Interactions between Zinc Deficiency and Environmental Enteropathy in Developing Countries

Abstract

Zinc deficiency affects one-fifth of the world’s population and leads to substantial morbidity and mortality. Environmental enteropathy (EE), a subclinical pathology of altered intestinal morphology and function, is almost universal among people living in developing countries and affects long-term growth and health. This review explores the overlapping nature of these 2 conditions and presents evidence for their interaction. EE leads to impaired zinc homeostasis, predominantly due to reduced absorptive capacity arising from disturbed intestinal architecture, and zinc deficiency exacerbates several of the proposed pathways that underlie EE, including intestinal permeability, enteric infection, and chronic inflammation. Ongoing zinc deficiency likely perpetuates the adverse outcomes of EE by worsening malabsorption, reducing intestinal mucosal immune responses, and exacerbating systemic inflammation. Although the etiology of EE is predominantly environmental, zinc deficiency may also have a role in its pathogenesis. Given the impact of both EE and zinc deficiency on morbidity and mortality in developing countries, better understanding the relation between these 2 conditions may be critical for developing combined interventions to improve child health.

Introduction

It has been apparent for several decades that a subclinical enteropathy is almost universal among people living in developing countries (1, 2). Enteropathy can be defined as histological abnormalities of the small intestinal mucosa that are visible under light microscopy (3). In developing countries, it appears that this pathology is driven by environmental factors and it has therefore been termed environmental enteropathy (EE) (4). Although the precise mechanisms underlying EE are yet to be elucidated, it is likely caused by poor sanitation and recurrent fecal ingestion, leading to chronic intestinal inflammation and subsequent histological changes (5). However, in developing countries, EE exists in complex overlap with other causes of enteropathy, such as HIV and micronutrient deficiencies (6). Deficiency of zinc, an essential micronutrient, may be interrelated with EE both as a consequence of the decreased absorptive capacity resulting from EE and/or as a potential contributor to EE through its effects on intestinal function, immunity, and inflammation. This review explores the interactions between enteropathy and zinc deficiency and discusses their implications for child health.

Zinc Deficiency

Since it was shown to be essential for human life more than 50 y ago (7), zinc has been found to have important roles in enzymatic processes, genetic expression, and cell stability (8). Deficiencies can lead to substantial morbidity, as exemplified by the rare autosomal recessive condition of impaired zinc metabolism, acrodermatitis enteropathica, which provides a clear clinical syndrome of severe zinc deficiency and is fatal without treatment (9). Although the consequences of mild zinc deficiency are less clearly identified, there is nonetheless considerable evidence that even mild deficiency compromises growth and immune function (7). Zinc deficiency is therefore estimated to be responsible for 1.7% of total deaths among children <5 y (~116,000 attributable deaths annually) (10).

Estimates of the prevalence of zinc deficiency and evaluation of its effects on health are hampered by the lack of a reliable biomarker of zinc status. Although the WHO, UNICEF, and the International Zinc Nutrition Consultative Group jointly recommend the use of serum zinc concentration for assessment of population zinc status (11), this measure is not reliable at the individual level and few large-scale studies have been conducted in developing countries. The risk of inadequate zinc intake in children has been evaluated using suggested estimates of zinc requirements in conjunction with data on food composition and dietary intakes across the world. From these estimates, it appears that 17.3% (95% CI: 15.9%, 18.8%) (10) of the world’s population is at risk of inadequate zinc intake and that this risk is highest in children under 5 y in developing countries (12).

The maintenance of zinc homeostasis relies on 2 processes occurring concurrently in the gastrointestinal tract: first, the absorption of exogenous dietary zinc and second, the gastrointestinal secretion then excretion or reabsorption of endogenous zinc (13). These processes are regulated by zinc transporters, permeable channels, and metallothioneins (14), which work to modulate net absorption and the size of the body’s exchangeable zinc pool. Although they are generally effective mechanisms, there are limits to this homeostatic control. Zinc absorption is affected by dietary intake as well as dietary promoters, such as animal proteins, and inhibitors, such as phytates. Phytates are present in plant products, particularly grains and legumes, and form insoluble complexes that limit zinc absorption (13). They are of particular importance in developing countries, where largely plant-based, low-animal protein diets are common (15). The control of endogenously secreted zinc is less well understood; however, available data suggest that the quantity of zinc secreted with each meal may be considerable and that efficient reabsorption is critical to the maintenance of normal zinc balance (13, 16).

