Enhancement of Non-heme Iron Absorption by Anchovy (Engraulis

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Enhancement of non-heme iron absorption by anchovy (Engraulis japonicus) muscle protein hydrolysate involves a nanoparticle-mediated mechanism Haohao Wu, Suqin Zhu, Mingyong Zeng, Zunying Liu, shiyuan dong, Yuanhui Zhao, Hai Huang, and Martin Lo J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf5018719 • Publication Date (Web): 29 Jul 2014 Downloaded from http://pubs.acs.org on July 31, 2014

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Journal of Agricultural and Food Chemistry

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Enhancement of non-heme iron absorption by anchovy (Engraulis japonicus) muscle

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protein hydrolysate involves a nanoparticle-mediated mechanism

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Haohao Wu 1, Suqin Zhu 1, Mingyong Zeng 1,*, Zunying Liu 1, Shiyuan Dong 1, Yuanhui

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Zhao 1, Hai Huang 2, Y. Martin Lo 3

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College of Food Science and Engineering, Ocean University of China, 5 Yushan Road, Qingdao, Shandong Province, 266003, China

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School of Aquatic Products, Rizhao Polytechnic, No.16 north of Yantai Road, Rizhao, Shandong Province, 276826, China

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Department of Nutrition and Food Science, University of Maryland, College Park, MD 20742, USA

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Corresponding author. Fax: +86 0532 82032400; E-mail address: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT

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The mechanisms by which meat enhances human absorption of non-heme iron remain

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unknown. Recently, anchovy (Engraulis japonicus) muscle protein hydrolysate (AMPH) was

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found to mediate the formation of nano-sized ferric hydrolysis products in vitro. The current

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paper evaluates the effects of AMPH on the bioavailability and the intestinal speciation of

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non-heme iron in rats, followed by an investigation of cellular uptake pathways of in

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vitro-formed AMPH-stabilized nano-sized ferric hydrolysis products (ANPs) by polarized

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human intestinal epithelial (Caco-2) cells. The hemoglobin regeneration efficiencies in

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anemic rats followed the order of ferric citrate (9.79 ± 2.02 %) < commercial bare α-Fe2O3

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nanoparticles (16.37 ± 6.65 %) < mixture of ferric citrate and AMPH (40.33 ± 6.36 %) ≈

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ferrous sulfate (40.88 ± 7.67 %) < ANPs (56.25 ± 11.35 %). Percentage contents of intestinal

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low-molecular-weight iron in the groups of FC+AMPH, FeSO4 and ANPs were significantly

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lower than the corresponding hemoglobin regeneration efficiencies (P < 0.05), providing

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strong evidence for the involvement of nano-sized iron in intestinal iron absorption from

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FC+AMPH, FeSO4 and ANPs. Calcein-fluorescence measurements of the labile iron pool of

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polarized Caco-2 cells revealed the involvement of both divalent transporter 1 and

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endocytosis in apical uptake of ANPs, with endocytosis dominating at acidic extracellular pH.

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Overall, AMPH enhancement of non-heme iron absorption involves a nanoparticle-mediated

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mechanism.

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KEYWORDS

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Iron bioavailability; Muscle protein; Anchovy; Rats; Caco-2 cell; Nanoparticle. 2


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ABBREVIATIONS USED

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AMPH, anchovy muscle protein hydrolysate; ANPs, in vitro-formed nano-sized ferric

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hydrolysis products stabilized by anchovy muscle protein hydrolysate; BPDS,

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bathophenanthrolinedisulfonic acid; CDTA, trans-1,2-cyclohexanediaminetetraacetic acid;

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DFB, deferoxamine mesylate; DMT1, the divalent metal transporter-1; DTPA, diethylene

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triamine pentaacetic acid; EDTA, ethylenediaminetetraacetic acid; EDX, energy dispersive

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X-ray spectroscopy; FC, ferric citrate; FC+AMPH, mixture of ferric citrate and anchovy

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muscle protein hydrolysate; FeSO4, ferrous sulfate; HRTEM, high resolution transmission

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electron microscopy; LMW, low-molecular-weight; Nano-α-Fe2O3, commercial bare

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α-Fe2O3 nanoparticles; NTA, nitrilotriacetic acid; TEM, transmission electron microscopy.

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3



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INTRODUCTION

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After birth, humans have to acquire iron from the diet to support growth and

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compensate iron loss; over two billion people worldwide suffer from iron deficiency. While

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dietary iron can be found in the form of heme or non-heme iron, interventions to reduce iron

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deficiency usually focus on increasing non-heme iron absorption, which is greatly influenced

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by various dietary components. Inhibitors of non-heme iron absorption in humans include

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phytates, oxalates, polyphenols, fibers, calcium, copper and zinc; ascorbic acid and muscle

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foods (i.e. meat, poultry, and fish) enhance nonheme iron absorption.1 The enhancing effect

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of muscle foods on non-heme iron absorption is usually attributed to certain “meat factors”

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(e.g. peptides, acidic polysaccharides, and L-α-glycerophosphocholine), which may react

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with non-heme iron in the intestinal lumen, thereby increasing iron solubilization and

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absorption.2-4 However, the chemical nature of such soluble non-heme iron has still not been

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fully elucidated.

