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Poult Sci 2008. 87:2596-2604. doi:10.3382/ps.2008-00117
© 2008 Poultry Science Association
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METABOLISM AND NUTRITION

Kinetics of Manganese Absorption in Ligated Small Intestinal Segments of Broilers1

S. P. Bai*,{dagger},{ddagger}, L. Lu*,{dagger}, X. G. Luo*,{dagger},2 and B. Liu*,{dagger}

* Mineral Nutrition Research Division, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, P. R. China; {dagger} State Key Laboratory of Animal Nutrition, Beijing 100193, P. R. China; and {ddagger} Institute of Animal Nutrition, Sichuan Agricultural University, Yaan 625014, P. R. China

2 Corresponding author: wlysz{at}263.net


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Two experiments were conducted with 28-day-old male commercial broilers to study mechanisms of Mn absorption and the effect of Mn treatment on divalent metal transporter 1 (DMT1) mRNA levels in ligated segments from different intestinal regions of broilers. The results from experiment 1 showed that the amount of Mn absorption was asymptotic with respect to time within 40 min after perfusion of the duodenal, jejunal, and ileal segments of broilers with 2.18 mmol/L of Mn as MnSO4. In experiment 2, a kinetic study of Mn absorption was performed with duodenal, jejunal, and ileal loops perfused with solutions containing 0, 0.13, 0.27, 0.54, 1.09, 2.18, 4.37, or 8.74 mmol/L of Mn as MnSO4. Manganese concentrations in perfusates were determined at 5 min after perfusion. In the control group and in the group treated with 2.18 mmol/L Mn as MnSO4, DMT1 mRNA levels of ligated intestinal regions at 30 min after perfusion were analyzed by real-time reverse transcription PCR. The kinetic curves of Mn absorption showed that Mn absorption was a carrier-mediated process in the duodenum and jejunum. The maximum absorption rate (Jmax) in duodenal segments was greater (P < 0.05) than that in the jejunum (94.08 vs. 81.17 nmol/cm per min). There was no significant difference (P = 0.85) in the Michaelis-Menten constant (Km) values between the duodenum and jejunum (3.41 vs. 3.60 mmol/L). In the ileum of Mn-deficient broilers, the most probable mechanism of Mn absorption was a nonsaturable diffusion process, and the diffusive constant (P; means ± SE) was 2.42 x 10–2 ± 5.22 x 10–4 cm2/min. The DMT1 mRNA levels in the duodenum and jejunum of broilers were greater (P < 0.001) than the level in the ileum. The DMT1 mRNA level in the small intestine of broilers in the Mn treatment group decreased significantly (P < 0.001) compared with that of the control. The different mechanisms of Mn absorption found in different intestinal segments suggest that the ileum is the main site of Mn absorption in the small intestine of broilers.

Key Words: manganese absorption • divalent metal transporter 1 • small intestinal loop • broiler


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The physiological and nutritional functions of Mn have been investigated intensively in animals (Schroeder et al., 1966; Hurley, 1981; Grieger, 1999; Keen et al., 1999). However, there have been few observations (which have not been in agreement) on Mn absorption in the small intestine of animals. Thomson et al. (1971), who studied Mn absorption by using whole-animal models, concluded that diffusion was a major portion of the absorptive process, although other mechanisms were also at work. Similar findings were reported by Yu et al. (1994). In contrast, Testolin et al. (1993), using isolated perfused rat intestine, found that Mn absorption was a carrier-mediated saturable process. The same result was reported by Garcia-Aranda et al. (1983), who used in situ loops of rat intestine. Therefore, Mn absorption may be the type of function that is a nonsaturable diffusive process, a saturable process, or a mediated component of the above 2 processes in the small intestine of animals. Manganese deficiency is often characterized as a species-specific problem. Thus, poultry are known to have relatively high requirements for Mn in comparison with mammals. It is generally accepted that chicks absorb Mn less efficiently than do mammals (Settle et al., 1968). The Mn concentration and components in the broilers’ diets were found to influence Mn absorption from the gut (Underwood, 1981; Halpin et al., 1986). Research in our laboratory using everted intestinal sacs (Ji et al., 2006a) and ligated loops (Ji et al., 2006b) has shown that the ileum is the main intestinal absorption site in the small intestine of Mn-deficient birds. However, the mechanisms of Mn absorption in different intestinal regions of broilers are not known.

