Kinetics of Zinc Absorption by In Situ Ligated Intestinal Loops of Broilers Involved in Zinc Transporters1

Kinetics of Zinc Absorption by In Situ Ligated Intestinal Loops of Broilers Involved in Zinc Transporters1 Y. Yu,*† L. Lu,*† X. G. Luo,*†2 and B. Liu*† *Mineral Nutrition Research Division, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100094, China; and †State Key Laboratory of Animal Nutrition, Beijing 100094, China value was higher in the duodenum than in the jejunum (1.44 ± 0.33 vs. 0.51 ± 0.17 mM, P = 0.012). Similarly, the maximum absorption velocity (Jmax) value was greater in the duodenum than in the jejunum (5.32 ± 1.46 vs. 2.57 ± 0.39 nmol/min per cm, P = 0.069), whereas absorption in the ileum occurred with a nonsaturable diffusion process and had a diffusive constant (P) of 5.72 × 10−3 ± 1.1 × 10−4 cm2/min. Moreover, the mRNA levels of metallothionein, zinc transporter 1, and Zn transporter 5 were lower in the ileum than in the duodenum or jejunum in the Zn-supplemented group, further indicating that Zn absorption in the ileum occurred mainly by a nonsaturable diffusive pathway. The Zn fluxes were significantly higher (P < 0.005) in the ileum than in the other 2 segments at different Zn concentrations. This research suggests that the ileum is the main site of Zn absorption and that the mechanism involved is nonsaturable diffusion, which is different from that in the other 2 segments, depending on the regulation of Zn transporter expression.

Key words: kinetics, zinc absorption, zinc transporter, small intestine, broiler chicken 2008 Poultry Science 87:1146–1155 doi:10.3382/ps.2007-00430

INTRODUCTION Zinc is an essential trace element involved in many physiological functions because of the role it plays in numerous metalloenzyme systems (Vallee and Falchuk, 1993; Gaither and Eide, 2001). For good health, there is an elaborated homeostatic regulation in absorption, storage, and secretion of Zn within the whole body (Krebs, 2000). Central to maintaining this homeostasis are the processes of Zn absorption and secretion in the small intestine (SI; King et al., 2000). Zinc intestinal absorption had been

©2008 Poultry Science Association Inc. Received October 19, 2007. Accepted February 23, 2008. 1 Supported by the National Basic Research Program of China, Beijing, P.R. China (project no. 2004CB117501), Basic Science Research Program, Chinese Academy of Agricultural Sciences, Beijing, China (project no. ywf-td-4), and the Chinese Academy of Agricultural Sciences Foundation for First-Place Outstanding Scientists, Beijing, P.R. China. 2 Corresponding author: [email protected]

studied extensively in in vitro or in vivo systems (Seal and Heaton, 1983; Hempe and Cousins, 1992; Reeves et al., 2001), but the mechanisms involved are not fully understood. Accumulating evidence has suggested that Zn intestinal absorption in rats (Steel and Cousins, 1985; Oestreicher and Cousins, 1989), pigs (Tacnet et al., 1990), or Caco-2 cells (Raffaniello et al., 1992; Finley et al., 1995) is a saturable, carrier-mediated process combined with a nonsaturable, diffusive mechanism, although the intestines or the cells being detected were not specifically separated into 3 segments in these research reports. Condomina et al. (2002) studied Zn absorption in 3 intestinal segments of rats, but the results of kinetic absorption, which indicated that Zn intestinal absorption was a saturable, carrier-mediated process, were inconsistent with those obtained previously. Moreover, no reports are available on the kinetics of Zn absorption and the major site involved in the transport process in chicken intestines. The Zn stores in avians easily become deficient because the dietary requirement of Zn for avians is increasing with the extremely rapid growth rate or production rate

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ABSTRACT Two experiments were conducted to study the kinetics of Zn absorption and Zn transporter mRNA levels in the duodenum, jejunum, and ileum of broilers to investigate the main site of Zn absorption in the small intestine, the absorption mechanisms, and whether those transporters were involved. In experiment 1, we compared Zn absorption in 3 segments and at different postperfusion time points, and found that Zn absorption increased linearly within 30 min and was higher (P < 0.05) in the ileum than in the other 2 segments. In experiment 2, intestinal loops were perfused with solutions containing 0, 0.077, 0.154, 0.308, 0.616, 1.232, or 2.464 mmol/ L of ZnSO4ⴢ7H2O, and Zn concentrations in perfusates were determined at 30 min after perfusion. The mRNA levels of transporters in 3 intestinal loops from the control group and the 0.616 mmol of Zn/L group were analyzed. The kinetic curves showed that Zn transported to the duodenum and jejunum depended on a saturable carriermediated process. The Michaelis-Menten constant (Km)

