In Vitro Bioaccessibility and Bioavailability of Iron from Potatoes with

Article pubs.acs.org/JAFC

In Vitro Bioaccessibility and Bioavailability of Iron from Potatoes with Varying Vitamin C, Carotenoid, and Phenolic Concentrations Christelle M. Andre,*,† Danièle Evers,† Johanna Ziebel,† Cédric Guignard,† Jean-Francois Hausman,† Merideth Bonierbale,‡ Thomas zum Felde,‡ and Gabriela Burgos‡ †

Environmental Research and Innovation Department, Luxembourg Institute of Science and Technology, 5, Avenue des Hauts-Fourneaux L-4362 Esch/Alzette, Luxembourg ‡ International Potato Center, Avenida La Molina 1895, Lima 12, Lima, Peru ABSTRACT: The bioaccessibility and bioavailability of iron from 12 Andean potato clones were estimated using an in vitro gastrointestinal digestion procedure and the Caco-2 cell line as a model of human intestine, with ferritin formation as a marker of iron absorption. We first showed that 63.7% (for the genotype CIP_311422.016) to 79.0% (for the genotype CIP_311575.003) of the iron is released from the potato tuber matrix during in vitro gastrointestinal digestion and is therefore available at the intestinal level. On average, 32 and 24.5% of the hydrophilic bioactive components, vitamin C and chlorogenic acid, respectively, were also bioaccessible from boiled tubers. Intestinal absorption of intrinsic iron from potato tubers could not be detected using our in vitro Caco-2 cell model. When an extrinsic source of iron (20 μM FeCl3 and 1 mM ascorbic acid) was added to the digestion mixture, iron absorption varied from 1.8 to 8% for the genotypes CIP_311422.016 and CIP_311624.021, respectively, as compared to the reference control. Principal component analysis revealed negative relationships between bioavailable iron values and phenolic concentrations, whereas vitamin C concentrations were positively associated with the ferritin values. Further controlled intervention trials would be needed to conclusively assess the bioavailability of intrinsic iron from potato tubers. KEYWORDS: bioaccessibility, bioavailability, iron, potato, vitamin C, carotenoid, phenolic compound, in vitro digestion, biofortification, Caco-2 cell

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INTRODUCTION Potato (Solanum tuberosum sp.) is one of the most important crops in the world. In some regions such as the high Andes of South America, it constitutes the main staple food crop. The potato tuber provides energy through its high starch content, is a source of proteins of high biological value, and contains notable amounts of vitamins C, B6, and B3, potassium, phosphorus, and magnesium. Potato tubers also contain moderate amounts of (non-heme) iron, though due to its high consumption level it may be considered as a valuable source of iron.1 Environmental growing conditions and soil types in particular, are strong determinants of iron concentrations in potato tubers, although large genetic variation and inheritance patterns have been observed in the germplasm, indicating that breeding for increased iron content has good potential.1−4 Potato tubers are also an important source of health-promoting compounds, especially of phenolic compounds and carotenoids.5,6 These phytochemical compounds have received much attention due to their prospective effects on the prevention of various chronic diseases such as cancer and cardiovascular and degenerative diseases.7 The potato phenolic profile is dominated by chlorogenic acid and rutin, whereas lutein, violaxanthin, neoxanthin, and zeaxanthin predominate the carotenoid profile.8−11 It is also noteworthy that cultivar-specific genetic determinants and cultivation factors, including the farming system (organic vs traditional for instance), may also strongly affect important quality parameters and nutritional components of potato tubers.12,13 Iron deficiency represents one of the most widespread forms of micronutrient malnutrition.14 Iron deficiency anemia still © XXXX American Chemical Society

persists worldwide, especially in regions where consumption of meat and therefore heme iron intake is low. More than 90% of the affected populations live in developing countries. Iron deficiency during childhood and adolescence impairs physical and mental development, while for pregnant women it is associated with multiple adverse outcomes for both mother and infant, including increased risk of maternal mortality and low birth weight.15 In adults, iron deficiency reduces the capacity for physical and mental work.14 Biofortification of staple crops has tremendous potential to help alleviate these deficiencies. Potato biofortification represents a promising way to increase the iron and zinc status in affected human populations with high intake of potato.16,17 Three-fold to 4-fold variations in iron concentrations of peeled potato tubers (from 9−37 and 17−63 μg/g dry weight basis) have been reported in diverse potato germplasm grown in multiple locations in Peru (South America)1 and in the United States,18 respectively. However, determining the extent to which potato-consuming populations will benefit from increased iron concentrations in biofortified potato tubers requires further knowledge on its bioaccessibility and bioavailability. Non-heme iron bioavailability (the amount of iron that can be absorbed and used for physiological functions) is indeed strongly determined by interactions between iron and other compounds present in the human diet. Non-heme iron can be found in food as ferrous (Fe(II)) Received: June 30, 2015 Revised: September 18, 2015 Accepted: October 4, 2015

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DOI: 10.1021/acs.jafc.5b02904 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 1. Tuber appearance of the 12 iron biofortified potato clones under investigation.

vitamin C, phenolic compounds, and carotenoids were first measured following an in vitro gastrointestinal digestion procedure28 in order to get a better picture of the potential interactions that could explain the observed variations in the bioavailability of iron from potato tubers.

or ferric iron (Fe(III)). Ferric iron, the likely predominant form of iron in potato tubers,19 must be first reduced to Fe(II) to be taken up by the divalent metal ion transporters of the intestinal epithelial cells.20 Intracellular iron concentration, storage, and detoxification are further under the control of the ferritin protein. Non-heme iron absorption may be enhanced by simultaneous intake of vitamin C, citric acid, and animal proteins, whereas phytates, calcium, and phenolic compounds may inhibit the absorption. Potato tubers contain significant amounts of vitamin C, which is a potent enhancer of iron uptake in humans. Phenolic compounds, also present in potato, may, on the other hand, reduce in general iron uptake, through the formation of insoluble complexes with ferric iron in the gastrointestinal tract.21 A recent study showed however that some phenolic compounds may promote iron absorption, stressing the complexity of the interactions of this large family of compounds.22 Given the time and expense required for in vivo trials, in vitro models have been developed to estimate iron bioavailability from food.23 In combination with simulated human gastrointestinal digestion, the Caco-2 cell model has been demonstrated to be effective in comparing iron bioavailability from diverse bean, rice, and maize genotypes.24−26 The Caco-2 cell line is derived from a human colorectal adenocarcinoma. These cells may undergo differentiation and polarization to form monolayers of intestinal epithelial cells. Ferritin level in cells is a sensitive marker of iron uptake and enables measurement of iron availability in vitro.24 The purpose of this study was to compare the in vitro iron availability from diverse iron biofortified International Potato Center (CIP) potato clones grown in the Peruvian Andes and, thereby, to provide key information to breeders seeking to increase bioavailable iron from potato. Caco-2 cells were used as a model of human intestine, with ferritin formation as a marker of iron absorption.27 The bioaccessibility of iron,

