Effect of lettuce biofortified with iodine by soil fertilization on iodine concentration in various tissues and selected biochemical parameters in serum of Wistar rats

journal of functional foods 14 (2015) 479–486

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Effect of lettuce biofortified with iodine by soil fertilization on iodine concentration in various tissues and selected biochemical parameters in serum of Wistar rats Aneta Kopec´ a,*, Ewa Pia˛tkowska a, Renata Biez˙anowska-Kopec´ a, Mirosław Pysz a, Aneta Koronowicz a, Joanna Kapusta-Duch a, Sylwester Smolen´ b, Roksana Rakoczy b, Łukasz Skoczylas c, Teresa Leszczyn´ska a, Iwona Ledwoz˙yw-Smolen´ d a

Department of Human Nutrition, Faculty of Food Technology, University of Agriculture in Krakow, Balicka 122, 30-149 Krakow, Poland b Unit of Plant Nutrition, Institute of Plant Biology and Biotechnology, Faculty of Biotechnology and Horticulture, University of Agriculture in Krakow, al. 29 Listopada 54, 31-425 Krakow, Poland c Department of Fruit, Vegetable and Mushroom Processing, Faculty of Food Technology, University of Agriculture in Krakow, Balicka 122, 30-149 Krakow, Poland d Unit of Biochemistry, Institute of Plant Biology and Biotechnology, Faculty of Biotechnology and Horticulture, University of Agriculture in Krakow, Al. 29 Listopada 54, 31-425 Krakow, Poland

A R T I C L E

I N F O

A B S T R A C T

Article history:

In this study, for the first time, we evaluated the effect of lettuce biofortified with iodine

Received 27 August 2014

on iodine content in various tissues and selected biochemical parameters in the serum of

Received in revised form 11

Wistar rats. Significantly higher concentrations of iodine were measured in urine, faeces,

February 2015

hearts, and kidneys of rats fed the control diet (C diet) and the diet containing biofortified

Accepted 13 February 2015

lettuce (BFL) compared to rats fed a diet containing non fortified-control lettuce or a diet

Available online 2 March 2015

with a lower level of iodine (equal to the content in control lettuce). Significantly higher concentrations of iodine were measured in the liver and femoral muscle of rats fed the BFL

Keywords:

diet compared to the other experimental groups. Thyroid hormones, thyroid stimulating

Iodine

hormone, aspartate aminotransferase, alanine aminotransferase and selected genes’ mRNA

Biofortification

activities were not affected by the presence of biofortified lettuce in the diet compared to

Lettuce

the control group. Biofortified lettuce may be considered as a potential source of iodine in

Rats

the prevention of deficiency of this trace element. © 2015 Elsevier Ltd. All rights reserved.

1.

Introduction

Iodine is a trace element which is essential for health and the development of humans and animals, particularly mammals

(Dong, Liu, Wang, Xi, & Chen, 2009; WHO, 2014). This trace element is necessary for the biosynthesis of thyroid hormones: thyroxin (T4) and triiodothyronine (T3). Iodine deficiency contributes to a wide spectrum of diseases: from endemic goiter to impaired memory and cognitive function/mental disorder

* Corresponding author. Department of Human Nutrition, Balicka 122, 30-149 Krakow, Poland. Tel.: +48126624818; fax +48126624812. E-mail address: [email protected] (A. Kopec´). http://dx.doi.org/10.1016/j.jff.2015.02.027 1756-4646/© 2015 Elsevier Ltd. All rights reserved.

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journal of functional foods 14 (2015) 479–486

