Evaluation of Iodine Bioavailability in Seaweed Using in Vitro

Subscriber access provided by UNIVERSITY OF CONNECTICUT

Article

EVALUATION OF IODINE BIOAVAILABILITY IN SEAWEED USING IN VITRO METHODS Raquel Domínguez González, Gabriela M Chiocchetti, Paloma Herbello-Hermelo, D. Velez, V. Devesa, and Pilar Bermejo-Barrera J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02151 • Publication Date (Web): 30 Aug 2017 Downloaded from http://pubs.acs.org on August 30, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 33

Journal of Agricultural and Food Chemistry

EVALUATION OF IODINE BIOAVAILABILITY IN SEAWEED USING IN VITRO METHODS

M. Raquel Domínguez-González#, Gabriela M. Chiocchetti&, Paloma Herbello-Hermelo#, Dinoraz Vélez&, Vicenta Devesa&, Pilar Bermejo*#

#

Department of Analytical Chemistry, Nutrition and Bromatology, Faculty of Chemistry,

Health Research Institute of Santiago de Compostela (IDIS), Universidade de Santiago de Compostela, 15782 – Santiago de Compostela, Spain &

Instituto de Agroquímica y Tecnología de Alimentos (IATA-CSIC), Av. Agustín Escardino

7, 46980 Paterna (Valencia), Spain.

Corresponding Author: ORCID iD: 0000-0001-5864-6144 *

e-mail adress: [email protected] (Pilar Bermejo-Barrera)

Telephone Number: 34600942346 Fax Number: 34981547141

E-mail adresses: M. Raquel Domínguez-González: [email protected] Gabriela M. Chiocchetti: [email protected] Paloma Herbello-Hermelo: [email protected] Dinoraz Vélez: [email protected] Vicenta Devesa: [email protected]

1

ACS Paragon Plus Environment


1

ABSTRACT

2

Due to the high levels of iodine presents in seaweed the ingestion of large amount of this type

3

of food can produce excessive intake of iodine. However, the food after ingestion suffers

4

different chemistry and physical processes that can modify the amount of iodine that reaches

5

the systemic circulation (bioavailability).Studies on the bioavailability of iodine from food are

6

scarce and indicate that the bioavailable amount is generally lower than ingested. Iodine in

7

vitro bioavailability estimation from different commercialized seaweed has been studied using

8

different in vitro approaches (solubility, dialyzability and transport and uptake by intestinal

9

cells). Results indicate that iodine is available after gastrointestinal digestion for absorption

10

(bioaccessibility: 49-82%), being kombu the seaweed with the highest bioaccessibility. The

11

incorporation of dialysis cell cultures to elucidate bioavailability modifies the estimation of

12

the amount of iodine that may reach the systemic circulation (dialysis: 5-28%; cell culture: ≤

13

3%). The paper discusses advantages and drawbacks of these methodologies for iodine

14

bioavailability in seaweed.

15

Keywords: seaweed, iodine, solubility, dialyzability, bioavailability, Caco-2 cells, HT29-

16

MTX cells.

17

2


Page 2 of 33

Page 3 of 33


18

INTRODUCTION

19

Iodine is an essential element, necessary for synthesis of the thyroid hormones, thyroxine (T4)

20

and triiodothyronine (T3). It is found in nature in various chemical forms: inorganic salts

21

(iodides and iodates), inorganic diatomic iodine and organic mono or diatomic iodine.

22

Deficiencies in iodine, which affects about two thousand million people worldwide, cause

23

endemic goitre, the most visible sign, and central nervous system damage, causing mental

24

retardation in children1.

25

For the general population the major source of iodine is food, being seafood products

26

the type of food with the highest content. Concentration of iodine in fish may reach up to 15

27

mg/kg fresh weight (fw)2; whereas in shellfish vary between 0.32 and 135 mg/kg fw3,4.