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MATERIALS AND METHODS

Chemicals. Solvents [of analytical or high-performance liquid chromatography (HPLC) grade as required] were obtained from VWR International (Leuven, Belgium). Carotenoid standards (lutein, neoxanthin, violaxanthin, zeaxanthin, antheraxanthin, β-cryptoxanthin, β-carotene, and lutein-5,6-epoxide) were purchased from Carotenature (Lupsingen, Switzerland). Phenolic compounds (chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, caffeic acid, gallic acid, and rutin) were purchased from Sigma-Aldrich (St. Louis, MO). Kaempferol-3-rutinoside and petanine were obtained from ExtraSynthese (Genay, France). All digestive enzymes were purchased from Sigma-Aldrich (St. Louis, MO). Nitric acid (HNO3) and H2O2 for the mineral analyses were obtained from LGC Standards (Molsheim, France) and Fisher Scientific (Tournai, Belgium), respectively. Cell culture media, unless otherwise specified, were purchased from Invitrogen (Groningen, The Netherlands). Plant Materials. Twelve diploid (2n = 2x = 24) potato clones, from the biofortified potato population developed at the International Potato Center (CIP), were used for this study (Figure 1). Samples of the 12 clones were obtained from a field trial planted in April 2013 at CIP’s experimental station in Huancayo (Peru, 3280 m above sea level) and harvested in September 2013. Recommended agronomical practices were applied. Five to ten representative mature tubers were collected at harvest, washed, and shipped to the Luxembourg Institute of Science and Technology (LIST), where they were stored in incubators at 10 °C prior to sampling and analysis. Tubers were washed thoroughly with acidified water (pH 3) in order to remove any soil residues and avoid mineral contamination of the samples. They were further rinsed with demineralized water. For cooking, demineralized water was added in stainless steel pots and brought to boiling on induction plates. Unpeeled tubers were cooked for 10−15 min. Once cooked, tubers were peeled using ceramic knives to avoid B


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column temperature was 50 °C, and the flow rate was 0.75 mL/min. The eluents were 0.1% trifluoroacetic acid in water (A) and 0.1% trifluoroacetic acid in acetonitrile (B). The gradient was as follows: 0 min, 5% B; 9.27 min, 5% B; 13.53 min, 14% B; 22.60 min, 35% B; 23 min, 95% B; 25 min, 95% B; 26 min, 5% B. Phenolic compounds were detected at 280, 320, 360, or 520 nm according to their absorption maximum. For quantification, a six-point calibration curve was used. Furthermore, a validation standard was injected after every tenth injection. Anthocyanins were quantified as petanin equivalents. Carotenoid Analysis. Extraction. Approximately 0.75 g of material was mixed with 5 mL of acetone. This mixture was homogenized using a vortex for 30 s and shaken on ice for 30 min to allow extraction of the lipophilic compounds. After centrifugation at 5000g for 15 min, the supernatant was collected. The extraction was repeated on the pellet using the same extraction solvent. Both supernatants were combined, evaporated to dryness under a gentle stream of nitrogen, and resuspended in 1 mL of MTBE:MeOH (1:1). The samples were filtered (0.2 μM) into an HPLC amber vial and stored at −80 °C prior to analysis. For the intestinal filtrates, a 4 mL aliquot was first mixed with 1 mL of 30% methanolic KOH and incubated for 30 min on ice. Carotenoids were extracted by addition of 4 mL of hexane:acetone (1:1, v/v), shaken for 1 min, and centrifuged for 2 min at 5000g at 4 °C to hasten phase separation. The hexane phase was collected and transferred into a second tube. The extraction process was repeated with 4 mL of hexane and the combined hexane phases were dried under a stream of nitrogen using a TurboVapLV apparatus (Biotage, Sweden). The residue was redissolved in 500 μL of MTBE/MeOH (1:1/v:v), filtered through a 0.2 μm PVDF syringe filter and stored at −80 °C until analysis. UPLC-DAD Analysis. For the carotenoid quantification, an Acquity UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm particle size, Waters) was used as described by Kaulmann et al.30 The eluents were (A) ammonium acetate (50 mM):methanol (60:40, v/v) and (B) acetonitrile:dichloromethane (80:20, v/v), and the gradient was as follows: 0 min, 42% B, flow rate 0.35 mL/min; 4 min, 47% B; 11 min, 47% B; 13 min, 80% B, flow rate 0.40 mL/min; 18 min, 85% B; 29 min, 85% B, flow rate 0.35 mL/min; 30 min 42% B. The injection volume was 2.5 μL. Carotenoids were detected and quantified at 442 nm (violaxanthin, neoxanthin), 447 nm (lutein, antheraxanthin), and 453 nm (zeaxanthin, β-carotene) using six-point calibration curves. Determination of Iron Content. Raw and cooked samples (about 1 g of fresh weight) were mineralized in 7 mL of nitric acid (HNO3 for trace analysis minimum 67%) and 3 mL of H2O2 (30% w/ w). For the mineralization of intestinal filtrates (3 mL), a mixture of 1.75 mL of HNO3 and 0.75 mL of H2O2 was used. Acid mineralization was then performed in PFA tubes in a microwave oven (Anton Paar Multiwave Pro, Graz, Austria) by increasing temperature and pressure until 200 °C and 30 bar. At the end of the procedure, samples were diluted with demineralized water up to 25 mL for potatoes and up to 10 mL for filtrates, and kept at 4 °C prior to analysis. Blanks for all samples and a certified reference material for potatoes (spinach, NCS ZC 73013, LGC standards, France) were included at each mineralization cycle for quality control. The extraction was performed in triplicate for each clone on boiled and digestion materials. Samples were analyzed by inductively coupled plasma mass spectrometry (ICPMS, PerkinElmer Elan DRC-e,Waltham, MA) as described by Lefèvre et al.31 The concentrations in aluminum and chrome were also determined to detect any potential contamination and were below 2 and 0.04 ppm, respectively. Cell Viability. Cytotoxicity of the potato digestion mixtures was determined using the resazurin/Alamar Blue assay. Cells were first seeded into 96-well plates at 1 × 104 cells per well and allowed to attach at 37 °C for 18 h. Cells were then exposed to digests for 24 h. Culture medium was then removed and 100 μL of cell culture medium containing 500 μM resazurin was added to each well. After 2−3 h, the formation of resorufin by cellular metabolic activity was recorded using a microplate fluorometer (excitation wavelength 530 nm, emission at 590 nm, 37 °C). A negative control with no cells was used for background subtraction. The percentage of cell viability was calculated using the nontreated cells as positive control (100% cell viability). The