Recently, a great deal of research interest has focused on the cellular mechanism involved in Mn transport. The divalent metal transporter 1 (DMT1; also termed the divalent cation transporter 1 or natural resistance-associated macrophage protein-2) is a protein recently shown to play a pivotal role in Mn uptake in the small intestine (Gunshin et al., 1997; Roth et al., 2002; Roth and Garrick, 2003). Solubilized Mn released from the stomach into the duodenum is transported across the microvilli into enterocytes via the DMT1 in mammals (Canonne-Hergaux et al., 1999; Trinder et al., 2000; Knopfel et al., 2005). Overexpression of DMT1 in cultured cells results in an increase in Mn transport (Forbes and Gros, 2003), and incubation with anti-DMT1 antibody blocks Mn uptake (Conrad et al., 2000). Those findings are strongly supported by studies of the Belgrade (b) rat, which possesses a point mutation in DMT1 (G185R) that impairs Mn metabolism (Chua and Morgan, 1997).

The objective of the present study was to research the mechanisms of Mn absorption by investigating the kinetics of Mn absorption in ligated segments from different intestinal regions of the broilers. We hypothesized that the different mechanisms of Mn absorption in different intestinal regions produced our previous finding that the ileum was the main site of Mn absorption in the small intestine of broilers (Ji et al., 2006a,b). To investigate the potential role of DMT1 in intestinal Mn transport, the expression profile of DMT1 and the effect of Mn treatment on DMT1 mRNA level were evaluated in different intestinal regions of broilers.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Birds, Diets, and Treatments

The present study consisted of 2 independent experiments. Male Arbor Acres broilers (1 d old; Arbor Acres Poultry Breeding Co., Beijing, China) were housed in electrically heated and thermostatically controlled cages with fiberglass feeders and a 24-h constant-light schedule. The birds were allowed ad libitum access to the diet and tap water containing 95.90 µg of Ca/mL, 40.73 µg of Mg/mL, 0 µg of Cu/mL, 0 µg of Fe/mL, 0 µg of Mn/mL, and 1.3 µg of Zn/mL. Before the age of 22 d, the birds received an Mn-supplemented corn-soybean meal diet (Table 1Go) formulated to meet or exceed all nutrient requirements of broilers recommended by the NRC (1994). The level of Mn in the diet was 116.80 mg/kg by analysis. To study the mechanisms of Mn absorption in different intestinal regions of Mn-deficient broilers, all birds were fed a basal diet containing 18.40 mg of Mn/kg (Table 1Go) for 1 wk from d 22, during which Mn stores in the body were depleted (Ji et al., 2006a,b).


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  		Table 1. Composition of the broilers diets in the experiments
 
Experiment 1 was conducted to determine the response in Mn absorption with time after perfusion of ligated segments from different intestinal regions of the birds. Twelve male Arbor Acres broilers at the age of 28 d, weighing an average of 1,072 g after an overnight fast (12 h), were allotted randomly to 1 of 6 replicate cages (2 chicks per replicate cage). In each replicate, the intestinal loops of one broiler were perfused with saline solution without addition of Mn to deduct the endogenous Mn, and the intestinal loops of another were perfused with a solution containing 2.18 mmol of Mn/L (120 mg of Mn/L; Luo et al., 1991) as MnSO4. The duodenum, jejunum, and ileum of the small intestine of each bird were used as one replicate of the ligated loops of the corresponding intestinal segments. Time 0 of each perfusion was the time when the intestinal perfusate was injected into the intestinal loops. The perfusate (0.2 mL) was collected at 5, 15, 30, and 40 min after injection (Ji et al., 2006b).

