Characterization of Key Factors of Anchovy (Engraulis japonicus) Meat

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Characterization of Key Factors of Anchovy (Engraulis japonicus) Meat in the Nanoparticle-Mediated Enhancement of Non-Heme Iron Absorption Yaqun Zou, Liang Zhao, Guangxin Feng, Yu Miao, Haohao Wu, and Ming-Yong Zeng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04547 • Publication Date (Web): 04 Dec 2017 Downloaded from http://pubs.acs.org on December 5, 2017

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Journal of Agricultural and Food Chemistry

Characterization of Key Factors of Anchovy (Engraulis japonicus) Meat in the Nanoparticle-Mediated Enhancement of Non-Heme Iron Absorption Yaqun Zou a,∥, Liang Zhao a,∥, Guangxin Feng a, Yu Miao b, Haohao Wu a,*, Mingyong Zeng a,*

a

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

b

Department of Clinical Laboratory, The Affiliated Hospital of Qingdao University, Qingdao, Shandong Province, 266003, China ∥

*

These authors contributed equally to this work.

Authors to whom correspondence should be addressed; E-mails:

[email protected] (Wu, H.); [email protected] (Zeng, M.); Tel.: +86-532-8203-2400 (Wu, H.); +86-532-8203-2783 (Zeng, M.).

1

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ABSTRACT

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Anchovy (Engraulis japonicus) meat (AM) has been shown to promote non-heme iron

3

absorption via a ferric oxyhydroxide nanoparticle (FeONP)-mediated mechanism. Here,

4

formulation modifications of an egg-white-based AIN-93G diet with AM fractions resulted

5

hemoglobin regeneration efficiencies in anemic rats following an order control (23.69 ±

6

3.99%)

7

de-haemed-AM-protein-replacement of egg white (45.88 ± 4.76%) ≈ AM-lipid-replacement

8

of soybean oil (43.14 ± 3.48%) ≈ chondroitin-sulfate-replacement of ~2.5% corn starch

9

(39.92 ± 1.88%) < L-α-phosphatidylcholine-replacement of ~29% soybean oil (53.42 ±

10

2.04%), with nano-sized iron enriched in proximal-small-intestinal contents by these AM

11

fractions. The calcein-fluorescence-quenching assay in polarized Caco-2 cells revealed good

12

iron absorption from FeONPs coated with AM peptides, L-α-phosphatidylcholine,

13

L-α-lysophosphatidylcholine, and chondroitin sulfate, with the latter two disfavoring

14

endocytosis thereby inducing relatively weaker iron absorption. These results suggest

15

peptides, phospholipids and mucopolysaccharides released during AM digestion are key

16

factors promoting non-heme iron absorption.

17

18

18 MΩ.cm) from a Milli-Q plus system (Merck

66

Millipore, Göttingen, Germany) was used throughout this work for all purposes such as

67

sample preparation, glassware rinsing and animal drinking. All glassware was rinsed with

68

10% HCl and water before used in the experiments to avoid mineral contamination.

69

Preparation of Protein and Lipid Fractions of Anchovy Meat. The protein fraction of

70

anchovy meat was obtained by a trichloroacetic acid (TCA)-acetone procedure.10 Briefly,

71

lyophilized anchovy meat was homogenized in 10 volumes (w/v) of ice-cold acid acetone

72

containing 10% TCA and 0.07% β-mercaptoethanol. The homogenate was kept at −20 °C for

73

24 h before being centrifuged at 8,000 g for 15 min at 4 °C. The pellet was washed at least

74

three times with 5 volumes of ice-cold acetone (w/v) to remove residual TCA, before being

75

further washed with 7 volumes (w/v) of 95% ethanol for the purpose of removing

76

phospholipid. The residual acetone and ethanol was removed by overnight evaporation in the

77

fume hood, and the pellet was then ground thoroughly to a very fine powder to produce the

78

anchovy meat protein (AMP).

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The lipid fraction of anchovy meat was prepared following the Bligh and Dyer method.11

80

Briefly, 20 g lyophilized anchovy meat was homogenized with a mixture of 80 mL water,

81

100 mL chloroform and 200 mL methanol for 2 min. After another 100 mL of chloroform

82

was added, the solution was re-homogenized for 30 s, and 100 mL water was added with 5



83

re-homogenization once again for 30 s. After suction filtration, the chloroform layer in the

84

final biphasic system was collected, and was evaporated to constant weight under nitrogen to

85

produce the anchovy meat lipid (AML).