metal contamination and pureed in a mortar and pestle. Homogenized samples were further submitted to the in vitro digestion protocol, while representative samples were frozen in liquid nitrogen, and kept at −80 °C until chemical analysis. In Vitro Digestion. The in vitro digestion protocol was performed as described by Miranda et al.28 Approximately 10 g of boiled potato flesh underwent in vitro digestion. The digestions were performed in triplicate. Salivary digestion was first simulated by adding 5 mL of αamylase in NaCl 0.9% (450 U/g of potato flesh) at pH 6.9 and shaking for 10 min at room temperature. For the gastric step, pepsin (6500 U/ g of potato flesh) and 25 mL of NaCl 0.9% were added. The pH was decreased to 2 with HCl. The mixture was then shaken for 1 h at 37 °C. For the duodenal step, the pH was increased to 5.5 using NaHCO3 1 M. Bile extract (11 mg/g of potato flesh) and pancreatin (1.8 mg/g of potato flesh) were then added and the incubation volume was set to 50 mL. The digestion mixture (pH 7) was then further shaken for 2 h at 37 °C. The final digestion extract was centrifuged (5000g for 20 min at 4 °C) and the supernatant was filtered (PVDF, 0.2 μm). Digests were then flash frozen in liquid nitrogen and kept at −80 °C until further analysis. A control was made using the digestive enzymes in the same conditions without plant material. For cytotoxicity and iron uptake experiments, the potato digests were quickly thawed by mixing in a water bath at 37 °C, directly appropriately diluted, and added onto the Caco-2 cells. Vitamin C Analysis. Extraction. The extraction of vitamin C was conducted as described previously by Andre et al. 9 Briefly, approximately 750 mg of boiled material was weighed in a 15 mL Falcon tube and 4 mL of an aqueous solution containing 5% metaphosphoric acid and 1% dithiothreitol were added. The dithiothreitol allows the reduction of dehydroascorbic to ascorbic acid and thereby quantifying ascorbic acid as total vitamin C content. Therefore, we quantified the total vitamin C, which includes both molecules. After shaking the samples for 1 h at 4 °C, the samples were centrifuged (5000g, 15 min, 4 °C). The supernatant was collected and the extraction was repeated. The pooled supernatants were filtered (0.2 μm) and analyzed by high-performance liquid chromatography coupled with a diode array detector (UPLC-DAD). The extraction was done in triplicate. For the analysis of potato digests, 0.5 mL of digest was mixed with 0.5 mL of an aqueous solution containing 10% metaphosphoric acid and 2% dithiothreitol, shaken for 30 s at 4 °C, centrifuged (5000g, 5 min, 4 °C), and filtered (0.2 μm) prior to analysis. UPLC-DAD Analysis. The quantification was carried out using a Waters Acquity UPLC system (Milford, MA) equipped with a photodiode array detector. An aliquot of 5 μL was injected onto an Acquity UPLC HSS T3 column (2.1 × 100 mm, 1.8 μm particle size, Waters, Milford, MA) at 40 °C and a flow rate of 0.5 mL/min. The eluents were 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The gradient was as follows: 0 min, 0% B; 1.5 min, 0% B; 2 min, 100% B; 5 min, 100% B; 5.5 min, 0% B; 7 min, 0% B. Ascorbic acid was detected at 243 nm according to its absorption maximum and quantified using a six-point calibration curve. Furthermore, a validation standard was injected after every tenth sample injection. Phenolic Compound Analysis. Extraction. The extraction of phenolic compound was conducted as described previously by Andre et al.8 Briefly, approximately 750 mg of fresh or boiled material was weighed in a 15 mL Falcon tube and 10 mL of methanol/water/acetic acid (80:19.5:0.5, v/v/v). This mixture was homogenized using a vortex for 30 s and shaken for 2 h at room temperature. After centrifugation at 5000g for 15 min at 4 °C, the supernatant was collected and evaporated to dryness in a SpeedVac concentrator (Heto, Thermo Electron Corp., Waltham, MA). Polyphenols were resuspended in 1 mL of an aqueous solution containing 5% methanol and filtered through a 0.2 μm PVDF syringe filter prior to UPLC-DAD injection. For the analysis of potato digests, 1 mL of filtered digest was kept in an amber HPLC vial prior to analysis. UPLC-DAD Analysis. The quantification was carried out as reported by Deußer et al.,29 using the same equipment and column described for the vitamin C analysis. The injection volume was 10 μL, the C