(Dong et al., 2009; Melse-Boonstra & Jaiswal, 2010). Developing fetuses are most susceptible to the lack of iodine which leads to numerous neurological disorders beginning with lowered intelligence quotient scores to severe mental retardation (cretinism). Children are particularly liable to iodine deficiency in every stage of development (Melse-Boonstra & Jaiswal, 2010; Skeaff, 2011; Walker et al., 2007). According to the current World Health Organization (WHO) data, the deficiency of iodine was ascertained in 1993 in 110 countries around the World. In 2013 this number decreased to 31 countries. Deficiency of iodine is still common in Asia, Africa and Eastern Europe. It has been estimated that about 1.88 billion people suffer from it (WHO/UNICEF, 2007; WHO, 2014). Fortification of salt with iodine has been a major reason for the reduction of iodine deficiency in many countries in Europe (i.e. Austria, Finland, Poland, Switzerland, Norway), Asia (i.e. China, Indonesia, India), and Latin America (i.e. Ecuador, Peru, Venezuela) mainly due to relatively high levels of salt intake in daily diets (7.9–16.0 g NaCl/day), (Andersson, de Benoist, & Rogers, 2010; WHO, 2008, WHO, 2014). However, this level of salt consumption exceeds the amount recommended by WHO (5 g NaCl/day). Excessive salt intake is one of the major causes of cardiovascular diseases (i.e. hypertension, stroke, atherosclerosis) and other diseases such as stomach cancer (WHO, 2008; Maillot & Drewnowski, 2012). Global economic and social effects of these disorders are enormous. For that reason WHO established the “2008–2013 Action Plan for the Global Strategy for the Prevention and Control of Noncommunicable Diseases”. This particular programme included guidelines for reducing table salt consumption as well as the development/identification of alternative ways of iodine reduction in the human daily diet (WHO, 2008; WHO, 2014). One of these methods (apart from iodized mineral water, milk, flour, bread etc.) can be plant fortification-biofortification with iodine trace element during plant cultivation (Blasco et al., 2008; Caffagni et al., 2011; Smolen´, Roz˙ek, Ledwoz˙yw, & Strzetelski, 2011; Strzetelski, Smolen´, Roz˙ek, & Sady, 2010; Ujowundu et al., 2010; Voogt, Holwerda, & Khodabaks, 2010). The objective of this study was to evaluate the effect of the addition of lettuce biofortified with iodine in KI form, on iodine content in selected tissues, lipid profile, thyroid hormone concentration and mRNA expression of selected genes involved in iodine metabolism in Wistar rats.

mg dm−3 of soil): N-90, P-70 and K-200, based on the results of soil analysis. This part of the study included: 1) control lettuce – growing without iodine fertilization and 2) lettuce – grown with soil fertilization with potassium iodide (KI). Potassium iodide was applied twice: before cultivation and as a top-dressing 21 days after planting seedlings in a total dose of 5 kg I/ha and 2.5 kg I/ha respectively. On April 18, 2012 seedlings were transferred into the soil with spacing of 30 cm × 30 cm. The experiment was arranged in a split-plot design with four replications of 5 m × 1.5 m (7.5 m2) plots. For the animal study, randomly selected full grown lettuce heads were collected at harvest (30th May 2012) from the middle part of each plot, individually for both treatments.

2.2.

Fresh samples of lettuce were frozen and freeze dried with lyophilizer (Christ Alpha 1–4, Gefriertrocknungsanlangen, Germany). In these prepared samples the chemical composition was measured. Total proteins (method no. 950.36), raw fat (method no. 935.38), total dietary fibre (method no. 991.43) and ash (method no. 930.05) were measured according to the AOAC (2006) methods. Carbohydrates were calculated using the equation: Carbohydrates = 100-(protein + crude fat + dietary fibre + ash); (Fortuna, Juszczak, & Sobolewska-Zielin´ska, 2003). In order to analyze iodine content, air dried lettuce samples were ground in a variable speed rotor mill Pulverisette 14 FRITSCH (Idar-Oberstein, Alemania, Germany) using 0.5 mm a sieve. Digestion of 0.5 g samples of lettuce in the mixture of 10 cm3 65% nitric acid (HNO)3 (superpure, Merck, Whitehouse Station, NJ, USA) and 0.8 cm3 70% HClO4 (superpure, Polskie Odczynniki Chemiczne, Gliwice, Poland) was conducted in the microwave system CEM MARS-5 Xpress (CEM World Headquarters, Matthews, NC, USA). The content of iodine was analyzed by the cold vapour generation technique with the use of high-dispersion Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES; Prodigy spectrometer, Leeman Labs, New Hampshire, MA, USA) (Vtorushina, Saprykin, & Knapp, 2008, 2009). A similar method was used for the determination of iodine in the experimental diets for rats.

2.3.

2.

Materials and methods

2.1.

Plant material

Lettuce ‘Melodion’ cv. was cultivated in the spring season of 2012 in a field experiment on heavy soil (24% sand, 23% dust and 53% loam) characterized by: pH(H2O) 6.02, pH(KCl) 4.97, EC (electrical conductivity) 0.10 mS cm−1 and the content of: organic matter −2.33%, N as a ammonium nitrate (NH4NO3; NO3− and NH4+ in proportion 1:1) −13.4 mg, P-32.7 mg, K-168.6 mg, Mg194.2 mg, Ca-2089.3 mg and S-41.4 mg in 1 dm3 of soil. One day before plant seedling into the field, mineral fertilizers: ammonium nitrate, potassium chloride and potassium monophosphate were introduced into soil in order to supplement the deficiency of nutrients to the optimal level for lettuce (in