28

Seaweeds have an inherent biologic capacity to concentrate iodine from the sea, consequently,

29

concentrations up to 6138 mg/kg (dry weight, dw) have been found in commercialized

30

samples5,6,7. Milk and dairy products may also contain relevant concentrations of iodine

31

(milk: 0.017-0.365 mg/kg; dairy products: 0.044-1.36 mg/kg)8. In fact, due to their high

32

consumption, they are the most important source of iodine in many countries9,10. In addition

33

to dietary sources, various mineral supplements and medical preparations can further increase

34

iodine intake.

35

Most studies identify deficiencies in iodine intake11, however, in some cases high

36

intakes above the recommended values have also been described. In certain susceptible

37

population groups (individuals with pre-existing thyroid disease, the elderly, fetuses and

38

neonates or patients with other risk factors), ingestion of iodine above the recommended

39

levels might increase the risk of developing iodine-induced thyroid dysfunction12. Moreover,

40

it has been shown that chronic exposure to high levels of iodine also affects healthy adult and

41

child population13.The ingestion of large amounts of seaweed, marine fish, ground beef

42

containing thyroid tissue, iodized water, bread, salt and iodide-containing dietary supplements 3



43

can produce excessive intake of iodine14. Daily iodine intake from nori, wakame and kelp in

44

Japanese population can be estimated at 1.2 to 1.3 mg/day15, much higher than the daily

45

intake recommended by the Scientific Committee on Food of the European Commission(600

46

µg/day14). Yeh et al.6 also show that a regular consumption of brown seaweed kombu (0.76-

47

0.96 g) by Taiwanese population entails high iodine intakes (up to 4.8mg/day).

48

Assessment of exposure to iodine is carried out in most cases by performing an

49

evaluation of the contents of the element in foodstuffs. However, the food after ingestion

50

suffers different processes that can modify the quantity of iodine that finally reaches the

51

systemic circulation. The digestion process allows the release of iodine from food in the

52

lumen where it may interact with other dietary compounds forming complexes that could

53

affect its subsequent intestinal absorption. Furthermore, the absorption may be modified by

54

competition of iodine and other elements or compounds in the food matrix or diet for the

55

same transport mechanisms. Studies on the amount of iodine from food that may reach the

56

systemic circulation (bioavailability),16,17are scarce and indicate that the bioavailable amount

57

is generally lower than that ingested. It is therefore necessary to consider this variable in the

58

exposure assessment of iodine through food products.

59

The aim of this study is to estimate the bioavailability of iodine present in different

60

types of seaweed. For this purpose, the iodine solubility and dialyzability values after a

61

simulated gastrointestinal digestion, and the iodine transport and uptake by intestinal

62

epithelial cells (co-cultures Caco-2/HT29-MTX) have been evaluated.

63

MATERIALSANDMETHODS

64

Instrumentation. Perkin Elmer Nex-IonTM 300X Inductively Coupled Plasma Mass

65

Spectrometer (ICP-MS) (Massachusetts, USA) equipped with a SC2 DX autosampler from

66

Elemental Scientific (Omaha, USA) and Ethos Plus microwave laboratory station from

67

Milestone(Cinisello Balsamo, MI, Italy) with 100 mL closed Teflon vessels and Teflon 4


Page 4 of 33

Page 5 of 33


68

covers, HTC adapter plate and HTC safety springs (Milestone,Cinisello Balsamo, MI, Italy)

69

were used for iodine determination. Cellu Sep H1 high grade regenerated cellulose tubular

70

membranes (molecular weight cut-off 10 kDa, 50 cm length, diameter dry 25.5 mm and a

71

volume to length ratio of 5.10 mL/cm) from Membrane Filtration Products Inc. (Seguin, TX,

72

USA)were used for dialyzability assays. Six-well plates with polyester membrane inserts (24

73

mm diameter, pore size 0.4 µm, Transwell®, Corning, Cultex,NY, USA) and Millicell®-ERS

74

voltohmmeter (Millipore Corporation, Massachusetts, USA) were employed for transport and

75

uptake cell assay. A freezing point osmometer, Automatic Micro-Osmometer Type 15 Löser,

76

Löser Messtechnik, Germany) was used for adjusting osmolarity of the bioaccesible fractions.

