43.9 years (n=11) regardless of the length of treatment. We also compared the status of hepatitis B surface antigen (HBsAg), hepatitis C virus (HCV) antibody, cirrhosis, and HCC differentiation between the two groups of patients (i.e., WD with HCC versus HCC patients from LCS-J). Eleven WD patients were HBsAg-negative, one was HBsAgpositive, and the status was unknown in 13. In contrast, 15.0–16.8% of the HCC patients from the LCS-J were HBsAg-positive. Seven WD patients with HCC were HCV-negative, and the HCV status was unknown in 18. In contrast, 69.6–76.0% of the LCS-J subjects were positive for HCV antibody. All the WD patients had cirrhosis, whereas 70.6–84.5% of patients in LCS-J had the condition. Among the 25 WD patients, 12 were of the trabecular type, one was

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mixed acinar and anaplastic pattern with striking cellular atypia, and 12 were unknown. Well, moderately, and poorly differentiated HCC in the WD patients occurred with frequencies of 57.1%, 28.6%, and 14.3%, respectively; the frequencies were 29.5%, 58.5%, and 10.7% in LCS-J subjects, respectively. Among the 25 WD patients, four were diagnosed with HCC at autopsy, and 10 patients died before or immediately after diagnosis of HCC, suggesting that patients with WD have a higher risk for HCC. Thus, WD patients should be periodically examined for HCC. Uneven hepatic copper distribution in WD For 12 WD patients, levels of copper were measured in multiple liver samples simultaneously. Uneven distributions of hepatic copper concentration were observed in these patients. The ranges of copper concentrations (in ng/mg dry weight) in multiple samples from 12 patients were 67–415, 94–1,072, 252–482, 497–770, 585–794, 764–1,091, 634–875, 824–1,252, 870–939, 1,252–1,367, 1,540–1,697, and 1,670–5,634. In two male patients, the copper concentration was higher in the right lobe than in the left lobe; unfortunately, for the other 10 patients, information about the origin of the different samples was not available. Consistent with the report on Western WD patients (Faa et al. 1995), we concluded that the uneven copper distribution is not associated with any particular ethnicity. In WD patients with HCC, copper concentrations could be lower or higher than either those found in non-cancerous liver tissue or normal liver tissue; copper concentrations were not dependent on the period of treatment or the type of chelating agents used. In the current study, the location of HCC was described in 17 patients: in the right lobe in seven male and two female patients; in the left lobe in three male and two female patients; in the middle in one male patient; and distributed throughout the whole liver in two male patients. These observations suggest that abnormal copper levels in the liver may be associated with the development of HCC. Interestingly, it was reported that the generation of oxygen/ nitrogen species and unsaturated aldehydes from copper overload in the WD liver causes mutations in the p53 tumor suppressor gene (Staib et al. 2003). The limitation of this study was that we were able to

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analyze the data from only 25 HCC patients with WD. A large cohort study is necessary to better understand the relation between copper accumulation and hepatic cell carcinogenesis. References Aydinli M, Harmanci O, Ersoy O, Iskit AT, Ozcebe O, Abbasoglu O, Bayraktar Y. Two unusual cases with Wilson’s disease: hepatoma and fulminant hepatitis treated with plasma exchange. J Natl Med Assoc. 2006;98:1989–91. Buffet C, Servent L, Pelletier G, Rondot P, Etienne JP. Hepatocellular carcinoma in Wilson’s disease. Gastroenterol Clin Biol. 1984;8:681–2. Cao LY, Yu GQ. Hepatocellular carcinoma in hepatolenticular carcinoma. Med J CASC. 2002; 4: 63. Cheng WS, Govindarajan S, Redeker AG. Hepatocellular carcinoma in a case of Wilson’s disease. Liver. 1992;12:42–5. Członkowska A, Tarnacka B, Litwin T, Gajda J, Rodo M. Wilson’s disease-cause of mortality in 164 patients during 1992–2003 observation period. J Neurol. 2005;252:698–703. Faa G, Nurchi V, Demelia L, Ambu R, Parodo G, Congiu T, Sciot R, Van Eyken P, Silvagni R, Crisponi G. Uneven hepatic copper distribution in Wilson’s disease. J Hepatol. 1995;22:303–8. Girard PF, Vachon A, Tommasi M, Paliard P, Rochet M, Barthe J. Hepatolenticular degeneration and primary cancer of the liver. Lyon Med. 1968;219:1395–400. Guan R, Oon CJ, Wong PK, Foong WC, Wee A. Primary hepatocellular carcinoma associated with Wilson's disease in a young woman. Postgrad Med J. 1985;61:357–9. Imhof M, Lehmann L, Wasmer HP, Kroib A, Baumer F. Morbus Wilson end primares Leberzellkarzinom. Munch Med Wschr. 1985;127:1001–2. Iwadate H, Ohira H, Suzuki T, Abe K, Yokokawa J, Takiguchi J, Rai T, Orikasa H, Irisawa A, Obara K, Kasukawa R, Sato Y. Hepatocellular carcinoma associated with Wilson’s disease. Intern Med. 2004;43:1042–5. Kamakura K, Kimura S, Igarashi S, Fujiwara K, Toshitsugu O. A case of Wilson’s disease with hepatoma. Nippon Naika Gakkai Zasshi. 1975;64:232–8. Kodama H, Murata Y, Mochizuki D, Abe T. Copper and ceruloplasmin metabolism in the LEC rat, an animal model for Wilson disease. J Inherit Metab Dis. 1998;21:203–6.

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Kodama H, Sato E, Gu YH, Shiga K, Fujisawa C, Kozuma T. Effect of copper and diethyldithiocarbamate combination therapy on the macular mouse, an animal model of Menkes disease. J Inherit Metab Dis. 2005;28:971–8. Kumagi T, Horiike N, Michitaka K, Hasebe A, Kawai K, Tokumoto Y, Nakanishi S, Furukawa S, Hiasa Y, Matsui H, Kurose K, Matsuura B, Onji M. Recent clinical features of Wilson’s disease with hepatic presentation. J Gastroenterol. 2004;39:1165–9. Kumagi T, Horiike N, Abe M, Kurose K, Iuchi H, Masumoto T, Joko K, Akbar SF, Michitaka K, Onji M. Small hepatocellular carcinoma associated with Wilson’s disease. Intern Med. 2005;44:439–43. Lygren T. Hepatolenticular degeneration (Wilson’s disease) and juvenile cirrhosis in the same family. Lancet. 1959;1:275–6. Madden JW, Ironside JW, Triger DR, Bradshaw JP. An unusual case of Wilson’s disease. Q J Med. 1985;55:63–73. Masuda R, Yoshida MC, Sasaki M, Dempo K, Mori M. High susceptibility to hepatocellular carcinoma development in LEC rats with hereditary hepatitis. Jpn J Cancer Res. 1988;79:828–35. Ozçay F, Canan O, Bilezikçi B, Torgay A, Karakayali H, Haberal M. Effect of living donor liver transplantation on outcome of children with inherited liver disease and hepatocellular carcinoma. Clin Transplant. 2006;20:776–82. Polio J, Enriquez RE, Chow A, Wood WM, Atterbury CE. Hepatocellular carcinoma in Wilson’s disease. Case report and review of the literature. J Clin Gastroenterol. 1989;11(2):220–4. Qian GJ, Qu ZQ, Chen H. A case of hepatocellular carcinoma in hepatolenticular degeneration. Di Er Jun Yi Da Xue Xue Bao. 2000;5:465. Savas N, Canan O, Ozcay F, Bilezikci B, Karakayali H, Yilmaz U, Haberal M. Hepatocellular carcinoma in Wilson’s disease: a rare association in childhood. Pediatr Transplant. 2006;10:639–43. Staib F, Hussain SP, Hofseth LJ, Wang XW, Harris CC. TP53 and liver carcinogenesis. Hum Mutat. 2003;21:201–16. The liver cancer study group of Japan. Reports of the follow-up survey of primary liver cancer. Report of the 17th follow-up survey of primary liver cancer. Hepatol. 2007:48:117–140. Terao H, Itakura H, Nakata K, Kono K, Muro T, Furukawa R, Kusumoto M, Munehisa T, Nagataki S,

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Ishii N, Koji T, Hirama M. An autopsy case of hepatocellular carcinoma in Wilson’s disease. Acta Hepatologica Japonica 1982;23:439–445. Walshe JM, Waldenström E, Sams V, Nordlinder H, Westermark K. Abdominal malignancies in patients with Wilson’s disease. QJM. 2003;96:657–62. Wang X, He GY, Han YZ, Yang RM. Hepatocellular carcinoma in Wilson disease-case report and a review of literatures. Chinese Journal of Gastroenterology and Hepatology. 2000;9:236–238. Chinese. Wilkinson ML, Portmann B, Williams R. Wilson’s disease and hepatocellular carcinoma: possible protective role of copper. Gut. 1983;24:767–71. Xu R, Bu-Ghanim M, Fiel MI, Schiano T, Cohen E, Thung SN. Hepatocellular carcinoma associated with an atypical presentation of Wilson's disease. Semin Liver Dis. 2007; 27:122–7. Yamaguchi A, Matsuura A, Arashima S, Kikuchi Y, Kikuchi K. Mutations of ATP7B gene in Wilson disease in Japan: identification of nine mutations and lack of clear founder effect in a Japanese population. Hum Mutat. 1998;1:S320–2. An invited paper presented in the symposium “Is copper involved in carcinogenesis/carcinostatics?”. Carcinogenesis in LEC rat and patients with Wilson disease Norikazu Shimizu and Tsugutoshi Aoki Second Department of Pediatrics, Toho University School of Medicine, Tokyo, Japan

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low-serum ceruloplasmin levels and high urinary copper excretion. The hepatic copper content increases remarkably. Prognosis of Wilson disease is very poor without treatment. However, after the introduction of copperchelating agents, the patient’s prognosis improves dramatically. Now, Wilson disease is treatable. The copper-chelating agents, such as D-penicillamine and trienthine and/or zinc sulfate are very effective. LEC rats The Long–Evans cinnamon (LEC) rats, which are a mutant inbred strain isolated from Long–Evans rats, develop hepatitis and hepatocellular carcinoma in an autosomal recessive pattern of inheritance. They designate from peculiar cinnamon-like coat color. They were discovered in 1983 at the Center for Experimental Plants and Animals of Hokkaido University, Sapporo, Japan. Approximately the spontaneous hepatitis of LEC rats is characterized by a rather sudden onset at approximately 4 months after birth. About 40% of them die of fulminate hepatitis within 1 week of manifestation of jaundice. The remaining 60% of rats survive and develop chronic hepatitis. Liver cancer develops in all of LEC rats surviving longer than 1 year. Most are histologically classified into well-differentiated hepatocellular carcinomas. LEC rats also demonstrate many clinical and biochemical features of Wilson disease, including a low-serum ceruloplasmin level, a high urinary copper excretion. And copper-chelating agents prevent the onset of hepatitis in LEC rats. Pathophysiology of LEC rats

Corresponding Author: E-mail: [email protected] Wilson disease Wilson disease is an autosomal recessive disorder resulting from the defective functioning of copper transport P-type ATPase (ATP7B). It reveals a reduction in the rate of copper incorporation into ceruloplasmin and a reduction in the biliary excretion of copper. Copper accumulates in the liver, brain, cornea, kidney, and others. The major clinical signs and symptoms of the disease are cirrhosis, extrapyramidal signs, and Kayser– Fleischer rings. The incidence of Wilson disease is one in 35,000 to 45,000 live birth in Japan (Aoki et al. 1966). The typical biochemical features of these patients are

Keyword of LEC rats study is copper. The copper content in the liver of LEC rats is significantly higher than control rats in 0-day-old animals. Then, the copper content in the LEC rats liver markedly in creased, becoming more than 50 times higher than in the liver of age-matched control rats of 15 weeks (Shimizu et al. 1997). The serum aspartate transaminase levels rises to more than 1,200 IU, and the ALT level reaches up to 500 IU in this period (Shimizu et al. 1997). And the renal copper concentration of LEC rats markedly increases from around 15 weeks (Fig. 1) (Shimizu et al. 1997). Between the age of 16 and 23 weeks, 90% of LEC rats have acute hepatitis, and about 30% of them die with acute hepatic failure. The copper concentration in LEC rat liver reaches a toxic level; the hepatocytes become

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necrotic. As a result, non-ceruloplasmin-binding copper overflows into the peripheral blood, and these copper accumulate in extrahepatic organs such as kidney. The remaining 60% of LEC rats survive and develop chronic hepatitis. And liver cancer arises in all of rats surviving longer than 1 year. Most are histologically classified into well-differentiated hepatocellular carcinoma (Mori et al. 1994). The chronic liver injury due to hepatic copper accumulation promotes hepatocarcinogenesis of LEC rats. Copper ions produce oxygen radicals. They cause cell damage, DNA damage including the formation of 8-hydroxydeoxyguanosine, and protein–DNA crosslinking in chromation (Nackerdien et al. 1991; Toyokoshi et al. 1994; Sone et al. 1996). Copper ions also interact directly with DNA and induce mutations (Sone et al. 1996). These affects may be involved in the evolution of liver cancer in LEC rats. The copperchelating agents such as D-penicillamine and trienthine inhibited the development of hepatitis and liver cancer (Sone et al. 1996; Jong-Hon et al. 1993).

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Genetics of Wilson disease and LEC rats The gene mutation of LEC rats is partial deletion of Wilson disease gene in rat (atp7b). At least 900-bp deletion from 3′-end coding region of atp7b gene was detected (Wu et al. 1994). It removes the information encoding the conserved adenosine triphosphate (ATP)-binding domain. More than 200 kinds of mutations, such as deletions, insertions, nonsense mutations, missense mutations, and splicing mutations of ATP7B gene, are reported in Wilson disease patients (Butler et al. 2001). Although the correlation between genotype and phenotype in Wilson disease remain unclear, the patients who have mutations and make truncated protein indicate a hepatic phenotype (Shimizu et al. 1997; Shimizu et al. 1995). The LEC rats are good animal model of hepatic type of human Wilson disease. Conclusion From biochemical features and gene mutation of LEC rats, the LEC rats are not only the animal model of hepatitis and liver cancer but also the animal model of Wilson disease. Although human hepatocellular carcinoma is a well-recognized complication of cirrhosis, a relationship has not been recognized between hepatocellular carcinoma and Wilson disease. Probably, Wilson disease patients died before reaching an age at which hepatocellular carcinoma would have developed. The same carcinogenic mechanism with LEC rats must be arose in the liver of Wilson disease patients. Authors expect that the prevalence of liver cancer in Wilson disease may increase as survival improves. In these studies, we conclude that LEC rats are a rodent model for Wilson disease. Thus, LEC rats will serve as an important model of investigation about the mechanism and prevention for this problem. References

Fig. 1 Age-related changes of copper content in the liver, kidney, and whole brain of LEC rats and control (LEA) rats during development. Open circles are LEC rats and closed circles are LEA rats (Shimizu et al 1997).

