Concurrent repletion of iron and zinc reduces intestinal oxidative damage in iron- and zinc-deficient rats

Abstract

Aim: To understand the interactions between iron and zinc during absorption in iron- and zinc-deficient rats, and their consequences on intestinal oxidant-antioxidant balance.

Methods: Twenty-four weanling Wistar-Kyoto rats fed an iron- and zinc-deficient diet (< 6.5 mg Fe and 4.0 mg Zn/kg diet) for 4 wk were randomly divided into three groups (n = 8, each) and orally gavaged with 4 mg iron, 3.3 mg zinc, or 4 mg iron + 3.3 mg zinc for 2 wk. At the last day of repletion, 3 h before the animals were sacrificed, they received either 37 mBq of (55)Fe or (65)Zn, to study their localization in the intestine, using microautoradiography. Hemoglobin, iron and zinc content in plasma and liver were measured as indicators of iron and zinc status. Duodenal sections were used for immunochemical staining of ferritin and metallothionein. Duodenal homogenates (mitochondrial and cytosolic fractions), were used to assess aconitase activity, oxidative stress, functional integrity and the response of antioxidant enzymes.

Results: Concurrent repletion of iron- and zinc-deficient rats showed reduced localization of these minerals compared to rats that were treated with iron or zinc alone; these data provide evidence for antagonistic interactions. This resulted in reduced formation of lipid and protein oxidation products and better functional integrity of the intestinal mucosa. Further, combined repletion lowered iron-associated aconitase activity and ferritin expression, but significantly elevated metallothionein and glutathione levels in the intestinal mucosa. The mechanism of interactions during combined supplementation and its subsequent effects appeared to be due to through modulation of cytosolic aconitase, which in turn influenced the labile iron pool and metallothionein levels, and hence reduced intestinal oxidative damage.

Conclusion: Concurrent administration of iron and zinc corrects iron and zinc deficiency, and also reduces the intestinal oxidative damage associated with iron supplementation.

Figure 1

Zn reduces uptake of ⁵⁵Fe and Fe reduces uptake of ⁶⁵Zn during combined administration: A representative microautoradiogram of the duodenal mucosa of ⁵⁵Fe in Fe (top left), Fe + Zn (top right), ⁶⁵Zn in Zn (bottom left) and Fe + Zn (bottom right) groups. Iron and zinc deficient rats were orally administered 37mBq of ⁵⁵Fe and 4.0 mg Fe and/or 37mBq of ⁶⁵Zn and 3.3 mg Zn. Presence of black spots in the intestinal mucosa indicates the presence of the radioactivity (× 100).

Figure 2

Zn reduces ferritin but enhances metallothionein abundance in the intestinal mucosa: A representative photomicrograph of the immunohistochemical localization of ferritin (top panel) and metallothionein (bottom panel) in the intestinal mucosa of Fe, Zn and Fe + Zn repleted groups. The staining intensity indicates the abundance of ferritin and metallothionein (× 250).

Figure 3

Iron increases intestinal oxidative stress and lowers functional integrity: Concentrations of thiobarbituric acid-reactive substances (TBA-RS) and protein carbonyls (A and B) as indicators of oxidative stress, and activities of alkaline phosphatase and lys-ala-dipeptidyl aminopeptidase (C and D), as markers of mucosal functional integrity, in the intestinal mucosa of iron and zinc deficient rats treated with iron and/or zinc. Fe: Iron administered group; Zn: Zinc administered group; Fe + Zn: Iron and zinc administered group; Number of animals: 7 in each group. Vertical columns and error bars represent mean and SD respectively; Bars with different superscripts are significantly different with P < 0.05 among groups.

Figure 4

Changes in antioxidant enzyme activities due to iron and zinc repletion: Activity levels of superoxide dismutase (SOD, A and B), catalase (C) and glutathione peroxidase (Gpx, D) in the small intestinal mucosa of rats during repletion with iron and/or zinc. Total SOD activity indicates the activity of Cu, Zn-SOD and Mn-SOD. Fe: Iron administered group; Zn: Zinc administered group; Fe + Zn: Iron and zinc administered group; Number of animals: 8 in each group. Vertical columns and error bars represent mean and SD respectively; bars with different superscripts are significantly different with P < 0.05.

Figure 5

Altered intestinal mucosal redox status: Changes in the levels of GSH (Panel A) and GSSG (Panel B) in the intestinal mucosa at the end of repletion for 2 wk with iron and/or zinc. Vertical columns and error bars represent mean and SD respectively; bars with different superscripts are significantly different with P < 0.05.

Figure 6

Intestinal aconitase activity in response to iron and/or zinc repletion: Response of intestinal cytosolic (Panel A) and mitochondrial (Panel B) aconitase to intestinal iron content and as a functional indicator of oxidative stress after 2 wk of oral iron and/or zinc administration. Fe: Iron administered group; Zn: Zinc administered group; Fe + Zn: Iron and zinc administered group. Number of animals :is 8 in each group. Vertical columns and error bars represent mean and SD respectively; bars with different superscripts are significantly different with P < 0.05.