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MATERIALS AND METHODS Birds, Diets, and Experimental Design This study was approved by the Animal Research Center at the Veterinarian Office of Beijing. Arbor Acres commercial male broilers (Huadu Broiler Breeding Corp., Beijing, China) were used in 2 experiments. Birds were randomly housed in stainless-steel suspended cages with fiberglass feeders and plastic waterers, and were fed a corn-soybean meal basal diet (90.49 mg of Zn/kg of diet; Table 1) from d 1 to 21 but a semipurified diet (12.58 mg of Zn/kg of diet; Table 1) after d 21 to deplete the body Zn stores. All other nutrients in the 2 diets met or exceeded the NRC (1994) requirements for broilers. The birds were allowed ad libitum access to feed and to tap water containing undetectable Zn before d 21, but had access to deionized water after d 21. Chicks were managed following the guidelines suggested by Arbor Acres Farm in Beijing. At 28 d of age, after an overnight fast, 80 birds were weighed (1,062 g, means) and allotted randomly to 8 replicate groups of 2 chicks for 5 treatments in experiment 1, or 70 birds were weighed (1,008 g, means) and allotted randomly to 10 replicate groups of 1 chick

Table 1. Composition of two diets for 1- to 28-d-old broilers in 2 experiments Amount (%) Item Ingredient Ground yellow corn Soybean meal Fish meal DL-Methionine Corn starch Casein Cellulose Soybean oil Calcium carbonate Calcium hydrogen phosphate Sodium chloride Micronutrients Nutrient composition ME (MJ/kg) CP3 (%) Lysine (%) Methionine (%) Methionine + cysteine (%) Calcium3 (%) Nonphytate phosphorus (%) Zinc3 (mg/kg)

Basal diet (d 1 to 21)

Semipurified diet (d 22 to 28)

54.65 34.82 3.50 0.18

3.60 1.26 1.30 0.30 0.391 12.58 21.66 1.30 0.57 0.95 1.04 0.45 90.49

66.00 23.00 5.01 1.50 1.50 1.12 0.30 1.572 13.22 19.33 1.69 0.62 0.72 0.90 0.40 12.58

1 Provided (per kilogram of diet): vitamin A (all-trans retinol acetate), 13,500 IU; vitamin D3 (cholecalciferol), 3,600 IU; vitamin E (all-rac-αtocopherol acetate), 33 IU; vitamin K (menadione Na bisulfate), 6 mg; thiamine (thiamine mononitrate), 4.5 mg; riboflavin, 10.5 mg; vitamin B6, 6 mg; vitamin B12, 0.03 mg; calcium pantothenate, 18 mg; niacin, 60 mg; folic acid, 1.8 mg; biotin, 0.165 mg; choline (choline chloride), 700 mg; Cu (CuSO4ⴢ5H2O), 8 mg; Zn (ZnSO4ⴢ7H2O), 60 mg; Mn (MnSO4ⴢH2O), 100 mg; Fe (FeSO4ⴢ7H2O), 80 mg; I (KI), 0.35 mg; Se (Na2SeO3), 0.15 mg. 2 Provided per kilogram of diet: vitamin A (all-trans retinol acetate), 13,500 IU; vitamin D3 (cholecalciferol), 3,600 IU; vitamin E (all-rac-αtocopherol acetate), 33 IU; vitamin K (menadione Na bisulfate), 6 mg; thiamine (thiamine mononitrate), 4.5 mg; riboflavin, 10.5 mg; vitamin B6, 6 mg; vitamin B12, 0.03 mg; calcium pantothenate, 18 mg; niacin, 60 mg; folic acid, 1.8 mg; biotin, 0.165 mg; choline (choline chloride), 700 mg; K (KCl), 3,000 mg; Mg (MgSO4ⴢ7H2O), 600 mg; Cu (CuSO4ⴢ5H2O), 8 mg; Fe (FeSO4ⴢ7H2O), 80 mg; Mn (MnSO4ⴢH2O), 100 mg; I (KI), 0.35 mg; Se (Na2SeO3), 0.15 mg. 3 Determined values.

for 7 treatments in experiment 2. The average BW of birds used for each treatment were nearly equal to avoid the effect of weight in the results. Experiment 1 was performed to obtain an optimal sampling time of ligated loops after perfusion according to the change in Zn absorption with time, and to understand the major absorption site in broiler intestines. The 5 sampling time points were 5, 15, 30, 45, and 60 min after perfusion, respectively. To exclude the effect of endogenous Zn, 3 intestinal segments of each bird in each replication were injected with solutions without Zn, and 3 intestinal segments of another bird in each replication were injected with solutions containing 0.616 mmol of Zn/L (40 mg of Zn/L) as ZnSO4ⴢ7H2O. This amount of Zn was chosen because of the Zn requirement for broilers (NRC, 1994). Each intestinal segment of one bird was considered as one replication of the corresponding intestinal segment. Experiment 2 was conducted to elucidate the mechanisms of Zn absorption in 3 intestinal segments of broilers