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Journal of Agricultural and Food Chemistry toxicity of the 12 potato tubers digests has been evaluated. As a conclusion, the digests will be diluted 5 times before our iron absorption experiments. At this concentration, there was no significant difference with the negative control (100% cell viability) for any potato clones. Cell Culture and Ferritin Measurement. The TC-7 Caco-2 subclone cells (ATCC No. HTB-37) were a generous gift from Monique Rousset (INSERM, Paris, France). Cells were seeded (50 000 cells/cm2) into six well plates (VWR) and grown in Dulbecco’s modified Eagle’s medium + GlutaMAX (DMEM, Invitrogen, Halle, Belgium) with 10% fetal bovine serum (FBS, Gibco, Halle, Belgium), 1% penicillin/streptomycin (Gibco), and 1% nonessential amino acids (Sigma-Aldrich, St. Louis, MO). Cells were maintained in FBS at 37 °C in an incubator with 10% CO2/90% O2 under constant humidity. Protocol 1. Cells were maintained in DMEM supplemented with 10% FBS, which was changed every couple of days. Cells were used for experiments 14 days after seeding. The integrity of the monolayer was verified by optical microscopy. At 48 h prior to the experiment, the growth medium was removed from culture wells, the cell layer was washed with phosphate buffered saline (PBS), and the growth medium was replaced with minimum essential medium (MEM) at pH 7.0. The MEM was supplemented with 10 mmol/L PIPES, 1% antibiotic/ antimycotic solution, 4 mg/L hydrocortisone, 5 mg/L insulin, 5 μg/L selenium, 34 μg/L triiodothyronine, and 20 μg/L epidermal growth factor, as described by Glahn et al.24 Caco-2 cells were treated with or without 20 μM FeCl3 and 1 mM ascorbic acid to induce ferritin synthesis, as well as with or without solutions containing 20% intestinal filtrate diluted in MEM (v/v). Incubation was done for 4 h. The treatment was then removed and replaced by MEM for 20 h. Protocol 2. After seeding, cells were maintained in DMEM supplemented with 2% FBS, which was changed every couple of days. Cells were used for experiments 14 days after seeding as described in protocol 1. At 48 h prior to the experiment, the growth medium was replaced with supplemented MEM. Caco-2 cells were treated with or without 4 μM FeCl3 and 1 mM ascorbic acid to induce ferritin synthesis, with or without 100 μM FeSO4, as well as with or without solutions containing 10 or 20% intestinal filtrate diluted in MEM (v/v). Incubation was done for 24 h. Cell Harvest and Ferritin Analysis. Cells were lysed in ice-cold extraction buffer: 50 mM HEPES pH 7.6, 50 mM KCl, 50 mM NaF, 5 mM NaPPi, 5 mM EDTA, 5 mM EGTA. Inhibitor cocktail (P-8340, Sigma-Aldrich, St. Louis, MO) and Triton X-100 (1%) were added extemporaneously. The lysate was centrifuged (20 min/16000g/4 °C). Ferritin levels were determined using the human ferritin enzymelinked immunosorbent assay (ELISA) kit provided by Biovendor (Brno, Czech Republic). The plates were read at 450 nm with a luminometer (Synergy 2, Biotek Instruments, Inc.; Drongenbos, Belgium). Results were normalized by total protein concentration measured by spectrophotometry using a Bradford kit (Biorad) and bovine serum albumin (BSA) as a standard. Statistical Analyses. Pearson and Spearman Rank correlation coefficients were determined on (log-transformed, when necessary) data to evaluate relationships between variables. ANOVA and Tuckey tests were carried out on the bioavailable iron (ferritin) values. Principal component analysis (PCA) was performed on standardized data to evaluate the contribution of the different variables (iron, bioactives) to iron bioavailability. For these purposes, the statistical software R (www.r-project.org, version 2014) and SigmaPlot 12.5 (Systat Software Inc., San Jose, CA) were used.

Table 1. Concentration of Iron in Boiled Tubers, Bioaccessible Iron, and Percentage of Iron Released during in Vitro Digestion of 12 Experimental Potato Clonesa accession no.

skin and flesh colorb

CIP_311200.003 CIP_311575.003 CIP_311575.013 CIP_311422.014 CIP_311422.016 CIP_311422.019 CIP_311624.021 CIP_311097.055 CIP_311420.074 CIP_311611.079 CIP_311623.105 CIP_311623.123

P/Cp Y/Y Y/Y P/Cr P/Cp P/Cp R/Y C/Cp C/Cp C/Cr R/Yr C/C

total iron in boiled tissue (μg/g DW) 21.29 25.64 23.44 27.96 31.03 39.47 26.87 31.95 33.64 23.75 36.96 35.50

± ± ± ± ± ± ± ± ± ± ± ±

0.48 1.16 1.86 0.96 2.80 3.69 1.02 3.64 0.51 1.17 0.71 5.79

bioaccessible iron (μg/g DW)

% release

± ± ± ± ± ± ± ± ± ± ± ±

74.33 79.00 68.05 77.33 63.73 72.29 66.29 64.01 70.14 72.06 75.51 64.49

15.83 20.25 15.95 21.62 19.78 28.53 17.81 20.45 23.59 17.12 27.91 22.89

1.45 2.61 1.02 0.49 2.73 0.39 1.97 2.43 0.93 1.25 1.31 2.46

Data are expressed on a dry weight basis and represent mean ± SD (n = 3). bPrimary (in uppercase) and secondary skin color/primary (in uppercase) and secondary flesh colors: P, purple; R, red; Y, yellow; C, cream. a

concentration that is similar to the unique highest value reported for the native Andean landrace CIP_703274.1 The percentage of iron bioaccessibility, i.e. the amount of iron that is released during digestion, was genotype-dependent and varied from 63.7 to 79.0%, with an average value of 70.6% (Table 1). There was a strong positive relationship between the iron concentrations in the boiled tuber and the iron recovered after in vitro digestion (r = 0.93, p < 0.01), suggesting that tuber iron values are good indicators of the bioaccessible fraction. To our knowledge, this is the first report on the bioaccessibility of iron from potato. In vitro bioaccessibility of iron has been evaluated in various cereals and legumes, showing values, in general, that are lower than the ones presented here for potato. Lestienne et al.32 reported iron bioaccessibility of about 10% from whole pearl millet flour, which increased to 24% after dephytinization. The bioaccessibility of iron from fava bean, soybean, and rice has also been shown to increase from 6.3 to 31.5%, from 6.2 to 30.7%, and from 5.6 to 19%, respectively, after phytate degradation through germination.33 Wheat iron bioaccessibility was low (4.6%), with no improvement seen after dephytinization.33 It is worth stressing that our study was performed on cooked and pureed material, which may not be the case for the above-mentioned studies. Our in vitro digestion procedure allowed us to estimate that the consumption of 200 g of potato tubers triggers mean iron concentrations of 20 μM at the intestinal level (considering that potato is diluted in 1 L of gastrointestinal fluids). It is worth noting that these values represent the total bioaccessible iron content and do not reflect the bioavailable iron. Also, this measurement does not give any information about iron species. On top of this, potato is a complex matrix containing numerous components that may improve or inhibit iron absorption.6 Absorption of non-heme iron can be improved or impeded by the binding of iron to compounds such as chelators and ligands that create complexes. Vitamin C, along with citric acid, has been described as a potent enhancer of iron uptake.34 On the other hand, oxalic acid, phytates (as described earlier), calcium, and zinc, as well as some phenolic compounds, may reduce iron absorption.28 Although less well-documented, carotenoids may interact with minerals including iron and form precipitates that