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One economical meat source that could be used to combat iron deficiency in

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developing countries is the anchovy, a small marine pelagic fish harvested in huge quantity

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worldwide, mainly for non-food uses.5 Recently, we demonstrated that anchovy (Engraulis

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japonicus) muscle protein hydrolysate (AMPH) could mediate the formation of nano-sized

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ferric hydrolysis products within a pH range similar to that of human digestive lumen;6 thus,

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AMPH might enhance non-heme iron absorption in humans via a nanoparticle-mediated

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mechanism. The current research examined the effects of AMPH on the bioavailability and

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the intestinal speciation of non-heme iron in rats, followed by an investigation of cellular 4


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uptake pathways of in vitro-formed AMPH-stabilized nano-sized ferric hydrolysis products

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(ANPs) in polarized human intestinal epithelial (Caco-2) cells.

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MATERIALS AND METHODS

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Materials. Japanese anchovy, Engraulis japonicus (5−8 cm total length), caught in the

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Yellow Sea by Allen Ship Service Co. Ltd. (Shandong, China) were frozen immediately at

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−35 °C on board and transported in a Styrofoam-insulated box to the laboratory refrigerator

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(−20 °C) within 4 h. Trypsin from bovine pancreas with a declared activity of 250 U/mg was

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purchased from Sinopharm Chemical Co. Ltd. (Shanghai, China). Nano-α-Fe2O3 powder with

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declared diameters of 20-30 nm was purchased from Beijing Nachen Co. Ltd. (Beijing,

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China). Bathophenanthrolinedisulfonic acid (BPDS), deferoxamine mesylate (DFB),

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ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), phytic acid sodium salt,

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diethylene triamine pentaacetic acid (DTPA) and trans-1,2-cyclohexanediaminetetraacetic

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acid (CDTA) sodium selenite, hydrocortisone, and triiodothyronine were obtained from

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Sigma-Aldrich Co. (St. Louis, USA). Calcein acetoxymethylester, cell culture mediums,

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insulin, and epidermal growth factor were purchased from Gibco (Grand Island, USA). All

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other reagents used in this study were commercially available and of analytical grade.

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Preparation of AMPH and ANPs. AMPH was obtained as described previously.7

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Briefly, 25% (w/v) homogenates of anchovy meat was hydrolyzed by trypsin with a 25:1

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substrate to enzyme ratio (w/w) at 37 °C and pH 8 for 4 h with continuous magnetic stirring,

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followed by heat inactivation of the enzyme in a boiling water bath for 10 min, and the

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reaction mixture was then centrifuged at 10000g for 20 min. The supernatant was stored at 5



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4 °C for no more than 48 h and filtered through 0.22 μm cellulose acetate filters before use.

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Total protein concentration in the supernatant was determined according to Lowry’s method

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using bovine serum albumin as the standard protein.8 ANPs were prepared according to the procedure described by Wu et al

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with slight

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modification. Briefly, one volume of freshly prepared ferric chloride (20 mM) was added

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dropwise into ten volumes of magnetic stirred AMPH solution (20 g peptide/L) with the

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solution pH maintained at 7.0 ± 0.2 using 1 M sodium hydroxide. The obtained sample

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solution was stored at 4 °C for no more than 24 h and filtered through 0.22 μm cellulose

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acetate filters before use. For the structural characterization of ANPs, sample solutions were

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dropped onto carbon-coated copper grids, allowed to air-dry, and then examined in a JEM

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1200 EX (JEOL, Tokyo, Japan) electron microscope. Iron content in the sample solution was

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determined by flame atomic absorption spectroscopy using a Shimadzu AA-6800

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spectrometer (Kyoto, Japan).

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Animal Experiments. All animal handling was conducted according to the principles

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and guidelines outlined in National Institute of Health (NIH) Guide for the Care and Use of

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Laboratory Animals, and approved by the Ethical Committee of Animal Care and Use at

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Ocean University of China (Permit No: 20001013). Sprague-Dawley rats were obtained from

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Shandong Lukang Pharmaceutical Group Co., Ltd (Shandong, China). Pelletized purified

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AIN-93G-based diets were prepared by TROPHIC Animal Feed High-tech Co., Ltd.