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Animal Studies. Sprague-Dawley rats were obtained from Shandong Lukang

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Pharmaceutical Group Co., Ltd. (Shandong, China). Animals were housed in a

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light-controlled (light/dark cycle with 12 h/12 h) and temperature-controlled (20 ~ 25 °C)

89

room, and had ad libitum access to diet and Milli-Q water. All experiments were carried out

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ethically according to the principles of the Guide for the Care and Use of Laboratory Animals

91

of the Institute for Laboratory Animal Research of the National Research Council and were

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approved by the Committee on the Ethics of Animal Experiments of Ocean University of

93

China.

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Egg white-based, modified AIN-93G purified diets were supplied by TROPHIC Animal

95

Feed High-tech Co., Ltd. (Nantong, China) in pelletized form. The Fe-deficient diet

96

contained ∼10 mg/kg iron derived from raw materials. The Fe-sufficient diet contained ∼35

97

mg/kg iron in the form of ferric citrate (FC) and ∼10 mg/kg iron derived from raw materials.

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The Fe-repletion diets contained ∼10 mg/kg iron derived from raw materials and ∼25 mg/kg

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exogenously added iron. Ferric citrate was used as the exogenous iron source, except for the

100

ferrous sulfate (FeSO4) group in which FeSO4 was used instead. In the AMP diet, egg white

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solid in the formula was substituted with an equivalent amount of AMP. In the AML diet,

102

soybean oil in the formula was substituted with an equivalent amount of AML. The PC diet

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contained 20 g/kg PC in substitution for an equivalent amount of soybean oil in the formula. 6


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In the chondroitin sulfate group, 10 g/kg chondroitin sulfate was used to substitute an

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equivalent amount of cornstarch in the formula. Iron contents in the pelletized diets were

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measured using flame atomic absorption spectroscopy on a Shimadzu AA-6800 spectrometer.

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The iron bioavailability was evaluated by a hemoglobin (Hb) regeneration assay with

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anemic rats.12 Female rats (n = 15) breastfeeding male pups (16 days old; n = 68) were

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housed individually in stainless-steel cages with four to five pups each on the Fe-deficient

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diet ad libitum for a week. The pups were then weaned at 23 days old and transferred into

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individual stainless-steel cages. These just-weaned rats were randomly allocated to two

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groups: the Fe-depleted group (n = 64) on the Fe-deficient diet and the control group (n = 4)

113

on the Fe-sufficient diet. After a Fe-depletion period of 14 days, 20 µL of fresh rat blood was

114

sampled by tail vein incision, diluted with EDTA, and analyzed on a XN-9000 hematology

115

analyzer (Sysmex, Kobe, Japan). The mean Hb concentrations of the control group and the

116

Fe-depleted group were 122.8 ± 25.9 and 49.38 ± 9.0 g/L, respectively. Rats in the

117

Fe-depleted group were randomized into eight groups (eight rats each group) by stratification

118

on hemoglobin concentration and body weight. The rats in each group were fed on the

119

Fe-deficient diet or one of the five Fe-repletion diets, i.e. the FeSO4 diet (the positive

120

control), the AMP diet, the AML diet, the PC diet, and the chondroitin sulfate diet, for 13

121

days (the repletion period) ad libitum. The control group continued on the Fe-sufficient diet

122

throughout the repletion period. The experimental plan is shown in Figure 1.

123

Food intake of each rat was recorded manually every day during the repletion period.

124

Adequate diet with a known weight was given to each rat between 8:30 and 9:00 a.m., and in 7



125

the next day, the remained diet was reweighed to calculate daily food intake. Body weight

126

was also recorded for each rat at the beginning and end of the repletion period. Blood

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samples (20 µL) of all rats were once again taken by tail vein incision at the end of the

128

repletion period to determine the hemoglobin concentrations.

129 130

Assuming a blood volume of 6.7% body weight and an iron content of 0.335 in Hb, total Hb iron pool (mg) in rat was calculated as follows:12

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Hb iron pool (mg) = body weight (kg) × 0.067 × Hb concentration (g/L) × 3.35;

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The Hb regeneration efficiency was calculated as follows:

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Hb regeneration efficiency (%) = [final Hb iron pool (mg) − initial Hb iron pool (mg)] /

134

total iron consumed (mg) × 100.