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RESULTS AND DISCUSSION Concentration and Bioaccessibility of Iron, Vitamin C, Carotenoids, and Phenolics in Boiled Potatoes. The total iron concentrations in boiled peeled potato tubers ranged from 21.29 μg/g dry weight (DW) for the clone CIP_311200.003 to 39.47 μg/g DW for the clone CIP_311422.019, with an average value of 29.9 μg/g DW (Table 1). Five of the biofortified clones evaluated showed iron concentrations above 30 μg/g, a D


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Journal of Agricultural and Food Chemistry could potentially reduce their absorption.35 In this study, we focused on the predominant potato phytochemicals, i.e. vitamin C, phenolic compounds, and carotenoid compounds. As was done for iron, we determined the levels of these compounds in boiled material tubers well as in intestinal digests. The average total vitamin C concentration in boiled potato tubers was 526 μg/g DW (Table 2). About 32% of this content

intestinal digest. This is likely due to the in vitro digestion method we used, which created favorable conditions for watersoluble compounds and limiting ones for the production of mixed micelles, essential for lipophilic compounds such as carotenoids. The bioaccessibility of carotenoids (micellarization) is largely enhanced when dietary fat is consumed together with carotenoids. In addition, the concentrations in β-carotene were already low in boiled potatoes. Indeed, the β-carotene level is usually negligible in the tubers of commercial potato varieties whereas it is detected in low but quantifiable amounts in some native Andean cultivars.8 The phenolic profile of potato extracts was dominated by chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, caffeic acid, rutin, kaempferol-3-rutinoside, and ferulic acid as a minor component (Table 4). The presence of anthocyanins was also detected in purple-fleshed clones. The mean bioaccessibility value for total chlorogenic acids (sum of the three chlorogenic acid isomers) was 24.5%, with clones CIP_311623.123 and CIP_311611.079 showing the highest bioaccessibility values (82 and 65%, respectively). Similarly, the bioaccessibility of total chlorogenic acids evaluated in another study on apple cultivars ranged from 32 to 57%.40 Using two Luxembourg-grown potato varieties, Miranda et al.28 showed however higher concentrations of chlorogenic acids in the intestinal filtrate than in boiled potatoes. It is worth noting that some components such as rutin and kaempferol-3-rutinoside were not detectable in boiled tubers of some genotypes under investigation but were released during digestion, suggesting that a traditional solvent extraction on boiled material may also underestimate the presence of phenolic compounds that are actually bioaccessible. The bioaccessibility of anthocyanins across the purple-fleshed clones ranged from 40 to 81%, which is similar to the recovery rate observed in chokeberries.41 Much lower bioaccessibility values have however been reported in the literature with regard to anthocyanins, due to their high instability in the mild alkaline intestinal environment.40 Discrepancies could be explained by the variability of the in vitro digestion protocols and also by the type of anthocyanins present in the plant matrix. Interestingly, most anthocyanins present in purple-fleshed potato tubers are acylated with hydroxycinnamic acids, which are known to increase the stability of the compounds.8 No significant overall correlation was evident between iron bioaccessibility and vitamin C, carotenoid, and phenolic concentrations. However, the highest iron bioaccessibility was observed in the clone CIP_311575.003 that also presented the highest vitamin C concentration, while the lowest bioaccessibility values were observed in three purple-fleshed clones with high levels of anthocyanins and chlorogenic acid (CIP_311422.016, CIP_311097.055, and CIP_311420.074). Evaluation of the in Vitro Bioavailability of Iron from Boiled Potatoes. We used an in vitro Caco-2 cell culture model to evaluate iron bioavailability from potato tubers by measuring ferritin formation in the cells (i.e., a measure of cell iron uptake) following exposure to digests of the samples. The toxicity of the digests of the 12 genotypes was first evaluated and the cell viability was above 70% for all accessions when used at 20% on the cells. Following classical culture conditions (protocol 1), ferritin values for the control (MEM only) were about 9 ng/mg of protein. As expected, ferritin synthesis was induced up to 190 ng/mg of protein in Caco-2 cells supplemented with 20 μM FeCl3 and 1 mM vitamin C. These values are in agreement with those described in the

Table 2. Concentration of Vitamin C in Boiled Potato Tubers and Bioaccesible Vitamin C of 12 Experimental Potato Clonesa accession no. CIP_311200.003 CIP_311575.003 CIP_311575.013 CIP_311422.014 CIP_311422.016 CIP_311422.019 CIP_311624.021 CIP_311097.055 CIP_311420.074 CIP_311611.079 CIP_311623.105 CIP_311623.123 a

vitamin C in boiled tissue (μg/g DW)

bioaccessible vitamin C (μg/g DW)

± ± ± ± ± ± ± ± ± ± ± ±

281.80 ± 29.63 402.52 ± 67.88 179.18 ± 4.72 106.00 ± 4.19 156.16 ± 19.10 132.71 ± 7.28 112.38 ± 20.60 80.48 ± 5.63 89.22 ± 2.70 138.18 ± 14.02 156.81 ± 11.31 166.15 ± 1.49

754.43 873.46 413.02 335.58 411.30 369.26 333.80 496.21 532.16 656.35 412.52 718.05

33.25 37.70 17.71 27.57 55.50 19.73 23.32 16.98 35.80 13.45 51.27 73.56

Data are expressed on a dry weight basis ± SD (n = 3).