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(Nantong, China). The Fe-deficient diet contained ~10 mg/kg of iron derived from raw

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materials. The Fe-sufficient diet contained ~25 mg/kg of iron in the form of ferric citrate (FC) 6


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and ~10 mg/kg of iron derived from raw materials. The Fe-repletion diets contained ~10

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mg/kg of iron derived from raw materials, and ~20 mg/kg of iron in forms of ferrous sulfate

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(FeSO4), ANPs, FC, mixture of FC and AMPH (FC+AMPH), and commercial bare α-Fe2O3

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nanoparticles (Nano-α-Fe2O3). The iron compounds were incorporated into the diets during

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preparation of the wet ingredient mixtures. FeSO4, FC or Nano-α-Fe2O3 equivalent to 20 mg

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iron was mixed with 1 kg of the other dry ingredients and 200 mL of Milli-Q water to obtain

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the wet ingredient mixture. When incorporating the solution form of AMPH (18 g peptide/L)

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and ANPs (1.8 mM iron and 18 g peptide/L), 200 mL of either of them was mixed with 1 kg

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of the other dry ingredients to make the wet ingredient mixture. After being pelletized, the

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wet ingredient mixture was dried overnight at 40°C in a drying oven. Iron contents in the

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dried pelletized diets were determined by flame atomic absorption spectroscopy using a

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Shimadzu AA-6800 spectrometer (Kyoto, Japan). Rats received Milli-Q water ad libitum

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throughout the experiments. Body weight was recorded at the beginning and end of each

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experimental period.

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The iron bioavailability was measured in anemic rats using a hemoglobin regeneration

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bioassay.9,10 Female Sprague-Dawley rats (n = 12) breastfeeding male pups (16 days old; n =

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52) were housed in individual stainless steel cages with four to five pups each and fed a

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Fe-deficient diet ad libitum for a week. On the weaning day, 52 male rats (23 days old) were

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transferred into individual stainless steel cages under controlled conditions at temperatures of

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22 ± 2 °C and relative humidities of 55 ± 10% with a light/dark cycle of 12 h/12 h. The

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just-weaned rats were stratified according to body weight and within each stratum were 7



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randomly allocated at random to two groups: the Fe-depleted group (n = 48) fed a

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Fe-deficient diet ad libitum and a control group (n = 4) fed a Fe-sufficient diet ad libitum.

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After a Fe-depletion period of 27 days, 20 µL of fresh rat blood sampled by tail vein incision

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was diluted with EDTA and subsequently analyzed using a Cell-Dyn 1600 hematology

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analyzer (Abbott Laboratories, Abbott Park, USA). The mean hemoglobin concentrations of

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the Fe-depleted group and the control group were 71.1 ± 13.8 g/L and 153.8 ± 13.9 g/L,

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respectively. Rats in the Fe-depleted group were stratified by the hemoglobin concentrations

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and randomized into six groups (eight rats each). The rats in each group consumed one of the

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five Fe-repletion diets containing FeSO4, ANPs, FC, FC+AMPH or Nano-α-Fe2O3, or the

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Fe-deficient diet for 13 days (the repletion period) ad libitum. Rats in the control group

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remained on the Fe-sufficient diet during the repletion period.

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Periodically, daily food intake of each rat was manually recorded. A known and

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adequate amount of a diet was given to a rat in the feeding container between nine and ten

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o’clock in the morning. After 24 h, the diet remaining in the feeding container and at the

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bottom of the cage was reweighed; calculating the differences showed the rat’s daily food

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intake.

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At the end of the repletion period, blood samples were once again taken by tail vein

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incision. The rats were anaesthetized with ether before their livers were taken, rinsed with

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saline and stored at -20 °C. Iron concentration in the liver was measured by flame atomic

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absorption spectroscopy using a Shimadzu AA-6800 spectrometer (Kyoto, Japan). Hemoglobin iron pool (mg), assuming a total rat blood volume of 6.7 % body weight

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and an iron content in hemoglobin of 0.335 %, was calculated as follows:10 Hemoglobin iron pool (mg) = body weight (kg) × 0.067 × hemoglobin concentration

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(g/L) × 3.35.

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The hemoglobin regeneration efficiency was calculated based on the following equation:

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Hemoglobin regeneration efficiency (%) = [final hemoglobin iron pool (mg) – initial

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hemoglobin iron pool (mg)] / total iron consumed (mg) × 100. The relative biological value was calculated based on the hemoglobin regeneration

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efficiencies: Relative biological value (%) = hemoglobin regeneration efficiency of each animal /

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average hemoglobin regeneration efficiency of the FeSO4 group × 100.

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Iron speciation analysis of rat intestinal contents was carried out as previously described

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by Kapsokefalou and Miller11 and Simpson et al.12 with some modifications. Male

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Sprague-Dawley rats (53 days old; n = 40) weighing 290−310 g were stratified according to

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body weight and within each stratum were randomly distributed into five groups (eight rats

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each). The rats were fed the Fe-deficient diet for a week before they were daytime fasted for

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9 h. The fasted rats in each group then consumed one of the five Fe-repletion diets for 30 min

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ad libitum and were anesthetized with ether ninety minutes after consumption of the meal.

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The rats were killed by cervical dislocation before an abdominal midline incision was

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performed. After the small intestine was removed from the carcass, content in the proximal

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one-third segment was flushed with saline into a centrifuge tube and further fractionated into 9



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two fractions using Millipore filters (Göttingen, Germany): the 0.22-µm filtrate and the

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3-kDa (≈ 1 nm) filtrate. Iron in the intestinal content, 0.22-µm filtrate and 3-kDa filtrate were

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determined by flame atomic absorption spectroscopy using a Shimadzu AA-6800

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spectrometer (Kyoto, Japan).