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of commercial strains, or with Zn deficiency, and many factors limit Zn absorption in feed (Lonnerdal, 2000). Use of the Zn-deficient chicken as experimental model can reflect Zn absorption in the animal intestine sensitively. In recent years, many researchers have focused on the identification, cloning, and characterization of several Zn transporters (ZnT) that might be involved in Zn transport. Zinc transporter 5 has been implicated in the transport of Zn from the intestinal lumen into the enterocytes (Cragg et al., 2005), metallothionein (MT) and ZnT 2 are believed to function in the transport and sequestration of Zn by intracellular vesicles (Palmiter et al., 1996; Davis et al., 1998), and ZnT 1 can regulate the efflux of Zn from the enterocytes into the serosa (Palmiter and Findley, 1995; Liuzzi and Cousins, 2004). Knowledge of these transporters provides a clear understanding of cellular Zn transport, but does not account for Zn transport in an intact gastrointestinal tract. Whether these genes are involved in Zn absorption in the animal intact gut has not been elucidated previously with a kinetic study, which could characterize the different transporters according to kinetic parameters. The purposes of the present study were to investigate the kinetics of Zn absorption to elucidate the mechanisms of absorption, and to identify the major absorption site of Zn in the SI of chickens by using in situ ligated loops. This operation system has been shown to be rapid and useful in predicting the absorptive response in rats and chicks (Hempe and Cousins, 1989; Ji et al., 2006a). Moreover, we examined the mRNA expression of MT, ZnT 1, ZnT 2, and ZnT 5, according to the results of the kinetic study, to test whether these genes were involved in Zn absorption in the SI of chickens and to better understand their mode of action.

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by studying the kinetics of Zn absorption and transcription of ZnT. The 7 Zn supplemental concentrations were 0 (0 mM), 5 (0.077 mM), 10 (0.154 mM), 20 (0.308 mM), 40 (0.616 mM), 80 (1.232 mM), and 160 mg/L (2.464 mM). Each intestinal segment of one bird was considered as one replication of the corresponding intestinal segment. The optimal sampling time observed in experiment 1 was adopted in this experiment.

Preparation of Perfusion Solutions

Ligated Loop Procedure Chickens were fasted overnight and anesthetized by wing venous injection of sumianxin (a complex anesthetic, 0.1 mL/kg of BW). The abdomen was opened by midline incision. The duodenum was incised 1 cm distal to the pyloric sphincter, the jejunum was incised just anterior to the remnant of yolk stem, and the ileum was incised just anterior to the ileocecal junction (Melvin, 1984). Plastic cannulas were inserted into 3 incisions and secured by sutures. Loose ligatures were then placed 12 cm distal to the above-mentioned tight ligatures to separate different intestinal segments. The isolated segments were flushed out with 40 mL of warm saline followed by 20 mL of air, and the loose ligature of each intestinal segment was tightened (Hempe and Cousins, 1989). A syringe without needle was then inserted into the cannula of each intestinal segment, and the 3.5-mL dose of Zn was injected. After administration of the dose, the syringe was removed and the cannula was clamped by hemostatic forceps. The intestine was put back into the abdomen cavity. The anesthetized birds were warmed with infrared lamps to maintain their body temperature and laid on gauze pads wetted with warm saline to maintain their body humidity.

Determination of Zn and Phenol Red Concentrations in Perfusion Solutions In the 2 experiments, 2-mL perfusion solutions were collected with a syringe at each time point and frozen (−20°C) until the concentrations of Zn and phenol red

