Manganese (Mn) is an essential trace element and is necessary for catalytic processes and acts as a promoter of various enzymes including the brain-specific glutamine synthesis, superoxide dismutase and pyrurate carboxylase (Welder and Denman, 1984; Keen et al., 2000).
Although (Mn) is in an essential trace elements for brain function, but manganese toxicity has been reported to cause brain disorder and also disturbs heme synthesis (Hundell, 1999; Erikson et al., 2004). However, there is currently no exact recommended dietary allowance for this element although an estimated safe and adequate daily dietary intake of 2-5 mg per day has been recommended (Hurley and Keen, 1987). Inhalded manganese is also well absorbed through respiratory system (Thompson et al., 2006; Rao et al., 2003). Manganese shares many similarities with iron from different point of views including structural, biochemical and physiological functions (Reid et al., 2006). Previous investigation showed that the amount of manganese absorbed in healthy young woman is strongly associated with iron status which gives greater insight into manganese absorption and retention in humans (Finley, 1999).
Right now there is little known about manganese absorption by human body and the exact mechanism by which manganese absorbed, by intestine cells is still matter of speculation. The major aim of this investigation was to study and compare manganese and iron absorption by rat everted gut sac and the effects of various factors on this process.
MATERIALS AND METHODS
All chemicals used in this project were reagent grade and purchased from Sigma Chemical Company UK. Deionized water was used throughout this investigation. Laboratory glassware was soaked overnight in 10% HNO3 and washed three times with distilled water. Male wistar rats purchased from Tehran Pasteur Institute (Iran) and were kept in faculty animal house of Isfahan university medical sciences (Isfahan, Iran) at standard conditions and fed on basal diet and water until their weights reached between 200-250 g.
Animals were fasted 24 h prior to the experiments and they were killed by cervical dislocation. Small intestine was removed, cleaned from debris, washed, blotted dried and weighted. The intestine cut into small pieces and proximal duodenum, proximal jejunum and distal ileum were prepared (Moshtaghie et al., 1996).
The segments were everted. The everted gut sacs were filled up with Kerebs Ringer Phosphate (KRP) medium, pH 7.4 and suspended in kerebs ringer phosphate medium with or without iron and/or manganese. The incubation mixture was capped and gassed with O2/CO2 = 95.5 on water bath shaker at 30°C. At the time of intervals the reaction mixture was removed and the concentration of iron and/or manganese inside the sacs was determined. Iron determination was carried out using spectrophotometry and phenanthrolin reagent was used as chromogen (Brithenham, 1979).
Manganese Concentration was determined using flameless-atomic absorption Zemman (Model 23030).
Preparation of iron and manganese citrate complex: Separate stock standard solutions of FeCl3, 6H2O (3.0 mM) and MnCl2, 4H2O (3.0 mM), were prepared in deionized water and mixed with equal vol of 60.0 mM citric acid. The solutions were adjusted to pH 7.4 with 1 M NaOH and Made up to final concentration of 1.5 mM iron and manganese.
Earlier reports have shown that iron absorption occurs through duodenum. In order to study the possible major site of manganese absorption various parts of small intestine were chosen: the proximal 8 cm of duodenum, the proximal 11 cm of jejunum and the distal 14 cm of ileum. Variations in the absorptive surface of three segments compensated by differences in their length. To follow this, EGS was prepared and incubated in kerebs ringer phosphate medium including 100 mg L-1 Mn, for 1 h. It shows that the absorption of Mn by duodenum is approximately 12-14% higher than jejunum and/or lleum respectively (Fig. 1).
|| Mn absorption by the small intestinal segments
||The effect of incubation time on Fe () and Mn () absorption by rat EGS
Next experiments were carried out to study and compare the effects of other factors which may necessary for iron and manganese uptake by EGS. The effects of incubation time on this process was studied first. To follow this, EGS was prepared and incubated into two series of volumetric flasks in kerebs ringer phosphate medium containing 100 mg L-1 Fe and/or Mn as complex with citric acid (1:20).
At intervals time EGS was removed from medium and washed with saline. The iron and manganese containing solution inside the sac were determined and the results show that maximum Fe and Mn uptake occurs within 30-60 min of incubation time, respectively (Fig. 2). Significant reduction by 30% in iron absorption was seen when Mn was added to the medium. The level of iron uptake was then decreased, suggesting that the mucosal cells gradually lose their abilities to take up iron (Fig. 2).
