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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

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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]

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ACS Paragon Plus Environment


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ABSTRACT

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

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

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

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

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

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vitro bioavailability estimation from different commercialized seaweed has been studied using

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

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

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

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

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

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

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bioavailability in seaweed.

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

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MTX cells.

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INTRODUCTION

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

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

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

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

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

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retardation in children1.

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

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

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

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

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

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

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

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

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

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iodine intake.

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

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

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

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

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

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

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

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



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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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exposure assessment of iodine through food products.

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

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

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

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

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MATERIALSANDMETHODS

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

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

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

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


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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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pure water (resistivity 18MΩcm) collection.

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Chemicals. For the alkaline solubilisation of seaweed tetramethylammonium

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

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

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

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

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

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

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

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



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

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

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

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Environmental Studies (Ibaraki, Japan).

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

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

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

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

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

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

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

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hydroxyethylpiperazine-N′-2-ethanesulfonic

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

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

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

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

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

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Biorad (California, USA).

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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

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Valencia(Spain),they

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

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

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

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at -20ºC until analysis.

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

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

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are show in table 1.

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In vitro digestion procedures

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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also obtainedto control possible contamination.

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

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Solubility = [A/B] × 100(Equation1)

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

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

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

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

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

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

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

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

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

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-20 ºC until measurements.

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

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

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

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

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

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

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

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equation(Equation2): 157

D (%) =

[ I ] dialyzate [ I ]total

x100 ( Eq.2) 158

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

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Cell transport and uptake assays

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

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

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

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

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

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

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

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

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

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a density of 5–6 × 104 cells/cm².

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

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

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

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

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

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

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

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MTX cells between passages 15 and 24.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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concentration was determined in the aliquots to evaluate apparent permeability coefficients

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(Papp) using Equation 3.

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

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

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

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concentration in the apical compartment (mg/L).

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

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

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

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

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

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

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

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

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

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

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

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

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


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

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

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

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

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point osmometer (Löser, Germany).

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

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

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

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

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

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

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Cellular transport (%) =

[ I ]transporte d [ I ]added

x100 ( Equation 4)

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

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

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beginning of the experiment.

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

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

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

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

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

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apical compartment.

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Iodine determination. Iodinedetermination was performed by ICP-MS using

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

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



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

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

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

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

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

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triplicate the CRM NIES-09.

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

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

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

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

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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 (

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