Experiment 2 was carried out to investigate the response of Mn absorption to different Mn concentrations and the effect of Mn treatment on the DMT1 mRNA expression in ligated loops of different small intestinal segments of birds. Eighty male Arbor Acres broilers at the age of 28 d, weighing an average of 1,121 g after an overnight fast (12 h), were allotted randomly to 1 of 10 replicate cages (8 birds per replicate cage). One bird per cage was allocated to 1 of 8 treatments. The 8 treatments of different Mn concentrations in perfusates were 0, 0.13, 0.27, 0.54, 1.09, 2.18, 4.37, and 8.74 mmol/L of Mn as MnSO4 respectively. The duodenum, jejunum, and ileum of each bird were used as one replicate of the ligated loops of the corresponding intestinal segments. Manganese concentration of perfusates in ligated loops perfused with Mn-free solution after dosing were too low to be detected in experiment 1; therefore, there was no need to deduct the endogenous Mn from intestinal secretions in this experiment. Because Mn absorption increased linearly within 5 min after perfusion, standard conditions of the sample collection (Ji et al., 2006b) at 5 min after perfusion were adopted to study the kinetics of Mn absorption compared with Mn concentration. All the mucosal samples of ligated segments from different intestinal regions of broilers in the control group (perfused with Mn-free solution) and the group treated with 2.18 mmol/L of MnSO4 were collected at 30 min after perfusion. The ligated segments from different intestinal regions were excised and flushed with ice-cold saline. Mucosal samples were scraped and immediately frozen in liquid N and stored at –70°C until analysis.

Constituents of the Intestinal Perfusate

To avoid the effect of the other contents in the diet on Mn absorption, a simple saline solution was used as the intestinal perfusate (Ji et al., 2006a,b). Phenol red (20 mg/L) was used as a nonabsorbable indicator. Because the pH values of chymes in the duodenum, jejunum, and ileum of 28-d-old broilers were measured as 6.0, 6.0, and 7.0, respectively (Zhang, 2002), the perfusates injected into the duodenal and jejunal loops were buffered with 15.5 mmol/L of morpholinoethanesulfonic acid, and the perfusates injected into the ileal loops were buffered with 15.5 mmol/L of Tris(hydroxymethyl) aminomethane based on the pH values indicated above. A recent study from our laboratory had confirmed that the morpholinoethanesulfonic acid and Tris(hydroxymethyl) aminomethane had no effect on Mn absorption in the ligated segments from different intestinal regions of broilers under our experimental conditions (Ji et al., 2006b). Inorganic divalent Mn as MnSO4 was added to the saline medium to obtain the desired Mn concentrations. All chemicals used were of reagent grade, except for morpholinoethanesufonic acid and Tris(hydroxymethyl) aminomethane, which were of biochemical grade (Beijing Jingke Chemical Reagent Co., Beijing, China).

Perfusion Protocol

The fasted birds were anesthetized by intravenous injection of sodium pentobarbital (20 mg/kg of BW), and the small intestine was exposed via a longitudinal abdominal incision. The duodenum was ligated 1 cm distal to the pylorus, the jejunum was ligated just anterior to the remnant of the yolk stem, and the ileum was ligated just anterior to the ileocecal junction (Melvin, 1984; Ji et al., 2006b). Loose ligatures were then placed next to and 12 cm distal to the above ligatures to isolate different regions of the intestine. The isolated segments were rinsed with 30 mL of warm saline to eliminate food residues and debris (Hempe and Cousins, 1989). The distal loose ligatures of duodenal segment, jejunal segment, and ileal segment, respectively, were secured. The remaining loose ligature of each intestinal segment was tightened around a needle-like plastic cannula inserted into the proximal end of the loop, and 3.5 mL of perfusate was injected with a calibrated syringe. After administration of the dose, the cannula was obturated and the proximal ligature was secured. The intestine was returned to the body cavity after perfusion. The anesthetized birds were warmed with infrared lamps to maintain their body temperature.

Calculations of Mn Absorption and Methods of Assays

Manganese absorption was determined by measuring the disappearance of Mn from the perfusion solution containing different concentrations of Mn (Ji et al., 2006a). Manganese concentrations in samples of the diet, the tap water, and the fluid content in loop tissue were determined by inductively coupled Ar plasma spectroscopy (IRIS Intrepid II, Thermo Electron Co., Waltham, MA). A nonabsorbable reference indicator (phenol red) was used to correct the changes in Mn concentrations resulting from water absorption or intestinal secretion (Ji et al., 2006a,b). The concentrations of phenol red in initial and final fluid contents were determined by measuring absorbency at 520, 560, and 600 nm with a ultraviolet-visible spectrophotometer (Model Cary-100, Varian, Palo Alto, CA). The 3-wavelength correction was applied, because phenol red concentration tends to be overestimated in samples if absorbency is measured at 560 nm only (Schedl et al., 1966). Final volumes of perfusates in different intestinal loops were calculated by the change in concentration of the phenol red, as described by Ji et al. (2006a).