Fe II and Fe III uptake by EGS was studied and compared. It was found that
iron as Fe II complex with citric acid 1:20 being absorbed more than Fe III
(Fig. 3). The Fe II absorption was 25% Fe II higher than Fe
|| Fe (II) () and Fe (III) () absorption by rat EGS
||Manganese Mn ()
and iron Fe ()
uptake by rat EGS. Effect of concentration
||Effect of citric acid on manganese uptake by rat EGS
|| Effect of glucose on manganese uptake by rat EGS
||Effect of on manganese on iron absorption by rat EGS
In order to determine whether iron and manganese uptake by EGS was dependent
on the concentration of these elements, two series of volumetric flasks containing
varying concentrations of metals were selected and next experiment was performed.
Addition of varying concentration of Fe and/or Mn (0-200 mg L-1)
to the reaction mixtures showed that there was a gradual increases in iron and/or
Mn uptake by everted gut sacs which is up to 100 mg L-1 (Fig.
Citric acid has been previously reported to enhance iron absorption through
|| Effect of iron on manganese absorption by rat EGS
In this project when the effect of citric acid on Mn absorption was investigated,
there was a 25% elevation in Mn absorption (Fig. 5). Addition
of 5 mM glucose to the reaction mixtures also enhanced Mn uptake by 20% (Fig.
6). Finally the intestinal interactions between Mn and Fe were studied (Fig.
7). It shows the effect of Mn (100 mg L-1) on intestinal absorption
of iron and Fig. 8 shows the effect of identical concentration
of Fe (100 mg L-1) on Mn uptake by everted gut sac. Addition of Mn
reduced Fe absorption by approximately 48%, respectively.
A previous study by this laboratory found that manganese interferes with iron uptake by transferrin. Since transferrin also has been reported to play an important role in iron and cadmium uptake by intestine (Moshtaghie et al., 1996) it is obvious that there should be possibility of the interaction between Mn and Fe absorption.
Initial examination in this project showed that the uptake of manganese in duodenum section of intestine is much higher than ileum and jejunum Fig. 1. Secondly, it has been reported that high dietary iron depress manganese absorption (Finely, 1999) and addition of iron to an intestinal perfusate decreased manganese absorption (Thomson et al., 2006). These findings are in good agreement with the present data in this manuscript that uptake of either iron and/or manganese by rat everted gut sac is a time and dose dependent processes (Fig. 2 and 3) and addition of iron to the incubation medium lead to the reduction of manganese uptake by intestinal cells. Heilig et al. (2005) reported that intestinal manganese uptake is upregulated by iron deficiency and is thought to be mediated by Divalent Metal Transporter 1 (DMT1), an iron regulated factor known to play a role in dietary absorption. Interestingly, the reduction in manganese uptakes by gut in the presence of iron is much higher than the reduction of iron uptake in the presence of manganese. It seems that the existence of iron could be a major regulator in manganese uptake by rat everted gut sac.
These findings consistent with the recent observations by Thompson et al. (2006) that individuals chronically exposed to manganese are at high risk for neurotoxin effects of this metal absorption from the lung to the blood and it be modified by iron status. High dietary iron levels inversely affect intestinal uptake of manganese.
On the other hand it has been reported that manganese inhibited iron absorption both in solutions and in a diet. Fractional iron absorption is strongly dose dependent. Suggesting a direct competitive inhabitation of manganese and iron absorption (Rosssander-Hulten et al., 1991). These observations are in agreement with our findings that the absorption of both metals by EGS is not only a time dependent process but also depends on the concentration of either Mn or Fe in the solutions. Also in confirmation to all these findings is the report of Thompson et al. (2006) that high dietary iron levels inversely affect intestinal uptake of Mn.
Some dietary factors including carbohydrate have been reported to influence manganese absorption (Lee and Johnson, 1988). Data of present investigation showed that addition of glucose have increasing effects on either iron and/or manganese absorption by intestine. It could be assumed that either glucose or carbohydrate containing glucose could be involved in the production of chemical energy (ATP). This might be needed for active transport process. Previous findings by Slon (1965) showed that some cations including potassium and calcium transportation across intestine membrane required energy which is provided by ATPase located in the membrane following releasing of energy from ATP molecules (Kristin and Hamish, 1988). It seems that manganese absorption may however follow this mechanism.
We think at this point more investigations should be done to elucidate deeply the exact mechanism by which manganese absorption performs by intestine.