Excretion of dietary zinc in mammals (rats) fed overdoses of zinc lactate and infected with tapeworms

Zinc is present in all biological systems and, owing to its versatile physicochemical properties, performs various functions in the body. Zinc is involved in the metabolism of insulin (Khoobbakht et al., 2020), carbohydrates, lipids and proteins. Zinc forms part of the digestive enzyme, and it is an important antioxidant which removes free radicals and protects cell membranes against lipid peroxidation. It significantly contributes to the growth and develop-ment of the individual as well as the proper functioning of the immune and reproductive systems (Williams, 2012; Gonçalves-Neto et al., 2011). Zinc is released from food in the form of free ions. This occurs primarily in the distal duodenum and proximal jejunum. Approximately 60 % of absorption occurs in the duodenum, 30 % in the ileum, 7 % in the jejunum, and roughly 3 % in the appendix and colon (Hotz et al., 2004). Zinc is absorbed by enterocytes through the basolateral membrane and then passes into the portal circulation. The portal system directs the zinc to the liver, from where it is released into the bloodstream, eventually reaching other tissues (Hotz et al., 2004). However, if zinc is ingested in high amounts, it is absorbed at the expense of other trace elements, and this may be expressed as a reduction in their uptake. This leads to a disruption of the metabolic processes that are dependent on these elements (Ferguson et al., 1995).

Zinc excretion takes place mainly through feces, which contains unabsorbed zinc from food consumption, zinc present in intestinal epithelial cells, as well as endogenous zinc, which is excreted into the intestine by the gallbladder, pancreas, and gastroduodenal secretions (Erdman et al., 2012). Approximately 70 to 80 % of ingested zinc is excreted in this way (Davies & Nightingale, 1975). There is also evidence that zinc exerts a negative effect on ecosystems, basic soil functions and species diversity among microorganisms. This can, in turn, negatively affect the conversion of nutrients in the soil. In order to ensure protection of the environment and public health, a number of steps have been taken to reduce heavy metal emissions in recent years. However, such steps have not been applied to zinc. Zinc continues to be a common additive in compound feed. For example, zinc is given to newly weaned piglets in order to reduce the incidence of diarrhea (Jensen-Waern et al., 1998) and improve both growth parameters and daily weight gain. However, such use of zinc leads to its accumulation in the environment and also to the development of bacterial resistance in LA-MRSA (livestock associated methicillin resistant Staphylococcus aureus (Rosenbach, 1884)). In Denmark, an increase in soil zinc concentrations of more than 45 % was recorded between 1998 and 2014. Based on those results, European agencies (EMA, EFSA) are reviewing the current permitted levels of zinc in compound feed (e.g. Directive 2016/1095) (Jensen et al., 2018).

Zn in animals is taken up orally and subsequently absorbed and excreted mainly by the digestive tract. Since gastrointestinal helminths (including tapeworms) are present in the digestive tract, they can influence zinc metabolism significantly. According to many authors, tapeworms of the genus Hymenolepis can accumulate significant amounts of metal ions taken up by the host (Sures et al., 2002; Jankovská et al., 2018).

Our study, therefore, focused on comparing the levels of zinc ingested and excreted by rats infected with tapeworms to those ingested and secreted by uninfected rats. In addition to being given the standard dose of zinc, some rats received a zinc lactate additive. Thus, it has been hypothesized that tapeworms will significantly affect the excretion of zinc in the feces of a host.

The authors confirm that the ethical policies of the journal, as noted on the journal’s author guidelines page, have been adhered to and the appropriate ethical review committee approval has been received. The authors confirm that they have followed EU standards for the protection of animals used for scientific purposes [and feed legislation, if appropriate]. All experiments with laboratory animals were conducted in compliance with the current laws of the Czech Republic, Act No. 246/1992 coll. on the protection of animals against cruelty and EC Directive 86/609 EEC.The study was approved by the ethic committee of Czech University of Life Sciences Prague.