Analysis in plant material

Animal study

Five week old Wistar rats (n = 32, male), were purchased from Animal Husbandry in Brwinów, Warsaw, Poland. All experimental procedures were approved by the First Local Ethical Committee on Animal Testing at the Jagiellonian University in Krakow (Poland). Before the experiment, the rodents were acclimatized for 7 days on standard laboratory chow. After this period the rats were randomly divided into four experimental groups (n = 8). The average body mass of the rats at the beginning of the experiment was 121 ± 10 g. During the experimental period, rodents were fed with diets based on AIN93G diets (Reeves, 1997). The composition of experimental diets is shown in Table 1. In the C-diet, the mineral mixture contained the iodine in an amount recommended by Reeves (1997). In the diet containing biofortified lettuce (BFL) the only source of iodine was lettuce (mineral mixture was without iodine).

journal of functional foods 14 (2015) 479–486

Table 1 – Composition of experimental diets [g/kg]. Ingredient g/kg

C diet

BFL

DI

DIL

Corn starch Saccharose Casein Soybean oil Fibre Mineral mix Vitamin mixd choline chloride TBHQc Control lettuce Biofortified lettuce Iodine mg/kg

532.486 100 200 70 50 35d 10 2.5 0.014 0 0 0.246

500.646 100 200 70 40.24e 35a 10 2.5 0.014 0 46.1 0.231

532.486 100 200 70 50 35b 10 2.5 0.014 0 0 0.071

501.756 100 200 70 39.13f 35a 10 2.5 0.014 46.1 0 0.070

a

Mineral mix without iodine; in these diets the source of iodine was biofortified lettuce or control lettuce. b In mineral mix the level of iodine was the same as in controlnot biofortified lettuce. c tert-butylhydroquinone. d According to AIN-93G. e 9.76 g of fibre was delivered from biofortified lettuce. f 10.87 g of fibre was delivered from control lettuce. C diet, control AIN-93G diet; BFL, diet containing biofortified lettuce as the source of iodine; DI, diet prepared based on AIN-93G diet with the same level of iodine as in diet with control lettuce; DIL, diet containing control lettuce.

Biofortified lettuce was added to the BLF diet to obtain the same amount of iodine as in the C-diet. In the DI diet, the mineral mixture contained the same level of iodine as the diet with non biofortified-control lettuce. The source of iodine in the DIL diet was only non biofortified, control lettuce (in the DIL diet the mineral mixture was without iodine). The rodents were housed separately in stainless steel metabolic cages at 25 °C and 12/12 h light/dark cycle. During the experiment, animals had free access to water and diets. The intake of diets was recorded every day. Body weight gain was recorded during the whole experiment on a weekly basis. Urine and faeces were collected between the 5–10th, 13–17th, 20–24th and 27th–31st days of the experiment to determine iodine excretion. All samples were stored at −20 °C until the analysis. At the end of the experiment (after 5 weeks), fasted rats were anaesthetized with thiopental (Biochemie GmbH, Vienna, Austria). Blood was obtained by heart puncture and collected in plain test tubes. Blood samples were collected to obtain serum by centrifugation (1500 × g, 15 min). Livers, kidneys, thyroid glands and hearts were dissected, washed in 0.9% sodium chloride dried with laboratory tissue paper and weighed. Serum and tissue samples were kept frozen at −80 °C until the analysis.

2.4.

Analysis in serum and blood

Serum was analyzed for the concentration of total cholesterolTC; (cat no. Liquick Cor-CHOL60 2–204, PZ Cormay S.A., Lublin, Poland), HDL-cholesterol (cat no. Cormay HDL 2–052, PZ Cormay S.A.), and triacylglycerols-TAG (cat no. Liquick Cor-TG60 2–253, PZ Cormay S.A.). The differences between TC and HDL were used for calculations of the LDL + VLDL level (Friedewald, Fredrick, & Levy, 1972). The concentration of thiobarbituric acid

481

reactive substances (TBARS) was measured with an OxiTekTBARS kit (cat no. 850-287-KI01, Zeptometrix, Buffalo, NY, USA). The level of triiodothyronine (T3) and thyroxine (T4) was measured with Mouse/Rat kits (cat no. T3043T-100; T4044T100; respectively, Calbiotech, Spring Valley, CA, USA). The level of the thyroid stimulating hormone (TSH) was measured with a Rat kit (cat no. CEA463Ra, Cloud-Clone Corp., Houston, TX, USA). The level of glucose was measured in the whole blood with a glucometer (Accu-chek, Roche Diagnostic, Mannheim, Germany). The activity of aspartate aminotransferase (AST) or alanine aminotransferase (ALT) in the serum was measured using Alpha Diagnostic kits (Alpha Diagnostic, Warsaw, Poland; cat no. A6661-050, A6624-050, respectively).

