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Comparison of Absorption Characteristics of Iron Glycine Chelate and Ferrous Sulfate in Caco-2 Cells.

Byline: Wen-Qiang Ma1, Jing Wu, Zhao Zhuo, Hong Sun, Min Yue and Jie Feng

Abstract

The study was conducted to compare the absorption characteristics of iron glycine chelate (Fe-Gly) and ferrous sulfate (FeSO4) in Caco-2 cells. Effects of several factors, including concentration, time and temperature on iron (Fe) transport were investigated. In Caco-2 cell model, Fe transport amount in Fe-Gly treatment was higher than that in FeSO4 from the apical side (AP) to the basolateral (BL) side. However, there was no significant difference between the FeSO4 and Fe-Gly treatments in the direction of BL to AP. Similar results were also found on Fe transport rate of Fe-Gly and FeSO4 across Caco-2 monolayers. The apparent permeability coefficient of Fe-Gly was significantly higher than that of FeSO4 in the direction of AP to BL. The ratio of the apparent permeability coefficient between AP to BL and BL to AP was greater than 1.0 for both forms of Fe resources.

The Fe transport amount in Fe-Gly treatment was higher than that in FeSO4 treatment, no matter the incubation temperature was 37degC or 4degC. However, it significantly decreased, when the incubation temperature dropped from 37degC to 4degC. The results indicate that Fe of Fe-Gly can be easily absorbed than FeSO4 in Caco2 cells. (c) 2013 Friends Science Publishers

Keywords: Fe-Gly; FeSO4; Caco-2 Cell; Concentration; Time; Temperature

Introduction

Iron (Fe) plays an important role in physiological functions, for instance, carries oxygen, forms a part of the oxygen- carrying proteins (hemoglobin and myoglobin) and serves as a cofactor in enzymes that involving in oxidation- reduction reactions (Glahn et al., 2002; Kloots et al., 2004). In terms of oxygen transport and cellular respiration, Fe is an indispensable element for fish (Javed and Saeed, 2010). In order to meet the growth requirement, an addition of 38 to 80 mg of Fe was given to the diet of swine or poultry (Kratzer et al., 1994; 1998). However, studies have shown that a relatively low absorption of nonheme Fe in maize, soybeans and wheat, ranging from 2 to 20 (Percent) (Hallberg et al.,1997; Tapiero et al., 2001). Therefore, many Fe additives were used in the animal diets to provide enough Fe source, such as Fe sulfate, Fe carbonate, Fe proteinate, and Fechelate.

The bioavailability values vary greatly among different Fe sources. Compared with ferrous sulfate (FeSO4), chelated or proteinated source of Fe had higher bioavailability which was 125 to 185 (Percent) at the same level of FeSO4 (Henry et al., 1995). Langini et al. (1988) reported that the Fe absorptions of weanling rats which given infant formula labeled with [59Fe] glycine and [59Fe] sulfate were 30.9 and 15.8 (Percent) , respectively. Layrisse et al. (2000) found that the absorption rate of Fe in iron glycine chelate (Fe-Gly) was almost two times of that in FeSO4. We also reported a better bioavailability in ferrous glycine than ferrous sulfate in piglet (Feng et al.,2007; 2009; Ma et al., 2012). However, the data of absorption characteristics between Fe-Gly and FeSO4 is still limited.

Caco-2 line that derived from the human colon adenocarcinoma is able to differentiate spontaneously (Sanchez et al., 1996). It can present many absorption characteristics analogous to intestinal cells during culture, such as formatting a monolayer of the cells and expressing several morphological and functional characteristics of the mature enterocyte (Rousset, 1986; Artursson, 1990; Gan et al., 1997). Caco-2 cell model has been used extensively in numerous of biological, biochemical and toxicological studies, as well as in the intake or transport study of trace elements, such as iron, zinc, copper, chromium, and so on (Zodla et al., 2005; He et al., 2008; Villarroel et al., 2011). Thus, the purpose of the present study was to compare the absorption characteristics between Fe-Gly and FeSO4 using the Caco-2 cell lines.