77

Other equipment included: Boxcult incubator situated on a Rotabit orbital-rocking platform

78

shaker (Selecta, Barcelona, Spain); ORION 720A plus pH-meter with a glass–calomel

79

electrode (ORION, Cambridge, UK);cellulose acetate syringe filters (0.45 µm) (Millipore,

80

Massachusetts, USA);fluorescence microplate reader (PolarSTAR OPTIMA, BMG-Labtech,

81

Germany); PowerWave HT microplate scanning spectrophotometer (BioTek Instruments,

82

Vermont, USA), MilliQ water-purification system (Millipore, Massachusetts,USA) for ultra-

83

pure water (resistivity 18MΩcm) collection.

84

Chemicals. For the alkaline solubilisation of seaweed tetramethylammonium

85

hydroxide (TMAH)25% (m/m) from Merck (Germany) in water was used. Digestive enzymes

86

(porcine pepsin and pancreatin), bile salts (approx. 50% sodium cholate and 50%sodium

87

deoxycholate) and piperazine-N,N’-bis(2-ethanesulfonicacid) disodium salt (PIPES) were

88

obtained from Sigma Chemicals(Madrid, Spain). HCl and NaCl were acquired from Panreac

89

(Barcelona, Spain) and ammonium hydrogen carbonate from Merck (Darmstad, Germany).

90

Standard solutions of I-and IO3-were prepared from potassium iodide (99.5%) and potassium

91

iodate (99.7-100.4%), both from Merck (Darmstad, Germany). Standard stock solutions of

92

1000µg/mL of iodinated amino acids 3-iodo-L-tyrosine (MIT) and 3,5-diiodo-L-tyrosine 5



Page 6 of 33

93

dehydrate (DIT) (both from Sigma-Aldrich-Fluka, Madrid, Spain) were prepared dissolving

94

0.1g of each compounds in 100mL of 0.01M NaOH/MeOH (1:1). The NIES-09, Sargasso, a

95

certified reference material of brown seaweed was supplied by the National Institute of

96

Environmental Studies (Ibaraki, Japan).

97

The Caco-2 cell line (human colon carcinoma) was acquired from the European Collection of

98

Cell Cultures (ECACC, number 86010202, Salisbury, UK). The HT29-MTX cell line was

99

kindly provided by Dr. Técla Lesuffleur (Institut National de la Santé et de la Recherche

100

Médicale, INSERM UMR S 938, Paris, France).All reagents for maintaining the cell culture

101

and their transport assays were provided by Hyclone (Fisher, Madrid, Spain).Dulbecco’s

102

modified Eagle’s medium (DMEM) containing 4.5 g/L glucose and 0.87 g/L glutamine; fetal

103

bovine serum (FBS); non-essential amino acids (NEAA); sodium pyruvate; N-2-

104

hydroxyethylpiperazine-N′-2-ethanesulfonic

105

solution;amphotericin B;tripsyn/EDTA (ethylene diamine tetraacetic acid) solution;minimum

106

essential medium (MEM); Hanks’ balanced salt solution (HBSS);PBS free of Ca2+ and Mg2+;

107

HBSS free of Ca2+ and Mg2+. Sodium resazurin (7-hydroxy-3H-phenoxazin-3-one-10-oxide

108

sodium salt), bovine serum albumin (BSA) and lucifer yellow(LY) were acquired from Sigma

109

(Madrid, Spain).Bio-Rad Protein Assay kit for protein determination was obtained from

110

Biorad (California, USA).

111

acid

(HEPES);

penicillin/streptomycin

Seaweed samples. Three species of brown algae were purchased in food stores of included species of brown algae: Undaria pinnatifida (wakame), Hizikia

112

Valencia(Spain),they

113

fusiforme (hijiki) and Laminaria japonica (kombu). The samples were cookedin MilliQ

114

water;20g of seaweed sample (dried weigh (dw)) and 500g of MilliQ water were boiled

115

according to the instructions indicated in the food packaging. Samples were ground and store

116

at -20ºC until analysis.