2.5.

Iodine content in urine, faeces and selected tissues

Collected samples of urine were adjusted to the same volume before analysis. The faeces, kidney, liver, heart and femoral muscles were freeze-dried. After freeze-drying, organs were weighed and crushed in mortar and pestle. Thus prepared samples (particle size about 1 mm) were used for measurements of iodine content. The content of iodine in these samples was analyzed by the cold vapour generation technique with the use of ICP-OES Prodigy spectrometer (Leeman Labs) (Vtorushina et al., 2008, 2009) after sample digestion in the mixture of 10 cm3 65% HNO3 (superpure, Merck) and 0.8 cm3 70% HClO4 (superpure, Polskie Odczynniki Chemiczne) in the microwave system CEM MARS-5 Xpress (CEM World Headquarters).

2.6.

Crude lipid levels in liver

The crude fat content was determined by the Soxhlet method with a Soxtec Avanti’s 2050 Auto Extraction Unit (Tecator Foss, Hillerød, Sweden) as was previously reported (Kopec´, Pia˛tkowska, Leszczyn´ska, & Koronowicz, 2013).

2.7.

Gene expression

RNA was isolated from the thyroid glands and livers with a commercially available kit (cat no. 036–100, Total RNA Mini Plus A&A Biotechnology, Gdynia, Poland). Its concentration was measured by a spectrophotometer (Multiskan Go, Thermo Scientific, Waltham, MA, USA) using absorbances at 260 and 280 nm. For cDNA synthesis mRNA was reverse transcribed with the use of a TranScriba cDNA Synthesis Kit, (cat no. 4000-100 A&A Biotechnology). cDNA was subjected to real time PCR in a reaction of a mixture containing the TaqMan Gene Expression Master mix (cat no.4369016, Invitrogen, Life Technologies, Oslo, Norway) and primers for the following genes: Dio1 (deiodinase iodothyronine type 1), E2f1 (E2F transcription factor 1), thyroid hormone receptor alpha (Thra) and thyroid hormone receptor beta (Thrb) with fluorescent marked starters (Invitrogen, Life Technologies). The thermal profile of the PCR reaction included initial denaturation for 15 min at 95 °C, followed by 40 amplification cycles of denaturation for 1 s at 95 °C, annealing for 20 s at 60 °C, and elongation for 20 s at 72 °C. RT-PCR reaction was performed with the use of the CFX96 Touch™ Deep Well Real-Time PCR Detection System (Bio Rad, Hercules, CA, USA). Expression rates were calculated as the normalized

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journal of functional foods 14 (2015) 479–486

Table 2 – Chemical composition of lettuce used for experiment [g/100 g d.m.]. Ingredient

Control lettuce

Biofortified lettuce

Protein Crude fat Carbohydrates Dietary fibre Ash Iodine [mg/100 g d.m.]

20.79 ± 0.06a 2.76 ± 0.007a 36.17 ± 0.39a 26.14 ± 0.80a 14.13 ± 0.15a 0.12 ± 0.034a

20.78 ± 0.02a 2.72 ± 0.005a 39.52 ± 0.92a 23.46 ± 0.90a 13.50 ± 0.14a 0.50 ± 0.087b

Values in rows with different letters (a, b) are significantly different, P ≤ 0.05.

threshold cycle (CT) difference between the control and the sample with adjustment for amplification efficiency relative to the expression level of the housekeeping gene 18S.

2.8.

Statistical analysis

The data were presented as mean ± SD. One-way, nonparametric analysis of variance by ranks (Kruskal–Wallis test) was applied for testing the difference at P ≤ 0.05 (Statistica v. 10.0, StatSoft, Inc., Tulsa, OK, USA). The Mann–Whitney U test with a Bonferroni adjustment was used for testing the differences between experimental treatments.

3.

Results

Biofortification of lettuce with iodine did not affect basic chemical composition when compared to the control lettuce (Table 2). Iodine content significantly increased in the biofortified lettuce when compared to the control lettuce. The body weight gain, feed efficiency ratio (FER), heart and kidney weights were not affected by various dietary treatments. Liver weight was significantly higher (P ≤ 0.05) in the rats fed the control diet when compared to the liver of the rats fed the BFL diet as well as the DIL diet (Table 3). The level of iodine in urine was affected by various dietary treatments. The significantly highest concentration of iodine was measured in the urine of rats fed the C diet when compared to the rats from the rest of the experimental groups in