Materials and Methods Cells

Caco-2 cells during 20-40 serial passages (Shanghai Institute of Biochemistry and Cell Biology, SIBS, CAS) were cultured according to the method described by Mazariegos et al. (2004). Briefly, cells were cultured in 25 cm3 flasks with Dulbecco's minimum essential medium (DMEM, Gibco) which supplemented with 10 (Percent) fetal bovine serum (FBS), 4.5 g/L glucose, 2 (Percent) L-glutamine, 1 (Percent) non-essential amino acids and 100 U/L penicillin/streptomycin. The incubation condition was 37degC,5 (Percent) CO2 and 90 (Percent) relative humidity. Suspended the Caco-2 cells, which were in the logarithmic growth phase and reseeded them on the polycarbonate membrane of 6-Transwell plug-in Petri dish at a density of 1x104 cells /cm2.

Assessment of Caco-2 Model

The cells were evaluated for study use on the 20-22th days. Caco-2 cells were similar to the epithelial cells morphologically (observed by inverted phase contrast microscope, Olympus CKX41, Japan), transmembrane resistance value (466.75 (omega) cm2) and mannitol permeation rate (0.85 (Percent) ) could meet the requirements of tightness, integrity and permeability. Besides, cells differentiated well with polarity, which tested by alkaline phosphatase activity. Therefore, Caco-2 model could be used for intestinal absorption in vitro.

Fe Solutions

Fe-Gly and FeSO4 were made into 1 moL/L stock solution with double-stilled water. Filtrated the stock solution with aseptic Millipore filter and diluted into different multiple with D'Hanks buffer. Atomic absorption spectrometry (Shimadzu AA-6501; Paleologos et al., 2002) was used for the measurement of Fe concentration.

Transport Assays in Caco-2 Cells We conducted the bidirectional transport of FeSO4 and Fe-Gly using caco-2 monolayers prepared as above. Transports from the apical side (AP) to the basolateral side (BL): Sample solutions (1.5 mL) were placed on the apical side of Caco-2 cells, served as the donor chamber, while the basolateral side was supplemented with D'Hanks (2.6 mL) buffer as a receiver. Whereas, transports from BL to AP: BL with 2.6 mL sample served as the donor chamber and AP with 1.5 mL D'Hanks buffer as the receiver. Triplicate wells were used for each treatment.

Transport Study on Concentration

Sample solutions (1.5 mL) containing different Fe concentration (0.5, 5, 10 and 20 (Mu)mol/L) were placed on the side of the donor chamber, while the side of the receiver chamber was supplemented with 2.6 mL D'Hanks buffer.

The experiment was performed in the oscillation sink with 37degC constant temperature and at a speed of 50 rpm.

Transport of Fe across the cell monolayer was tested by withdrawing 200 (Mu)L of sample from the receiver chamber at different time points, and an equivalent amount of pre-warmed D'Hanks buffer was immediately given to replace this volume. The experiment was ended after 120 min.

Transport Studies on Time and Temperature

As for the study of transports across Caco-2 monolayers on time and temperature, we collected 200 (Mu)L of sample from the receiver chamber at the specific time points (30, 60, 90 and 120 min) and just changed the temperature condition from 37degC to 4degC, respectively. The other details were same as the different Fe concentration study.

Calculations

Apparent permeability coefficient (Papp, cm/s) Papp was calculated by the following formula Where, dQ/dt ((Mu)moL/min) is the flux rate of mass transport across the monolayers, A is the surface of insert membrane(4.7 cm2), C0 is the initial concentration ((Mu)moL/mL) of sample in the donor chamber.

The following equation was applied to revise the values for minimizing sampling errors. Where, TRcum is the revised mass transport across the monolayers, An is the transport amount, which is actual measured, VSn is the volume of collected sample, VR is the volume of the donor chamber.