Environmental Enteropathy

The small intestine has 2 main functions: digestion and absorption of dietary nutrients and barrier defense against external substances (17). Barrier function is derived from both mechanical and immunological components together with the defense provided by the resident intestinal microbiota. EE affects the vast majority of people in developing countries (18), where subclinical impairments in these critical small intestinal functions are emerging as important determinants of long-term growth and health (4–6). It has been postulated that EE is caused by poor sanitation and recurrent fecal ingestion leading to chronic T-cell–mediated intestinal inflammation (19) and the subsequent histological changes of villous atrophy, crypt hyperplasia, inflammatory cell infiltrate, and increased intestinal permeability (5). These pathological changes in gut morphology and function begin early in infancy and have been linked to stunting, which has long-term effects on physical and cognitive function and economic productivity (5, 6, 10). The gold standard of endoscopy and biopsy to diagnose EE is both impractical and unethical in otherwise healthy children and surrogate measures are therefore generally used to assess small intestinal structure and function. A dual-sugar absorption test, in which concentrations of lactulose and mannitol are measured in urine following an oral challenge, has been used in several prior studies (20, 21). Lactulose, a large disaccharide, can pass only slowly through intercellular spaces (the paracellular pathway) in the healthy intestine and increased lactulose absorption (and subsequent urinary excretion) therefore indicates impaired barrier function. In contrast, the small monosaccharide mannitol passes through numerous small, water-filled pores in the enterocyte membranes (the transcellular pathway) and a decrease in mannitol indicates reduced absorptive capacity of the small intestine, suggestive of villous damage. The lactulose-mannitol (L:M) ratio in urine can therefore be used as a surrogate marker of small intestinal structure and function.

The Impact of EE on Zinc Deficiency

It is well recognized that gastrointestinal health (including maintenance of mucosal integrity) and nutritional status are interrelated (17, 22) and EE is known to affect absorption of macronutrients (23). However, few studies have explored the impact of EE on human zinc status, aside from 2 informative observational studies using zinc isotopes to assess zinc homeostasis in rural Malawian children (24, 25). One study reported that apparently healthy children consuming a high-phytate, low-animal protein diet (resulting in low fractional absorbed zinc) had unexpectedly high fecal endogenous zinc excretion. Because children would be expected to have low fecal endogenous zinc in the context of low intakes of absorbable zinc, the authors speculated that this may have been a consequence of EE impairing the ability to reabsorb endogenously secreted zinc (24). Subsequently, the same investigators selected 25 young children at risk of EE (due to poor hygiene or previous malnutrition) and demonstrated that L:M ratios were positively associated with endogenous fecal zinc excretion, with the effect apparently linear and predominantly due to low mannitol absorption (25). They concluded that perturbed zinc homeostasis was associated with EE, predominantly due to reduced absorptive capacity arising from disturbed intestinal architecture. Studies in celiac disease, which is characterized by similar histological changes to EE, similarly show a correlation between impaired gut function and absorption of zinc (26) and increased losses of fecal endogenous zinc (27).

Contrary to these findings, a Guatemalan study of poor peri-urban infants found no relationship between L:M and serum zinc status despite one-third of participants having evidence of EE (28). Similarly, a study of Brazilian children living in a shantytown found no relationship between L:M and serum zinc (29). However, given that serum zinc is a poor indicator of overall, whole-body zinc status, these studies do not rule out a relation between zinc and EE, as was demonstrated in the intensive Malawian studies that used more dynamic markers of zinc status.

Zinc and the L:M Ratio: A Two-Way Relationship?

Although it is plausible that the impaired absorptive capacity arising from EE leads to disturbed zinc balance, it also appears that impaired zinc balance can affect small intestinal function. Animal studies show that zinc deficiency impairs the function of gastrointestinal digestive enzymes, which may reduce absorptive capacity. Early studies in zinc-deficient rats demonstrated marked pathological changes in pancreatic cells and the cytoplasm of absorptive cells (30) and more recent studies showed decreased activity of peptidase (31), maltase, amylase, and protease when rats were fed a zinc-deficient diet (32). Therefore, a self-perpetuating cycle may occur, with EE leading to zinc deficiency and zinc deficiency exacerbating consequent malabsorption of both macro- and micronutrients, further reducing the availability of dietary zinc (33).