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After blood sampling, all rats were fasted for 9 h and subsequently fed on corresponding

136

Fe-repletion diets for 30 minutes ad libitum. All animals were then anesthetized with ether

137

before an abdominal midline incision was performed. Their livers were removed from the

138

carcass, rinsed with saline, and stored at −20 °C. The small intestine was also taken, and the

139

content in the proximal one-third segment was flushed into a centrifuge tube with saline. The

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intestinal contents were further fractionated into two fractions, i.e. the 0.22 µm filtrate and

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the 3 kDa (≈ 1 nm) filtrate, using 0.22-µm polyethersulfone syringe filters (Jinteng, Tianjin,

142

China) and Amicon Ultra-15 centrifugal filter units with 3-kDa cut-off regenerated cellulose

143

membranes (Merck Millipore, Göttingen, Germany), respectively. Polyethersulfone and

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regenerated cellulose membranes are believed to bind little ferric and ferrous ions considering

145

their absence of strong acidic ligands. Iron concentrations in the liver, the intestinal content, 8


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the 0.22 µm filtrate and the 3 kDa filtrate were determined by flame atomic absorption

147

spectroscopy on a Shimadzu AA-6800 spectrometer.

148

Cellular Uptake Studies. Polarized Caco-2 cells were used for iron uptake experiments

149

according to Glahn et al. (1998).13 Caco-2 cells from the Cell Bank of the Chinese Academy

150

of Sciences (Shanghai, China) were incubated at 37 °C in a thermostat incubator with a

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humid atmosphere containing 95% air/5% CO2. High-glucose (4.5 g/L) DMEM

152

supplemented with 4 mM glutamine, 10% fetal bovine serum, 1 mM sodium pyruvate, 25

153

mM HEPES, and 1% penicillin-streptomycin mixture was used to routinely maintain the

154

cells, and renewed every 2 days.

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Cells were seeded at a density of 5 × 104 cells/cm2 in collagen-coated 24-well plates (BD

156

Biosciences, San Joes, CA, USA). After reaching confluence, the cells were maintained in

157

completed DMEM for another 12 days. The medium was then changed to a serum-free MEM

158

supplemented with 20 µg/L epidermal growth factor, 4 mg/L hydrocortisone, 5 mg/L insulin,

159

and 34 µg/L triiodothyronine, and after 24-h incubation, the medium was aspirated. The cells

160

were washed three times with HBSS, and stained with 1µM Calcein-AM in HBSS at 37 °C

161

for 30 minutes. After three time-washing with HBSS again, 970 µL Tyrode solution (137

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mM NaCl, 5.5 mM D-glucose, 2.7 mM KCl, 1.8 mM CaCl2, and 1 mM MgCl2) buffered with

163

20 mM MES (pH 5.5) or MOPS (pH 7.0), 10 µL water or BPDS (100 µM), and 20 µL

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sample solution containing 1 mM iron were sequentially added, and the plates were

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immediately incubated in a Synergy H4 hybrid microplate reader (Bio-Tek, Winooski, VT,

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USA) at 37 °C for 210 minutes, with calcein fluorescence (485 nm excitation, 530 nm 9



167

emission) recorded every 5 minutes. Finally, the assay solution was removed, and cell

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viability was measured using the MTT-reduction test according to Takahashi and Abe

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(2002).14

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The sample solutions of FeSO4 and FeONPs containing 1 mM iron were freshly prepared

171

before use. The simulated digestion of AMP and AML were performed as described in our

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previously study.9 The preparation of FeONPs was carried out by dropwise addition of

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freshly prepared 2 mM FeCl3 into equal volume of vortex-stirred MOPS buffer (60 mM, pH

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7.0) containing PC (5 g/L), LPC (2.5 g/L), chondroitin sulfate (0.2 g/L), the AMP digest (8

175

g/L), or the AML digest (4 g/L). The dynamic light scattering (DLS) measurements were

176

carried on a Zetasizer Nano ZS 90 (Malvern Instruments, Herrenberg, Germany) equipped

177

with a 633 nm He−Ne laser using a constant scattering angle of 90° at 25 ± 0.1 °C. For

178

transmission electron microscopy (TEM) characterization, sample solutions were dropped

179

onto carbon-coated copper grids, allowed to air-dry, and then examined with a JEM 1200 EX

180

(JEOL, Tokyo, Japan) electron microscope at 100 kV.