weeks 1, 2 and 4 (Table 4). In week 3 we measured the highest iodine content in the C and BFL groups when compared to the rats fed DI as well as DIL diets. Additionally, there were no statistically significant differences between the C and BFL groups. The highest level of iodine in faeces was measured also in the C group when compared to the rest of the experimental groups in weeks 2, 3 and 4. There were no statistically significant differences in iodine content in faeces between the C group and the BFL groups in the first week (Table 4). The significantly higher content of iodine was measured in the kidneys of rats from the C group when compared to the rest of the experimental groups. Significantly higher concentrations of this trace element were measured in the liver and the femoral muscle of rats fed the BFL diet when compared to the rest of the experimental groups (Table 4). The level of iodine in the hearts of rats was significantly lower in the DI and DIL groups when compared to the content of this trace element in the heart of rats from the C and BFL groups. The level of TC and LDL + VLDL was significantly higher in the serum of the rodents fed diets with biofortified and control lettuce when compared to the C group (Table 5). HDL concentration in the serum of the rats was not affected by various dietary treatments. The TAG level was significantly higher in the serum of rats fed the DI diet when compared to the C, BLF, and DIL groups. The content of TBARS was significantly lower in the serum of the rats fed diets with the addition of biofortified and control lettuce when compared to the C group. Concentration of crude fat in the liver was significantly lower in the group fed the diet containing control lettuce (DIL) when compared to the other experimental groups. The level of T3 was significantly higher in the serum of rodents from the DI group when compared to the BFL and DIL groups. The level of T4 and TSH was not affected by various dietary treatments. Concentration of glucose in the blood was significantly lower in groups fed diets with biofortified or control lettuce (Table 5). The activity of ALT and AST was not affected by various dietary treatments. Expression of mRNA of Dio1, E2f1 and Thrb in the thyroid gland of rats was not affected by various dietary treatments. The mRNA expression of Thra was significantly higher in the thyroid gland of the rats fed the BFL diet when compared to the group of rodents fed the DIL diet. Expression of mRNA of Dio1, E2f1 and Thrb was significantly higher in the livers of the rats fed the DI and DIL diets when compared to the animals

Table 3 – Body gain, feed efficiency ratio, liver, heart and kidney weights of experimental rats. Treatment

C-group

BFL

DI

DIL

Body gain [g] FERa Liver [g] Heart [g] Kidneys [g]b

179 ± 9a 0.346 ± 0.02a 11.57 ± 0.71b 1.03 ± 0.08a 2.32 ± 0.29a

175 ± 22a 0.337 ± 0.04a 10.10 ± 1.13a 1.02 ± 0.11a 2.29 ± 0.23a

174 ± 12a 0.336 ± 0.02a 10.38 ± 0.67ab 1.10 ± 0.11a 2.21 ± 0.16a

169 ± 17a 0.326 ± 0.03a 9.79 ± 0.97a 1.00 ± 0.09a 2.17 ± 0.20a

Values in rows with different letters (a, b) are significantly different, P ≤ 0.05. a FER feed efficiency ratio (g) body weight gain/diet consumed (g). b Weight of both kidneys. C diet, control AIN-93G diet; BFL, diet containing biofortified lettuce as the source of iodine; DI, diet prepared based on AIN-93G diet with the same level of iodine as in diet with control lettuce; DIL, diet containing control lettuce.

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journal of functional foods 14 (2015) 479–486

Table 4 – Iodine content in urine, faeces and selected tissues in rats. Treatment

C-group

BFL

DI

DIL

3

Urine [µg/dm ] Week 1 Week 2 Week 3 Week 4 Faeces [µg/kg d.m.] Week 1 Week 2 Week 3 Week 4 Tissues [mg/kg d.m.] Kidneys Liver Heart Femoral muscle