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Cellular Uptake Experiment. Caco-2 cells were obtained from the Cell Bank of the

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Chinese Academy of Sciences (Shanghai, China), and seeded at a density of 5×104 cell/cm2

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in collagen-treated 24-well plates (BD Biosciences, San Jose, CA, USA). The cells were

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routinely maintained in high glucose (4.5 g/L) Dulbecco’s Modified Eagle Medium

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supplemented with 10% heat-inactivated fetal bovine serum, 25 mM HEPES, 4mM

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glutamine and 1 mM sodium pyruvate at 37 °C in an incubator with a 95% air/5% CO2

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atmosphere at constant humidity, and the medium was renewed every two days. After

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reaching confluence (2-3 days post-seeding), the cells were maintained in complete

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Dulbecco’s Modified Eagle Medium for another 12 days to differentiate completely, and

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further incubated for 24 h in serum-free minimum essential medium supplemented with 4

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mg/L hydrocortisone, 5 mg/L insulin, 5 µg/L selenium, 34 µg/L triiodothyronine, and 20 µg/L

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epidermal growth factor. Following this, the medium was aspirated, and the cells were stained

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with 1 µM calcein acetoxymethylester in Hanks’ balanced salt solution for 30 min at 37 °C

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before being washed three times with HBSS. The cells were then incubated in Tyrode

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solution (137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 5.5 mM D-glucose)

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buffered with 20 mM MES (pH 5.5) or MOPS (pH 7.0) containing FeSO4, ANPs, or any of

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1:1 ferric chelates of NTA, phytate, EDTA, DTPA, CDTA and DFB at the concentration 10


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equivalent to 20 µM iron in a Synergy H4 Hybrid Microplate Reader (Bio-Tek, Winooski, VT,

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USA) at 37 °C for 2.5 h, and calcein fluorescence (485 nm excitation, 530 nm emission) was

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monitored every 5 min. To trap and detect ferrous iron, 100 µM BPDS was used, and the

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absorbance of the ferrous tris(BPDS) complex was measured at 536 nm (molar extinction

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coefficient, ε =1.927 × 104 L mol-1 cm-1). Stock solution of FeSO4 (1 mM) was freshly

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prepared before use. Stock solution of ferric chelates (1:1 molar ratio) of NTA, phytate,

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EDTA, DTPA, CDTA, and DFB was prepared by mixing of equal volume of chelators (2 mM)

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and freshly prepared FeCl3 (2 mM).

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Statistical Analysis. Data are expressed as the mean ± standard deviation (SD).

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Differences between groups were tested for significance by the least significant difference

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(LSD) mean comparison using the SPSS software program (SPSS Inc., Chicago, IL, USA).

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The relation between variables was determined by the Pearson correlation test. The

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probability level < 0.05 was considered statistically significant.

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RESULTS AND DISCUSSION

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To evaluate the structural properties of ANPs, i.e. the products of ferric hydrolysis in the

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presence of AMPH in vitro at room temperature, we used transmission electron microscopy

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(TEM), high resolution transmission electron microscopy (HRTEM), electron diffraction, and

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energy dispersive X-ray spectroscopy (EDX). TEM imaging (Figure 1A) showed that the

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average particle size ranged from 10 to 20 nm, and this size was greater than pure two-line

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ferrihydrite (2−4 nm).13 Whole area EDX analysis (Figure 1B) of one of the particles in

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Figure 1A revealed three peaks (0.7, 6.4, and 7.1 keV) that corresponded to the Lα, Kα, and 11



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Kβ lines of iron, respectively, so the particles in the TEM image were iron-containing. In

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addition, the EDX spectra also showed prominent peaks for copper, sodium, potassium,

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chloride, sulfur, carbon, oxygen, phosphorus and silicon. The copper obviously represented

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the supporting copper grid, and the silicon peak was presumably due to contamination.

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Spurious silicon peaks have been recognized as a problem in EDX analysis of biological

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specimens due to silicon contamination from oils or greases during the course of specimen

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preparation or from the vacuum system of the electron microscope.14 Any remaining peaks

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would presumably represent true compositions of AMPH or the specimen support film. The

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HRTEM image (Figure 1C) revealed no recognizable lattice fringe, suggesting the

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amorphous property of ANPs. The selected area electron diffraction (Figure 1D) showed a

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two-ring pattern similar to that of 2-line ferrihydrite with the obtained d-spacing values (2.90

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and 1.97 Å), a little higher than typical corresponding values of the latter (~2.5 and 1.5 Å).

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ANPs are likely to be a poorly crystalline ferric oxyhydroxide similar to 2-line ferrihydrite. It

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is known that poorly crystalline iron oxides are prone to electron beam damage,15 so the

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actual structure of ANPs might vary somewhat in size and crystalline phase from that

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observed under TEM, HRTEM and electron diffraction.