117 6


Page 7 of 33


118

Microwave assisted alkaline digestion (MAE) procedure. This procedure was

119

developed previously by our research group 5 .The ICP-MS instrumental operating conditions

120

are show in table 1.

121

In vitro digestion procedures

122

Solubility assays. The simulated in vitrodigestion usedwas a modification of the

123

procedure described by Laparra et al.18. Cooked seaweed sample (0.25 g) were weighed in

124

Erlenmeyer flask, 20 g of ultrapure water was added and 6 mol/L HCl was added until to

125

obtain at pH 2. Gastric solution (10% m/v pepsin in 0.1 mol/L HCl) was added to provide 1

126

mg of pepsin/g sample. Afterwards ultrapure water was added sample up to 25 g, and the

127

sample was incubated in a shaking water bath (120 strokes/min) at 37 °C for 2 h.

128

Subsequent the pH was increases to pH 6.5 using 1 mol/LNH4HCO3. The intestinal solution

129

(0.4% m/v pancreatin and 2.5% m/v bile extract in 0.1 mol/L NH4HCO3) was added to

130

obtain0.25 mg of pancreatin and 1.5 mg of bile extract per gramof seaweed, and again the

131

sample was incubated at 37 °C for 2 h. After the intestinal step, the pH was adjusted to 7.2

132

using 0.5mol/LNH4OH. In order to separate the soluble (bioaccessible) phase and the

133

residue,the digest was transferred to a polypropylene centrifuge tube and centrifuged at 10000

134

rpm for 30 minutes at 4°C.Both fractions,soluble and residual, were kept at -20 ºC before

135

measurements. In vitro digestion assays were performed by triplicate. Reagent blanks were

136

also obtainedto control possible contamination.

137

Iodine solubility was determined using the following equation (Equation1):

138

Solubility = [A/B] × 100(Equation1)

139

where A is the concentration of iodine in the soluble fraction after application of the in vitro

140

digestion, and B is the concentration of iodine in the cooked seaweed.

7



141

Dialyzability assays. To evaluate the iodine dialyzability, Cellu Sep® H1 high grade

142

regenerated cellulose tubular membranes were used. At the beginning of the intestinal stage of

143

seaweed, the dialysis membranes filled with 20 mL of a 0.15 N PIPES solution (pH 7.5

144

adjusted with HCl) were placed inside the flasks. After intestinal digestion, membranes were

145

removed and their outer surface was rinsed with ultra-pure water and the membrane

146

containing solution (dialyzate) and the residual (non-dialyzable fraction) were transferred to

147

polyethylene vials and separately weighted.Both, dialyzate and residual fractions, were kept at

148

-20 ºC until measurements.

149

Dialyzability of iodine species standards (species found in the seaweeds under study),I-

150

30µg/mL, MIT 0.4 µg/mL and DIT 6µg/L,was also evaluated. Firstly, dialysis membranes

151

filled with PIPES solution were placed inside aqueous solutions of iodine species (25 mL).

152

After 2 h, membranes were removed and dialyzatefractions were analyzed. Afterwards, the

153

assay was performed subjecting the standards of the iodine species to the gastrointestinal

154

digestion in presence of the membranes, following the protocol described above for seaweeds.

155

Iodine bioaccessibility expressed as dialyzability (D) was calculated using the following

156

equation(Equation2): 157

D (%) =

[ I ] dialyzate [ I ]total

x100 ( Eq.2) 158

159

where [I]dialyzateand [I]total are the iodine concentrations in the dialyzate fraction.