Tapeworms have generally been shown to reduce the amount of zinc in the feces of a host. However, infected rats in the control groups (00 and 0T) excreted more Zn than did rats without tapeworms (6.3 %); this was the case even during the second week of the study. For the remaining weeks, rats in group 0T excreted less Zn in feces than did rats in group 00 (the most dramatic difference was observed in the fifth week: 8.5 %); however, differences between these two groups were not considered significant (Fig. 1).

Fig. 1

Fig. 2

When comparing the groups that are provided with high amounts of zinc in their feed (M0; MT), it is evident that the tapeworm does have an effect on the excretion of this metal in the feces. Throughout the entire six weeks of study, rats of the MT group excreted less zinc in the feces than did uninfected rats (M0). In the first week, only insignificant differences between the two groups were observed. During the next five weeks, however, differences in Zn levels between the two groups were determined to be statistically significant. These differences were evaluated as more significant (p ^˂ 0.01) in the 3rd (28.4 %), 5th (27 %), and 6th (24.9 %) week (Fig. 2).

The values of fecal zinc excretion for the entire duration of the experiment indicate that the MT group excreted over 20 % less zinc than did the M0 group. Differences in Zn levels between groups 00 and 0T were not considered significant; rats with tapeworms excreted less zinc in feces than did rats without tapeworms. The M0 group excreted the excreted the highest levels of zinc of all groups, while the MT group excreted the lowest levels. Thus, the difference in zinc levels between groups M0 and MT was significantly greater (p ^˂ 0.01) than it was between groups 00 and 0T. Tapeworm infection had the strongest effect on zinc levels in the feces of rats fed high doses of zinc (Table 3).

Zinc is an element that is essential for the proper functioning of animal metabolism, but it can also be toxic in high doses. Insufficient nutritional intake of zinc is relatively common in some areas, so dietary supplements, among them zinc, are used to complement the diet. These supplements contain various zinc compounds, some of which are well absorbed, while others are not. In our study, we chose zinc lactate as a dietary zinc supplement. Hotz et al. (2004), state that in order to focus future research on the intake and excretion of zinc, there is a need to clarify the role gastrointestinal helminths play in these processes. This was the main objective of our study.

Gastrointestinal parasites come into contact with ingested food before it is absorbed by the host’s digestive tract, and this has an effect on the amount of nutrients available to the host. Krebs (2000) states that in humans and animals, two factors are most important for maintaining zinc homeostasis - endogenous intestinal excretion and the amount of zinc absorbed by the intestine.

According to Hall et al. (2008), tapeworms impair the absorption of nutrients, especially in the final portion of the ileum, of the ileum, and this impairment can lead to anemia. In addition, according to Hotz et al. (2004), approximately 30 % of zinc is absorbed in the ileum, and Johnson (1989) reported levels as high as 60 %. Wada et al. (1985) report that approximately 53 % and 49 % of zinc is absorbed at dietary doses of 16.5 mg/day and 5.5 mg/day, respectively. In contrast, Jackson et al. (1984) state that when dietary zinc is increased, the levels of absorbed zinc decrease. When levels of dietary zinc were doubled, the absorption rate was 45 % for the first 4 days and decreased to 32 % during the following 4-day period. Another study also proves the extraordinary ability of the body to regulate zinc intake. King et al. (2000) report that the body absorbs up to 100 % of ingested zinc when a diet is lacking in this metal. When rats were fed 0.5 mg of Zn per day, the absorption rate decreased to 55 % (King et al., 2000).

In our experiment, control rats from groups 0T and 00 received an average of 1.71 mg and 1.75 mg of Zn per day, respectively. Groups 0T and 00 excreted 70 % and 74 % of ingested zinc, respectively. Rats in experimental group M0 excreted, on average, the highest levels of ingested zinc from all the groups, namely 87 % (Table 3); the highest levels (94 %) were eliminated in the final week. This confirms that the organism responds to a higher content of zinc in the diet by excreting higher amounts in the feces. The study of Cadkova et al. (2013) demonstrates how the type of ingested Zn can play an important role in the absorption process.

The authors administered lead to rats in two different forms - lead acetate and phytobound Pb (Pistia stratiotes (Linné, 1753)). Their results show that 53 % of the ingested lead was excreted in the feces within 24 hours of exposure. In our study, from 65 % (MT group) to 87 % (M0 group) of ingested zinc was excreted 24 hours after exposure.