Quantitative Real-Time PCR Procedure In experiment 2, intestinal segments of chickens from the control and the 0.616 mmol of Zn/L groups were immediately excised and rinsed with ice-cold saline after collecting the perfusion solutions, and the intestinal mucosa was scraped from the underlying submucosa with a sterile glass slide. Samples were then frozen in liquid nitrogen for determination of the mRNA levels of MT, ZnT 1, ZnT 2, and ZnT 5 by real-time PCR. Total RNA was isolated from the intestinal mucosa by using the TRIzol reagent following the manufacturer’s instructions (Invitrogen Life Technologies, Carlsbad, CA). Ribonucleic acid integrity was assessed by agarose gel electrophoresis, and the gel was stained with ethidium bromide. Firststrand cDNA was synthesized from 3 ␮g of total RNA by using the SuperScript III Two-Step Reverse Transcript kit (Invitrogen Life Technologies). The detailed operational process was performed according to the manufacturer’s instructions. Real-time PCR analysis was carried out by using the SYBR Green qPCR detection system and the ABI 7000 HT real-time thermocycler (Applied Biosystems Inc., Foster City, CA). Gene-specific primers were designed by using the software Primer Premier 5.0 and oligo 6.0 (Table 3). Real-time PCR reaction conditions consisted of 50°C for 2 min; 95°C for 10 min; 40 cycles of 95°C for 15 s; 60°C for 1 min; 95°C for 15 s; 60°C for 15 s; and 95°C for 15 s. The reaction system was 15 ␮L (Huang et al., 2007). The identity of the PCR products was confirmed by comparison of the PCR-amplified DNA with the predicted fragment size. All samples were measured in triplicate. A nontemplate control sample was used for each PCR run to check the contaminating genomic DNA and primer-dimer formation. A standard curve was constructed with serial dilutions of cDNA, and the level of each gene mRNA was given as a relative copy number normalized to β-actin mRNA by using the following formulas: (MT/β-actin) and (ZnT 1, ZnT 2, ZnT 5/βactin) × 100.

Statistical Analysis Data from both experiments were analyzed by one-way ANOVA with the GLM procedure of SAS (SAS Institute,

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Because the pH values of chymes in the duodenum, jejunum, and ileum of 28-d-old broilers were previously determined to be 6.0, 6.0, and 7.0, respectively (Zhang, 2002), the solutions injected into the duodenal and jejunal loops were buffered with 15.5 mmol/L of morpholineoethanesulfonic acid, and the solutions injected into the ileal loops were buffered with 15.5 mmol/L of Tris at the pH mentioned above. Inorganic Zn ion as ZnSO4ⴢ7H2O was added to the medium as treatments to obtain the desired Zn concentrations. Phenol red acted as a nonabsorbable marker in the luminal medium; it was used to correct the changes in Zn concentration resulting from water absorption or intestinal secretion. The content of phenol red in perfusion solutions was 20 mg/L (Schedl et al., 1966). All chemicals used were biochemical grade.

were analyzed. Zinc concentrations in diets and perfusion solutions were determined by inductively coupled Ar plasma spectroscopy (Model IRIS Intrepid II, Thermal Jarrell Ash, Waltham, MA; Ji et al., 2006a). Validation of the mineral analysis was conducted by using bovine liver powder [GBW (E) 080193, National Institute of Standards and Technology, Beijing, China] as a standard reference. The concentrations of phenol red in perfusion solutions were assayed by measuring absorbency at 520, 560, and 600 nm with an ultraviolet-visible spectrophotometer (Model Cary 100, Varian Inc., Palo Alto, CA; Steel and Cousins, 1985). Final volumes of solutions, absorption percentages, and velocities of Zn were calculated according to the equations outlined in Table 2.

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MECHANISMS OF ZINC ABSORPTION IN BROILER INTESTINE Table 2. Formulas used for calculating final volume of perfusion solution and absorption percentage, absorption velocity of Zn by in situ ligated loops of broilers Term

Formula1

Symbol

Final volume of perfusion solution (mL)

VF

Experiment 1: absorption percentage of Zn (%)

UP

Experiment 2: absorption velocity of Zn (nmol/min per cm)

UV

VF = UP =

CP(1) × VI CP(2)

CZn(1) × VI − CZn(2) × VF × 100 CZn(2) × V1

UV =

CZn(1) × VI − CZn(2) × VF T×L

1

2003). If the variances were significant, differences between mean values were ascertained by using Duncan’s multiple range test method. In addition, regression analysis was used to fit the data in experiment 1 to determine the optimal sampling time of the ligated loops. The differences in kinetic parameters were analyzed by the t-test procedure of SAS. The differences in mRNA levels between the control group and the group supplemented with 0.616 mmol of Zn/L at the same intestinal segment were also analyzed by Student’s t-test. The level for significant differences was P < 0.05. A kinetic analysis of Zn absorption was performed by fitting the data obtained from experiment 2 to the following equations: first-order equation (nonsaturable diffusive component, equation [1]), Michaelis-Menten equation (saturable process, equation [2]), or 2 components including both equations mentioned above (a saturable process plus a nonsaturable diffusive component, equation [3]; Condomina et al., 2002): JZn = PA, JmaxA , or Km + A

[2]

JmaxA + PA, Km + A

[3]

JZn =

JZn =

[1]

where JZn and its maximum velocity, Jmax, are given in nanomoles per minute per centimeter, Km is the Michaelis-

Menten constant in millimolar per liter concentration, P is the diffusive constant in square centimeters per minute, and A is the millimolar per liter concentration of Zn. The fits of experimental data to the equations were carried out by using a nonlinear least squares regression program (SigmaPlot v. 9.0, SPSS Inc., Chicago, IL). To select the best kinetic model of Zn absorption in this research, the Akaike information criterion (AIC; Akaike, 1986; Gagne and Dayton, 2002) was used. We also considered the coefficient of variation of the parameter obtained after each fit.

