Discovery of human zinc deficiency and studies in an experimental human model

The importance of zinc for human health was first documented in 1963. During the past 25 y, deficiency of zinc in humans due to nutritional factors and several disease states has now been recognized. The high phytate content of cereal proteins is known to decrease the availability of zinc, thus the prevalence of zinc deficiency is likely to be high in a population consuming large quantities of cereal proteins. Alcoholism, malabsorption, sickle cell anemia, chronic renal disease, and chronically debilitating diseases are now known to be predisposing factors for zinc deficiency. A spectrum of clinical manifestations ranging from mild to severe degree have now been recognized in human zinc-deficiency states. Zinc is required for many biological functions including DNA synthesis, cell division, and gene expression. It is required for the activity of many enzymes in biological systems. Recent studies indicate that zinc is needed for cell-mediated immunity.     

Evaluation of the EDTA-washed diet for use in the experimental production of zinc deficiency in human subjects

Ethylenediaminetetraacetate (EDTA) is known to bind zinc (Zn) and other metals. EDTA-washed soy protein-based diet has been extensively used as a dietary model for the production of Zn-deficiency in human subjects as well as in experimental animals. The present study was conducted to test this diet for possible contamination with EDTA (inhereted by washing procedure) and also to evaluate it for its acceptability and palatibility by human subjects. In the first experiment, EDTA-washed soy protein-based diets were prepared as previously described (repeated washing with EDTA-sodium salt followed by washing with deionized water and filteration), but 14C-EDTA was used. Scintillation counter analysis showed no traces of 14C in the prepared diet (100% of the 14C activity was recovered in the filtrate). In the second experiment, the diets were prepared using inactive EDTA, then fed to 10 volunteers for sensory evaluation. Results of this trial showed that the palatibility and acceptability of these diets are generally very poor. In conclusion, the use of EDTA-washing procedure did not result in any contamination of the soy assay protein with the EDTA. However, the present dietary model may not be used to induce zinc deficiency in human volunteers on a long term basis since the palatibility of the washed product is poor.     

Dietary vitamin E deficiency and zinc metabolism.

In adult rats, the influence of vitamin E deficiency on zinc metabolism in general and specifically in 15 tissues was studied. After 50 days, we found evidence of vitamin E deficiency and at this time point we injected a tracer amount of 65Zn. During the next 18 days the zinc status was unaffected. The zinc metabolism, however, was altered: the apparent retention increased and the biological half life was prolonged. On day 68, the changes in various tissues varied. Some tissues were affected in zinc concentration (higher in plasma and spleen; lower in cerebrum, fur and tail), others in specific activity (higher in pancreas; lower in cerebellum). The different effects may reflect differences in tissue response on impairement, caused by vitamin E deficiency.     

Dietary zinc deficiency decreases glutathione S-transferase expression in the rat olfactory epithelium

Zinc deficiency leads to olfactory and gustatory dysfunction, but little is known about the underlying molecular mechanism of this phenomenon. We examined the effect of dietary zinc deficiency on the rat olfactory epithelium. Immunoreactivities of glutathione S-transferase (GST) mu, neuron-specific enolase (NSE) and proliferating cell nuclear antigen (PCNA), and in situ hybridization of GST mu mRNA in the olfactory epithelia were examined under different dietary zinc intake conditions. Adult male rats were fed a zinc-deficient (ZD) diet (0.5 mg zinc/kg diet), whereas control rats, including pair-fed (PF) and zinc-adequate (ad libitum consumption, AL) groups, were fed a zinc-adequate diet (58 mg zinc/kg diet) for 7 wk. We also examined the effect of zinc replacement (ZR) by subsequently feeding half of the ZD group a zinc-adequate diet for 5 wk after the initial 7-wk deprivation. No significant differences in immunoreactivity for NSE in olfactory epithelial receptor cells or for PCNA in basal cells were noted among groups. Intense GST mu immunoreactivity and hybridization signals were observed in olfactory supporting cells of AL, PF and ZR groups, but very minimal or no such signal was noted in ZD rats. Our findings indicated that zinc deficiency reduces GST mu expression in the supporting cells of rat olfactory epithelia but does not affect receptor cell proliferation or maintenance.     