Aoki T, Suzuki M, Fujioka Y, Shimizu N, Fuji H, Nakazono H, Kawase C, Yamaguchi Y, Arashima S, Matsuda I, Arima M. Nationwide survey of clinical features of Wilson’s disease in Japan. In: Lam STS, Pang CCP, editor. Neonatal and perinatal screening, the Asian pacific perspective. Hong Kong: The Chinese University Press; 1996. pp. 25–8.

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Butler P, McIntyre N, Mistry PK. Molecular diagnosis of Wilson disease. Mol Genet Metab. 2001;72:2223–30. Jong-Hon K, Togashi Y, Kasai H, Hosokawa M, Takeichi N. Prevention of spontaneous hepatocellular carcinoma in Long–Evans cinnamon rats with hereditary hepatitis by administration of D-penicillamine. Hepatology. 1993;18:614–20. Mori M, Hattori A, Sawaki M, Tsuzuki N, Sawada N, Oyamada M, Sugawara N, Enomoto K. The LEC rat: a model for human hepatitis, liver cancer, and much more. Am J Pathol. 1994;144:200–4. Nackerdien Z, Rao G, Cacciuttolo MA, Gajewski E, Dizdaroglu M. Chemical nature of DNA–protein cross-links produced in mammalian chromatin by hydrogen peroxide in the presence of iron or copper ions. Biochemistry. 1991;30:4873–9. Shimizu N, Kawase C, Nakazono H, Hemmi H, Shimatame H, Aoki T. A novel splicing mutation in Japanese patients with Wilson disease. Biochem Biophys Res Commun. 1995;217:16–20. Shimizu N, Fujii Y, Saito Y, Yamaguchi Y, Aoki T. Age-related copper, zinc, and iron metabolism in Long–Evans cinnamon rats and copper-eliminating effects of D-penicillamine and trienthine-2HCl. J Trace Elem Exp Med. 1997;10:49–59. Sone H, Maeda M, Wakabayashi K, Takeichi N, Mori M, Sugimura T, Nagao M. Inhibition of hereditary hepatitis and liver tumor development in Long–Evans cinnamon rats by the copper-chelating agent trientine dihydrochloride. Hepatology. 1996;23:764–70. Toyokoshi S, Sagripanti JL. Increased 8-hydroxyguanosine in kidney and liver of rats continuously exposed to copper. Toxicol Appl Pharmacol. 1994;126:91–7. Wu J, Forbes JR, Hai Shiene Chen, Cox DW. The LEC rat has a deletion in the copper transporting ATPase gene homologous to the Wilson disease gene. Nature Genet. 1994;7:541–545. An invited paper presented in the symposium “Is copper involved in carcinogenesis/carcinostatics?” The effects of hepatic copper overload on development of hepatocellular carcinoma in hepatitis C virus -positive patients with chronic liver disease Masaaki Ebara Department of Gastroenterology and Hepatology, Kameda Medical Center, Kamogawa, Chiba, Japan

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Corresponding Author: E-mail: [email protected] MRI diagnosis of hepatocellular carcinoma Magnetic resonance imaging (MRI) diagnosis of hepatocellular carcinoma (HCC) gave us an important clue to study the relationship between trace elements and hepatic carcinogenesis (Ebara et al. 1991, 1999). Metastatic liver cancer and hepatic hemangioma, which are representative liver tumors, are visualized as a lowsignal-intensity pattern on a T1-weighted image and a high-signal-intensity pattern on a T2-weighted image almost without exception. However, small HCCs are often visualized as a high-signal-intensity pattern on a T1-weighted image and a low-signal-intensity pattern on a T2-weighted image adversely. The pattern of a high-signal intensity on T1weighted image is considered to be a characteristic of an HCC and is seen in around 40% of HCC, which are 3 cm or less in diameter (Ebara et al. 1986). Steatosis is considered one of the causes of a highsignal-intensity pattern on a T1-weighted image, which can be differentiated by fat-suppression methods (Ebara et al. 1986). However, another cause must be considered in a case of HCC without steatosis. We suspected the participation of the paramagnetic metal ion as a cause of shortening T1 relaxation time and examined histologic specimen by using various metal stainings. We applied metal staining of copper, iron, and manganese, which may contain relatively more in liver parenchyma to HCC and the surrounding liver parenchyma. In cases of HCC showing a high-signal-intensity pattern on T1weighted image, positive rate of the copper staining was significantly higher in cancerous portion than in the surrounding liver parenchyma (Ebara et al. 1991). Furthermore, we examined metal quantity in cancerous tissue and the surrounding liver parenchyma by using particle induced X-ray emission (PIXE) methods (Khaliquzzaman et al. 1981). A histologic specimen in a target chamber was exposed to accelerated proton beams. Histologic specimen was obtained by using a 21-gauge aspiration biopsy needle, naturally dried and fixed on a carbon tape for PIXE examination. We measured the quantity of various metals in cancerous tissue and the surrounding liver parenchyma. In case of HCCs showing a high-intensity pattern on T1-weighted image, copper was accumulated

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more in cancerous tissue than in the surrounding liver parenchyma, although there was no such difference in iso or low-intensity pattern. Furthermore, there was no such difference in the content of iron and zinc. Trace elements in chronic liver diseases In Japan and some European countries, patients with HCC have hepatitis C virus (HCV) in 70% or more. Therefore, we examined the quantity of metals in the liver of patients with C-type chronic hepatitis (Hatano et al. 2001). As for quantity of copper, zinc, and iron in the liver of patients with C-type chronic hepatitis, copper alone increased according to the grade of hepatic fibrosis. However, there was no such correlation with zinc and iron. We analyzed quantity of copper, zinc, and iron in the liver of patients with HCC in relation to the etiology of chronic liver diseases. Copper was accumulated more in HCVpositive patients than in HBs-Ag positive patients, although there was no significant differences among other etiologies. We compared the quantity of copper, zinc, and iron in cancerous tissue with the surrounding liver parenchyma (Ebara et al. 2000). Copper was accumulated more in the cancerous tissue than in the surrounding liver parenchyma. In contrast, zinc and iron were accumulated more in the surrounding liver parenchyma than in cancerous tissue. Carcinogenicity of copper When following up 206 nodules of 6 mm or more in diameter seen as high-signal intensity on T1-weighted image in cirrhotic liver, we found that these lesions had changed into apparent HCCs in 6% in 1 year, 15% in 2 years, and 18% in 3 years. The areas of copper accumulated in the order of quantity were the surrounding liver parenchyma, dysplastic nodules that are border-line lesion of HCC, and HCC (Ebara et al. 2003). However, there was no such significant differences in zinc and iron. Comparing metal quantity in the liver parenchyma between with and without coexistence of HCC, copper was accumulated more in the liver coexisting with HCC (Ebara et al. 2003). However, there was no such significant differences in zinc and iron. According to the Cox proportional hazard model, the factor influencing coexistence of HCC was copper quantity in the liver parenchyma

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alone (Ebara et al. 2003). There was no significant influence on the coexistence of HCC in zinc, iron, age, or the grade of liver dysfunction. Radicals on copper accumulation in liver It is reported that 50% of Long–Evans rats with a cinnamon-like coat color (LEC rats), which are animal models of Wilson disease, die of acute hepatitis due to copper accumulation in the liver, and 40% suffer from HCC (Yoshida et al. 1987). Copper in the liver of LEC rats combines with metallothionein (MT) to form copper–thionein. A cuprous-ion binding with MT changes to a cupric ion in the action of a hydrogen peroxide by Fenton reaction (Sakurai et al. 1993). By this reaction, a hydroxyl radical is produced, and it oxidizes deoxyguanosin of deoxyribonucleic acid (DNA) and forms 8-hydroxy-2-deoxy-guanosin (8-OHdG; Kasai et al. 1984). The oxydative DNA cleavage by the copper is assumed in this way. According to the quantitative analysis of 8-OHdG in the tissue, 8-OHdG was contained more in the surrounding liver parenchyma than in normal liver parenchyma and most in HCC. We compared the distribution of metal-binding protein in each of normal liver, non-cancerous liver parenchyma, and HCC (Kasai et al. 1984). MT was contained mostly in the form of zinc MT in normal liver. Copper MT was slightly increased in the noncancerous liver and extraordinarily increased in HCC. We showed the distribution of copper and zinc, which are bound to MT in various tissues (Ebara et al. 2000; Kubo et al. 2005). MT was present in the form of ZnMT in all normal liver, Zn, Cu-MT was dominantly present in non-cancerous liver parenchyma, and CuMT was present mostly in HCC. However, MT was not detected at all in HCC 4 cm or more in diameter. Measuring the quantity of hydroxyradical in MT by electron spin resonance, no production of hydroxyradical was found in the normal liver, and hydroxyradical increased in non-cancerous liver parenchyma and much more in HCC. In addition, no hydroxyradical was produced in the ferritin of these tissues. Relationship between metals and 8-OHdG in the liver was studied by using 8-OHdG immune staining. We defined positive examples of when a hepatocyte nucleus is stained more than 30%. As for the copper, the positive rate of 8-OHdG was significantly higher in high-quantity group than low-quantity group when

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dividing into the two groups from the mean quantity. However, such a difference was not found in zinc and iron. Relationship between prognosis of HCC patients and accumulation of trace elements in the liver We analyzed the relationship between quantity of metal in the liver and their prognosis in 91 HCV positive patients with HCC that had been treated with radiofrequency tumor ablation or percutaneous ethanol injection. In this paper, we excluded patients who had received blood transfusion, patients with cancer of other organs, chronic nephritis, anemia, or are heavy drinkers. There was not significant relationship between zinc quantity and survival rates. In addition, no significant relationship was found between iron quantity and survival rates. The survival rate was significantly better in the group with low content of copper than in the group with high content of copper. Factors influencing survivals of patients with HCC that had been treated by local ablation were calculated by using Cox-proportional hazards model. Sixty or more years old in age, hepatic dysfunction, and high copper content in the liver were found to be significantly worsening factors influencing the survival rate. Chemoprevention of trientine hydrochloride, a copper-chelating agent for HCC

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administration of hydrochloric acid trientine, copper and ceruloplasmin in serum were significantly decreased in both groups. When comparing metal content in liver parenchyma before and 3 months after the administration of hydrochloric acid trientine, copper was decreased in both groups, but zinc and iron did not change quantitatively (Fukuda et al. 2004). We compared recurrence rates of HCC occurring at different sites from initially treated HCCs with and without administration of hydrochloric acid trientine. As the recurrence rate with administration of hydrochloric acid trientine tended to be lower than without administration, there is a potential of prevention for the recurrence of HCC. Conclusion Copper accumulation in liver may be related with hepatocarcinogenesis. Administration of copper-chelating agents may have a potential of chemoprevention for hepatocellular carcinoma in patients with chronic liver diseases caused by hepatitis C virus. Acknowledgments The author would like to thank Drs. S. Okabe, H. Sakurai, K. Suzuki, M. Yukawa, and other colleagues for their great contribution to this paper. References

The recurrence rate from the untreated site in patients with HCCs of 3 cm or less in diameter, which had been treated by percutaneous ethanol injection or radiofrequency ablation, was 60% in 3 years and 70– 80% in 5 years. The recurrence rate tended to be higher in high-copper-content group than in lowcopper-content group. Therefore, we performed a randomized clinical trial of the recurrence prophylactic treatment by using the hydrochloric acid trientine, which is a copper-chelating agent. We enrolled 24 patients with HCC of 3 cm or less in diameter, which had been completely ablated by percutaneous ethanol injection or radiofrequency ablation in this trial. We randomly divided into two groups of hydrochloric acid trientine, 250 and 750 mg in dosage, and analyzed the results. The drug was administered to 12 patients in each group everyday for 12 weeks. While administering hydrochloric acid trientine, copper was discharged in urine in both groups. After

Ebara M, Ohto M, Watanabe Y, Kimura K, Saisho H, Tsuchiya Y, Okuda K, Arimizu N, Kondo F, Ikehira H, Fukuda N, Tateno Y. Diagnosis of small hepatocellular carcinoma: correlation of MR imaging and tumor histologic studies. Radiology. 1986;159: 371–7. Ebara M, Watanabe S, Kita K, Yoshikawa M, Sugiura N, Ohto M, Kondo F, Kondo Y. MR imaging of small hepatocellular carcinoma: effect of intratumoral copper content on signal intensity. Radiology. 1991;180:617–21. Ebara M, Fukuda H, Kojima Y, Morimoto N, Yoshikawa M, Sugiura N, Satoh T, Kondo F, Yukawa M, Matsumoto T, Saisho H. Small hepatocellular carcinoma: relationship of signal intensity to histopathologic findings and metal content of the tumor and surrounding hepatic parenchyma. Radiology. 1999; 210:81–8.