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Statistical Analysis. Statistical analyses were done using SPSS software version 19.0

182

(SPSS, Inc., Chicago, USA). Data are presented as means ± standard deviations. The mean

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differences were compared by one-way ANOVA with least significant difference (LSD). All

184

the differences were considered to be significant at P < 0.05.

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RESULTS AND DISCUSSIONS

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Details and results of the Hb regeneration assay in this study are displayed in Tables 1 and

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S1. Diet intake and weight gain during the repletion period did not differ between rats of the

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control group and any of the other five repletion groups (Table S1). The formulation

189

modifications in this study produced no apparent abnormal behaviors in the experimental

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rats.

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Iron in the body is mainly present in Hb (65%) and iron stores (i.e. ferritin and

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hemosiderin, 30%).15 During the repletion period, intestinally absorbed iron is generally used

193

for Hb repletion prior to iron storage.8 Iron contents of the liver, a main site of iron storage, in

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the repletion groups of control, FeSO4, AMP, AML and chondroitin sulfate were at the same

195

level (Figure 2), so iron bioavailabilities in these groups could be evaluated by comparing

196

their Hb regeneration efficiencies. A significantly higher Hb regeneration efficiency was

197

observed for the FeSO4 group, compared to the control group (Table 1), validating the better

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iron absorption from a diet fortified with the “gold standard” FeSO4 than with ferric citrate.

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The AMP group showed a Hb regeneration efficiency as high as that of the FeSO4 group

200

(Table 1), so in comparison with egg white, AMP seemed to enhance non-heme iron

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absorption. There are a variety of hemeproteins such as hemoglobin, myoglobin and

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sarcoplasmic enzymes (e.g. cytochromes and catalases) in the muscle tissue. Heme can be

203

removed from hemeproteins by acid acetone extraction.16 In this study, no heme was detected

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in AMP (data not shown), owning to the bulky acid acetone used during the preparation

205

process. The interference of heme iron should thus not be considered when evaluating

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non-heme iron bioavailability in the AMP group. 11



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The PC group showed the highest levels of both Hb regeneration efficiency and liver iron

208

concentration (Table 1 and Figure 2), indicating a high potential of PC to boost non-heme

209

iron absorption. There are a variety of food sources (e.g. seeds, eggs and marine fish) rich in

210

phospholipids. However, refined vegetable oils, which account for a majority of dietary fat in

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the industrialized countries,17 contain little phospholipids due to the degumming process

212

during oil refinery. Anchovy is a kind of small, pelagic marine fish, and AML has been

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previously found to contain a considerable amount of PC.9 The AML group displayed a

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significantly higher Hb regeneration efficiency than the control group (Table 1), so compared

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to refined soybean oil, AML facilitated non-heme iron absorption in the diet. The

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consumption of marine fish therefore seems to favor non-heme iron absorption at least in part

217

by means of phospholipids.

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Mucopolysaccharides (also called glycosaminoglycans) in muscle are mainly found in the

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connective tissue that binds muscle fibers together. There have been several studies showing

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the promoting effect of mucopolysaccharides on non-heme iron absorption in Caco-2

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cells.18-20 However, no such effect was observed in young women on meals supplemented

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with an amount of mucopolysaccharides expected to be present in 150 g beef muscle.21 The

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mucopolysaccharide content in muscle is generally very low,22 so one possible reason for the

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contradicting in vitro and in vivo results is that the amount of mucopolysaccharides used in

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the in vivo study was too low. In this study, about 1/40 of corn starch in the AIN-93G

226

formulation was replaced by chondroitin sulfate, a predominant muscle mucopolysaccharide,

227

in the chondroitin sulfate diet. This diet induced a significantly higher Hb regeneration 12


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efficiency than that the control group (Table 1), so in comparison with corn starch, an

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adequate amount of chondroitin sulfate could markedly promote non-heme iron absorption.

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In fact, mucopolysaccharides are abundant in food sources like tendons, ligaments and

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cartilage;22 nevertheless, these animal tissues are largely excluded from the modern diet

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which includes a large amount of processed meat, especially in the Western world and newly

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industrialized countries.17 The inclusion of more such tissues in the diet may therefore help to

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promote dietary non-heme iron absorption.