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Details and results of the hemoglobin regeneration bioassay are shown in Table 1. It is

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known that body iron is distributed in hemoglobin (65%), iron stores (ferritin and

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hemosiderin, 30%), myoglobin (3.5%) and other heme proteins (1.5%).16 Liver iron stores

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did not show significant increase in all Fe-repletion groups during the repletion period

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(Figure 2), so most of the iron absorbed should be used to generate new hemoglobin 12


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molecules, amounts of which could be calculated from changes of blood volumes and

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hemoglobin concentrations. The negative changes of the hemoglobin concentrations in

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groups of Fe-deficient, FC and Fe-sufficient were the result of a marked increase in blood

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volume, which is proportional to body weight, during fast growth of the anemic rat. As

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shown in Table 1, body weights and hemoglobin concentrations varied independently among

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the groups, so to measure iron bioavailability by hemoglobin regeneration in anemic rats, it is

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necessary to calculate the hemoglobin regeneration efficiencies from body weight gain, diet

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intake, diet iron content, and hemoglobin concentration.

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As shown in Table 2, the hemoglobin regeneration efficiency of FC+AMPH was as

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high as that of FeSO4, a gold standard in iron bioavailability studies, and significantly higher

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than that of FC alone (P < 0.05). Apparently, AMPH effectively enhanced iron absorption

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from FC. ANPs displayed an even higher hemoglobin regeneration efficiency than

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FC+AMPH and FeSO4, although not statistically significant (P = 0.06 and 0.12, respectively).

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Since crystalized bare iron oxide nanoparticles feature simple production process and low

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cost, we examined the bioavailability of iron from Nano-α-Fe2O3, a commercial product with

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declared size similar to ANPs. The hemoglobin regeneration efficiency of Nano-α-Fe2O3 was

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found to be significantly lower than that of ANPs (P < 0.05). Interestingly, the relative

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biological value of Nano-α-Fe2O3 (39.0 ± 15.8%) was also found to be significantly lower

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than those of previously reported ZnO-, MgO-, CaO- or FePO4-doped iron oxide

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nanopowders, namely Fe2O3/ZnO/MgO (81.8−100.1%), Fe2O3/ZnO/CaO (72.4−92.6%),

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Fe2O3/ZnO (68.0−87.5%), and Fe2O3/FePO4 (69.0−87.4%),9 which might be explained by the 13



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higher iron dissolution rates of these latter iron oxide nanostructures in gastric acid due to

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doping with compounds more soluble in dilute acid than iron oxide.17

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To elucidate whether nano-sized iron involved iron absorption in vivo, we analyzed

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iron speciation in the luminal content of the proximal one-third segment of small intestine,

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where dietary iron is mainly absorbed.18 Iron in the 3-kDa filtrates were regarded as the truly

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dissolved iron species or low-molecular-weight (LMW) iron, whereas that in the 0.22-µm

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filtrate was supposed as total soluble iron including both LMW iron and nano-sized colloidal

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iron. As shown in Table 2, percentage contents of intestinal LMW iron in the groups of

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FC+AMPH, FeSO4 and ANPs were significantly lower than the corresponding hemoglobin

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regeneration efficiencies (P < 0.05), so LMW iron alone was unlikely to account for total iron

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absorption in these groups. This provided strong evidence for the involvement of nano-sized

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iron in intestinal iron absorption from FC+AMPH, FeSO4 and ANPs.

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LMW iron in the intestinal tract might be ferrous iron or ferric chelates. Except for

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FeSO4, the other four iron compounds in the present study are all in the form of ferric iron.

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The reducing power of tert-butylhydroquinone, the fat-soluble antioxidant added in the

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AIN-93G diet, might not contribute to the formation of intestinal LMW iron due to its very

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low solubility in the aqueous phase. Although meat digestion products are capable of

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reducing non-heme ferric iron to the ferrous form in vitro,19 AMPH and ANPs did not

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increase the percentage content of LMW iron (Table 2), which might be explained by the

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oxidation of protein sulfhydryl groups during preparation and storage of AMPH and

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pelletized diets.20 There are usually considerable amounts of residual phytates in the 14


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commercial starch that comprises an important part of the AIN-93G diet,21 so iron phytate

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chelates might account for part of the intestinal LMW iron in the present study. It is also

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possible that some vitamins added in the AIN-93G diet could form LMW ferric complexes in

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the intestinal lumen.22

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Ferric hydrolytic polymers could be stabilized at the nanoscale by biomacromolecules

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(e.g. polysaccharides and peptides) derived from some common food components (e.g. starch,

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carrageenan, pectin, alginate, casein, whey, and muscle protein) or gastrointestinal secretions

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(e.g. mucins).6,23-27 The percentage contents of nano-sized iron in the rat proximal small

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intestinal tracts were calculated by subtraction of LMW iron from total soluble iron (Table 2),

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and were found to be over 30% in all five groups, suggesting that AIN-93G diet could induce

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a high basal level of intestinal nano-sized iron. This might be attributed to the digestion

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products of maltodextrin, starch and casein in the basal formulation of AIN-93G diet or

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gastrointestinal secretions. Interestingly, the percentage of nano-sized iron for the FeSO4

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group was also significantly higher than that of its LMW counterpart (P < 0.05), which might

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be explained by the formation of nano-sized iron via rapid in situ oxidation and hydrolysis of

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ferrous iron bound to soluble macromolecules in the presence of swallowed oxygen in the