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Ebara M, Fukuda H, Hatano R, Saisho H, Nagato Y, Suzuki K, Nakajima K, Yukawa M, Kondo F, Nakayama A, Sakurai H. Relationship between copper, zinc and metallothionein in hepatocellular carcinoma and its surrounding liver parenchyma. J Hepatol. 2000;33:415–22. Ebara M, Fukuda H, Hatano R, Yoshikawa M, Sugiura N, Saisho H, Kondo F, Yukawa M. Metal contents in the liver of patients with chronic liver disease caused by hepatitis C virus. Reference to hepatocellular carcinoma. Oncology. 2003;65: 323– 30. Fukuda H, Ebara M, Okabe S, Yoshikawa M, Sugiura N, Saisho H, Kondo F, Yukawa M. Metal contents of liver parenchyma after percutaneous ethanol injection or radiofrequency ablation in patients with hepatocellular carcinoma before and after trientine hydrochloride therapy. J Lab Clin Med. 2004;143:333–9. Hatano R, Ebara M, Fukuda H, Yoshikawa M, Sugiura N, Kondo F, Yukawa M, Saisho H. Accumulation of copper in the liver and hepatic injury in chronic hepatitis C. J Gastroenterol. Hepatol. 2000;15:786–91. Kasai H, Nishimura S. Hydroxylation of deoxyguanosine at the C-8 position by ascorbic acid and other reducing agents. Nucleic Acids Res 1984;12:2137–45. Khaliquzzaman M, Zaman MB, Khan AH. Trace element analysis in biological materials by external beam PIXE. Nucl Instrum Methods. 1981;181:209– 15. Kubo S, Fukuda H, Ebara M, Ikota N, Saisho H, Nakagawa H, Ozawa T, Yukawa M, Kato K, Satoh T, Watayo T, Sakurai H. Evaluation of distribution patterns for copper and zinc in metallothionein and superoxide dismutase in chronic liver diseases and hepatocellular carcinoma using high-performance liquid chromatography (HPLC). Biol Pharm Bull. 2005;28:1137–41. Sakurai H, Nakajima K, Kamada H, Satoh H, Otaki N, Kimura M, Kawano K, Hagino T. Coppermetallothionein distribution in the liver of Long Evans cinnamon rats: Studied on Immunohistochemical staining, metal determination, gel filtration and electron spin resonance spectroscopy. Biochem Biophys Res Commun 1993;192:893–8. Yoshida M, Masuda R, Sasaki M, Takeichi N, Kobayashi H, Dempo K. New mutation causing

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hereditary hepatitis in laboratory rat. J Hered 1987; 78:361–5. An invited paper presented in the symposium “Is copper involved in carcinogenesis/carcinostatics?” Copper transports and carcinostatics: copper transporters regulate the cellular pharmacology of cisplatin Roohangiz Safaei, Ph.D. and Stephen B. Howell, M.D. Moores UCSD Cancer Center, University of California, San Diego, La Jolla, CA 92093, USA Corresponding Author: [email protected] Introduction The platinum (Pt)-containing drugs cisplatin (DDP), carboplatin (CBDCA), and oxaliplatin (L-OHP) are widely used for the treatment of common cancers. These drugs are highly polar and require specific transporters for their passage across the plasma membrane (Gately and Howell 1993; Wang and Lippard 2005). Cu homeostasis in cells is maintained by the orchestrated efforts of the major Cu importer, CTR1, the metallochaperones ATOX1, CCS, and COX17, and the exporters ATP7A and ATP7B. This system ensures Cu delivery to the Cu-requiring centers and detoxification of the toxic-free Cu (Pena et al. 1999). CTR1 exists as an integral membrane homotrimer that forms a pore through which the Cu (I) flows inward after interactions with the metalbinding sequences at the N-terminal extracellular and membrane-spanning domains. Absence of CTR1 is lethal to embryos due to the essential role of Cu in mesodermal differentiation and organogenesis (Haremaki et al. 2007). In cultured cells, CTR1 is most essential when environment levels of Cu are low (Nose et al. 2006). CTR1 also appears to function as a sensor of metal toxins as indicated by its downregulation in the presence of toxic levels of Cu (Holzer and Howell 2006). Metallochaperones COX17, CCS2, and ATOX1 mediate the transfer of internalized Cu to the mitochondria, cytosol, and the secretory compartments, respectively. ATOX1 binds Cu(I) via a single metal binding sequence Cys-X-X-Cys and transfers it to similar sequences in the N-terminal cytosolic domain of

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ATP7A and ATP7B at the trans-Golgi network (TGN). These P-type ATPases sequester Cu in the TGN where it is transferred to secretory cuproproteins. Several motifs of the ATP7A and ATP7B molecules, including the conserved CPC motif in the sixth transmembrane domain, are required for translocation of Cu across the vesicular membrane and for Cu-induced trafficking of these proteins toward the plasma membrane. Mutations in ATP7A and ATP7B are the cause of Menkes and Wilson’s diseases, respectively (Linz and Lutsenko 2007). A large body of evidence supports a direct role for the Cu transporters and chaperones in regulating the uptake, intracellular distribution, and efflux of platinum-containing drugs. Attention was initially called to this phenomenon by the finding of parallel changes in the cellular pharmacology of Cu and DDP in cell lines that had been selected independently for resistance to these agents. This led to investigation of the role of Cu homeostasis system in the regulation of cellular pharmacology of Pt-containing drugs (Katano et al. 2002; Samimi et al. 2004a; Komatsu et al. 2000). Cells selected for resistance to DDP are often found to be cross-resistant to Cu and vice versa. Reduction in the initial influx of Cu in Cu-resistant cells is paralleled by decreased uptake of DDP; reduced initial uptake of DDP in the DDP-resistant cells is accompanied by reduced accumulation of Cu. CTR1 Studies in whole cells lacking the expression of CTR1 or in cells engineered to over-express this transporter showed that CTR1 modulates the cytotoxic effects and accumulation levels of Cu and the platinum-containing drugs cisplatin (DDP), carboplatin (CBDCA), and oxaliplatin (L-OHP) in a similar manner. Loss of CTR1 expression in yeast cells and mouse embryonic fibroblasts reduced the uptake of DDP, CBDCA, and LOHP by ~65% and diminished the sensitivity of cells to the cytotoxic effects of DDP and CBDCA (Lin et al. 2002; Ishida et al. 2002; Holzer et al. 2006). Forced expression of human CTR1 in ovarian carcinoma A2780 cells markedly increased the uptake of the platinum-containing drugs and Cu (Holzer et al. 2004a). hCTR1-mediated influx of DDP appears to be more important during the initial phase of accumulation. A difference in accumulation was detectable as early as 3 min; by 5 min, CTR1-expressing cells contained 5.5-

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fold more DDP than controls; and by 60 min, the different in uptake was 2.4-fold (Holzer et al. 2004a). Confocal microscopy and Western blot analysis has demonstrated that exposure to even quite low concentrations of DDP trigger the rapid downregulation of CTR1; even a concentration as low as 0.5 μM DDP downregulates CTR1 within 15 min (Holzer and Howell 2006). Thus, DDP triggers downregulation of its own major influx transporter. Cu also triggers degradation of CTR1 but requires much higher concentrations, and Cu-induced degradation occurs more slowly (Holzer et al. 2004b). DDP-induced downregulation of CTR1 requires endocytosis, the 20S proteasome, and possibly the ubiquitin ligase NEDD4-L (Liu et al. 2007). Current evidence suggests that there is a major difference in the way in which CTR1 mediates the influx of Cu and DDP. Whereas Cu appears to pass through a pore formed by the trimeric CTR1 complex, DDP appears to be ingested by endocytosis. The finding that the ATOX1 also plays a role in controlling the downregulation of CTR1 by DDP (Safaei et al. 2007b) and the demonstration of a requirement for CTR1 in the regulation of FGF-related signaling events in the embryo (Haremaki et al. 2007) suggest that CTR1 may regulate the permeability of the cell to DDP indirectly via effects on other transporters. ATOX1 ATOX1 interacts with ATP7A (Banci et al. 2005) and ATP7B (Ralle et al. 2004) and is believed to transfer Cu from CTR1 to these proteins. Fibroblasts from transgenic ATOX1−/− mice were found to be markedly more sensitive to the cytotoxic effect of Cu and accumulated higher levels of Cu due to their inability to efflux Cu (Hamza et al. 2001). This confirmed that ATOX1 is required for the efflux of Cu. When exposed to DDP for various periods of time, ATOX1−/− cells showed less sensitivity to DDP, lower rates of DDP uptake into both whole cells and DNA than controls (Linz and Lutsenko 2007). Surprisingly, ATOX1−/− cells were impaired in their ability to downregulate CTR1 during exposure to either Cu or DDP, suggesting that ATOX1 also functions in the regulation of the uptake of DDP (Safaei et al. 2007b). ATP7A and ATP7B The two heavy metal ATPases, ATP7A and ATP7B, are reported to play distinctly different roles in the

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transport of Cu. The ubiquitously expressed ATP7A is mainly involved in the synthesis of the cuproproteins in the secretory compartment and the distribution of Cu throughout the body. In contrast, while the liverspecific ATP7B is involved in the loading of Cu onto ceruloplasmin, it seems to function mainly to detoxify excess Cu (reviewed in Linz and Lutsenko 2007). The two proteins also have distinct subcellular localization and trafficking patterns (Linz and Lutsenko 2007). Upregulation of ATP7A and ATP7B has been observed in tumors and cell lines that had acquired resistance to DDP (Safaei 2006). Transfection of ovarian cells with ATP7A or ATP7B increased resistance to DDP, CBDCA, and L-OHP and also the rates of efflux of these drugs. ATP7A-transfected cells were more resistant to the all three platinum-containing drugs but nevertheless accumulated more rather than less platinum, suggesting that ATP7A functions in the intracellular sequestration and detoxification of the platinum-containing drugs (Katano et al. 2002; Samimi et al. 2004a; Komatsu et al. 2000; Katano et al. 2003; Katano et al. 2004). Analysis of the subcellular distribution of ATP7B (Katano 2004) and ATP7A (Samimi et al. 2004b) after DDP exposure in the 2008 ovarian carcinoma cells showed that DDP and Cu both caused ATP7B to traffic toward the plasma membrane, but only Cu was able to induce the centripetal trafficking of the ATP7A in these cells. Studies of the ability of ATP7B to transport DDP were performed in vesicles isolated from insect Sf9 cells over-expressing ATP7B. The Sf9 cells were engineered to express either a wild-type ATP7B or a transport-defective mutant of this protein in which the CPC motif in the sixth transmembrane domain was converted to CPA. When vesicles expressing either the wild type or mutant forms of ATP7B were incubated with DDP, it was found that, while both bound equal amounts of DDP on their surface, only the wild-type form transported DDP into the vesicles in an ATP-dependent manner. Like Cu, DDP stimulated the formation of an acylphosphate intermediate of ATP7B. Kinetic studies showed that DDP was not as good a substrate for ATP7B as Cu (Safaei et al. 2007a). Conclusions Emerging data supports a direct role for the transporters and chaperones of the Cu homeostasis system

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in the transport and intracellular distribution of the three commonly used platinum-containing drugs and suggest that the metal-binding sequences of these proteins are likely to be involved. CTR1 appears to enhance the initial influx of DDP, probably via an endocytotic mechanism, and ATP7A and ATP7B modulate DDP efflux. As CTR1 has been reported to be linked with the FGF signaling system (Haremaki et al. 2007); it is likely that its DDP- and Cu-induced downregulation triggers signaling events that may impact survival programs within the cell. This concept is particularly intriguing, as the two efflux proteins, ATP7A and ATP7B, are also regulated by hormones and signaling events (Linz and Lutsenko 2007). It is important to note that all studies to date have utilized cell lines in tissue culture; little is currently known about the importance of these Cu homeostasis proteins as determinants of response to treatment with the platinum-containing drugs in patients. Given the evidence that Cu transporters and chaperones can modulate the cellular pharmacology of the platinum drugs, it will be of interest to investigate how these drugs alter Cu homeostasis in patients undergoing therapy. Acknowledgments This review was supported by the NIH grant CA78648-08 and a grant from the Clayton Medical Research Foundation, Inc. The production of 64Cu at Washington University School of Medicine was supported by the National Cancer Institute grant R24-CA86307. References Banci L, Bertini I, Ciofi-Baffoni S, Chasapis CT, Hadjiliadis N, Rosato A. An nmr study of the interaction between the human copper(i) chaperone and the second and fifth metal-binding domains of the menkes protein. FEBS J 2005;272:865–71. Gately DP, Howell SB. Cellular accumulation of the anticancer agent cisplatin: a review. Br J Cancer 1993;67:1171–6. Hamza I, Faisst A, Prohaska J, Chen J, Gruss P, Gitlin JD. The metallochaperone atox1 plays a critical role in perinatal copper homeostasis. Proc Natl Acad Sci U S A 2001;98:6848–52. Haremaki T, Fraser ST, Kuo YM, Baron MH, Weinstein DC. Vertebrate ctr1 coordinates morpho-

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genesis and progenitor cell fate and regulates embryonic stem cell differentiation. Proc Natl Acad Sci U S A 2007;104:12029–34. Holzer AK, Katano K, Klomp LW, Howell SB. Cisplatin rapidly down-regulates its own influx transporter hctr1 in cultured human ovarian carcinoma cells. Clin Cancer Res 2004a;10:6744–9. Holzer AK, Samimi G, Katano K, et al. The copper influx transporter human copper transport protein 1 regulates the uptake of cisplatin in human ovarian carcinoma cells. Mol Pharmacol 2004b;66:817–23. Holzer AK, Manorek GH, Howell SB. Contribution of the major copper influx transporter ctr1 to the cellular accumulation of cisplatin, carboplatin, and oxaliplatin. Mol Pharmacol 2006;70:1390–4. Holzer AK, Howell SB. The internalization and degradation of human copper transporter 1 following cisplatin exposure. Cancer Res 2006;66:10944–52. Ishida S, Lee J, Thiele DJ, Herskowitz I. Uptake of the anticancer drug cisplatin mediated by the copper transporter ctr1 in yeast and mammals. Proc Natl Acad Sci USA 2002;99:14298–302. Katano K, Kondo A, Safaei R, et al. Acquisition of resistance to cisplatin is accompanied by changes in the cellular pharmacology of copper. Cancer Res 2002;62:6559–65. Katano K, Safaei R, Samimi G, Holzer A, Rochdi M, Howell SB. The copper export pump atp7b modulates the cellular pharmacology of carboplatin in ovarian carcinoma cells. Mol Pharmacol 2003;64:466–73. Katano K, Safaei R, Samimi G, et al. Confocal microscopic analysis of the interaction between cisplatin and the copper transporter atp7b in human ovarian carcinoma cells. Clin Cancer Res 2004;10:4578–88. Komatsu M, Sumizawa T, Mutoh M, et al. Coppertransporting p-type adenosine triphosphatase (atp7b) is associated with cisplatin resistance. Cancer Res 2000;60:1312–6. Lin X, Okuda T, Holzer A, Howell SB. The copper transporter ctr1 regulates cisplatin uptake in saccharomyces cerevisiae. Mol Pharmacol 2002; 62:1154–9. Linz R, Lutsenko S. Copper-transporting ATPases atp7a and atp7b: Cousins, not twins. J Bioenerg Biomembr 2007;39:403–7. Liu J, Sitaram A, Burd CG. Regulation of copperdependent endocytosis and vacuolar degradation of the