256.12 ± 47.71c 221.28 ± 36.35c 202.12 ± 40.51b 222.63 ± 35.61c

118.02 ± 28.51b 201.28 ± 27.68b 190.58 ± 51.00b 163.30 ± 19.73b

65.07 ± 22.05a 40.95 ± 25.88a 47.75 ± 18.87a 45.78 ± 11.34a

71.21 ± 32.58a 35.18 ± 13.96a 45.21 ± 8.10a 46.83 ± 11.83a

2183.81 ± 555.82b 2917.84 ± 514.28c 3428.25 ± 365.73c 3975.18 ± 402.03c

2064.09 ± 499.18b 1905.65 ± 434.24b 2167.87 ± 631.64b 3363.90 ± 683.71b

717.57 ± 202.48a 782.19 ± 176.03a 1010.40 ± 219.36a 1174.63 ± 263.93a

808.38 ± 343.70a 710.24 ± 246.87a 906.86 ± 229.54a 1171.93 ± 258.79a

24.80 ± 3.38c 3.28 ± 0.71b 5.91 ± 0.94b 1.50 ± 0.20b

20.32 ± 2.62b 5.11 ± 0.98c 5.88 ± 0.73b 2.21 ± 0.41c

4.17 ± 1.43a 1.53 ± 0.24a 1.62 ± 0.25a 0.60 ± 0.03a

4.56 ± 0.60a 1.90 ± 0.43a 1.82 ± 0.34a 0.42 ± 0.15a

Values in rows with different letters (a, b, c) are significantly different, P ≤ 0.05. C diet, control AIN-93G diet; BFL, diet containing biofortified lettuce as the source of iodine; DI, diet prepared based on AIN-93G diet with the same level of iodine as in diet with control lettuce; DIL, diet containing control lettuce.

fed the C or BFL diets (Table 6). mRNA expression of Thra was significantly higher in the liver of the rats fed the DI and DIL diets when compared to the group fed the C diet.

4.

Discussion

Basic chemical composition of the biofortified lettuce was not affected by soil fertilization with iodine. The biofortified lettuce had a higher content of iodine in comparison to the control lettuce (Table 2). According to the current dietary recommendations we should consume 5 portions of vegetables and fruits per day (WHO/FAO, 2003). Our results shows that a 100 g of dried, biofortified lettuce will deliver about 500 µg of iodine, and only 30 g will deliver the Recommended Nutrient Intake (RNI) or the Recommended Daily Allowance (RDA) for this trace element (RNI or RDA is about 150 µg I/day for adults

(WHO/UNICEF, 2007)). It has been previously suggested that foliar or soil fortification of various plants (tomato, potato, radish, carrot, fluted pumpkin) with different forms of iodine (potassium iodide, potassium iodate) may improve the content of this trace element in plants and can be an alternative way to increase the content of iodine in an average daily diet (Caffagni et al., 2011; Kiferle, Gonzali, Holwerda, Real Ibaceta, & Perata, 2013; Strzetelski et al., 2010; Ujowundu et al., 2010). Some authors have reported that the iodine biofortification in lettuce increased its content. Blasco, Leyva, Romero, & Ruiz (2013) reported that the addition of iodine in the form of KIO 3 significantly increased the concentration of this trace element in the leaves of lettuce cultivated under controlled environmental conditions. Also Voogt et al. (2010) reported that the addition of iodine to the nutrients’ solution significantly increased the content of iodine in lettuce grown in water culture. Additionally these authors reported that iodine in the form of I− was accumulated better than in the form of IO3−.

Table 5 – Selected biochemical parameters in rats fed experimental diets. Treatment

C-group

BFL

DI

DIL

TC [mmol/L] HDL [mmol/L] LDL + VLDL [mmol/L] TAG [mmol/L] TBARS [nmol/mL] T3 [µg/dL] T4 [ng/mL] TSH [ng/mL] Glucose [mg/dL]* ALT [U/L] AST [U/L] Crude fat in liver [g/100 g f.m.]

1.44 ± 0.26a 0.92 ± 0.28a 0.43 ± 0.17a 0.48 ± 0.17a 25.25 ± 11.07c 2.14 ± 0.58ab 7.48 ± 0.44a 4.38 ± 0.20a 122 ± 12.0b 24.22 ± 6.73a 59.36 ± 10.44a 3.61 ± 0.78b

1.64 ± 0.24b 1.10 ± 0.13a 0.73 ± 0.29b 0.40 ± 0.08a 12.27 ± 3.96ab 2.06 ± 0.45a 7.58 ± 0.81a 4.28 ± 0.20a 107 ± 7.4a 17.57 ± 3.25a 52.59 ± 6.61a 3.48 ± 0.64b

1.49 ± 0.30ab 0.96 ± 0.24a 0.46 ± 0.16a 0.65 ± 0.14b 21.54 ± 6.89bc 2.57 ± 0.38b 8.28 ± 1.17a 4.35 ± 0.13a 122 ± 6.0b 23.79 ± 7.56a 63.18 ± 11.25a 3.91 ± 0.87b

1.77 ± 0.38b 1.04 ± 0.17a 0.80 ± 0.36b 0.48 ± 0.09a 9.68 ± 3.12a 1.81 ± 0.44a 7.55 ± 0.94a 4.27 ± 0.24a 109 ± 7.1a 19.42 ± 5.63a 59.69 ± 11.60a 2.57 ± 0.27a

Values in rows with different letters (a, b, c) are significantly different, P ≤ 0.05. * In whole blood. C diet, control AIN-93G diet; BFL, diet containing biofortified lettuce as the source of iodine; DI, diet prepared based on AIN-93G diet with the same level of iodine as in diet with control lettuce; DIL, diet containing control lettuce.