DMT1 mRNA Expression Analysis

Divalent metal transporter 1 mRNA levels in mucosal samples of different intestinal segments were analyzed by quantitative real-time reverse transcription-PCR (Tchernitchko et al., 2002). The total RNA was extracted with the use of Trizol reagent (Invitrogen, Carlsbad, CA) per the manufacturer’s instructions. One microgram of the total RNA was reverse-transcribed to cDNA by using SuperScript III Two-Step Reverse Transcript kit (Invitrogen). Divalent metal transporter 1 and β-actin were amplified in the ABI SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) from the cDNA with specific primers. The following quantitative real-time reverse transcription-PCR primers were used: β-actin (GenBank L08165) forward 5'-GAGAAATTGTGCGTGACATCA-3', reverse 5'-CCTGAACCTCTCATTGCCA-3'; DMT1 forward 5'-AGCCGTTCACCACTTATTTCG-3', reverse 5'-GGTCCAAATAGGCGATGCTC-3'. A pair of DMT1 primers amplified the 2 chicken DMT1 isoforms (GenBank EF635922 [GenBank] and EF635923 [GenBank] ; Bai et al., 2008). Amplification was initiated by 2- and 10-min incubations at 50 and 95°C, respectively, followed by 40 cycles at 95°C for 15 s and 60.5°C for 1 min. To quantify DMT1 and β-actin, we used the standard curve method described by Tchernitchko et al. (2002). Relative standard curves were obtained by plotting the cycle thresh-old (Ct) obtained after PCR amplification of serial dilutions of a steady quantity of a plasmid containing the corresponding cDNA. Because the expression levels of β-actin mRNA were identical in the different intestinal regions of the chicken, β-actin was used as the endogenous control (Mete et al., 2005). The PCR products of DMT1 were normalized to the β-actin level and were then amplified 10 times.

Kinetic and Statistical Analysis

The kinetic analysis of Mn absorption compared with Mn concentrations in small intestinal loops was carried out by fitting the following equations according to the method of Condomina et al. (2002): nonsaturable diffusion equation [1], saturable process equation [2], or the sum of both equations (a saturable process and a nonsaturable diffusion, equation [3]).


Formula


Formula


Formula

where JMn and its maximum rate of Mn absorption, Jmax, are given in nmol/min per cm; Km is the Michaelis-Menten constant in mmol/L; P is the diffusivity constant in cm2/min; and A is the concentration of Mn in perfusate in mmol/L.

The fits of equations to experimental data were performed by using a nonlinear least squares regression program (SigmaPlot v. 4.0, Jandel Scientific, Erkratch, Germany). To select the best kinetic model of Mn absorption in our experimental conditions, the Akaike information criterion (AIC) was used (Gagne and Dayton, 2002). The model with the smallest AIC was deemed the best model because it minimized the difference from the given model to the "true" model.

Data from 2 experiments were subjected to 2-way ANOVA by using PROC GLM of SAS (SAS Institute, 2003). The model in experiment 1 included time, region, and their interaction. The model in experiment 2 included Mn concentration, region, and their interaction. A logarithmic transformation was applied to the data on Mn absorption percentages before statistical analysis. The effect of postperfusion time on Mn absorption was analyzed by nonlinear least squares regression analysis (Condomina et al., 2002). The differences in the kinetic parameters obtained for ligated segments from different intestinal regions were analyzed by a t-test procedure (SAS Institute, 2003). Significance was set at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Experiment 1: Changes of Mn Absorption with Time in Different Small Intestinal Segments of Broilers After Perfusion