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yeast copper transporter, ctr1p, by the rsp5 ubiquitin ligase. Traffic 2007;8:1375–84. Nose Y, Rees EM, Thiele DJ. Structure of the ctr1 copper trans'pore'ter reveals novel architecture. Trends Biochem Sci 2006;31:604–7. Pena MM, Lee J, Thiele DJ. A delicate balance: Homeostatic control of copper uptake and distribution. J Nutr 1999;129:1251–60. Ralle M, Lutsenko S, Blackburn NJ. Copper transfer to the n-terminal domain of the Wilson disease protein (atp7b): X-ray absorption spectroscopy of reconstituted and chaperone-loaded metal binding domains and their interaction with exogenous ligands. J Inorg Biochem 2004;98:765–74. Safaei R, Otani S, Larson BJ, Rasmussen ML, Howell SB. Transport of cisplatin by the copper efflux transporter ATP7B. Mol Pharmacol 2007a; 73:461–8. Safaei R, Rasmussen ML, Francisco KS, Howell SB The copper chaperone atox1 is involved in the intracellular sequestration of cisplatin. In: Proc Amer Assoc Cancer Res, 2007b, pp. 1330. Safaei R. Role of copper transporters in the uptake and efflux of platinum containing drugs. Cancer Lett 2006;234: 34–9. Samimi G, Katano K, Holzer AK, Safaei R, Howell SB. Modulation of the cellular pharmacology of cisplatin and its analogs by the copper exporters atp7a and atp7b. Mol Pharmacol 2004a;66:25–32. Samimi G, Safaei R, Katano K, et al. Increased expression of the copper efflux transporter atp7a mediates resistance to cisplatin, carboplatin and oxaliplatin in ovarian cancer cells. Clin Cancer Res 2004b;10:4661–9. Wang D, Lippard SJ. Cellular processing of platinum anticancer drugs. Nat Rev Drug Discov 2005;4:307–20. An invited paper presented in the symposium “Zinc and the common cold” Zinc and the common cold George A. Eby George Eby Research, 14909-C Fitzhugh Road, Austin, TX 78736, USA Corresponding Author: [email protected]

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Some early and modern moments in treating respiratory disease with zinc Zinc has a long history of use in treating upper respiratory disease. As early as 1901, astringent zinc sulphocarbolate nose drops were used in the treatment of chronic nasal catarrh, while menthol and eucalyptol were used to treat acute nasal catarrh (Merck 1901). Astringent zinc sulfate-soaked nasal packings in conjunction with intranasal electricity (3–5 mÅ) prevented allergy and common cold symptoms for up to a year (Shields 1936). This technique was originally used by Franklin to treat hay fever (Franklin 1931). Others later reported intranasal zinc ionization as a clinical treatment for vasomotor rhinitis (Gray 1961; Weir 1967). In 1984, Eby, Davis, and Halcomb showed that astringent zinc gluconate lozenges shortened common colds by 7 days, but in 2006, the same team showed that non-astringent zinc orotate lozenges and zinc gluconate nasal spray had no effect on common colds (Eby et al. 1984; Eby and Halcomb 2006). For over 100 years, there has been much difference in technique and results. Why is there so much difference in results? Since the initial 1984 paper, there have been several dozen original papers and over one hundred journal commentaries about zinc lozenges and common colds. Trial results have been mixed, with some reporting strong efficacy while others produced worsened results and some produced modest or null results. There are several reasons for these mixed results.

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glycinate releases <57% iZn, while zinc gluconate citrate and zinc citrate and zinc orotate release 0% iZn (Eby 2004). Computation of metal speciation is complex and requires the use of the US National Institute of Technology (NIST) solution chemistry data bases and the skills of solution chemists. Guy Berthon, PhD, retired Directeur de Recherche CNRS Laboratoire de Chimie Bioinorganique Médicale, Université Paul Sabatier, Toulouse, France and editor of “Handbook of Metal-Ligand Interactions in Biological Fluids - Bioinorganic Medicine” performed critical zinc speciation computations, and they have been published (Eby 2004). The sum of all positively charged zinc species used in clinical trials of zinc lozenges for common colds is termed iZn. At sufficiently low pH, nearly all zinc compounds release iZn, which represents good bioavailability at stomach acid pH. At physiologic pH 7.4, the pH of the oral and nasal tissues, iZn is reduced (or eliminated) from all zinc compounds except zinc acetate. Weak bonds between a metal and ligand produce mainly ionic and neutral species (zinc gluconate, sulfate, chloride, and acetate). Strong bonds between metal and ligand produce mainly neutral (zinc orotate) and/or negative species (zinc citrate). Nonsoluble metal ligand compounds (zinc oxide, stearate, palmitate, and oleate) are non-ionic. Amounts of metal/ ligand species of soluble metal compounds are calculable, and variables include metal and ligand stability constants, pH and pK values, concentrations, and temperature. Consequently, some zinc lozenges did not release any active ingredient. Mouth–nose biologically closed electric circuit:

Solution chemistry: One major difference is the solution chemistry of the zinc lozenges compositions used. Ionized zinc (iZn) is astringent, antirhinoviral, (Korant 1974; Merluzzi 1989), reduces intercellular adhesion molecule-1 (ICAM-1) (Prasad 2008), increases INF-γ (Driessen 1995; Berg 2001), and protects cell plasma membranes (including mast and goblet cells) in a dose–response manner (Pasternak 1987). Neutral and negatively charged zinc species do not have these beneficial characteristics and are non-astringent to the mouth. Zinc acetate release 100% iZn at physiologic pH. Zinc gluconate releases 72% iZn, zinc gluconate

The second major reason involves the mouth–nose biologically closed electric circuit (BCEC). The BCEC was first described in 1983 by Björn E W Nordenström, MD, Professor Emeritus of Diagnostic Radiology, Karolinska Institute and Hospital of the Karolinska Institute in Stockholm Sweden and chairman of the Nobel Prize Selection Committee for Physiology and Medicine. (Nordenström 1983) The mouth–nose BCEC is part of a large number of human BCECs, which remain largely unstudied in medicine, but are vital in recovery from certain diseases. In the mouth-nose BCEC, electrons flow from nose to mouth, and metallic ions flow from mouth to nose.

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The mouth–nose BCEC can be reversed using exogenous electricity (Franklin 1931; Shields 1936). The mouth–nose BCEC is the only BCEC observable externally in the human body, requiring only a volt meter sufficiently accurate to detect the 1 to 2 mV fluctuation resulting from the respiratory rhythm. Had the mouth–nose BCEC not existed, the cure for the common cold, as zinc sulphocarbolate nose drops, would likely have been described in 1901 (Eby and Halcomb 2006). There have been both positive (Hirt et al. 2000; and Mossad 2003) and negative (Turner 2001; Belongia 2001; Eby and Halcomb 2006) reports of zinc gluconate used intranasally to treat common colds. Nasal application is not believed to be efficacious against common colds without electricity driving iZn into the nasal epithelium to reverse the BCEC. However, zinc nose drops, sprays, and gels may produce benefits observed by blocking mast cell degranulation, thus benefiting rhinitis as has been occasionally reported since 1901. Contact of zinc with the olfactory bulb can cause excruciating pain and persistent anosmia, which may become permanent unless immediately removed by continued nasal irrigation until pain subsides (Eby 2006). Zinc lozenges have never been associated with any side effects other than taste issues. Voltages between interior of mouth and interior of nose vary from 60 to 120 mV, which may be important to the rate of recovery using zinc lozenges. Resistances between the interior of the mouth and the interior of the nose vary from 1,000 to 500,000 Ω. Low resistances (1,000–20,000 Ω) are found in people that have very frequent colds and nasal allergies. Mid-range resistances (40,000 to 60,000 Ω) are found in most people. Very high resistances (100,000 to 500,000 Ω) are found in people that never have cold symptoms or allergy symptoms (Eby 2006). Other reasons for difficulties: Zinc gluconate was flavor-unstable and extremely bitter in common candy base formulations “requiring” flavor-masking with food acids or other agents. Some manufacturers also used very low doses of zinc to limit astringency and bitterness. An important 1987 in vitro test of ionic zinc compounds against rhinoviruses (Geist 1987) appears sabotaged by the addition to the culture medium of 30 mmol magnesium chloride, which is known to increase rhinoviral release

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by 8- to 310-fold (Fiala 1968). Similarly, 100 mMol magnesium chloride throat lozenges have been reported to greatly worsen and lengthen common colds (Eby 2006). Changing methods of clinical trial re-analysis, wherein a variety of criteria (including total zinc) was substituted for iZn dose–response criteria, thus denying efficacy of zinc lozenges (Caruso et al. 2007), was particularly harmful. Others have written meta-analyses that did not consider solution chemistry, reporting mainly a “head-count” of successful and unsuccessful reports; therefore, they incorrectly reported lack of efficacy. Meta-analysis of 15 zinc lozenge clinical trials (1984–2007) Two solution-chemistry-based meta-analyses of all zinc lozenges for common colds clinical trials showed that there was strong dose–response linearity in results when daily iZn was considered. While lozenges that had little or no iZn produced no benefits, lozenges having a daily iZn value of 80 (equivalent to 10 mg iZn lozenges used eight times per day) shortened colds by 4 days, and lozenges having a daily iZn value of 150 (equivalent to 18 mg iZn lozenges used 8.3 times per day) shortened colds by 5 to 7 days (Eby 2004; Eby 2008). Daily iZn intake from lozenges was found to be directly associated with reductions in durations of common colds by two measures: median (n=14, r= 0.772, P=0.0012) and mean (n=13, r=0.813, P= 0.0007) durations. A review of these studies (Eby 2004; Eby 2008) further showed that most single ligand (zinc gluconate and zinc acetate) compositions succeeded and most multi-ligand (flavor-masked zinc gluconate and zinc acetate) compositions failed. All other zinc compounds failed. Astringent, mildly chalky, and drying (non-bitter) zinc gluconate lozenges were successful (Eby et al 1984; Al-Nakib 1987). Astringent and drying zinc acetate lozenges were successful in three trials of identical compositions (Petrus 1998; Prasad 2000; Prasad 2008). Slightly astringent and drying zinc gluconate glycine lozenges were equivocally successful in four trials due to their low iZn content. Non-astringent, non-drying zinc gluconate with citric acid as flavor mask lozenges failed. Non-astringent and non-drying zinc orotate lozenges with zinc gluconate nasal spray failed. Non-astringent and non-drying zinc acetate lozenges flavor masked with

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tartaric acid and sodium bicarbonate to produce effervescent lozenges lengthened colds by more than 4 days, most likely due to strongly negatively charged zinc species binding with nasal ionic zinc to neutralize the species. Non-astringent and non-drying zinc acetate lozenges (flavor masked by cooking hard candy, zinc acetate, and several fatty food acids) failed. Non-astringent zinc aspartate lozenges failed. Zinc lozenges should dissolve in 20 to 30 min with a minimum of saliva produced to keep iZn salivary concentration over 5 mMol. Lozenges should always be used each 2 h for optimal performance. Fick’s Laws of Membrane Permeability apply (Eby 1994). Take home message Comparison of clinical trial results using daily iZn values is the correct way to review the literature. Concerning commercial zinc lozenges, there are over 30 different zinc lozenges marketed in the USA, but nearly all have formulations and additives (usually citric acid) that eliminate astringency, iZn, and efficacy. A current listing of effective and non-effective zinc lozenges is maintained at http://zinc-lozenges.com (accessed March 17, 2008). The future of effective zinc lozenges appears bleak to non-existent. Clinical trials of properly manufactured 14 to 20 mg zinc acetate lozenges used 10 times per day (daily iZn values of 140 to 200) are needed. Separately, we should revisit zinc ionization techniques and learn more about the possibility of preventing common colds and rhinitis for a year with two 15-min zinc ionization office treatments (Shields 1936), paying careful attention to the risk of inducing anosmia if zinc contacts the olfactory bulb (Eby and Halcomb 2006). We should be very concerned about switching decision criteria for efficacy by common cold authorities from iZn dose– response criteria to other criteria as has recently occurred (Caruso 2006). We should be extremely concerned about sabotaging research, as what appears to have occurred in a pivotal study by common cold authorities of the antiviral effects of zinc (Geist 1987). We should be very concerned about the future possibility of sabotaging clinical trial results by addition of magnesium to research lozenges. We should be concerned about common cold authorities’ longstanding negativity toward zinc lozenges for colds. We should be very concerned by the lack of efficacy in non-astringent commercial zinc lozenges, when a cure

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for common colds using astringent zinc lozenges is possible.

References: Al-Nakib W, Higgins PG, Barrow I, Batstone G, Tyrell DA. Prophylaxis and treatment of rhinovirus colds with zinc gluconate lozenges. J Antimicrob Chemother. 1987;20:893–901. Belongia EA, Berg R, Liu K. A randomized trial of zinc nasal spray for the treatment of upper respiratory illness in adults. Am J Med. 2001;111:103–8. Berg K, Bolt G, Andersen H, Owen TC. Zinc potentiates the antiviral action of human IFN-alpha tenfold. J Interferon Cytokine Res. 2001;21:471–4. Caruso TJ, Prober CG, and Gwaltney JM Jr. Treatment of naturally acquired common colds with zinc: a structured review. Clin Infect Dis. 2007;45:569–74. Driessen C, Hirv K, Kirchner H, and Rink L. Zinc regulates cytokine induction by superantigens and lipopolysaccharide. Immunology. 1995;84:272–7. Eby GA, Davis DR, Halcomb WW. Reduction in duration of common colds by zinc gluconate lozenges in a double blind study. Antimicrob Agents Chemother. 1984;25:20–24. Eby GA. Do zinc lozenges shorten common colds? An assessment of dose response criteria from all clinical trials from 1984 – 2007 and discussion of bias sources. Pending. Eby GA, Halcomb WW. Ineffectiveness of zinc gluconate nasal spray and zinc orotate lozenges in common-cold treatment: a double-blind, placebocontrolled clinical trial. Altern Ther Health Med. 2006;12:34–8. Eby GA. Handbook for curing the common cold: the zinc lozenge story. George Eby Research, Austin, Texas; 1994. Available at: http://george-eby-research. com/html/handbook-for-curing-the-common-cold. html. Accessed October 18, 2007. Eby GA. Rescue treatment and prevention of asthma using magnesium throat lozenges: Hypothesis for a mouth-lung biologically closed electric circuit. Med Hypotheses. 2006;67:1136–41. Eby GA. Zinc lozenges: cold cure or candy? Solution chemistry determinations. Biosci Rep. 2004;24:23–39. Fiala M. Plaque formation by 55 rhinovirus serotypes. Appl Microbiol. 1968;16:1445–50.

