Research paper
Effect of zinc depletion/repletion on intestinal iron absorption and iron status in rats

https://doi.org/10.1016/j.jnutbio.2021.108800Get rights and content

Abstract

Iron and zinc deficiencies likely coexist in general population. We have previously demonstrated that zinc treatment induces while zinc deficiency inhibits iron absorption in intestinal cell culture models, but this needs to be tested in vivo. In the present study we assessed intestinal iron absorption, iron status (haemoglobin), red blood cell number, plasma ferritin, transferrin receptor, hepcidin) and tissue iron levels in zinc depleted, replete and pair fed control rats. Zinc depletion led to reduction in body weight, tissue zinc levels, intestinal iron absorption, protein and mRNA expression of iron transporters, the divalent metal ion transporter-1, hephaestin and ferroportin, but elevated the intestinal and liver tissue iron levels compared with the pair fed control rats. Zinc repletion led to a significant weight gain compared to zinc deficient rats and normalized the iron absorption, iron transporter expression, tissue iron levels to that of pair fed control rats. Surprisingly, haemoglobin levels and red blood cell number reduced significantly in zinc repleted rats, which could be due to rapid weight gain. Together, these results indicate that whole body zinc status has profound influence on growth, intestinal absorption and systemic utilization of iron, mediated via modulation of iron transporter expression.

Introduction

Iron and zinc deficiencies likely coexist in general populations. Phytic acid-rich plant foods have low bioavailability of both iron and zinc [1]. Therefore, populations consuming a phytate-rich diet with high prevalence of anemia are also expected to be at risk of zinc deficiency [2,3]. Supplementation of both iron and zinc together is a simple therapeutic alternative, however their combined supplementation in humans showed negative interactions, but the magnitude and direction of these interactions are conflicting [4], [5], [6], [7]. In particular, the interactions are evident only when iron and zinc are supplemented in solution but not in food [8]. Though competitive interaction at a transport protein level during intestinal absorption is hypothesized, the underlying mechanisms remain elusive [9,10]. Studies in animal models indicate accumulation of iron but reduction in haemoglobin during zinc deficiency [11,12]. Furthermore, cross sectional studies found higher prevalence of anemia and iron deficiency in subjects with zinc deficiency [13], [14], [15]. A positive association between serum zinc levels, haemoglobin and markers of iron status was also observed in humans [16,17]. Interestingly, alternate supplementation of iron and zinc significantly improved haemoglobin and iron status compared to iron supplementation alone [18]. Zinc supplementation has been shown to induce erythropoiesis, while zinc deficiency reduced the erythroid cell mass in animal models [19,20]. These observations together suggest that zinc interacts at multiple sites of iron metabolism including intestinal absorption and its utilization.

Intestinal iron uptake at the apical surface of enterocytes is mediated through duodenal cytochrome B-dependent reduction of ferric iron followed by uptake of ferrous iron via divalent metal ion transporter -1 (DMT1) [10,[21], [22], [23]. At the basolateral side of enterocytes, ferrous iron is exported via ferroportin (FPN1), and is then oxidized to ferric iron by hephaestin (HEPH) [10,21,22,24]. FPN1 is the only iron exporter so far identified. In previous studies we have demonstrated that zinc induces uptake and transcellular transport of iron in intestinal cells via Pi3K-dependent induction of DMT1 and HEPH expression [9,[25], [26], [27], [28]. Further we also demonstrated that zinc deficiency down regulates iron uptake and expression of DMT1, FPN1 and HEPH [27]. Based on these results and in the context of observed associations between zinc and iron status in humans, we hypothesized that whole body zinc status serves as an important modulator of intestinal iron absorption [10]. However, this needs to be tested in vivo.

In order to further understand the zinc status induced alterations in iron metabolism and underlying mechanisms, we studied the intestinal iron absorption and iron status during zinc depletion and repletion in rat models.

Section snippets

Materials

Control and zinc deficient diet were obtained from MP Biomedical, USA. 55Fe was obtained from American radio chemicals, USA. DMT1, FPN1, HEPH antibodies were procured from Alpha Diagnostics, USA. β-Actin antibody was from Abcam, USA. All other chemicals were purchased from Sigma-Aldrich, Bangalore, India unless specified.

Animal experiment

Animal experiments were conducted as per the guidelines of Indian Council of Medical Research, India. Experimental protocols and procedures were approved by the Institutional Animal Ethics Committee (IAEC) of National Institute of Nutrition, Hyderabad (IAEC-P31F/III-IAEC/NIN/12/2016/PR/WKY). The flow chart of animal experimentation was provided in supplementary Figure 1. Briefly, the weanling male Wistar/Kyoto rats (n=27) were supplied by the National Centre for Laboratory Animal Sciences, NIN,

Effect of zinc depletion/repletion on body weights and plasma zinc levels

The food intake in ZnD group was reduced during the first 3 days of feeding; 11.3±2.1 g as opposed to 38.0±4.0 g in control rats. Therefore, the control group was pair fed with 12 g/d diet throughout the experiment (pair fed control). The body weights of ZnD rats (77.5±5.3 g) were significantly reduced compared to pair fed control rats (118.2±7.6 g) at the end of 4-week depletion period (Fig. 1). The body weights of zinc replete (ZnR; 118±7.6 g) and pair fed control (135.44±8.7 g), but not ZnD

Discussion

Although iron and zinc interact negatively with the intestinal absorption of the other metal when supplemented together [5], [6], [7], several lines of evidence indicate a positive association of zinc with iron status, possibly mediated via regulation of intestinal iron absorption and its utilization [10,20,31]. In the current study, we demonstrated that zinc deficiency inhibits intestinal iron absorption by inducing mucosal retention of iron via down regulation of iron transporter expression.

Conclusions

The above results suggest that zinc status has a profound influence on iron absorption and utilization and that this is mediated at least in part via regulation of iron transporter expression. The fact that both pancreatic and intestinal zinc content correlates with intestinal iron absorption and iron transporter expression suggests cross-talk between pancreatic zinc excretion with intestinal iron absorption. Despite the favorable changes in iron metabolism observed during zinc repletion, the

Declaration of conflict of interest

The funder had no role in experimentation, interpretation of results, manuscript writing or its submission to the journal.

Funding

This study was supported in part by intramural grants of NIN-Indian Council of Medical Research (12/BS03) and Department of Biotechnology [BT/PR12128/PEN/20/960/2014], Govt of India. K.P. (SRF) and PSY (JRF) are supported by University Grants Commission, Govt of India.

Author contribution

KP, PS and RP designed the research and interpreted the results; K.P. carried out the experiments. RPL contributed to the animal experiments, carried out ELISA measurements. PCM and PSY contributed to the mineral estimations; all authors contributed to writing the manuscript, reviewed and approved the manuscript for submission.

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