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journal of functional foods 14 (2015) 479–486

Table 6 – Selected relative gene expression in the liver and in the thyroid gland of experimental rats [relative mRNA levels]. Treatment Thyroid gland Dio1 E2f1 Thra Thrb Liver Dio1 E2f1 Thra Thrb

C-group

BFL

DI

DIL

1.05 ± 0.05a 1.10 ± 0.10a 1.01 ± 0.05ab 1.04 ± 0.07a

1.10 ± 0.09a 1.18 ± 0.09a 1.48 ± 0.09b 1.12 ± 0.13a

1.05 ± 0.09a 1.10 ± 0.05a 1.03 ± 0.06ab 1.04 ± 0.06a

0.99 ± 0.07a 1.13 ± 0.05a 0.99 ± 0.04a 1.02 ± 0.04a

0.84 ± 0.09a 1.04 ± 0.09a 0.98 ± 0.11a 0.92 ± 0.09a

0.93 ± 0.11a 1.12 ± 0.15a 1.05 ± 0.15ab 0.92 ± 0.11a

1.15 ± 0.07b 1.40 ± 0.09b 1.21 ± 0.06bc 1.11 ± 0.08b

1.16 ± 0.03b 1.34 ± 0.02b 1.19 ± 0.04bc 1.08 ± 0.03b

Values in rows with different letters (a, b, c) are significantly different, P ≤ 0.05. C diet, control AIN-93G diet; BFL, diet containing biofortified lettuce as the source of iodine; DI, diet prepared based on AIN-93G diet with the same level of iodine as in diet with control lettuce; DIL, diet containing control lettuce; Dio1, deiodinase iodothyronine type 1; E2f1, E2F transcription factor 1; Thra, thyroid hormone receptor alpha; Thrb, thyroid hormone receptor beta.

In this study for the first time, compared to available literature, we evaluated the effect of lettuce biofortified with iodine on the iodine bioavailability in an animal study. The body gain, feed efficiency ratio (FER), heart and kidney weights were not affected by various dietary treatments. It can be suggested that the addition of freeze-dried control or biofortified lettuce had no effect on these parameters and was safe for animals. The liver weight was significantly lower (P ≤ 0.05) in the rats fed the diet containing biofortified or control lettuce. It can be suggested that the presence of various bioactive compounds in lettuce, especially polyphenolic compounds and dietary fibre, could result in a lower content of crude fat which may have influenced the lower weight of this organ. Additionally, we found that the content of crude lipids was significantly lower in the livers of rats fed the control lettuce and tended to be lower (p = 0.07) in the group fed the lettuce biofortified with iodine when compared to the control group. Lower content of crude lipids in the livers could also cause lower weight of these organs. What is more, in the serum of rats from the groups fed with biofortified or control lettuce the concentration of the TC and the LDL + VLDL cholesterol increased significantly. What is probable is that polyphenolic compounds and short chain fatty acids (product of fermentation of fibre in the colon) could inhibit the triacylglycerols and cholesterol accumulation in the liver and the concentration of the TC and the LDL + VLDL cholesterol significantly increased in the serum of rats fed diets containing biofortified or control lettuce. The level of TBARS significantly decreased in the serum of rats fed diets containing biofortified and control lettuce. What is likely the presence of polyphenolic compounds and carotenoids decreased the oxidation of lipids in rodents which may have led to a lower TBARS content. The level of glucose was also affected by the presence of both types of lettuce in the diets of the experimental rats. This can be explained not only by the presence of natural antioxidants in the diets but also by the presence of dietary fibre. It is well known that dietary fibre decreases the digestion of starch in the intestine and slows down the absorption of glucose, giving the hypoglycaemic effect (Galisteo, Duarte, & Zarzuelo, 2008; Weickert & Pfeiffer, 2008). The activity of ALT and AST was not affected by the various dietary treatments (Table 5). The activity of these enzymes