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Franklin P. Treatment of hay fever by intranasal zinc ionization. BMJ 1931:1115–6. Available at: http:// george-eby-research.com/gif/zinc-ionization.jpg. Accessed December 31, 2007. Geist FC, Bateman JA, Hayden FG. In vitro activity of zinc salts against human rhinoviruses. Antimicrob Agents Chemother. 1987;31:622–4. Gray G. Zinc ionization and vasomotor rhinitis. Med J Aust. 1961;48:548–50. Hirt M, Nobel S, Barron E. Zinc nasal gel for the treatment of common cold symptoms: a double-blind, placebo-controlled trial. Ear Nose Throat J. 2000;79:778–80, 782. Korant BD, Kaurer JC, and Butterworth BE. Zinc ions inhibit replication of rhinoviruses. Nature (Lond). 1974;248:588–90. Merck. Manual of the Materia Medica, Part II, Formulas, Merck, New York; 1901. p. 125. Available at: http://george-eby-research.com/gif/merck-1901zinc-nasal-spray.pdf. Accessed December 31, 2007. Merluzzi VJ, Cipriano D, McNeil D, Fuchs V, Supeau C, Rosenthal AS, Skiles JW. Evaluation of zinc complexes on the replication of rhinovirus 2 in vitro. Res Commun Chem Pathol Pharmacol. 1989;66:425–40. Mossad SB. Effect of zincum gluconicum nasal gel on the duration and symptom severity of the common cold in otherwise healthy adults. QJM. 2003;96: 35–43. Nordenström BE. Biologically closed electric circuits. Clinical, experimental and theoretical evidence for an additional circulatory system. Stockholm: Nordic Medical Publications;1983. pp. 112–172. Pasternak C. A novel form of host defense: membrane protection by calcium and zinc ions. Biosci Rep. 1987;7:81–91. Petrus EJ, Lawson KA, Bucci L R, Blum K. Randomized, doublemasked, placebo-controlled, clinical study of the effectiveness of zinc acetate lozenges on common cold symptoms in allergytested subjects. Curr Ther Res. 1998; 59:595–607. Prasad AS, Fitzgerald JT, Bao B, Beck FWJ, Chandrasekar PH. Duration of symptoms and plasma cytokine levels in patients with the common cold treated with zinc acetate. A randomized, double-blind, placebo-controlled trial. Ann Int Med. 2000;133: 245–52. Prasad AS. Zinc, ICAM-1, and the common cold. J Infect Dis. 2008. In press. Abstract available at:

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http://www.angelfire.com/nd/isterh/2007conference/ glimpse.html Shields C. The zinc ionization treatment of hay fever. The Practitioner. Jan–June 1936;645–8. Available at: http://george-eby-research.com/html/zincionization-allergy-colds-1936.pdf. Accessed December 31, 2007 Turner RB. Ineffectiveness of intranasal zinc gluconate for prevention of experimental rhinovirus colds. Clin Infect Dis. 2001; 33: 1865–70. Weir CD. Intranasal ionization in the treatment of vasomotor nasal disorders. J Laryngol Otol. 1967;81:1143–50. An invited paper presented in the symposium “Zinc and the common cold” as part of ISTERH/NTES/ HTES ‘07” (Hersonissos, Crete-Greece, 21–26 October 2007), a joint meeting that constituted the VIIIth Conference of the International Society for Trace Element Research in Humans (ISTERH), the IXth Conference of the Nordic Trace Element Society (NTES), and the VIth Conference of the Hellenic Trace Element Society (HTES).

Treatment of the common cold with zinc: effect on pro-inflammatory cytokine (Soluble Interleukin-1 receptor antagonist) and ICAM-1 *A complete manuscript has been recently published: Prasad AS, Beck FWJ, Bao B, Snell D, Fitzgerald JT. The J. Inf. Dis. 197:795–802, 2008. Ananda S Prasad1, Frances WJ Beck1, Bin Bao1, Diane Snell1, and James T. Fitzgerald2 1 Wayne State University School of Medicine, Detroit, MI 48201 USA 2 University of Michigan Medical School, Ann Arbor, MI 48109 USA Corresponding author: Ananda Prasad Department of Internal Medicine 1122 Elliman Bldg., 421 East Canfield Wayne State University School of Medicine Detroit, MI 48201 Phone: 313–577–1597 Fax: 313–577–1128 E-mail: [email protected]

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Keywords Zinc, Cold, Cough, sIL-1ra, ICAM-1 Introduction Adults and children in the USA experience two to six episodes of common cold per year (Dingle 1964; Gwaltney et al. 1966), and the morbidity and loss of working hours due to this are substantial. The complications of the common cold include otitis media, sinusitis, and exacerbations of reactive airway diseases. The clinical syndrome of the common cold is caused by a variety of different viruses (Turner 2001). The rhinoviruses are the most frequent. The effect of zinc lozenges on the duration or severity of common cold symptoms has been examined in at least 14 different studies since 1984 when Eby et al. (1984) reported for the first time its efficacy in treatment of this disorder. Later trials gave inconclusive results (Turner 2001). Results of trials in which no effect of zinc was demonstrated were criticized as having inadequate sample sizes or using inadequate doses of zinc or formulations of zinc that reduced the release of zinc ions from the lozenge. In some studies, a significant effect of zinc lozenges for the treatment of common cold was criticized for inadequate blinding either by the use of poorly matched placebos or because the active preparation was associated with a high incidence of adverse effects (Turner 2001). A direct correlation between daily dosage of all positively charged zinc species at physiologic pH released from lozenges and reductions in duration of common cold has been hypothesized (Eby 1995). Zinc gluconate and zinc acetate have very low chemical stability and release positively charged zinc ions in aqueous solutions at physiological pH, while stronger complexes do not. We hypothesize that the beneficial effects of zinc may be due to its antioxidant and anti-inflammatory properties and its effect on intercellular adhesion molecule (ICAM-1), which is known to be a major cellular receptor for rhinovirus (Novick et al. 1996; Turner et al. 1999). Methods We recruited 50 volunteers from Detroit Medical Center, Michigan, to participate in a randomized, placebo-controlled trial of the efficacy of zinc acetate

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lozenges in treating the common cold. Participants were medical students, house staff, and employees at Wayne State University who were older than 18 years of age. Participants were informed of the placebocontrolled, double-blind nature of the study, and the study protocol was approved by the Human Investigation committee of Wayne State University. We recruited healthy volunteers who were free of any illness for various laboratory tests as controls. Our methods of study were similar to those published earlier (Prasad et al. 2000). The lozenges were cherry oil flavored Fast Dry (Eby 2004) zinc acetate lozenges manufactured by F & F Foods of Chicago, IL, USA. The active lozenges contained 13.3 mg of zinc as zinc acetate. One hundred percent of the zinc was available at physiologic pH 7.4 in positively charged, ionic form. The placebo and zinc lozenges were identical in weight, appearance, flavor, and texture. Participants were given 50 lozenges and were asked to dissolve one lozenge in their mouths every 2 to 3 h while awake for as long as they had cold symptoms. They were instructed to take no other cold preparations during the study period. Our primary end point was the average duration of cold symptoms. Secondary end points were plasma levels of (a) zinc, (b) the anti-inflammatory cytokines, soluble interleukin 1 receptor antagonist (sIL-Ira) and soluble tumor necrosis factor receptor 1 (sTNF-R1), and (c) the plasma adhesion molecules, soluble vascular endothelial cell adhesion molecule (sVCAM-1), and soluble intercellular adhesion molecule-1 (sICAM-1). Base line data were obtained and participants returned to the clinic on the fifth day for a blood draw and again for the final visit within 1 day of resolution of cold symptoms. At this time, they returned unused lozenges. This was done to check adherence and confirm that cold symptoms had resolved. Comparability in taste between zinc and placebo was tested in the participants at the beginning and at the end of the trial. We compared the changes in outcomes before and after intervention for the zinc and placebo groups. If the changes were normally distributed in both groups (Shapiro–Wilk test), we used unpaired t test to compare the mean changes. If the changes were not normally disturbed, the differences were examined using the nonparametric Wilcox on rank sum test. Multivariate analysis of variance with repeated meas-

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ures was used to determine the effect of treatment x time on severity scores. Fisher’s exact test was used to determine group differences in adverse effects. χ2 analysis was performed to determine group differences in correctly identifying lozenges at baseline and after treatment. Statistical analyses were completed using JMP IN software (version 5.1.2; SAS institute) on a MacBook Pro computer.

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symptoms lasted for 3.54±0.96 days in the zinc group and cold symptoms lasted for 7.39±0.98 days in the placebo group (p<0.0001). The duration of cough and nasal discharge were also significantly shorter in the zinc group. Table 1 Duration of common cold symptoms all subjects

Results The average duration of cold symptoms was 4.0 days in the zinc group and 7.1 days in the placebo group (p <0.0001). The duration of cough, nasal discharge, and muscle ache were significantly shorter in the zinc group compared to the placebo group. Whereas in 56% of the zinc group, the cold was completely resolved on day 4, no subject in the placebo group was free of cold symptoms on day 4. Repeated measures analysis of severity score indicated a significant effect for treatment × time between the zinc and placebo groups over 10 days (p=0.0002). At baseline, the average severity score of the zinc and placebo groups were 8.32 vs 7.78, respectively. By day 4, the average severity score of the zinc and placebo group were 3.45 vs 5.61, respectively. In the zinc group, at the beginning, only one subject identified the lozenge as “certainly zinc” and two subjects identified as “probably zinc.” Thus, three subjects out of 25 in this group (12%) were correct. At the end of the study, only one subject identified as “certainly zinc,” and another subject identified as “probably zinc.” Thus, two subjects (8%) were correct. In the placebo group, at the beginning, one subject said that the lozenge was “certainly placebo” and one other subject identified this as “probably placebo.” Thus, two subjects in this group (8%) were correct. At the end of the study, none of the subjects (0%) identified the placebo lozenge correctly. Contingency analysis of correct by group at the beginning by chi-square test (Fisher’s exact test), gave p=1.0. At the end of the study, the same test gave the p=0.5. Thus, there was no difference between the two groups. From these data, we concluded that the blinding of the lozenges were adequate. In addition, we analyzed the effect of clinical variables of 22 completely blinded subjects in zinc group and 23 in placebo group. Of the completely blinded groups, cold

Over all symptoms Nasal discharge Cough Muscle ache

Zinc group (n=25)

Placebo group (n=25)

P

4.00±1.04 (3.57–4.42) 3.00±1.63 (2.32–3.67) 2.16±1.70 (1.45–2.86) 0.80±1.22 (0.29–1.30)

7.12±1.26 (6.59–7.64) 4.56±3.01 (3.31–5.80) 5.08±2.97 (3.85–6.30) 2.00±2.25 (1.06–2.93)

<.0001 .02 <.0001 .02

Mean±SD (95% CI, days). Only significant effects are presented in this table. CI Confidence interval

The zinc group and placebo group did not differ significantly in the incidences of any of the side effects such as diarrhea, constipation, sweet taste, sour taste, bitter taste, after taste, dry mouth, mouth irritation, or bad taste. No subject complained of either abdominal pain or vomiting. Adherence to therapy was determined by lozenge count. The average number of lozenges taken daily was 6.9 in the zinc group and 6.5 in the placebo group. Plasma zinc increased significantly in the zinc group. Plasma sIL-1ra decreased significantly in the zinc group after treatment but increased in the placebo group post-treatment. A comparison of the mean changes (post vs pre) in plasma sTNF-R1 and plasma sVCAM-1 in the two groups were not statistically significant. Plasma sICAM-1 decreased significantly after treatment in the zinc group. Discussion Our results showed that the mean duration of cold symptoms, cough, nasal discharge, and muscle ache, and severity score were significantly decreased in zinctreated group in comparison to the placebo group. The blinding of therapy was adequate. The mean change in plasma sIL-1ra showed a significant decrease in the zinc group, whereas in the

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placebo group, the level of mean change in plasma sIL-ra showed a positive increase. Between-group mean change difference was statistically significant. IL-1ra is an anti-inflammatory cytokine and functions as a specific inhibitor of IL-1α and IL-1β inflammatory cytokines and is produced by monocytes and macrophages (Opal and De Palo 2000). The synthesis of IL-1ra and IL-1β are differentially regulated at their own promoter sites in the macrophages and monocytes. Our results suggest that the common cold viruses increased oxidative stress, which activated macrophages and monocytes resulting in increased production of both inflammatory cytokine IL-1β (not assayed here) and anti-inflammatory cytokine IL-1ra. We also assayed another anti-inflammatory cytokines sTNF-R1, which functions as a specific inhibitor of tumor necrosis factor activity on target tissues. sTNFR1 levels also decreased but non-significantly in the zinc-treated group. Thus, the decrease in sIL-1ra and sTNF-R1 only in the zinc group suggests that zinc decreased the oxidative stress resulting in decreased activation of monocytes and macrophages. Our previous studies have shown that zinc is an antioxidant (Prasad et al. 2007; Prasad et al. 2004). Many of the symptoms observed in the common cold resemble the effects of proinflammatory cytokines. Fever, lack of appetite, leukocytosis, hypoferremia, and induction of acute phase reactant proteins are known effects of interleukin-1 production by monocytes and macrophages. We previously showed that interleukin-1β production by mononuclear cells is increased in zinc-deficient subjects and is normalized by zinc supplementation suggesting that zinc modulates the proinflammatory cytokines released by monocytes and macrophages (Beck et al. 1997). Infection and oxidative stress activate nuclear factorkB (NF-kB), which results in increased generation and gene expression of inflammatory cytokines, such as TNF-α, IL-1β, and IL-8, and adhesive molecules, such as VCAM-1 and ICAM-1 (Prasad et al. 2004; Prasad et al. 2007). Zinc decreases oxidative stress and induces a zinc-dependent transcription factor A-20 in monocytesmacrophages, which inhibits NF-kB activation via TNF receptor associated factor (TRAF) pathway (Prasad et al. 2004). Zinc decreases oxidative stress by several mechanisms (Prasad et al. 2004, 2007). Inflammatory cytokines, generated by activated monocytes and macrophages, are also known to

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generate greater amounts of reactive oxygen species (Prasad 2007). Zinc supplementation to healthy human subjects aged 20–50 years and the elderly (55–80 years) reduced the concentration of the oxidative stress markers such as oxidative-stressrelated by-products malondialdelyde (MDA), 4hydroxyalkenals (HAE), and 8 hydroxydeoxyguanine in plasma, inhibited the ex vivo induction of TNF-α and IL-1β mRNA in mononuclear cells, and provided protection against TNF-α induced nuclear factor-kB activation in isolated peripheral blood mononuclear cell (Prasad et al. 2004; Bao et al. 2003). In this study, we observed that only the zinc group showed a significant decrease in plasma ICAM-1 levels, which suggests that zinc decreased the generation and gene expression of ICAM-1. Rhinovirus HRV-14 “docks” with ICAM-1 on the surface of somatic cells (Turner 2001; Novick et al. 1996). Thus, zinc may be in effect acting as an antiviral agent by reducing ICAM-1 levels. Another possibility is that zinc ions may complex with ICAM-1 preventing rhinovirus binding to cells (Novick et al. 1996). We conclude that zinc acetate preparation, as used in our study, was significantly effective in decreasing the mean duration of cold symptoms. We propose that the beneficial clinical effects seen in the zinc group were due to antioxidant and anti-inflammatory effects of zinc. We also suggest that a decrease in the ICAM-1 level in the plasma due to zinc therapy may have decreased the docking of the cold viruses on the surface of somatic cells. Acknowledgments This research is funded in part by NIH grant no. 5 R01 A150698-04 and The George and Patsy Eby Foundation, Austin, TX, USA. References Bao B, Prasad AS, Beck FWJ, Godmere M. Zinc modulates mRNA levels of cytokines. Am J Physiol (Endocrinol Metab). 2003;285:E1095–102. Beck FWJ, Prasad AS, Kaplan J, Fitzgerald JG, Brewer GJ. Changes in cytokine production and T cell subpopulations in experimentally induced zinc-deficient humans. Am J Physiol (Endocrinol Metab). 1997;272: E1002–7.