RESULTS Time Course of Zn Absorption and the Major Absorption Site in Broiler Intestines The results of Zn influx varying with time are shown in Figure 1A. Comparison of the absorption percentages at each time point in 3 segments revealed that the values in the ileum were significantly higher than those in the duodenum (P < 0.0001) at 5 min, and were higher (P < 0.03) than those in the duodenum and jejunum at the remaining time points. It seems that the ileum is the major absorption site of Zn. Zinc absorption in the jejunum was obviously higher (P < 0.002) than that in the duodenum at all time points except at 30 min; no difference was found between the jejunum and duodenum at 30 min. Additionally, we could observe that Zn influx increased with a prolonged postperfusion time in the 3 segments.

Table 3. Parameters of oligo-nucleotide primer pairs for the β-actin, MT, ZnT 1, ZnT 2, and ZnT 5

Gene name

GenBank accession number

β-Actin

NM_205518

MT

NM_205275

ZnT 1

AJ619980

ZnT 2

XM_423325

ZnT 5

NM_001031419

Primer sequence F 5′-GAG AAA TTG TGC GTG ACA TCA-3′ R 5′-CCT GAA CCT CTC ATT GCC A-3′ F 5′-AAG GGC TGT GTC TGC AAG GA-3′ R 5′-CTT CAT CGG TAT GGA AGG TAC AAA-3′ F 5′-TGC GAG TGC CTT CTT CCT-3′ R 5′-AAG GAG CTG TCA GGT CTG TAA T-3′ F 5′-TCT TCT CCG TGC TGG TGC T-3′ R 5′-CGG CGT TGA AAT CCA TCC-3′ F 5′-ATG CTG TTG TGG GAT GTA-3′ R 5′-TTG TCT TGG CTG GTC CTC-3′

Product size (bp) 152 163 131 99 159

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CP(1), CP(2) = initial and final concentrations (mg/L) of phenol red, respectively; VI = initial volume (mL) of injected dose; CZn(1), CZn(2) = Zn concentration (mmol/L) of initial and final perfusion solution, respectively; T = sampling time (min) after initiation of dosing; L = length (cm) of ligated intestinal segment.

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The change in tendencies for Zn absorption in the duodenum and jejunum (data at 30 min were deleted) with time was best fit to a quadratic model [Y (absorption percentage) = 0.1051 +1.1134X (postperfusion time) − 0.0114X2 (P < 0.003, R2 = 0.9829); Y = 2.9486 + 1.7597X − 0.0193X2 (P < 0.026, R2 = 0.9748)], whereas the tendency in the ileum was best fit to an asymptotic model [Y = 58.6915 − 56.9417e−0.0758X (P < 0.002, R2 = 0.9874)]. Therefore, we could deduce that the time points of maximum absorption were 49, 46, and 60 min in the duodenum, jejunum, and ileum, respectively. The amount of absorption at any time in each intestinal segment was then expressed as a fraction of the amount of absorption at the time point of maximum absorption in the corresponding intestinal segment (Figure 1B). We concluded that the absorption at 30 min was greater than 85 to 90% of what it would be at the time point of maximum absorption in each segment. For this reason, the subsequent experiment was carried out for 30 min.

Kinetic Absorption of Zn in the Ligated Duodenum, Jejunum, and Ileum The kinetic absorption of Zn was investigated within a wide Zn concentration range (Figure 2). Zinc influx in the ileum was 1 to 5 times (P < 0.005) greater than those in the duodenum and jejunum at each Zn concentration. This result was consistent with the conclusion of experiment 1, which indicated that the ileum was the major absorption site of Zn in the SI of broilers. No regular changes were found between the duodenum and jejunum at different Zn concentrations. Regression analysis showed that the best fit in the duodenal segments and the jejunal segments was to equation [2] (Figure 2A and 2B), whereas in the ileum, the fit to equation [1] gave the best result (Figure 2C). This meant that Zn absorption was a saturable, carrier-mediated process in duodenal segments and jejunal segments, in con-

trast to the ileum, where it could be described as a nonsaturable, diffusive process. The kinetic parameters obtained, Jmax, Km, and P, are outlined in Table 4. The Km value was higher in the duodenum than in the jejunum (1.44 vs. 0.51 mM, P = 0.012). Similarly, the Jmax value was greater in the duodenum than in the jejunum (5.32 vs. 2.57 nmol/min per cm), but the difference was not significant. The diffusive constant (P) in the ileum was 5.72 × 10−3 cm2/min.