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As shown in Table 2, iron speciation in the luminal content of proximal small intestine,

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where iron absorption mostly occurs,23 was analyzed to provide information on the

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mechanism of iron absorption. Iron in the 0.22 µm and 3 kDa filtrates of luminal contents

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were regarded as total soluble iron and low-molecular-weight (LMW) iron, respectively.

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Apart from LMW iron, the remaining part of total soluble iron was supposed to be nanosized

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colloidal iron.9 The control group displayed a similar level of total soluble iron as the

241

chondroitin sulfate group; nevertheless, iron bioavailability from the control diet was

242

remarkably lower than that from the chondroitin sulfate diet (Table 1). In comparison with

243

the control group, a significantly higher level of nanosized iron was observed in the

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chondroitin sulfate group (Table 2), which could be owning to the very high capacity of

245

chondroitin sulfate in mediating the formation of FeONPs,9 so chondroitin sulfate facilitated

246

iron absorption most likely by promoting the intraluminal formation of FeONPs.

247

The control, AML and PC groups had a similar level of LMW iron, but their levels of

248

nanosized iron followed the order of control < AML < PC (Table 2), corresponding to that of 13



249

iron bioavailabilities in these groups, so AML and PC seemed to promote non-heme iron

250

absorption by mediating the formation of more intraluminal FeONPs. In fact, in our

251

previously study, the simulated digests of AML and PC showed high capacities to mediate

252

the formation of FeONPs via phospholipid-based mixed micelles.9 Peptides in the simulated

253

digest of AMP have also displayed a considerable potential to mediate the formation of

254

FeONPs.9 In this study, the AMP group had a higher level of nanosized iron than the control

255

group, with their levels of LMW iron being similar (Table 2), so FeONPs should also

256

contribute to AMP’s enhancement of non-heme iron absorption.

257

Peptides, phospholipid-based mixed micelles and mucopolysaccharides may endow

258

distinct surface properties of FeONPs, and thereby influence the bioavailability and cellular

259

uptake mechanism of FeONPs. Beside endocytosis, a major cellular uptake pathway of

260

nanoparticles, FeONPs could also been absorbed via the divalent transporter 1 (DMT-1)

261

pathway after being reduced into Fe2+ ions by duodenal cytochrome b.8 In this study, iron

262

uptake from FeONPs in the calcein-loaded polarized Caco-2 cells was assayed based upon

263

the quenching of calcein fluorescence following iron influx into the labile iron pool, and

264

endocytosis was characterized by blocking the DMT-1 pathway with BPDS, a strong ferrous

265

chelator. Figures 3 and 5 show the kinetics of cellular calcein fluorescence in mediums with

266

an initial concentration of 20 µM iron in the form of FeSO4 or FeONPs. They generally

267

followed a negative exponential patter (r2 > 0.99) except for some cases in neutral medium.

268

BPDS was found to completely block iron uptake from FeSO4, validating its effectiveness in

269

antagonizing DMT-1 dependent iron absorption. At the end of the iron uptake experiment, no 14


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significant reduction of cell viability was observed for all of these FeONPs according to the

271

results of MTT assay (Figure S1), suggesting no acute toxicity of these FeONPs in polarized

272

Caco-2 cells.

273

Dietary iron is mainly absorbed through the duodenum,23 where after a meal, the median

274

pH falls to about 5.5 then lasting for at least 4 h of postprandial period.24 As described in our

275

previous report, FeONPs templated by the digests of AMP and AML have been prepared in

276

vitro and characterized by DLS and TEM.9 The iron oxyhydroxide cores of FeONPs

277

templated by the digests of AMP and AML had sizes of 15−30 nm and 20−30 nm,

278

respectively, and the AMP-digest-mediated FeONPs displayed a higher tendency to form

279

aggregates than the AML-digest-mediated ones.9 In this study, at the duodenum-like pH of

280

5.5, there was no significant difference in cellular iron uptake between the

281

AMP-digest-mediated FeONPs and the AML-digest-mediated ones, both of which induced

282

calcein fluorescence quenching as high as over 40% of that induced by FeSO4, and a larger

283

extent of endocytosis was observed for the former (Figure 3a). In contrast, at the extracellular

284

pH of 7.0, iron absorption from the AMP-digest-mediated FeONPs was a little weaker than

285

that from the AML-digest-mediated ones (P < 0.05), with a larger extent of endocytosis

286

observed for the AML-digest-mediated FeONPs instead (Figure 3b). Less robust iron