160

Cell transport and uptake assays

161

Cell culture maintenance and seeding. Caco-2 cells were cultured in DMEM

162

supplemented with 10% (v/v) FBS, 1% (v/v) NEAA, 1 mM sodium pyruvate, 10 mM HEPES,

163

100 U/mL of penicillin, 0.1 mg/mL of streptomycin and 0.0025 mg/L of amphotericin B

164

(DMEMc). The maintenance of HT29-MTX cells was done in DMEM supplemented with

8


Page 8 of 33

Page 9 of 33


165

10% (v/v) FBS, 1 mM sodium pyruvate, 10 mM HEPES, 100 U/mL of penicillin, 0.1 mg/mL

166

of streptomycin and 0.0025 mg/L of amphotericin B (HT-DMEMc).

167

The cells were incubated at 37 °C at 95% relative humidity and a CO2 flow of 5%. The

168

medium was changed every 3 days. When the cell monolayer reached 80% confluence, the

169

cells were detached with a solution of trypsin (0.5 g/L) and EDTA (0.22 g/L) and reseeded at

170

a density of 5–6 × 104 cells/cm².

171

All the transport assays were carried out in 6-well plates with polyester membrane inserts

172

(Transwell). The cells resuspended in HT-DMEMc were seeded (5.5 × 104 cells/cm², 1.5

173

mL) on the apical side to produce co-cultures of Caco-2/HT29-MTX (80/20). Then 2 mL of

174

HT-DMEMc was added to the basolateral chamber and cells were maintained in conditions

175

described before (11–12 days post-seeding). Throughout cell differentiation, transepithelial

176

electrical resistance (TEER) was measured with a Millicell®-ERS voltohmmeter to evaluate

177

the progress of the monolayers.Caco-2 cells wereused between passages 11 and 20 and HT29-

178

MTX cells between passages 15 and 24.

179

Cell viability assays. In order to work under sublethal conditions, the effect of the

180

iodine species present in seaweed on the viability of Caco-2 and HT29-MTX cells was

181

evaluated using sodium resazurin (Sigma). Cells were seeded in 24-well plates at a density of

182

2.5×104 cells/cm2with 1 mL of DMEMc or HT-DMEMc, depending on the cell type. After

183

differentiation took place, the cells were exposed for 24 h to various concentrations of I-(1,

184

10, 100 and 1000 mg/L), IO3- (1, 10, 100 and 1000 mg/L), MIT(0.1, 1, 5, 10 mg/L) and

185

DIT(0.1, 1, 5, 10 mg/L) prepared in MEM supplemented with 1 mM sodium pyruvate, 10 mM

186

HEPES, 100 U/mL of penicillin, 0.1 mg/mL of streptomycin and 0.0025 mg/L of

187

amphotericin B. After exposure, cells were incubated with resazurin solution following the

188

protocol described by Rocha et al. (2011)19.

9



189

Determination of apparent permeability coefficients (Papp) of iodine species.

190

Transport of standards of I- (20 mg/L), IO3- (20 mg/L), MIT (10 mg/L) and DIT (10 mg/L)

191

was studied. Iodine standards prepared in HBSSwith 10 mM HEPES were added to the apical

192

compartment(1.5 mL) and HBSS-10 mM HEPES to the basolateral compartment (2 mL). At

193

the various assay times (30, 60, 90 and 120 min), aliquots (600 µL) were obtained from the

194

basolateral compartment and replaced with an equal volume of HBSS-10 mM HEPES. Iodine

195

concentration was determined in the aliquots to evaluate apparent permeability coefficients

196

(Papp) using Equation 3.

197

Papp = (dC/dt) (Vr/AC0) (Equation3)

198

where dC/dt is the flow of iodine species (mg/s); Vr is the volume of the basolateral

199

compartment (2 mL); A is the surface of the cell monolayer (4.67 cm2); C0 is the initial iodine

200

concentration in the apical compartment (mg/L).