Serum alkaline phosphatase and serum zinc levels in the diagnosis and exclusion of zinc deficiency in man

In the present study we monitored serum zinc (Zn) and serum alkaline phosphatase (AP) levels during Zn supplementation in (A) a young zinc depletion syndrome (ZDS) patient with severe Zn deficiency, (B) three acrodermatitis enteropathica (AEP) patients with mild Zn deficiency and (C) 7 elderly and 3 younger patients without Zn deficiency. In (A) serum Zn and serum AP values were low, but following parenteral Zn the parameters rose to normal levels (r = +0.79, p less than 0.001). In (B) serum Zn and serum AP levels decreased when oral Zn was stopped, but shortly returned to normal upon reinitiation of Zn. In (C) Zn therapy caused a rise in the serum Zn concentration whereas the serum AP activity decreased (r = -0.01, p greater than 0.1). The results suggest that serial determinations of serum Zn and serum AP during Zn supplementation may be a valid tool in the diagnosis of severe and mild Zn deficiency as well as in the exclusion of the diagnosis.     

Dietary zinc deficiency in rodents: effects on T-cell development, maturation and phenotypes

Zinc deficiency is one of the leading risk factors for developing disease and yet we do not have a clear understanding of the mechanisms behind the increased susceptibility to infection. This review will examine the interrelationships among the hypothalamus-pituitary-adrenal stress axis, p56(lck), and T-cell maturation in both zinc deficiency and responses during zinc repletion. We will highlight differences between the adult mouse model (wasting malnutrition) and growing rat model (stunting malnutrition) of dietary zinc deficiency and discuss the use of various controls to separate out the effects of zinc deficiency from the associated malnutrition. Elevated serum corticosterone in both zinc deficient and pair-fed rats does not support the hypothesis that zinc deficiency per se leads to corticosterone-induced apoptosis and lymphopenia. In fact, the zinc deficient rat does not have lymphopenia. Thymocytes from zinc deficient mice and rats have elevated levels of p56(lck), a signalling protein with a zinc clasp structure, but this does not appear to affect thymocyte maturation. However, post-thymic T-cell maturation appears to be altered based on the lower proportion of splenic late thymic emigrants in zinc deficient rats. Fewer new T-cells in the periphery could adversely affect the T-cell repertoire and contribute to immunodeficiency in zinc deficiency.     

Effect of experimental zinc deficiency and repletion on some immunological variables in guinea-pigs

1. Cellular and humoral immune responses were studied in guinea-pigs fed on zinc-deficient (ZnD), Zn-adequate (control) and Zn-replete diets containing 1.25, 50 and 100 mg Zn/kg diet respectively. 2. It was found that the ZnD guinea-pigs had significantly decreased ability to elicit delayed-type hypersensitivity (DTH) response against sheep erythrocytes as compared with controls on the 9th day of immunization. This was further substantiated by histological examination of DTH-positive skin sections. 3. A significant reduction in direct splenic plaque-forming-cell response and haemagglutinating-antibody titre was also observed in ZnD guinea-pigs. 4. Serum electrophoretic studies revealed a highly disordered protein profile with a significantly depressed value for gamma-globulin. 5. Zn repletion of the previously ZnD group resulted in marked, though incomplete, restoration of immunological responses.     

Roles for cell death in zinc deficiency

Studies of zinc deficiency (ZD) have become important for demonstrating that nutritional imbalances can readily induce programmed cell death (PCD) or apoptosis in a variety of kinds of cells. In mice, ZD caused a 300% increase in the amount of apoptosis among pre T-cells, which was a major cause of thymic atrophy that alters host defense. Embryogenesis was significantly altered in ZD mice due to increased apoptosis in the neural crest, optic, and head regions. Insufficient zinc initiated PCD in hepatocytes, glioma, kidney, monocytes, fibroblasts, and testicular cells, demonstrating the scope of this phenomenon. New forms of cell death continue to emerge. For example, autophagy is initiated by starvation and various nutritional and metabolic imbalances. Autophagy is a form of PCD whereby the cell digests some of its own organelles to provide needed nutrients. Understanding the interplay between these different forms of cell death and nutritional imbalances is very important because of their profound impact on development, growth, immune function, and health.     