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intestinal tract. Due to their tendency to aggregate and sediment in aqueous solutions, bare

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iron oxide nanoparticles are difficult to disperse into stable suspensions within the nanoscale

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range unless very efficient dispersing techniques (e.g. probe ultrasonication) are employed,28

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so it is unsurprising that Nano-α-Fe2O3 induced a significantly lower level of intestinal

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nano-sized iron than ANPs (P < 0.05). 15



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The involvement of intestinal nano-sized iron in iron absorption from FC+AMPH,

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FeSO4 and ANPs raises questions about how gut epithelial cells absorb intraluminal

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nano-sized iron. Usually, exogenous iron enters the cellular labile iron pool before it is stored

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in ferritin, exported from the cell, or used to synthesize iron proteins.29 Calcein, a

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fluorescence probe for the labile iron pool, has been frequently used to monitor cellular iron

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uptake in real time.30,31 We thus used calcein-loaded polarized Caco-2 cell monolayers as a

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gut epithelial model to investigate the intestinal iron absorption from ANPs, a mimetic for

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intraluminal nano-sized iron. As shown in Figure 3, iron uptake from FeSO4 and ANPs at

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concentrations equivalent to 20 µM iron was monitored by the quenching of calcein

308

fluorescence, i.e. the incorporation of exogenous iron into the cellular labile iron pool, under

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two pH conditions (5.5 and 7.0) simulating those in the proximal and distal small intestine.32

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BPDS, a strong ferrous chelator, was used to block iron absorption via the divalent metal

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transporter 1 (DMT1). As expected, cellular iron uptake from FeSO4 was completely blocked

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by BPDS at pH 5.5 and 7.0 (data for the latter pH not shown), while iron uptake from ANPs

313

was only partially blocked at both pH values, suggesting that both DMT1-dependent and

314

DMT1-independent mechanisms are involved in iron absorption from ANPs.

315

The two best known DMT1-independent iron uptake mechanisms are by means of

316

heme carrier protein 1 and endocytosis. Heme carrier protein 1 is specific to heme iron

317

uptake,33 so endocytosis, a very common mechanism for cellular entry of nanoparticles,

318

attracted much attention in recent studies on intestinal absorption of nano-sized iron. Work by

319

Theil and colleagues demonstrated that iron absorption from ferritin (i.e., ferrihydrite-like 16


Page 16 of 33

Page 17 of 33


320

nanostructures encapsulated by a protein cage) is independent of heme iron and ferrous salts

321

in both women and rat intestinal segments,34 and a paper published later by Antileo et al.

322

revealed an endocytic pathway of ferritin in Caco-2 cells using TEM and fluorescence

323

confocal microscopy.35 The elegant studies of Pereira et al. and Powell et al.,36,37 utilizing

324

Caco-2 cells and mice, proved that intestinal uptake of luminally hydrolyzed nano-sized iron

325

invokes a nanoparticulate endocytic pathway. Thus, endocytosis could be the mechanism for

326

DMT1-independent apical uptake of ANPs.

327

The calcein fluorescence changes in Figure 3 also showed a more noticeable uptake of

328

iron from ANPs at pH 5.5 in the presence of BPDS than that observed at pH 7.0, suggesting a

329

lower intraluminal pH in the proximal small intestine favors the endocytic uptake of ANPs.

330

Acidic extracellular pH enhancement of endocytosis due to the proton-induced change of

331

cellular membrane properties has been well-documented,38 so it is not surprising that in the

332

present study an extracellular pH of 5.5 was more favorable for the endocytic uptake of ANPs

333

compared to pH 7.0. The marginal inhibition of iron absorption from ANPs by BPDS at pH

334

5.5 indicated the dominant role of the endocytic pathway at acidic extracellular pH. Iron

335

uptake from FeSO4 at pH 7.0 without the presence of BPDS was less evident than that at pH

336

5.5 after about 20 min of incubation, which could be explained by the relatively fast

337

oxidation rate of ferrous iron at pH 7.0.39

338

Ferric iron must be reduced to the ferrous form by duodenal cytochrome-b in the

339

apical membrane of enterocytes before being absorbed by the DMT1 pathway, so the part

340

played by the DMT1-dependent pathway in apical uptake of ANPs depends on the 17



Page 18 of 33

341

reducibility of ANPs. As shown in Figure 5, the reducibility of ferric chelates of NTA,

342

phytate, EDTA, DTPA, CDTA, and DFB to polarized Caco-2 cells followed the reversed

343

order of ferric stability constants of the chelators,40 and ANPs were as reducible as ferric

344

NTA, which is the most reducible of these ferric chelates.