287

absorption was observed for FeSO4 compared to these FeONPs in neutral medium (Figure

288

3b), possibly owning to the fast autooxidation of Fe2+ at pH ≥ 6.0.25 These results indicate

289

that FeONPs coated with peptides and phospholipid-based mixed micelles can be well

290

absorbed in the small intestine via both the endocytosis and DMT-1 pathways. 15



291

In order to characterize the respective effects of PC and LPC in the coatings on iron

292

absorption from FeONPs, commercially available purified PC and LPC were used to prepare

293

FeONPs. DLS results for PC and LPC in aqueous solutions buffered with MOPS (pH 7.0)

294

before and after the addition of FeCl3 are shown in Figures 4a and 4b, respectively. The blank

295

samples of PC and LPC displayed scattering signals around 164 nm and 142 nm, respectively,

296

which should be owning to the presence of PC vesicles and LPC multilamellar aggregates in

297

these systems.26,27 After addition and hydrolysis of FeCl3, FeONPs formed in the presence of

298

PC and LPC displayed hydrodynamic sizes smaller than those of vesicles or aggregates in the

299

blank samples. FeONPs templated by PC displayed only one DLS peak at 91 nm, while those

300

templated by LPC gave two maximum DLS peaks at 21 and 59 nm, respectively. TEM

301

imaging revealed well dispersed FeONPs templated by PC and LPC with size ranges of 40-65

302

nm and 3-9 nm, respectively (Figures 4c and 4d). The DLS peak of PC-mediated FeONPs

303

seem to reflect the hydrodynamic diameters of monodispersed FeONPs in the system. The

304

first and second DLS peaks of LPC-mediated FeONPs were probably scattering signals from

305

monodispersed and agglomerated particles, respectively. PC and LPC might form vesicle-like

306

membrane structures around FeONPs, and compared to PC, LPC usually forms more curved

307

structures,25 which could be the reason for the smaller FeONPs templated by LPC. The

308

LPC-mediated FeONPs seemed to be more adhesive than the PC-mediated ones, so that DLS

309

signals of agglomerated particles were observed for the former rather than the later.

310

At the extracellular pH of both 5.5 and 7.0, iron from the PC-mediated FeONPs was better

311

absorbed than that from the LPC-mediated ones (Figures 5a and 5b). In acidic medium, 16


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endocytosis and DMT-1 almost equally contributed to the cellular iron uptake from

313

PC-mediated FeONPs (Figure 5a), while in neutral medium, iron from the PC-mediated

314

FeONPs was absorbed almost completely via endocytosis (Figure 5b). There was no and a

315

minor contribution of endocytosis to cellular iron uptake from the LPC-mediated FeONPs in

316

mediums at the pH of 5.5 and 7.0, respectively (Figures 5a and 5b). Apparently, the PC

317

coating favored the endocytic uptake of FeONPs especially under near neutral conditions,

318

while the LPC coating on FeONPs disfavored endocytosis particularly in acidic medium.

319

Unlike PC, LPC is a kind of nonbilayer-forming lipid, and its incorporation into cellular

320

membrane has been shown to create a positive curvature to prevent the endocytic process.28,29

321

The fusion of LPC coating with cellular membrane may occur during the interaction of

322

FeONPs with apical microvillus, which might therefore inhibit the endocytic pathway of iron

323

absorption from the LPC-mediated FeONPs.

324

FeONPs templated by chondroitin sulfate were found to precipitate at pH 5.5 in this study

325

(data not shown), probably because the induced deprotonation of the alcoholic OH groups

326

cannot take place at pH ≤ 6.0.30 Therefore, chondroitin sulfate might aid the solubilization of

327

ferric iron only under near neutral conditions like those in the jejunum.31 At the extracellular

328

pH of 7.0, there was no contribution of endocytosis to cellular iron uptake from the

329

chondroitin sulfate-mediated FeONPs (Figure 5b), so the chondroitin sulfate coating seems to

330

disfavor the endocytic uptake of FeONPs. Chondroitin sulfate has been shown to specifically

331

bind to PC in cellular membrane, and this restricts the lateral diffusion of PC,32 probably

332

thereby blocking the endocytic process. 17



333

In

summary,

the

present

study

indicates

that

peptides,

Page 18 of 33

phospholipids,

and

334

mucopolysaccharides released during the digestion of muscle foods should serve as key

335

“meat factors” enhancing non-heme iron absorption. We posit that, in the small intestine,

336

these “meat factors” mediate the formation of FeONPs, iron from which can be readily

337

absorbed by intestinal epithelial cells via DMT-1 or endocytosis. Peptides and PC in the

338

coatings are more effective in aiding iron absorption from FeONPs than LPC and chondroitin

339

sulfate, owning to endocytosis inhibition by the latter ones. Our study, for the first time,

340

reports a particularly good activity of dietary PC in promoting iron absorption, which might

341

be inspiring for the application of PC in preventing or correcting iron deficiency anemia.