Marginal zinc deficiency exacerbates experimental colitis induced by dextran sulfate sodium in rats

We investigated the impact of Zn status on the maintenance of mucosal homeostasis. Rats were fed diets containing different amounts of Zn (30, 10, 5, <1 mg Zn/kg diet) for 21 d. Serum Zn concentrations were lower in rats fed marginally Zn-deficient (MZD; 5 mg Zn/kg diet) and severely Zn-deficient (<1 mg/kg) diets but not in those fed the marginally Zn-adequate diet (10 mg/kg) or the Zn-adequate (ZA; 30 mg/kg) group (P < 0.05). However, organ weights, colonic epithelial cell proliferation, and crypt fission did not differ between the MZD and ZA groups. We then evaluated whether MZD modulated dextran sulfate sodium (DSS)-induced colonic inflammation by administering 2% DSS to the MZD and ZA groups for 7 d. Myeloperoxidase activity and TNFα production increased in response to DSS in the MZD group (P < 0.03). Colonic permeability in the 2 groups did not differ after DSS administration. In a culture experiment using isolated mesenteric leukocytes, TNFα production was higher (P < 0.05) and TNF receptor type I (TNFR1) expression was detected in culture medium containing 20 and 30 μmol/L of Zn compared with culture medium lacking Zn supplementation. These results suggest that MZD exacerbated colitis by modulating the immune response through the impairment of TNFα production and TNFR1 expression rather than through the impairment of epithelial barrier function.     

Dietary zinc deficiency and repletion modulate metallothionein immunolocalization and concentration in small intestine and liver of rats

Metallothionein (MT) functions in zinc (Zn) homeostasis and dietary Zn affects tissue MT concentration. The objective of this study was to investigate the effects of dietary Zn deficiency and 24-h Zn repletion on MT immunolocalization and concentration in the small intestine and liver of growing rats. Three-week-old rats fed Zn-deficient diet (< 1 mg Zn/kg) for 16 d had no MT staining in either small intestine or liver. After 24-h Zn repletion with control diet (30 mg Zn/kg), strong MT staining was observed in intestinal Paneth cells and surface epithelial cells in the proliferative regions of villi. Pair-fed control rats had strong MT staining in liver that was localized around central veins. After 24-h energy repletion, the hepatic MT staining diminished. Furthermore, Zn-deficient rats had significantly reduced intestinal (57%) and hepatic (61%) MT concentrations but unaffected Zn concentrations compared with controls that consumed food ad libitum. Zn repletion for 24 h restored intestinal and hepatic MT concentrations and reduced hepatic Zn concentration. Pair-fed control rats had elevated MT concentration in liver that was normalized by energy repletion. There was a significant positive correlation between tissue Zn and MT concentrations in liver (r = 0.60, P = 0.0001), but not in small intestine. In summary, MT immunolocalization and concentration in rat small intestine and liver were responsive to changes in Zn status, supporting the role of MT in Zn metabolism. Cell-type-specific localization of MT in small intestine after dietary Zn manipulations indicates a function of Zn and MT in gut immunity and intestinal mucosal turnover, and the pattern of hepatic MT distribution with energy restriction may be linked to detoxification processes.     

金黄地鼠实验性缺锌模型的初步研究

采用幼小金黄地鼠，自由摄取缺锌饲料或添加６０ｍｇ／ｋｇ双硫腙饲料，结果动物均可出现腹泻，毛稀疏，无光泽，足趾溃烂，有出血点，活动减少，血清锌、铜浓度降低等缺锌现象，双硫腙组粪锌显著增加。说明两种方法复制成缺锌模型，而采用自由摄取双硫腙饲料法较灌胃法更简单，方便。     

Release of zinc from maternal tissues during zinc deficiency or simultaneous zinc and calcium deficiency in the pregnant rat