The increased intestinal permeability that characterizes EE is hypothesized to arise predominantly from recurrent fecal ingestion and subclinical enteric infection (5, 34). However, zinc may also have a crucial role in maintaining intestinal barrier function. In vitro, zinc-deficient endothelial cells show increased transfer of albumin across monolayers, which returns to normal following zinc supplementation (35). Furthermore, when barrier dysfunction is induced using the cytokine TNFα, physiologic doses of zinc are protective and exhibit a dose-response relationship (36). Similar results have been shown in studies of piglets that experience decreased feed intake and diarrhea, associated with increased intestinal permeability and decreased villous height, when abruptly weaned at 4 wk of age (37). High-dose zinc supplementation during weaning leads to dramatic reductions in the symptoms of diarrhea and improvements in L:M ratios (37, 38). Both these studies demonstrated reduced lactulose recovery as the factor mediating improvements in the L:M ratio, suggesting that the benefit of zinc predominantly occurs through reduced intestinal permeability. By evaluating mRNA and subsequent protein expression, Zhang et al. (38) observed the effect of zinc to be at the level of the tight junctions, with higher amounts of occludin and ZO-1 among pigs given supplemental zinc.

Other animal studies confirm the effect of zinc on tight junctions. Rats with pharmacologically induced colitis had a reduced number of tight junction complexes opening following high-dose supplemental zinc (39), and high-dose zinc given to malnourished guinea pigs with increased intestinal permeability prevented loss of barrier function; morphological assessment suggested this was through tight junction modification (40). Interestingly, in a recent study of Caco-2 cell lines under adequate zinc conditions, the addition of supra-physiological zinc improved barrier function through remodeling of tight junctions (41), raising the possibility that a dose-response relation may exist irrespective of zinc status.

A number of randomized controlled trials have studied zinc supplementation in populations expected to have a high prevalence of both zinc deficiency and EE. Apparently healthy rural Gambian children with high rates of stunting (likely due to EE) were given supplemental zinc at a dose of 70 mg twice weekly for 15 mo. Zinc had no impact on growth and no significant effect on L:M ratios; however, intestinal permeability to lactulose was significantly lower in the supplemented group (42). Bangladeshi children with both acute and persistent diarrhea associated with a markedly abnormal L:M, who were randomized to receive 5 mg/(kg · d) of zinc or placebo had a significant reduction in lactulose excretion following zinc supplementation (43). In a subset of Brazilian children selected by low weight or persistent diarrhea (29), a 2-wk course of zinc sulfate (20 mg/d) in addition to vitamin A also led to decreased intestinal permeability. Results suggested that this was the effect of zinc rather than vitamin A due to its demonstrable impact on lactulose excretion. Taken together, in 3 different populations with probable EE, zinc supplementation appeared to reduce intestinal permeability (as measured by lactulose recovery) but had no apparent impact on intestinal absorptive capacity (as measured by mannitol recovery), consistent with findings from animal studies. Other micronutrients, in particular vitamin A (44), are also likely to play a role in enteropathy, although a detailed discussion of their effects is outside the scope of this review.

Zinc and Gastrointestinal Infections

Zinc supplementation is known to have important benefits in diarrheal disease, which remains a major cause of morbidity and mortality among children in developing countries (45). In meta-analyses, preventive zinc supplementation reduced the incidence of diarrhea by 13% (46) and zinc supplementation in children with diarrhea reduced hospital admissions by 23% and all-cause mortality by 46% (47). The exact mechanism underlying these benefits is unknown; however, improved intestinal barrier function, enhanced immune function, and anti-inflammatory effects are all likely to be important. Using in vitro gastrointestinal models of Escherichia coli infection, zinc oxide was shown to reduce bacterial adhesion, internalization, and disruption of cell membranes and reduce tight junction permeability induced by the bacteria (48). Bacterial translocation from the gut to the systemic circulation, arising because of increased intestinal permeability, is hypothesized to be an important part of the pathogenesis of EE, causing a chronic inflammatory state that impairs growth and development (5). High-dose zinc oxide given to piglets prior to ingesting components of E. coli had protective effects, including decreased bacterial translocation from the small intestine to the mesenteric lymph nodes (49). Zinc supplementation led to improved L:M ratios in children with shigellosis (50); however, in contrast to other studies (29, 42, 43), the improvement was seen through increased mannitol excretion, with nonsignificant improvement in lactulose excretion.