increases when the hepatocytes are damaged by various factors. It can be suggested that the level of lettuce in the diets did not affect liver functions and was safe for rodents. Iodine excretion in the urine and faeces was affected by various dietary treatments. It was found that in the C and BFL groups, in which the rats were fed a proper dose of iodine, the excretion of iodine in urine and faeces was highest when compared to the DI and DIL groups (Table 4). It can be suggested that the biofortified lettuce is a good source of bioavailable iodine, which is an important finding in this study. However, we also measured lower iodine contents in urine and faeces of the rat group fed biofortified lettuce when compared to the control group. The presence of fibre and phenolic compounds could cause lower absorption and bioavailability of iodine from lettuce. On the other hand it was found that iodine contents in the liver and femoral muscle of rats fed lettuce biofortified with iodine were significantly higher than in the control rats. It has been reported that the iodine nutrition in a population of various ages is assessed based on its excretion with urine (Andersson et al., 2010; WHO, 2014). It has been previously reported that iodine nutrition in rats can also be evaluated by the content of its urine, faeces and organs (Kirchgessner, He, & Windisch, 1999; Windisch, He, & Kirchgessner, 1999). Kirchgessner et al. (1999) reported that iodine excretion with urine and faeces increased with the higher content of this trace element in the diet. Additionally, these authors reported that the iodine concentration in various organs increased with the higher level of iodine in the diet. In our study we also found that the iodine contents in urine, faeces and various organs depended on dietary content (Table 4). Some authors suggested that iodine from various food products, including fortified products, has a high bioavailability of about 99% (Fordyce, Stewart, Ge, Jiang, & Cave, 2003; Weng, Yan, Hong, Qin, & Xie, 2009). The level of TSH was not affected by various dietary treatments. This may suggest that the T3 and T4 were synthesized in sufficient amounts in all experimental groups. Additionally, in groups with deficiency of iodine in diets (DI, DIL) the excretion of iodine with urine and faeces was lower to protect the organism from iodine deficiency. The significantly lower content of T3 in the serum of rats fed the BFL and DIL diets

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when compared to the rats fed the DI diet may be explained by the presence of naturally occurring substances in lettuce which may decrease or inhibit absorption of iodine or the form of iodine in lettuce. On the other hand, the highest level of T3 in rats from the DI group may be explained by an increased production of T3 from T4 to keep a proper level of this hormone in the serum. T3 is produced by the deiodination of T4. In this metabolic pathway the enzyme deiodinase type 1 is involved. What is more, hepatic deiodinase type 1 is responsible for the content of the T3 in the blood (Bianco & Kim, 2014; Konturek, 1994; Schneider et al., 2006). It can be suggested that T4 concentration in the serum of rats from the DI and DIL diet was not increased because T4 was used for the production of T3. It has been postulated that the level of T3 is pretty stable in the deiodinases deficient mice for example with disruption of Dio1 (D1KO mice; with deficiency of deiodinase type 1) and Dio2 gene (D2KO mice with deficiency of deiodinase type 2). In these animals the content of T4 was increased (Schneider et al., 2001, 2006). In this study we also found that mRNA expression of Dio1 (deiodinase iodothyronine type 1), E2f1 (E2Ftranscription factor 1), thyroid hormone receptor alpha (Thra) and thyroid hormone receptor beta (Thrb) expression significantly increased in the liver of the animals fed the DI and DIL diets. E2F1 gene encodes protein from the family E2F which play an important role in cell cycle especially in proliferation and apoptosis (El-Darwish, Parvinen, & Toppari, 2006). Dio1 encodes enzymes responsible for deiodination of T4 to T3 (Bianco & Kim, 2014). Thra and Thrb encode proteins (receptor α; TRα and receptor β; TRβ, respectively) responsible for the proper function of T3. TRα is responsible for the activation of T3 in the heart, brain white and brown adipose tissues. TRβ is necessary for the regulation of the TSH level and cholesterol metabolism in the liver (Erion et al., 2007; Liu, Kogai, Schultz, Mody, & Brent, 2012). It can be suggested that deficiency of iodine in diets of rats from the DI and DIL groups caused a higher expression of mRNA of these genes because the organism would like to keep the proper level of T3. Additionally, we have found that TC and LDL + VLDL levels increased significantly in the serum of the rats from the DIL group when compared to the C-group. It can be suggested that apart from the dietary fibre mechanism of action, the higher expression of mRNA of Thrb caused the higher level of TRβ. TRβ could change the metabolism of cholesterol in the liver and finally increase the level of LDL + VLDL in the serum of rats. In conclusion, rats fed a diet containing lettuce biofortified with iodine had a higher content of iodine in urine, faeces, selected organs as well as in the femoral muscle when compared to rats fed the control lettuce. Thyroid hormones, TSH, ALT, AST and selected genes’ mRNA activity were not affected by the presence of this lettuce in the diet. Biofortified lettuce may be considered as a potential source of iodine in daily diets but more studies need to be performed.

Acknowledgement This work was financed by the 2012–2015 Polish National Science Center – grant no. DEC-2011/03/D/NZ9/05560 “I and Se

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biofortification of selected vegetables, including the influence of these microelements on yield quality as well as evaluation of iodine absorption and selected biochemical parameters in rats fed with vegetables biofortified with iodine”.

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