Earlier studies have shown that diets that increase tissue catabolism reduce the teratogenic effects of zinc deficiency. The hypothesis that zinc may be released from body tissues when the metabolic state is altered was further tested. Nonpregnant Sprague-Dawley females were injected with 65Zn; after equilibration, the two major pools of zinc, bone and muscle had different specific activities, muscle being much higher. Females were mated and fed diets adequate in zinc and calcium, deficient in zinc alone or deficient in both zinc and calcium. Calculations using weight loss, zinc content of maternal bone and muscle and total zinc content of the fetus at term indicated that most of the zinc in the fetus at term in both the zinc-deficient and zinc-calcium-deficient groups came from breakdown of maternal muscle in the last 3 d of pregnancy. The relatively small amount of additional zinc released from bone in the zinc-calcium-deficient rats early in pregnancy was sufficient to prevent abnormal organogenesis. Specific activity of zinc in the zinc-deficient and zinc-calcium-deficient fetuses was equal and high, indicating that most zinc in these fetuses came from maternal tissues and from the same maternal sources in both groups. In contrast, specific activity of zinc in the fetuses from rats fed adequate zinc and calcium was less than 30% of that in either of the deficient groups; this is consistent with the hypothesis that most zinc accrued by these fetuses came directly from the diet.     

Dietary prophylaxis of selenium deficiency

By studying the specific features of selenium exchange in subjects with equal dietary selenium intakes, but with different contents of methionine, the authors have established that the diet containing the optimum methionine level has a pronounced selenium-sparing effect and may be used as one of the modes to prevent selenium deficiency states in the areas having insufficient environmental selenium levels.     

The effect of severe zinc deficiency on serum levels of albumin, transferrin, and prealbumin in man

Concentrations of three serum transport proteins, albumin, transferrin, and prealbumin, were determined in seven patients with severe zinc deficiency. Zinc deficiency was manifested not only by depressed serum zinc concentrations, but also by skin lesions typical of zinc deficiency that corrected with zinc supplementation only. Concentrations of all three serum proteins were significantly depressed in zinc-deficient patients compared to healthy controls, and levels of all three proteins improved or corrected with a short period of zinc supplementation as the sole form of therapeutic intervention. Prealbumin levels dropped and corrected most rapidly, probably due in part to its short half-life of 2 days. This study demonstrates that zinc plays an important role in protein metabolism in man and is necessary for the maintenance of normal levels of certain transport proteins. These results support the possibility that zinc deficiency may alter tissue availability of other nutrients such as vitamin A or iron through its effect on transport proteins.     

Laboratory diagnosis of zinc deficiency

Zinc status in human subjects is assessed by measurement of zinc in plasma, erythrocytes, neutrophils, lymphocytes, and hair. Available data indicate that zinc in neutrophils and the assay of activity of alkaline phosphatase in neutrophils may be the best tools for the diagnosis of zinc deficiency. Measurement of zinc in the plasma is simple and readily available in many laboratories. Plasma zinc is useful provided the plasma is unhemolyzed and conditions, such as infections, acute stress, myocardial infarction and intravascular hemolysis, are ruled out. Inasmuch as hair and erythrocytes turn over slowly, their zinc levels do not reflect recent changes with respect to zinc status. Other useful parameters for assessment of zinc status include metabolic balance studies, urinary zinc excretion. Cu:Zn ratio, zinc tolerance test, and measurement of activities of zinc-dependent enzymes in suitable biological samples.     

Immunobiology of gestational zinc deficiency

The trace element zinc is an essential micronutrient for the proper functioning of the immune system. Zinc deficiency leads to impaired function of the unspecific and specific immune response and consequently to an increased susceptibility to bacterial, viral and fungal infections. Immunological defects are not only seen in pronounced but even in marginal and moderate zinc deficiency. Lack of zinc is especially harmful for the development of the immune system, which stresses the importance of a balanced zinc level during pregnancy. However, gestational zinc deficiency due to an imbalance between intake and increased requirements is a common problem world-wide. In animals, gestational zinc deficiency results in reduced thymic and spleen size and depressed active and passive immunity in the infant. For example, depressed immunoglobulin levels, altered antibody repertoire, reduced proliferative response of lymphocytes and diminished neutrophil functions have been reported. Interestingly, immune defects caused by prenatal zinc deficiency, such as depressed antibody levels and lymphocyte proliferation, may even persist in subsequent generations and are not reversible by postnatal zinc administration. Since gestational zinc deficiency is a common problem throughout all cultures and socioeconomic levels, it might have immense consequences for the health status of the population. Based on a summary of the immunobiology of zinc, this article reviews the significance of zinc deficiency during pregnancy and the effect of gestational zinc deficiency on passive and active immunity in the infant. It provides a rational basis for both immunological laboratory investigations and field studies, such as large community-based zinc supplementation trials in pregnant women.