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Dingle JH, Badger GF, Jordan WS Jr. Illness in the home: study of 25,000 illnesses in a group o f Cleveland families. Cleveland, Ohio: Western Reserve University Press; 1964. 398 pp. Eby GA, Davis DR, Halcomb WW. Reduction in duration of common cold by zinc gluconate lozenges in a double-blind study. Antimicrob Agents Chemother. 1984;25:20–4. Eby GA. Linearity in dose-response from zinc lozenges in treatment of common colds. J Pharm Tech. 1995;11:110–22. Eby GA. Zinc lozenges: cold cure or candy? Solution chemistry determinations. Bioscience Reports. 2004;24:23–39. Gwaltney JM Jr, Hendley JO, Simon G, Jordan WS Jr. Rhinovirus infections in an industrial population. I. The occurrence of illness. N Engl J Med. 1966;275:1261–8. Novick SG, Godfrey JC, Godfrey NH, Wilder HR. How does zinc modify the common cold? Med Hypotheses. 1996;46:295–302. Opal SM, De Palo VA. Anti-inflammatory cytokines. Chest. 2000; 117: 1162–72. Prasad AS, Fitzgerald JT, Bao B, Beck FWJ, Chandrasekar PH. Duration of symptoms and plasma cytokine levels in patients with the common cold treated with zinc acetate. Annals Int Med. 2000;133:245–52. Prasad AS, Bao B, Beck FWJ, Kucuk O, Sarkar FH. Antioxidant effect of zinc in humans. Free Rad Bio Med. 2004;37:1182–90. Prasad AS. Zinc: mechanisms of host defense. J Nutr. 2007; 137: 1345–9. Prasad AS, Beck FWJ, Bao B, Fitzgerald JT, Snell DC, Steinberg JD, Cardozo LJ. Zinc supplementation decreases incidence of infections in the elderly: Effect of zinc on generation of cytokines and oxidative stress. Am J Clin Nutr. 2007;85:837–44. Turner RB. The treatment of rhinovirus infections: progress and potential. Antiviral Res. 2001;49:1–4. Turner RB, Wecker MT, Pohl G, Witek TJ, McNally E, St. George R, Winther B, Hayden FG. Efficacy of Tremacamra, a soluble intercellular adhesion molecule 1, for experimental rhinovirus infection. A randomized clinical trial. JAMA. 1999;281:1797–804. An invited paper presented in the symposium “The role micronutrients (iodine, iron and zinc) in intellectual development”

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Essentiality of zinc for human neuropsychological performance Harold H Sandstead1 and James G Penland2 Department of Preventive Medicine and Community Health, The University of Texas Medical Branch, Galveston, TX 77555–1109 USA 2 USDA ARS Grand Forks Human Nutrition Research Center, Grand Forks, ND 58201 1

Corresponding Author: [email protected] Zinc (Zn) deficiency appears to have a prevalence of at least 20.5% (Wuehler et al. 2005). Thus, adverse effects of Zn deficiency on neuropsychological function (NP) are of high importance (Frederickson et al. 2005; Sandstead et al. 2000). Evidence that Zn deficiency impairs human NP was first described in experimental subjects (ss) that were rapidly depleted of Zn. Abnormalities included confusion, ataxia, depression, hallucinations, paranoia, dysgeusia, and dysosmia (Henkin et al. 1975). Patients fed parenterally without Zn also had abnormal NP (Kay et al. 1976). In this paper, we summarize our work on the requirement of Zn for human NP. In all but the first study, Zn deficiency was the consequence of the ss selected diet. A powerful evaluation tool was the Cognition Psychomotor Assessment System (CPAS) developed by Penland (2000). Some studies included a design feature for the correction of simultaneous but unidentified micronutrient deficiencies that interfere with the physiological response to Zn treatment (Sandstead 1999; Solomons et al. 1999) was the administration of a broad mixture of the micronutrients (M) identified as essential by the US National Academy of Sciences (NAS FNB Expert Committee, 1989). The dose was 50% of the Recommended Dietary Allowance or Estimated Safe and Adequate Daily Dietary Intake. The first study (Penland 1991) involved 14 men fed with four diets providing 1–4 mg Zn/2,000 kcals in random order, for 35 days each, and a control diet providing 10 mg Zn/2,000 kcals at the beginning and end. Low Zn decreased (p<0.05) the performance of nine of 15 commonly used psychomotor, attention, perception, memory, and reasoning tasks (Golden 1981; Lachman et al. 1979; Lezak 1995; Posner 1978).

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The second study (Darnell and Sandstead 1991) measured effects of treatment (Rx) on short-term memory of 56 women (44 experimental and 12 controls) with good health, regular menses, and normal hemoglobin (Darnell and Sandstead 1991) in a study of short term memory (Wechsler 1981). The experimental ss had serum ferritin (SF) <20 ng/ml and the controls had SF >30 ng/ml. The 8-week daily Rx were 30 mg Zn and M (ZM), 30 mg Fe and M (FM), 30 mg each of Zn and Fe and M (ZFM), and M. The controls were given M. Short-term memory was measured on the eight day of the menstrual cycle using the Wechsler Memory Scale (Wechsler 1981). Many ss resigned. Even so, the experimental ss displayed increases in memory (p<0.05) over baseline after ZM (n=11), FM (n=6), and ZFM (n=6) but not after M (n=6). However, the 11 controls given M also improved (p<0.05). Evidence of Zn deficiency among preschool Chinese (Chen et al. 1985) led to a RCT of Zn in 740 low-income, 6- to 9 year-old Chinese from Shanghai, Chongqing, and Qingdao (Penland et al. 1997; Sandstead et al. 1998). Rx was 20 mg Zn and M (ZM), 20 mg Zn (Z), or M 6 days/week, for 10 week given at school. Knee height (KH) was measured by a digitized device (Stallings and Cronk 1993), and NP was measured by CPAS-R (revised). At baseline, almost all had normal serum ferritin (SF) and plasma Zn (PZ). The Rx effect on percent change KH was highly significant in Chongqing and Qingdao, and group comparisons showed ZnM>Z and M (Bonferroni, p<0.05). The Rx effect on percent change NP is shown in Table 1. Based on the above a similar RCT was done in 359 low-income 6- to 9-year-old Mexican Americans from Brownsville, TX, USA (Egger et al. 1999; Penland 2000; Penland et al. 1999). Rx was placebo (P), M, 20 mg Zn and M (ZM), and 24 mg Fe and M (FM), given at school five times per week for 10 weeks. Breakfast and lunch were also offered. Before Rx, no ss was anemic, the 25th % for SF=14 ng/ml (low), the 10th % for PZ=12.44 µmol/l (normal) and the 50th % for HZ=1.65 μmol/g (low). In girls with HZ< 1.65 μmol/g, the Rx effect on percent-change triceps skin fold and fat free mass was highly significant and group comparisons showed ZM > P, M and FM (Tukey–Kramer test, p<0.05). In girls with baseline PZ<75th % the Rx effect on NP for oddity recognition

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(with co-variables: Ln baseline HZ, Ln baseline SF, Ln post Rx PZ) was highly significant, and group comparisons showed ZM>M but marginally>P and FM (Tukey–Kramer test, p<0.05). Table 1 Zinc and micronutrient treatment effects on cognitive and psychomotor functions of 740 low-income Chinese children, aged 6–9 years (Penland et al. 1997; Sandstead et al. 1998) Task (measure)

Zn

ZnM

M

p value

Tapping single key, number of taps Tracking circular path, percent on target Continuous vigilance, percent correct hitsb Oddity (shapes) trials to criterionc

7.71± 0.83Aa 28.4± 2.03Aa

6.72± 0.87Aa 37.0± 2.16Ba

3.44± 0.84Ba 28.3± 2.14Aa

0.0009

−4.92± 1.93Aa −4.55± 1.16Aa

3.66± 2.02B −8.98± 1.20Ba

−0.12± 2.00AB −4.13± 1.19Aa

0.0092

0.0048

0.0067

Ages are mean±SEM. Means with different letters (A or B) differ significantly (p<0.05). a Mean significantly different from zero (p<0.05) b Minus sign indicates a decrease in performance from baseline c Minus sign indicates a decrease in the number of trials (improvement) required to accomplish the task

Based on the above a RCT was done in 209, adequate-income, 11- to 13-year-old Americans from Grand Forks, ND, USA (Penland et al. 2005). The Rx was 0 (P), 10, or 20 mg Zn (gluconate), given five times per week at school for 10–12 weeks. Baseline PZ was with few exceptions normal. The CPAS-R found efficacy of 20 mg Zn>P, but 10 mg Zn=P. An RCT was done in 60 experimental and 20 control, 19–39 years, adequate-income, healthy premenopausal women from Galveston, TX, USA (Penland et al. 2002). At baseline, all had a regular 24–28 days menstrual cycle, and none were anemic; food frequencies of the experimental ss were consistent with Zn deficiency (Yokoi et al. 2003, 2007), and SF >5 and <18 ng/ml; in controls, SF> 30 ng/ml. The Rx were M, ZM (30 mg Zn) and FM (30 mg Fe). Rx was self-administered for 16 weeks with crossover of ZM and FM at 8 weeks. Preliminary results are shown in Table 2. Discussion These experiments show that Zn deficiency as commonly encountered can impair brain function

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without the affected persons, their family, co-workers, or physicians being aware of disability. These studies and research that relates diet and Fe stores to exchangeable Zn and Zn turnover rates (Yokoi et al. 1994, 2003, 2007) suggest that Zn deficiency is more common than the current estimate of prevalence. Research is needed that focuses on effects of Zn nutriture on health across the range that is commonly consumed and bioavailable. Because physiological functions reflect the adequacy of Zn nutriture, experiments are needed to define adequacy of typical diets for function and the effectiveness of public health efforts to improve Zn nutriture. Table 2 Change NP function of women after treatment (Penland et al. 2002) Tasks

Perception set comparison, react time Memory object recognition, percent correct Reasoning oddity, objects, react time Psychomotor skills tracking, circular path

Treatments FeM M

ZnM

94±2a

96±2a,b 0.02

102±4b

p value

100±1a 106±3a,b 108±2b 0.04 93±2a,b 98±2b

90±2a

105±2a 99±1b

107±1a 0.007

0.03

Mean±standard deviation. Means with different superscripts (a or b) are different by Tukey’s test (p<0.05).

Acknowledgments This study was supported by the International Lead Zinc Research Organization, the Thrasher Foundation, the Gerber Foundation, the General Nutrition Products Company, the USDA Coop- agreement 58-1235-2-151 and grant 97-35200-4577, US Army Grant DAMD1795C5112. Neither author had a financial or professional conflict of interest. References Chen XC, Yin TA, He JS, Ma QY, Han ZM, Li LX. Low levels of zinc in hair and blood, pica, anorexia, and poor growth in Chinese preschool children. Am J Clin Nutr. 1985;42:694–700. Darnell LS, Sandstead HH. Iron, zinc and cognition of women. Am. J. Clin. Nutr. 1991;53:P-16. Egger N, Sandstead H, Penland J, Alcock N, Plotkin R, Rocco C, Dayal H, Zavaleta A. Zinc supplementa-

tion improves growth in Mexican-American children. Exp Biol. 1999;A246. Frederickson CJ, Koh JY, Bush AI. The neurobiology of zinc in health and disease: Nat Rev Neurosci. 2005;6:449–62. Golden C. Handbook of Clinical Neuropsychology. New York: Wiley-Interscience; 1981. Henkin RI, Patten BM, Re PK, Bronzert DA. A syndrome of acute zinc loss. Cerebellar dysfunction, mental changes, anorexia, and taste and smell dysfunction. Arch Neurol. 1975; 32:745–51. Kay RG, Tasman-Jones C, Pybus J, Whiting R, Black H. A syndrome of acute zinc deficiency during total parenteral alimentation in man. Ann Surg. 1976;183:331–40. Lachman R, Lachman J, Butterfield E. Cognitive Processing and Information Processing: An Introduction. Hillsdale, NJ: Erlbaum; 1979. Lezak M. Neuropsychological assessment. New York: Oxford University Press; 1995. NAS FNB Expert Committee. Recommended dietary allowances: recommended dietary allowances. Washington, DC: National Academy Press; 1989. 283 p. Penland J, Egger N, Ramanujam V, Dayal H, Sandstead H. Zinc (Zn) and iron (Fe) repletion improves cognitive function of mildly deficient women. FASEB J. 2002;16:A974. Penland J, Lukaski H, Gray J. Zinc affects cognition and psychosocial function of middle-school children. FASEB J. 2005;19:A973. Penland JG. Cognitive performance effects of low zinc (Zn) intakes in healthy adult men. FASEB J. 1991;5:A938. Penland JG. Behavioral data and methodology issues in studies of zinc nutrition in humans. J Nutr. 2000;130:361S–364S. Penland JG, Sandstead HH, Alcock NW, Dayal HH, Chen XC, Li JS, Zhao F, Yang JJ. A preliminary report: effects of zinc and micronutrient repletion on growth and neuropsychological function of urban Chinese children. J Am Coll Nutr. 1997;16:268–72. Penland JG, Sandstead HH, Egger NG, Dayal HH, Alcock NW, Plotkin R, Rocco C, Zavaleta A. Zinc, iron and micronutrient supplementation effects on cognitive and psychomotor function of MexicanAmerican school children: FASEB J. 1999;13:A921. Posner M. Chronometric Explorations of the Mind. Hillsdale, NJ: Erlbaum; 1978.