345

Some previous studies have revealed that reduction rates of ferric chelates by plant

346

roots (Arachis hypogeae L.) and yeast (Saccharomyces cerevisiae) were inversely related to

347

the ferric stability constants,41,42 and our data confirmed the previous observations in human

348

intestinal cells. The reducibility of iron oxides is greatly dependent on their crystalline phases

349

(e.g. ferrihydrite, lepidocrocite, maghemite, goethite, and hematite) and surrounding organic

350

substances. Ferrihydrite is the most reducible phase of iron oxides. The phase of intestinal

351

nano-sized iron in the present study should be similar to ANPs, a poorly crystalline ferric

352

oxyhydroxide like 2-line ferrihydrite. Iron-binding organic substances such as AMPH in the

353

present study might enhance reduction dissolution of iron oxides via solubilizing ferric iron

354

from the oxide surface or complexation of ferrous iron.43 The high reducibility of ANPs to

355

polarized Caco-2 cell monolayers could thus be easily understood. In

356

summary,

our

data

suggest

that

intestinal

nano-sized

iron

involves

357

AMPH-enhancement of non-heme iron absorption in rats. While both DMT1 and endocytosis

358

play parts in the intestinal absorption of luminal nano-sized iron, endocytosis dominates at

359

acidic extracellular pH levels.

360

18


Page 19 of 33

361


ACKNOWLEDGEMENTS

362

Financially supported by the National Natural Science Foundation of China (No.

363

31371758), National Key Technology Research and Development Program of China (No.

364

2012BAD28B05), and China Scholarship Council (No. 201306330015).

365

REFERENCES

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salts in women and rat intestinal segments. J Nutr. 2012, 142, 478-483.

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Fernández, J.; Núñez, M. T. Endocytic pathway of exogenous iron-loaded ferritin in

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intestinal epithelial (Caco-2) cells. Am. J. Physiol. Gastrointest. Liver. Physiol. 2013,

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K.; Prassmayer, L.; Lönnerdal, B.; Brown, A. P.; Powell, J. J. Caco-2 cell acquisition of

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dietary iron(III) invokes a nanoparticulate endocytic pathway. PLoS. ONE. 2013, 8,

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(37) Powell, J. J., Bruggraber, S. F., Faria, N., Poots, L. K., Hondow, N,, Pennycook, T. J.,

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Latunde-Dada, G. O., Simpson, R. J., Brown, A. P., Pereira, D. I. A nano-disperse

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fluidity, lipid-phase order and the membrane resting potential. Biochim. Biophys Acta.

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2013, 1828, 2672-2681.

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(39) Morgan, B.; Lahav, O. The effect of pH on the kinetics of spontaneous Fe(II) oxidation

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(40) Martell, A. E.; Smith, R. M. (Eds.), NIST Standard Reference Database 46: NIST

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critically selected stability constants of metal complexes: version 8, National Istitute of

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Standards and Technology, Gaithersburg, 2004.

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(41) Römheld, V.; Marschner, H. Mechanism of iron uptake by peanut plants. I. FeIII reduction, chelate splitting, and release of phenolics. Plant. Physiol. 1983, 71, 949–954.

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(42) Lesuisse, E.; Raguzzi, F.; Crichton, R. R. Iron uptake by the yeast Saccharomyces cerevisiae: involvement of a reduction step. J. Gen. Microbiol. 1987, 133, 3229–3236.

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(43) Wu, H.; Yin, J. J.; Wamer, W. G.; Zeng, M.; Lo, Y. M. Reactive oxygen species-related

488

activities of nano-iron metal and nano-iron oxides. J. Food. Drug. Anal. 2014, 22, 86–

489

94.

490

491

25



Page 26 of 33

492

Figure captions

493

Figure 1. Structural characterization of in vitro-formed nano-sized ferric hydrolysis products

494

stabilized by anchovy muscle protein hydrolysate (ANPs). A) Transmission electron

495

microscopy images collected from a drop of ANPs. B) Whole area energy dispersive X-ray

496

analysis of one of the particles in “A” showing elemental compositions. Peaks for sodium

497

(Na), copper (Cu), potassium (K), silicon (Si), phosphorus (P), sulphur (S), chlorine (Cl),

498

carbon (C), and oxygen (O) are identified. C) High resolution transmission electron

499

microscopy. D) Selected area electron diffraction.

500

Figure 2. Liver iron concentrations at the end of the iron-repletion period. Abbreviations: FC,

501

ferric citrate; FC+AMPH, the mixture of ferric citrate and anchovy muscle protein

502

hydrolysate, FeSO4, ferrous sulfate; ANPs,

503

products stabilized by anchovy muscle protein hydrolysate; Nano-α-Fe2O3, commercial bare

504

α-Fe2O3 nanoparticles. Values are means ± standard deviations of a representative experiment

505

(n = 4 for the Fe-sufficient group; n = 8 for the other groups). Groups not sharing a common

506

letter are significantly different (P < 0.05).

507

Figure 3. Iron uptake by polarized Caco-2 cell monolayers in the presence of 20 µM iron at

508

extracellular pH values of 5.5 and 7.0. Abbreviations: FeSO4, ferrous sulfate; ANPs, in

509

vitro-formed

510

bathophenanthrolinedisulfonic acid. Cellular uptake of exogenous iron into the labile iron

511

pool was measured as the quenching of calcein fluorescence. BPDS is a strong ferrous

512

chelator to block the divalent metal transportor-1 pathway. Data are means of three

513

independent experiments with the standard deviations less than 1.6.