Manganese absorption at varying times after perfusion was expressed as a percentage of the amount of Mn perfused in ligated segments from different intestinal regions. The Mn concentrations in fluid contents of ligated loops dosed with the Mn-free solution were too low to be detected at any time; therefore, there was no need to deduct the endogenous Mn from intestinal secretions in the experiments. The absolute amount of Mn absorbed increased with time in the ligated duodenum, jejunum, and ileum injected with the solution containing 2.18 mmol/L of Mn, whereas the rate of Mn absorption decreased (Figure 1Go). However, there was no significant difference (P = 0.63) in the amount of Mn absorption between 30 and 40 min after perfusion in ligated segments from different intestinal regions of broilers. The Mn absorption in ligated ileal loops was greater (P < 0.01) than that in duodenal and jejunal loops of broilers. There were no significant differences (P > 0.05) between the duodenal loops and jejunal loops (Figure 1Go). Nonlinear least squares regression analysis showed that there was an asymptotic time-dependent increase in Mn absorption in the ligated duodenum (r = 0.95, P < 0.001), jejunum (r = 0.89, P < 0.001), and ileum (r = 0.92, P < 0.001; Figure 1Go). The maximum Mn absorption percentages in the ligated duodenum, jejunum, and ileum, as extrapolated by the nonlinear regression, were 5.73, 6.73, and 74.30%, respectively. The results showed that the disappearance of Mn from the intestinal lumen could be considered as a first-order process within 5 min after perfusion. For this reason, standard conditions of the sample collection at 5 min after perfusion were adopted in experiment 2 to study the kinetics of Mn absorption compared with Mn concentration in ligated intestinal segments.


Figure 1
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  		Figure 1. Changes in Mn absorption with postperfusion time in ligated duodenal, jejunal, and ileal loops of broilers perfused with a simple solution containing 2.18 mmol/L of Mn as MnSO4. Manganese absorption was analyzed at 5, 15, 30, and 40 min after perfusion and expressed as a percentage of the amount of manganese perfused in the corresponding intestinal loops. The values are means for 6 replicate intestinal loops, with SE represented by vertical bars. The continuous lines are nonlinear regression lines. The equations in the duodenum, jejunum, and ileum were Y = 5.73%[1 – exp –(0.0554 x t)] (r = 0.95, P < 0.001), Y = 6.73%[1 – exp –(0.0528 x t)] (r = 0.89, P < 0.001), and Y = 74.30%[1 – exp –(0.114 x t)] (r = 0.92, P < 0.001), respectively, where Y represents the percentage of the amount of Mn perfused in ligated segments from different intestinal regions, and t is the postperfusion time.

 
Experiment 2: Kinetics of Mn Absorption Compared with Mn Concentrations in Different Small Intestinal Segments of Broilers

The kinetic analysis of Mn absorption rates in ligated intestinal loops was carried out by graphic means (Figure 2Go). The regression analysis showed that in the ligated duodenum and jejunum, the best fits were for the saturable process equation (AIC = 3.95 and 3.72, respectively), whereas in the ligated ileum, the fit to the nonsaturable process (AIC = 4.06) gave the best result (Table 2Go and Figure 2Go). Those findings suggested that Mn absorption was a carrier-mediated process in the broiler duodenum and jejunum, in contrast to the ileum, where it could be described as a nonsaturable diffusion process. The maximum absorption rate (Jmax; mean ± SE) in the duodenal loops (94.08 ± 9.54 nmol/ cm per min) was greater (P < 0.05) than that in the jejunum (81.17 ± 7.75 nmol/cm per min). There was no significant difference (P = 0.85) with the Michaelis-Menten constant (Km) values (means ± SE) between the duodenum (3.41 ± 0.77 mmol/L) and jejunum (3.60 ± 0.75 mmol/L). In the ileum of Mn-deficient broilers, the diffusive constant (P; mean ± SE) of the perfusion process of Mn absorption was 2.42 x 10–2 ± 1.43 x 10–4 cm2/min. The slopes of kinetics curves, indicating the relative efficiencies of Mn absorption, decreased with the increase in Mn concentration in ligated duodenal and jejunal loops, whereas they remained steady in the ligated ileum of broilers.


Figure 2
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  		Figure 2. Kinetic curves of Mn absorption in ligated segments from different intestinal regions of birds evaluated. A 3.5-mL solution, containing 0 to 8.74 mmol/L of Mn as MnSO4, was injected in the ligated segments from different intestinal regions of the broilers. Samples of the perfusates in different intestinal loops were collected at 5 min after injection, and their Mn concentrations were examined by inductively coupled Ar plasma spectroscopy after correcting the volumes (see the Materials and Methods section). The kinetic curves of Mn absorption in the duodenum and jejunum are described by equation [2] (Michaelis-Menten, a saturable process), and the kinetic curve in the ileum is described by equation [1] (a nonsaturable diffusion).