mRNA Expression of MT, ZnT 1, ZnT 2, and ZnT 5 in Different Intestinal Segments For the control group, MT and ZnT 1 mRNA levels in the duodenal segments were 2 to 3 times (P < 0.0005) those in the jejunum and ileum (Figure 3), whereas expression of the 2 genes was similar in the jejunum and ileum. The ZnT 5 mRNA level in the ileum was the same as that in the duodenum and was higher (P = 0.019) than that in the jejunum. For the 0.616 mM Zn-supplemented group, the changes in MT and ZnT 1 mRNA levels in the 3 segments were similar to those in the control group, whereas no difference was found for ZnT 5 mRNA expression in the 3 intestinal segments. In addition, MT, ZnT 1, and ZnT 5 mRNA levels were lowest in the ileum among the 3 intestinal segments. Comparison of gene expression between the control and the Zn-supplemented groups indicated that Zn supplementation increased (P < 0.02) MT mRNA levels in the 3 segments and decreased (P = 0.035) the ZnT 5 mRNA level in the ileum. Although no differences were found, Zn supplementation decreased ZnT 1 and ZnT 2 mRNA levels in the duodenum and ileum and the ZnT 5 mRNA level in the duodenum, but increased expression of the 3 genes in the jejunal segments. Different Zn concentrations had no effect on the ZnT 2 mRNA level in different segments.

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Figure 1. Time course of Zn absorption in the ligated duodenum, jejunum, and ileum of broilers. Absorption percentage was analyzed at each time point (A) and normalized for 100% absorption at the time point of maximum absorption (B; Bronner and Yost, 1985). Values were expressed as means ± SD, n = 6 to 8. a–cLowercase letters indicate significant differences (P < 0.05) within different segments but at the same time point. The curves corresponding to the duodenum, jejunum, and ileum were described by quadratic, quadratic, and asymptotic models, respectively (A).

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DISCUSSION Plots of Zn absorption percentage against time showed that there were quadratic time-dependent changes in Zn absorption in the duodenal loops and jejunal loops, indicating that absorption decreased when the postperfusion time was too long, whereas the asymptotic time-dependent increase in Zn absorption in the ileal loops suggested that Zn absorption reached the equilibrium when the time

was prolonged. The perfused intestines seemed to retain the ability to regulate Zn absorption. Zinc absorption obviously increased at the preliminary phase, because the intestines from broilers with deficient Zn stores were very sensitive. However, absorption decreased or reached equilibrium when the time was prolonged because the negative regulation in Zn homeostasis began to function or the absorption ability of intestines was gradually lost. The time points of maximum absorption were 49, 46, and

Table 4. Kinetic and statistical parameters, obtained from fitting the experimental data by a nonlinear least squares regression1 Kinetic parameter2 Segment Duodenum Jejunum Ileum

Jmax (nmol/min per cm)

Km (mM)

P (cm2/min)

5.32 ± 1.46 2.57 ± 0.39 —

1.44 ± 0.33 0.51 ± 0.17b — a

— — 5.72 × 10−3 ± 1.1 × 10−4

Statistical parameter3 R2

AIC

0.92 0.90 0.99

1.15 1.05 1.10

Mean values within a column with different superscript letters were different (P < 0.05). Values were expressed as means ± SE, n = 8 to 10. 2 Jmax represents the maximum absorption velocity of Zn; Km is the Michaelis-Menten constant; P is the diffusive constant. 3 2 R is the coefficient of determination; AIC represents Akaike’s information criterion. a,b 1

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Figure 2. Kinetic absorption of Zn in ligated duodenum (A), jejunum (B), and ileum (C), respectively. The curves corresponding to absorption in the duodenum and jejunum were described by equation [2] (saturable, carrier-mediated pathway), and the curve of the ileum was described by equation [1] (nonsaturable diffusion). The value at each concentration was mean ± SD, n = 8 to 10.