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Sandstead HH. Improving study design. Am J Clin Nutr. 1999;70:110–1. Sandstead HH, Frederickson CJ, Penland JG. History of zinc as related to brain function. J Nutr. 2000;130:496S–502S. Sandstead HH, Penland JG, Alcock NW, Dayal HH, Chen XC, Li JS, Zhao F, Yang JJ. Effects of repletion with zinc and other micronutrients on neuropsychologic performance and growth of Chinese children. Am J Clin Nutr.1998;68:470S–475S. Solomons NW, Ruz M, Gibson RS. Single-nutrient interventions with zinc: Am J Clin Nutr. 1999;70:111–3. Stallings V, Cronk C. Clinical use of the knee height measuring device to detect growth deficiency. Am J Human Biol. 1993;5:623–32. Wechsler D. 1981. WAIS-R Manual: New York, Psychological Corporation. Wuehler SE, Peerson JM, Brown KH. Use of national food balance data to estimate the adequacy of zinc in national food supplies: methodology and regional estimates. Public Health Nutr. 2005;8:812–9. Yokoi K, Alcock NW, Sandstead HH. Iron and zinc nutriture of premenopausal women: associations of diet with serum ferritin and plasma zinc disappearance and of serum ferritin with plasma zinc and plasma zinc disappearance. J Lab Clin Med. 1994;124:852–61. Yokoi K, Egger, NG, Ramanujam VM, Alcock NW, Dayal HH, Penland JG, Sandstead HH. Association between plasma zinc concentration and zinc kinetic parameters in premenopausal women. Am J Physiol Endocrinol Metab. 2003;285:E1010–20. Yokoi K, Sandstead HH, Egger NG, Alcock NW, Sadagopa Ramanujam VM, Dayal HH, Penland JG. Association between zinc pool sizes and iron stores in premenopausal women without anaemia. Br J Nutr. 2007;98:1214–23. An invited paper presented in the symposium “The role micronutrients (iodine, iron and zinc) in intellectual development”. Recent developments on WHO recommendations for the prevention and control of iron, iodine, and zinc deficiencies Lisa M. Rogers Department of Nutrition for Health and Development, World Health Organization, 1211 Geneva 27, Switzerland

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Corresponding Author: [email protected] Along with vitamin A deficiency, the World Health Organization (WHO) recognizes that the most common micronutrient deficiencies worldwide include deficiencies in iron and iodine, with zinc deficiency recently being recognized as a common deficiency in some populations. The most nutritionally vulnerable groups include young children and pregnant and lactating women. Young children have increased requirements due to their rapid development, particularly in the first 2 years of life when growth and brain development are peaking. Pregnant and lactating women also have increased micronutrient requirements due to fetal growth and the production of breast milk. The purpose of this article is to present recent developments in WHO recommendations for the prevention and control of iron, iodine, and zinc deficiencies. Iron deficiency Anemia is the most prevalent micronutrient disorder among women and young children (WHO 2002). It has been estimated that 1.6 billion people (~25% of the total population) are anemic worldwide (WHO and CDC Atlanta, in press). Iron deficiency is often considered to be the main cause of anemia. It has been associated with adverse effects on physical and mental development and increased risk of morbidity in infants and young children. In woman of reproductive age, iron deficiency anemia has been associated with an increased risk of these women giving birth to babies with intrauterine growth retardation and babies of low birth weight. Therefore, preventing iron deficiency and providing additional iron to infants and young children and women of childbearing age who are iron-deficient should be a public health priority. For all children 6–23 months of age, where the anemia prevalence is above 40%, the recommended preventative dosage is 2 mg/kg body weight per day (Table 1; WHO, UNICEF, and UNU 2001). If the infant is of low birth weight, the dosage remains the same, but supplementation may be initiated as early as 2 months of age.

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Table 1 Supplementation for the prevention of iron deficiency in non-malaria-endemic regions (WHO, UNICEF, and UNU 2001) Age group

Indications for supplementation

Iron dosage schedule

Duration

Low-birthweight infants 6–23 months of age

Universal supplementation

2 mg/kg body weight/day

Diet void of foods fortified with iron or anaemia prevalence >40% Anaemia prevalence >40% Anaemia prevalence >40% Anaemia prevalence >40% Universal supplementation Anaemia prevalence >40%

2 mg/kg body weight/day

From 2 to 23 months of age From 6 to 23 months of age

24–59 months of age School-age children Women

Pregnant women Lactating women

2 mg/kg body weight/day

3 months

30 mg/day

3 months

60 mg/day

3 months

60 mg/day

Throughout pregnancy 3 months postpartum

60 mg/day

For the treatment of anemia, 25 mg iron daily for 3 months in children <2 years of age is recommended (Stoltzfus and Dreyfus 1988). After completing 3 months of therapeutic supplementation, infants should continue preventative supplementation. For maximal effectiveness, iron supplementation programs should be linked with other programs, such as those targeting hookworm control, immunizations, malaria prophylaxis, etc. Recently, there has been concern about the use of iron supplements in malaria-endemic areas. Two large trials were conducted and supported by WHO in Zanzibar, Tanzania and in Nepal to evaluate the impact of zinc and/or iron–folic acid supplementation on the mortality and severe morbidity of preschool children (Sazawal et al. 2006; Tielsch et al. 2006). In Nepal, where exposure to malaria is low, there was no difference in the incidence of common infections or mortality between children receiving iron–folic acid, with or without zinc, and those who received zinc alone or placebo (Tielsch et al. 2006). However, in Zanzibar, an area of intense transmission of Plasmodium falciparum malaria, routine iron–folic acid supplementation, with or without zinc, was associated with an increased rate of severe adverse events in supplemented children who were not iron-defi-

cient (Sazawal et al. 2006). This led to a WHO Expert Consultation in 2006 to review the scientific evidence on the safety and efficacy of different ways of administering iron to control iron deficiency and iron deficiency anemia and to provide guidance on the safest, most feasible, and effective ways of delivering additional iron to control iron deficiency and anemia in malaria-endemic regions (WHO 2007). The Expert Consultation recommended that, although the health benefits of additional iron are well recognized in all iron-deficient children, universal iron supplementation should not be implemented in children <2 years of age in malaria-endemic regions without screening individuals for iron deficiency (WHO 2007). Iron preparations (i.e., powders, crushable tablets, and fat-based spreads) administered through home fortification of complementary foods for infants and young children in malaria-endemic areas also cannot be recommended at this time since their safety has not been demonstrated in this population. However, additional iron may be provided to infants and young children in malaria-endemic areas as processed complementary foods fortified with iron, as the iron would be consumed in smaller amounts throughout the day and would likely avoid the potential adverse effects of a large bolus of iron taken in a single dose (WHO 2007). The potential harm of universal iron supplementation in malaria-endemic regions does not diminish the utility of adequate iron therapy when iron deficiency is diagnosed. Infants and young children who have malaria and are diagnosed with iron deficiency or severe anemia should be treated with antimalarials and iron therapy, which should always be administered with food (Table 2; WHO 2007). As folate deficiency is not known to be a problem for infants and young children, and because folic acid may potentially interfere with the efficacy of antifolate antimalarial drugs, supplemental folic acid or foods fortified with folic acid should not be given to infants and young children in areas where antifolate antimalarial drugs are being utilized. It was further emphasized that strategies to control iron deficiency should be carried out along with the use of insecticide-treated nets and vector control for the prevention of malaria and treatment of malaria with antimalarial therapy. Exclusive breastfeeding for the first 6 months of life should be promoted, followed by the consumption of fortified complementary foods.

Cell Biol Toxicol (2009) 25:153–184 Table 2 Strategies to control iron deficiency in infants and young children in malaria-endemic regions (WHO 2007)

Age group

Strategy

Low-birth-weight Delay cord clamping infants less than Exclusive breastfeeding 6 months of age Infection/parasite control Iron supplementation for 3 months starting at 2 months of age, given in conjunction with preventative measures to control malaria, such as use of insecticide-treated bed nets and vector control for the prevention of malaria Treatment of malaria with antimalarial drugs Normal birth Delay cord clamping weight infants Exclusive breastfeeding less than Infection/parasite control 6 months of age Iron supplementation for 3 months starting at 2 months of age only to infants with detected iron deficiency or with clinical symptoms of severe anaemia, given in conjunction with preventative measures to control malaria, such as use of insecticide-treated bed nets and vector control for the prevention of malaria Treatment of malaria with antimalarial drugs Infants and young Breastfeeding with adequate children complementary feeding Infection/parasite control 6–24 months Processed complementary foods fortified with of age iron preferred. If not available, then iron supplementation for 3 months only to those with detected iron deficiency or with clinical symptoms of severe anaemia, given in conjunction with preventative measures to control malaria, such as use of insecticidetreated bed nets and vector control for the prevention of malaria Treatment of malaria with antimalarial drugs

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Most countries where iodine deficiency disorders have been identified as a public health problem now have national USI programs in place; however, in some countries, iodized salt still does not reach the entire population, especially the most susceptible groups (young children and pregnant and lactating women). Countries are encouraged to first assess and categorize their level of USI and use the results as a basis for their strategy to control iodine deficiency as necessary. WHO and UNICEF recently published guidelines for categorizing countries based on the proportion of household use of iodized salt at the national level (Andersson et al. 2007). Guidelines are then provided for deciding when and how to plan for additional iodine intake in children 7–24 months of age and in pregnant and lactating women. When iodized salt is not utilized by at least 90% of households, iodine supplementation of these groups may be necessary. In these areas, it has been recommended that pregnant and lactating women receive either 250 µg of iodine per day or 400 mg per year, using an iodized oil preparation (Andersson et al. 2007). Where complementary foods fortified with iodine are not available, it is recommended that children 7–24 months of age receive 90 µg iodine per day or 200 mg per year, using an iodized oil preparation (WHO and UNICEF 2007). It is critical to monitor all prevention and control programs, whether they are based on fortification or supplementation. This will ensure that the additional iodine intakes are effective in reducing the deficiency while preventing excessive intakes. Monitoring should include both the assessment of iodine status and coverage of the intervention (WHO, UNICEF, and ICCIDD 2007). Zinc deficiency

Iodine deficiency Iodine deficiency exists in most parts of the world due to low dietary intakes, as there are low concentrations of iodine in foods grown in iodine-poor soil. Universal salt iodization (USI) is the preferred strategy for the sustainable elimination of iodine deficiency to ensure adequate intakes of iodine by all individuals. However, iodine supplements can be useful as a temporary solution in areas of severe iodine deficiency where USI cannot be rapidly implemented.

Zinc deficiency has been recognized as an important cause of morbidity in developing countries, and there is increasing interest and need for quantifying the global prevalence of zinc deficiency. A Joint Interagency Meeting on Zinc Status Indicators was recently held by WHO, UNICEF, the International Atomic Energy Association (IAEA), and the International Zinc Nutrition Consultative Group (IZiNCG). For populations, serum or plasma zinc is recommended as the best available biomarker of zinc deficiency, as it is a good reflection of zinc intake (WHO/UNICEF/ IAEA/IZiNCG 2007). When assessing serum zinc

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concentrations, it is important to control for time of day in which the sample is collected, recent intake, age, sex, and presence of any infections or inflammation, as all of these factors are known to affect serum zinc. It was further recommended that a serum zinc cut-off of 65 µg/dl (9.9 µmol/l) be used for the population assessment of children under 10 years of age, for those samples collected in the morning hours during a nonfasted state. Although serum zinc cut-offs for other population groups have been proposed, an interagency consensus has not yet been achieved. If the population prevalence of deficiency is >20%, it is considered that the population is at elevated risk of zinc deficiency of public health concern (WHO/ UNICEF/IAEA/IZiNCG 2007). These recommendations will enable the initiation of work to identify countries and regions in which children under 10 years of age may be at increased risk for zinc deficiency and areas in which zinc interventions should be considered a priority. It has been demonstrated that zinc supplements given for 10–14 days during an acute episode of diarrhea reduces the duration and severity of the episode (Bahl et al. 2001) and lowers the incidence of diarrhea in the following 2–3 months (Bhutta et al. 1999). Zinc supplementation given during and until cessation of diarrhea had a significant and beneficial impact on the clinical course of acute diarrhea, reducing both its duration and severity. Furthermore, when 10–20 mg zinc was given daily for 10–14 days, there was a reduced number of episodes of subsequent cases of diarrheal diseases in the following 2–3 months after the supplementation regimen. An in-depth review of these studies led WHO and UNICEF to jointly recommend that children be given 20 mg per day of zinc supplementation for 10–14 days (10 mg per day for infants under 6 months of age) along with other appropriate measures for the treatment of diarrhea (WHO and UNICEF 2004).

Future directions Micronutrient research is continuously providing further insight into the optimal strategies for combating deficiency disorders and the research agenda continues to grow. The effects of iron and folic acid supplementation on malaria-associated morbidity are incompletely understood. Continued research is needed on the impact of various types of iron preparations, doses,

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duration, and frequency of administration on both iron uptake and metabolism and gut microflora and the immune system. Additionally, affordable and fieldfriendly indicators and tools for screening young children are needed. A better understanding of the consequences of exceeding the upper limits of iodine intake and careful monitoring of iodine intervention programs are needed. With respect to zinc, a close examination of the impact of zinc supplementation on morbidity, mortality, and growth is needed, and future studies should evaluate how to safely deliver iron and zinc together to avoid interference between these two micronutrients. Acknowledgments The author is a staff member of the World Health Organization. The author alone is responsible for the views expressed in this publication and they do not necessarily represent the decisions, policy, or views of the World Health Organization.