201

Paracellular transport.The participation of the paracellular pathway in transport of

202

iodine species was evaluated by a modulation of the cell junctions, incubating the cell

203

monolayer for 5 min with 5 mM EDTA in PBS without Ca2+ and Mg2+ in the apical side and

204

HBSS free of Ca2+ and Mg2+ in the basolateral side. Then the standard of iodine species [I- (20

205

mg/L), IO3- (20 mg/L), MIT (10 mg/L) and DIT (10 mg/L)], prepared in medium consisting of

206

50% of HBSS without Ca2+ and Mg2+ and 50% of HBSS with Ca2+ and Mg2+, both

207

supplemented with 10 mM HEPES, was added to the apical compartment. The acceptor

208

medium was collected at various times (15, 30 and 45 min) and the concentration of iodine

209

was determined in order to evaluate the Papp (Equation 3). The efficiency of the EDTA in

210

modulating the cell junctions was monitored by determining the Papp of Lucifer Yellow (LY),

211

which was added at a concentration of 100 µM to the apical compartment. The transport of

212

LY to the basolateral side was measured with a fluorescence microplate reader Polar STAR

213

OPTIMA at excitation/emission wavelengths of 485/520 nm. 10


Page 10 of 33

Page 11 of 33


214

Iodine transport and uptake from seaweed samples by intestinal cells. The

215

bioaccessible fractions of the seaweeds obtained previously were inactivated by heating for 5

216

min at 100 ºC. Glucose (final concentration 1 g/L, Sigma) was added to facilitate cell viability

217

and NaCl (5 mM) was used to adjust the osmolarity to 290±25 mOsm/kg using a freezing

218

point osmometer (Löser, Germany).

219

Treated bioaccessible fraction (1.5 mL) was added to the apical chamber and 2 mL of HBSS-

220

10 mM HEPES to the basolateral compartment. After 2 h, the medium was collected from the

221

basolateral compartment to determine the quantity of iodine transported, which was corrected

222

per mg of protein. Protein content was determined by Bio-Rad Protein Assay kit, following

223

the manufacturer’s instructions and using a standard curve of BSA (0.2–1.0 mg/mL).

224

Percentage of transport across the monolayer was calculated using Equation 4.

225

Cellular transport (%) =

[ I ]transporte d [ I ]added

x100 ( Equation 4)

226

where [I]transported is the concentration of iodine present in the basolateral compartment at the

227

end of the assay, and [I]added is the concentration of iodine added to the apical side at the

228

beginning of the experiment.

229

Evaluation of the integrity of the cell monolayer. Cell monolayer integrity was

230

evaluated during the assay by measuring (a) TEER during the transport and (b) LY passage to

231

the basolateral compartment. The transport assays were only considered valid if, a) the TEER

232

values did not vary by more than 25% from those values observed at the beginning of the

233

experiment, and b) the transport of LY did not exceed 2% of the total amount added to the

234

apical compartment.

235

Iodine determination. Iodinedetermination was performed by ICP-MS using

236

conditions listed in Table 1. TMAH extracts were diluted before ICP-MS measurement.

237

Tellurium (2 mg/L) was used as an internal standard. He was used in the collision cell at a 11



238

flow rate of 80 mL/min in order to obtain the best sensitivity and linear ranges. The

239

calibration was perform in the concentration range of 0 and 500µg/L.

240

Limit of detection (LOD) and a limit of quantification (LOQ) of ICP-MS determinations,

241

were calculated based on the 3.SD/10.SD criterion (SD standard deviation of eleven

242

measurements of reagent blank). Accuracy of the method was assessed by analysing by

243

triplicate the CRM NIES-09.

244

Statistical analysis. The statistical analysis by one-factor analysis of variance

245

(ANOVA) with the Tukey HSD post hoc multiple comparison test or using the Student t-test

246

(SigmaPlot version 12.0) was used. All analysis was performed by triplicate. Statistical

247

significance was accepted for p 10×10-6 cm/s. The permeability values obtained

353

in the present study for inorganic iodine species and MIT indicate that these species undergo

354

moderate absorption (20-70%); whereas DIT is poorly absorbed (

Evaluation of Iodine Bioavailability in Seaweed Using in Vitro

Recommend Documents