remained in the intestinal digest. This reduction is likely due to the instability of the compound at the pH and temperature conditions of the in vitro digestion procedure and/or the reduced extent of release from the potato matrix. To our knowledge, this is the first report on vitamin C bioaccessibility from potato tubers. Higher levels of degradation have been observed in tomatoes,36 broccoli,37 and blended fruit juice,38 showing bioaccessibility values of 0, 9, and 15%, respectively, which could be due to differences in the matrix composition but also in the gastrointestinal digestion protocol. The potato carotenoid pattern was dominated by lutein and zeaxanthin, followed by antheraxanthin, violaxanthin, neoxanthin, and β-carotene (Table 3). Their identification was confirmed by comparison of their retention times and UV spectra with those of authentic standards. The clones CIP_311575.003, CIP_311575.013, and CIP_311623.105 presented the highest total carotenoid contents and the highest zeaxanthin concentrations in particular. The bioaccessibility of lutein and zeaxanthin in the 12 biofortified clones ranged from 53 to 160% and from 24 to 388%, respectively. The bioaccessibility of lutein and zeaxanthin in the yellow clones (CIP_311575.003, CIP_311575.013, and CIP_311623.123) ranged from 76 to 82% for lutein and from 24 to 55% for zeaxanthin. In a recent study, Burgos et al.39 estimated that the bioaccessibility of lutein and zeaxanthin in yellow fleshed potatoes ranged from 33 to 71% and from 51 to 71%, respectively. Interestingly, the gastrointestinal digestion process allowed release of zeaxanthin from the potato matrix of five clones to a greater extent than that observed in the respective boiled samples. Chemical extraction from boiled material may therefore underestimate carotenoid amounts that can be released during digestion and that are actually bioaccessible. On the other hand, the efficiency of micellarization or bioaccessibility of the other carotenoids could not be determined, as they were below the detection limit in the E


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Table 3. Concentration of Carotenoid in Boiled Potato Tubers and Bioaccessible Carotenoids of 12 Experimental Potato Clonesa accession no. CIP_311200.003 boiled bioaccessible CIP_311575.003 boiled bioaccessible CIP_311575.013 boiled bioaccessible CIP_311422.014 boiled bioaccessible CIP_311422.016 boiled bioaccessible CIP_311422.019 boiled bioaccessible CIP_311624.021 boiled bioaccessible CIP_311097.055 boiled bioaccessible CIP_311420.074 boiled bioaccessible CIP_311611.079 boiled bioaccessible CIP_311623.105 boiled bioaccessible CIP_311623.123 boiled bioaccessible a

antheraxanthin (μg/g DW)

β-carotene (μg/g DW)

neoxanthin (μg/g DW)

ndb nd

0.56 ± 0.02 nd

0.34 ± 0.04 nd

0.33 ± 0.08 nd

19.62 ± 1.86 10.90 ± 5.61

0.40 ± 0.04 nd

5.83 ± 0.70 nd

0.61 ± 0.08 nd

2.09 ± 0.26 nd

2.57 ± 0.13 2.06 ± 0.42

23.65 ± 2.09 5.70 ± 5.81

nd nd

1.41 ± 0.16 nd

0.36 ± 0.01 nd

0.67 ± 0.05 nd

36.9

4.10 ± 0.13 2.16 ± 0.29

1.74 ± 0.19 1.50 ± 0.53

0.29 ± 0.02 nd

nd nd

0.32 ± 0.02 nd

0.30 ± 0.05 nd

25.8

6.56 ± 0.17 5.61 ± 0.44

nd 4.39 ± 0.55

nd nd

nd nd

nd nd

nd nd

24.8

5.08 ± 0.23 4.10 ± 1.71

0.90 ± 0.08 3.51 ± 1.50

0.47 ± 0.04 nd

nd nd

0.52 ± 0.05 nd

0.67 ± 0.12 nd

24.7

4.22 ± 0.61 6.77 ± 0.72

2.62 ± 0.13 5.11 ± 1.64

0.31 ± 0.05 nd

nd nd

0.39 ± 0.09 nd

0.27 ± 0.08 nd

29.1

4.10 ± 0.51 3.78 ± 0.42

2.65 ± 0.52 2.73 ± 0.27

nd nd

0.77 ± 0.06 nd

0.34 ± 0.03 nd

0.00 ± 0.00 nd

22.8

5.62 ± 0.39 4.41 ± 0.24

nd 2.63 ± 0.66

nd nd

nd nd

0.48 ± 0.02 nd

0.63 ± 0.14 nd

24.9

3.37 ± 0.97 3.01 ± 0.81

6.58 ± 1.19 6.30 ± 1.15

0.41 ± 0.09 nd

nd nd

nd nd

nd nd

25.4

2.35 ± 0.27 1.94 ± 0.99

41.50 ± 2.23 11.87 ± 4.71

0.38 ± 0.13 nd

0.67 ± 0.05 nd

nd nd

0.43 ± 0.03 nd

22.7

3.98 ± 0.25 3.03 ± 0.70

14.05 ± 2.62 7.44 ± 1.30

0.54 ± 0.04 nd

nd nd

nd nd

0.43 ± 0.04 nd

DM (%)

lutein (μg/g DW)

zeaxanthin (μg/g DW)

violaxanthin (μg/g DW)

26.2

3.84 ± 0.21 2.80 ± 0.29

1.97 ± 0.12 2.32 ± 0.41

21.4

6.43 ± 0.21 5.31 ± 0.90

30.9

Data are expressed on a dry weight basis ± SD (n = 3). The dry matter content (DM) is also presented. bnd, compound not detected.

literature.28 There were no significant changes in ferritin concentrations when the cells were exposed to an in vitro digestion blank as compared to the control values, indicating that the variations in ferritin levels observed from potato digests are due to their constituents and not to the digestion enzyme mixture. Intrinsic iron bioavailability from the digests of the 12 genotypes was first evaluated. Ferritin concentrations were not significantly different from the baseline (control exposed to MEM only, data not shown). Similar results have previously been reported with this Caco-2 in vitro model and high phenolic-containing food such as beans.25,42 The influence of potato components on the uptake of extrinsic iron was then investigated. The data for the Caco-2 cell ferritin formation following exposure to the digests of the 12 genotypes are summarized in Figure 2. The data are presented as the percentage of the response of the positive control (exposure to digest blank containing 20 μM FeCl3 and 1 mM vitamin C). In the presence of 20% potato digest, the iron absorption ranged from 1.8% for the clone CIP_311422.016 to 8% for CIP_311624.021 as compared to the reference control. It

appears therefore that potato digests host components that may interact with iron and reduce its absorption. Significant differences were found between the genotypes, with CIP_311200.003 and CIP_311624.021 exhibiting higher in vitro iron bioavailability when compared with the remaining genotypes. On the other hand, the purple-fleshed clones with the highest levels of total anthocyanins and chlorogenic acid (CIP_311422.016, CIP_311097.055, CIP_311420.074, and CIP_311422.014) showed the lowest iron bioavailability. In order to identify the major contributors or inhibitors of the in vitro iron bioavailability, a principal component analysis (PCA) was performed on the total iron content, bioactive composition, and ferritin values (Figure 3). The first two principal components (PC) accounted for 51.37% of the total variance. There was no association between variation for cell ferritin and total iron content. Interestingly, cell ferritin formation was associated with total vitamin C content and was inversely related to the phenolic compound levels, in particular total anthocyanins, caffeic acid, rutin, and kaempferol-3-rutinoside, as well as to chlorogenic acid. F