514

Figure 4. Ferrous concentrations after 2-h incubation of 20 µM different forms of ferric iron

515

with polarized Caco-2 cell monolayers at an extracellular pH of 5.5. Abbreviations: ANPs, in

516

vitro-formed nano-sized ferric hydrolysis products stabilized by anchovy muscle protein

517

hydrolysate; CDTA, trans-1,2-cyclohexanediaminetetraacetic acid; DFB, deferoxamine

518

mesylate; DTPA, diethylene triamine pentaacetic acid; EDTA, ethylenediaminetetraacetic

519

acid; NTA, nitrilotriacetic acid. Ferric stability constants were obtained from the literature.40

520

Data are presented as means ± standard deviations of three independent experiments. Groups

521 522

not sharing a common letter are significantly different (P < 0.05).

AMPH-stabilized

in vitro-formed nano-sized ferric hydrolysis

nano-sized

ferric

hydrolysis

26


products;

BPDS,

Page 27 of 33


523

Table 1. Details and results of the hemoglobin regeneration bioassay. Group*

Fe-deficient

FeSO4

ANPs

FC

FC+AMPH

Nano-α-Fe2O3

Fe-sufficient

N

8

8

8

8

8

8

4

Diet iron (mg/kg)

10.5±1.5

32.4±2.3

30.5±0.7

29.8±2.0

28.8±0.2

31.8±2.4

36.8±2.5

Diet intake (g)

265±30.5a

419.5±70.3c

332.1±32.7ab

268.2±43.7a

380.6±70.0bc

267.2±16.9a

383.9±37.9b

Baseline BW (g)

222.7±21.5

221.0±20.1

221.4±39.2

222.4±38.8

215.0±17.2

219.9±11.8

297.5±33.9

c

ab

ab

ab

85.0±3.6ab

*

BW gain (g)

a

62.4±27.8

101.7±32.9

Baseline Hb (g/L)

71.1±11.9

71.4±16.8

†

Hb change (g/L)

524 525 526 527

-14.4±11.1

a

b

50±19.8

bc

109.7±37.2

79.7±25.9

88.7±28.9

73.7±10.5

70.5±15.3

71.9±12.7

b

a

b

52.8±19.8

-6.9±21.7

42.8±21.0

75.6±26.7

71.1±19.0

153.8±13.9 a

0.125±17.2

-25.3±23.8



FeSO4, ferrous sulfate; ANPs, in vitro-formed AMPH-stabilized nano-sized ferric hydrolysis products; FC, ferric citrate; FC+AMPH, the mixture of ferric citrate and anchovy muscle protein hydrolysate; Nano-α-Fe2O3, commercial bare α-Fe2O3 nanoparticles. Values are means ± SD. Means in a column without a common superscript letter (a, b and c) are statistically different, P < 0.05. *BW = body weight; Hb = hemoglobin. †

528

27



Page 28 of 33

529 530

Table 2. Hemoglobin regeneration efficiencies and percentage contents of intestinal iron species in different groups.

531 532 533 534 535 536 537

Percentage contents of intestinal iron species Hemoglobin Group regeneration Nano-sized Total soluble Low-molecular-weight 1 2 efficiency (%) iron iron3 iron FC 9.79±2.01a 43.18±4.49ef 12.44±3.47ab 30.74±6.49d Nano-α-Fe2O3 16.37±6.65abc 56.57±11.69fg 24.35±4.67cd 32.22±12.24de ef g bc 60.74±6.84 18.31±4.16 42.43±3.40ef FC+AMPH 40.33±6.36 40.88±7.67ef 59.01±9.02g 19.21±5.26bc 39.80±8.87def FeSO4 ANPs 56.25±11.35fg 61.24±11.01g 14.47±2.63ab 46.77±8.89f 1 2 3 Iron in the 0.22-µm filtrate. Iron in the 3-kDa filtrate. Calculated by subtraction of low-molecular-weight iron from total soluble iron. Abbreviations: FC, ferric citrate; Nano-α-Fe2O3, commercial bare α-Fe2O3 nanoparticles; FeSO4, ferrous sulfate; ANPs, in vitro-formed nano-sized ferric hydrolysis products stabilized by anchovy muscle protein hydrolysate; FC+AMPH, the mixture of ferric citrate and anchovy muscle protein hydrolysate. Values are means ± SD of a representative experiment (n = 8). Means in a column without a common superscript letter (a, b and c) are statistically different, P < 0.05.

538

28


Page 29 of 33

539


Figure 1

540

541

29



542

Figure 2

543 544

Page 30 of 33

30


Page 31 of 33

545


Figure 3

546

547

31



548

Page 32 of 33

Figure 4

549

550

32


Page 33 of 33

551


Table of Contents (TOC) Graphic

552

553

TOC. Enhancement of non-heme iron absorption in humans by fish possibly involves the

554

formation of intestinal nano-sized iron, which is likely to be apically absorbed by means of

555

divalent metal transporter-1 and endocytosis.

33


Enhancement of Non-heme Iron Absorption by Anchovy (Engraulis

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