342

ABBREVIATIONS USED

343

AMP,

344

bathophenanthrolinedisulfonic acid; DMT-1, the divalent transporter 1; FeONPs, ferric

345

oxyhydroxide nanoparticles; Hb, hemoglobin; LPC, L-α-lysophosphatidylcholine; LMW,

346

low molecular weight; PC, L-α-phosphatidylcholine; TEM, transmission electron

347

microscopy.

348

Supporting Information

349

This material is available free of charge via the Internet at http://pubs.acs.org.

350

Supplementary Table S1. Detailed Results of the Hb Regeneration Assay with Anemic Rats.

351

Supplementary Figure S1. The viabilities of polarized Caco-2 cells following the experiments

352

of iron uptake from FeONPs templated by the simulated digests of anchovy meat lipid

the

anchovy

meat

protein;

AML,

the

anchovy

18


meat

lipid;

BPDS,

Page 19 of 33


353

(AML),

354

L-α-lysophosphatidylcholine (LPC) and chondroitin sulfate (CS).

355

356

(1) Lopez, A.; Cacoub, P.; Macdougall, I. C.; Peyrin-Biroulet, L. Iron deficiency anaemia.

357 358 359

anchovy

meat

protein

(AMP),

L-α-phosphatidylcholine

(PC),

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particles in iron (III)-polysaccharide solutions. J. Inorg. Biochem. 1995, 58, 129−138.

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(31) Fuchs, A.; Dressman, J. B. Composition and physicochemical properties of fasted-state

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human duodenal and jejunal fluid: a critical evaluation of the available data. J. Pharm.

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Sci. 2014, 103, 3398−3411.

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(32) Satoh, A.; Toida, T.; Yoshida, K.; Kojima, K.; Matsumoto, I. New role of

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glycosaminoglycans on the plasma membrane proposed by their interaction with

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phosphatidylcholine. FEBS Lett. 2000, 477, 249−252.

444

Notes

445

This work was financially supported by National Natural Science Foundation of China (No.

446

31371758).

447

23



448

Figure captions

449

Figure 1. Schematic representation of the experimental plan. The variable n represents the

450

animal number assigned to a treatment group. Abbreviations: AML, the anchovy meat lipid;

451

AMP, the anchovy meat protein; CS, chondroitin sulfate; Hb, hemoglobin; PC,

452

L-α-phosphatidylcholine.

453

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

454

AML, the anchovy meat lipid; AMP, the anchovy meat protein; CS, chondroitin sulfate; LPC,

455

L-α-lysophosphatidylcholine; PC, L-α-phosphatidylcholine.

456

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

457

the form of FeSO4 or FeONPs templated by the simulated digests of anchovy meat fractions

458

at extracellular pH values of (a) 5.5 and (b) 7.0. Abbreviations: AML, the anchovy meat lipid;

459

AMP, the anchovy meat protein; BPDS, bathophenanthrolinedisulfonic acid; FeONPs, ferric

460

oxyhydroxide nanoparticles. Iron uptake was measured according to the quenching of cellular

461

calcein fluorescence following iron influx into the labile iron pool. BPDS is a strong ferrous

462

chelator used to antagonize the divalent metal transportor-1 pathway. Data are presented as

463

means of a representative experiment (n = 3) with standard deviations of less than 1.6.

464

Figure 4. Transmission electron microscopy images (a, b) and dynamic light scattering

465

measurements (c, d) of ferric oxyhydroxide nanoparticles synthesized in the presence of (a, c)

466

L-α-phosphatidylcholine (5 g/L) and (b, d) L-α-lysophosphatidylcholine (2.5 g/L) in a 30 mM

467

MOPS buffer at pH 7.0 with a FeCl3-loading concentration of 1 mM.