 

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  		Table 2. Kinetic and statistical parameters obtained after fitting Michaelis-Menten equations and nonsaturable diffusion equations to experimental data from different small intestinal segments1
 
Experiment 2: Effect of Mn Treatment on the Expression of DMT1 mRNA in Different Small Intestinal Segments of Broilers

To investigate the potential role of the DMT1, the DMT1 mRNA (GenBank EF635922 [GenBank] and EF635923 [GenBank] ) levels of the duodenal, jejunal, and ileal loops of broilers were determined in the control group (injection with Mn-free solution) and in the group treated with 2.18 mmol/L of MnSO4. Because the amount of Mn absorption was more than 80% of the maximum amount of Mn absorption in ligated segments from different intestinal regions at 30 min after perfusion, this time point was selected to study the changes in DMT1 mRNA levels of ligated intestinal loops. The DMT1 mRNA levels in the duodenum and jejunum were greater (P < 0.001) than that in the ileum of broilers; however, there was no difference (P = 0.26) in DMT1 mRNA expression between the duodenum and jejunum. The DMT1 mRNA levels in the duodenum and jejunum were, respectively, 3.3- and 2.9-fold that in the ileum (Figure 3Go). The DMT1 mRNA levels of ligated intestinal loops in the Mn treatment group decreased significantly (P < 0.001) in comparison with the control. The means of DMT1 mRNA levels were decreased by 44, 55, and 32% in the duodenum, jejunum, and ileum, respectively.


Figure 3
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  		Figure 3. Manganese treatment decreased divalent metal transporter 1 (DMT1) mRNA levels of the ligated segments from different intestinal regions of broilers, in comparison with the control treatment (perfused with the Mn-free solution). The ligated duodenal, jejunual, and ileal loops were perfused with solutions containing either 0 or 2.18 mmol/L of Mn as MnSO4. The mucosal samples of the ligated segments from different intestinal regions were collected at 30 min after perfusion. Total RNA of the mucosal samples was harvested and the DMT1 mRNA level was determined by quantitative real-time reverse transcription-PCR. The DMT1 mRNA level in each tissue was normalized by expression of the β-actin mRNA level and was then amplified 10 times. The black bars represent the means of the DMT1 mRNA levels of the control group, and the lighter gray bars represent the means of DMT1 mRNA levels of the Mn treatment group. The SE are represented by vertical error bars. The DMT1 mRNA levels of ligated segments from different intestinal regions in the Mn treatment group were significantly lower (P < 0.001) than those in the control group. The DMT1 mRNA levels in the duodenum and jejunum were greater (P < 0.001) than that in the ileum of the broilers.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The plots of Mn absorption percentage against the time after perfusion showed that there was an asymptotic time-dependent increase in Mn absorption in ligated segments from different intestinal regions, whereas the rate of Mn absorption decreased regardless of different intestinal segments. Those results are in agreement with the findings of Mn absorption and absorption of other mineral elements in mammals. Manganese absorption declined with time in the intestine of rat when an in vivo perfusion system was used (Thomson et al., 1971). The rate of intestinal Zn transport was also decreased with respect to time (up to 30 min) when using the brush border membrane from the piglet (Blakeborough, 1987). The finding that Mn absorption was greater in the ileum than in the duodenum and jejunum at varying times after perfusion in the present study is consistent with the previous results in our laboratory, which also suggested that the ileum is the main site of Mn absorption in the small intestine of broilers (Ji et al., 2006a,b). Manganese absorption in different intestinal loops of broilers involved rapid initial uptake and reached the equilibrium within 30 to 40 min after perfusion. The maximum Mn absorption percentages in the duodenal, jejunal, and ileal loops of the broilers (5.73, 6.73, and 74.3%, respectively), especially in the ileum, which, extrapolated by the nonlinear regression, were greater than the Mn absorption percentage (2.62%) in intact birds when radiotracer techniques were used (Wedekind et al., 1991). In our study, broilers were fed a corn-soybean basal diet unsupplemented with Mn for 1 wk, during which Mn stores in the body were depleted. However, Wedekind et al. (1991) used broilers fed a Mn-supplemented corn-soybean basal diet to investigate the amount of Mn absorption in the small intestine. Many components affecting the Mn absorption in the chyme, such as fiber, P, and other mineral elements, might explain the difference in the 2 experimental results (Underwood, 1981; Halpin et al., 1986).