Aside from reducing diarrheal disease, zinc supplementation reduces morbidity from childhood pneumonia by 19% (45), likely due to its impact on immune function. Even mild zinc deficiency impairs cell-mediated and antibody-mediated immune responses in mice (51). Zinc-deficient mice infected with nematodes have a higher worm burden, impaired T-cell and antigen-presenting cell function, and decreased Igs and eosinophils compared with zinc-adequate mice (52). In humans, mild zinc deficiency leads to decreased amounts of the thymic hormone thymulin, reduced IL-2, and impaired natural killer cell activity within 12 wk (53). A blunted immune response may also be a feature of EE due to chronic inflammation and altered intestinal mucosal architecture, which may partly account for the reduced efficacy of oral vaccines in developing countries (54). Studies in Bangladesh suggest that zinc supplementation may improve immune responses to the oral cholera vaccine (from 57% to 80%) through enhanced T- and B-cell–mediated seroconversion to vibriocidal antibodies (55, 56), suggesting that at least some of the pathology previously attributed to EE may be related to zinc deficiency.

Inflammation, Zinc Deficiency, and EE

Chronic inflammation arising from the subclinical pathology of EE leads to impaired growth (and, plausibly, anemia of inflammation) in children with EE (5)(A.J. Prendergast, S. Rukobo, B. Chasekwa, K. Mutasa, R. Ntozini, M.N. Mbuya, A. Jones, R.J. Stoltzfus, J.H. Humphrey, unpublished data). Zinc is a potent anti-inflammatory and antioxidant nutrient and deficiency leads to a proinflammatory state in the gastrointestinal tract and systemic circulation (53). In vitro work using Caco-2 cells showed that zinc depletion leads to massive neutrophil migration through the paracellular space through disruption of junctional complexes and the induction of chemokines that contribute to intestinal damage (57). Zinc-deficient rats had a higher content of nitrous oxide metabolites in the cecum (58) and upregulated uroguanylin and inducible NO synthase gene expression in response to inflammatory agents (59). Zinc deficiency may also be an important mediator of chronic inflammatory intestinal conditions such as celiac disease and inflammatory bowel disease (60) and is associated with overexpression of proinflammatory mediators in the esophageal mucosa, leading to the development of esophageal hyperplasia and squamous cell cancer (61).

Furthermore, there is increasing evidence that low zinc status underlies inflammation in several conditions, including cardiovascular disease (62), obesity (63), and age-related macular degeneration (64). Zinc induces the zinc finger protein A20, which inhibits activation of NK-κB, a transcription factor responsible for the generation of inflammatory cytokines (53), and decreases reactive oxygen species (65). Zinc deficiency is associated with raised inflammatory markers, cytokines, and inability to respond to oxidative stress (53, 65). Therefore, in EE, zinc deficiency is likely to exacerbate the systemic inflammation that has been shown to mediate stunting (A.J. Prendergast, S. Rukobo, B. Chasekwa, K. Mutasa, R. Ntozini, M.N. Mbuya, A. Jones, R.J. Stoltzfus, J.H. Humphrey, unpublished data).

In conclusion, there is some evidence that the relation between zinc deficiency and EE is bidirectional: EE may impair zinc homeostasis and zinc deficiency may contribute to EE by impairing intestinal barrier function, increasing the incidence of gastrointestinal infections, and inducing intestinal inflammation (Fig. 1). Once EE is established, zinc deficiency has been shown to increase malabsorption of nutrients (including zinc itself), impair adaptive immune function, and exacerbate chronic systemic inflammation, which may ultimately mediate impaired growth and development. Although EE likely arises from poorly defined environmental factors, zinc deficiency may contribute to several of the proposed pathways that underlie EE. Given the plausible impact of both EE and zinc deficiency on morbidity and mortality in developing countries, better understanding the relationship between these 2 conditions may be critical for developing combined interventions to improve child health.

FIGURE 1.

Hypothesized (or plausible) interactions between zinc deficiency and EE. A conceptual framework showing possible interactions between zinc deficiency and intestinal pathology, including pathways through which EE is thought to occur (solid arrows), pathways through which EE may contribute to zinc deficiency (solid bold arrows), and pathways through which zinc deficiency may contribute to or exacerbate EE (dashed arrows). EE, environmental enteropathy; GI, gastrointestinal.