Cadkova et al. (2013) also determined that lead acetate was excreted in greater amounts than lead in hyperaccumulator.

Although zinc was not evaluated in the study of Cadkova et al. (2013), we can assume, due to their similar physicochemical properties, that zinc, cadmium and lead (Das et al., 1997; Chaney, 2010), behave in a similar manner. For example, Decker et al. (1957) orally administered 2 mg of cadmium to rats. They found that 90 % of the administered dose was excreted in the feces. This amount is similar to the values of zinc excreted by feces. According to a study by Horakova et al. (2017), zinc, cadmium and lead are concentrated in the same areas of the tapeworm, namely the immature strobils, so it can be assumed that these metals are indistinguishable from one another for the tapeworm. In our experiment, zinc lactate was used to increase zinc content in feed. Zinc is a compound of metal and organic acid, such as lead acetate.

Based on the results of Cadkova et al. (2013), we can expect the amount of zinc excreted in the feces to be higher after the intake of zinc lactate than it would be after the intake of phytobound Zn.

This is confirmed by a comparison with the results of one of our previous publications (Sloup et al., 2018), where we evaluated how tapeworms affect the excretion of zinc and cadmium in the feces of rats fed Arabidopsis halleri (Hayek, 1908). The results indicated that the uninfected group that were fed A. halleri excreted 78 % and 70 % of zinc and cadmium in the feces, respectively. The group infected with tapeworms and fed A. halleri excreted 76 % and 61 % of zinc and cadmium in the feces, respectively. In our new study, rats from the M0 and MT groups excreted 87 % and 65 % of zinc in their feces respectively (Table 3). The levels of zinc excreted in the feces from ingested zinc lactate were actually higher than those from zinc ingested in the form of phytobound Zn. Isaure et al. (2015) state that zinc, cadmium and lead are also bound as a metal-organic acid complex in hyperaccumulators, including P. stratiotes and A. halleri.

This finding is unexpected, as the amount of zinc absorbed and excreted can be affected by phytic acid, which is considered an antinutritional agent that reduces the utilization of certain nutrients, including zinc (House et al., 1982). Moreover, according to Milne et al. (1984), phytic acid significantly reduces the absorption of Zn. In contrast, the bioavailability of zinc from zinc lactate is thought to be very high, even higher than that of zinc gluconate, which is very well-absorbed (Shengkui et al., 1994).

The MT group excreted 65 % of ingested zinc, while the infected group that were fed A. halleri excreted 76 % of ingested zinc (Sloup et al., 2018). This can be attributed to the ability of the tapeworm to absorb more zinc from a diet rich in zinc lactate, which has a high bioavailability, than it could from a diet with added A. halleri. However, it is also possible that the tapeworms of A. halleri received less zinc because in addition to Zn, the plants contained hazardous metals, namely cadmium, which the zinc lactate diet did not. Another reason could be tapeworm intoxication in the intestine of rats fed A. halleri. According to Scott and Koski (2000), a high zinc content has a negative effect on tapeworms. A relatively high intake of cadmium and zinc could also lead to a poisoning of the tapeworms in cases where rats are fed a diet with added A. halleri. This in turn could result in tapeworms with smaller dimensions. These smaller tapeworms would accumulate less zinc than would tapeworms that were exposed to high levels of zinc exclusively through a diet fortified with zinc lactate.

Although the entire complex of processes that ultimately determine how much zinc will be absorbed, excreted, and ultimately accumulated in an animal depends on a number of factors, tapeworms represent an integral and important part of such processes. The amount of metal in the diet is one of the most important factors for homeostasis. However, the type of ingested zinc, food composition and the quantity of other nutrients are also vital. This is evident from our results, which indicated that tapeworm presence had a significant effect on zinc excretion in rats fed high doses of zinc (M0 and MT groups) that was not evident in rats fed a standard diet containing normal amounts of the metal (groups 00 and 0T). Our hypothesis was thus partially confirmed.