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60 min in the duodenum, jejunum, and ileum, respectively, and Zn absorption increased linearly within 30 min. This result was similar to the conclusion of Smith et al. (1978), who found that the absorption of Zn was maximal at 40 min when the isolated, vascularly perfused rat intestine was used as a model. The experimental method used in the present study was an in vivo technique that has been widely used to study the kinetic absorption of metal elements (Bronner and Yost, 1985; Hempe and Cousins, 1989). With this technique, we were able to keep physiological conditions as normal as possible and directly quantify the influx of Zn across the intact intestinal membrane of the bird. Despite some disadvantages, this method was apparently capable of allowing the absorption of Zn in the SI of broilers to be monitored (Ji et al., 2006a). To our knowledge, information on the main intestinal absorption site of Zn in avians is scarce. This problem has been investigated principally in the rat by several researchers. However, the intestinal site at which Zn is absorbed most efficiently has remained controversial. Sorensen et al. (1998) demonstrated that the duodenum was the main absorption site of Zn in the rat intestine, whereas other reports indicated that the jejunum (Wang

et al., 2001) or the ileum (Antonson et al., 1979) was the main absorption site. Condomina et al. (2002) concluded that those authors who used low Zn concentrations in their experiments found that the distal segment was the major site involved in Zn transport, whereas those who used high concentrations found that transport was more effective in the proximal segment. In addition, the techniques used, the length of the experimental period, the species of animal, and the different physiological status of the animals in these studies may explain the discrepancies among the research reports. Preliminary research from our laboratory showed that the ileum was the major absorption site of Mn in broiler intestines (Ji et al., 2006a,b). Our data in 2 experiments consistently showed that the absorption of Zn was significantly greater in the ileum than in the duodenum and jejunum, implying that the mechanism of Zn absorption in the ileal segment might be different from that in the duodenum and jejunum. In the last few years, kinetic absorption has been used to study mechanisms of Zn absorption. Steel and Cousins (1985), using the luminal and vascular perfusion method, found that absorption of Zn by the intestine of the Zn-depleted rat showed evidence of both carrier-

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Figure 3. The mRNA levels of MT (A), ZnT 1 (B), ZnT 2 (C), and ZnT 5 (D) in the duodenum, jejunum, and ileum of broilers at different Zn concentrations. Values were means ± SD, n = 8 to 10. A,BMeans with different capital letters differed significantly (P < 0.05) within different segments but at the same Zn concentration (0 mg/L). a,bMeans with different lowercase letters indicate significant differences (P < 0.05) within different segments but at the same Zn concentration (40 mg/L). An asterisk (*) shows significant differences (P < 0.05) within different concentrations but at the same segment.

MECHANISMS OF ZINC ABSORPTION IN BROILER INTESTINE

protein in human ileal mucosa; moreover, ZnT 5 mRNA and protein were reduced in Caco-2 cells cultured in 200 ␮M Zn compared with 100 ␮M Zn. The data in experiment 2 showed that Zn supplementation decreased ZnT 5 mRNA levels in the duodenum and ileum, which was in agreement with previous reports, but increased the level of ZnT 5 mRNA in the jejunum. The reason for this discrepancy induced by the different segments is not yet clear. Metallothionein is especially prevalent in the liver, kidney, and intestine. It chelates the cytosolic Zn, limiting Zn passage from the enterocytes to portal circulation (Davis et al., 1998). Levenson et al. (1994) demonstrated that a high-Zn diet or parenteral Zn administration elevated the MT mRNA level in the rat intestine and resulted in a decrease in Zn absorption from the subsequent meal. In our study, Zn supplementation significantly increased MT mRNA levels in the 3 segments of the broiler intestine. This result was in agreement with data in the literature on mammals. Zinc transporter 2 mRNA is detectable in the SI, kidney, pancreas, and prostate. It is believed to function by storing Zn in intracellular vesicles (Liuzzi et al., 2003). Liuzzi et al. (2004) suggested that Zn deficiency reduced the ZnT 2 mRNA level to a nearly undetectable level in the mouse SI, based on real-time PCR. The same result was obtained from a report that found Zn deficiency tended to down-regulate the ZnT 2 mRNA level in the jejunum of the rat (Pfaffl and Windisch, 2003). Our result, which indicated that Zn supplementation increased the ZnT 2 mRNA level in the jejunum, was in accordance with the above-mentioned research, but that the level was decreased in the duodenum and ileum. The reason for the difference induced by different intestinal segments is still unclear. A role for ZnT 1 in the efflux of Zn from the enterocytes into the serosa is supported by its basolateral location in the intestine (Palmiter and Findley, 1995; Cousins and McMahon, 2000). Zinc supplementation increased the ZnT 1 mRNA level in the rat SI (Liuzzi et al., 2001; Devergnas et al., 2004), whereas other studies found that Zn supplementation reduced the ZnT 1 mRNA level in human ileal mucosa, the rat colon, and Caco-2 cells (Pfaffl and Windisch, 2003; Cragg et al., 2005). In this investigation, we knew that Zn supplementation decreased ZnT 1 mRNA levels in the duodenum and ileum but that it increased the ZnT 1 mRNA level in the jejunum. This partly explained the reason for the discrepancy among the above-mentioned research studies, because different intestinal segments were used The transcriptional regulation of ZnT 1 and the MT gene by Zn is mediated by metal response element-binding transcription factor-1, which is activated by Zn to bind to metal response elements in the gene promoter (Heuchel et al., 1994; Langmade et al., 2000). Additionally, MT, ZnT 1, and ZnT 5 mRNA levels in the ileum tended to be lower than those in the other 2 segments in the Zn-supplemented group, further indicating that the absorption of Zn in the ileum was princi-