24


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Page 25 of 33


468

Figure 5. Iron uptake by polarized Caco-2 cell monolayers in the presence of 20 µM iron in

469

the form of FeSO4 or FeONPs templated by commercially available purified

470

L-α-phosphatidylcholine,

471

extracellular pH values of (a) 5.5 and (b) 7.0. Abbreviations: PC, L-α-phosphatidylcholine;

472

LPC,

473

bathophenanthrolinedisulfonic acid; FeONPs, ferric oxyhydroxide nanoparticles. Iron uptake

474

was measured according to the quenching of cellular calcein fluorescence following iron

475

influx into the labile iron pool. BPDS is a strong ferrous chelator used to antagonize the

476

divalent metal transportor-1 pathway. Data are presented as means of a representative

477

experiment (n = 3) with standard deviations of less than 1.6.

L-α-lysophosphatidylcholine

L-α-lysophosphatidylcholine;

CS,

and

chondroitin

chondroitin

25


sulfate;

sulfate

at

BPDS,


Page 26 of 33

Table 1. Details and Results of the Hb Regeneration Assay with Anemic Rats. Diet iron

Exogenous iron

Diet protein

Diet lipid

Diet starch

Hb regeneration

(mg/kg)

compound

(200 g/kg)

(70 g/kg)

(397.5 g/kg)

efficiency (%) b

8

9.0 ± 2.2

−

Egg white

Soybean oil

Corn starch

−

FeSO4

8

30.6 ± 3.7

FeSO4

Egg white

Soybean oil

Corn starch

53.34 ± 3.77b

Control

8

43.0 ± 2.8

Ferric citrate

Egg white

Soybean oil

Corn starch

38.21 ± 9.14c

AMP

8

37.5 ± 1.4

Ferric citrate

AMP

Soybean oil

Corn starch

48.41 ± 7.44b

AML

8

31.3 ± 1.3

Ferric citrate

Egg white

AML

Corn starch

50.91 ± 5.86b

PC

8

29.3 ± 0.9

Ferric citrate

Egg white

Corn starch

65.91 ± 6.65a

CS

8

33.1 ± 2.7

Ferric citrate

Egg white

Soybean oil

Fe-sufficient group

4

46.0 ± 3.3

Ferric citrate

Egg white

Soybean oil

N Fe-deficient group

Fe-repletion groups a

a

PC/Soybean oil (w/w, 2/5)

CS/Corn starch (w/w, ~1/39) Corn starch

47.56 ± 7.54b −

Abbreviations: FeSO4, ferrous sulfate; AMP, anchovy meat protein; AML, anchovy meat lipid; PC,

L-α-phosphatidylcholine; CS, chondroitin sulfate. b

Values are means ± standard deviations. Means in a column without a common superscript letter (a, b

and c) are statistically different, P < 0.05.

26


Page 27 of 33


Table 2. Iron Speciation in the Luminal Contents of Proximal Small Intestine. Group

a

percentage contents of intestinal iron species b total soluble iron c low-molecular-weight iron d

nanosized iron e

Control

25.26 ± 9.51cd

17.13 ± 9.35b

8.13 ± 3.55c

FeSO4

53.48 ± 10.37ab

29.30 ± 1.58a

24.18 ± 11.95b

AMP

36.60 ± 3.99c

18.64 ± 9.78ab

17.97 ± 5.79b

AML

49.96 ± 0.48b

17.24 ± 6.56b

32.72 ± 6.08ab

PC

63.82 ± 12.68a

17.19 ± 1.74b

46.62 ± 14.42a

CS 26.08 ± 4.44d 5.26 ± 2.06c 20.82 ± 6.50b a Abbreviations: FeSO4, ferrous sulfate; AMP, anchovy meat protein; AML, anchovy meat lipid; PC, L-α-phosphatidylcholine; CS, chondroitin sulfate. b Values are means ± standard deviations. Means in a column without a common superscript letter (a, b, c and d) differ by P < 0.05. c Iron in the 0.22 µm filtrate. d Iron in the 3 kDa filtrate. e Calculated by subtraction of low-molecular-weight iron from total soluble iron.

27



Figure 1

28


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Page 29 of 33


Figure 2

29



Figure 3

30


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Page 31 of 33


Figure 4

31



Figure 5

32


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Graphic for table of contents

33


Characterization of Key Factors of Anchovy (Engraulis japonicus) Meat

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