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Table 4. Concentration of Phenolics in Boiled Potato Tubers and Bioccessible Phenolics in 12 Experimental Potato Clonesa accession no. CIP_311200.003 boiled bioaccessible CIP_311575.003 boiled bioaccessible CIP_311575.013 boiled bioaccessible CIP_311422.014 boiled bioaccessible CIP_311422.016 boiled bioaccessible CIP_311422.019 boiled bioaccessible CIP_311624.021 boiled bioaccessible CIP_311097.055 boiled bioaccessible CIP_311420.074 boiled bioaccessible CIP_311611.079 boiled bioaccessible CIP_311623.105 boiled bioaccessible CIP_311623.123 boiled bioaccessible a

total anthocyanins (μg/g DW)

chlorogenic acid (μg/g DW)

neochlorogenic acid (μg/g DW)

cryptochlorogenic acid (μg/g DW)

ferulic acid (μg/g DW)

rutin (μg/g DW)

kaempferol-3rutinoside (μg/g DW)

206.9 ± 168.6 167.0 ± 23.2

835.5 ± 207.3 139.9 ± 17.9

133.5 ± 61.9 8.7 ± 8.2

433.8 ± 162.3 5.7 ± 3.2

22.4 ± 9.9 5.6 ± 0.7

3.3 ± 1.9 1.0 ± 0.3

7.0 ± 1.6 5.9 ± 0.7

10.5 ± 3.8 9.7 ± 0.3

ndb nd

925.9 ± 102.8 190.9 ± 94.2

148.5 ± 4.2 4.8 ± 2.1

495.4 ± 37.2 53.2 ± 9.9

nd 9.9 ± 1.1

4.7 ± 2.3 1.0 ± 0.1

0.0 ± 0.0 3.0 ± 0.1

nd 4.3 ± 0.2

nd nd

474.1 ± 163.8 76.0 ± 7.3

116.9 ± 38.2 4.8 ± 1.7

326.5 ± 112.3 50.3 ± 21.9

19.0 ± 5.2 4.3 ± 0.4

2.1 ± 0.6 0.9 ± 0.0

nd 1.9 ± 0.0

nd 3.1 ± 0.1

637.24 ± 35.4 393.4 ± 26.8

2290.4 ± 338.5 756.61 ± 33.15

401.5 ± 60.2 102.5 ± 4.5

1254.9 ± 185.9 78.6 ± 13.8

32.8 ± 4.1 7.5 ± 0.5

1.7 ± 0.3 0.6 ± 0.1

3.8 ± 0.1 4.5 ± 0.3

44.1 ± 5.2 33.5 ± 3.8

744.5 ± 76.6 463.0 ± 74.9

2413.8 ± 104.7 448.4 ± 68.4

520.1 ± 26.2 8.1 ± 8.5

1552.1 ± 79.7 3.7 ± 0.2

47.5 ± 2.1 11.4 ± 1.3

2.6 ± 0.9 1.1 ± 0.6

11.4 ± 5.7 3.6 ± 1.6

33.0 ± 1.2 29.6 ± 5.1

310.2 ± 42.5 224.9 ± 13.8

1030.1 ± 109.8 167.3 ± 17.9

157.3 ± 21.4 18.7 ± 1.1

515.1 ± 62.0 5.5 ± 0.3

61.2 ± 1.3 10.5 ± 2.2

2.1 ± 0.1 1.6 ± 0.1

10.0 ± 1.3 7.0 ± 4.3

30.5 ± 4.7 23.7 ± 2.7

nd nd

663.2 ± 273.3 81.0 ± 24.0

131.1 ± 28.3 14.6 ± 2.2

382.7 ± 109.6 4.2 ± 0.2

8.6 ± 4.3 3.0 ± 1.1

2.2 ± 0.5 0.9 ± 0.2

8.1 ± 2.9 5.1 ± 1.8

13.0 ± 2.4 8.9 ± 2.2

728.7 ± 50.7 432.5 ± 51.06

3364.3 ± 402.7 660.1 ± 49.9

575.6 ± 55.5 71.0 ± 16.4

1828.8 ± 194.2 3.6 ± 0.3

68.2 ± 12.8 200.8 ± 18.9

3.5 ± 1.8 1.6 ± 0.2

9.1 ± 4.9 15.9 ± 1.9

7.0 ± 0.8 22.0 ± 2.1

1440.5 ± 302.7 578.3 ± 89.7

3027.8 ± 185.0 450.0 ± 10.2

324.1 ± 24.9 35.3 ± 10.5

1189.2 ± 73.7 8.3 ± 3.7

50.1 ± 10.0 9.5 ± 4.8

4.1 ± 0.2 1.4 ± 0.4

6.9 ± 0.3 4.8 ± 1.1

40.9 ± 0.9 29.0 ± 7.6

nd nd

828.2 ± 58.2 667.6 ± 17.4

61.1 ± 6.5 74.4 ± 9.9

257.1 ± 127.3 4.1 ± 0.3

19.8 ± 6.5 10.3 ± 2.3

4.6 ± 3.4 1.6 ± 0.3

nd 2.2 ± 0.1

nd 4.2 ± 0.3

nd nd

317.9 ± 63.2 283.6 ± 10.9

88.8 ± 4.8 31.5 ± 1.2

35.5 ± 37.7 2.8 ± 0.2

7.1 ± 0.9 9.1 ± 1.0

2.1 ± 0.1 1.1 ± 0.0

7.5 ± 0.4 4.1 ± 0.1

nd 10.8 ± 1.1

nd nd

507.5 ± 32.3 426.8 ± 61.0

83.3 ± 8.2 65.0 ± 14.3

268.6 ± 26.6 212.6 ± 47.8

45.6 ± 6.9 9.7 ± 0.8

6.2 ± 7.3 6.4 ± 6.5

nd 2.4 ± 0.1

nd 4.3 ± 0.2

caffeic acid (μg/g DW)

Data are expressed on a dry weight basis ± SD, respectively (n = 3). bnd, compound not detected.