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mediated and nonsaturable components. Tacnet et al. (1990) demonstrated Zn transport into the brush border membrane vesicles of the pig by a saturable, carriermediated process and an unsaturable pathway. The Km value was 0.215 mM, the Jmax was 17.2 nmol/min per mg of protein, and the value of P was 0.025, but the intestines being detected were not separated into 3 segments in these 2 studies. Condomina et al. (2002) demonstrated that the intestinal transport of Zn occurred by a saturable process in the rat. The Km values obtained in the proximal, mid, and distal segments were 10.78, 1.94, and 3.04 mM, respectively. The Jmax values were 8.39, 1.62, and 3.42 (mmol/cm2ⴢh)ⴢ103, respectively. In our study, AIC was used to choose an optimal model to describe the data of Zn absorption in different ligated intestinal loops. The model was considered to be the optimal model if the AIC value was minimal. We found that Zn absorption was a saturable, carrier-mediated process in the duodenum and jejunum of broilers, in contrast to the ileum, where it could be described as a nonsaturable, diffusive process. This result is consistent with the change in thickness of the 3 intestinal walls, in which the ileal segment is the thinnest; hence, the absorption of substance Zn in the ileum easily relies on a concentration-dependent diffusion because of its physiological structure. In contrast, specialized mechanisms are activated when simple diffusion cannot satisfy the requirement for uptake, as in the duodenum and jejunum. These uptake systems use integral membrane transport proteins to move Zn across the lipid bilayer of the plasma membrane (Tako et al., 2005). The Km values in the duodenum and jejunum were 1.44 and 0.51 mM, respectively, which were lower than those in the Zn-adequate rat mentioned above (Condomina et al. 2002), indicating that the affinity of Zn for carriers is related to the animal species, the kinds of carriers, or the body Zn stores. Additionally, in comparing the Km values, we could see that although there was high variability, the Km value in the jejunum was lower than that in the duodenum, which suggests that the carriers located at the proximal segment showed a higher affinity for Zn. Consequently, the Jmax value was also lower in the jejunum (2.57 nmol/min per cm) than in the duodenum (5.32 nmol/min per cm). The value of P in the ileum was 5.72 × 10−3 cm2/min. These differences observed in the kinetic parameters indicate that more than one Zn transporter might be implicated in Zn absorption in the duodenum and jejunum, because different transporters have different affinities and capacities. Many ZnT play an important role in Zn cellular absorption (Kambe et al., 2004). Zinc transporter 5 mRNA is abundantly expressed in the pancreas, ovary, kidney, and SI (Inoue et al., 2002). Apical localization of ZnT 5, when expressed in Caco-2 cells, coupled with a demonstrated ability of the splice variant to mediate cellular Zn absorption, indicated the involvement of this transporter in the transport of Zn from the intestinal lumen into the enterocytes (Cragg et al., 2002). Cragg et al. (2005) found that Zn supplementation reduced ZnT 5

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pally a nonsaturable, diffusive process in which the carriers played a limited role. Expression of ZnT 5 and ZnT 2 mRNA was higher in the jejunum than in the duodenum, whereas expression of MT and ZnT 1 mRNA was lower in the jejunum than in the duodenum. This suggests that the particular process by which Zn is transferred from the lumen to the vasculature may occur by a transcellular pathway involving brush border transport, intracellular diffusion, and basolateral transport and that the process may differ between the duodenum and jejunum, with absorption in both the duodenum and jejunum dependent on a saturable, carrier-mediated pathway. This may be the first report of direct Zn regulation of 4 genes expressed in 3 intestinal segments of birds. In conclusion, our results clearly showed that the ileum is the preferential site for Zn ion transport in the SI of chickens, which can be explained by the different absorption mechanisms in different intestinal segments. Zinc absorption was regulated by a nonsaturable, diffusive process in the ileum, which had the lowest mRNA levels of ZnT 5, ZnT 1, and MT among the 3 segments, whereas this process was via a saturable carrier-mediated pathway in the duodenum and jejunum, where ZnT 5, ZnT 1, and MT played an important role in Zn transport.

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