The transport rate is calculated using the following equation: Transport rate (Percent) = Fe transport amounts in the receiver chamber Fe transport amounts in the donor compartment '100 (Percent)

Statistical Analysis

Values presented in the study are given as the mean +- SEM. One-way analysis of variance (ANOVA) and paired comparison of LSD were used in analyzing the effects between different treatments in SPSS 11.5 software. P value of (Less than) 0.05 was considered significant, while (Less than) 0.01 were regarded extremely significant.

Results

Effect of Concentration on Fe Transport

The bidirectional transports of Fe-Gly and FeSO4 are concentration-dependent at the temperature of 37degC (Fig. 1).

Table 1: Apparent permeability coefficient (Papp) of Fe-Gly and FeSO4 at different concentrations across Caco-2 cell monolayers

Concentration (umo1/L)###Apparent permeability coefficient (Papp, x 10 -6cm/s)

###FeSO4###Fe-Gly

###AP - BL###BL - AP###R###AP - BL###BL - AP###R

0.5###4.70 +- 0.67 b###0.72 +- 0.05 B###6.50###10.40 +- 0.62 a###0.64 +- 0.08 u###16.14

5###3.37 +- 0.14 b###0.34 +- 0.02###9.99###6.74 +- 0.21 a###0.33 +- 0.04###20.71

10###2.39 +- 0.12 b###0.27 +- 0.0###8.96###5.48 +- 0.25 a###0.21 +- 0.03###25.43

20###1.60 +- 0.11 b###0.18 +- 0.03###8.77###3.55 +- 0.39 a###0.15 +- 0.04###23.57

Note: Experiments were conducted 120 mm at 37degC. The ab or czf3 represents the differences between Fe - Gly and FeSO4 treatments under the same concentration and direction, without common letters are considered significantly (P Less than 0.05). R represents the apparent permeability coefficient ratio

Fe transport amount in Fe-Gly treatment is higher than that in FeSO4 from AP to BL side, but there is no obvious difference in the direction of BL to AP. The same results were also found on Fe transport rate of Fe-Gly and FeSO4 across Caco-2 monolayers (Fig. 2).

Table 1 shows the Papp of Fe-Gly and FeSO4 at different concentrations across Caco-2 cell monolayers. The Papp of Fe-Gly is significantly higher than that of FeSO4 in the direction of AP to BL. Besides, the ratio of the papp value between AP to BL and BL to AP is greater than 1.0 for the both forms of iron resources.

Effect of Time and Temperature on Fe Transport

Fe transport amount of Fe-Gly and FeSO4 across Caco-2 monolayers are linear increased with the time prolonged (Fig. 3). The bidirectional Fe transport of Fe-Gly and FeSO4 are time-dependent at the temperature of 37degC. It also showed similar pattern as the concentration on Fe transportation, Fe transport amount in Fe-Gly is higher than in FeSO4 from AP to BL side.

Effect of temperature on Fe transport was shown in Table 2. Compared with FeSO4, Fe transport amount in Fe- Gly treatments were both higher when incubated in 37degC or 4degC. However, it decreased (P (Less than) 0.05) when the incubation temperature dropped from 37degC to 4degC.

Discussion

Iron amino acid chelate has been proven to be one of the original models for animals to absorb Fe compound supported by studies on Fe absorption mechanism (Saltman, 1965). Many studies have showed that Fe-Gly has a high bioavailability in the bodies of rats, human beings and other animals, compared with FeSO4, (Ashmead, 2001; Allen, 2002).