References Andersson M, de Benoist B, Delange F, Zupan J. Prevention and control of iodine deficiency in pregnant and lactating women and in children less than 2-years-old: conclusions and recommendations of the Technical Consultation. Public Health Nutr. 2007;101(12A):1606–11. Bahl R, Baqui A, Bhan MK, Bhatnagar S, Black RE, Brooks A, Cuevas LE, Dutta P, Frischer R, Ghosh S, Malhotra S, Penny M, Roy SK, Sachdev HPS, Sack DA, Sazawal S, Strand T, Fontaine O, Patwari AK, Raina N, Khanum S. Effect of zinc supplementation on clinical course of acute diarrhoea. Report of a Meeting, New Delhi 7–8 May 2001. J Health Popul Nutr. 2001;19(4):338–46. Bhutta ZA, Black RE, Brown KH, Gardner JM, Gore S, Hidayat A, Khatun F, Martorell R, Ninh NX, Penny ME, Rosado JL, Roy SK, Ruel M, Sazawal S, Shankar A. Prevention of diarrhoea and pneumonia by zinc supplementation in children in developing countries: pooled analysis of randomized controlled trials. Zinc Investigators’ Collaborative Group. J Pediatr. 1999;135(6):689–97. Sazawal S, Black RE, Ramsan M, Chwaya HM, Stolzfus RJ, Dutta A, Dhingra U, Kabole I, Deb S, Othman MK, Kabole FM. Effects of routine prophy-

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lactic supplementation with iron and folic acid on admission to hospital and mortality in preschool children in a high malaria transmission setting: a community-based, randomised, placebo-controlled trial. Lancet. 2006;367:133–43. Stoltzfus R, Dreyfus ML. Guidelines for the use of iron supplements to prevent and treat iron deficiency anemia. INACG, WHO, UNICEF, editors. Washington: IUNACG 1988. Tielsch JM, Khatry SK, Stoltzfus RJ, Katz J, LeClerq SC, Adhikari R, Mullany LC, Shresta S, Black RE. Effect of routine prophylactic supplementation with iron and folic acid on preschool child mortality in southern Nepal: a community-based, cluster-randomised, placebo-controlled trial. Lancet. 2006;367:144–52. WHO, UNICEF, UNU. Iron deficiency anaemia. Assessment, prevention, and control: a guide for programme managers. Geneva: WHO; 2001. WHO. The World Health Report 2002: Reducing risks, promoting healthy life. Geneva: WHO; 2002. WHO/UNICEF. Joint Statement. Clinical management of acute diarrhoea. UNICEF, WHO; 2004. WHO. Report of the World Health Organization technical consultation on prevention and control of iron deficiency in infants and young children in malaria-endemic areas. Lyon, France, 12–14 June 2006. de Benoist B, Fontaine O, Lynch S, Allen LH, editors. Food Nutr Bull. 2007;28(4):S489– 631. WHO/UNICEF. Joint Statement. Reaching optimal iodine nutrition in pregnant and lactating women and young children. Geneva: WHO; 2007. http://www.who.int/nutrition/publications/WHO Statement__IDD_pregnancy.pdf WHO/UNICEF/IAEA/IZiNCG. Conclusions of the Joint WHO/UNICEF/IAEA/IZiNCG Interagency Meeting on Zinc Status Indicators. Food Nutr Bull. 2007;28(3):S480–4. WHO, UNICEF, ICCIDD. Assessment of iodine deficiency disorders and monitoring their elimination: A guide for programme manages. 3rd edn. Geneva: WHO; 2007. WHO, CDC Atlanta. Worldwide prevalence of anaemia 1993–2005: WHO global database on anaemia. Geneva: WHO, in press. An invited paper presented in the symposium “The role micronutrients (iodine, iron and zinc) in intellectual development”.

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Iodine deficiency and intellectual development: the role of The International Council for the Control of Iodine Deficiency Disorders (ICCIDD) in the global push to eliminate iodine deficiency Michael B. Zimmermann Human Nutrition Laboratory, ETH Zürich, Switzerland and Division of Human Nutrition, Wageningen University, Wageningen, The Netherlands Corresponding Author: Email: [email protected] The iodine deficiency disorders Iodine deficiency has multiple adverse effects on growth and development in animals and humans. These are collectively termed the iodine deficiency disorders (IDD; Table 1) and are one of the most important and common human diseases (WHO/ ICCIDD/UNICEF 2001; Delange et al. 2007). They result from inadequate thyroid hormone production due to lack of sufficient iodine. Thyroid enlargement (goiter) is the classic sign of iodine deficiency and can occur at any age, even in the newborn. It is a physiologic adaptation to chronic iodine deficiency. As iodine intake falls, secretion of TSH increases in an effort to maximize uptake of available iodine, and TSH stimulates thyroid hypertrophy and hyperplasia. Initially, goiters are characterized by diffuse, homogeneous enlargement, but over time, nodules often develop. Iodine deficiency is associated with a high occurrence of multinodular toxic goiter, mainly seen in women older than 50 years (Laurberg et al. 1991). Iodine deficiency in pregnancy The most serious adverse effect of iodine deficiency is damage to the fetus. Severe iodine deficiency during pregnancy increases risk for stillbirths, abortions, and congenital abnormalities (Cobra et al. 1997). Iodine treatment of pregnant women in areas of severe deficiency reduces fetal and perinatal mortality and improves motor and cognitive performance of the offspring (Cao et al. 1994). Severe iodine deficiency in utero causes a condition characterized by gross mental retardation along with varying degrees of short stature, deaf mutism, and spasticity that is termed

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cretinism (Delange et al. 2007). The potential adverse effects of mild-to-moderate iodine deficiency during pregnancy are unclear. In Europe, several randomized controlled trials of iodine supplementation in mild-tomoderately iodine deficient pregnant women have been done (Zimmermann 2007). Iodine reduced maternal and newborn thyroid size and, in some, decreased maternal TSH. However, none of the trials showed an effect on maternal and newborn total or free thyroid hormone concentrations, the most important outcome, and none measured long-term clinical outcomes, such as maternal goiter, thyroid autoimmunity, or child development. Table 1 The iodine deficiency disorders, by age group Physiological Health consequences of iodine deficiency groups All ages

Fetus

Neonate Child and adolescent Adults

Goiter, including toxic nodular goiter Increased occurrence of hypothyroidism in moderateto-severe iodine deficiency; decreased occurrence of hypothyroidism in mild-to-moderate iodine deficiency Increased susceptibility of the thyroid gland to nuclear radiation Abortion Stillbirth Congenital anomalies Perinatal mortality Infant mortality Endemic cretinism Impaired mental function Delayed physical development Impaired mental function Iodine-induced hyperthyroidism Overall, moderate-to-severe iodine deficiency causes subtle but widespread adverse effects in a population secondary to hypothyroidism, including decreased educability, apathy, and reduced work productivity, resulting in impaired social and economic development.

Iodine deficiency in childhood Although iodine deficiency in utero impairs fetal growth and brain development, its postnatal effects on growth and cognition are less clear. Cross-sectional studies of moderate-to-severely iodine deficient children have generally reported impaired intellectual function and fine motor skills; two meta-analyses estimated that populations with chronic iodine deficiency experience a reduction in IQ of 12.5–13.5 points (Bleichrodt et al. 1987; Qian et al. 2005). In a recent controlled trial in 10to 12-year-old moderately iodine-deficient Albanian children who received 400 mg of iodine as oral iodized

oil or placebo, iodine treatment significantly improved information processing, fine motor skills, and visual problem solving compared to placebo (Zimmermann et al. 2006). Thus, in childrenbornandraisedinareasofiodine deficiency,cognitiveimpairmentisatleastpartiallyreversiblebyiodinerepletion.Datafromcross-sectionalstudieson iodineintakeandchildgrowtharemixed, with most studies finding modest positive correlations (Zimmermann et al. 2007). In iodine-deficient children, impaired thyroid function and goiter are inversely correlated with IGF-1 and IGFBP-3 concentrations (Wan Nazaimoon et al. 1996). Recent controlled trials found iodine repletion increased insulin-like growth factor (IGF)-1 and insulinlike growth factor binding protein (IGFBP)-3 and improved somatic growth in children (Zimmermann et al. 2007). Epidemiology of iodine deficiency The severity of iodine deficiency in populations is typically classified based on the median urinary iodine concentration (Table 2). Only a few countries— Switzerland, some of the Scandinavian countries, Australia, the USA, and Canada—were completely iodine sufficient before 1990. Since then, globally, the number of households using iodized salt has risen from <20% to >70%, dramatically reducing iodine deficiency (Delange et al. 2002). This effort has been spurred by a coalition of international organizations, including ICCIDD, WHO, MI, and UNICEF, working closely with national IDD control committees and the salt industry; this informal partnership was established after the World Summit for Children in 1990. It has been funded by Kiwanis International, the Gates Foundation, and country aid programs. In 2007, WHO estimated nearly two billion individuals have an insufficient iodine intake, including one third of all school-age children (de Benoist et al. 2008). The lowest prevalence of iodine deficiency is in the Americas (10.6%), where the proportion of households consuming iodized salt is the highest in the world (≈90%). The highest prevalence of iodine deficiency is in Europe (52.0%), where the household coverage with iodized salt is the lowest (≈25%), and many of these countries have weak or non-existent IDD control programs. The number of countries where iodine deficiency remains a public health problem is 47. However, there has been progress since 2003: 12 countries have progressed to optimal iodine status and

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the percentage of school-aged children at risk of iodine deficiency has decreased by 5%. However, iodine intake is more than adequate, or even excessive, in 34 countries, an increase from 27 in 2003 (de Benoist et al. 2008). In Australia and the USA, two countries previously iodine sufficient, iodine intakes are falling. Australia is now mildly iodine deficient (Li et al. 2006), and in the USA, the median UI is 145 µg/l, still adequate but half the median value of 321 µg/l found in the 1970s (Haddow et al. 2007). These changes emphasize the importance of regular monitoring of iodine status in countries to detect both low and excessive intakes of iodine.

Table 2 Epidemiological criteria for assessing iodine nutrition in a population based on median and/or range of urinary iodine concentrations (WHO/ICCIDD/UNICEF 2001; WHO 2007a).

Treatment and prevention of iodine deficiency

Pregnant women <150 Insufficient 150–249 Adequate 250–499 More than adequate ≥500 Excessivea Lactating womenb <100 Insufficient ≥100 Adequate Children less than 2 years old <100 Insufficient ≥100 Adequate

Salt fortification with iodine In nearly all regions affected by iodine deficiency, the most effective way to control iodine deficiency is through salt iodization (WHO/ICCIDD/UNICEF 2001). Salt iodization is the recommended strategy for control of IDD because: & & & & & &

Salt is one of few foodstuffs consumed by virtually everyone. Salt intake is fairly consistent through the year. In many countries, salt production/importation is limited to a few sources. Iodization technology is simple and relatively inexpensive to implement. The addition of iodine to salt does not affect its color or taste. The quantity of iodine in salt can be simply monitored at the production, retail, and household levels

WHO/UNICEF/ICCIDD recommends that iodine is added at a level of 20–40 mg iodine/kg salt, depending on local salt intake. Iodine can be added to salt in the form of potassium iodide (KI) or potassium iodate (KIO3). Because KIO3 has higher stability than KI in the presence of salt impurities, humidity, and porous packaging, it is the recommended form in tropical countries and those with low-grade salt. Salt iodization remains the most cost-effective way of delivering iodine and of improving cognition in iodine-deficient populations (Engle et al. 2007). Worldwide, the annual costs of salt iodization are estimated at US$ 0.02–0.05 per child covered, and the costs per child death averted are US$ 1,000 and per DALY gained are US$34–36 US (Caulfield et al. 2006).

Median urinary iodine (μg/l)

Iodine intake

School-aged children <20 Insufficient 20–49 Insufficient 50–99 Insufficient 100–199 Adequate 200–299 More than adequate >300 Excessive

Iodine nutrition

Severe iodine deficiency Moderate iodine deficiency Mild iodine deficiency Optimal Risk of iodine-induced hyperthyroidism in susceptible groups Risk of adverse health consequences (iodine-induced hyperthyroidism, autoimmune thyroid disease)

The term “excessive” means in excess of the amount required to prevent and control iodine deficiency. b In lactating women, the figures for median urinary iodine are lower than the iodine requirements because of the iodine excreted in breast milk. a

Iodine supplementation In some regions, iodization of salt may not be practical for control of iodine deficiency, at least in the short term. This may occur in remote areas where communications are poor or where there are numerous small-scale salt producers. In these areas, iodized oil supplements can be used (WHO/ICCIDD/UNICEF 2001). Usual oral doses are 200–400 mg iodine/year, and it is often targeted to women of child-bearing age, pregnant women, and children. Iodized oil given in the first and second trimesters of pregnancy decreased the prevalence of neurological abnormalities and improved developmental test scores through 7 years, compared with supplementation later in pregnancy or treatment after birth (O’Donnell et al. 2002). In countries or regions where a salt iodization program covers ≥90% of households and has been sustained for ≥2 years, and the median UI indicates iodine sufficiency, pregnant, and lactating women do not need iodine supplementation (WHO

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2007a). In iodine-deficient countries or regions that have weak iodized salt distribution, supplements should be given to pregnant women, lactating women, and infants (WHO 2007a). The International Council for the Control of Iodine Deficiency Disorders (ICCIDD) The ICCIDD is the only international organization specifically constituted to promote optimal iodine nutrition and the elimination of IDD (ICCIDD 2007) via a multidisciplinary global network consisting of over 600 specialists from more than 100 countries. They include scientists in the medical and nutrition fields, public health workers, development managers, technologists, communicators, economists, salt producers, other industry experts, and many others involved in fields related to iodine nutrition. All of them are committed to assisting governments and international agencies in developing national programs for the virtual elimination of IDD as a public health problem. ICCIDD was formed in 1985 with support from UNICEF, WHO, and the Australian government to bridge the gap between available knowledge and its application to attain iodine sufficiency for the millions at risk. ICCIDD has played a major role in communicating the IDD threat to decision makers of national governments and international agencies and to a wide variety of health professionals and planners. ICCIDD consultants participate in public policy development and advocacy, program development, implementation, and training by assisting countries with significant IDD problems to develop national IDD control programs, in cooperation with national governments, institutions, individuals, private industries, welfare agencies, major international agencies, and key bilateral aid-giving agencies (ICCIDD 2007).

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challenges (UNICEF 2007). An important strategy will be to strengthen national coalitions that include government partners, national and international agencies, the health-care sector, and salt producers. In the countries that have begun iodized salt programs, sustainability will become a major focus. These programs are fragile and require a long-term commitment from governments. In several countries where iodine deficiency had been eliminated, salt iodization programs fell apart, and iodine deficiency recurred (Dunn 2000). Children in iodine-deficient areas are vulnerable to even short-term lapses in iodized salt programs (Zimmermann et al. 2004). To this end, countries should monitor the state of their iodine nutrition every 3 years and report to the World Health Assembly on their progress (WHO 2007b). Advocacy should focus on damage to reproduction and cognitive development. Governments need to understand the serious impact of iodine deficiency; many still equate iodine deficiency with goiter, a cosmetic problem and thus a low priority. IDD is the single most important cause of preventable mental retardation worldwide, and elimination of IDD can contribute to at least five of the Millennium Development Goals: (1) eradicate extreme poverty and hunger; (2) achieve universal primary education; (3) reduce child mortality; (4) improve maternal health; and (5) develop a global partnership for development. The World Bank (McGuire and Galloway 1994) strongly recommends that governments invest in micronutrient programs, including salt iodization, to promote development, concluding: “Probably no other technology offers as large an opportunity to improve lives at such low cost and in such a short time.”

References Future perspectives The International Child Development Steering Group identified iodine deficiency as one of four key global risk factors for impaired child development where the need for intervention is urgent (Walker et al. 2007). But controlling IDD in the remaining third of the global population at risk will not be easy. Although the key contributors to successful national programs have been identified, reaching economically disadvantaged groups living in remote areas and convincing small scale salt producers to iodize their salt are major

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