Effect of Potato Digests on Iron Uptake Using IronDeprived Caco-2 Cells. In order to mimic anemic conditions and thereby increase the reactivity of the cells when exposed to iron, Caco-2 cells were cultured in low-iron media postseeding (see Protocol 2 under Material and Methods). The basal ferritin level was successfully reduced from 9 to 3 ng/mg of protein. Cells appeared much more reactive, showing an increase of ferritin expression up to 174 ng/mg of protein when exposed to 4 μM FeCl3 (+1 mM vitamin C), an induction similar to the one we previously obtained with 20 μM FeCl3 (and 1 mM vitamin C) following protocol 1. The value of 4 μM FeCl3 is also the average iron concentration found in potato digests after a 5-fold dilution as used in our Caco-2 experiments. Using four contrasting genotypes in terms of iron and bioactive composition (Figure 3, genotypes in bold), the intrinsic iron bioavailability from 20% potato digest was evaluated, with no significant induction of ferritin (data not shown), as previously described with protocol 1. It appears that even with iron-deprived cells the inhibition of iron uptake is strong. The addition of 4 μM FeCl3 and 1 mM ascorbic acid induced ferritin expression, with iron uptake ranging from 4%

for the clone CIP_311422.014 to 6.2% for CIP_311624.021 (Figure 4). Ferritin synthesis was further increased when the potato digests were diluted down to 10% of the media, confirming the inhibitory role of potato constituents. It is noteworthy that the yellow-fleshed clone CIP_311624.021 triggered consistently higher in vitro iron bioavailability as compared to its counterparts, suggesting a reduced level of inhibitors (including phenolic compounds) in that cultivar. Conclusions. We showed that more than 50% of the potato iron is released from the matrix during the in vitro digestion and is therefore available at the intestinal level. Hydrophilic bioactive components such as vitamin C and phenolic compounds in potato tubers are also bioaccessible to a large extent. The intestinal absorption of intrinsic iron from potato tubers could however not be detected using our in vitro Caco-2 cell model. Inhibition of iron uptake is typically reported for foods containing high levels of phenolic compounds, which could partly explain our results. Only human studies will provide a clear and valid percentage of iron absorption from potatoes. Indeed, a recent human study showed significant iron absorption from test meals based on pearl millet (7.5%),43 G


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Figure 4. Iron bioavailability from four potato clones as measured by ferritin expression in Caco-2 cells grown under low iron conditions. Ferritin expression was evaluated by an ELISA assay and normalized by total protein concentrations. Caco-2 cells were exposed to 10 or 20% potato digest supplemented with iron (4 μM FeCl3) and ascorbate (1 mM). The data are presented as percentages of the positive control (exposure to digest blank containing 4 μM FeCl3 and 1 mM vitamin C). Values are means ± SEM (n = 9, resulting from three independent experiments). There was no significant difference between the genotypes.

Figure 2. Iron bioavailability from 12 potato clones as measured by ferritin expression in Caco-2 cells. Ferritin expression was evaluated by an ELISA assay and normalized by total protein concentration. Caco-2 cells were exposed to potato digest supplemented with iron (20 μM FeCl3) and ascorbate (1 mM). The data are presented as percentage of the positive control (exposure to digest blank containing 20 μM FeCl3 and 1 mM vitamin C). Values are means ± SEM (n = 9, resulting from three independent experiments). Bar values with no letter in common are significantly different (p < 0.05).

multivariate analyses revealed opposing relationships between the bioavailable iron values (as determined by cell ferritin formation) and the phenolic concentrations. In our assay procedure, carotenoid concentrations did not influence iron bioavailability, whereas the potato vitamin C was positively correlated with the ferritin values. The determination of the iron content in cooked potato tubers was not a good indicator of iron bioavailability, as evaluated by our Caco-2 cell-based model. Indeed, the values of bioavailable iron are the result of various and complex interactions that could be attributed to endogenous iron, to phenolic compounds, to other non-

whereas this food matrix is known to contain high levels of iron absorption inhibitors such as phytates and phenolic compounds. Tako et al.42,44 also showed that the Caco-2 model predicted well animal iron absorption results for bean and pearl millet meals. The Caco-2 cell model is an advantageous preliminary in vitro screening method to efficiently identify promising clones in terms of iron bioavailability. Indeed, when an extrinsic source of iron was added (FeCl3) to the digestion mixture, ferritin synthesis (marker of iron uptake) was noticed to an extent depending on the genotype. Nonsupervised

Figure 3. Principal component analysis (PCA) resulting from the in vitro iron bioavailability evaluation of 12 potato clones. (A) Score plots of the 12 potato genotypes. (B) Loading plot showing the relationships between the 13 variables. H


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identified molecules, and to synergistic as well as antagonistic effects among all these components. Such models may help plant breeders interested in increasing the bioavailable levels of iron and/or reducing the amount and type of iron uptake inhibitors in staple food crops such as potato. Deeper metabolite profiling studies on potato tubers, including phytate measurements, should also be performed to better identify other potential iron absorption inhibitors. Controlled human intervention or animal trials will however be needed to improve our knowledge on iron absorption from potato.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Funding

This study was partly supported by the CGIAR Research Program on Agriculture for Nutrition and Health (CRPA4NH). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Aude Corvisy for her help in in vitro digestion and cell culture analyses, Boris Untereiner for his help in phytochemical extractions, and Walter Amoros for providing the biofortified potato clones in Peru.

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ABBREVIATIONS USED DW, dry weight; PCA, principal component analysis; MEM, minimum essential medium; SD, standard deviation; SEM, standard error of the mean; P, purple; R, red; Y, yellow; C, cream; nd, compound not detected; DM, dry matter

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REFERENCES

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In Vitro Bioaccessibility and Bioavailability of Iron from Potatoes with

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