Our results for the best kinetic models describing the Mn absorption mechanisms in the duodenum, jejunum, and ileum of broilers suggested that Mn absorption was a carrier-mediated process in the duodenum and jejunum but was a nonsaturable diffusion process in the ileum. In comparison with previous reports in mammals, however, the present results revealed some conflicting data on the Mn absorption process. Davidsson et al. (1991), who used human subjects to study Mn absorption from human milk, concluded that Mn absorption was a diffusion process, which was in agreement with a study of the rat intestine (Sahagian et al., 1967). In contrast, Thomson et al. (1971) and Yu et al. (1994), who studied Mn absorption by using animal models, concluded that diffusion was a major portion of the absorption process, although other mechanisms were also at work. None of the studies using whole animals as models clarified the role of the different intestinal segments. Thomson and Valberg (1972), using open-ended duodenal loops of the rat, found that Mn absorption displayed a saturable kinetics process in the different small intestinal segments. This result was consistent with the present report. Manganese transport in the proximal intestinal lumen of Fe-deficient rats has characteristics of a carrier-mediated process, but has characteristics of a diffusion process in the proximal intestine of Fe-overload rats (Thomson et al., 1971). Garcia-Aranda et al. (1983), using an in vivo perfusion system, suggested that Mn absorption in the rat ileum included both diffusive and saturable components. Leblondel and Allain (1999), who studied the uptake kinetics of Mn from the apical to the basolateral by using an intestinal Caco-2 cell line, showed saturable-type kinetics. The experimental models selected, the techniques used, the lengths of the experimental periods, and the different physiological conditions of animals in these studies might explain the discrepancies among the research reports. In situ-ligated intestinal loops had absorptive epithelial cells with an intact orientation, and the intestinal loops did not impair the functions of the circulatory and lymphatic systems. Therefore, this model was the most appropriate to study the different mechanisms of Mn absorption in ligated segments from different intestinal regions of the broilers.

The different mechanisms of Mn absorption between the duodenum, jejunum, and ileum have possibly provided insight into our previous findings, in which the main absorption site of Mn was the ileum in the small intestine of Mn-deficient broilers (Ji et al., 2006a,b). The Km values of Mn absorption kinetics in the duodenum and jejunum were lower than that in the proximal intestine of rats (4.50 mmol/L) when ligated intestinal loops were used, and the values of Jmax in the duodenum and jejunum of broilers were also lower than that of rats (Thomson et al., 1971; Garcia-Aranda et al., 1983). This finding was consistent with the report that chicks absorbed Mn less efficiently than did mammals (Settle et al., 1968). The diffusive constant of Mn absorption kinetics in the ileum of male broilers was greater than that in the rat ileum, which suggests that the ileum of birds has more permeability for Mn than does the ileum of rats (Thomson et al., 1971). To account for the passive diffusion of charged hydrophilic species through the lipid bilayers of plasma membranes, numerous investigators have focused extensively on the existence of aqueous channels or pores that penetrate the plasma membrane (Inouye, 1974; Wright and Pietras, 1974; Stein, 1986). The ionic Mn could diffuse through the channels for Ca2+ in the plasma membrane of the cells (Milanick and Frame, 1991; Fasolato et al., 1993). Alternatively, tight junctions acting as paracellular shunts have been shown to be the main route of passive ion permeation in the rabbit gall bladder epithelium, which has much in common with the intestinal epithelium (Fromter and Diamond, 1972; Oschman, 1980). Thus, in the passive diffusion of inorganic Mn through the ileum of broilers, it is reasonable to speculate that the metal might pass either intracellularly through aqueous channels in the plasma membranes or paracellularly through tight junctions.