Langini et al. (1988) feed weaning rats with infant formula food which added the same level of Fe-Gly or FeSO4 marked by isotope [59 Fe]. The study showed that the iron absorption rate of FeSO4 was 15.8 (Percent) ,while the rate of Fe-Gly was 30.9 (Percent) . Layrisse et al. (2000) reported that the

Table 2: Effect of temperature on transport of Fe-Gly and FeSO4 across Caco-2 monolayers (10 umo1/L, 37degC, 120 min)

Transport amount (pmol/cm2)###AP - BL

###37 C###4 C

FeSO4###172.34 +- 5.62 a###120.41 +- 2.79 b

Fe-Gly###394.5512 +-###31 a###278.55 +- 10.40 b

Note: Measurements without common letters in the same row are considered significantly different (P Less than 0.05)

Fe absorption rate of Fe-Gly was higher than that of FeSO4 by nearly 2-fold in human being body. Ashmead (2001) supplied Fe-Gly and FeSO4 as Fe fortification for anemia, at the dose of 5 mg/kg weight. After 28 days experiment, the apparent biological utilization rate of Fe-Gly was 90.9 (Percent) , but the rate of FeSO4 was only 26.75 (Percent) with plasma ferrtin were monitored as biomarker. Nielsen et al. (2005) conducted an experiment treating anemia with Fe-Gly offered as Fe fortificants for nutrition. After 6-week therapy, they found that the mean heme value of patients in the Fe- Gly treatment group was significantly higher than the value of inorganic iron group (12.1 +- 1.8:10.7 +- 1.7 g/dl). In America, Fe-Gly as one of new iron fortificants for nutrition has been applied to milks of infant and food (Fox et al.,1998; Giorgini et al., 2001).

This study was designed to compare the bidirectional transport of Fe-Gly and FeSO4 using Caco-2 cells. It proved that Fe of Fe-Gly can be easily absorbed than that of FeSO4 in Caco-2 cells. We found that the transepithelial transport of Fe-Gly and FeSO4 were concentration- and time- dependent from both directions. And the transport amounts were much greater in the direction of AP to BL than those in BL to AP transports. The Papp of Fe-Gly and FeSO4 decreased as the concentration increased and the Papp of transport from AP to BL divide by that in inverse direction is greater than 1.0 for the both two forms of iron sources. The culture temperature has significant effect on transport amount. These results suggest that the absorption of FeSO4 and Fe-Gly in Caco-2 cells is mainly through active transport. It was also found that the transport amount of Fe- Gly was notably higher than that of FeSO4 at the same concentration or temperature.

All the results taken into account, we speculate that Fe-Gly may have specific or non- specific intestinal active transit system, except for some ionic iron dissociating from Fe-Gly before uptake and being transported through the apcial membrane follow the same pattern as FeSO4 absorption.

The absorption mechanism of Fe-Gly is still unknown exactly. Zhu et al. (2006) proposed at least some iron dissociates from EDTA and is reduced just as simple inorganic iron at the brush border membrane of the enterocyte when study iron uptake by Caco-2 cells from NaFe-EDTA and FeSO4. Pizarro et al. (2002) found that Fe- Gly competes for the absorption pathway of nonheme Fe. They enter the common nonheme Fe pool and activate the same transporter on the cells for transportion. They concluded that one reason for better absorption of Fe-Gly is glycine can chelate and protect Fe from inhibitors.

In conclusion, our work confirmed that part of Fe-Gly absorbed by the same pattern as FeSO4 absorption mechanism, but it is likely that Fe-Gly is also using specific or non-specific intestinal active transit system.

For further study, transport kinetics of Fe-Gly and relevant proteins of Fe transporter should be tested for revealing the mechanism.

Acknowledgements

This work was supported by the Project supported by the National Natural Science foundation (No.31272398), New- Century Training Program Foundation for Talents from the Ministry of Education of China (No. NCET-10-0727), and the Natural Science Foundation for Distinguished Young Scholars of Zhejiang province, China (No. R3110085).

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To cite this paper: Ma, W.Q., J. Wu, Z. Zhuo, H. Sun, M. Yue and J. Fe, 2013. Comparison of absorption characteristics of iron glycine chelate and ferrous sulfate in Caco-2 cells. Int. J. Agric. Biol., 15: 372-376
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Author:Ma, Wen-Qiang; Wu, Jing; Zhuo, Zhao; Sun, Hong; Yue, Min; Feng, Jie
Publication:International Journal of Agriculture and Biology
Article Type:Report
Geographic Code:9CHIN
Date:Apr 30, 2013
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