Divalent metal transporter 1 is a protein recently shown to play a pivotal role in Mn transport in the small intestine of animals (Trinder et al., 2000). Divalent metal transporter 1 is responsible for solubilized Mn in the intestinal lumen crossing the microvilli into enterocytes (Trinder et al., 2000). Overexpression of DMT1 in cultured cells resulted in an increase in Mn transport (Forbes and Gros, 2003), and incubation with anti-DMT1 antibody blocked uptake of Mn (Conrad et al., 2000). In this study, the levels of DMT1 mRNA expression in the duodenum and jejunum were greater than that in the ileum, which suggested the DMT1 might play an important role in the carrier-mediated process of Mn transport in the duodenum and jejunum, whereas it might play a limited role in the ileum of Mn-deficient birds. The expression profile of DMT1 in the small intestine of broilers did not entirely agree with previous findings. Using 4 mynah and 4 chickens (strains not shown) fed a normal diet, Mete et al. (2005) studied the DMT1 expression profile in the small intestine and found that DMT1 expression was the greatest in the jejunum of 2 kinds of birds, whereas there was no difference in DMT1 expression between the duodenum and ileum in the mynah. In mammals, DMT1 mRNA expression was the greatest in the duodenum and was decreased toward the colon (Gunshin et al., 1997; Hubert and Hentze, 2002). The animal species and strains used, the positions of sampling, and the animal physiological conditions, especially the Mn2+ stores in the body, might explain the differences in the above results.

Efficiency of Mn absorption declined with an increase in Mn concentration in the diets of chicks (Settle et al., 1968). Divalent metal transporter 1 is responsible for solubilized Mn in the intestinal lumen crossing the microvilli into enterocytes (Trinder et al., 2000). Therefore, the change in DMT1 mRNA level in ligated intestinal loops treated with 2.18 mmol/L of Mn, in contrast to the control group, was studied to clarify the potential role of DMT1 in Mn transport in the small intestine of broilers. Our observation of the decreases in DMT1 mRNA levels in the intestinal loops when comparing the Mn and the control treatments might be the first report of direct Mn regulation of the DMT1 mRNA level in the small intestine of broilers. These results were identical to the finding in bacteria. The mRNA level of Mn transporter homolog, a homologous gene of DMT1, was decreased by the high Mn2+ condition and Mn transporter-responsive element functioned as an Mn-dependent repressor in the process (Que and Helmann, 2000). The present decrease in the DMT1 mRNA level after Mn treatment seems to be a response of the expression of DMT1 mRNA after the Fe treatment in mammals. Iron treatment decreased the DMT1 mRNA level through the Fe-responsive element (IRE)-mediated downregulation mechanism (Tchernitchko et al., 2002). The stability of DMT1 mRNA is affected through the Fe-dependent dissociation of an Fe regulatory protein from the IRE motif found in the 3' untranslated region of DMT1 mRNA (Tchernitchko et al., 2002). The Fe concentrations in the diet and the perfusate were the same for all birds in present study, so the decrease in the DMT1 mRNA level was not the reason for the Fe concentration. Those observations could reflect the view that there might be an Mn-dependent protein in the enterocytes of the broilers to regulate the expression of DMT1; however, further study will be needed to elucidate the mechanisms resulting in the lower DMT1 mRNA levels caused by the Mn treatment. Nevertheless, it is tempting to speculate that the Mn concentration affects Mn absorption in the small intestine of broilers by regulating the DMT1 mRNA level.

The present study clearly demonstrated that Mn absorption showed a saturable carrier-mediated process in the duodenum and jejunum, whereas a nonsaturable diffusion process was present in the ileum. The DMT1 mRNA levels in the ligated small intestinal segments suggested that DMT1 plays an important role in Mn absorption in the duodenum and jejunum but a limited role in the ileum in Mn-deficient broilers. The Mn treatment decreased mRNA levels of DMT1 in different ligated small intestinal segments, and this appeared to indicate that Mn concentration regulated DMT1 mRNA expression in the small intestine of the broilers.


   FOOTNOTES
 
1 Supported by the National Basic Research Program of China (project no. 2004CB117501; Beijing, P. R. China), Basic Science Research Program (project no. ywf-td-4; Beijing, P. R. China), the Key Program of the National Natural Science Foundation of China (project no. 30530570; Beijing, P. R. China), and the Research Program of the Key Laboratory of Animal Nutrition (project no. 2004DA125184G0812; Beijing, P. R. China). Back

Received for publication March 18, 2008. Accepted for publication July 14, 2008.


   REFERENCES
TOP
 ABSTRACT
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 MATERIALS AND METHODS
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